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ADVANCES IN PROTEIN CHEMISTRY Volume 56 Drug Discovery and Design

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ADVANCES IN PROTEIN CHEMISTRY EDITED BY FREDERIC M. RICHARDS

DAVID S. EISENBERG

Department of Molecular Biophysics and Biochemistry Yale University New Haven, Connecticut

Department of Chemistry and Biochemistry University of California, Los Angeles Los Angeles, California

PETER S. KIM Department of Biology Massachusetts Institute of Technology Whitehead Institute for Biomedical Research Howard Hughes Medical Institute Research Laboratories Cambridge, Massachusetts

VOLUME 56

Drug Discovery and Design EDITED BY EDWARD M. SCOLNICK Merck and Company, Inc., West Point, Pennsylvania

ACADEMIC PRESS San Diego London Boston New York Sydney Tokyo Toronto

This book is printed on acid-free paper. Copyright © 2001 by ACADEMIC PRESS All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the Publisher. The appearance of the code at the bottom of the first page of a chapter indicates the Publisher’s consent that copies of the chapter may be made for personal or internal use of specific clients. This consent is given on the condition, however, that the copier pay the stated per copy fee through the Copyright Clearance Center, Inc. (222 Rosewood Drive, Danvers, Massachusetts 01923) for copying beyond that permitted by Sections 107 or 108 of the U.S. Copyright Law. This consent does not extend to other kinds of copying, such as copying for general distribution, for advertising or promotional purposes, for creating new collective works, or for resale. Copy fees for pre-2000 chapters are as shown on the title pages. If no fee code appears on the title page, the copy fee is the same as for current chapters. 0065-3233/01 $35.00 Explicit permission from Academic Press is not required to reproduce a maximum of two figures or tables from an Academic Press chapter in another scientific or research publication provided that the material has not been credited to another source and that full credit to the Academic Press chapter is given.

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00 01 02 03 04 05 SB 9 8 7 6 5 4 3 2 1

CONTENTS

PREFACE

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xi

“Natural History” Clinical Trials: An Enduring Contribution to Modern Medical Practice EDWARD M. SCOLNICK, EVE E. SLATER, AND GEORGE W. WILLIAMS I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . II. Role of the Pharmaceutical Industry in Clinical Trials . . . III. Use of “Natural History” RCTs to Validate the Cholesterol Hypothesis and Support Changes in the Management of Other Conditions . . . . . . . . . . . . . . . . . . . . . . . IV. Development of a New Chemical Entity . . . . . . . . . . . V. “Natural History” RCTs: Some Considerations . . . . . . . VI. Patient Safety. . . . . . . . . . . . . . . . . . . . . . . . . . VII. Clinical Trials and the Practice of Medicine in the Age of Genomics . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Angiotensin-Converting Enzyme Inhibitors JOEL MENARD AND ARTHUR PATCHETT I. II. III. IV. V.

Introduction . . . . Peptide Inhibitors . Captopril . . . . . . Enalapril . . . . . . Lisinopril . . . . . .

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VI. Fosinopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Clinically Available ACE Inhibitors. . . . . . . . . . . . . . . . . . VIII. Contribution of ACE Inhibitors to the Growth of Physiological and Pathophysiological Knowledge . . . . . . . . . . . . . . . . . IX. Biological Advances in the Knowledge of ACE That Evolved in Parallel with the Drug Development Process . . . . . . . . . . . . X. Clinical Development Process of ACE Inhibitors in Hypertension XI. Benefits of ACE Inhibition Beyond the Fall in Blood Pressure . . XII. ACE Inhibitors and Congestive Heart Failure. . . . . . . . . . . . XIII. ACE Inhibitors and Myocardial Infarction . . . . . . . . . . . . . XIV. ACE Inhibitors, Coronary Heart Disease, and Atherosis . . . . . . XV. ACE Inhibitors and Prevention of Restenosis . . . . . . . . . . . . XVI. ACE Inhibitors and Renal Insufficiency . . . . . . . . . . . . . . . XVII. The Fallacy of the Concepts of Normotension and Hypertension and the Cardiovascular Protective Effects of ACE Inhibitors . . . XVIII. Surrogate End Points in Clinical Trials of ACE Inhibition: Are We Being Misled? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XIX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. Background and History . . . . . . . . . . . . . . . . . . . . . . . . II. Effects of Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . III. Mechanisms of the Cholesterol-Lowering Effects of Reductase Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Combination Therapy. . . . . . . . . . . . . . . . . . . . . . . . . . V. Safety and Tolerability. . . . . . . . . . . . . . . . . . . . . . . . . . VI. Outcome Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Mechanisms of the Reduction in Coronary Morbidity and Mortality VIII. Safety of HMG-CoA Reductase Inhibitors in the Megatrials . . . . IX. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

77 84

HMG-CoA Reductase Inhibitors ROGER ILLINGWORTH AND JONATHAN A. TOLBERT

89 89 91 98 100 102 105 108

Cyclooxygenase-2 Inhibitors ALAN S. NIES I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

115 116

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III. IV. V. VI. VII. VIII.

Assays for Cyclooxygenase-2 Selective Inhibitors . . . Selectivity of Cyclooxygenase Inhibitors . . . . . . . . Enzymology/Medicinal Chemistry . . . . . . . . . . . Clinical Development of Cyclooxygenase-2 Inhibitors Future Directions . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . .

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5α-Reductase Inhibitors JOHN D. MCCONNELL AND ELIZABETH STONER I. II. III. IV. V. VI. VII.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . Identification and Characterization of 5α-Reductase . Development of 5α-Reductase Inhibitors . . . . . . . . Clinical Studies in Men with Androgenic Disorders . . Clinical Studies in Women with Androgenic Disorders Other 5α-Reductase Inhibitors . . . . . . . . . . . . . . Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

Peroxisome Proliferator-Activated Receptor (PPAR)γ Agonists for Diabetes DAVID E. MOLLER AND DOUGLAS A. GREENE I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Mechanism of Action of Peroxisome Proliferator-Activated Receptor (PPAR)γ Agonists . . . . . . . . . . . . . . . . . . . III. Clinical Experience with PPARγ Agonists . . . . . . . . . . . IV. Conclusions and Future Directions . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Discovery and Clinical Development of HIV-1 Protease Inhibitors JOEL HUFF AND JAMES KAHN I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Selection and Validation of HIV-1 Protease as a Therapeutic Target . III. Development of HIV-1 Protease Inhibitors . . . . . . . . . . . . . .

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CONTENTS

IV. V. VI. VII. VIII. IX. X.

Structure-Based Design . . . . . . . . . . . . . . . . . . . Inhibitor Identification through Broad-Based Screening Mechanism-Based Strategy . . . . . . . . . . . . . . . . . Future Directions for Discovery . . . . . . . . . . . . . . HIV-1 Protease Inhibitors: The Clinical Perspective . . . Clinical Development Milestones . . . . . . . . . . . . . Issues of Ongoing Concern for the Clinical Use of HIV-1 Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . XI. Rational Treatment Combinations That Include HIV-1 Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . XII. Future Considerations for HIV-1 Protease Inhibitors. . . XIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

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Calcineurin Inhibitors and the Generalization of the Presenting Protein Strategy KURT W. VOGEL, ROGER BRIESEWITZ, THOMAS J. WANDLESS, AND GERALD R. CRABTREE I. Calcineurin, Calcineurin Inhibitors, and the Effects of Inhibition of Calcineurin . . . . . . . . . . . . . . . . . II. Inhibition by Immunophilin/Immunosuppressant Complexes: The Presenting Protein Strategy . . . . . . III. Generalization of the Presenting Protein Strategy . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . .

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Pure Selective Estrogen Receptor Modulators, New Molecules Having Absolute Cell Specificity Ranging from Pure Antiestrogenic to Complete Estrogen-Like Activities FERNAND LABRIE, CLAUDE LABRIE, ALAIN BÉLANGER, VINCENT GIGUERE, JACQUES SIMARD, YVES MÉRAND, SYLVAIN GAUTHIER, VAN LUU-THE, BERNARD CANDAS, CELINE MARTEL, AND SHOUQI LUO I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Women’s Health Needs . . . . . . . . . . . . . . . . . . . . . . . . . III. The Estrogen Receptors and Their Multiple Gene Activation Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . .

294 295 303

ix

CONTENTS

IV. Classes of Antiestrogens. . . . . . . . . . . . . . . . . . . . . . . . . V. Properties of EM-652 (SCH 57068) and EM-800 (SCH 57050) . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

313 319 357

Monoclonal Antibody Therapy JOHN W. PARK AND JOSEF SMOLEN I. II. III. IV. V. VI.

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . General Aspects of Monoclonal Antibody Therapy. . . . . Monoclonal Antibody Therapy in Organ Transplantation . Monoclonal Antibody Therapy in Cardiac Disease . . . . . Monoclonal Antibody Therapy in Infectious Diseases . . . Monoclonal Antibody Therapy in Rheumatologic and Autoimmune Diseases. . . . . . . . . . . . . . . . . . . . . VII. Monoclonal Antibody Therapy of Cancer . . . . . . . . . . VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . .

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477 553

Glucan Synthase Inhibitors as Antifungal Agents MYRA B. KURTZ AND JOHN H. REX I. II. III. IV. V. VI. VII.

Introduction and Background . . . . . . . . . . . . . . . The Fungal Cell Wall Is An Attractive Target . . . . . . . Early Research on Cell-Wall Active Agents . . . . . . . . The Pneumocandins: Mycology and Parasitology Collide Development of Amino Compounds . . . . . . . . . . . Current Compounds in Clinical Development . . . . . . Outlook. . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . .

AUTHOR INDEX SUBJECT INDEX

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PREFACE

In the past two decades, several important new medicines have been discovered and developed for patients. Major advances have been made in the treatment of hypertension, atherosclerosis, osteoporosis, diabetes, AIDS, and arthritis. Advances in biomedical science have provided the understanding of disease processes and the technology to foster these discoveries. This book recounts the basic and clinical work that led to some of the most important new treatments. With the advent of the genomic era in biomedicine, we can look forward to many more treatment advances.

Edward M. Scolnick

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“NATURAL HISTORY” CLINICAL TRIALS: AN ENDURING CONTRIBUTION TO MODERN MEDICAL PRACTICE BY EDWARD M. SCOLNICK,* EVE E. SLATER, AND GEORGE W. WILLIAMS Merck Research Laboratories, Rahway, New Jersey 07065

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Role of the Pharmaceutical Industry in Clinical Trials . . . . . . . . . . . . . . . . . . . III. Use of “Natural History” RCTs to Validate the Cholesterol Hypothesis and Support Changes in the Management of Other Conditions . . . . . . . . . . . IV. Development of a new Chemical Entity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. “Natural History” RCTs: Some Considerations. . . . . . . . . . . . . . . . . . . . . . . . . . VI. Patient Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Clinical Trials and the Practice of Medicine in the Age of Genomics . . . . . . . References. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

1 2 2 3 8 9 10 11

I. INTRODUCTION Uncertainty is an inextricable part of medical practice. Despite the significant medical advances of the past century, there is much that remains unknown. Strategies to reduce medical uncertainty and build evidence therefore have become critical to the advancement of medical knowledge and modern medical practice. Accordingly, a key question is: How do physicians and patients understand the merits of various medical interventions? The development of the randomized controlled trial (RCT) to evaluate the impact of medical interventions on the natural history of disease has provided answers to this question by improving the evidence base of medicine. Looking to the future, the advent of genomics coupled with advances in information technology promises to enhance the power of RCTs to generate medical knowledge about both existing and new therapies. To reap the fruits of genomics and other advances, however, RCTs—with the necessary precautions and safeguards—must continue unfettered. Although many examples of clinical investigation can be found throughout the history of medicine, the RCT emerged in the mid-20th century as the most powerful and scientifically sound way to establish the efficacy and safety of medicines. In 1948, Austin Bradford Hill used the statistical method of randomization with concealment of the allocation code to reduce biases related to selection and analysis of patients * Correspondence should be addressed to E. Scolnick, MRL, 126 East Lincoln Avenue, PO Box 2000, Rahway, NJ 07065. 1 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0065-3233/01 $35.00

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EDWARD M. SCOLNICK, EVE E. SLATER, AND GEORGE W. WILLIAMS

in his study of streptomycin for tuberculosis infection (1); Amberson had pioneered the method almost 20 years earlier (2). Today, as the gold standard by which the merits of drug therapy must be measured, RCTs provide the major scientific support for the contemporary practice of medicine. The data provided by these studies must be integrated with the physician’s clinical expertise through conscientious, explicit, and judicious use of current best evidence in making decisions about the care of individual patients (3). In other words, sound statistical principles on which clinical trials are based aid the treating physician in making daily decisions in the oft uncertain management or prevention of disease. II. ROLE OF THE PHARMACEUTICAL INDUSTRY IN CLINICAL TRIALS Research supported by academic, government, and philanthropic institutions and the pharmaceutical industry has always sustained physicians in their search for medical certainty. For the most part, academic and public institutions have focused on basic research, whereas industry has focused on applying the basic fundamentals to the development of medicines (4). In the latter context, the pharmaceutical industry, responsible for a large number of clinical trials, has made a great contribution to advancing scientific knowledge by establishing the benefit-to-risk profile of new medical treatments. Since evidence is critical to commercial success, pharmaceutical companies have, of necessity, developed an infrastructure to enable rigorous, long-term RCTs. Often, these trials are conducted to evaluate the effects of medicines on chronic diseases in which the rate of clinical events requires data collection over tens of thousands of patient-years for meaningful statistical analysis (5). Many RCTs are required by U.S. and other national regulatory authorities to demonstrate the beneficial effects of a therapy on the outcomes of a chronic disease; after many years of debate and discussion, similar standards for clinical research are now enforced worldwide (6). “Natural history” RCTs are those studies designed a priori to demonstrate that a medical intervention provides clinically meaningful and statistically significant reductions in the morbidity and/or mortality of a condition–in other words, the intervention changes the natural history of a disease. III. USE OF “NATURAL HISTORY” RCTS TO VALIDATE CHOLESTEROL HYPOTHESIS AND SUPPORT CHANGES IN THE MANAGEMENT OF OTHER CONDITIONS

THE

Recognized examples of important natural history RCTs include five large trials (7), which demonstrated that the sustained, substantial low-

“NATURAL HISTORY” CLINICAL TRIALS

3

ering of plasma cholesterol by certain members of the statin class significantly reduces cardiovascular morbidity and mortality. In the case of simvastatin, this was shown to reduce all-cause mortality (8). Despite decades of epidemiologic and animal studies and earlier clinical trials, the impact of lowering cholesterol was an area of medicine rife with controversy (9) before 4S and other endpoint trials were completed in over 30,000 patients (7). The so-called cholesterol controversy is now a matter of historical interest. Evidence painstakingly gathered through large clinical trials has also transformed the management of other medical conditions. Table I provides a small sample of some of the most important natural history trials of the past two decades (10–24). While it appears that a preponderance of studies have focused on cardiovascular conditions, others have been conducted (with major advances made) on other chronic disorders, including diabetes, osteoporosis, breast cancer, and certain forms of arthritis. Probably the most significant advance in ulcer management is the recognition of the causative role of H. pylori and the ability to eradicate the organism and reduce ulcer recurrence accordingly (25). In addition, although clinical outcome data like those in cardiovascular disorders are not as clearly established, medical care has benefited from the development of immunosuppressants for organ transplantation; of erythroid and myeloid cell growth factors for cancer chemotherapy, chronic renal failure and immunocompromised patients; and of H2 blockers for peptic ulcer disease (26–28). The demonstration of beneficial outcomes in RCTs, which constitutes the best available proof of the value of a medicine or of any medical intervention, results from a formidable clinical research effort. Unequivocal results from such trials demand the collection of extensive epidemiological data on the natural history of the condition under study. Careful design of the study ensures comparability of treatment groups at baseline and complete, uniform, unbiased ascertainment of outcomes during the trial. Rigorous, prospective statistical methods of data analysis, a specific study hypothesis, and a detailed protocol designed to test that hypothesis while preserving patient safety and confidentiality help to ensure the quality of the data. The operations required to conduct these investigations, mostly over long periods of time and involving numerous clinical centers located in many states or countries pose an enormous scientific and managerial challenge. IV. DEVELOPMENT OF A NEW CHEMICAL ENTITY The full development of a new chemical entity (NCE) from discovery to launch takes an average of 10 to 12 years and substantial financial

4 TABLE I Examples of Trials Demonstrating the Clinical Impact of Medical Interventions on the Natural History of Certain Illnesses a Condition

Intervention

Atherosclerotic cardiovascular disease

HMG co-A reductase inhibitors: Lovastatin Pravastatin Simvastatin

Acute myocardial

Fibrinolytic therapy

infarction (MI)

Primary and secondary prevention of coronary heart disease (CHD); reduced hospitalizations, percutaneous transluminal coronary angioplasties (PTCA), and coronary artery bypass graft surgeries (CABG); reduced all-cause mortality Reduced coronary and all-cause mortality

Streptokinase

Key trials

>30,000

7,8

GISSI-1 ISIS-2

>120,000

10

GUSTO IIb

>1,100

11

EPIC EPILOG PRISM PRISM-PLUS PURSUIT RESTORE

>33,000

12

GISSI-2/ISG

Reteplase

ISIS-3

Heparin

GUSTO

Glycoprotein IIb/IIIa platelet receptor antagonists: Abciximab Eptifibatide Tirofiban

Similar impact on death, nonfatal reinfarction as thrombolytic therapy Reduce infarction, revascularization, death (composite)

Total patients Reference

4S AFCAPS CARE LIPID WOSCOPS

Alteplase

Aspirin Primary PTCA PTCA, unstable angina, non-Q wave MI

Major effects demonstrated

Post-myocardial infarction

-blockade Timolol Propanolol

Reduced CHD and total mortality, reinfarction

Norwegian Timolol Study BHAT

>5,600

13

Congestive heart failure (CHF), other high-risk patients

Angiotensin-converting enzyme (ACE) inhibition Enalapril Captopril Ramipril

Reduction in cardiovascular, all-cause mortality; reduced hospitalizations and recurrent CHF; in patients post-MI, with CHF and with decreased left ventricular ejection fraction (LVEF)

CONSENSUS SAVE SOLVD AIRE HOPE

>23,000

14

Hypertension

-blockade Diuretics

Reduction in MI, stroke, chronic renal failure CHF

VACSGAA SHEP, others

>48,000

15

Diabetic nephropathy

ACE inhibitors Captopril

Prevented end-stage renal disease

Diabetic Collaborative Study Group

>400

16

Diabetic microvascular disease Osteoporosis

Near-normalization of blood glucose Alendronate

Prevented/delayed retinopathy, nephropathy, neuropathy Reduced spine, hip, and wrist fractures

DCCT

>1,400

17

Raloxifene

Reduced spine fractures

Alendronate Phase III >15,000 FIT 1 and 2 MORE

18,19

(continues)

5

6

TABLE I Continued Condition Acquired immune

Intervention Protease inhibitors:

deficiency syndrome

Indinavir

(AIDS)/human

Ritonavir

Major effects demonstrated Reduced hospitalization and mortality

Advanced HIV Disease Ritonavir

Total patients Reference >2,000

20

>3,000

21

Study AIDS Clinical Trials

immunodeficiency

Group 320 Study

virus (HIV) Benign prostatic hypertrophy

5-Reductase inhibitor: Finasteride

Breast cancer

Tamoxifen

Osteoarthritis and rheumatoid arthritis

Cox-2 inhibitors: Rofecoxib Celecoxib

a

Key trials

Reduced incidence of urinary retention and of the need for transurethral resections of the prostate (TURPs) Prevented breast cancer in high-risk women

Reduced GI side effects in treatment of osteoarthritis or rheumatoid arthritis versus nonsteroidal anti-inflammatory drugs (NSAIDs)

PLESS

>13,000 National Surgical Adjuvant Breast and Bowel Project P-1 Study >6,500 Studies to compare the incidence of upper GI tract perforations, ulcers bleeding in patients treated with rofecoxib, or endoscopic ulcers with celecoxib versus NSAIDs

Due to space limitations, trials and references cited above are just selected examples of natural history studies in a given therapeutic area.

22

23,24

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investment (29). Additionally, some 80% of compounds introduced into the clinic fail to reach later development. Despite the advent of combinatorial chemistry and high throughput screening technologies, total development and failure rates have not changed appreciably over the past decade (30). Traditionally, there have been four phases in clinical development that follow the extensive preclinical evaluation of the pharmacological characteristics, safety, and toxicity of a compound both in vitro and in animal models. In phase I, the pharmacokinetics and safety of a new molecule are studied in both single- and multiple-dose trials in healthy human volunteers. During the ensuing phase IIa, the actions of the molecule on the target physiological and/or biochemical parameters are assessed in patients affected by the condition in question, with use of parameters derived from basic research in physiology, biochemistry, and molecular biology. These studies determine if the compound actually causes the desired effect, for example, lowering blood pressure, blocking gastric acid secretion, decreasing markers of bone turnover, or, in human immunodeficiency virus (HIV)-infected patients, reducing blood viral RNA. Through the sustained progress of science, new and more sophisticated methods are being introduced continuously to assess the actions of drugs during phase IIa. Technologies range from functional magnetic resonance or positron emission tomography imaging for diseases of the central nervous system, to determine the dose response of receptor occupancy, to proteomic technologies and genomic chip arrays to analyze the actions of a compound on the intricate biochemical machinery of cells. Surrogate end points are often used as proxies in the analysis of drug action, narrowing the dose range of the drug to be studied and greatly accelerating the transition to more established clinical parameters in the subsequent phases of development. Phase IIb, typically studied in several hundred patients, establishes the dose range of the compound and may validate end point measures to be studied in later stages. Although these investigations usually last weeks to months, they can have durations of several years, particularly when the disease in question is chronic, as with osteoporosis. Finally, replicate multinational RCTs, termed phase III studies, are usually performed in 1,000 to 5,000 patients, end points such as changes in bone mineral density, the occurrence of ulcers demonstrated by endoscopy, or the lowering of blood cholesterol or blood pressure being used. These so-called pivotal studies constitute the foundation for achieving rigorous clinical proof of the efficacy and safety of a new compound and for submitting a compound for regulatory approval. But even large phase III trials are rarely designed as natural history trials. The latter require closely following thousands of patients, often

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for many years. Such studies are usually performed after initial regulatory approval. They provide the incontrovertible evidence of the clinical impact of the drug, firmly established through the observed effects on clinically relevant end points such as heart attacks, the need for surgical procedures or hospitalization, death, fractures, or bleeding from ulcers. Quality-of-life and cost utilization end points can also be measured in these studies (31). V. NATURAL HISTORY RCTS: SOME CONSIDERATIONS Despite the obvious benefits of RCTs in the generation of information about diseases, some issues need to be considered. When medical knowledge is lacking and the risk to patients does not constitute an ethical issue, RCTs are carried out with the inclusion of a placebo control group to quantify the efficacy and safety of the compound being investigated (32). For obvious ethical reasons, in some diseases such as HIV/AIDS or congestive heart failure, the trial must contrast the effects of different active compounds, comparing the effect of innovative therapy versus standard care. When demonstrating equivalence or noninferiority of the new agent to the comparator is the intended outcome, the design of the trial becomes even more challenging: the smaller the effect being sought, the larger the trial necessary to have sufficient detection and statistical power (33). Often forgotten is the fact that even multiple independent observational studies do not allow an inference of causality, a goal that requires properly conducted RCTs. It is only in relation to concurrent controls that the value of a treatment can be assessed with certainty. This point was exemplified by the recent study of estrogen replacement therapy that failed to demonstrate the expected reduction of cardiovascular risk in postmenopausal women with prior heart disease (34). Careful precautions should be taken to avoid the multiple causes of bias that can complicate the analysis of data derived from RCTs (35). Among them are ensuring the soundness of the clinical and laboratory infrastructure; ascertaining the quality and training of investigators participating at the study sites; obtaining the appropriate informed consent from patients and adopting processes that maintain confidentiality of information; disclosing potential conflicts of interest by investigators and others related to the study; and utilizing the expertise of the Institutional Review Boards that judge the appropriateness of study protocols. Frequently, RCTs are triple-blind, which means that neither the investigators, patients, nor sponsor has access to study results until the database is completed and locked. Oversight of the natural history RCT

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itself is normally performed by a steering committee and an independent data and safety monitoring board (DSMB) (36). The DSMB, whose primary function is to protect the safety of patients, monitors the results of ongoing trials and can recommend stopping them early if efficacy is convincingly demonstrated or if safety or ethical issues arise. Randomized controlled trials aim not only to demonstrate efficacy but also to detect potential safety issues associated with innovative therapies, which is accomplished through the strict obligation to report all adverse experiences (not just those considered to be drug-related) observed by the investigator or the patient. Quality control and quality assurance, in the form of elaborate monitoring and sophisticated auditing mechanisms, respectively, have been incorporated into modern RCTs by those directly involved in the trials and by government agencies that bear the responsibility for approving new drugs and protecting public health. Such procedures ensure the accuracy and completeness of reporting efficacy and safety data, as well as confidence in the results obtained. Site monitors, representing the sponsoring pharmaceutical company or contract research organization (CRO), make regular visits to trial centers to oversee the procedures used for collecting the data. In the case of Merck, for example, clinical trials involving more than 120,000 patients at some 7,400 sites worldwide were monitored during 1999 (Merck & Co., data on file). In addition, independent study auditors, who report directly to a senior manager separate and distant from those responsible for conducting the trial, compare trial data against source documents (clinical history, laboratory data, imaging studies electrocardiograms, etc.) to ensure authenticity of results. Furthermore, site audits by the U.S. Food and Drug Administration (FDA) and other regulatory agencies worldwide may occur at any time during a study. For most large pharmaceutical companies, several hundred clinical trials on a plethora of compounds are underway worldwide at any given time, involving thousands of investigators and many more patients. Although breaches in the conduct of investigators have occurred from time to time and have been reported in the lay press (37), these are rare transgressions (38). Appropriate measures are constantly being introduced by all those responsible for conducting clinical trials to ensure that such deviations do not occur and that if they do, that they are rapidly identified and rectified. VI. PATIENT SAFETY Each prospective participant in a trial is presented with all available information regarding potential benefits and risks and enters only after

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signing a consent form, which also pledges confidentiality of the patient’s identity. Reporting of untoward experiences identified by investigator or patient is obligatory. All study sites receive frequent periodic monitoring and auditing by the sponsor and regulatory agencies for adequacy of such reporting. Nonetheless, randomized clinical trials are not of themselves sufficient to determine all adverse consequences of drugs (39). Phase IV studies (requested additionally by regulatory agencies as a condition of approval) and phase V trials (initiated by firms to study new populations, obtain new indications, etc.), including the large end point trials discussed above, are similarly subject to the same safety surveillance requirements. As these studies result in an approximate tenfold increase in patient exposure to the drug under investigation, they also serve to identify rarer adverse events, which may occur at an incidence of 0.1% or lower. An additional necessary and powerful safety net comes from the postmarketing spontaneous report surveillance systems active in all countries in which a drug is marketed. Data on all untoward events, regardless of potential causality, are reported to the sponsor, the agency, or both and are organized by diagnostic codes and compared with epidemiologic databases to detect potential safety issues caused by a drug. VII. CLINICAL TRIALS AND THE PRACTICE OF MEDICINE IN THE AGE OF GENOMICS The achievements of the past are only a stepping stone for potential developments of the future. The widespread application of the knowledge derived from outcomes of the study of genes and their function— the so-called genomic revolution—combined with the RCT model promises to open new and needed opportunities for therapeutics. Recent advances in genomics are dramatically changing our understanding of the molecular mechanisms of disease, including the complex interplay of genetic and environmental factors. Moreover, they are providing powerful stimuli to discover new and original ways to modify biological function by disclosing thousands of potential targets for the development of novel drugs. Whereas the pharmaceuticals used today target the products of several hundred genes, the decoding of the human genome will increase the number of targets approximately tenfold (40). The knowledge generated through genomics should have great influence on the design of RCTs in the future, allowing for more accurate identification of eligible patients. Entry criteria for clinical trials will almost certainly specify certain gene sequences rather than traditional relatively gross phenotypic variables such as blood pressure, plasma

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cholesterol, or a history of vertebral fracture. As a result of such developments, clinical trials may become more complex but will yield more therapeutically precise information. In addition, by combining genomics and RCT approaches, it should also become possible to predict the most likely response of a patient to a prescribed drug. Such “pharmacogenetic profiles” should stimulate the design of new pharmaceuticals for use in specific genotypic subsets of the population to achieve an increased therapeutic specificity and, at the same time, decrease nonresponses and adverse events (41). Medical and ethical complexities, however, may arise from increasing “presymptom” or “predisease” diagnoses. The advances predicted from improving science undoubtedly will modify the way in which clinical trials are designed and performed to protect the participants, analyze the outcomes, and design new strategies for surveillance. Increased funds and manpower will likely be needed to carry out more sophisticated studies in even more intractable disease states. However, RCTs will almost certainly continue to provide the cornerstone information for the practice of medicine, which is becoming increasingly based on the outcomes of sound scientific research. The knowledge base generated through RCTs during the past 50 years will expand further because the information provided is essential to reduce the ever-present uncertainty of daily medical practice. Although enormous changes have occurred in the practice of modern medicine in the past 20 years, many as results of sophisticated natural history controlled trials, the full implementation of the genomics revolution will undoubtedly lead to even more profound and beneficial changes in the coming decades. ACKNOWLEDGMENTS The authors would like to thank Reynold Spector, M.D. for his invaluable contribution and assistance. Dr. Spector retired in 1999 from his position as executive vice president, clinical sciences, at Merck Research Laboratories. Since joining Merck in 1987, he participated in the design and implementation of some of the major trials outlined in this paper.

REFERENCES 1. Doll, R. (1992). BMJ 305, 1521. 2. Amberson, J. B., McMahon, B. T., and Pinner M. A. (1931). Am. Rev. Tuberc. 24, 401. 3. Wilson, M. C., Hayward, R. S. A., Tunis, S. R., Bass, E. R., and Guyatt, G. H. for the Evidence-Based Medicine Working Group. (1995). JAMA 274, 1630. 4. Mowery, D. C., and Teece, D. J. (1996). Strategic alliances and industrial research. In “Engines of Innovation: U.S. Industrial Research at the End of an Era” (R. Rosenbloom, and W. Spencer, eds.) Harvard Business School Press, Boston.

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5. Thomas, L. G. (1996). “Spare the Rod and Spoil the Industry: Vigorous Competition and Vigorous Regulation Promote Global Competitive Advantage.” Goizueta Business School, Emory University, Atlanta. 6. The International Conference on Harmonisation Process. Accessed from http://www. ifpma.org/ich4.html on 9 May 2000. 7. LaRosa, J. C., He, J., and Vupputuri, S. (1999). JAMA 282, 2340. 8. Scandinavian Simvastatin Survival Study Group. (1994). Lancet 344, 1383. 9. Oliver, M. F. (1991). Lancet 337, 1529. 10. Fibrinolytic Therapy Trialists’ (FTT) Collaborative Group. (1994). Lancet 343, 311. 11. The Global Use of Strategies to Open Occluded Coronary Arteries in Acute Coronary Syndromes (GUSTO IIb) Angioplasty Substudy Investigators. (1997). N. Engl. J. Med. 336, 1621. 12. Lincoff, A. M., Califf, R. M., and Topol E. J. (2000). J. Am. Coll. Cardiol. 35, 1103. 13. β-Blocker Heart Attack Trial Research Group. (1982). JAMA 247, 1707. 14. Garg, R., and Yusuf, S. (1995). JAMA 273, 1450. 15. Psaty, B. M. et al. (1997). JAMA 277, 739. 16. Lewis, E. J., Hunsicker, L. G., Bain, R. P., and Rohde, R. D., for the Collaborative Study Group. (1993). N. Engl. J. Med. 329, 1456. 17. Diabetes Control and Complications Trial Research Group. (1993). N. Engl. J. Med. 329, 977. 18. Black, D. M. et al., for the Fracture Intervention Trial Research Group. (1996). Lancet 348, 1535. 19. Ettinger B. et al., for the Multiple Outcomes of Raloxifene Evaluation (MORE) Investigators. (1999). JAMA 282, 637. 20. Palella, F. J. et al. and the HIV Outpatient Study Investigators. (1998). N. Engl. J. Med. 338, 853. 21. McConnell, J. D. et al., (1998). N. Engl. J. Med. 338, 557. 22. Fisher, B. et al. (1998). J. Natl. Cancer Inst. 90, 1371. 23. Langman, M. J. et al. (1999). JAMA 282, 1929. 24. Simon, L. S. et al. (1999). JAMA 282, 1921. 25. Laine, L., Hopkins, R. J., and Girardi, L. S. (1998). Am. J. Gastroenterol. 93, 1409. 26. Knoll, G. A., and Bell, R. C. (1999). BMJ 318, 1104. 27. Marsh, W. A., and Rascati, K. L. (1999). Clin. Ther. 21, 1443. 28. Jensen D. M. et al. (1994). N. Engl. J. Med. 330, 382. 29. DiMasi, J. A. (1996). Am. J. Ther. 3, 647. 30. The Pharmaceutical R&D Compendium: CMR International/Scrip’s Complete Guide to Trends in R&D (1999). (CMR International and PJB Publications Ltd., London, U.K. 31. Pedersen, T. R. et al. (1996). Circulation 93, 1796. 32. Passamani, E. (1991). N. Engl. J. Med. 324, 1589. 33. Ware, J. H., and Antman E. M. (1997). N. Engl. J. Med. 337, 1159. 34. Petitti, D. B. (1998). JAMA 280, 650. 35. Schulz, K. F., Chalmers, I., Hayes, R. J., and Altman, D. G. (1995). JAMA 273, 408. 36. Friedman, L. (1993). Stat. Med. 12, 42. 37. Eichenwald, K., and Kolata, G. (17 May 1999). The New York Times 38. DeMets, D. L. (1997). Controlled Clin. Trials 18, 637. 39. Temple, R. (1999). JAMA 281, 841. 40. Galas, D. J. (1998). Int. J. Pharm. Med. 12, 13. 41. Roses, A. D. (2000). Lancet 355, 1358.

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS BY JOEL MENARD* AND ARTHUR A. PATCHETT† *Faculté de Médecine, Université Paris, 75270 Paris CEDEX 06, France and †Medicinal Chemistry Department, Merck Research Laboratories, Rahway, New Jersey 07065

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. The Physiological and Physiopathological Background that Led to the Initiation of Research on ACE Inhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Angiotensin-Converting Enzyme (ACE). . . . . . . . . . . . . . . . . . . . . . . II. Peptide Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Bradykinin-Potentiating Peptides . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Animal and Clinical Studies With Teprotide . . . . . . . . . . . . . . . . . . . . . . . III. Captopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Carboxyalkanoylproline Derivatives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Captopril and Close Analogues. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. The Characterization of Captopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Metabolism and Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Clinical Approvals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Enalapril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Designing Inhibitors Without the Sulfhydryl Group. . . . . . . . . . . . . . . . . B. Enalaprilat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Enalapril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The Characterization of Enalapril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Clinical Approvals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Lisinopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Oral Testing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. The Characterization of Lisinopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Clinical Approvals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Fosinopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Phosphorus-Containing Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Fosinopril . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Clinical Approval . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Clinically Available ACE Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Contribution of ACE Inhibitors to the Growth of Physiological and Pathophysiological Knowledge . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Biological Advances in the Knowledge of ACE that Evolved in Parallel with The Drug Development Process. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . X. Clinical Development Process of ACE Inhibitors in Hypertension . . . . . . . . XI. Benefits of ACE Inhibition Beyond the Fall in Blood Pressure . . . . . . . . . . . XII. ACE Inhibitors and Congestive Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . XIII. ACE Inhibitors and Myocardial Infarction . . . . . . . . . . . . . . . . . . . . . . . . . . . XIV. ACE Inhibitors, Coronary Heart Disease, and Atherosis . . . . . . . . . . . . . . . . XV. ACE Inhibitors and Prevention of Restenosis . . . . . . . . . . . . . . . . . . . . . . . . . XVI. ACE Inhibitors and Renal Insufficiency. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XVII. The Fallacy of the Concepts of Normotension and Hypertension and the Cardiovascular Protective Effects of ACE Inhibitors . . . . . . . . . . . . . . . . . . . . XVIII. Surrogate End Points in Clinical Trials of ACE Inhibition: Are We Being Misled? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 13 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0065-3233/01 $35.00

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XIX. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Angiotensin-converting enzyme (ACE) inhibitors were first studied clinically in the 1970s. Their availability exemplified at the time the power of a new discovery paradigm in which assays using molecular targets replaced the use of animal models of disease to find and perfect bioactive compounds. The promise of this approach is to intervene with high selectivity at a unique and critically important step in a disease process. Generally, to be successful the rationale for target selection will draw on the findings of many basic research studies. This was certainly the case with ACE inhibitors. In order to understand why angiotensin I–converting enzyme (kininase II) was considered 30 years ago as an interesting target for medicinal chemistry, it is mandatory to focus our attention on this enzyme of the renin-angiotensin system (RAS) and to extract the RAS from the “mosaic” of factors that play a role in blood pressure control, as conceptualized by Irvine Page in his mosaic theory (1). Research linking the RAS to hypertension goes back to 1898, when Tigerstedt and Bergman described a rabbit kidney extract with pressor properties, which they called renin (2). By the 1960s the principal components of the RAS had been identified, and the biosynthetic pathway leading to angiotensin II had been characterized in broad outline. It was known that the enzyme renin was elaborated by the kidneys and that it acted on an α-globular protein called angiotensinogen to produce angiotensin I, which is the inactive decapeptide precursor of angiotensin II. The ACE generates angiotensin II by removing the Cterminal dipeptide of angiotensin I. Both angiotensin I and angiotensin II had been synthesized, and the blood pressure elevation properties of angiotensin II had been described. This early history of the RAS up to the 1960s, including the major contributions of Skeggs, Goldblatt, Braun-Menendez, Page, Bumpus, Peart and their colleagues has been extensively reviewed (3–5). The main elements of physiology and pharmacology acquired in the 1960s constitute the background of knowledge that made it possible to select the angiotensin I–converting enzyme as a target for hypertension and congestive heart failure treatment. Given the fact that ACE inhibitors were discovered by taking advantage of previous basic research on the physiology of sodium, potassium, and water homeostasis and blood pressure regulation, in a reciprocal way they have also advanced research in this field, and their availability has stimulated

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countless studies, which are being continued to this day to enrich our understanding of the RAS and its relevance to the etiology of and risk factors associated with cardiovascular and renal diseases. Many reviews on ACE inhibition have been previously written, especially those of B. Waeber, J. Nussberger, and H. R. Brunner in the 1990 and 1995 editions of “Hypertension: Pathophysiology, Diagnosis and Management,” edited by J. H. Laragh and B. M. Brunner (Raven Press Ltd, New York). We will try to assess the scientific literature by integrating as much biology, chemistry, pharmacology, and therapeutics as possible in order to better understand the progression from pathophysiological hypothesis to the practice of evidence-based medicine (6). There is permanently a gray area in medicine (7, 8); its size is decreased or its position is moved when new results and contradictory interpretations of observational and experimental data are analyzed, classified, synthesized, and debated (9). It is, at the same time, an exciting and frustrating procedure, since it can only provide temporary answers to permanent questions, or definitive answers to temporary questions. A. The Physiological and Physiopathological Background that Led to the Initiation of Research on ACE Inhibition At the end of the 1960s as well as in 2000, most concepts of hypertension were based on the hypothesis of a disequilibrium between vasoactive factors, that influence the level of blood pressure through vasoconstrictor or vasodilator properties. Initiated with norepinephrine (and the sympathetic nervous system) and angiotensin II (and the RAS), the vasoconstrictor hypothesis of hypertension was attractive for industrial chemists who designed drugs able to block the effects of these vasoconstrictors (10). Many other vasoconstrictor agents besides norepinephrine and angiotensin II exist for which inhibitors such as vasopressin, thromboxane A2, and endothelins (11–13) are sought. At the other extreme kallikreins (14), followed after 1970 by prostacyclin (15), atrial natriuretic factor (16), and nitric oxide (17) have been suspected to behave as hypotensive or antihypertensive mediators or hormones, because of a hypotensive effect after their administration or a rise in blood pressure after their blockade. Numerous agents may be demonstrated as potential targets for manipulating blood pressure levels through vasoconstriction or vasodilation, but, at the beginning of the 21th century, the main candidates are essentially those that have just been enumerated. In parallel with studies on vasoconstrictors and vasodilators have been studies on the kidney, which has been known for a long time as an endocrine organ able to produce vasodilator or vasoconstrictor sub-

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stances and to play a major role in the regulation of sodium, potassium, and water excretion according to perfusion pressure (18). Modeling of this concept is mainly the work of Guyton et al. (19). Sodium, potassium, and water exercise a major role in the homeostasis of the organism. They are finely regulated by hormones (vasopressin, aldosterone), and the need for a complex and simultaneous regulation of a low-volume, high-pressure compartment and a high-volume, low-pressure compartment is mainly dependent on the renal, cardiac, and vascular effects of these hormones (20). The renin–angiotensin system, in this context, is quite unique, since its main regulator, renin, is synthetized and secreted by the kidney under a sophisticated regulation by the juxtaglomerular cells. At this level, there are converging signals, which translate into a calcium-mediated modification of renin release; changes in volemia and sodium and chloride concentrations in the tubular fluid at the level of the macula densa; norepinephrine release; and prostacyclin, nitric oxide, and angiotensin II production (21). The complexity of this regulation, initially dissected as a competition between a “perfusion pressure” signal in the afferent arteriole and a chemical signal at the macula densa (22–24), had created the theory that hypertension could be due to an inappropriate secretion of renin (25, 26). Such a theory probably does not apply to the majority of hypertensive patients but has been exemplified by the hypertension induced by an excess of renin in the presence of a negative sodium balance, such as is observed in some forms of experimental or clinical hypertension (27, 28). The link between the pressor agent, angiotensin II, and the production of the hormone aldosterone, which produces a positive sodium balance and a negative potassium balance and which increases plasma volume and extracellular fluid, had been made by the discovery of the stimulating effect of angiotensin II on aldosterone secretion and excretion, a property not shared by the other pressor agents (29, 30). Shortly afterward, the development of the methodologies necessary to measure renin and aldosterone allowed the description of their rise during a low salt diet and their decrease during a high salt diet (31, 32). Some researchers attributed greater importance to the renin-angiotensin system for regulating sodium balance than to blood pressure levels (33), whereas others considered it to be equally important for the control of blood pressure and the regulation of sodium balance. This essential physiological system was more fully understood later on, when it was shown that the pressor and renal vasoconstrictor effects of angiotensin II were potentiated by a high salt diet, whereas its aldosterone-stimulating effect was blunted, the opposite being observed during a low salt

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diet (34, 35). An abnormal reactivity of the vascular, renal, and adrenal responses to angiotensin II is still today another theory to explain the genesis of some forms of hypertension (36). Within this context of a balance between vasoconstrictor and vasodilator factors and a renal equilibrium between ingestion and excretion of sodium, the discovery and characterization of a “hypertensin-converting enzyme” by Skeggs (37) and of a “dipeptidyl carboxypeptidase that converts angiotensin I and inactivates bradykinin” by Yang et al. (38) have revealed an attractive target for a pharmacological intervention in the RAS and in the kallikrein-kinin system through the inhibition of a single enzyme. The inhibition of this enzyme would be expected to increase bradykinin production and decrease angiotensin II formation. The result would be more vasodilation and less vasoconstriction, and at the same time, more natriuresis. After 30 years of intensive investigation, it is not yet possible to attribute the antihypertensive properties of ACE inhibition exclusively to the decrease in angiotensin II production, even if it is the most likely mechanism of action. The increase in bradykinin generation at some sites (kidney, heart, vessels) may contribute to the hemodynamic effect of ACE inhibition (39–43). It was expected, when orally active angiotensin II antagonists became available in the 1990s, that this dilemma would be easily solved if angiotensin II antagonists were as effective as ACE inhibitors. However, when angiotensin II type 1 and type 2 receptors were discovered, a new link between the vasodilator peptide bradykinin and the vasoconstrictor angiotensin II was described through the stimulation of type 2 angiotensin II receptors, especially in the kidney, to generate nitric oxide, bradykinin, prostacyclin, and cyclic guanosine monophosphate (44, 45). The role of angiotensin II has received more and more support to explain the changes in blood pressure and the prevention of cardiovascular or renal lesions, but participation of bradykinin is still possible, even during administration of angiotensin II antagonists. If today the blockade of the RAS seems to be very logical (Goldblatt et al. [46] had tested the in vivo administration of renin antibodies), many arguments were used in the early 1970s to raise concerns about pharmaceutical research directed toward the inhibition of the reninangiotensin system and the selection of the ACE as an antihypertensive target: (1) the interpretation that the renin-angiotensin-aldosterone system does not directly regulate blood pressure but is mainly involved in sodium balance equilibrium; (2) the consideration that renin secreted by the kidney is the rate-limiting enzyme in the angiotensin II biosynthetic pathway, whereas the ACE is in excess, active at the surface of vascular endothelial cells, and present in all vascular territories, espe-

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cially in the pulmonary capillary bed (47); (3) the absence of detectable activation of the system in the majority of hypertensive patients, especially given the normal or even low values reported for plasma renin activity (48); (4) the predominant role of the sympathetic nervous system in the control of blood pressure. At that time, the pharmacological interruption of the sympathetic nervous system had already provided major results in high blood pressure treatment, through the use of sympathectomy, ganglioplegics, reserpine, α-methyldopa, clonidine, alpha blockers and beta blockers. Conceptual opposition to the relative importance of both pressor systems was attenuated when a link was made between the sympathetic nervous system and the renin-angiotensin system by the discovery of a “brain” reninangiotensin system (49), for which a functional role is still under careful investigation through all the tools of modern biology, from immunohistochemistry and mRNA quantification to pharmacological blockade and antisense therapy. Interactions between angiotensin II and the sympathetic nervous system have been observed at different sites, and they remain one among the several determinants of the antihypertensive effect of the renin-angiotensin system blockade (50). B. The Angiotensin-Converting Enzyme (ACE) The angiotensin I–converting enzyme (ACE), designated peptidyl– dipeptidase A (E.C.3.4.15.1), is identical to the bradykinin-metabolizing enzyme kininase II (38). Its early history and initial characterizations have been reviewed (51–54). It was discovered by Skeggs and co-workers (55), and in their pioneering work they showed it to be inhibited by ethylenediaminetetraacetic acid (EDTA) (37), to remove a dipeptide from the carboxyl terminus of angiotensin I (then called hypertensin I [56]) and to be activated by sodium chloride (55). The fact that ACE is a Zn2+-containing peptidase was first reported by Das and Soffer in 1975 (57). When the currently available ACE inhibitors were synthesized in the mid-1970s and early 1980s, very little was known about the enzyme to help in the synthesis of inhibitors. Design strategies, which will be described later, relied almost entirely on the structures of peptidal inhibitors and substrates and on active site hypotheses generated in analogy with other Zn2+ metallopeptidases, especially carboxypeptidase A and thermolysin. Fortunately, the medical importance of ACE inhibitors has resulted in continuing, extensive studies of the enzyme’s structure and mechanism. Reviews by Soubrier et al. (59) and Corvol et al. (60, 61) summarize much of the recent progress.

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

19

Angiotensin-converting enzyme is found in mammalian tissue in two forms, the somatic enzyme and a testicular (germinal) enzyme with molecular weights in the range 150,000–180,000 and 90,000–110,000, respectively. When the somatic enzyme was cloned, the presence of two homologous domains each containing the same Zn2+ binding sequence was apparent (62, 63). Both domains are catalytically active (64), and commercially available ACE inhibitors are similarly effective inhibitors of both enzyme active sites. One can design selective inhibitors such as the recently described phosphinic acid–containing peptide RXP407, whose dissociation constant is three orders of magnitude lower on the N-terminal active site of ACE (65). However, no cardiovascular advantage has been demonstrated in doing so. Most natural substrates of the enzyme are hydrolyzed at comparable rates by the N- and C-terminal domains. An exception is N-acetylserylaspartyllysylproline, which is a natural regulator of stem cell proliferation that is hydrolyzed by the Nterminal domain 50-fold faster than by the C-terminal domain (66). The somatic enzyme is found in vascular endothelial cells and is present in especially high levels in the lung. It is also found in the kidney and brain, in intestinal brush border absorptive cells, and in circulatory mononuclear cells (61). The germinal enzyme corresponds to the Cterminal domain of the somatic enzyme. Both include a hydrophobic region in their carboxyl termini which helps anchor the enzymes onto the plasma membrane. Some ACE is also found in the plasma and other body fluids and it lacks this anchor sequence. Formation of soluble enzyme presumably involves posttranslational proteolytic release of the anchor motif. The ACE acts on bradykinin and angiotensin I as a dipeptidyl carboxypeptidase. It hydrolyzes in vitro a number of physiologically active peptides in addition to angiotensin I and bradykinin. They include the stem cell–modulating acetyltetrapeptide mentioned above, neurotensin, and enkephalins. The ACE also acts as an endopeptidase toward substance P by releasing Arg-Pro-Gly-NH2 from its C-terminus. Quite unexpectedly, its action on luteinizing hormone releasing hormone is to hydrolyze off its N-terminal tripeptide, Glu-His-Trp (67). Apart from the action of ACE on angiotensin I and bradykinin, the clinical importance, if any, of these other enzymatic activities is still to be determined. The active sites of ACE contain the sequence His-Glu-X-X-His, in which the histidines are considered to participate in Zn2+ binding and Glu in the catalytic mechanism. A third Zn2+ ligand is proposed to be a glutamic acid, and the fourth is the nucleophilic water molecule (62). This structural motif is present in a number of metallopeptidases,

20

JOEL MENARD AND ARTHUR A. PATCHETT

including the crystalline bacterial endopeptidase thermolysin, and a probable mechanism for the latter’s action has been proposed (68). In it Zn2+ polarizes the scissile amide carbonyl, and the catalytically important Glu acts as a base to increase the nucleophilicity of the Zn2+-bound water molecule by removing a proton from it. The Glu is also in position to transfer this proton to the leaving nitrogen. The general features of this mechanism no doubt also apply to ACE. To date it has not been possible to crystallize ACE, presumably even with the recently reported active deglycosylated testicular enzyme (69). II. PEPTIDE INHIBITORS The first important ACE inhibitors were snake venom peptides. Their isolations and characterizations have been described in comprehensive reviews by Ferreira (70, 71) and by Cushman and Ondetti (72–74). A. Bradykinin-Potentiating Peptides Ferreira’s interest in the physiology of bradykinin led him in the early 1960s to search for substances that would inhibit its in vivo inactivation. The venom of the Brazilian arrowhead viper Bothrops jararaca generates bradykinin in plasma, and Ferreira discovered that the venom itself contained substances capable of potentiating bradykinin-induced contractions of isolated guinea pig ileum. He and Rocha e Silva called the active fraction of this venom the bradykinin-potentiating factor (BPF) (75). Studies with BPF were continued by Ferreira in the laboratories of J. R. Vane, where it was also shown to increase the in vivo stability of bradykinin (76). Importantly, Ng and Vane established BPF as a potent inhibitor of angiotensin I conversion in the lung (77), and Bakhle in Vane’s department demonstrated that BPF blocked the formation of angiotensin II from angiotensin I in vitro (78). Subsequently, Erdos and co-workers (35) established the identity of the bradykinin-metabolizing enzyme kininase II with the ACE. Studies to isolate the peptide components of BPF were initiated by Ferreira and in the Squibb Institute by Ondetti and Cushman. Similar BPF activities were observed in the venom of the Japanese viper Agkistrodon halys blomhoffi, and the active peptides (A through E) in it were identified at Osaka University by Kato and Suzuki (79–81). Nine bradykinin-potentiating peptides (BPP5) were isolated from the Bothropos jararaca venom by Ferreira, Greene and co-workers (82,83).

21

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

The most active of these enzyme inhibitors when tested in vitro without preincubation was termed BPP5a, and its structure and synthesis were reported (84,85). Ondetti’s group isolated and determined the structures of six peptides from the same venom, and these entered the literature with SQ designations (86). In Table I the structures (1–4) of some of the more important snake venom peptides are shown, along with estimates of their relative bradykinin-potentiating activities in the guinea pig ileum assay (72). The most active of these inhibitors in that assay is SQ20,881 (BPP9a, later designated teprotide). Because BPP5a is a substrate of ACE, its activity in the guinea pig ileum assay is compromised, although its structure became of primary importance in the design of orally active ACE inhibitors. As inhibitors of rabbit lung ACE, the Ki’s of BPP5a, SQ20,858, and SQ20,881 were reported to be 0.09, 4.3 and 0.84 µM, respectively (72), and peptide C from the Agkistrodon viper is a competitive inhibitor of ACE, with Ki = 2 × 10–5M (72). B. Animal and Clinical Studies With Teprotide (SQ20,881) Teprotide’s capacity to block the in vivo generation of angiotensin II from angiotensin I was demonstrated by its inhibition of the latter’s pressor activity when both are given intravenously to rats (87). Teprotide was also shown to lower blood pressure in animal models of hypertension, especially those characterized by high circulating renin levels such as the two-kidney, one-clip rat model of renal hypertension (88). For reviews of teprotide pharmacology, see Cushman and Ondetti (72) and Ondetti and Cushman (73). TABLE I Important Snake Venom Peptide Inhibitors of Angiotensin-Converting Enzyme

Compound 1 2 3 4

Designations SQ20,881, BPP9a, teprotide SQ20,858,BPP10c C SQ20,475,BPP5a

Structure

Relative activity (%)a

100

11 3–5 23–24

a Relative activities are from Cushman and Ondetti (72). The activity of each peptide to increase the smooth muscle contractile action of bradykinin was determined relative to SQ20,881 (100%).

22

JOEL MENARD AND ARTHUR A. PATCHETT

In parallel with animal experiments, Vane and colleagues (89) showed that intravenous teprotide reduced the pressor activity of angiotensin I in humans. Nor was there any significant augmentation or reduction of angiotensin II’s hypertensive response. A biochemical proof of efficacy had been achieved, and clinical antihypertensive studies were begun. The first clinical data came from John Laragh’s group in 1974 (90). In 12 of 13 hypertensive patients, intravenous doses of 1 to 4 mg/kg of teprotide produced an immediate lowering of blood pressure. Three of the patients had malignant hypertension, six had renovascular hypertension, two had essential hypertension, and two had hypertension-associated renal failure. The only nonresponder was in the last group. In addition, the blood pressure–lowering effects of teprotide were augmented by the addition of either furosemide or chlorothiazide, whose sodium-displeting activity activates the RAS. Additional clinical studies, which confirmed and extended these findings, were reported by Johnson et al. (91), by Gavras et al. (92), by Case et al. (93), and by Hulthen and Hoffelt (94). Concerns were now set aside that ACE inhibitor use would be limited to the diagnosis and treatment of hypertensive patients with high renin levels. Neither were there any reports of serious, mechanismbased side effects, including kinin-related ones. Furthermore, as expected, the enhanced efficacy of ACE inhibitor/diuretic combinations had been confirmed in humans. The challenge was to design orally active ACE inhibitors. A number of laboratories recognized the significance of the teprotide clinical studies and set out to achieve that goal. III. CAPTOPRIL Captopril was announced in 1977 by the Squibb group led by Ondetti and Cushman (95,96), and the history of its design has been extensively reviewed (for example in 72,73, 97–102). Attempts to build metabolic stability into the peptide BPP5a were not successful, nor was a viable nonpeptide lead discovered in the Squibb sample collection (101). Captopril instead is one of the landmark achievements of rational drug design. A. Carboxyalkanoylproline Derivatives Publications by Byers and Wolfenden (103,104) stimulated the conceptual breakthrough that led to captopril. These authors introduced a new design concept, by-product inhibition, to explain the high potency of compounds such as L-benzylsuccinic acid as an inhibitor of car-

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

23

boxypeptidase A (Ki = 4.5 × 10–7M). In this design, potent active site affinities are attributed to molecules that combine part structures of the products formed in an enzymatic reaction. Since both carboxypeptidase A and ACE are zinc metallopeptidases, Ondetti and Cushman hypothesized their active sites and mechanisms to be similar. Therefore they decided to extend the carboxypeptidase A by-product design to ACE, with allowance for the latter’s hydrolytic removal of dipeptides, not amino acids, from its substrates. Ondetti and colleagues might have attempted by-product designs based on the C-terminal sequences of angiotensin I and bradykinin, but their work with the snake venom peptides fortunately caused them to continue with the essential functionalities of BPP5a. Initially they made and tested succinylproline (5 See Table II). It was a very weak ACE inhibitor (IC50 = 330 µM) but its potentiation of bradykinin in the excised guinea pig ileum test encouraged the synthesis of additional analogues. When an α-methyl group was introduced to mimic the penultimate Ala of BPP5a, the resulting compound 7 (Table II) blocked ACE with IC50 = 22 µM. It also produced a typical teprotidelike spectrum of activity on the guinea pig ileum, in as much as it was a

TABLE II ACE Inhibition by Carboxyalkanoylproline Derivativesa

Compound

R

IC50 (µM)b

5

330

6

70

7

22 4.9

8 9

1,200c

10

260

Data from Cushman et al. (96). Concentration required for 50% inhibition of ACE activity using the substrate Hip-His-Leu as described in reference 96. c Isomer a. a

b

24

JOEL MENARD AND ARTHUR A. PATCHETT

functional antagonist of angiotensin I and a potentiator of bradykinin without affecting the activities of angiotensin II and acetylcholine. As summarized in Table II, α-methylglutarylproline (8) was the best of additional analogues. Both it and compound 7, when given orally or parenterally, inhibited the pressor effect of angiotensin I in rats. In accord with their presumed relationship to BPP5a, the absolute configuration of the methyl group in these compounds corresponds to an L-alanine. Furthermore, succinyl derivatives of 11 naturally occurring amino acids, including L-Leu (angiotensin I) and L-Arg (bradykinin), established the superior potency of C-terminal proline, as had been earlier exemplified in the snake venom peptides (96). Unfortunately, however, none of the alkanoylproline derivatives were quite potent enough to be developed as drug candidates. B. Captopril and Close Analogues To increase potency, Ondetti et al. developed an active site hypothesis, which is schematically illustrated in Fig. 1, taken from their 1977 publication (95). The carboxyl group of by-product inhibitors was proposed to be within the ligand sphere of the enzyme’s catalytic Zn2+ ion. If so, it should be possible to improve inhibitor potency with carboxyl replacements having greater affinity for Zn2+. Various noncarboxyl ligands were explored without success, including nitrogen-containing functionalities (amines, amides, and guanidines) (95). However, thiol groups have exceptionally potent Zn2+-chelating properties, and their introduction led to the potency breakthrough. Compound 12 (Table III) inhibited ACE with an IC50 of 200 nM. Changing the sulfhydryl tether length did not improve activity, but introduction of a methyl group in a configuration equivalent to L-alanine gave an additional tenfold increase in potency (compound 14, Table III). Both a free proline carboxyl group and the amide group contributed significantly to the activity of compound 14 and its analogues. Clearly, compound 14 is an outstanding ACE inhibitor, and when good in vivo properties had been established, it entered development with the generic name captopril. C. The Characterization of Captopril Captopril (14) is a competitive inhibitor of ACE, with Ki = 1.7 × 10–9 M. Its inhibitory activity does not increase with time and is reversible on dilution. These properties and the analogue studies referenced above support its designation as an active site inhibitor not functioning by

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

25

FIG. 1. The proposed binding of substrates and inhibitors to the active sites of carboxypeptidase A and angiotensin-converting enzyme. Reproduced with permission from Ondetti et al. (95). Copyright 1977 American Association for the Advancement of Science.

TABLE III Mercaptoalkanoyl Proline Derivativesa

Compound

R

IC50 (µM)b

11

1.1

12

0.20

13

9.7

14

0.023

15

2.4 a b

Data from Cushman et al. (96). See footnote b of Table II.

26

JOEL MENARD AND ARTHUR A. PATCHETT

chelating and removing Zn2+ from the enzyme (96). Specifity is evidenced by IC50 values greater than 10–3 M in the inhibition of carboxypeptidases A and B, trypsin, and chymotrypsin. Leucine aminopeptidase is inhibited with an IC50 = 5.4 × 10–6 M (96). As is consistent with its mechanism of action, captopril orally and parenterally blocked the pressor activity of angiotensin I given iv to Sprague-Dawley rats without affecting the blood pressure elevation produced by angiotensin II. It also augmented the hypotensive activity of bradykinin but not that of acetylcholine. Oral doses as low as 0.1 to 1 mg/kg were adequate to produce dose-responsive blood pressure lowering in angiotensin I–challenged rats (95). In the high-renin Goldblatt two-kidney, one-clip renal hypertensive rat, immediate pressure lowering was observed with orally administered captopril (30 mg/kg), and an antihypertensive effect could be maintained for at least 10 months. Spontaneously hypertensive rats, which are considered to be a model of essential hypertension in humans, also responded to oral captopril, but a dose of 100 mg/kg was required. As expected in both models of hypertension, the addition of diuretics enhanced the antihypertensive activity of captopril (105). D. Metabolism and Pharmacokinetics [35S]-

and [14C]-captopril have indicated complex and Studies with extensive metabolism. The disulfide dimer of captopril is observed, as well as mixed disulfides with cysteine, N-acetylcysteine, and glutathione. There is covalent reversible binding to plasma proteins, which is similarly considered to involve disulfide linkages. S-Methylcaptopril and its sulfoxide are also formed (105). Captopril is well absorbed orally in rats (71%), in dogs (77%), and in monkeys (79%). In humans the oral data are 68% absorption, and 38% bioavailability. Absorption is rapid in all species, and excretion of captopril and its metabolites is predominantly in the urine (105). E. Clinical Approvals Clinical testing began in 1976 in a study in normal volunteers, in which the pressor activity of intravenous angiotensin I was inhibited by increasing oral doses of captopril ranging from 1 to 20 mg (106). The extensive clinical studies that led to its approvals have been reviewed (98, 107–111). Captopril was first approved in 1981 for use in hypertensive patients poorly responsive to multidrug therapy. It received FDA approval in 1982 for heart failure and in 1985 for general use in hypertension (101). For

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

27

the latter indication, doses are generally 25 to 50 mg bid or tid although dosage as high as 150 mg bid or tid is sometimes used. IV. ENALAPRIL Promising clinical reports on teprotide began to appear in 1974, and during that year, under the leadership of Dr. Charles S. Sweet ACE assays both in vitro and in vivo were initiated in the Merck Research Laboratories. Sweet provided the Merck group with expertise in the reninangiotensin system acquired at the Cleveland Clinic with F. M. Bumpus. Also in 1974, Patchett and Maycock began an exploratory project on enzyme inhibitor design. One of their first attempts was to extend Wolfenden’s by-product design (103,104) to the ACE target. They synthesized succinyl-L-proline and its derived hydroxamic acid, but their poor activities against ACE, 43% inhibition at 3.3 × 10–4M and 84% inhibition at 6.7 × 10–5 M, respectively, caused them to abandon the approach (112). Instead, the zinc metallopeptidase thermolysin and its natural product inhibitor phosphoramidon (113) were taken as better models for the design of ACE inhibitors. Indeed, the approach yielded potent inhibitors, but oral activity was not achieved (114). A. Designing Inhibitors Without the Sulfhydryl Group Design efforts were increased at Merck following the announcement of captopril in 1977. Its reported side effect profile, especially with the high doses used initially in clinical trials, included rash, loss of taste, and proteinuria (115). Because similar adverse experiences were known with penicillamine (116), Patchett et al. focused their efforts on the design of nonsulfhydryl ACE inhibitors (117). Furthermore, they felt that its twice daily or three times daily dosing might be a consequence of the well-known metabolic lability of SH groups. Unfortunately, attempts to replace the thiol group of captopril with imidazoles, tetrazoles, α-ketoacids, catechols, or sulfilimines led to only weakly active compounds (118). In their early efforts to apply the by-product design to ACE, Patchett and Maycock had synthesized only succinylproline, based on the findings of Byers and Wolfenden that benzylsuccinate was superior to benzylglutamate as an inhibitor of carboxypeptidase A. Similar active site topologies were assumed for these two enzymes. Evidently this was not the case, since Cushman et al. reported in 1977 (96) that glutarylproline (compound 6 Table II.) was more active than compound 5 (Table

28

JOEL MENARD AND ARTHUR A. PATCHETT

II) as an inhibitor of ACE. This report was of pivotal importance, in as much as Patchett and co-workers realized it would be possible to introduce an NH group into the glutarylproline analogue 8 (Table IV) to generate an N-alkylated AlaPro derivative and thereby complete the Cterminal part of a by-product design. Perhaps not unexpectedly, compound 16 (Table IV) was no more active within experimental error than compound 8, because the added polarity of the NH group could promote solvation and dissociation of the enzyme/inhibitor complex. Given the substrate specificity of the enzyme, it was not possible to achieve good activity by alkylating the NH group of the inhibitor. Thus, to increase lipophilicity, a methyl group was added α to the carboxyl. The result was a dramatic potency breakthrough, since compound 17 (Table IV), even as a mixture of two diastereomers, inhibited ACE with an IC50 of 90nM. Subsequent interpretations placed this methyl group and its higher congeners in the hydrophobic S1 pocket of the enzyme. Remarkably, methyl substitution α- to the glutaryl carboxyl group actually leads to a loss of inhibitory potency (compare compounds 6 and 10 Table II). Apparently, the NH group in compound 17 makes an important hydrogen bond with the enzyme, which is needed to direct the compound’s α-methyl group into the S1 binding site of the enzyme. B. Enalaprilat Extensive structural variations were then made α to the carboxyl group of compound 17 and these have been reviewed (112, 117–122). In agreement with analogues of the snake venom inhibitor BPP5a, hydrophobic arylalkyl groups afforded high enzyme inhibitory potencies. Furthermore, a preferred orientation of these groups was evident in the (S)-diastereomer 19 (Table IV), which is nearly 700 times as potent as the corresponding (R)-diastereomer 18 (Table IV). Compound 19, with an enzyme inhibitory activity roughly 10 times that of captopril, was given the name enalaprilat. Clearly, its high affinity for the enzyme, unlike that of captopril, is highly dependent on an important, critically positioned hydrophobic interaction. Enalaprilat is a slow-binding inhibitor of ACE, with an on-rate that is at least two orders of magnitude less than the diffusion-controlled rate. Its Ki is 2 × 10–10 M, and from its off-rate a half-life of 27 minutes was calculated for the enalaprilat-converting enzyme complex (119). Enalaprilat also very effectively potentiated the contractile effects of bradykinin on guinea pig ileum strips, with an EC50 of 77 pM (123). It attenuated the pressor activity of angiotensin I in rats with an intravenous ID50 of 8.2 µg/kg in rats and 6.4 µg/kg in dogs, and in these tests it was approx-

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

29

TABLE IV Potency Breakthrough in the By-Product Inhibitor Design

Compound

RCO

IC50a

8

4.9 × 10–6Mb

16

2.4 × 10–6M

17

9 × 10–8M

18

8.2 × 10–7M

19

1.2 × 10–9M

20

1.2 × 10–6M

a b

Patchett et al. (117). Cushman et al. (96)

imately 10 times as potent than captopril. However, the oral activity of enalaprilat is poor. An oral dose of 3 mg/kg in rats was needed to produce significant inhibition of intravenous angiotensin I, whereas captopril was effective at the 0.3 mg/kg level (124). Subsequently, the bioavailability of enalaprilat was estimated to be only 5% in rats and 12% in dogs (124). C. Enalapril To improve the oral properties of enalaprilat, Patchett and colleagues turned to a prodrug approach. Esters were made of only one and of both carboxyls, and fortunately only esters of the N-carboxyalkyl group were needed for good oral absorption. When the proline carboxyl is esterified, the resultant inhibitor would have been difficult to formulate, since closure to a diketopiperazine easily takes place. Although various esters were tried, none were clearly superior to the ethyl ester 20 (Table IV).

30

JOEL MENARD AND ARTHUR A. PATCHETT

Initial blood pressure lowering studies of compound 20 in rats and dogs have been summarized by Sweet, Gross and co-workers (123, 125). After thorough study of its in vivo properties, compound 20 as its maleate salt entered clinical studies as MK-421, with the generic name enalapril. To account for the good oral activity of enalapril, attention was drawn to the fact that enalaprilat is an anionic zwitterion at physiological pH (pKa’s 2.8, 3.5 and 7.6), whereas only the carboxyl of enalapril is ionized (pKa’s 3.0 and 5.4) (118,120). The possibility was considered that enalapril is absorbed from the intestine, as are bile acids and other carboxyl-containing compounds including the nonsteroid anti-inflammatory drugs. Later, Amidon and colleagues published data supporting peptide transport in the oral absorption of enalapril (126,127). D. The Characterization of Enalapril As a prodrug, enalapril is essentially inactive as an ACE inhibitor. Thus, it must be enzymatically converted to enalaprilat, and liver homogenates of rats, dogs, rhesus monkeys, and humans have that capacity (128). In humans and in all nonhuman species except the rhesus monkey, deesterification is the only metabolism which is observed. In monkeys, a small amount of the desproline metabolite of enalaprilat has been detected (128). Absorption of enalapril is reported to be 39% in rats, 64% in dogs, and 60–70% in humans (128). E. Clinical Approvals The phase I clinical testing of enalapril began in 1980 in a study in which its efficacy to inhibit intravenously administered angiotensin I was determined. Oral doses as low as 2.5 mg produced a substantial decrease in ACE, activity and lowering was evident even 21–24 hours after the drug was given (129). Phase II and phase III trials began in 1981, and the first approval to use enalapril in hypertension came in 1984 and in heart failure in 1986. V. LISINOPRIL Lisinopril was synthesized in the course of systematically varying each structural unit in enalaprilat. Commercially available amino acids were introduced in the Ala position; results with some of them are summarized in Table V.

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

31

TABLE V Angiotensin-Converting Enzyme Inhibition by Central Amino Acid Analogues of Enalaprilat

Compound

Xa

IC50(M)

21 22 23 24

Glycine L-Alanine α-Methylalanine N-Methyl-L-alanine

25 26 27 28

L-Valineb L-Phenylalanine L-Glutamic L-Lysineb

acid

2.3 × 10–7 3.8 × 10–9 2.5 × 10–6 1.0 × 10–7 7.8 × 10–8 5.2 × 10–8 1.3 × 10–6 1.2 × 10–9

a Patchett (118). Unless otherwise noted, data were obtained on the isomer mixture formed by reductive alkylation of the proline dipeptide with 2-keto-4-phenylbutyric acid. b Single isomer.

In agreement with the enzyme’s specificity, acidic amino acids and Nalkylated amino acids afforded poor inhibitors. The discovery that lysine in the penultimate position provided good inhibition was at the time unprecedented in the ACE substrate or snake venom peptide literature, and this development was pursued with additional analogues (Table VI). Excellent activity was obtained with arginine and with higher and lower homologues of lysine. Even the ε-N-acetyllysine analogue 33 (Table VI) was active. A. Oral Testing All potent ACE inhibitors generated in the Merck program were evaluated both intravenously and orally in the rat angiotensin I pressor assay. Thus, a decision did not have to be made concerning the likely oral activity of compound 28 (Table V). This lysine analogue is doubly zwitterionic and is more polar than the poorly bioavailable enalaprilat. Logically, its testing might have been delayed until enalapril-like prodrugs had been made of it. Fortunately, they were only made later, since remarkably, compound 28 itself displayed an oral ID50 of 0.19 mg/kg in the rat angiotensin I pressor assay, in which enalapril was comparably active with an oral ID50

32

JOEL MENARD AND ARTHUR A. PATCHETT

TABLE VI Angiotensin-Converting Enzyme Inhibition by Basic Amino Acid Analogues of Enalaprilat

Compound 29 30 31 32 33 34 a

R

IC50(M)a

–(CH2)3NH2 –(CH2)4NH2 –(CH2)5NH2 –(CH2)4N(CH3)2 –(CH2)4NHCOCH3 –(CH2)3NH(C=NH)NH2

4.5 × 10–9 1.2 × 10–9 5.2 × 10–9 9.1 × 10–9 1.6 × 10–8 6.4 × 10–9

Patchett (118).

of 0.29 mg/kg. In dogs challenged with angiotensin I, both enalapril and the lysine analogue 28 were similarly active at an oral dose of 0.3 mg/kg, with durations of action that exceeded 6 hours (117). In fact, unlike enalapril, the monoethyl ester of compound 28 provided no oral activity improvement nor was it of interest for detailed studies because it was considered that a good alternative to enalapril would be one that did not require prodrug activation. Further testing validated its selectivity and efficacy (130), and it was selected for development with the designation MK521 and the generic name lisinopril. B. The Characterization of Lisinopril Lisinopril is a potent competitive inhibitor of rabbit lung ACE with a Ki of 1 × 10–10 M, which is slightly better than enalaprilat’s Ki of 2 × 10–10 M (131). It forms a tighter binding complex with ACE than either captopril or enalaprilat. From their dissociation rates from rabbit lung ACE at pH 7.5, the enzyme inhibitor half-lives were calculated to be captopril 9 minutes, enalaprilat 27 minutes, and lisinopril 105 minutes (131). Animal studies with lisinopril have been summarized by Sweet and Ulm (132). Perhaps as a result of its high polarity, lisinopril undergoes no metabolism either in animal species or in humans and is excreted solely by the kidneys (133). Bioavailability in humans is about 25%. Lisinopril’s duration of action is superior to enalapril and it is clearly a once-a-day drug (134).

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

33

The probable basis for the reasonably good oral bioavailability of lisinopril was first suggested by analogue studies. The ornithine-containing compound 29 (Table VI) showed good oral activity in the rat angiotensin I pressor assay, but the N-dimethyl and N-acetyl analogues 30 (Table VI) and 33 (Table VI) did not, nor did the arginine derivative 34 (Table VI) (118). The ethyl ester of lisinopril also did not appear to improve oral activity, as noted above. Uptake on a facilitated transport system, possibly via a peptide transporter, was inferred, although the oral activity of lisinopril was not improved by introducing amide or thia functionality into the lysine side chain (118). Nonetheless, absorption of lisinopril from the rat jejunum is concentration-dependent and is inhibited by the dipeptide TyrGly. Such data are consistent with the involvement of a peptide carrier system in its slow, steady absorption (135). C. Clinical Approvals Phase I testing of lisinopril began in 1980. Single oral doses of 1.25 to 40 mg were given to normotensive volunteers, and at intervals thereafter pressor responses to intravenous angiotensin I were recorded. Lisinopril produced a noticeable inhibition of angiotensin I with oral doses as low as 1.25 mg, and efficacy persisted for 21 to 24 hours at the higher doses (133). Clinical trials in patients with mild to moderate essential hypertension demonstrated significant blood pressure lowering with once-a-day lisinopril in oral doses generally from 10 to 80 mg (134,136,137). In doses of 2.5 to 20 mg/day, lisinopril is effective in treating patients with congestive heart failure who are also taking diuretics and digitalis (138,139). Regulatory approval was given in the United States in 1987 for its antihypertensive use and in 1993 for its use in congestive heart failure. VI. FOSINOPRIL A third structural class of clinically available ACE inhibitors is represented by a single member, fosinopril. The design of this phosphorusbased inhibitor had its ultimate precedent in the natural product phosphoramidon, which is a potent inhibitor of the zinc endopeptidase thermolysin (113). As noted above, the Merck group had worked briefly with this design (114) but abandoned it following the discovery of the carboxyalkyldipeptides. Obtaining good oral activity is a formidable challenge with phosphonic and phosphinic acid–based inhibitors. Kerenewsky et al. and DeForest et al. of Petrillo’s group at Squibb solved

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the problem with a lysine-containing compound (140–142) reminiscent of lisinopril and with prodrugs of highly active phosphinic acids culminating in fosinopril. A. Phosphorus-Containing Inhibitors Phosphonic acids have greater affinity than carboxylic acids for the active site of the ACE, as judged by a comparison of succinylproline (5) with the phosphonic acid 36 (Table VII). However, surprisingly, the lower homologue 35 is more active than 36, and introduction of the important methyl group in captopril and enalapril led to decreased activity in compound 37 (Table VII). As expected, significant potency increases were obtained by conversion to phosphinic acids, whose substituted alkyl groups were directed toward the S1 subsite (98,143). A TABLE VII Phosphonic and Phosphinic Acid Inhibitors of Angiotensin-Converting Enzyme

Compound

RCO

IC50(µM)a 8.4

35 36

48

37

18

38

3.3

39

0.88

40

0.18

a

Petrillo and Ondetti (98).

35

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

butyl chain was optimal for aryl placement (compound 40 Table VII), presumably to compensate for a shortened phosphorus-to-proline distance and a binding mode that did not utilize the S1′ site. Compound 40 became the focus of additional analogues, since its intravenous activity was a third that of captopril despite being 10 times weaker as an ACE inhibitor (144). To increase intrinsic potency the Squibb group turned to lipophilic proline derivatives, as they had in the design of the captopril analogue zofenopril (144,145). Perhaps again reflecting a different binding mode, lipophilic substituents on proline produced greater activity enhancements in this series than they had with sulfhydryl and Ncarboxyalkyl inhibitors. Data on several of them are shown in Table VIII. Compound 45 (Table VIII) as the orally bioavailable prodrug 46 (Table VIII) was selected for development as fosinopril (144). B. Fosinopril The active form of fosinopril, compound 45 (fosinoprilat), is a tightbinding inhibitor with a Ki of 1.5 nM versus rabbit lung ACE (144). Fosinopril given orally is equipotent with captopril as an inhibitor of the pressor activity of angiotensin I in rats (146). It is effective orally in the TABLE VIII Angiotensin-Converting Enzyme Inhibition by 4-Substituted Proline Phosphinic Acidsa

Compound

R

X

41 42 43 44 45

H H H H

46

(CH3)2CH—CH

H





CH3CH2C—O



O a

Krapcho et al. (144).

Y

IC50(nM)

H H H S-Ph X,Y = SCH2CH2S H C6H11 C6H11 H

180 20 2 7 11

C6H11

NA

H

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two-kidney, one-clip high-renin rat model at 10 and 30 mg/kg. As expected, blood pressure lowering is less in the spontaneous hypertensive rats at these dosages, and efficacy is enhanced with added hydrochlorothiazide (147). Fosinopril was tested in rats, dogs, and cynomologus monkeys, and in all species its duration of action was longer than that of captopril. Following oral administration of radiolabeled fosinopril to humans, approximately 75% of the dose in plasma and urine was present as fosinoprilat. The remainder was a β-glucuronide (15–20%) and a monohydroxylated metabolite of fosinoprilat (148). In dogs, esterase metabolism was observed mainly in the gut wall and to a lesser extent in the liver (149). When given orally to humans in capsules, the oral absorption of fosinopril sodium averaged 36% and that of fosinoprilat 29% (148). Most ACE inhibitors are eliminated primarily via the kidney. However, fosinoprilat is excreted about equally in the bile and urine (148). Biliary excretion can compensate for compromised renal function, and thus blood levels of fosinoprilat remain relatively constant in patients with varying degrees of renal impairment (150). C. Clinical Approval Reviews of clinical studies with fosinopril in the treatment of essential hypertension (151,152) and heart failure (153,154) are available. It is approved for both indications in the United States and is used in antihypertensive therapy at a recommended initial dose of 10 mg once per day, which may be increased to 80 mg once per day. For the treatment of heart failure, the initial dose is also 10 mg, which is often increased to a maintenance dose of 20–40 mg once per day. VII. CLINICALLY AVAILABLE ACE INHIBITORS Salient properties of the ACE inhibitors that have been described above are summarized in Table IX. Numerous other ACE inhibitors have been synthesized and evaluated since captopril was announced in 1977. As of 1996, 16 were in use worldwide (157), all of which are variations of the designs exemplified in Table IX. Reviews are available that describe dosage, pharmacokinetics, metabolism, and routes of elimination of many of them (111,156–159). In addition to those in Table IX, five additional ACE inhibitors are currently in use in the United States. Their chemical structures and generic

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37

TABLE IX Prototype Angiotensin-Converting Enzyme Inhibitors Generic name

Usual daily antihypertensive dose rangea

Captopril Enalaprilat Enalapril maleate Lisinopril

25–50 mg bid or tid p.o. 1.25 mg iv every 6 h 10–40 mg p.o. qid 20–40 mg p.o. qid

Fosinopril

20–40 mg p.o. qid

a

Comments SH-containing, renal excretion Intravenous use only, renal excretion Prodrug, renal excretion Oral uptake likely via peptide transport, renal excretion Prodrug, renal, and biliary excretion

Physicians Desk Reference (155), which should be consulted for details.

names are shown in Fig. 2. They are all extensions of the enalapril and in the case of perindopril the phenethyl group of enalapril has been replaced by a propyl group. Their recommended clinical dose ranges and several key references to their use in treating hypertension and heart failure are indicated in Table X. Another ACE inhibitor benazepril is available in combination with amlodipine. VIII. CONTRIBUTION OF ACE INHIBITORS TO THE GROWTH OF PHYSIOLOGICAL AND PATHOPHYSIOLOGICAL KNOWLEDGE The experimental and clinical use of synthetic peptides (BPP5a, saralasin, teprotide) and angiotensin II antibodies provided crucial information on the physiological role of the renin-angiotensin system in the control of blood pressure (175). Its importance is dependent on the sodium balance: the more negative is the balance, the more important is renin dependency (90, 92, 176, 177). This is true in animals and in humans (178, 179), and it is true however one inhibits the RAS. Ironically, angiotensin II antagonists (saralasin) were administered to humans before ACE inhibitors (teprotide), but orally active angiotensin II antagonists only appeared in clinical investigation in the 1990s, whereas orally active ACE inhibitors have been successfully studied in humans from 1980 to the present. As noted earlier, the teprotide clinical studies established two important properties of ACE inhibitors: (1) their effectiveness is not limited to patients with high renin levels, and (2) as expected, diuretics enhance their efficacy in lowering blood pressure. The RAS is very complex physiologically. Thus, when interventions are made in it, the results are often difficult to interpret in a definitive

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FIG. 2. Additional ACE inhibitors available in the United States

way, and this problem is well exemplified in studies comparing saralasin and teprotide (180). Teprotide was more effective than saralasin in lowering blood pressure in low-renin hypertensive patients; this was interpreted as evidence of agonistic properties of saralasin. It could also be due to the bradykinin accumulation induced by the ACE inhibitor teprotide, in addition to its interruption of angiotensin II generation (181). At the same time, the finding in rats that beta blockers inhibit TABLE X Antihypertensive Dose Ranges as Monotherapy for the Angiotensin-Converting Enzyme Inhibitors Shown in Figure 2 Generic name Moexipril Perindopril Quinapril Ramipril Trandolapril a

Usual daily oral dose range (mg)a

References

7.5–30 4–16 10–80 2.5–20 1–4

160–162 163–165 166–168 169–171 172–174

Physicians Desk Reference (155), which should be consulted for details.

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39

renin secretion (182) has been used as one explanation, among others, for the antihypertensive effects of beta blockers in humans (183). The New York research group, which has made many important contributions to this field by testing the acute effects of RAS inhibitors in hypertension and later in congestive heart failure, has also reported that the antihypertensive effect of propranolol was proportional to pretreatment plasma renin activity levels. Because of this association between pretreatment plasma renin and their antihypertensive effect, beta blockers could be credited with being the first orally active inhibitors of the RAS (183). However, up to now the direct participation of the decreased renin secretion observed during their administration has still been a disputable explanation of their blood pressure lowering activity. Whatever the interpretation, we have learned that a normal plasma renin activity does not preclude the participation of the RAS in blood pressure control and that an appropriate use of diuretics is always able to convert a low-renin profile into a high-renin profile, providing the logic for developing fixed-dose combinations of beta blockers and diuretics, ACE inhibitors and diuretics, and angiotensin II antagonists and diuretics (184). The birth of a revised and better substantiated concept of an equilibrium between volume and renin in determining blood pressure levels (185) encouraged the industry to invest more and more in the inhibition of the RAS as a potential treatment of high blood pressure and congestive heart failure. In 2000, ACE inhibition and angiotensin II antagonism have both been successfully developed. Renin inhibitors with useful oral activity and duration of action have still not been developed, and it is very likely that if successful, they will not fundamentally change our current views of the treatment of hypertension and of its pathophysiology (33, 186, 187). It is needless to emphasize that the development of orally active ACE inhibitors was a necessary step to progress from research-oriented pharmacological probes to patient-oriented drugs. These drugs were necessary to investigate the long-term effects of a chronic inhibition of the RAS. A point that deserves attention is the absence of tachycardia during the fall in blood pressure induced by an acute or a chronic blockade of the RAS. This counterregulation, which is very marked after administration of hydralazine, minoxidil, and fast-acting calcium blockers, is an indirect measurement of an overactivity of the sympathetic nervous system, which limits a drug-induced primary hypotensive effect (188). Blockade of the RAS may suppress this compensatory activation of the sympathetic nervous system, for instance, after administration of short-acting dihydropyridines or arteriolar vasodilators (189–191). Such a stimulation cannot be considered as desirable in the profile of

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an ideal antihypertensive drug and is not observed during the inhibition of the RAS. Another counterregulation observed during treatment with guanethidine, alpha blockers, clonidine, methyldopa, hydralazine, and minoxidil is a positive sodium balance, which limits the initial fall in blood pressure (192). Because of the blockade of the intrarenal RAS and the decrease in aldosterone production, such a positive sodium balance does not exist during treatment with an ACE inhibitor (193). The absence of sympathetic nervous system stimulation (194) and the neutral or slightly negative sodium balance induced by ACE inhibition are two fundamental observations, which certainly contribute to the longterm beneficial effects of these drugs in addition to the decrease in blood pressure. IX. BIOLOGICAL ADVANCES IN THE KNOWLEDGE OF ACE THAT EVOLVED IN PARALLEL WITH THE DRUG DEVELOPMENT PROCESS The chemical synthesis of ACE inhibitors has facilitated the purification of ACE by affinity chromatography with immobilized lisinopril and has therefore contributed to the cloning of ACE (195). Subsequent to the development of ACE inhibitors, the most important discovery was this determination of the structure of the gene coding for ACE, which has opened the way for many unexpected findings, whose consequences are not yet completely explored. The presence of two genes, two active sites, and an allelic polymorphism (I/D) associated with high (D/D) or low (I/I) levels of plasma ACE and tissue ACE has initiated new theories on the pathophysiological importance of ACE (196, 197). Increased expression of ACE in macrophages, vascular smooth muscle cells, and fibroblasts may participate in the initiation and development of cardiac, vascular, and renal lesions. The D/D polymorphism of ACE has been proposed as a candidate risk factor for diseases affecting the target organs of patients with high blood pressure after the initial publication of a case control study by Cambien et al. who found an excess of D alleles in male subjects affected by a myocardial infarction (not revealed by sudden death) (198). The ACE plasma levels of D/D subjects are higher than those of I/I subjects, and their tissue expression of ACE in various tissues and in pathological circumstances is also higher (199–201). A meta-analysis performed by Staessen et al. has included a multitude of more or less concordant results, which were produced in a follow-up study to Cambien’s observations (202). From their analysis, Staessen et al. concluded that there was increased risk associated with D/D polymorphism, whereas Keavney et al., in the largest cohort study

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

41

so far reported, did not reach the same conclusions, and their results contradicted the results of the initial report, that is, the risk ratio for myocardial infarction with the D/D genotype seems to lie in the range of 1.0 to about 1.1 (203). The conclusions achieved about myocardial infarction cannot be extrapolated to other organ diseases, and the small amount of ACE present within the renal vascular beds may explain why the progression of renal insufficiency may be faster in D/D than in I/I subjects (204). The findings of Keavney et al. (203) illustrate the need for much larger populations than is customary for studies of candidate genes, without undue emphasis on retrospectively defined groups. Although this has already been suggested, large-scale genotyping to specifically prescribe ACE inhibitors to D/D subjects would in fact deprive of treatment the largest fraction of the population that have a functional RAS whatever their plasma or tissue levels of plasma angiotensinogen, renin, and ACE may be. In current randomized studies (205), genotyping may differentiate D/D from I/I treated subjects, either by confirming a higher absolute risk in D/D patients or by demonstrating a more important relative risk reduction in the I/I subjects. Another contribution of gene cloning is the expression in transfected CHO cells of the N-terminal and C-terminal part of ACE (206). It has also allowed a more in-depth characterization of ACE inhibitors at its two active sites in the presence of different substrates, including Nacetyl (SDKP), whose hemoregulatory properties and physiological role are now being defined and which may become a target for future therapeutic research (207–210). In the meantime, the measurement of Nacetyl SDKP in plasma and urines may be a tool for the follow-up evaluation of patients’ compliance with ACE inhibitors (211). X. CLINICAL DEVELOPMENT PROCESS OF ACE INHIBITORS IN HYPERTENSION Physiological and pharmacological knowledge of the RAS was extremely helpful in the early characterization of new ACE inhibitors in humans (212, 213). Vascular reactivity to a carefully selected dose of angiotensin I has been the main test used to monitor the in vivo blockade of ACE, and to demonstrate its presence, its magnitude, and its duration. Such a test was performed initially by intraarterial measurements of blood pressure, and later by a noninvasive finger-pulse method. These investigations were crucial in selecting doses (214, 215) for blood pressure lowering in hypertensive patients (216, 217).

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Pharmacokinetic–pharmacodynamic studies (218, 219) coupling the measurement of active drug plasma levels and the placebo-corrected fall in blood pressure later allowed application of the Emax model to describe the blood pressure effects of ACE inhibitors and to compare the reproducibility of first-dose effects versus effects following repeated administration (219–224). This approach, which demonstrated an escape to alpha blockade (225), a drug treatment of hypertension that was later on eliminated by the ALLHAT study (226, 227), has shown the absence of such an effect during chronic ACE inhibition and has confirmed the large interindividual variability of the Emax from one hypertensive patient to another. The reactive rise of renin levels after ACE inhibition is another way of looking at dose-response curves of ACE inhibition in mildly sodium-depleted normotensive volunteers (228, 229), and it has been used to look at the additive effects of ACE inhibition and angiotensin II antagonism after combined single-dose administration. Because of the heterogeneity of hypertension, the blood pressure response to blockade of the RAS is quite variable from one patient to another, which complicates the search for the optimal daily dose of ACE inhibitors and the optimal dosage interval. Many factors influence the magnitude and the duration of the blood pressure response to ACE inhibition (230): • The basal activity of the RAS, which depends on sodium balance, age, and underlying diseases • The pharmacokinetic profile of the active drug and the interindividual variability of its pharmacokinetic constants, especially the half-life of its elimination from the plasma and tissue compartments (231, 232) • The persistence of a residual inhibition at the active sites of ACE following intake of the last dose • The counterregulations induced by the fall in angiotensin II, which stimulates renin release and increases de novo angiotensin I production, which competes with inhibitor at the active sites of ACE (233) • Possibly the amount of genetically determined plasma and tissue ACE The multiple difficulties encountered in the quantification of ACE inhibitor blood pressure effects have importantly contributed to a general improvement in the antihypertensive drug development process leading up to registration by health authorities (234, 235). Definition of the dose-range that is effective in blood pressure lowering has become more and more precise with time (236). It became obvious that a placebo-controlled investigation of the effect of ACE inhibitors on

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

43

blood pressure should be performed at the time of the peak effect and also at trough, just before ingestion of the next dose (237). It was learned, once more, that protocols based on ascending doses always favor the highest dose because of the time effect and of the presence of resistant hypertensive patients who will never respond to the therapeutic approach whatever the dose. The parallel group design, later on, was the only one used, especially for the development of angiotensin II antagonists. The number of patients included for each dose in each group must be large enough to differentiate 2 to 5 mmHg falls of blood pressure with a standard deviation of around 5 to 7 mmHg, which makes it necessary to include about 100 patients per group (238). Selfmeasurement or ambulatory measurement of blood pressure could make the dose differentiation easier by increasing the precision of the measurement and by the absence of a placebo effect, but the choice of the method of measurement is still debated (239–241). Most development programs are built on dose-finding protocols aimed at defining the minimal once-daily dose that is able to decrease blood pressure more than a placebo for 24 hours. This objective looks safe and reasonable (242). If a high daily dose of a drug induces specific adverse effects above a certain safety threshold, as did hydralazine and captopril, it is obvious that the definition of the minimal effective dose is the best strategy. If, within a large range, there is no toxic effect of the compound and if there is no dose-related side effect, such as those observed with calcium blockers, the search for the maximally effective dose becomes more appealing. Captopril was unfortunately a perfect example of the unexpected adverse effects (agneusia, proteinuria, leucopenia) linked to an excessive increase in daily doses. The objective was to prolong the duration of action up to 24 hours, whereas in reality, 12.5 mg captopril tid was effective but difficult to accept by some patients and difficult to advertise, in comparison with once-daily drugs such as diuretics (236). Indeed, one may still wonder if a twice daily administration of many ACE inhibitors in some hypertensive patients would not allow us, as in congestive heart failure, to reach more easily a maximal effect instead of the once-daily administration that is preferred to improve treatment compliance (243). Contradictory results have also been published in favor of the once-daily prescription dosage of ACE inhibitors in the morning or in the evening (244, 245). As to the use of crossover designs, with all their weaknesses they can be suitable for testing different doses in the same individual. They require an extremely precise methodology and placebo periods and treatment periods of sufficient duration (246–248). They also need to be used with a constant sodium diet (249).

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Finally, the use of factorial designs, concluded by a response-surface analysis, is the latest approach used to select the most appropriate combination doses of two classes of drugs that allows complete pharmacological manipulation of the RAS, namely diuretics and ACE inhibitors (250, 251). At the end of the clinical development programs, the once-daily recommended dose of ACE inhibitors extends from 2 mg (trandolapril) to 20 mg (enalapril) and 50 mg (captopril). Because of the difficulties encountered in precisely measuring blood pressure and because of the interindividual variability of the renin dependency, a head-to-head comparison of two ACE inhibitors or of an ACE inhibitor and an angiotensin II antagonist is difficult to perform and is extremely dependent on the dose range selected for the study. It is even more difficult if a surrogate end point is selected, such as the demonstration of a favorable effect on morbidity and mortality. A few examples of the influence of dose selection on results are already available from studies on hypertension and congestive heart failure. Captopril 25 or 50 mg once daily was selected for the CAPPP study, and this dose, which cannot cover 24 hours, may have contributed to the generation of an unnecessary debate about the lack of prevention of cerebrovascular accidents by ACE inhibitors in comparison with other antihypertensive drugs (252). A 20-mg twicedaily dose of enalapril in congestive heart failure seemed initially to be more risky than a 50 mg tid dose of captopril because it produces a fall in kidney perfusion pressure of longer duration than that produced by captopril (253), whereas a progressive increase from low to high doses of enalapril has, later on, demonstrated large beneficial effects in this indication (254). It is possible that the 50-mg dose of losartan in the Elite II study provided similar blockade of the RAS, as was achieved with 50 mg tid captopril (255). A higher dose of losartan, however, may provide more effective blockade but will need to be studied. It is important to realize how difficult it is to define the in vivo inhibition of ACE. The enzyme may be explored for its N-terminal active sites by measurement of a constant and maximally increased level of N-acetyl SDKP in plasma and urine (211), but the residual activity of the C-terminal sites is probably not being measured. The methods for in vitro measurement of plasma ACE, except perhaps that described by Nussberger et al. (256), do not appropriately quantify global ACE inhibition. Moreover, the consequences of enzyme blockade are modified by secondary activation of the RAS (233). Residual amounts of angiotensin II secondary to a reactive rise in renin and angiotensin I may explain why the administration of an angiotensin II antagonist still has an additive effect on blood pressure and possibly on the heart, the vessels, and the kidney when added to certain doses of ACE inhibitors (228, 229, 257).

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

45

A persistence of angiotensin II production, during ACE inhibition may also come from the production of angiotensin II by vascular and cardiac chymase (258–260), and there is still doubt concerning the origins of angiotensin II during ACE inhibition. Whatever the explanation, a combined blockade of the RAS should be more effective but under certain circumstances of low blood pressure or negative sodium balance, more risky than ACE inhibition or angiotensin II antagonism alone. In the absence of dose-dependent adverse effects, the present issue is to know how to inhibit ACE as much as possible, wherever it is located. The dose of ACE inhibitors prescribed should be able to bind to all active sites, either present at their physiological site, mainly endothelial cells, or abnormally present and functioning at the level of hypertrophied or ischemic hearts, atherosclerotic vessels, inflammatory infiltrate of the renal interstitium, or damaged glomeruli. The less angiotensin II is available at the level of vascular smooth muscle cells and other cells equipped with angiotensin II receptors, the more effective an ACE inhibitor or an Ang II antagonist is likely to be. For instance, to block neointimal development, the dose of drug necessary for vascular protection was considerably greater than the dose required to elicit a hemodynamic effect (261). XI. BENEFITS OF ACE INHIBITION BEYOND THE FALL IN BLOOD PRESSURE The randomized controlled clinical trials performed by Freis and his colleagues at the Veterans Administration Hospitals have provided some of the first solid evidence that moderate permanent hypertension has an improved prognosis when actively treated by sodium depletion (hydrochlorothiazide), by interruption of the sympathetic nervous system (reserpine) and with a vasodilator (hydralazine) (262). In parallel, the beneficial effects of this triple therapy were demonstrated in spontaneously hypertensive rats by the spectacular prevention and cure of their cardiac, vascular, and renal lesions (263). The possibility of obtaining greater benefit through the use of an ACE inhibitor came soon after the observation that these drugs were effective in reducing blood pressure in normal and high renin models of experimental hypertension (264). Captopril was not effective in the low-renin model of DOCA rats, which provides good evidence against a major role of bradykinin in the mechanism of action of ACE inhibitors (265), but they are very effective in decreasing the blood pressure of spontaneously hypertensive rats, despite the absence of any detectable abnormality of their RAS (95). Very soon after the discovery of captopril, it was reported that interruption of triple therapy or atenolol treat-

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ment of spontaneously hypertensive rats is accompanied by a rapid return of blood pressure toward its initial level, whereas it remains low for a long time after the interruption of a chronic captopril treatment (266–268). Sen et al. had previously selected cardiac hypertrophy as an end point in their investigation of the organ damage linked to high blood pressure. They demonstrated that similar falls in blood pressure induced by a vasodilator and α-methyldopa can induce different reductions in cardiac weight (269). The regression of left ventricular hypertrophy was more marked when the sympathetic nervous system was blocked than when it was stimulated by vasodilators. Others have also shown that ACE inhibition was much more effective than triple therapy or vasodilators in promoting regression of left ventricular hypertrophy (270) and on the profile of myosin isoforms (271). These experimental observations have been, more or less, confirmed at the level of patients, with different ACE inhibitors (272–274). Additional experimental evidence that ACE inhibitors might have specific benefits of their own in protecting target organs of hypertension comes from the work of Linz et al. (275), who reported prevention of cardiac hypertrophy without detectable fall in blood pressure in a rat model of aortic banding during treatment with low-dose ramipril. However, an answer to this important question is still pending results of the LIFE Study (276). This study compares an angiotensin II antagonist and a beta blocker, two antihypertensive drugs that have different hemodynamic effects despite the fact that they both inhibit the RAS, the first at the level of angiotensin II action and the second at the level of renin secretion. Another example includes the specific effects of ACE inhibition on the prevention of renal insufficiency. In comparison with triple therapy, the reduction of intraglomerular pressure induced by ACE inhibitors (277) as well as by angiotensin II antagonists (278), is much more marked, and it protects the kidney from glomerular, vascular, and tubulointersitial lesions observed in various experimental models, especially renal ablation and diabetes (279). Besides the fall in efferent arteriole resistance, the interruption of the RAS inhibits tubulointerstitial inflammation and the production of cytokines, which are observed in these various experimental models (280, 281). A large amount of experimental evidence is accumulating, year after year, in favor of a protective effect of target organs during ACE inhibition, presumably by preventing the end-organ damaging properties of angiotensin II independently of its classical effect on blood pressure (282). The absence of sympathetic nervous system stimulation, the absence of sodium retention, and more importantly, the existence of a slightly negative sodium balance through an intrarenal effect of ACE

ANGIOTENSIN-CONVERTING ENZYME INHIBITORS

47

inhibition on sodium reabsorption are the usual explanations of the beneficial effects arising from blockade of the RAS. However, multiple actions of angiotensin II at the cellular level apparently provide good reasons to consider angiotensin II as a major determinant of cardiac, vascular, and renal diseases, and the advantages of RAS blockade may be attributed to the neutralization of these tissue effects mediated by a balance between AT1 and AT2 receptors (261, 283–288). Once more, the participation of bradykinin is suggested by some results (289). Before opposing two interpretations of a phenomenon that is not yet completely demonstrated (cardiovascular and renal benefits independent of a fall in blood pressure), it is worthwhile to reemphasize that the clinical measurement of blood pressure by physicians or nurses is an insensitive and imprecise method for investigating the human organism’s hemodynamics. Failure to detect a fall in blood pressure by this method does not eliminate a hemodynamic effect. The cellular actions of angiotensin II (290) and its hemodynamic effects are so closely linked that attributing cardiovascular changes to one at the expense of the other is probably an intellectual exercise more than a realistic approach. In this context, the results of the Heart Outcomes Prevention Evaluation (HOPE) study will certainly influence our future use of ACE inhibitors and possibly also angiotensin II antagonists (291, 292). In the HOPE study, the cardiovascular benefit of ACE inhibition was assessed with an oral dose of 10 mg ramipril. This ACE inhibitor, which was tested in up to a 50-mg dose in normal volunteers (293), is prescribed in hypertension at a 2.5- to 5-mg oral dose. The 9,000 subjects with a high cardiovascular risk included in this study were not selected according to their blood pressure levels, which were not titrated to target blood pressures (except for detecting hypotension). Ramipril was administered versus a placebo for 4 years. The impressive relative risk reduction reported for all major cardiovascular events, including strokes, constitutes the first human demonstration of a global cardiovascular protective effect of ACE inhibition. Whatever its mechanism, whether angiotensin II suppression or increase in bradykinin levels, beneficial effects have been observed in the presence of a minimal fall in blood pressure (3/2 mmHg), with all the reservations previously made on blood pressure measurement methodology taken into account. In the context of the rules of evidence-based medicine, the extrapolation of the HOPE study results in an interesting matter for debate. On the basis of the experimental and clinical data so far available, a class effect can be reasonably accepted (294). For instance, captopril (295), benazepril (296), and ramipril (297), all have been

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superior to placebo for the prevention of renal insufficiency. A class effect seems to be acceptable, but the influence of the dose is much more difficult to analyze (294). Experimental and clinical data do suggest a dose-dependent effect of ACE inhibition on cardiovascular lesions: enalapril and renal damage (298), lisinopril and cardiac lesions (299), trandolapril and experimental atherosclerosis (300, 301), ramipril and cardiac hypertrophy (275), and lisinopril and human congestive heart failure (302). To answer the question of the ACE inhibitor dose useful for cardiovascular risk prevention beyond a detectable blood pressure effect, a 1.25 mg daily dose of ramipril is currently being tested in the Diabhycar study (205). Results will be available in 2001, and they will be extremely important in determining the dose of an ACE inhibitor that should be used for cardiovascular damage prevention. The patients included in the Diabhycar study are similar to those reported in HOPE in that they have non–insulin-dependent diabetes and permanent microalbuminuria (303). Therefore, a negative result with this low dose of ramipril will support the prescription of high doses of ACE inhibitors. A risk reduction of about 15% will favor a dosedependent effect of ACE inhibition on cardiovascular risk. A 25% risk reduction will support the prescription of low doses of ACE inhibitors. XII. ACE INHIBITORS AND CONGESTIVE HEART FAILURE Congestive heart failure studies in humans led to the discovery of the sodium-retaining factor now known as aldosterone (304) and the characterization of increased renin levels in the renal venous blood of these patients (305). Subsequently, activation of the renin-angiotensin aldosterone system was demonstrated experimentally in models of low- and high-output cardiac failure (306). Acute and chronic administration of the nonapeptide SQ 20881 was used by A. C. Barger and his group to investigate the role of the RAS system in conscious dogs after constriction of the pulmonary artery or thoracic inferior vena cava. They demonstrated that restoration of blood pressure is initially dependent on circulating angiotensin II and in later stages on an increase in plasma volume (307). An imbalance between renin secretion and sodium status is present during congestive heart failure, which explains why the management of this condition requires the appropriate use of two pharmacological tools, namely diuretics, including aldosterone antagonists (255, 308), and inhibitors of the RAS. Once more, the role of bradykinin cannot be ruled out by these experiments, but we have learned that the bradykinin B2 antagonist HOE-140 does not modify the vascular effects of enalapril in patients with congestive heart failure

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(309). After the demonstration of hemodynamic and symptomatic improvements induced by the short-term and long-term use of captopril (310, 311), Furberg and Yusuf pooled the results of placebo-controlled trials of both vasodilators and ACE inhibitors. Despite the absence of overall effects of vasodilators on survival, they suggested that ACE inhibitors may influence the vital prognosis beneficially (312). The CONSENSUS study was performed in 253 patients with severe congestive heart failure. They were randomized to receive either placebo (n = 126) or enalapril (n = 127), from 2.5 mg bid to 20 mg bid with a follow-up period that averaged 182 days. By the end of the study, there had been 68 deaths in the placebo group and 50 in the enalapril group (p = .003) (313). Later, enalapril was shown to be superior to hydralazine–isosorbide dinitrate (314, 315). With the data included in the overview of Garg et al. (316), it is possible to calculate that 18 patients need to be treated for 90 days to avoid one death or one hospitalization for congestive heart failure (95% confidence interval [CI] 16–23). This meta-analysis includes 32 trials with the ACE inhibitors captopril, enalapril, lisinopril, quinapril, ramipril, and perindopril. It is likely that high doses (for instance, lisinopril 35 mg daily) are more effective than low doses (lisinopril 5 mg daily) (302). Treating 30 patients for 4 years with a high dose of lisinopril (95% CI 16–509) will avoid one hospitalization for cardiovascular reasons or one death in comparison with a low dose, without increasing the number of adverse effects requiring withdrawal from treatment. XIII. ACE INHIBITORS AND MYOCARDIAL INFARCTION Coronary artery ligation in rats was initially used in research on the pathophysiology of congestive heart failure. This provided an animal model in which a wide range of infarct sizes and left ventricular dysfunction could be produced. Using this methodology, Pfeffer et al. demonstrated with long-term captopril therapy an improvement in ventricular pump function and an attenuation of ventricular dilation (317). When drug treatment was started 14 days after experimental myocardial infarction and administered for 1 year, 1-year survival was dependent on the size of the infarct, the most important benefit being observed in rats with moderate (20–40%) myocardial infarction. The second step in the research process was a placebo-controlled 1-year study performed on 59 patients with anterior myocardial infarction. Pfeffer et al. translated their animal data into a clinical investigation, which demonstrated that ventricular enlargement is progressive after anterior myocardial infarction. Captopril can attenuate this progres-

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sion, reduce filling pressures, and improve exercise tolerance (318). The last step was the performance of several large-scale controlled trials, whose overview has been performed by a research group independent of the industry (254, 319–322). To understand the use of ACE inhibitors in this indication, we can now rely on a systematic overview of individual data from 100,000 patients included in randomized trials (320). Thirty-day mortality was 7.1% among patients allocated to ACE inhibitors and 7.6% among control subjects, corresponding to a 7% relative risk reduction (95% CI 2–11). There is no doubt concerning the class effect based on results with enalapril, lisinopril, captopril, zofenopril, ramipril, and trandolapril. The majority of the lives saved (200 of 239) were saved within the first week, most of them in the first 2 days after randomization. The absolute benefit was particularly great in some high-risk groups, especially those with an anterior myocardial infarction. Treatment is associated with increased persistent hypotension (17.6 versus 9.3%) and renal dysfunction (1.3 versus 0.6%). These complications were observed in both the higher and lower fatality risk groups. For this reason, two alternate treatment strategies are currently proposed. In the first one, all patients who have a systolic blood pressure above 100 mmHg without a contraindication for the use of ACE inhibitors should be treated within 1 or 2 days after infarction with an orally administered ACE inhibitor. The survival benefit is independent of the additive use of other proven therapies (reperfusion, aspirin, beta-blockers). Another strategy is more selective. It proposes selecting patients at higher risk of death to avoid exposing the lower-risk patient to the adverse effects of treatment (323). A second meta-analysis was based on data from individual patients in five long-term randomized trials, which assessed ACE inhibitors in 12,763 patients with left ventricular dysfunction or heart failure. The relative risk reduction of death, with a follow-up period exceeding 4 years, is 20% (13–26%). This means it is necessary to treat 26 patients for 5 years (95 % CI 19–41) to avoid one death. If the combined end point used to assess the results is death plus reinfarction plus hospitalization for heart failure, it is necessary to treat 14 patients for 4 years to avoid one such event (95% CI 12–18). XIV. ACE INHIBITORS, CORONARY HEART DISEASE, AND ATHEROSIS An influential proposal came out of a retrospective review of 219 dossiers of hypertensive patients by Brunner et al. (324). They con-

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cluded that heart attacks and strokes were more frequently observed in high-renin (11%) and normal-renin (14%) patients than in low-renin (0%) patients. Like all retrospective case studies and especially like innovative hypotheses, these results have been heavily challenged. However, the data of Brunner et al. have been reinforced by the observations made by Alderman et al. (325) in a cohort of 2902 hypertensive patients followed for 3.6 years, namely, that plasma renin activity (separate from urinary sodium levels) was independently and directly associated with the incidence of myocardial infarction (55 cases). However, another cohort study in which renin was measured in normotensive people did not find any predictive value in renin measurements (326). The influence of age, posture, and sodium intake complicates plasma renin activity investigations, which may explain why, three decades after the original hypothesis, renin measurements are not routinely performed. The hypothesis that activation of the “circulating” RAS induces vascular damage still remains plausible (272). The role of the RAS has been extended to “local” systems, based on overexpression of various of its components in most target organs of hypertension (282, 327, 328). Cardiac and vascular chymases are other enzymes responsible for a local and vasculotoxic generation of angiotensin II (260). As for hypertension, congestive heart failure, and myocardial infarction, there are physiological and pharmacological rationales for the use of ACE inhibitors in patients with asymptomatic and symptomatic (angina pectoris) coronary heart disease. In sodium-depleted conscious dogs, it was shown, after injection of teprotide or saralasin, that activation of the RAS during sodium depletion contributes to circulatory homeostasis through vasoconstriction. Inhibition of the RAS lowers peripheral vascular resistance and arterial blood pressure and increases cardiac output and coronary blood flow but has no effect on myocardial contractility or energetics (329). These studies, performed in normal animals, as well as those performed in subjects with normal coronary arteries (330), are not directly relevant to the treatment of atherosclerotic coronary arteries of humans whose vascular wall functions are severely impaired. However, potential explanations in favor of the expected beneficial effects of decreasing production of angiotensin II and possibly of increasing bradykinin accumulation are the presence of an endothelial dysfunction (331); vasospasms that are facilitated by potentiation of the sympathetic nervous system and the RAS (332); inflammatory response and expression of procoagulant factors influenced by angiotensin II; and overexpression of the RAS genes, especially ACE itself. Prevention of atherosclerosis has now been reported in various animal models during treatments

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with both ACE inhibitors and angiotensin II antagonists (333), but the use of ACE inhibitors to prevent acute angina pectoris or to improve exercise tolerance has not been consistently demonstrated (334, 335). The main argument in favor of a beneficial effect of ACE inhibition on coronary heart diseases comes from the pooled results of the SOLVD treatment trial, the SOLVD prevention trial, and the SAVE, AIRE, and TRACE studies, which indicate a 21% (95% CI, 11–29%, p ≤ .001) relative risk reduction for myocardial infarction associated with ACE inhibitor therapy. Enalapril (SOLVD) significantly reduced hospitalization for unstable angina, and captopril (SAVE) reduced revascularization procedures (291). In patients treated for 38 to 42 months with enalapril or captopril and selected on the basis of a reduction in ejection fraction with or without heart failure, it is necessary to treat 49 patients to avoid one myocardial infarction (95% CI 32–117). A traditional approach in cardiovascular medicine is to contrast results with patients managed in primary prevention and patients managed after a cardiovascular accident (secondary prevention). The highest absolute risk is present during secondary prevention, which may explain why, for statistical reasons, the demonstration of a significant reduction of cardiovascular risk is achievable within a few years. It is conceivable that, within the natural course of atherosclerosis, ACE inhibition becomes effective only at a late stage of the disease as a result of the tissue effects of ACE inhibitors on endothelial function, inflammatory reactions, plaque instability, and hypercoagulation phenomena. At an earlier stage, these abnormalities could be absent or not yet be of sufficient magnitude to be beneficially influenced by a decrease in angiotensin II (or the accumulation of bradykinin). This is why results obtained in high-risk subjects are not easily applied to people with a lower risk; the demonstration would need many more patients followed for a longer time. It may happen that the early stages of atherosclerotic lesions are not initially angiotensin- or bradykinin-dependent, but at these early stages of the disease the hemodynamic effects of ACE inhibition are present. The ACE inhibitors may have their own beneficial effects on the natural evolution of the atherosclerotic lesions, possibly by modifying stress-mediated endothelial functions (336, 337). XV. ACE INHIBITORS AND PREVENTION OF RESTENOSIS Angiotensin-converting enzyme inhibitors have been tested for possible prevention of the restenosis that frequently occurs after coronary dilation. These studies were undertaken based on an experimental

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model involving endothelial denudation and injury induced by balloon catheterization of the rat carotid artery (338). Unfortunately, the translation of encouraging animal results to humans in the MERCATOR and MARCATOR studies yielded negative results (339, 340). Once more, there were experimental arguments in favor of local interruption of the RAS (341) or in favor of the accumulation of bradykinin (342), but interpretations are complex, with large differences from one species to another, and the participation of other angiotensin II–generating enzymes such as chymase. ACE inhibitors need now to be tested after coronary stenting. The increasing use of stents reduces the incidence of restenosis but still creates local lesions characterized by early thrombus formation, acute inflammation, and neointimal growth (343). A pharmacological approach in association with stenting in conceivable, and ACE inhibition looks promising, especially if it is confirmed that the D allele of ACE is a risk factor for restenosis (344, 345) by influencing local expression of the enzyme (199). XVI. ACE INHIBITORS AND RENAL INSUFFICIENCY The work of Brenner et al. has provided much of the experimental evidence supporting the concept of ACE inhibition as a specific tool to oppose the progressive deterioration of renal function that occurs after initial renal injury from various causes (346). The prevention of intraglomerular hypertension and its consequences on proteinuria and mesangial cell function is the main effect of decreased angiotensin II formation or of type 1 angiotensin II receptor blockade (277, 278). It is also possible that ACE inhibitors protect the kidney from the direct tropic and proinflammatory effects of angiotensin II (280, 281). The prevention of glomerular capillary hypertension is especially effective in rats with diabetes mellitus, thereby avoiding the development of glomerular structural injury and proteinuria (279). In normotensive diabetic patients, ACE inhibition reduces microalbuminuria (347), and careful measurements of blood pressure do detect a fall in blood pressure. Likewise in their main hypertension trials, it was demonstrated that enalapril, captopril, benazepril, and ramipril prevent a progressive fall in glomerular filtration rate and reduce renal deaths, in comparison with a placebo or with another antihypertensive agent (295–297, 348). However, ACE inhibitors fail to lower proteinuria in low-renin experimental models, such as DOCA hypertension (265, 349). They also can severely impair the renal function of stenotic kidneys, both in the one-clip, two-kidney model of experimental hypertension and in humans (350–353).

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Renal insufficiency is a late complication of hypertension (354). This is why the choice of the antihypertensive drug selected as the first-line treatment of a condition that will persist for many years is so important. From this viewpoint, the fall in blood pressure induced by ACE inhibitors might have beneficial renal effects, in addition to those induced by any decrease in perfusion pressure of the kidneys, especially in diabetic patients (222, 355–358). It is unknown if, independently of the hemodynamic effect, at an early stage of hypertension and before the initiation of a progressive decrease in renal function, a local decrease in angiotensin II (or increase in bradykinin) explains or directly participates in a so-called renoprotective effect (359). XVII. THE FALLACY OF THE CONCEPTS OF NORMOTENSION HYPERTENSION AND THE CARDIOVASCULAR PROTECTIVE EFFECTS OF ACE INHIBITORS

AND

The normative approach to the practice of medicine, based on the definition of thresholds, is a different paradigm from the continuous distribution of most biological parameters and their associated risks, as described by physiologists and epidemiologists (360–362). Blood pressure, cholesterol, and renin have a logarithmic gaussian distribution in populations. Renin dependency, for instance, may be considered as a constant feature of all humans except when they have a positive sodium balance, which more or less mimics schematic animal models such as DOCA hypertension (349). In this extreme situation, cardiac, renal, and vascular damages may be directly induced by the excess of salt itself, in the absence of any functional RAS (363). Acceptance of the continuous distribution of blood pressure and renin activity levels in human populations is a crucial step forward in understanding the advantages of ACE inhibition. We should not think of ACE inhibition in terms of treating an arbitrarily defined disease, hypertension, but instead should orient this enzyme inhibition toward its primary biological definition: the suppression of angiotensin II effects or eventually, the potentiation of bradykinin, as suggested by some. In the logarithmic linear model, which is generally proposed to describe the association between blood pressure level and cardiovascular and renal diseases (364), the imperfections of blood pressure measurements and the small number of events reported in the lowest blood pressure range make it difficult to precisely quantify the relationship between cardiovascular events and blood pressure in the absence and in the presence of treatment. It is certainly possible to live 1440 minutes per day with a blood pressure lower by 1–5 mmHg without detecting

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such a small change by the methods of measurement used in the daily practice of physicians. Nevertheless, a different physiological status can be created with beneficial cardiovascular effects. Low doses of an ACE inhibitor or an angiotensin II antagonist may offer end-organ protection in hypertension and have a beneficial effect in the absence of a fall in blood pressure (275), but it is very unlikely that complete normalization of cardiac, vascular, and renal lesions can be obtained in the absence of any fall in blood pressure; in experimental models, the lower the blood pressure, the less are the lesions of target organs (365). Huge clinical consequences are implied by this interpretation of the available experimental literature. It opens the way to inhibit the RAS independently of the control of high blood pressure, whatever might be the selected threshold for “high.” Let us formulate the problem this way. A global cardiovascular risk of 20% in 10 years is frequently proposed as a threshold to initiate treatment of hypertension or hypercholesterolemia (366–369). Such a risk is present in many people above 70 years of age even if their systolic blood pressure is 140 mmHg or lower. In the absence of clinical intolerance or long-term adverse effects, would it be worthwhile to inhibit ACE in these older people? The objective would be to have the beneficial effects of angiotensin II suppression, even if the risk has been mainly initiated along the aging process by factors other than elevated blood pressure (hypercholesterolemia, prolonged tobacco use, diabetes, high homocysteinemia, proinflammatory profile…)? The occurrence of an unexpected sodium or volume depletion in normotensive people treated with ACE inhibitors can be considered as a rare but potentially dangerous situation. Is the risk of occurrence of such an event (sodium depletion and functional renal insufficiency) out of proportion to the calculated cardiovascular benefits obtained from randomized controlled trials? In the end, the uncertainty concerning the health benefits or risks of a more and more frequent use of RAS inhibition brings us back to the physiological question: In the absence of salt, the RAS is a necessity, but when salt is largely available to all, it becomes a luxury and perhaps a risk factor for all. Should we restrict salt use or block the RAS or cautiously combine both approaches according to individual decisions and organizational opportunities? XVIII. SURROGATE END POINTS IN CLINICAL TRIALS OF ACE INHIBITION: ARE WE BEING MISLED? In the field of hypertension, despite the unfortunate and unexpected adverse effects of practolol, tienilic acid, and mibefradil, it has been generally considered that fall in blood pressure was an acceptable sur-

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rogate end point. Between 1964 and 1999, randomized controlled hypertension trials always demonstrated the superiority of all tested antihypertensive drugs over placebo or the equality between different drugs in reducing cardiovascular morbidity and mortality (370). Unfortunately, in 2000, doxazosin, a drug that was registered, tested in a small controlled trial (n = 371), and recommended as a first-line treatment (372), has been demonstrated to worsen the cardiovascular prognosis in comparison with the reference treatment, a low daily dose of chlorthalidone (227). As previously mentioned, there were indications from pharmacokinetic-pharmacodynamic studies that the antihypertensive effect of alpha blockade could diminish with time, probably because of a positive sodium balance, which tends to neutralize the initial hemodynamic effects of the alpha blocker (225). This class of drug had previously failed in the treatment of congestive heart failure. The worsened outcome in hypertensive patients using an alpha blocker in comparison with chlorthalidone is mainly explained by the occurrence of more cases of congestive heart failure. In addition to the initial risks of hypotension that were observed with prazosin, the positive sodium balance that accompanies alpha blocker use should have discouraged the prescription of alpha blockers regardless of the promotion of their ancillary properties on lipid and glucose metabolism (373–376). What about ACE inhibitors in hypertension? We do have a multitude of short-term randomized trials comparing their efficacy and their tolerability with those of all the other antihypertensive drugs (377). The ACE inhibitors have been marketed for hypertension on the exclusive basis of this proof of efficacy on blood pressure and on their safety profile in postmarketing surveys (378–380). Two adverse effects were attributed to bradykinin accumulation: (1) the extremely rare angioneurotic edema; and (2) cough, a nondose-dependent side effect, which is the most frequent adverse effect of this class of drug and which generally disappears when treatment is interrupted (381). These drugs should also be avoided in pregnant women, especially because of the reports of some cases of neonatal anuria (382–384). The collection of data on 2500 to 5000 patients during the clinical development programs before registration and then the worldwide pharmacological vigilance systems have been considered as sufficient to expose millions of people, for decades, to many new antihypertensive drugs. Concerning ACE inhibitors, in addition to their use in hypertension, convincing data have been accumulated on their efficacy in the treatment of severe conditions, such as congestive heart failure, renal insufficiency and post–myocardial infarction. However, the development of evidence-based medicine has encouraged the view that cheap drugs (diuretics and beta

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blockers), which have demonstrated their benefits to reduce not only blood pressure but also cardiovascular morbidity and mortality, should be preferred to recent expensive drugs, which have not been tested in morbidity-mortality trials in mild to moderate essential hypertension (385). The conclusion of the meta-analysis of Psaty et al. (385) in 1997 was the following: “Diuretics and beta blockers—inexpensive antihypertensive agents—have been proved to be both safe and effective in large long-term randomized clinical trials. The clinical rationale for withholding safe, effective and proven therapeutics must be compelling.” However, we and many others have done so since 1980. On the one side are those who practice medicine on the basis of a physiopathological interpretation of the control of blood pressure, derived from the equilibrium between sodium balance and the RAS (386). They also consider that ACE inhibition and low doses of diuretics are better tolerated than all previous antihypertensive drugs, which contributed to improved patient compliance with physicians’ prescriptions and to simplified disease management (387). On the other side are those who pay more attention to cost effectiveness. In their judgment there have been no “hard” data demonstrating that first-line treatment of hypertension with ACE inhibitors has a benefit/risk ratio superior or even equal to that of the antihypertensive drugs previously tested in large-scale trials of hypertension. They also assume that the cost/benefit ratio using ACE inhibitors is not favorable (388). Both assumptions are closely linked in their argumentation (389, 390). This debate is reflected in the fluctuating practice recommendations issued in various countries (372, 391). It is true that in many situations other than hypertension treatment, an apparent beneficial effect on a surrogate end point has been accompanied by severe adverse effects and increased mortality (392, 393). In medicine, the unexpected occurrence of severe adverse effects has on several occasions prevented confirmation of initial optimistic approaches based on pathophysiology, for instance, in placebo-controlled mortality trials of antiarrythmic or inotropic drugs and vasodilators (394–396). Whatever the disease, a mortality-morbidity study needs to collect a number of terminal end points whose incidence will be sufficient to give statistical-significance to a relative risk reduction of 5, 10, or 20% under the influence of one treatment in comparison with a placebo or with other treatments. The number of necessary events is dependent on the magnitude of the therapeutic effect and of the initial risk assessment. Using high-risk patients leads to a collection of more fatal and nonfatal cardiovascular events and makes it easier to obtain a statistically significant result within a reasonable time. In addition, high-risk patients are generally fragile

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patients. A drug investigation in such a population, if it does not induce adverse effects is indirect evidence in favor of the safety of the treatment when applied to healthier people in the general population. However, extrapolation of the results to low-risk patients remains what it is—an extrapolation. If the size of the benefit decreases, the relative importance of a small risk increases (397). For a physiologist, severe congestive heart failure is an extreme disorder of the renin-sodium balance. It is the exacerbated situation of any human when he or she regulates sodium balance through an unavoidable activation of the RAS. The physiopathologist will look at the continum from hypertension to congestive heart failure and will consider that this extreme situation brings to the forefront two essential aspects of treatment: the validity of the conceptual approach to improve hemodynamics by manipulating sodium balance and angiotensin II effects; and the safety of the pharmacological intervention demonstrated by its tolerability in the most severely ill patients. The natural history of the disease from hypertension to left ventricular hypertrophy to systolic dysfunction and to congestive heart failure should be interrupted as soon as possible (398), and the manipulation of the RAS and the sodium balance is the same, with various degrees of difficulties, according to the severity and the complexity of the clinical situation. On the contrary, an evidence-based approach will consider that the disorder of the reninsodium axis in congestive heart failure does not bring any insight concerning the hemodynamics of uncomplicated essential hypertension and the benefits expected from its treatment by blockade of the RAS. The beneficial effects of ACE inhibition in congestive heart failure treatment are so impressive that they make it impossible to detect rare and severe adverse effects, whose reality will only be unmasked in the absence of immediate and massive benefits. There is no basis to assume that results obtained in congestive heart failure provide for its prevention by prescribing at an early stage of mild hypertension a therapeutic approach that may be better than others 20 to 40 years after its initiation. Unfortunately, that is precisely the dilemna of any long-term drug prescription, and one may wonder about the future over 30 years of 50year-old patients with mild, uncomplicated hypertension treated with an alpha blocker. In the same way, one can also look at the results of ACE inhibition in renal insufficiency. The decrease in the rate of deterioration of renal function verifies experimental concepts and is reassuring for the prevention of long-term renal function deterioration in hypertensive patients. In untreated hypertensive patients, cerebrovascular and cardiac events occured quite frequently before renal insufficiency, which

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requires time to appear; renal insufficiency is a late complication of hypertension in the absence of malignant nephrosclerosis. Therefore, there is no practical way to design a controlled trial on the prevention of renal insufficiency in mild to moderate uncomplicated permanent hypertension. There are not enough events and there is need for much too long a follow-up period. If the results obtained in patients with renal insufficiency are extrapolated to those without renal insufficiency, we can consider that in the very long term, ACE inhibition is more likely to protect renal function than any other antihypertensive drugs with practically no chance of deteriorating it. From the viewpoint of evidence-based medicine, in the absence of positive results on total mortality in patients with severe renal insufficiency, beneficial effect on renal function is not a sufficient end point, and it should be accompanied by the absence of adverse effects on other functions that could contribute to mortality. Another approach is to challenge the concept of renal insufficiency as an entity and to observe that the influence of the RAS on the evolution of this syndrome encompasses too many clinical presentations. A treatment that is very effective in patients with glomerulopathies may be ineffective, unnecessary, and possibly contraindicated in patients with polycystic diseases or interstitial nephritis, or it may be specifically targeted toward diabetic nephropathy (399). For the last 20 years, the translation of scientific data into practice recommendations has been moving between these two extreme concepts of medicine. Therefore, too many patients are exposed to the most expensive drug instead of this therapeutic approach being specifically targeted toward those who will benefit from it, for instance, those who have microalbuminuria (399). This scientific debate has emerged within an explicit or implicit economic debate (386, 389, 390). Minimization of health expenditures is desirable for third-party payers, who take into account absolute costs and opportunity costs and look at the extra money that would be used in fields of therapy other than hypertension if the treatment of hypertension were made more cost-effective. Maximization of profits, on the contrary, is an objective of the pharmaceutical industry. When the relatively short duration of patents is taken into account, it is difficult to postpone drug registration and marketing while awaiting the results of 5-year mortality-morbidity studies. Industrial profits are used to try to meet the needs of untreated diseases, and this necessary permanent investment in research is a valid argument, which is parallel for tomorrow to the argument of the opportunity costs given by the third-party payers for today. Profits are needed for reinvestment in the many other fields of therapy that have not the maturity of hypertension management (400).

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Initiation of mortality-morbidity studies at the middle of phase III clinical studies without delaying the registration process seems to be an acceptable compromise in the present context. Limitations to these studies are coming from the facts that (1) ethically, placebo-controlled trials can no longer be justified in hypertensive patients whose systolic blood pressure is permanently above 160 mmHg; (2) practically, the comparison between two active drugs requires a huge number of patients even when they are at a 2% annual cardiovascular risk, such as diabetic patients over 50 years old or patients with microalbuminuria or left ventricular hypertrophy. Even more, the head-to-head comparison of two different strategic approaches to hypertension treatment does not allow one to attribute a difference to the beneficial effect of one or to the detrimental effect of the other (401, 402). For this reason, the treatment that has demonstrated beneficial effects on mortality and morbidity needs to be present in these comparisons (especially lowdose diuretics). Another issue is the synergism of drugs that are successively combined to achieve blood pressure control. Adding a diuretic to a beta blocker or an ACE inhibitor is a pharmacologically justified choice; adding a beta blocker to an ACE inhibitor in the absence of diuretic seems to be less justified (226). It is paradoxical that ACE inhibitors will remain for 25 years as the only antihypertensive therapy that has not been tested, as a first-line therapy, in a mortality-morbidity placebo-controlled study, performed in patients with uncomplicated permanent hypertension who are less than 80 years old. This may be due to the historical context in which they were developed: a more urgent perceived need for treatment of cardiac and renal insufficiency; a huge effort made at the same time to demonstrate in placebo-controlled studies the benefits of 3-hydoxy-3methylglutaryl-CoA (HMG-Co-A) reductase inhibitors in the primary and also secondary prevention of cardiovascular events; and the discovery of angiotensin II antagonists less than 15 years after ACE inhibitors. In the year 2000, because of the possibility that an increase in bradykinin induces modifications of endothelial functions and plays a role in the mechanism of action of ACE inhibitors (403), the beneficial results obtained in the prevention of major cardiovascular events in high-risk subjects cannot be securely extrapolated to angiotensin II antagonists. Nevertheless, the physiological and physiopathological background of the RAS is strongly in favor of the predominant importance of angiotensin II suppression or neutralization. Type 2 angiotensin II receptor stimulation, which seems to occur during type I angiotensin II blockade, cannot yet be considered to be relevant to the practice of evidence-based medicine, even if it apparently provides

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some advantages that could favorably replace the absence of bradykinin accumulation induced by ACE inhibitors. XIX. CONCLUSION The use of ACE inhibitors has been extended from the two indications that were predicted from the physiological studies performed before the 1970s, hypertension and congestive heart failure, to new indications: prevention of renal disease progression; prevention of left ventricular enlargement after myocardial infarction; and prevention of all cardiovascular events in high-risk patients defined by age, previous cardiovascular accident, diabetes, proteinuria, or microalbuminuria. Use of ACE inhibition has not been successful in preventing restenosis after coronary dilation and has not yet been tested after stenting. Prevention of dementia has not been studied. In normotensive or borderline hypertensive subjects, long-term cardiovascular damage secondary to a risk factor such as high blood pressure and others (hypercholesterolemia, tobacco use, homocystinemia, or the vascular aging process itself) might be prevented by inhibition of the RAS. This seems to be a conceivable but unreasonable objective for at least two reasons: the scientific demonstration of a benefit is extremely difficult to achieve, and the number of subjects who could be classified as needing drug therapy would finally include the largest part of the population, which can be regarded as an excessive and potentially dangerous medicalization of society. The biological basis that has made possible the development of this unique therapeutic progress still needs to be enlarged. We do not yet know the relative participation of bradykinin in the long-term effects of ACE inhibition; comparative studies with angiotensin II antagonists are already being undertaken on a large scale, but all necessary precautions must be taken to be sure that the doses of the compounds to be compared are equipotent in terms of blockade of the RAS. If we are quite certain that ACE immobilized at the surface of the endothelial cells is physiologically more important than is the circulating enzyme (197), we do not know yet what is the relative importance of its Nand C-terminal active sites. We do not know how they possibly affect the activity of each other. We do not know the physiological role of the Nacetyl SDKP, a relatively specific substrate of the N-terminal active site. We may still discover other natural substrates for endothelial and epithelial ACE. We do not fully understand the basis of differences in the various daily doses of ACE inhibitors as usually prescribed, from trandolapril 2 mg/day to lisinopril 80 mg/day, and the consequences of dose choices.

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We do not know if residual angiotensin II during ACE inhibition is produced by a residual ACE activity or by a chymase. We do not know if angiotensin II neutralization by a combination of an ACE inhibitor and an angiotensin II antagonist can reinforce, without excessive risk, the blockade of the renin-angiotensin system and its benefits. We do not know if the inhibition of neutral endopeptidase at the same time as inhibition of ACE can provide an improved benefit/risk ratio (404). We do not know if ACE inhibition can improve glucose metabolism, as suggested in HOPE (303), or can protect from cancer, as suggested by the retrospective review of the hypertensive patients followed up at Glasgow (405). Finally, we do not know the impact of the D/I polymorphism, especially through differences in the cellular expression of ACE, on the cardiovascular and renal prognosis or on the therapeutic results. It is reassuring to observe that, after learning so much from the discovery of ACE inhibitors, so many questions remain unanswered, and therefore so many possibilities for progress should still be achievable within the next decade. ACKNOWLEDGMENTS The very excellent, skillful preparation of this manuscript by Ms. Janice Gwaldis and Mrs. Joelle Schalma is gratefully acknowledged. The experimental and clinical investigations of one of us (JM) have been supported by INSERM and the Association Claude Bernard from 1967 to 2000.

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HMG-CoA REDUCTASE INHIBITORS BY D. ROGER ILLINGWORTH* AND JONATHAN A. TOBERT† *Division of Endocrinology, Diabetes, and Clinical Nutrition (L465), The Oregon Health Sciences University, Portland, Oregon 97201 and †Merck Research Laboratories, Rahway, New Jersey 07065

I. Background and History . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Effects on Lipoproteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Chronopharmacology . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Dose-Response Relationship . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Comparative Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Mechanisms of the Cholesterol-Lowering Effects of Reductase Inhibitors . . . IV. Combination Therapy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Diet . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Drugs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Safety and Tolerability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Myopathy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Liver. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other Safety Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Outcome Studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Mechanisms of the Reduction in Coronary Morbidity and Mortality . . . . . VIII. Safety of HMG-CoA Reductase Inhibitors in the Megatrials. . . . . . . . . . . . . A. Noncardiovascular Deaths . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Treatment Intensity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Effects on Other Vascular Beds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. BACKGROUND AND HISTORY It has been clear for several decades that an elevated concentration of plasma cholesterol attributable to increased concentrations of atherogenic lipoproteins (low-density lipoproteins [LDL] and remnant lipoproteins) is a major risk factor for the development of coronary heart disease (CHD) (Castelli et al., 1992; Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1993). The cholesterol biosynthetic pathway (Fig. 1) was a natural target in the search for drugs to reduce plasma cholesterol concentrations in the hope that these treatments would reduce the risk of CHD. However, early attempts to reduce cholesterol biosynthesis were disastrous. Triparanol, which inhibits the penultimate step in the pathway, was introduced into clinical use in the mid-1960s but was withdrawn from the 77 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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FIG. 1. Cholesterol biosynthesis pathway.

market shortly thereafter owing to the development of cataracts and various cutaneous adverse effects, including ichthyosis (Kirby, 1967). These side effects are now known to be attributable to tissue accumulation of the sterol desmosterol, which is a late-stage lipid-soluble intermediate in the cholesterol biosynthetic pathway. The triparanol experience set the stage for subsequent skepticism about the safety of any pharmacological attempts to reduce plasma concentrations of cholesterol-rich atherogenic lipoproteins by inhibiting the cellular biosynthesis of cholesterol, which in the liver would be predicted to reduce lipoprotein synthesis.

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FIG. 1. (continued)

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FIG. 1. (continued)

3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase is the ratelimiting enzyme in the cholesterol biosynthetic pathway (Fig. 1). In contrast to desmosterol and other late-stage lipid-soluble intermediates, HMG is water-soluble, and there are alternative metabolic pathways for its breakdown when HMG-CoA reductase is inhibited so that there is no buildup of potentially toxic precursors. Therefore, of the more than 30 enzymes involved in the biosynthesis of cholesterol, HMG-CoA reductase was a natural target. Substances that have a powerful inhibitory effect on this enzyme, including ML236B (compactin), were first discovered by Endo in a fermentation broth of Penicillium citrinum in the

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FIG. 1. (continued)

1970s during a search for substances with antifungal properties (Endo et al., 1976; Endo et al., 1977). Although no HMG-CoA reductase inhibitor has been shown to have useful antifungal activity, the possibility that an agent that inhibited the rate-limiting step in the biosynthetic pathway could have useful lipid-lowering properties was quickly appreciated by Endo and others. Compactin was shown to lower plasma cholesterol in the rabbit (Watanabe et al., 1981), monkey (Kuroda et al., 1979), and dog (Tsujita et al., 1979). However, some investigators were led astray by the fact that compactin did not lower serum cholesterol in the rat (Fears et al.,

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1980), which was later shown to be the result of massive induction of HMG-CoA reductase in rat liver (Singer et al., 1988; Bergstrom et al., 1998). The dog was found to be a good animal model (Alberts et al., 1980), especially when pretreated with the bile acid sequestrant cholestyramine added to the dog chow. The prototype compound compactin was developed by the Japanese company Sankyo and was shown to be highly effective in reducing concentrations of total and low-density lipoprotein (LDL) cholesterol in the plasma of adult patients with heterozygous familial hypercholesterolemia (Mabuchi et al., 1981; Mabuchi et al., 1983). In 1978 Alberts, Chen, and others at Merck Research Laboratories discovered mevinolin (later known as lovastatin) in a fermentation broth of Aspergillus terreus and showed it to be a potent inhibitor of HMG-CoA reductase (Alberts et al. 1980). Clinical trials in healthy volunteers were begun in April 1980, and lovastatin was quickly shown to be dramatically effective for lowering LDL cholesterol in these volunteers, with no obvious adverse effects (Tobert et al., 1982a; Tobert et al., 1982b). Clinical trials with compactin had been proceeding, but for reasons that have never been made public (but that were believed to include serious animal toxicity), were stopped in September 1980. Because of the close structural similarity between compactin and lovastatin, the clinical development of lovastatin was also promptly suspended, and additional animal safety studies were initiated. In 1982, Bilheimer and Grundy in Dallas and Illingworth in Portland, Oregon began to test the effect of lovastatin in small groups of patients with severe familial hypercholesterolemia refractory to existing therapy and obtained dramatic reductions in LDL cholesterol (Bilheimer et al., 1983; Illingworth and Sexton 1984). Most of the patients treated by one of us (DRI) in these early studies have continued to take lovastatin to this day. Thompson in London, England found that lovastatin considerably enhanced the hypolipidemic effect of apheresis in patients with severe familial hypercholesterolemia (Thompson et al., 1986). When the additional animal safety studies revealed no evidence for the phenomenon that had apparently caused the studies with compactin to be stopped, the lovastatin development program was reinitiated, initially in patients at high risk of myocardial infarction, in 1984. It quickly became apparent that lovastatin was as effective and well tolerated in patients with familial (Havel et al., 1987) or polygenic (Lovastatin Study Group II 1986) hypercholesterolemia as had previously been reported in healthy volunteers (Tobert et al., 1982a). Lovastatin first became available for prescription in the United States in 1987 and in other countries shortly thereafter. Simvastatin, which differs from lovastatin only in that it has an additional side chain methyl

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FIG. 2. Structures of the six marketed HMG-CoA reductase inhibitors.

group, was approved for marketing first in Sweden in 1988. Pravastatin followed in 1991, fluvastatin in 1994, atorvastatin in 1997, and cerivastatin in 1998. At least two further HMG-CoA reductase inhibitors are in clinical development. As noted above, lovastatin is a fermentation product and simvastatin and pravastatin are semisynthetic compounds related to lovastatin, whereas the other three HMG-CoA reductase inhibitors are totally synthetic products. The structures of these drugs are shown in Fig. 2. Prior to 1987, the lipid-lowering armamentarium was limited essentially to dietary changes (reductions in saturated fats and cholesterol), the bile acid sequestrants (cholestyramine and colestipol), nicotinic acid (niacin), the fibrates, and probucol. Unfortunately, all of these treatments have limited efficacy or tolerability or both. Substantial reductions in LDL cholesterol (up to 47%) accompanied by increases in HDL cholesterol of up to 32% could be achieved by the combination of a lipid-lowering diet, a bile acid sequestrant, and the subsequent addition of nicotinic acid (Illingworth et al., 1981). However, this therapy is not easy to administer or tolerate and was therefore often unsuc-

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cessful, except in specialist lipid clinics. The fibrates (gemfibrozil, fenofibrate, bezafibrate, and others) produce a moderate reduction in LDL cholesterol accompanied by an increase in high density lipoprotein (HDL) cholesterol and a substantial reduction in triglycerides. Because they are well tolerated, these drugs have been more widely used. Probucol produces only a small reduction in LDL cholesterol but also reduces HDL cholesterol, which, because of the strong inverse relationship between HDL cholesterol level and CHD risk, is generally considered undesirable. This drug is no longer marketed in most countries. With the introduction of lovastatin in 1987, physicians were for the first time able to obtain large reductions in plasma cholesterol concentrations in hypercholesterolemic patients with very few adverse effects and excellent patient compliance. At the maximal recommended dose of 80 mg daily, lovastatin is capable of reducing LDL cholesterol by 40% on average (Lovastatin Study Group II, 1986; Havel et al., 1987; Lovastatin Study Group III, 1988), and the vast majority of patients experience no adverse effects. For these reasons, lovastatin was rapidly accepted by prescribers and patients, and was an immediate commercial success. It is perhaps ironic that at one time lovastatin was considered for development as an orphan drug; because of fears of toxicity based on the experience with compactin, it was initially expected to be appropriate therapy only in patients at extremely high risk. After these concerns were resolved by numerous animal safety studies and an intensive clinical development program, the efficacy and tolerability of the drug paved the way for its success. Today HMG-CoA reductase inhibitors (statins) account for the large majority of prescriptions for lipid-lowering drugs in most countries. As discussed in a subsequent section of this chapter, the widespread acceptance of this drug class is due not only to excellent efficacy and tolerability but also to the publication of several very large intervention trials, which have unequivocally demonstrated the effectiveness of the first three members of the class—lovastatin, simvastatin, and pravastatin—in reducing coronary morbidity and mortality. II. EFFECTS ON LIPOPROTEINS The characteristic effect of inhibitors of HMG-CoA reductase is a profound reduction in LDL cholesterol. The maximal doses of the most effective drugs in this class, simvastatin and atorvastatin, produce reductions by about half in the plasma levels of this atherogenic lipoprotein (Heinonen et al., 1996; Ose et al., 1998; Stein et al., 1998b). The full

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FIG. 3. Lovastatin nonfamilial hypercholesterolemia study total cholesterol levels (mean ± SE) in group 5 (N = 17 to 20 at each point). Abbreviation “b.i.d.” indicates bis in die (“twice per day”); abbreviation “q.p.m.” indicates quaque post meridiem (“once daily with the evening meal”).

therapeutic effect is obtained by 4 to 6 weeks, with at least 75% of the ultimate effect apparent by 2 weeks after starting therapy. When treatment is stopped, the recovery back to baseline mirrors the onset of the therapeutic effect, as shown in Fig. 3 from an early study with lovastatin. In addition, there is a moderate reduction in plasma triglycerides, which may be greater in patients with combined hyperlipidemia (Stein et al., 1998d), although the effects of regression to the mean should not be ignored when comparing data in patients with high triglycerides with data for patients with normal triglyceride levels. In addition, HDL cholesterol increases modestly, and there are also small increases in apolipoprotein Al, the major protein of HDL. Although it has not yet been established that increasing HDL cholesterol reduces the risk of coronary events, there is a strong inverse relationship between HDL cholesterol and coronary disease (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1993), probably because of its role in transporting cholesterol from peripheral tissues back to the liver. Effects on HDL cholesterol may contribute to the reductions in coronary morbidity and mortality produced by simvastatin (Scandinavian Simvastatin Survival Study Group, 1994; Pedersen et al., 1998a), particularly in patients with low HDL cholesterol. Accompanying these changes is a large reduction in apolipoprotein B, the principal protein of both LDL and very low density lipoprotein (VLDL). The fact that apolipoprotein B falls nearly as much as LDL

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cholesterol indicates that the number of LDL particles is reduced, as each particle contains one molecule of apolipoprotein B. There is little if any effect on apolipoprotein All or Lp(a). A. Chronopharmacology Except for atorvastatin, which may be administered at any time of day, HMG-CoA reductase inhibitors are most effective when given in the evening. Other than hypnotics, very few drugs are given in the evening, but evening dosing of HMG-CoA reductase inhibitors has been shown to be more effective than morning dosing (Lovastatin Study Group IV, 1990; Saito et al., 1991), probably because in humans the rate of cholesterol biosynthesis is greater during the night than during the day (Jones and Schoeller, 1990). All of the statins except for atorvastatin have plasma half-lives of approximately 2 hours (Plosker and McTavish, 1995; Lea and McTavish, 1997; McClellan et al., 1998). The much longer half-life of atorvastatin (about 20 hours) (Cilla et al., 1996) allows for morning dosing. B. Dose-Response Relationship Within the currently recommended dosage ranges of the inhibitors of HMG-CoA reductase, the relationship between response (expressed as percent reduction in LDL cholesterol) and dose is loglinear, as is the case for most drugs. The dose-response curves in large comparative studies are generally parallel (Farmer et al., 1992; Mitchel for the Lovastatin Pravastatin Study Group, 1993; Ose et al., 1995; Illingworth et al., 1996), which also is typical. The slope of the dose-response curves is such that each doubling of the dose yields an additional mean reduction in LDL cholesterol of approximately 6% in most large studies with multiple dosage regimens (not confounded by titration) (Farmer et al., 1992; Mitchel YB for the Lovastatin Pravastatin Study Group, 1993; Illingworth et al., 1994a; Ose et al., 1995; Illingworth et al., 1996; Illingworth and Tobert, 1994b), including the Expanded Clinical Evaluation of Lovastatin (EXCEL) (Bradford et al., 1991), which randomized over 8000 patients to placebo or one of four dosage regimens of lovastatin and which because of its size provided values essentially free of random errors. Figure 4, from the study of Illingworth et al., (1996), demonstrates these relationships. It follows that an absolute 6% difference in LDL cholesterol reduction at the same dose of any two reductase inhibitors implies a twofold potency difference.

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FIG. 4. HMG-CoA reductase inhibitor dose-response relationships.

C. Comparative Efficacy Inhibitors of HMG-CoA reductase are the most effective means of lowering blood cholesterol, but it is clear that the members of the class are not all therapeutically equivalent. Since we last addressed this topic in 1994 (Illingworth and Tobert, 1994b) and 1996 (Pedersen and Tobert, 1996b), two new statins have been marketed, atorvastatin (Yee and Fong, 1998), introduced in 1997, and cerivastatin in 1998 (Stein, 1998a; Stein, 1998c). In addition, the dosage range of simvastatin has been extended to 80 mg daily in the United States and several other countries, with applications pending in others. Similarly, the dosage range for fluvastatin has been extended to 80 mg in some countries. Statins may be broadly divided according to efficacy, that is, the reduction in LDL cholesterol attainable across the usual recommended dosage range. As shown in Fig. 5, pravastatin, fluvastatin, and cerivastatin comprise the low-efficacy group, while atorvastatin and simvastatin are clearly much more effective and may be designated high-efficacy statins. Lovastatin occupies an intermediate position. The maximal recommended doses of the agents in the low-efficacy group are less effective than the usual starting doses of the two drugs

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FIG. 5. Comparative efficacy of statins: mean percent reduction in LDL cholesterol at usual starting dose (filled bar) and titration range (open bar).

in the high-efficacy group. The source of the information in Fig. 5 is predominantly the product circulars. However, the product circular for atorvastatin lists efficacy values based on a clinical pharmacology study with only 11 subjects taking the maximal 80 mg dose (Nawrocki et al., 1995), which is insufficient for an accurate estimate. There are remarkably few published data on the lipid-lowering efficacy of the maximal 80-mg dose of atorvastatin. However, a literature source with a larger sample size (189 patients) taking atorvastatin 80 mg is available (Heinonen et al., 1996). In this study, atorvastatin 80 mg produced a mean reduction in LDL cholesterol of 52% at 1 year, but 55% has been used in the figure to allow for the fact that with all statins efficacy tends to decline slightly at 1 year relative to studies of a few weeks in duration, presumably because of adherence to treatment (and possibly dietary compliance) declining over time. This may exaggerate the effect of atorvastatin 80 mg, because a recent study (Pitt et al., 1999) reported a mean decrease in LDL cholesterol of only 46% in 164 patients after 18 months, although this result was confounded by the fact that 22% of the patients were taking lipid-lowering therapy at baseline. On balance, however, the maximal dose of atorvastatin probably produces a mean reduction in LDL cholesterol of slightly more than the 47% obtainable with the maximal dose of simvastatin (Ose et al., 1998; Stein et al., 1998b). On the other hand, higher doses of simvastatin appear to increase HDL cholesterol more than equivalent doses of atorvastatin (Crouse et al., 1999).

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III. MECHANISMS OF THE CHOLESTEROL-LOWERING EFFECTS OF REDUCTASE INHIBITORS Although HMG-CoA reductase inhibitors lower plasma cholesterol concentrations by first inhibiting cholesterol synthesis at the rate-limiting step, the mechanism of the reduction in apolipoprotein B–containing particles is not simply due to a reduction in cholesterol biosynthesis. Inhibition of HMG-CoA reductase leads to a reduction in the regulatory sterol pool, which leads in turn to an up-regulation of HMG-CoA reductase (Brown and Goldstein, 1980), other enzymes of cholesterol biosynthesis (Balasubramaniam et al., 1977; Bergstrom et al., 1984), and the LDL receptor (Kovanen et al., 1981; Ma et al., 1986). The increases in activity of these proteins are largely mediated through transcription (Liscum et al., 1983; Goldstein and Brown, 1990) and by changes in the rate of degradation (Faust et al., 1982; Goldstein and Brown, 1990). Although most of these results have been obtained in animals, some data are available on the effects on human liver. Reihner et al. treated 10 patients undergoing elective cholecystectomy with pravastatin 20 mg twice daily for 3 weeks prior to surgery (Reihner et al., 1990). A liver specimen was obtained from each patient at operation, and the activities of rate-determining enzymes in cholesterol metabolism as well as LDL receptor binding activity were determined. Relative to an untreated control group, pravastatin reduced plasma total cholesterol by 26% and LDL cholesterol by 39%. Microsomal HMG-CoA reductase activity, analyzed in vitro in the absence of the inhibitor, was increased 11.8-fold, and the expression of LDL receptors increased by 180%. These changes indicate that HMGCoA reductase inhibitors lower plasma cholesterol by increasing the uptake of LDL via the LDL receptor. Kinetic studies also provide evidence that these drugs decrease production of apolipoprotein B–containing lipoproteins by the liver (Arad et al., 1992). Consistent with this view is the fact that atorvastatin when used at high doses (80 mg/day) (Marais et al., 1997) and simvastatin (Raal et al., 1997) have been shown to reduce LDL cholesterol concentrations by 25–30% in patients with homozygous familial hypercholesterolemia. IV. COMBINATION THERAPY A. Diet A diet low in saturated fat and cholesterol has been the traditional mainstay of therapy and should continue to accompany statin therapy, as the effects are additive (Hunninghake et al., 1993). However, perhaps

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because diet is a major determinant of the average blood cholesterol levels of populations, there has been a tendency to overestimate the efficacy of lipid-lowering diets in individuals. In the Scandinavian Simvastin Survival Study (4S), dietary therapy alone produced an average 2% reduction of serum cholesterol (Scandinavian Simvastatin Survival Study Group, 1994), a typical result in a large trial (69), and 28% of the patients receiving dietary therapy plus placebo suffered a major coronary event over the 5.4 years of the study. Although a diet low in saturated fat and cholesterol is quite effective in metabolic ward studies, adherence to the diet is difficult for many patients, which leads to much smaller reductions in outpatients preparing their own meals. Many outpatient trials have overestimated the efficacy of dietary therapy through the use of designs that are readily confounded by regression to the mean. The largest randomized controlled trial of intensive dietary therapy in outpatients found that the National Cholesterol Education Program (NCEP) step II diet lowered LDL cholesterol by 5% and HDL cholesterol by 6% (Hunninghake et al., 1993). Fortunately, the inadequacy of dietary therapy alone is now increasingly recognized (Corr and Oliver, 1997); the current NCEP guidelines (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in adults, 1993) no longer specify a 6-month trial of dietary therapy for CHD patients and instead recommend that “at the time of hospitalization for an acute coronary event, consideration can be given to initiating drug therapy at discharge if the LDL cholesterol level is 130 mg/dl (3.4 mmol/liter)”. Even so, on discharge from the hospital, hypercholesterolemic post–myocardial infarction patients are still often put on such a diet with no additional hypolipidemic therapy. All too frequently, the coordination among health care providers is such that follow-up evaluation and treatment of hypercholesterolemia in such patients are inadequate, which results in failure to prevent recurrent coronary events. B. Drugs It is well established that HMG-CoA reductase inhibitors and bile acid sequestrants can be used together safely, with a greater reduction in LDL cholesterol than is obtainable when either drug is used alone. Unfortunately, bile acid sequestrants are often poorly tolerated, which limits the usefulness of this combination. Relatively low doses of niacin, when used in combination with statins, produce a very attractive effect on the lipoprotein profile (Gardner et al., 1996; Stein et al., 1996); the ability of niacin to substantially increase HDL cholesterol is additive, with the profound reduction in LDL cholesterol produced by the statin, and there is also a moderate reduction in triglycerides. However,

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niacin, like bile acid sequestrants, is poorly tolerated, producing flushing in most patients and a variety of more serious adverse effects. In addition, the risk of myopathy during statin therapy is increased by concomitant use of niacin (Tobert, 1988). Drugs of the fibrate class have an effect on lipoproteins somewhat similar to that of niacin, although the reduction of LDL cholesterol and triglycerides is larger and the increase in HDL cholesterol smaller. Concomitant use of fibrates with statins produces little or no further decline in LDL cholesterol beyond that obtainable with the statin alone, but effects on HDL cholesterol and triglycerides are approximately additive (Illingworth and Bacon, 1989; Hutchesson et al., 1994; Ellen and McPherson, 1998). Investigators experienced in the management of lipid disorders have also used fibrates or niacin concomitantly with HMG-CoA reductase inhibitors (Illingworth and Bacon 1989; Horsmans et al., 1992; Da Col et al., 1993; Wiklund et al., 1993; Davignon et al., 1994; Jacobson et al., 1994; Kehely et al., 1995; Stein et al., 1996); severe myopathy was avoidable in these studies by stopping drug treatment in patients at the first sign of muscle symptoms accompanied by increases in creatine kinase, although moderate increases in creatine kinase and muscle pain were observed in a few patients. The absence of myopathy under the closely monitored conditions of a clinical trial involving perhaps about 100 patients treated for a few weeks should not be interpreted to indicate that it will not occur in routine clinical use. The concomitant use of fibrates or niacin with HMG CoA reductase inhibitors is not generally recommended (Pierce et al., 1990; Duell and Illingworth, 1993), although many physicians have used such combination therapy successfully (Shepherd, 1995). However, because fibrates increase the risk of myopathy (Pierce et al., 1990; Raimondeau et al., 1992; Duell et al., 1998; Pogson et al., 1999), the use of this combination requires careful consideration. In particular, diabetics, who are likely to need it the most, are also probably more susceptible to myopathy, especially when renal function is impaired. If one of these combinations must be used, drug doses should be as low as possible and niacin (if it can be tolerated) may be preferred over a fibrate, because the risk of myopathy is probably lower and the overall effect on the lipid profile more favorable (Stein et al., 1996). V. SAFETY AND TOLERABILITY A major reason for the widespread adoption of HMG-CoA reductase inhibitors is their excellent patient tolerability, which enhances compli-

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ance. However, the fact that these drugs inhibit the rate-limiting step in a critical biochemical pathway led to considerable early apprehension about their safety and tolerability. However, hundreds of clinical trials and over 10 years of experience in clinical use have abundantly demonstrated that HMG-CoA reductase inhibitors produce no adverse effects in the vast majority of patients for whom they are prescribed. As is true of most widely used drugs, there have been sporadic reports of a large variety of possible serious adverse effects, but it is clear from the massive amount of data from randomized trials (especially for lovastatin, simvastatin, and pravastatin) that the only important adverse effects of the class are asymptomatic but marked increases in hepatic transaminases in about 1% of patients and rarely, myopathy, which are discussed in more detail below. The largest trial conducted to evaluate the safety of any member of the class is the EXCEL (Bradford et al., 1991), which randomized over 8000 patients to placebo or one of four dosage regimens of lovastatin, including the maximal dose of 80 mg daily. This study confirmed doserelated increases in transaminases, ranging from the placebo rate (0.2%) at 20 mg, to 1.9% at 80 mg. The low incidence of myopathy was also confirmed, with only one case among 4,933 patients receiving lovastatin 20 or 40 mg daily and four cases among 1,663 patients receiving the maximal dose of 80 mg daily. In addition, in the case of lovastatin, simvastatin, and pravastatin, safety data are available from intervention trials designed to evaluate the effects of these drugs on cardiovascular end points, each of which have included several thousand patients studied for approximately 5 years (Scandinavian Simvastatin Survival Study Group, 1994; Shepherd et al., 1995; Sacks et al., 1996; Downs et al., 1998; The Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998). However, of these trials, detailed safety information is available only from the 4S Group (Pedersen et al., 1996a). This study confirmed the excellent safety profile of simvastatin. Only 8 patients of 2,221 allocated to simvastatin were discontinued owing to marked but asymptomatic increases in transaminases, as compared with 5 of 2,223 in the placebo group. There was only one case of myopathy, which was reversible with cessation of therapy. Slit-lamp examination of the lens provided no evidence for cataractogenesis, which had been a concern early in the history of this drug class. No new or unexpected adverse effects emerged. A. Myopathy In the context of therapy with an inhibitor of HMG-CoA reductase, the term myopathy is used to indicate unexplained muscle pain or weak-

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ness and a creatine kinase value exceeding 10 times the upper limit of normal. It is a condition of sudden onset that has been reported most commonly within the first few weeks of treatment or shortly after the introduction of an interacting drug, but sometimes myopathy occurs after several years of treatment for no obvious reason. For example, the sole case of myopathy in 4S occurred after 4 years of treatment with simvastatin 20 mg (Pedersen et al., 1996a). It is important that patients and physicians be aware of the risk of myopathy, so that unexplained muscle pain or weakness can be properly reported. Creatine kinase elevated to 10 times the upper limit of normal in such a patient establishes the diagnosis. Therapy with simvastatin should be discontinued promptly, whereupon there is almost always a prompt recovery without sequelae. If myopathy is not recognized and drug treatment is not discontinued, it may proceed to frank rhabdomyolysis and acute renal failure. Renal insufficiency and serious acute illnesses appear to aggravate the risk of myopathy. The incidence of myopathy with inhibitors of HMG-CoA reductase has been reported to be less than 0.1% in large clinical trials (Bradford et al., 1991; Pedersen et al., 1996a; The Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group 1998). The risk is probably slightly higher at maximal doses of the most effective statins, simvastatin (Ose et al., 1998; Stein et al., 1998b) and atorvastatin, although data on atorvastatin are lacking. The mechanism that produces myopathy is unknown. It is clearly a class effect, because it has been reported with all statins, and in rats the myopathic effects can be prevented by mevalonate (Smith et al., 1991). The risk of myopathy is considerably enhanced by the concomitant use of cyclosporine and to a lesser extent, fibrates or nicotinic acid (Tobert 1988; Tobert et al., 1990; Illingworth, 1991). This effect of cyclosporine was recently confirmed for both simvastatin and pravastatin by examination of the FDA postmarketing surveillance database (Gruer et al., 1999). The mechanism by which this drug increases the risk of statin-induced myopathy is not clear, but it probably involves an increase in the inhibitory activity of plasma HMG-CoA reductase through inhibition of cytochrome P450 3A4, inhibition of p-glycoprotein, or some other mechanism. This increase has been reported for lovastatin (Olbricht et al., 1997), simvastatin (Arnadottir et al., 1993), pravastatin (Regazzi et al., 1993; Olbricht et al., 1997), and cerivastatin (Muck et al., 1999). The effect on fluvastatin appears to be smaller (Goldberg and Roth, 1996), and data on atorvastatin have not been published. Both fibrates and niacin can cause myopathy when given alone (Litin and Anderson, 1989; Magarian et al., 1991).

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There is currently no explanation for why three classes of lipid-lowering drugs (HMG-CoA reductase inhibitors, fibrates, and niacin) that have quite different pharmacological properties can all, rarely, cause myopathy. Unlike the case with cyclosporine, there is no evidence that fibrates can elevate plasma HMG-CoA reductase inhibitory activity, so the interaction with these drugs appears to be pharmacodynamic as opposed to pharmacokinetic. Myopathy in patients receiving niacin and an HMC-CoA reductase inhibitor is more likely to occur in the setting of niacin-induced hepatotoxicity, resulting in impaired hepatic metabolism of the HMG-CoA reductase inhibitor with a resultant increase in plasma concentrations. The first published report of myopathy due to treatment with an HMG-CoA reductase inhibitor was in a cardiac transplant patient who was also taking gemfibrozil (Norman et al., 1988). Subsequent experience has shown that low dosages of HMGCoA reductase inhibitors can be used in transplant patients receiving cyclosporine (Barbir et al., 1991; Ballantyne et al., 1992; Yoshimura et al., 1992; Arnadottir et al., 1994; Vanhaecke et al., 1994; Holdaas et al., 1995; Pflugfelder et al., 1995). The temporary suspension of therapy with an inhibitor of HMG-CoA reductase is advisable when any acute serious medical condition supervenes. Atherosclerotic disease is a chronic process, and there is no evidence that brief cessation of therapy is harmful. In addition, there are some pharmacokinetic interactions that can increase the risk of myopathy. These depend on the metabolic pathways of the various statins (Gruer et al., 1999). However, the number of cases reported is small (Gruer et al., 1999). Although the mechanism or mechanisms responsible for myopathy in patients receiving statins have not been precisely defined, evidence suggests that the development of myopathy results from an underlying predisposition to myopathy in a given patient who is then treated with statins, which at higher doses or when used in combination with fibrates lead to myopathy. Evidence in favor of an underlying factor being present that predisposes to myopathy is supported by personal (DRI) observations of myopathy in two patients who have been treated with statins for hypercholesterolemia but in whom the hypercholesterolemia was exacerbated by unrecognized hypothyroidism. Myopathy and muscle dysfunction are a recognized feature of severe hypothyroidism, and the view that this predisposing factor led to the development of myopathy when patients were treated with statins is supported by the fact that after correction of their hypothyroidism, retreatment with statin therapy for their underlying genetic hypercholesterolemia was well tolerated and did not lead to any recurrence of muscle dysfunction.

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B. Liver The liver is the target organ for inhibitors of HMG-CoA reductase, and all drugs in this class tend to increase transaminase levels (Witztum, 1996). Alanine aminotransferase increases more than aspartate aminotransferase, and this differential effect is so reproducible that when the latter exceeds the former, some cause other than an effect of an HMGCoA reductase inhibitor on the liver is much more likely. In the case of simvastatin and lovastatin, which are closely related, these increases are usually seen between 3 and 12 months of therapy (Bradford et al., 1991; Pedersen et al., 1996a), and are dose-related (Bradford et al., 1991). All currently marketed statins recommend monitoring of liver function tests for variable periods of time after starting therapy. As with myopathy, the molecular basis for this effect is unclear. In addition, although transaminase levels persistently increased above three times the upper limit of normal have traditionally been regarded as harbingers of hepatotoxicity with inhibitors of HMG-CoA reductase, it has not been possible to test this hypothesis because of the difficulty of designing an ethically defensible study. Hepatitis has been an extremely rare event in clinical studies with HMG-CoA reductase inhibitors. For example, there was one case of nonviral hepatitis among 2,221 patients in the simvastatin group in 4S, compared with two in the placebo group. It is still uncertain whether hepatitis, as opposed to marked but asymptomatic increases in transaminases, is a real adverse effect of this class of drugs. As in 4S, hepatitis with negative viral serology and no other known cause can occur, so that attribution of such cases reported during postmarketing surveillance to statin therapy is generally uncertain. This situation is in contrast to rhabdomyolysis, which is also rare but almost never idiopathic, and which often appears within the first few weeks of statin therapy. Therefore, unlike hepatitis, the causal relationship between inhibitors of HMG-CoA reductase and myopathy has been established beyond question. C. Other Safety Issues 1. Lens Some HMG-CoA reductase inhibitors produce subcapsular lens opacities in dogs (Gerson et al., 1989; Gerson et al., 1991; von Keutz and Schluter, 1998), although the doses required to produce opacities give rise to plasma concentrations of HMG-CoA reductase inhibitory activity much higher than those produced by therapeutic doses in humans (Gerson et al., 1989). Together with the triparanol

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experience (Kirby, 1967) indicated above, these animal toxicology findings led to early concern about possible cataractogenic effects on the human lens (Tobert, 1988). For some time after lovastatin was initially marketed, the product circular recommended slit-lamp examination of the lens to monitor for any adverse effects. However, this issue has been carefully examined in several long-term clinical studies with simvastatin and lovastatin, none of which produced any evidence for any adverse effects on the lens (Schmidt et al., 1990; Laties et al., 1991; Chylack et al., 1993; Lovastatin Study Groups I through IV, 1993; Schmidt et al., 1994; Harris et al., 1995; Pedersen et al., 1996a). This is consistent with a recent report that neither simvastatin nor lovastatin has any effect on cholesterol concentrations in any region of the lens (Mitchell and Cenedella, 1999). Finally, there is no epidemiological association between cataract and use of HMGCoA reductase inhibitors (Cumming and Mitchell, 1998). There is no longer any concern about adverse effects of these drugs on the human lens. Follow-up studies on two patients with familial hypercholesterolemia, who were found to have subcapsular lens opacities in 1985 and who have been treated with lovastatin (80 mg/day) for almost 15 years, have demonstrated regression of the lens opacities and support the view that in patients with familial hypercholesterolemia, these findings, like the presence of a corneal arcus, are attributable to high plasma concentrations of LDL cholesterol and that reduction in LDL concentrations leads to a reversal of lipid deposition, as has been observed in tendon xanthomas (Illingworth et al., 1990). 2. Central Nervous System Effects Some years ago there was speculation based on in vitro and animal experiments and an uncontrolled clinical report (Schaefer, 1988) that the greater hydrophilicity of pravastatin would confer a better safety profile, especially with regard to sleep disturbances and other central nervous system (CNS) adverse events, than that of the more lipophilic inhibitors simvastatin and lovastatin. This hypothesis has been carefully tested and found to be unsupported in several well-designed comparative trials (Eckernas et al., 1993; Harrison and Ashton, 1994; Kostis et al., 1994; Partinen et al., 1994; Gengo et al., 1995), most of which have previously been reviewed in detail (Illingworth and Tobert 1994). Furthermore, in 4S the frequency of adverse events in the simvastatin and placebo groups was very similar (Pedersen et al., 1996a). In particular, there was no difference in the incidence of CNS adverse effects. As was the case with the lens, concern about possible CNS adverse

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effects has been laid to rest by means of a series of carefully conducted clinical studies. 3. Long-Term Safety The long-term safety of simvastatin has been established by 4S, in which the median follow-up period was 5.4 years (Scandinavian Simvastatin Survival Study Group, 1994), and detailed safety data are available (Pedersen et al., 1996). Detailed 5-year safety data are also available for lovastatin, although in this case from an uncontrolled extension trial (Lovastatin Study Groups I through IV, 1993). Abbreviated safety data are available from the 5- to 6-year megatrials with pravastatin (Shepherd et al., 1995; Sacks et al., 1996; the Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998) and lovastatin (Downs et al., 1998). None of these studies revealed any previously unknown adverse effects, and in all of them the incidences of the cardinal adverse effects of the class, myopathy and increased transaminases, were barely distinguishable from the placebo rate, confirming the excellent tolerability and safety of these drugs. 4. The Cholesterol Controversy Before the advent of HMG-CoA reductase inhibitors, cholesterol-lowering interventions had repeatedly been shown to reduce CHD events in patients with and without prior CHD (Coronary Drug Project 1975; Lipid Research Clinics Program, 1984; Frick et al., 1987; Buchwald et al., 1990). Expert panels in Europe and in the United States therefore recommended dietary changes and where appropriate, addition of drugs to reduce elevated cholesterol levels (Expert Panel on Detection, Evaluation and Treatment of High Blood Cholesterol in Adults, 1993; Pyörälä et al., 1994), especially in patients with CHD. However, the role of lipid-lowering drugs was challenged (Davey Smith and Pekkanen, 1992; Oliver, 1992), mainly because prior to 1994 no clinical trial had convincingly shown that lowering cholesterol reduced mortality. Furthermore, overviews of these trials of treatments from the pre–statin era had suggested that survival is not improved, particularly in the absence of established CHD, because the observed reduction of CHD deaths appeared to be offset by an apparent increase in noncardiac mortality, including cancer and violent deaths (Muldoon et al., 1990; Oliver 1992). However, the publication in 1994 of the results of the Scandinavian Simvastatin Survival Study (4S) marks a turning point in medical thinking. As shown in Fig. 6, there was an unequivocal 30% reduction in all-cause mortality (p = .0003), due to a 42% reduction in coronary deaths. These effects on mortality were accompanied by a 34% reduc-

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Fig. 6. Effects of simvastatin on survival in 4S.

tion in major coronary events (nonfatal myocardial infarction plus CHD death) and a 37% reduction in revascularization procedures. Importantly, there was no suggestion of any offsetting increase in noncardiovascular mortality (see below). VI. OUTCOME STUDIES The purpose of altering plasma lipoprotein levels is to reduce the risk of coronary events. The results of outcome trials are available for lovastatin (Downs et al., 1998), simvastatin (Scandinavian Simvastatin Survival Study Group 1994), and pravastatin (Shepherd et al., 1995; Sacks et al., 1996; The Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998). Three of these trials, 4S, Cholesterol and Recurrent Events (CARE), and LIPID, studied patients with CHD, whereas the West of Scotland Coronary Revention Study Group and the Air Force Coronary Atherosclesosis Prevention Study (AFCAPS) evaluated the benefits of therapy in patients without known CHD. The main results of these trials are summarized in Tables Ia and Ib. The results of these five trials are remarkably consistent. In each case, the primary hypothesis was met, with a high degree of statistical significance. In all of the trials, there was a highly significant reduction in major coronary events (defined slightly differently in the various trials, but essentially nonfatal myocardial infarction plus coronary death). As shown in Table Ia, the effect of simvastatin on this end point was somewhat greater than that of pravastatin in patients with CHD, possibly because of the greater effect of simvastatin on LDL cholesterol. Other possible explanations include the fact that there were appreciable numbers of patients in

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TABLE IA Risk Reductions Observed in Statin Megatrials in CHD Patientsa Reductions

4S (1994) CARE (1996) LIPID (1998)

Statin (dose, mg)

N

LDL-C (long-term) (%)

Simvastatin 20–40 Pravastatin 40 Pravastatin 40

4,444 4,159 9,014

35 28 25

CHD eventsb (%)

CHD deaths (%)

Total deaths (%)

37 24 23

42 NSc 24

30 NSc 23

a 4S (Scandinavian Simvastatin Survival Study Group 1994), CARE (Sacks et al., 1996), and LIPID (The Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group 1998) b Nonfatal MI + CHD death. c Not significant.

both CARE and especially LIPID who stopped taking active treatment or were randomized to placebo but received statin therapy from their own physicians outside the trial. This would tend to attenuate the risk reduction achieved. Only the trials in patients with CHD had adequate power to evaluate fatal events, and only 4S and LIPID demonstrated a significant effect of treatment on coronary and total mortality. The beneficial effects of statin treatment in these trials did not depend on a large effect in a small subgroup. Effects in subgroups have been explored in detail, especially in 4S, in which significant effects were found in nearly all subgroups examined (Scandinavian Simvastatin Survival Study Group, 1994, 1995; Kjekshus et al., 1995; Miettinen et al., 1997; Pyörälä et al., 1997). There was no good evidence that any subgroup was resistant to the effects of treatment, but because of the inevitable loss of statistical power due to smaller numbers, significant effects could not have been expected in all cases. For example, in the TABLE IB Risk Reductions Observed in Statin Megatrials in Patients Without Known CHDa Reductions

WOS (1995) AFCAPS (1998)

Statin (dose, mg)

N

LDL-C (long-term)(%)

CHD eventsb(%)

Pravastatin 40 Lovastatin 20–40

6,595 6,605

26 NAc

31 40

WOS (Shepherd et al., 1995) and AFCAPS (Downs et al., 1998). Nonfatal MI + CHD death. c 25% at 1 year. a

b

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trials in patients with CHD, in 4S (Scandinavian Simvastatin Survival Study Group 1994) and CARE (Sacks et al., 1996) the reduction in major coronary events in women was statistically significant and at least as great as in men, whereas in the Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group (1998), there was a favorable trend in women but not a statistically significant result. Beneficial trends, statistically significant in many cases, were also generally observed in subgroups divided by age, baseline lipids, smoking history, history of hypertension or diabetes, and concomitant therapy with aspirin or beta-blockers. Further evidence on the effects of statin therapy in different patient types will likely be provided by the results of the Heart Protection Study, which has randomized 20,536 patients to simvastatin 40 mg or placebo and which is scheduled for completion in 2001 (MRC/BHF Heart Protection Study Collaborative Group, 1999). Despite the plethora of evidence provided by these trials and the publication of national guidelines (Expert Panel on Detection Evaluation and Treatment of High Blood Cholesterol in Adults 1993; Pyörälä et al., 1994), many patients still go untreated, even those with CHD (Bowker et al., 1996; EUROASPIRE Study Group 1997; Pearson and Peters, 1997). Unfortunately, some practitioners are slow to change their clinical approach despite unequivocal results from clinical trials. As yet there are no completed megatrials with the newer statins. A study that compared atorvastatin versus angioplasty in 341 patients with stable CHD over 18 months was recently reported (Pitt et al., 1999). There were fewer ischemic events in the atorvastatin group, but the difference did not quite reach statistical significance. Furthermore, a previous study had shown a significantly lower incidence of such events in a group treated with conventional medical therapy, because angioplasty can be complicated by myocardial infarction and other acute coronary events (RITA-2 trial participants, 1997). VII. MECHANISMS OF THE REDUCTION IN CORONARY MORBIDITY AND MORTALITY Simvastatin, pravastatin, and lovastatin have been shown to slow the progression of coronary atheromatous lesions by 2 years after starting treatment, compared with standard care (Blankenhorn et al., 1993; Multicentre Anti-Atheroma Study [MAAS] investigators, 1994; Waters et al., 1994; Jukema et al., 1995). These studies generally reported fewer new lesions and/or total occlusions in the treated group, but regression is less well established, although it may occur in some patients. All of these stud-

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FIG. 7. Effects of simvastatin on coronary artery minimal lumen diameter in the Multicentre Anti-Atheroma Study (MAAS).

ies followed patients for about 2 years, except for the MAAS (MAAS investigators 1994), which had a 4-year treatment duration. Figure 7 illustrates the steady progression of disease in this study in the group receiving only dietary therapy and the slowing of progression in the group allocated to simvastatin 20 mg daily. These effects are clearly not limited to inhibitors of HMG- CoA reductase, as they have also been achieved by dietary therapy alone or with cholestyramine (Watts et al., 1992), colestipol and niacin in combination (Blankenhorn et al., 1987), or ileal bypass surgery (Buchwald et al., 1990) and by multiple risk factor intervention (Haskell et al., 1994). Pravastatin and lovastatin also have beneficial effects on carotid lesions as measured by ultrasound (Furberg et al., 1994; Salonen et al., 1995). Progression of coronary atherosclerotic lesions clearly predicts subsequent coronary events (Waters et al., 1993). In the megatrials, the impact of inhibitors of HMG-CoA reductase on CHD appears to begin after approximately 1 year of therapy and to increase steadily thereafter as exemplified by 4S (Scandinavian Simvastatin Survival Study Group 1994). The claim by the WOS investigators (Shepherd et al., 1995) that pravastatin produced an earlier effect than

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simvastatin in 4S was not borne out by the results of subsequent studies with this drug (Sacks et al., 1996; the Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998). As noted above, HMG-CoA reductase inhibitors slow the progression of atherosclerosis, and progression of coronary atherosclerotic lesions clearly predicts subsequent coronary events. Coronary lesions may stabilize as their lipid core shrinks or at least does not further enlarge, avoiding plaque rupture, which triggers intramural hemorrhage and intraluminal thrombosis, which in turn may cause coronary events (Davies, 1996; Fuster et al., 1996). This is most likely the main cause of the improved survival and reduction of coronary morbidity observed in the statin intervention trials. Platelets are more prone to aggregate when hypercholesterolemia is present (Carvalho et al., 1974), and some reports suggest that platelet aggregation can be returned toward normal by inhibitors of HMG-CoA reductase (Kaczmarek et al., 1993; Notarbartolo et al., 1995), although this was not found to be the case in the well-controlled study by Broijersen et al., (1997). Antiplatelet therapy has been proved to reduce the risk of CHD events (Antiplatelet Trialists’ Collaboration, 1994). Hypercholesterolemia is associated with abnormal vascular reactivity (Vita et al., 1990; Seiler et al., 1993), and there is some evidence that lipid lowering with HMG-CoA reductase inhibitors (Treasure et al., 1995; O’Driscoll et al., 1997; Simons et al., 1998) and cholestyramine (Leung et al., 1993) can partially reverse this effect within a few months. Improvement in endothelial-dependent vasodilation has also been observed within 24 hours after LDL apheresis in patients with familial hypercholesterolemia; this supports the view that high plasma concentrations of LDL adversely affect endothelial function and impair vasodilation (Tamai et al., 1997). However, the Coronary Artery Reactivity After Treatment with Simvastatin (CARATS) study, a large, well-controlled study, provided no evidence for an effect, despite a mean 40% reduction in LDL cholesterol with simvastatin 40 mg daily (Hodgson et al., 1996; Vita et al., 2000). Furthermore, a link between vascular reactivity and coronary events has yet to be established. VIII. SAFETY OF HMG-COA REDUCTASE INHIBITORS IN THE MEGATRIALS A. Noncardiovascular Deaths In the five statin megatrials, the reductions in plasma cholesterol were greater than in the earlier trials, which had raised concerns about a possible increase in noncardiac mortality associated with cholesterollowering therapy. Thus, if these concerns were applicable to choles-

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terol-lowering therapy in general, more noncardiovascular deaths would have been expected in the statin-treated groups. However, no such effect was seen. Very similar numbers of noncardiovascular deaths were observed in the treated groups as compared with the placebo groups, as shown in Table II. In the five trials combined, there were 491 and 547 non-CHD deaths (including 400 and 436 noncardiovascular deaths) in the patients randomized to statin and placebo, respectively, indicating no adverse effect of statin therapy. Thus, these five trials support the conclusion of the meta-analyses of Gould et al., (1995) and Gordon (1995) that although there may be risks associated with certain drugs (especially obsolete hormonal treatments), cholesterol lowering is not hazardous per se. B. Cancer In the five statin megatrials, which collectively randomized over 30,000 patients and had treatment periods of approximately 5 years, new cancers were recorded in over 2000 patients. The numbers of patients with cancer in the active treatment and placebo groups were very similar, as shown in Table III. There was an excess of breast cancer in the pravastatin group in CARE (12 cases vs. 1 in the placebo group), but this is probably an anomalous finding in a small subgroup, as it was not replicable in 4S, LIPID, or AFCAPS, all of which included an appreciable number of female patients (WOS excluded women). The total number of breast cancers in these TABLE II Mortality in 4S,a WOS,b CARE,c LIPID,d and AFCAPS e All

CHD

Non-CHD

Non-CV

Study

Statin

N

Drug

Pbo

Drug

Pbo

Drug Pbo

Drug Pbo

4S CARE LIPID WOS AFCAPS

Simva Prava Prava Prava Lova

4,444 4,159 9,014 6,595 6,605

182 180 498 106 80

256 196 633 135 77

111 96 287 41 11

189 119 373 61 15

71 75 211 65 69

67 84 260 74 62

46 68 167 56 63

49 73 200 62 52

1,046 1,297

546

757

491

547

400

436

Total

30,817

Scandinavian Simvastatin Survival Study Group 1994. Shepherd et al. (1995). c Sacks et al. (1996). d The Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group 1998). e Downs et al. (1998). a b

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TABLE III Patients with Cancer in 4S,a WOS,b CARE,c LIPID,d and AFCAPS e (excluding nonmelanoma skin cancer) All cancer

Breast cancer

Study

Statin

N

Drug

Placebo

Drug

4S CARE LIPID WOS AFCAPS

Simva Prava Prava Prava Lova

4,444 4,159 9,014 6,595 6,605

89 172 379 116 252

96 161 399 106 259

3 12 10 0 13

6 1 10 0 9

3,0817

1,008

1,021

38

26

Total

Placebo

Scandinavian Simvastatin Survival Study Group 1994. Shepherd et al. (1995). c Sacks et al. (1996). d The Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group 1998). e Downs et al. (1998). a b

three studies combined was 38 in the statin groups and 26 in the placebo groups, an unremarkable difference. Newman and Hulley (1996) questioned the safety of HMG-CoA reductase inhibitors because very high doses increased the background tumor incidence in rodent lifetime carcinogenicity studies. This information is not new and has been available in the package circulars since the first member of the class (lovastatin) was approved in 1987. The accompanying editorial (Dalen and Dalton, 1996) found the evidence presented by Newman and Hulley unconvincing and noted that the predictive value of the rodent bioassay is low. It is also important to note that all the HMG-CoA reductase inhibitors are completely negative in a comprehensive battery of genotoxicity tests, whereas except for estrogens, all known organic chemical human carcinogens identified by the International Agency for Research on Cancer (IARC) are genotoxic (Shelby, 1988). Although the five statin megatrials combined provide no evidence that these drugs are carcinogenic, to exclude the possibility that any agent is a human carcinogen would require a trial of at least 20 years in duration, which is not feasible for many reasons. Newman and Hulley called for trials of much longer duration than these 5-year studies. However, it would be difficult to design ethically defensible studies of longer than 5 years. Extending any of the previous 5-year trials to 10 years or longer would have delayed important medical information

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and would have led to unnecessary mortality and morbidity in the placebo groups. Newman and Hulley suggest waiting for patients to develop CHD or other atherosclerotic disease before treating them, but this has several disadvantages, including the fact that the first manifestation of CHD is frequently fatal (Tunstall Pedoe et al., 1994). Prescribers must weigh the proved substantial benefit in patients with or at risk for CHD against a theoretical long-term risk, which is not supported by the available 5year data in over 30,000 patients but is impossible to disprove. Most practicing physicians have arrived at the conclusion that the proven benefit greatly outweighs the theoretical risk. IX. FUTURE DIRECTIONS A. Treatment Intensity In the five statin megatrials, the greatest percent reduction in coronary mortality was recorded in 4S. In this study, even though the risk of CHD death was reduced by 42% in the simvastatin group, 111 patients in this group (5%) still died of their disease in the 5.4 years of the study. Therefore, while the results of 4S and the other megatrials are certainly impressive, much more needs to be done. Epidemiological studies indicate that plasma cholesterol remains a risk factor for CHD at levels of 4 mmol/liter (155 mg/dl) or below (Szatrowski et al., 1984; Chen et al., 1991) but suggest that reduction of plasma cholesterol to this level or below is likely to be beneficial. Coronary angiographic studies with inhibitors of HMG-CoA reductase have consistently shown that the progression of atherosclerotic lesions is slowed but not arrested (Blankenhom et al., 1993; MAAS investigators, 1994; Waters et al., 1994; Jukema et al., 1995). Figure 7 shows this effect in MAAS (MAAS Investigators, 1994), with simvastatin 20 mg daily over 4 years. As long as some lesions are progressing in some patients, coronary events must be expected. More aggressive reduction of LDL cholesterol offers the hope of further slowing, or even reversing, lesion progression. In patients who have received a saphenous vein coronary artery bypass graft, aggressive lipid lowering to achieve LDL cholesterol below 100 mg/dL has been shown to produce clinical benefits in the form of reduced progression of atherosclerosis in the grafts and a 29% reduction in repeat revascularization procedures (Post Coronary Artery Bypass Graft Trial Investigators, 1997). In 4S there was no evidence of any threshold below which further reduction of LDL cholesterol was futile (Pedersen et al., 1998a).

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Although other analyses (Sacks et al., 1998; West of Scotland Coronary Prevention Study Group, 1998) of statin megatrials have suggested such a threshold, this conclusion was based largely on the analysis of results in small subgroups. Furthermore, meta-analyses of lipid-lowering intervention trials show that the reduction in coronary risk is proportional to the degree of lipid lowering achieved during the trial (Law et al., 1994; Gordon, 1995; Gould et al., 1995, 1998). Thus, it is likely that the more LDL cholesterol can be lowered, the greater the chance of escaping a coronary event such as myocardial infarction. However, there are currently no data on the effects of reducing LDL cholesterol beyond the long-term 35% average achieved in 4S with simvastatin 20–40 mg daily. Reducing LDL cholesterol by more than 35% would probably further reduce coronary risk. The “more is better” hypothesis is being tested in several ongoing megatrials. Pending the outcome of these trials, given the excellent and now well established safety profile of the statins, adequate doses should be prescribed (up to the currently recommended maximum dosage levels), especially in CHD patients in whom large reductions of LDL cholesterol are often needed to achieve desirable levels. For example, the U.S. National Cholesterol Education Program (NCEP) goal for CHD patients is an LDL cholesterol of 100 mg/dl (2.6 mmol/liter) or less (Expert Panel on Detection, Evaluation, and Treatment of High Blood Cholesterol in Adults, 1993) which generally requires reductions of 30% or more. The same goal has recently been recommended for diabetic patients with or without CHD (American Diabetes Association, 1998). The introduction of atorvastatin in 1997 and the extension of the dosage range of simvastatin to 80 mg in 1998 has provided therapy that can bring most patients to NCEP treatment target levels. B. Effects on Other Vascular Beds Although atherosclerosis exerts its most important effects on the coronary vessels, other vascular beds are frequently affected, such as the carotid arteries, the aorta, and the vessels of the legs. However, the possibility that effective lipid lowering could have beneficial effects on atherosclerotic diseases other than CHD has received relatively little attention. Although there is some evidence that hypercholesterolemia is a risk factor for stroke, especially in younger patients (Prospective Studies Collaboration, 1995), the epidemiologic data are not nearly as extensive or unequivocal as for CHD. The first evidence that HMG-CoA reductase inhibitors could reduce the risk of cerebrovascular effects was provided by 4S, in which there was a significant 28% reduction in the

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risk of the combined end point of stroke and transient ischemic attack (Scandinavian Simvastatin Survival Study Group 1994; Pedersen et al., 1998b). Although this was a post hoc analysis, significant reductions in the risk of stroke were subsequently obtained in the pravastatin megatrials CARE (Sacks et al., 1996) and LIPID (Long-Term Intervention With Pravastatin in Ischaemic Disease (LIPID) Study Group, 1998). These results are consistent with studies showing that inhibitors of HMG-CoA reductase slow or reverse the progression of carotid atherosclerotic lesions as measured by utrasound (Furberg et al., 1994; Salonen et al., 1995) and with the finding that the incidence of new or worsening carotid bruits was reduced in the simvastatin group in 4S (Pedersen et al., 1998b). Interestingly, Pedersen et al. have also reported that the incidence of new or worsening intermittent claudication was reduced by simvastatin in 4S (Pedersen et al., 1998b). An earlier meta-analysis (Atkins et al., 1993) of lipid-lowering intervention trials found no clear effect on stroke, but none of the trials included were designed for this purpose, and most of the interventions were relatively ineffective for lowering cholesterol. However, recent meta-analyses of the statin trials (Crouse et al., 1997; Hebert et al., 1997; Bucher et al., 1998) have found a significant effect of cholesterol-lowering treatment on cerebrovascular events in patients with preexisting coronary disease. In view of these data, investigators of ongoing clinical trials with lipid-lowering interventions (Cholesterol Treatment Trialists’ [CTT] Collaboration, 1995) are likely to incorporate stroke and other noncoronary vascular end points into their data analysis plans. These effects on cerebrovascular events and on intermittent claudication suggest that simvastatin and other effective lipid-lowering treatments may have a general antiatherosclerotic effect not limited to the coronary bed. Definitive evidence on the effects of statin therapy in stroke prevention and peripheral vessel disease is likely to be provided by the Heart Protection Study (MRC/BHF Heart Protection Study Collaborative Group, 1999). As noted above, this UK study has randomized over 20,000 patients aged up to 80 to simvastatin 40 mg or placebo, and the 5-year treatment period is scheduled for completion in 2001. Among these patients are 3288 patients with a history of cerebrovascular disease. Because of its size and the broad array of patient types randomized, this study should also provide reliable evidence of the effect of simvastatin on coronary morbidity and mortality in women, elderly patients, patients with low levels of LDL and HDL cholesterol, patients with peripheral vascular disease, and diabetic patients with or without coronary disease (MRC/BHF Heart Protection Study Collaborative Group, 1999).

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CYCLOOXYGENASE-2 INHIBITORS BY ALAN S. NIES* AND MICHAEL J. GRESSER† *Department of Clinical Sciences, Merck Research Laboratories, Merck & Co., Inc., Rahway, New Jersey and †Department of Biochemistry and Molecular Biology, Merck Frosst Centre for Therapeutic Research, Merck Frosst Canada & Co., Kirkland, Quebec, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Assays for Cyclooxygenase-2 Selective Inhibitors . . . . . . . . . . . . . . . . . . . . . . A. In Vitro Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Preclinical In Vivo Assays . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Selectivity of Cyclooxygenese Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Enzymology/Medicinal Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Clinical Development of Cyclooxygenase-2 Inhibitors . . . . . . . . . . . . . . . . . A. Selectivity in Humans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Efficacy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Safety . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Colon Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Alzheimer’s Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IX. References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Nonsteroidal antiinflammatory drugs (NSAIDs) have long been used to treat pain, fever, and inflammation. These drugs are widely used and effective for these indications, but their use is associated with various side effects, one of the most serious of which is NSAID-induced gastropathy, which is estimated to cause more than 100,000 hospitalizations and 16,000 deaths in the United States each year, approximating the death rate due to the acquired immunodefiency syndrome (AIDS) (Wolfe et al., 1999). Gastropathy caused by NSAIDs, thus constitutes a major public health problem and is one of the most prevalent serious adverse drug effects in industrialized societies. The biochemical target of NSAIDs is the enzyme cyclooxygenase (Cox), which catalyzes the synthesis of prostaglandin G2 (PGG2) from arachidonic acid and its conversion to prostaglandin H2 (PGH2), which is the precursor of all the prostanoids. Prostanoids are known to be involved not only in the pathophysiology of inflammation, pain, and fever but also in the important physiologic processes of blood clotting, homeostasis of the gastrointestinal tract, and maintenance of renal blood flow. It therefore 115 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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appeared that the major adverse effects of NSAID therapy were mechanism-based, and efforts to separate the beneficial from the adverse effects of NSAIDs over the years were not successful. In the late 1980s evidence began to appear that pointed to the existence of a second, inducible cyclooxygenase (Cox-2) gene, which is expressed at sites of inflammation. By the early 1990s, evidence for the existence of Cox-2 was conclusive, and several pharmaceutical companies had by this time initiated efforts to discover and develop Cox-2–selective inhibitors. These efforts were based on the hypothesis that such compounds would have the analgesic, antipyretic, and anti-inflammatory effects of NSAIDs but would not induce gastropathy or other adverse effects of NSAIDs (Vane, 1994; van Ryn and Pairet, 1997). Now that two of these new Cox2 inhibitors are in clinical use, and more soon will be, the hypothesis has been validated. For example, rofecoxib is as effective as NSAIDs in treating pain, fever, and inflammation and yet has no demonstrable effect on Cox-1 at and above the clinically effective doses. As is consistent with the Cox-2 hypothesis, the incidence of gastropathy among patients taking rofecoxib in clinical studies was not different from that among patients taking placebo and was markedly lower than among patients taking nonselective Cox inhibitors (discussed below). This chapter deals with aspects of the discovery and development of Cox-2 inhibitors, which, in the case of rofecoxib, resulted from efforts that were initiated in July of 1992 and culminated in the launch of the drug in the United States in June of 1999. Some of the literature on the discovery of Cox-2 and its biology, as well as pharmacological studies with Cox-2 inhibitors will be covered, and readers are referred to recent reviews that cover the literature of these areas thoroughly (Vane et al., 1998; Pairet et al., 1999). II. BACKGROUND Preparations from plant materials containing salicylate have been used to treat pain and fever (Stone, 1763) from ancient times into the nineteenth century, when synthetic salicylic acid was synthesized in Germany and used for the same purposes. Acetyl salicylate, aspirin, synthesized as a prodrug for salicylate, was introduced by Bayer in 1899 (Dreser, 1899) and is still widely used to treat pain, fever, and inflammation. In low doses aspirin is also used by many to reduce the incidence of heart attacks by an antithrombotic effect (Antiplatelet Trialists’ Collaboration, 1994). Eventually, other drugs with therapeutic effects similar to those of aspirin were introduced, including indomethacin, ibuprofen, and naproxen. These

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came to be known as nonsteroidal anti-inflammatory drugs (NSAIDs) but their mechanism of action remained unknown (Flower, 1974). In 1964, it was reported that NSAIDs blocked release of a rabbit aorta contracting substance from guinea pig lung (Vane, 1964), which was later shown to be thromboxane A2, a prostanoid (Hamberg et al., 1975). Subsequent investigations showed that aspirin inhibited prostanoid release from other tissues as well (Vane, 1971; Smith and Willis, 1971; Ferreira et al., 1971). In due course cyclooxygenase, which we now know as Cox-1, was purified, characterized, and found to catalyze the conversion of arachidonic acid to PGG2 and its further conversion to PGH2 (Hemler et al., 1976), and aspirin was shown to irreversibly inhibit Cox-1 by direct acetylation of the enzyme (Roth et al., 1975). Around 1990 a second, inducible Cox mRNA, Cox-2, was identified (Rosen et al., 1989; O’Banion et al., 1991; Xie et al., 1991; Sirois and Richards 1992; Masferrer et al., 1992; Kujubu et al., 1991; Lee et al., 1992; O’Sullivan et al., 1993) and the human Cox-2 cDNA described (Hla and Neilson, 1992; Kennedy et al., 1993). With this discovery it became clear that earlier reports of increases in prostaglandin synthesis by fibroblasts challenged with the proinflammatory cytokine IL-1 (Raz et al., 1988, 1989), and by human and mouse macrophages challenged with bacterial endotoxin (Fu et al., 1990; Masferrer et al., 1990) could be rationalized in terms of induction of Cox-2 expression. Cox-2 gene expression was found to be triggered by proinflammatory stimuli in cells in culture and in inflamed tissues in animals, whereas Cox-1 expression was unaffected by these stimuli. Also, Cox-2 expression could be suppressed by anti-inflammatory cytokines and by dexamethasone, which do not affect Cox-1 expression. A detailed review of these discoveries has appeared recently (Pairet et al., 1999). These and other observations led to formulation of the hypothesis that Cox-1derived prostanoids are involved in such physiological functions as homeostasis of the gastrointestinal (GI) tract, via a cytoprotective effect, and in regulation of platelet aggregation and kidney function, whereas Cox-2 is responsible for prostaglandin synthesis at sites of inflammation and perhaps is also involved in pain and fever (Vane, 1994; van Ryn and Pairet, 1997). A corollary of this hypothesis is that NSAIDs have their beneficial effects in pain and inflammation by inhibiting Cox-2 and their adverse effects on the GI tract and elsewhere by inhibiting Cox-1. Given the efficacy of NSAIDs and the incidence of NSAID gastropathy, several companies began designing assays to determine the Cox-2 versus Cox-1 selectivity of inhibitors in order to test the selectivity of existing NSAIDs and to discover highly selective Cox-2 inhibitors to use in testing the indicated hypothesis.

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III. ASSAYS FOR COX-2–SELECTIVE INHIBITORS A. In Vitro Assays Several assays have been described by investigators in the field and they are used for different purposes. Assays involving purified Cox-1 and Cox-2 are used for convenience and speed or to obtain an estimate of the relative intrinsic potency and selectivity of inhibitors. Selectivity based on intrinsic affinity for the catalytic site permits the development of structure–potency relationships that one can interpret in terms of structural features of the inhibitors and the catalytic sites of the target enzymes (Bayly et al., 1999). Cell-based assays are used to determine whether the inhibitors can enter cells and inhibit Cox, as well as to determine inhibitor potency in a more biologically relevant environment than provided by purified enzyme assays. At Merck, CHO cell lines that overexpressed human Cox-1 or Cox-2 were developed and used for screening cell-based assays (Kargman et al., 1996). As inhibitors with increased selectivity for Cox-2 were made by medicinal chemists, it became more and more difficult to differentiate them on the basis of their potency as Cox-1 inhibitors, because in many cases no inhibition was observed in the current Cox-1 assays. It was considered important to be able to distinguish weak from weaker Cox-1 inhibitors, because the adverse effects of NSAIDs were ascribed to inhibition of Cox-1. In order to be certain to avoid these effects, it would be necessary to ensure that therapeutically efficacious doses of Cox-2 inhibitors would not significantly inhibit Cox-1 in any tissue. This led to the development of a U937 cell microsome assay at low (0.1 µM) arachidonate concentration (Riendeau et al., 1997a). Cox inhibitors tend to be significantly more potent in this assay than in others used to assess Cox-1 potency because all of the Cox inhibitors known are competitive with arachidonate. Thus, very low arachidonate concentrations would favor the inhibitor gaining access to and inhibiting Cox-1. This point could be relevant to Cox-1 inhibition in an in vivo setting, where the drug might act on the enzyme in a tissue, such as the gastric mucosa, in which arachidonic acid concentration is low (Hamilton et al., 1999; Doyle et al., 1989; Preclik et al., 1992; Warner et al., 1999). As a measure of the functional selectivity of Cox inhibitors in an in vitro or ex vivo setting, human whole blood assays were used. The PGE2 produced in human whole blood challenged with bacterial endotoxin arises from monocyte-derived Cox-2, and thromboxane B2 produced in clotting blood arises from platelet Cox-1 (Patrignani et al., 1994; Brideau et al., 1996). These assays are widely used and are the generally accepted standards for selectivity of Cox inhibitors relevant to an in vivo setting

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(Brooks et al., 1999). The functional selectivity assessed in this manner is usually considerably less than the intrinsic affinity difference for a selective inhibitor in binding to the two enzymes, largely because of the timedependent nature of the inhibition (Ouellet and Percival, 1995). The inhibitors bind to Cox at the catalytic site, in competition with the substrate, in a rapidly reversible manner at fairly low affinity. Cyclooxygenase to which a potent inhibitor is bound at the catalytic site undergoes a relatively slow change of state to a form to which the inhibitor is bound with high affinity (Riendeau et al., 1997b). In order for the inhibition of potent inhibitors to develop fully, time must pass, during which the enzyme can still catalyze prostaglandin synthesis. Under conditions of high arachidonic acid concentration, the time for the inhibition to develop fully can be quite long (Ouellet and Percival, 1995; Riendeau et al., 1997a; Chan et al., 1999). Since Cox-2 expression is induced as a result of the insult that also results in inflammation and release of arachidonic acid from esterified stores, the inhibitor does not have time to bind to the enzyme before it can bind its substrate and begin producing prostaglandins. Thus, intrinsic affinity differences greater than one-thousandfold can be reduced to functional selectivities of tenfold or less when assessed by the whole blood assays. An early appreciation of this factor influenced the group at Merck to place considerable emphasis on the U937 cell microsome assay described above, which provided a way to select Cox-2 inhibitors with the weakest Cox-1 potencies. This resulted in compounds that not only are very selective for Cox-2 and weak against Cox-1 in the human whole blood assays but also will be very weak against Cox-1 encountered in any tissue in which the arachidonate concentration might be lower than it is in the peripheral blood cells examined in the whole blood assays. The complete profile of rofecoxib using these in vitro assays has been described recently (Chan et al., 1999). B. Preclinical In Vivo Assays The in vivo assays for the Cox-2 inhibitors are essentially those used historically for NSAIDs to evaluate both their desired effects on inflammation, pain, and fever and their undesired effects, mainly on GI lesions. The primary in vivo assay for anti-inflammatory efficacy is the carrageenan-induced rat paw edema assay, and 51Cr fecal excretion is used to test for damage to the intestinal mucosa in rats and in squirrel monkeys. Additional tests for efficacy are the endotoxin-induced pyresis in rats and squirrel monkeys for control of fever and the carrageenaninduced rat paw hyperalgesia assay for analgesic efficacy. These in vivo assays have been described (Chan et al., 1995; Chan et al., 1997). The rat

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paw edema assay involves injecting carrageenan into the foot pad of a rat, normally 1 hour after dosing with the compound to be tested or vehicle, and measuring the swelling of the paw by the method of Archimedes, using a plethysmometer. The challenge is similar in the hyperalgesia assay, but after paw swelling has been allowed to develop for a set time, mechanical pressure is applied to the injected paw, and the amount of pressure required to induce vocalization by the rat is measured. In the 51Cr fecal excretion test, 51Cr-labeled red blood cells are injected IV into the animal dosed with drug or vehicle, and appearance of 51Cr in the feces is monitored. In order to test for efficacy in chronic inflammation, the rat adjuvant-induced arthritis model is used (Fletcher et al., 1998; Chan et al., 1999). In this assay, arthritis is induced by injection of Freund’s adjuvant into one of the foot pads of a rat, and the animals are dosed daily with the compound being tested or vehicle. After 14 and 21 days, swelling in a noninjected paw is measured, thymus and spleen weights are recorded, and radiographic joint integrity is assessed. The results of a number of Cox-2 inhibitors have been reported in the publications cited above describing the assays, including a recent complete profile of rofecoxib (Chan et al., 1999). IV. SELECTIVITY OF COX INHIBITORS With the in vitro and in vivo assays described above in place, it was possible to test existing NSAIDs for selectivity in order to obtain an idea of how much selectivity might be needed to achieve efficacy without GI lesions. It turned out that the majority of NSAIDs are nonselective or somewhat Cox-1 selective (Brideau et al., 1996; Pairet et al., 1999; Warner et al., 1999). Compounds that had some selectivity for Cox-2 and were in clinical use or were studied before the discovery of Cox-2 include nimesulide, DuP 697, etodolac, flosulide, and meloxicam. The agent NS-398 from Taisho Pharmaceuticals Co. was selected by screening for compounds that inhibited prostaglandin synthesis in inflamed tissue but not in noninflamed tissue in an attempt to discover a GI-sparing NSAID (Futaki et al., 1993; Futaki et al., 1994). This compound turned out to be a selective Cox-2 inhibitor, and its discovery illustrates the virtue of the classical pharmacological approach of finding therapeutic agents with desired properties even if the underlying mechanisms are not understood. Tabulations of the Cox-1 and Cox-2 human whole blood IC50 values, as well as the Cox-1 IC50 values from the U937 microsome assay at low arachidonate concentration and selectivity ratios from cell-based assays, are shown in Table I for these compounds,

TABLE I Data for Selected Cox Inhibitors on Potency, Selectivity, Efficacy, and GI Tolerability Assays Described in Text IC50(µM) CHO Compound

Cellsb

Cox-1/Cox-2

IC50(nM)

HWBc Cox-1

HWBd Cox-2

Cox-1/Cox-2

U937

micrd

Rat paw ED50e

0.1 µM AA

(mg/kg)

0.16 ± 0.01

0.46 ± 0.06

0.35

19.8 ± 0.2

2.0

Meloxicam

300

1.43 ± 0.43

0.70 ± 0.28

2

143 ± 55

2.9

L-745,337

~800

>30

9.7 ± 2.0

>3

2,780 ± 280

2.0

~1,000

8.96 ± 2.45

2.79 ± 0.49

3.2

1,200 ± 260

8.0

36

6.65 ± 0.93

0.87 ± 0.18

7.6

52.3 ± 8.7

3.3

90

4.10 ± 1.25

0.56 ± 0.12

300

4.8 ± 1.2

0.47 ± 0.07

Indomethacin

Etodolac Celecoxib Nimesulide NS-398 DuP 697 Valdecoxib

0.7

117 ± 37

~3.0

10

300 ± 120

~3.0

7.3

30

1.2 ± 0.4

0.06 ± 0.01

20

7.1 ± 3.4

1.5

not determined

26.1 ± 4.3

0.87 ± 0.11

30

250

—

Rofecoxib

>830

18.8 ± 0.9

0.53 ± 0.02

35

2,000 ± 500

1.5

Flosulide

810

32 ± 2

0.75 ± 0.18

43

1,005 ± 70

~3.0

MK-0663 DFU

>617 >1,000

116 ± 18 >100

1.09 ± 0.11 0.28 ± 0.04

106 > 300

12,130 ± 2516 12,600 ± 2400

0.6 1.1

Data are presented as means ± SEM. The ratio of the Cox-1 and Cox-2 IC50 values from the assay described in the text using engineered CHO cell lines expressing human Cox-1 or human Cox-2. c The Cox-1 and Cox-2 IC values, and their ratio, from the human whole blood (HWB) assays described in the text. 50 d The Cox-1 IC values from the assay described in the text using U937 cell microsomes with 0.1 µM arachidonic acid. 50 e The ED values for the rat paw edema assay, described in the text, to test the in vivo anti-inflammatory potency of the compounds. 50 a b

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FIG. 1. Chemical structures of compounds in Table I.

whose structures are shown in Fig. 1. Where available, IC50 values for the rat paw edema assay are given as well. A tabulation of the selectivities of these and other Cox inhibitors has appeared recently (Warner et al., 1999), as has a thorough review of the clinical tests for efficacy and GI tolerability for the Cox-2 inhibitors (Pairet et al., 1999). The data indicate that a fairly high degree of selectivity, as assessed by the human whole blood assays, is needed for a Cox-2 inhibitor to be GI-sparing at its efficacious dose. This conclusion is borne out for the most part by the clinical studies. Clinical results for rofecoxib and celecoxib are considered in a later section of this chapter. In a number of clinical studies meloxicam has been found to be as effective in treating osteoarthritis as diclofenac and piroxicam (Goei et al., 1995; Hosie et al., 1995; Hosie et al., 1996; Lindén et al., 1996), and in large-scale GI tolerability trials, meloxicam was found to be better tolerated than effective doses of diclofenac and piroxicam (Goei et al., 1995; Dequeker et al., 1998), although not completely free of GI side effects. Results from a number of clinical studies with nimesulide are consistent with the conclusion that it is similar in efficacy and GI tolerability to other NSAIDs (Davis

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and Brogden, 1994). Etodolac at 600 mg daily was found to be similar in efficacy to other NSAIDS (Spencer-Green, 1997), and several GI tolerability studies indicate that it is superior in this regard to other NSAIDs but not completely free of adverse GI effects (Lanza et al., 1987; Lanza et al., 1986; Van Eeden et al., 1990). These GI tolerability studies involved small numbers of patients and were short-term. In a small trial, flosulide was found to be similar in efficacy to naproxen in the treatment of osteoarthritis and to cause a lower incidence of adverse GI effects (Bjarnason et al., 1997). However, its development has been discontinued owing to peripheral edema (Brunel et al., 1995). The results from the clinical studies with Cox inhibitors that were shown by the assays described earlier to be somewhat Cox-2 selective led to the conclusion that a high degree of selectivity would be needed to avoid the adverse GI effects associated with inhibition of Cox-1. This conclusion is also supported by the results of the fecal 51Cr excretion assay in rats. A single 3 mg/kg dose of meloxicam caused a threefold increase in 51Cr excretion, and a single 10 mg/kg dose of etodolac caused a twofold increase in the rat assay described earlier. Nimesulide in a single 10 mg/kg dose had no effect, and this compound was not tested at higher doses. By contrast, L-745, 337 had no effect at 100 mg/kg, and rofecoxib and DFU had no effect at 100 mg/kg twice per day over 5 days. Thus, rofecoxib had no effect on fecal 51Cr excretion in rats at over 100-fold its ED50 for the rat paw edema assay in the same species. The clinical studies do suggest that superior GI tolerability is associated with Cox-2 selectivity, and the results with more selective compounds such as celecoxib and rofecoxib, described below, further support this conclusion. It is difficult to carry out a rigorous study that can justify the conclusion that serious adverse GI events among patients treated with a Cox-2 inhibitor are statistically no more common than among patients receiving placebo. This is because of the need to treat large numbers of patients for a long time to observe enough adverse events among both groups to yield a confidence interval for the difference that is tight enough to remove any possibility of a clinically important difference. This is compounded by the ethical issue of administering placebo for long periods of time to patients in need of treatment. However, an outcomes study in patients with arthritis comparing a selective Cox-2 inhibitor with an NSAID is justified to test the hypothesis that serious GI events are less frequent with the Cox-2 inhibitor, and indeed such trials have recently been reported. The findings support the hypothesis (Bombardier et al., 2000; Silverstein et al., 2000). Ultimately, this issue will be settled only by epidemiological-type studies based on large numbers of patients in a clinical use/postmar-

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keting setting. Since no significant adverse effects, ascribable to inhibition of Cox-1, have been observed among patients taking rofecoxib, there is no reason to expect that a drug with greater Cox-2 selectivity than rofecoxib would have an advantage over rofecoxib. It is, however, interesting to consider whether even highly selective Cox-2 inhibitors, in some tissue in which low arachidonate concentrations exist, could inhibit Cox-1 significantly, leading to possible adverse effects. There is not at present any reason to believe that this occurs, although prostaglandin synthesis is known to be limited by arachidonate availability in the gastric mucosa (Hamilton et al., 1999; Doyle et al., 1989; Preclik et al., 1992) and possibly in other tissues as well. For this reason, it is interesting to examine the relative potencies of the Cox-2 inhibitors listed in Table I in the U937 cell microsome assay at low arachidonate concentration. This allows a further differentiation among the Cox-2 selective inhibitors, and this Cox-1 potency at low arachidonate concentration could prove to be important in the long run. In case additional Cox-2 selectivity should prove advantageous, the second-generation Cox-2 inhibitors in development have been chosen for greater selectivity compared with their predecessors. V. ENZYMOLOGY/MEDICINAL CHEMISTRY Cyclooxygenase is a single polypeptide of 72 kDa containing a heme residue at the catalytic site. Four amphipathic helices are at the mouth of a channel leading to the heme, and it is in this channel that NSAIDs bind. Three of the helices are thought to insert into one leaflet of phospholipid bilayer membranes, and arachidonic acid probably enters the channel directly from the membrane rather than from an aqueous phase. Human Cox-1 and Cox-2 are 61% identical in amino acid sequence (Otto et al., 1993), and in the catalytic site, as revealed by the crystal structures of Cox1 (Picot et al., 1994) and Cox-2 (Luong et al., 1996; Kurumbail et al., 1996; McKeever et al., Brookhaven Protein Databank, accession number 2CX2), there are very few differences in residues present (Wong et al., 1997). The main differences in the NSAID binding sites are illustrated in Fig. 2 (see color insert) (see Wong et al., 1997; Fig. 1, panels A and B, p. 9281). Referring to the residue numbers for Cox-1, Arg 120 is important for binding of NSAIDs having a carboxylic acid residue (Mancini et al., 1995; Greig et al., 1997; Bhattacharyya et al., 1996), Ser 530 is the site of acylation by aspirin (DeWitt et al., 1990), and Tyr 385 is thought to form a free radical during the catalytic mechanism and to be involved in the formation of a radical at C-13 of arachidonic acid during the cyclooxygenase reaction (Shimokawa et al., 1990). Mutagenesis studies have shown that by substituting only two

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amino acid residues in Cox-1 (His-513 to Arg, and Ile-523 to Val) with the corresponding residues from Cox-2 resulted in inhibitor sensitivities of the mutant enzyme toward some Cox-2 selective inhibitors that resembled Cox-2 more than Cox-1. The crystal structure of Cox-1 was published only after efforts to discover Cox-2 selective inhibitors had been initiated, and the coordinates were not in the public domain until some time after the initial publication, so this information was not available to most of the investigators in the field until their efforts were well advanced. The structural information was useful in rationalizing the selectivities of inhibitors: and in guiding chemical research efforts to produce backup Cox-2 inhibitors with increased selectivities (Bayly et al., 1999). The catalytic mechanism suggested by Ruf is still essentially the model used by those concerned with mechanism (Dietz et al., 1988; Smith et al., 1992). In order to be catalytically active, the enzyme has to be oxidatively boosted up into a higher oxidation state than that in which it is found in the absence of lipid hydroperoxides. Like 5-lipoxygenase, the enzyme requires the presence of at least low concentrations of lipid hydroperoxides in order to remain catalytically active, and it can be rendered inactive by the addition of peroxidases that remove the lipid hydroperoxides from the medium (Smith et al., 1992). For the Cox-2 selective inhibitors that have been studied carefully, the details of the catalytic mechanism of Cox appear to be relatively unimportant, because these inhibitors bind with similar affinities and kinetics whether the heme group is present or not (Houtzager et al., 1996). It is possible that further investigations will show that some Cox-2 inhibitors bind preferentially to some states of the enzyme, but there is not yet any compelling evidence indicating such behavior. The peroxidase activity of Cox occurs at a site distinct from the cyclooxygenase site, and the two activities can be ablated by various means independently of each other (Smith et al., 1992). The inhibitors that are the subject of this chapter bind at the cyclooxygenase site and do not inhibit the peroxidase activities of Cox-1 or Cox-2. A useful insight into the subtle differences between the catalytic sites of the two enzymes was provided by the observations that while the cyclooxygenase activities of both were inhibited by aspirin, aspirinacetylated Cox-2 has an increased 15-oxygenase activity, which can be inhibited by some Cox inhibitors (Mancini et al., 1994; Lecomte et al., 1994). Aspirin is known to acetylate Ser-530 of Cox-1 and Ser-516 of Cox-2. Apparently, in the acetylated Cox-2 catalytic site there is still room for arachidonate to enter in an extended conformation, which permits catalysis of 15-hydroperoxyeicosatetraenoic acid formation but not of PGG2 formation. This led to the conclusion that the catalytic site of Cox-2 must be slightly larger than that of Cox-1, a conclusion later

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supported by comparison of the crystal structures of the two enzymes. Medicinal chemists accordingly began adding steric bulk to known nonselective Cox inhibitors in order to achieve Cox-2 selectivity, as well as to the few selective inhibitors identified in order to increase selectivity. Efforts along these lines have been described (Black et al., 1996; Lau et al., 1997). In following up the indomethacin series, considerable selectivities were achieved, but the series was discontinued because of insufficient efficacy. In reviewing available indomethacin analogues in their sample collection, the Merck group identified the compound L583,916, which showed good selectivity and had been a development candidate during the program that some decades earlier had resulted in the discovery and development of indomethacin. The reasons for the lack of full efficacy of the selective Cox-2 inhibitors in this series are not understood, but may be due in part to the fact that the rate of development of inhibition by these compounds is, in general, slower than for other selective Cox-2 inhibitors. The compounds DuP 697, flosulide, nimesulide, etodolac, and NS398, as shown in Table I, turned out to be Cox-2 selective inhibitors when examined by the assays described earlier and received considerable attention from various chemistry groups. Work on several structural series of Cox-2 selective inhibitors has been recently described from the medicinal chemistry point of view (Prasit and Riendeau, 1997) A compound from the program at DuPont, DuP 697, received much attention, and rofecoxib and several other selective Cox-2 inhibitors of current interest can legitimately be considered to have originated from this series. The progress from this lead to rofecoxib has been described recently (Dubé et al., 1999; Prasit et al., 1999). In this series, it was found that substitution of a sulfonamide for the methyl sulfone, in general, gave an increase in potency but at the expense of a loss in Cox-2 selectivity, especially as assessed by the sensitive U937 cell microsome Cox-1 assay at low arachidonate concentration. It is still uncertain whether this additional selectivity criterion at low substrate concentration will prove to be advantageous in avoiding adverse effects in patients, but it is very unlikely to constitute a deficit for these highly selective Cox-2 inhibitors. Other aspects of the medicinal chemistry programs occupied much time and effort in the selection and development of Cox-2 inhibitors, but they are not reviewed here because they involve issues common to most drug discovery programs. These issues include bioavailability, pharmacokinetics, distribution of compound to the target tissues, metabolism, inhibition of drug metabolizing enzymes, induction of gene expression, and adverse effects due to known or unknown mechanisms other than inhibition of Cox-1. Many compounds were eventually identified that were potent and highly Cox-2 selective but either failed in development or

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failed to meet the criteria for development for reasons other than Cox-2 potency and selectivity. Flosulide and L-583,916 are examples of such compounds. Ultimately, it proved much more difficult to address these issues than the issues of potency and selectivity. This is because in many cases the mechanisms by which compounds exert their undesired effects are not known, and predictive in vitro assays are not available. VI. CLINICAL DEVELOPMENT OF CYCLOOXYGENASE-2 INHIBITORS The clinical development of rofecoxib began in late 1994, and the development of celecoxib began in early 1995. The hypothesis that led the development of the Cox-2 inhibitors was based on the extensive information available on NSAIDs that inhibited both Cox-1 and Cox-2. Preclinical information suggested that inhibition of Cox-1 was responsible for the GI toxic effects of NSAIDs, including ulcers and their complications, namely bleeding, perforation, and obstruction. The beneficial effects of NSAIDs were postulated to be due to inhibition of Cox-2. The clinical program, therefore, was designed to test the hypothesis that a highly selective inhibitor of Cox-2 would suppress the pathological effects of prostanoids (e.g., inflammation, pain, and fever) without producing the GI toxicity associated with inhibition of Cox-1. If Cox-1–derived prostanoids do not contribute significantly to the symptoms or pathogenesis of acute and chronic inflammation, then a selective Cox-2 inhibitor, free of dose-limiting Cox-1–associated toxicity, should demonstrate clinical efficacy comparable with that of the existing NSAIDs for a variety of clinical disorders. A. Selectivity in Humans The first step in the clinical development of rofecoxib was to determine the degree of selectivity when the drug was administered to humans. As outlined above, the assessment of selectivity of Cox inhibitors for the two isoforms is dependent on the systems used and in vitro systems may not reflect the degree of selectivity of the enzyme inhibitors in vivo. Two clinical models were used to assess the selectivity of rofecoxib in humans. For both tests, tissues were obtained from patients or volunteers receiving either rofecoxib or NSAIDs, and the ability of these tissues to synthesize prostanoids ex vivo was determined. The simplest of these models is the whole blood assay, which was mentioned earlier in this chapter as one of the tests than can be used for evaluation of Cox selectivity (Patrignani et al., 1994). To use this test in the clinical setting, two samples of blood are drawn from individuals receiving a Cox inhibitor. One sample is allowed

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to clot and the other is heparinized. When blood clots, the serum contains thromboxane B2 (TXB2) derived from platelets activated during the clotting process. Since platelets contain predominantly, if not exclusively, Cox1 and do not have the capacity to induce Cox-2, the amount of TXB2 in serum is an index of Cox-1 activity. If the heparinized aliquot of blood is incubated with bacterial endotoxin (LPS), monocytes in the blood are activated and induce Cox-2, which results in the production of prostaglandins that are secreted into the plasma. The measurement of plasma prostaglandin E2 (PGE2) produced by whole blood incubated with lipopolysaccharide for 24 hours at 37°C is an index of Cox-2 activity. Using this whole blood assay, we have shown that rofecoxib has little to no effect on Cox-1 activity at single doses up to 1,000 mg and at multiple doses of 375 mg/day for 10 days. Bleeding time is also not affected by these doses of rofecoxib, which are at least 20-fold higher than doses required for therapeutic effects; in contrast, the NSAIDs produce dose-related inhibition of Cox-1 activity within their therapeutic dose range (Ehrich et al., 1999). Recent market entries that have claimed to be selective for inhibition of Cox-2, such as nabumetone and meloxicam, also inhibit Cox-1 when given at their usual therapeutic doses (Patrignani et al., 1994; Tegeder et al., 1999). Unlike rofecoxib, these drugs are not sufficiently selective to spare Cox-1 at their clinical doses. The degree of inhibition of Cox-2 produced by rofecoxib and NSAIDs at their clinically useful doses is approximately the same. The second model used during the development of rofecoxib to determine selectivity in humans was the assessment of the ability of gastric mucosal biopsies obtained from subjects receiving therapeutic doses of rofecoxib or an NSAID (naproxen) to synthesize prostaglandins (PGE2 and PGF2α) ex vivo. Normal gastric mucosa contains predominantly, if not exclusively, Cox-1 and is the target organ for the toxicity of NSAIDs. As pointed out above, gastric mucosa may be particularly sensitive to Cox inhibition because of its low arachidonate concentration. Gastric mucosa was biopsied before and after normal volunteers received placebo, rofecoxib 25 mg or 50 mg daily, or naproxen 500 mg twice daily for 5 days. The last biopsy was obtained 4–5 hours after the last dose. Synthesis of PGE2 was measured as an index of Cox activity. The data indicate that naproxen but not rofecoxib inhibited gastric Cox activity. This model indicates that rofecoxib does not affect Cox-1 in the stomach at and above clinically used doses, whereas this enzyme is markedly suppressed by naproxen (Cryer et al., 1999). These assays for selectivity indicate that rofecoxib has a sufficiently low affinity for Cox-1 that no inhibition of this enzyme is detectable in humans at concentrations achievable with any dose tested up to 1,000 mg, 80 times the starting dose for osteoarthritis. Thus, rofecoxib maintains a high

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degree of selectivity in humans, in contrast to the NSAIDs. Data with celecoxib indicate that it, too, is highly selective, but somewhat less selective than rofecoxib. Published information indicates that celecoxib can achieve concentrations that inhibit Cox-1 as assessed by the whole blood assay at and slightly above the clinical doses (McAdam et al., 1999). B. Efficacy 1. Osteoarthritis The Cox-2 inhibitors, rofecoxib and celecoxib, are as effective as the NSAIDs in the treatment of osteoarthritis. Rofecoxib 12.5–25 mg once daily showed efficacy statistically equivalent to diclofenac 50 mg three times daily and ibuprofen 800 mg three times daily (Saag et al., 1998; Cannon et al., 1998). Celecoxib 200 mg daily showed effects similar to naproxen 500 mg twice daily in the treatment of osteoarthritis. 2. Rheumatoid Arthritis Celecoxib is approved for the treatment of rheumatoid arthritis at doses up to 200 mg twice daily. These doses produced effects that were similar to naproxen 500 mg twice a day. In clinical development, rofecoxib has shown efficacy in the therapy of rheumatoid arthritis. Rofecoxib is currently in phase III for the therapy of rheumatoid arthritis. 3. Analgesia Rofecoxib is approved for the treatment of acute pain and dysmenorrhea at a dose of 50 mg for up to 5 days. The clinical studies indicate that rofecoxib shows efficacy similar to that produced by the maximum analgesic doses of naproxen and ibuprofen (Ehrich et al., 1999). The pain settings in which rofecoxib has been tested include acute postoperative dental pain, the pain of dysmenorrhea for up to 3 days, and postoperative pain for 5 days following surgical replacement of the knee or hip. In contrast, celecoxib is not approved in the United States for the treatment of acute pain, and it appears to be less effective when given acutely than rofecoxib, ibuprofen, or naproxen. The explanation for the differences between rofecoxib and celecoxib in acute pain is not known. 4. Fever Studies in animals clearly indicate that prostaglandins can produce fever when injected into the central nervous system and that Cox-2 induction is responsible for the fever that occurs with bacterial endotoxin and cytokines. To address the potential for an antipyretic effect in humans, Schwartz et al. (1999) administered rofecoxib or ibuprofen to young adults who presented to an infirmary for acute, nonbacterial

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febrile illness. The results indicated that rofecoxib 25 mg and ibuprofen 400 mg were comparably antipyretic and promptly reduced fever over the 6-hour period after dosing. Although the Cox-2 inhibitors have not been approved for the reduction of fever, it is important for clinicians to recognize the antipyretic effect, since the Cox-2 inhibitors could mask fever that could be a sign of infection. C. Safety 1. Gastrointestinal The major hypothesis on which the clinical development programs of rofecoxib and celecoxib were based was that the Cox-2 inhibitors would be safer than NSAIDs in regard to GI toxicity. The clinical development of rofecoxib was very extensive in testing this hypothesis. The first part of the test was to determine the effects of rofecoxib on prostaglandin synthesis by gastric mucosa, which is catalyzed by Cox-1. As outlined above, rofecoxib at doses of up to 50 mg had no effect on prostaglandin synthesis in the gastric mucosa, whereas an NSAID, naproxen 500 mg twice daily, markedly inhibited gastric prostaglandin synthesis. Since gastric prostaglandins are cytoprotective, sparing their synthesis should translate into a safer drug for the GI tract. a. Endoscopy Trials. The gastroduodenal effects of rofecoxib were determined first in healthy volunteers and subsequently in patients with osteoarthritis. In a 7-day double-blind, randomized, controlled endoscopy study, normal volunteers were given placebo, rofecoxib 250 mg/day, ibuprofen 800 mg three times per day, or aspirin 650 mg four times per a day. The individuals were endoscoped prior to and after 1 week of therapy with these drugs. In this study, the effect of rofecoxib on the development of erosions or ulcers was similar to the result with placebo, whereas ibuprofen and aspirin produced significantly more lesions (Lanza et al., 1999). These data are all the more impressive when one realizes that the dose of rofecoxib in this study is 10 to 20-fold higher than the approved clinical doses for osteoarthritis, whereas the NSAID doses were within the therapeutic range. In two identical double-blind, randomized, controlled 6-month studies in patients with osteoarthritis, rofecoxib 25 and 50 mg (twice the therapeutic range for osteoarthritis) was compared with a placebo and ibuprofen 800 mg three times per day (a standard dose for osteoarthritis). Endoscopies were performed at baseline, 6 weeks, 12 weeks, and 24 weeks. Placebo was continued only for the first 3 months, since it was considered unethical to continue patients without adequate therapy for their osteoarthritis for 6 months. Acetaminophen was allowed for rescue analgesia in all groups. Each of

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these studies indicated that rofecoxib at both doses was markedly less toxic than ibuprofen and was similar to placebo. When the two endoscopy studies were combined, statistical equivalence to placebo could be shown with the 25-mg dose of rofecoxib (Laine et al., 1999; Hawkey et al., 1999). Thus, the data from endoscopy trials are consistent with the gastric biopsy data, indicating that rofecoxib does not affect the stomach to produce lesions. b. Other Studies of Gastrointestinal Effects. Damage to the gastrointestinal tract by NSAIDs includes loss of mucosal integrity, which results in an increase in the permeability of the small intestine. This effect impairs intestinal function, and the damage occurs beyond the reach of the endoscope. The effect of rofecoxib 25 and 50 mg daily (twice the doses approved for osteoarthritis) on intestinal permeability was compared with the effects of indomethacin 50 mg three times per day and placebo in healthy volunteers in a double-blind randomized crossover study. Intestinal permeability was assessed by determining the intestinal absorption of ethylenediaminetetracetic acid (EDTA) which usually is not absorbed, and L-rhamnose, which normally is absorbed. The degree of EDTA absorption was determined by measuring the ratio of EDTA to L-rhamnose excreted in the urine. The results indicate that neither dose of rofecoxib increases the absorption of EDTA as compared with placebo, and both doses produced significantly less change than indomethacin (Bjarnason et al., 1998). A second study to assess the effects of rofecoxib on portions of the GI tract not visualized by the endoscope was the red blood cell loss study. Healthy volunteers had their red blood cells labeled with 51Cr and were then treated for 4 weeks with rofecoxib 25 mg daily or 50 mg daily, ibuprofen 800 mg three times daily, or placebo. Red blood cell loss into the feces, which is a marker of bleeding anywhere along the GI tract from the mouth to the anus, was determined by measuring the fecal excretion of radioactivity over a 4-week period. The findings from this study indicate that both doses of rofecoxib were statistically equivalent to placebo, whereas ibuprofen differed significantly from placebo and rofecoxib in producing an increase in excretion of radioactivity, indicating an increase in GI blood loss (Hunt et al., 1998). The hypothesis that Cox-2 inhibitors are less toxic than NSAIDs on the GI tract because of sparing Cox-1–dependent GI prostaglandin synthesis is born out by these data. Furthermore, data from the rofecoxib phase II and III studies have been combined in order to compare the effects of rofecoxib with those of NSAIDs on clinical outcomes relevant to GI toxicity. In this prespecified, combined analysis of the clinical development program, rofecoxib produced a significantly reduced incidence of clinical GI ulcers, bleeds, and perforations compared with the

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NSAID comparators (relative risk 0.45) (Langman et al., 1999). Thus, the surrogate end points of intact gastric mucosal prostaglandin synthesis, normal upper GI endoscopy, normal intestinal permeability, and lack of fecal red blood cell loss were predictive of the clinical outcomes seen in the phase II and III studies. The celecoxib program showed roughly similar findings, with fewer endoscopic lesions and probably fewer ulcers in their phase III development program, although no prespecified analysis has been reported. Recently, two large outcomes trials have been completed confirming the reduced risk of ulcers and their complications in patients with arthritis treated with a Cox-2 inhibitor as compared with a NSAID (Bombardier et al., 2000; Salverstein et al., 2000). 2. Renal Since Cox-2 is constitutively present in the kidney of experimental animals and humans and can be induced by maneuvers such as salt depletion (Harris et al., 1998), effects of Cox-2 inhibitors on renal function might be expected to occur. Preclinical studies indicated that all Cox-2 inhibitors reduce sodium excretion in dogs similarly to NSAIDs. In the clinical development of rofecoxib, special studies were performed to determine the effects of the drug on various aspects of renal function. The renal sodium study evaluated the effects of rofecoxib 50 mg daily on sodium balance versus placebo and indomethacin 50 mg three times per day. Renal function was determined in healthy elderly subjects, from 60 to 80 years old, in balance on a 200 mmol sodium per day diet during 14 days of therapy. Rofecoxib and indomethacin were found to cause equivalent transient sodium retention over the first 72 hours but not at later time points. After 72 hours, a new steady state was reached with indomethacin, with the net retention of sodium persisting over the 14 days of the study. With rofecoxib, however, the sodium that had been retained over the first 72 hours was gradually lost over the ensuing 11 days, so that by the end of 2 weeks, the volunteers on rofecoxib were not different from those receiving placebo but were different from those receiving indomethacin. In this study, indomethacin produced a small reduction in glomerular filtration rate (GFR), but there was no significant effect of rofecoxib on GFR (Catella-Lawson et al., 1999). A second study determined the effects of rofecoxib on GFR in normal volunteers older than 65 years in balance on a sodium-restricted (60 mmol/day) diet. The results of this study indicated that rofecoxib 12.5 mg and 25 mg daily was comparable to indomethacin 50 mg three times daily in producing a small reduction of GFR (Swan et al., 1999). Similar findings on sodium retention and GFR have been reported with celecoxib in young healthy volunteers on a restricted sodium diet (Rossat

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et al., 1999). Clinical trials with rofecoxib and celecoxib indicate that Cox2 inhibition can result in mild degrees of sodium retention, particularly at higher doses, an effect that is not distinguishable from that with NSAIDs. This effect manifests as mild edema in a small number of patients. These findings from special renal studies and the clinical trial data indicate that inhibition of Cox-2 does not eliminate the renal effects of NSAIDs because Cox-2–derived prostanoids are involved in normal renal function. However, the kidney contains considerably more Cox-1 than Cox-2, and the localization of the two isoforms is different. It is not yet known whether the Cox-2 inhibitors will be safer in subgroups of patients prone to develop acute renal failure with NSAIDs, such as those patients with severe volume depletion, congestive heart failure, or hepatic cirrhosis with ascites (Bosch-Marcé et al., 1999). Also, it is not known whether rare events, such as interstitial nephritis or papillary necrosis, will occur with long-term use of Cox-2 inhibitors, although studies in animals suggest that such events may be related to Cox-1 inhibition, since only Cox-1 is found in the papilla. Therefore, Cox-2 inhibitors may not produce these serious adverse effects (Khan et al., 1998). 3. Articular Cartilage There is some concern, particularly in Europe, that certain NSAIDs may actually have a deleterious effect on articular cartilage in patients with osteoarthritis. An effect on cartilage has been predominantly suggested with indomethacin, although the data are not convincing (Doherty and Jones, 1995). This does not appear to be a concern with all NSAIDs, however, and diclofenac is not thought to have this problem. Thus, if there is a deleterious effect on cartilage with some NSAIDs, it is not likely to be mechanism-related. In order to determine whether Cox-2 inhibition is different from NSAIDs on articular cartilage, the effect of rofecoxib 12.5 and 25 mg was compared with that of diclofenac 50 mg three times a day in patients with osteoarthritis. Over a treatment period of 1 year, the joint space of the knee was measured with standardized radiographs of patients with osteoarthritis. A small and similar narrowing of the joint space occurred with both rofecoxib and diclofenac therapy. This degree of narrowing of the joint space is consistent with the known progression of osteoarthritis indicating that neither rofecoxib nor diclofenac has the deleterious effect of accelerating articular cartilage loss. VII. FUTURE DIRECTIONS A. Colon Cancer Epidemiological evidence indicates that patients who have taken longterm NSAID therapy or aspirin have a reduced incidence of colon cancer.

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Additionally, patients with familial adenomatous polyposis, an autosomaldominant disease characterized by numerous small intestinal and colonic polyps with a nearly universal progression to colon cancer, have a favorable response to NSAIDs. Administration of NSAID (usually sulindac) to patients with this disorder reduces the number and size of polyps (DuBois et al., 1996). Recent biochemical evidence indicates that colon polyps and colon cancer are frequently associated with induction of Cox-2 in the lesion as assessed by expression of Cox-2 mRNA and protein. Such induction appears to correlate with growth of the lesion, and inhibition of Cox2 correlates with apoptosis of the involved cells (Gupta and DuBois, 1998). The link between Cox-2 and polyps has been most extensively studied in an animal model of FAP, the adenomatous polyposis coli gene knockout mouse, which develops multiple intestinal polyps. In this model, both sulindac and a Cox-2 inhibitor are effective in producing regression of adenomas. When this knockout mouse was crossbred to a Cox-2 knockout mouse to produce a double adenomatous polyposis coli/Cox2 knockout, the development of intestinal polyposis was markedly reduced (Oshima et al., 1996). However, despite the firm experimental basis and encouraging clinical studies, prophylaxis with NSAIDs to prevent colon cancer in patients who have polyps or in the population in general is not justified because of the GI toxicity of such therapy. With the introduction of Cox-2 inhibitors, it now is feasible to test the hypothesis that inhibition of Cox2 over prolonged periods of time would be protective against the development of colon polyps. Since it is well accepted that colon polyps are precancerous lesions, the prevention of polyps should translate into a reduction in the incidence of colon cancer. Several studies using rofecoxib or celecoxib have been contemplated or begun. Both Cox-2 inhibitors are being studied in patients with familial adenomatous polyposis, and results with celecoxib have recently been reported (Steinbach et al., 2000). Also, there are plans to evaluate the ability of Cox-2 inhibitors to reduce the recurrence of polyps in patients who have had sporadic colonic polyps surgically removed. Ultimately, one would like to determine whether therapy with Cox-2 inhibitors could be used prophylactically in high-risk patients to prevent the development or recurrence of colon cancer. B. ALZHEIMER’S DISEASE The normal brain contains Cox-2, and brains from patients with Alzheimer’s disease have been found on autopsy to have induction of Cox-2 (Pasinetti and Aisen, 1998). Epidemiologic studies have found

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that individuals who have received NSAIDs chronically have a reduced incidence of Alzheimer’s disease (McGeer et al., 1996). The hypothesis to explain this effect is that inflammation in areas of the Alzheimer plaque enhances the progression of the disease and Cox-2 induction is, in part, responsible for the inflammation. It is believed, not that Cox-2 is etiologic in Alzheimer’s disease, but that inflammation speeds the time course of the disease. It is not possible to use NSAIDs to test the hypothesis prospectively because of their serious GI toxicity. However, the Cox-2 inhibitors could be used to test the hypothesis that interruption of the inflammation by inhibition of Cox-2 in the brain can delay progression of Alzheimer’s disease. Several studies are ongoing with rofecoxib and celecoxib to investigate this potential use. No data are yet available, but this is an exciting possibility for Cox-2 inhibitor therapy. VIII. CONCLUSIONS The hypothesis that NSAIDs exert their beneficial effects in pain and inflammation by inhibiting Cox-2 and their adverse effects on the GI tract and elsewhere by inhibiting Cox-1 has been validated. Clinical and postmarketing studies now ongoing will reveal whether the degree of Cox-2 selectivity available in current drugs is sufficient to completely eliminate serious GI adverse effects ascribable to inhibition of Cox-1. Should these studies indicate a need for even greater selectivity than is provided the currently available Cox-2 inhibitors, the next generation of compounds in development has additional selectivity to address this requirement, as indicated by the values for valdecoxib and MK-0663 (Table I) for example. As is seen in Table I, some of the drugs and compounds in development differ considerably more in their Cox-1 potencies as shown by the U937 cell microsome assay at low arachidonate concentration than they do when assessed by the Cox-1 whole blood assay. Time will tell whether this lower potency against Cox-1 at low arachidonate concentration correlates with a lower incidence of adverse effects. Beyond a certain selectivity threshold, Cox-2 inhibitors will be differentiated according to factors other than Cox-1 inhibitory potency. Just as some individual patients do better on one particular NSAID than on others, some Cox-2 inhibitors will perhaps prove to be more effective and better tolerated with some patients than with others, for reasons that might not become clear soon or ever but will not have to do with Cox-2 potency or selectivity. Although the discovery and development of the Cox-2 inhibitors proceeded unusually quickly from development of the hypothesis to launch of the first drugs, the process did not make heavy use of what might be

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considered advanced drug discovery technology. The Cox-1 (Langenbach et al., 1995) and Cox-2 (Morham et al., 1995; Dinchuck et al., 1995) knockout mice have interesting phenotypes but certainly did not provide clear support for the hypothesis that was the basis for the discovery programs. As mentioned earlier, the crystal structures became available too late to be a significant factor in selecting for development the drugs currently available, although computational modeling techniques were applied in the backup programs. The program at Merck did not involve the preparation and testing of large combinatorial libraries but did involve preparation of large quantities of key intermediate compounds for the rapid synthesis of structural analogues in each of the series being explored. The assays were not particularly sophisticated or rapid. Indeed, the cell-based assays that were the workhorses of the in vitro assay program involved immunoassay techniques that were rather cumbersome and had low throughput. The U937 cell microsome assay run at low arachidonate concentration was demanding and involved fairly large numbers of controls because of the difficulty in maintaining constant low concentrations of arachidonate in the assay medium. It is therefore interesting to ask why this program proceeded so rapidly from initiation to launch. The most important factors were without doubt the vast clinical experience with NSAIDs, including much experience with the serious, mechanism-based adverse effects, and the several structural classes of Cox inhibitors already known. The availability of Cox-1 to provide a convenient counterscreen predictive of the main adverse effects to be avoided provided a reliable guide for the medicinal chemistry program. Naturally, many of the potent and highly selective Cox-2 inhibitors prepared in the course of the program either did not give full efficacy, for reasons that are not understood, or caused adverse effects unrelated to inhibition of Cox-1. In most cases the mechanisms of these other adverse effects are not known, and no in vitro assays were available to guide the chemistry program toward compounds that would not cause these effects. Fortunately, it was possible to escape these liabilities of many of the compounds studied by moving to a structural series in which compounds free of these effects could be prepared. Thus, the availability of numerous structural leads was a major factor in the rapid progress of the Cox-2 inhibitor drug discovery programs. The main rate-limiting factor in this program, as in most others, was a lack of knowledge of the basic biological mechanisms that control compound absorption, distribution, metabolism, and excretion, as well as the mechanisms by which compounds induce the expression of numerous genes or modulate the function of numerous biological processes that they turn out to influence when they are subjected to close scrutiny in preclinical development. Drug discovery programs would no doubt all proceed much

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more rapidly if more were known about basic biological processes and if sample collections were available that contained several structural classes of compounds capable of modulating the function of any potential drug target in the desired manner. In the case of Cox-2 selective inhibitors, the latter requirement was met but the preceding requirement was not. There is a greater need for new technologies and strategies to advance our understanding of basic biology to aid drug discovery than there is for new technologies to increase the rate at which we can screen samples and carry out the other operations of drug discovery. When we possess sample collections that contain several compounds with different structures all of which have the desired effect on any potential drug target and when we generate sufficient knowledge of the detailed mechanisms by which bioactive compounds exert their biological effects, then all drug discovery programs can be even faster than those that led to the Cox-2 inhibitors. REFERENCES Antiplatelet Trialists’ Collaboration (1994). BMJ 308, 81–106. Bayly, C. I., Black, W. C., Léger, S., Ouimet, N., Ouellet, M., and Percival, M.D. (1999). Bioorg. Med. Chem. Lett. 9, 307–312. Bhattacharyya, D.K., Lecomte, M., Rieke, C.J., Garavito, M., and Smith, W.L. (1996). J. Biol. Chem. 271, 2179–2184. Bjarnason, I., Macpherson, A., Rotman, H., Schupp, J., and Hayllar, J. (1997). Scand. J. Gastroenterol. 32, 126–130. Bjarnason, I., Sigthorsson, G., Crane, R., Simon, T., Hoover, M., Bolognese, J., and Quan, H. (1998). Am. J. Gastroenterol. 93, 1670 (Abstract #246). Black, W. C., Bayly, C., Belley, M., Chan, C.-C., Charleson, S., Denis, D., Gauthier, J. Y., Gordon, R., Guay, D., Kargman, S., Lau, C. K., Leblanc, Y., Mancini, J., Ouellet, M., Percival, D., Roy, P., Skorey, K., Tagari, P., Vickers, P., Wong, E., Xu, L., and Prasit, P. (1996). Bioorg. Med. Chem Lett. 6, 725–730. Bombardier, C., Laine, L., Reicin, A., Shapiro, D., Burgos-Vargas, R., Davis, B., Day, R., Ferrazin, A., Hawkey, C., Hochberg, M., Kvien, T. K., Schnitzer, T., Weaver, A., for the VIGOR Study Group (2000). N. Engl. J. Med., 343, In Press. Bosch-Marcé, M., Claria, J., Titos, E., Masferrer, J. L., Altuna, R., Poo, J. L., Jiménez, W., Arroyo, V., Rivera, F., and Rodés, J. (1999). Gastroenterology 116, 1167–1175. Brideau, C., Kargman, S., Liu, S., Dallob, A. L., Ehrich, E. W., Rodger, I. W., and Chan, C.-C. (1996). Inflamm. Res. 45, 68–74. Brooks, P., Emery, P., Evans, J., Fenner, H., Hawkey, C. J., Patrono, C., Smolen, J., Breedveld, F., Day, R., Dougados, M., Ehrich, E. W., Gijon-Banos, J., Kvien, T., van Rijswijk, M. H., Warner, T., and Zeidler, H. (1999). Rheumatol. 38, 779–788. Brunel, P., Hornych, A., Guyene, T. T., Sioufi, A., Turri, M., and Menard, J. (1995). Eur. J. Clin. Pharmacol. 49, 193–201. Cannon, G., Caldwell, J., Holt, P., McLean, B., Zeng, Q., Ehrich, E., Seidenberg, B., Bolognese, J., and Daniels, B. (1998). Arth. Rheum. 41 (9 Suppl), S196 (Abstract). Catella-Lawson, F., McAdam, B., Morrison, B. W., Kapoor, S., Kujubu, D., Antes, L., Lasseter, K. C., Quan, H., Gertz, B. J., and FitzGerald, G. A. (1999). J. Pharmacol. Exp. Ther. 289, 735–741.

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5α-REDUCTASE INHIBITORS BY JOHN D. McCONNELL* AND ELIZABETH STONER† *University of Texas Southwestern Medical Center, Department of Urology, Dallas, Texas, 75390, and †Merck & Co., Inc., Clinical Research, Endocrinology and Metabolism, RY33-524, Rahway, New Jersey 07065.

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Identification and Characterization of 5α-Reductase . . . . . . . . . . . . . . . . . . A. Identification of 5α-Reductase . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Discovery of a Genetic Deficiency of 5α-Reductase in Humans . . . . . . . C. Identification of Two Isozymes of 5α-Reductase . . . . . . . . . . . . . . . . . . . III. Development of 5α-Reductase Inhibitors. . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rationale for Development of 5α-Reductase Inhibitors . . . . . . . . . . . . . B. Chemistry . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Pharmacokinetics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Pharmacodynamics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Clinical Studies in Men with Androgenic Disorders . . . . . . . . . . . . . . . . . . . A. Benign Prostatic Hyperplasia. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Prostate Cancer. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Androgenetic Alopecia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Clinical Studies in Women with Androgenic Disorders . . . . . . . . . . . . . . . . A. Androgenetic Alopecia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Hirsutism. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Other 5α-Reductase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Other Type 2 5α-Reductase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Type 1 5α-Reductase Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Type 1/Type 2 (Dual) 5α-Reductase Inhibitors . . . . . . . . . . . . . . . . . . . . D. Natural Products with 5α-Reductase Inhibitory Activity . . . . . . . . . . . . . VII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

143 144 144 145 146 148 148 148 150 150 151 151 161 162 169 169 169 171 171 171 172 172 173 174

I. INTRODUCTION Definitive evidence that dihydrotestosterone (DHT) is a potent androgen with its own important physiological and pathophysiological actions, separate from those of testosterone, was provided by two reports in 1974 of a rare inborn disorder of male phenotypic sexual differentiation caused by a deficiency in 5α-reductase, the enzyme that converts testosterone to DHT. The reduction in the conversion of testosterone to DHT by 5α-reductase, which underlies this syndrome, leads to a specific developmental defect in the formation of the male external genitalia and the prostate. Males with this genetic disorder exhibit a striking phenotype, in which the internal genitalia are normal 143 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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but the external genitalia are feminized. However, the marked increases in circulating androgens, especially testosterone, that occur during puberty produce virilization in the 5α-reductase-deficient males. These subjects are otherwise healthy, but they have sparse facial and body hair and appear to be protected against the development of benign prostatic hyperplasia (BPH), prostate cancer, and androgenetic alopecia (AGA) in later life. Thus, while DHT is clearly necessary for normal male genital development in utero, in adulthood DHT appears to have no major physiological role but rather is implicated in a variety of androgen-dependent disorders. Taken together, these findings provided a clear rationale for the development of an inhibitor of 5α-reductase in the treatment of DHT-dependent disorders that afflict men. Finasteride was developed as the first orally active, specific inhibitor of 5α-reductase for clinical use. Clinical studies in men with BPH demonstrated that treatment with finasteride reduced prostate size, improved urinary symptoms, and reduced the risk of developing serious BPH-related outcomes, including acute urinary retention (AUR) and the need for surgery, confirming the effects of DHT on the prostate. Additional studies also demonstrated that finasteride is an effective treatment in men with AGA. Several small studies have also suggested it is moderately efficacious in women with hirsutism. A number of other inhibitors of 5α-reductase are also presently in development for the treatment of BPH and AGA. This review examines recent advances in the development and clinical uses of 5α-reductase inhibitors in the treatment of disorders mediated by DHT, an important physiologic and pathophysiologic androgen. II. IDENTIFICATION AND CHARACTERIZATION OF 5α-REDUCTASE A. Identification of 5α-Reductase The 5α-reductase enzyme was initially identified in the early 1950s by Schneider and co-workers in studies examining the metabolism of desoxycorticosterone in rat liver slices (Schneider and Hortsmann, 1951; Schneider, 1952). Subsequent studies (Forchielli and Dorfman, 1952; Tomkins, 1957; McGuire and Tomkins, 1960; McGuire et al., 1960) demonstrated that the enzyme catalyzed the reduction of a variety of steroid substrates, including testosterone, which can be reduced to DHT (Fig. 1). The realization that 5α-reductase plays an important role in androgen action began in the early 1960s, when DHT was demonstrated to be a more potent androgen than testosterone in bioassays in the prostate

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FIG. 1. Androgen metabolic pathways. (Kaufman, 1999.)

(Saunders, 1963). Further studies demonstrated that administration of radiolabeled testosterone led to an accumulation of DHT in the nuclei of ventral prostate cells (Bruchovsky and Wilson, 1968; Anderson and Liao, 1968). Moreover, DHT was shown to bind preferentially to specific nuclear (androgen) receptor proteins (Mainwaring, 1969; Fang et al., 1969). Developmental studies showed that 5α-reductase activity in mammalian embryos was high in the primordia of the prostate and external genitalia prior to their virilization but very low in wolffian duct structures, suggesting that the enzyme was critical for the development of the normal male phenotype during embryogenesis (Wilson and Lasnitzki, 1971; Wilson, 1972; Siiteri and Wilson, 1974). Taken together, these data suggested that conversion of testosterone to DHT by 5α-reductase was a critical step in male sexual differentiation and focused attention on the role of 5α-reductase in androgen physiology and pathophysiology. B. Discovery of a Genetic Deficiency of 5α-Reductase in Humans Definitive evidence of the role of 5α-reductase in androgen physiology was provided by two reports in 1974 describing subjects who had genetic mutations affecting expression of the enzyme, resulting in a reduction in DHT formation (Imperato-McGinley et al., 1974; Walsh et

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al., 1974). Males who were homozygous for this inherited defect in 5αreductase were born with a specific phenotypic form of pseudohermaphroditism. Marked increases in circulating androgens during puberty produced virilization and normal libido in these men. Females who were homozygous for the 5α-reductase deficiency were reported to be phenotypically normal but could be detected by biochemical assay (Katz et al., 1995). C. Identification of Two Isozymes of 5α-Reductase 1. Identification of Two Isozymes Initial attempts to purify 5α-reductase were hindered by the extreme insolubility of the enzyme (Russell and Wilson, 1994). This problem was overcome in 1989 when the technique of expression cloning in Xenopus oocytes was successfully used to isolate cDNA encoding rat liver 5α-reductase. These studies ultimately led to the discovery that two isozymes of 5α-reductase exist in humans, which are coded for by two separate genes (Andersson et al., 1991; Harris et al., 1992; Russell and Wilson, 1994), referred to as types 1 and 2 based on the order of their discovery. Type 1 5α-reductase is prominent in the liver and in sebaceous glands of the skin, whereas type 2 5α-reductase is prominent in the genitourinary tract, including the prostate, and in the liver (Harris et al., 1992; Thigpen et al., 1993; Russell and Wilson, 1994). The type 2 isozyme has also been shown to be localized in root sheaths of scalp hair follicles (Eicheler et al., 1995; Bayne et al., 1999). Genetic experiments confirmed that the mutations in subjects with 5α-reductase deficiency occurred within the gene encoding for the type 2 isozyme (Andersson et al., 1991; Russell and Wilson, 1994). The type 1 5α-reductase isozyme was demonstrated to have a neutral optimum pH (6.5–7.5), whereas the type 2 isozyme had a more acidic optimum pH (5.5) (Harris et al., 1992) (Table I). Moreover, the type 1 5α-reductase isozyme was 50- to 100-fold less sensitive to the specific 5α-reductase inhibitor finasteride and had a 25-fold higher affinity for testosterone as compared with the type 2 5α-reductase isozyme (Harris et al., 1992) (Table I). 2. Physiological and Pathophysiological Roles Type 2 5α-reductase–deficient men were reported to develop sparse facial and body hair and to be protected against the development in later life of BPH, prostate cancer, and AGA (Imperato-McGinley et al., 1974; Walsh et al., 1974). These findings provide strong evidence that although type 2 5α-reductase activity clearly plays an important role in normal male genital development in utero, in adulthood it appears to

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TABLE I Biochemical Characteristics of Human Type 1 and Type 2 5α-Reductase a Parameter

Type 1 5α-reductase

pH optima Km, µM (testosterone) Finasteride IC50, nM a

6.5–7.5 7.7 670

Type 2 5α-reductase 5.5 0.3 4.2

From Harris et al., 1992.

have no useful physiological role but rather is involved in a variety of androgen-dependent disorders. Type 1 5α-reductase may also play a specific role in androgen physiology and pathophysiology. The discovery that there are high levels of type 1 5α-reductase activity in sebaceous glands of the skin, particularly in the acne-prone regions of the face and scalp, led to the suggestion that type 1 5α-reductase may play an important role in the regulation of sebum secretion and that it may also be involved in disorders of sebum secretion, including acne (Thiboutot et al., 1995). In contrast, type 2 5α-reductase appears to play little or no role in sebum production, since sebum output is normal in type 2 5α-reductase–deficient subjects (Imperato-McGinley et al., 1993). It is well established that sebum production is androgen-regulated and that androgens have marked acnegenic activity (Hamilton, 1941). However, the identity of the androgen (or androgens) responsible for stimulating sebum production and the formation of acneiform lesions, as well as the source of this androgen (adrenal, gonadal, target tissue) are not well understood. The above findings suggest that either DHT generated locally in the sebaceous glands by type 1 5α-reductase or circulating DHT may be important in the development of acne. Recently, homologous recombination in mouse embryonic stem cells was used to disrupt the gene encoding for type 1 5α-reductase, resulting in type 1 5α-reductase–deficient mice (Mahendroo et al., 1996). Male mice lacking type 1 5α-reductase appeared normal; however, the absence of the type 1 5α-reductase isozyme in female mice caused a parturition defect and a decrease in litter size. The reduction in litter size was shown to be due to a failure of androgen reduction by 5αreductase, which in turn caused an increased production of estrogens, leading to fetal death in midgestation (Mahendroo et al., 1997). This finding indicated that type 1 5α-reductase normally acts to protect against estrogen toxicity during pregnancy. The parturition defect was demonstrated to be due to impaired cervical ripening in late gestation due to failure to catabolize progesterone in the cervix, implying that

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the type 1 isozyme also normally plays an important role in cervical progesterone catabolism at the end of pregnancy (Mahendroo et al., 1999). The relevance of these findings to species other than mice, including humans, is not known at this time. To date, there have been no reports of a type 2 5α-reductase knockout animal model. III. DEVELOPMENT OF 5α-REDUCTASE INHIBITORS A. Rationale for Development of 5α-Reductase Inhibitors The first specific model for predicting the long-term effects of chronic pharmacologic inhibition of 5α-reductase was provided by the clinical features that define the syndrome of 5α-reductase deficiency in adult males (normal libido, prostate remaining small throughout adulthood, scant facial and body hair growth, and no vertex thinning or recession of the temporal hair line). Based on these clinical observations, it was postulated that distinct and specific physiologic processes are mediated by each of the androgenic hormones testosterone and DHT (Imperato-McGinley et al., 1979). While testosterone is the major circulating androgen, DHT is the apparent active androgen in the prostate and in skin. Thus, spermatogenesis, libido, and maintenance of muscle mass appear to be mediated primarily by testosterone. Growth of the prostate gland and the development of AGA appear to be processes primarily controlled by DHT. Taken together, these findings provided a clear rationale for the development of an inhibitor of 5α-reductase in the treatment of DHT-dependent disorders, including BPH, AGA, and prostate cancer. B. Chemistry It was the initial observation of the lack of prostate gland growth in men with a genetic deficiency of 5α-reductase that prompted the search for a 5α-reductase inhibitor for use in the treatment of adult males with BPH. A number of steroids compete with testosterone for 5α-reductase. Some endogenous substrates, such as progesterone, inhibit the reduction of testosterone in vitro but are metabolized too rapidly to be effective inhibitors in vivo. The structural requirements for synthetic steroidal 5α-reductase inhibitors include a stable configuration in the A ring of the steroid molecule that mimics the transition state in the conversion of testosterone to DHT (Fig. 2) (Liang et al., 1983; Liang et al., 1984; Rittmaster, 1994). This allows the inhibitor to bind tightly to the active site of the enzyme. In the search for 5α-reductase inhibitors, early

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FIG. 2. Inhibition by finasteride of type 2 5α-reductase–mediated conversion of testosterone to dihydrotestosterone. (From Gormley, 1991.)

studies focused on 3-oxosteroid derivatives of testosterone. The 3-oxosteroids were potent inhibitors of the enzyme, but they lacked systemic activity owing to rapid inactivation by 5α-reductase. The next major step in the search for a clinically useful 5α-reductase inhibitor came with the development of 4-aza-3-oxosteroid derivatives of testosterone that were orally active inhibitors of 5α-reductase (Rasmusson, 1987). However, these compounds were not specific for 5α-reductase inhibition; they also possessed weak antiandrogen activity. Enzyme inhibitor studies using human prostate tissue as the source of steroid 5α-reductase led to the discovery of finasteride (MK-906), or N-(1,1dimethylethyl)-3-oxo-4-aza-5α-androst-1-ene-17β-carboxamide (Fig. 2), the first highly specific, potent, orally active 5α-reductase inhibitor to be developed for clinical use that lacked affinity for the androgen receptor (Rittmaster, 1994). The drug was shown to have no intrinsic androgenic, estrogenic, progestational, or other steroidal properties, and it did not affect the physiologic actions of testosterone (Liang et al., 1983; Liang et al., 1984; Brooks et al., 1986; Rittmaster, 1994). More recent studies demonstrated that finasteride is a potent inhibitor of the type 2 isozyme (the predominant isozyme in the human prostate), with little activity against the type 1 form, the primary isozyme in skin (Table I) (Harris et al., 1992).

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C. Pharmacokinetics Finasteride is orally active and highly bioavailable (about 80%). It is rapidly absorbed after oral administration, with peak plasma levels occurring 1 to 2 hours after drug intake, and approximately 90% of circulating finasteride is bound to plasma proteins (Ohtawa et al., 1991; Carlin et al., 1992). Finasteride is the major component circulating in the plasma (Carlin et al., 1992). The serum half-life of the drug is approximately 6 hours, although second-order kinetics are consistent with the formation of an irreversible complex, resulting in slow, gradual (several days) return of serum DHT to baseline after discontinuation of the drug. After oral administration, finasteride is extensively metabolized in the liver by hydroxylation at the tert-butyl group (ϕ-hydroxyfinasteride), followed by further oxidation to the corresponding acid (finasteride-ϕoic acid), with ϕ-aldehyde finasteride as an intermediate. Each of the three steps of this oxidative pathway is mediated by cytochrome P450 3A4 (Huskey et al., 1995). The major metabolites, ϕ-hydroxyfinasteride and finasteride-ϕ-oic acid, possess minimal activity (<20%) as inhibitors of 5α-reductase and are excreted mainly via the bile (Ohtawa et al., 1991). Finasteride does not appear to affect the cytochrome P450–linked drug metabolism enzyme system. Compounds that have been tested in humans have included antipyrine, digoxin, propranolol, theophylline, and warfarin, with no drug interactions of clinical importance being identified (U.S. package insert for Proscar, 1999). D. Pharmacodynamics A single oral dose of finasteride 5 mg markedly reduces serum DHT (to about 70% below baseline) in men within hours of dosing (Gormley et al., 1990). Complete reduction of serum DHT levels does not occur, in part owing to the ongoing activity of type 1 5α-reductase in the liver, which is not significantly affected by finasteride. Daily dosing produces a similar and persistent suppression of serum DHT, and tachyphylaxis to the effects on DHT is not observed with chronic administration (Stoner et al., 1994). In association with the marked reduction of DHT, a small (about 15%) increase in serum testosterone is observed. Serum estradiol levels increase similarly to, and are correlated with, this small increase in serum testosterone, as testosterone is the primary substrate for formation of estradiol in men (Fig. 1). However, the ratio of serum testosterone to estradiol is unaltered. Despite the significant changes in serum levels of DHT, serum levels of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) increased slightly (by about 10%) but remained within normal limits with finasteride treatment, and the

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response of the latter two hormones to gonadotropin-releasing hormone was not affected (Rittmaster et al., 1992). No clinically relevant changes in serum free testosterone or sex hormone–binding globulin were reported in men treated for 6 months with finasteride (Tenover et al., 1989). These findings indicate that finasteride does not affect regulation of the hypothalamic-pituitary-testicular axis. Chronic administration of finasteride also had no effect on circulating levels of cortisol, prolactin, thyroid-stimulating hormone, or thyroxine. No effects on lipid profiles or glucose tolerance have been demonstrated. IV. CLINICAL STUDIES IN MEN WITH ANDROGENIC DISORDERS A. Benign Prostatic Hyperplasia 1. Role of DHT in Growth of Prostate Gland The important role of androgens in the growth of the prostate gland has been known for more than a century. Two studies conducted in the 1890s demonstrated that BPH did not develop in men who had been castrated and that castration could promote regression of BPH (Cabot, 1896; White, 1895). These findings suggested that testicular hormones play a critical role in the pathogenesis of BPH. Studies using luteinizing hormone–releasing hormone analogues, which markedly reduce the production of testosterone in the testes, demonstrated that androgen deprivation could lead to a significant reduction in prostate volume and could reverse some of the clinical manifestations of BPH (Schröder et al., 1986; Peters and Walsh, 1987). These findings clearly established the beneficial effects of androgen deprivation in the treatment of BPH and provided the basis for the treatment of BPH by hormonal modulation. However, androgen deprivation using analogues of luteinizing hormone-releasing hormone also resulted in a relatively high incidence of erectile dysfunction, loss of libido, and impotence, markedly limiting their use in otherwise healthy men with BPH. Clearly a better therapeutic approach was needed, one that could target selectively the specific androgenic mechanism underlying the disease. 2. Natural History and Anatomic Progression of Benign Prostatic Hyperplasia Benign prostatic hyperplasia is diagnosed pathologically by the presence of nonmalignant prostatic nodules and clinically by signs and symptoms of urinary obstruction resulting from the abnormal growth (Fig. 3). The prevalence of BPH increases with age. Autopsy studies suggest the presence of BPH in nearly half of men in their fifties and a large proportion of men over 80 years of age (Berry et al., 1984). Clinically, symptoms

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FIG. 3. Cross section of normal prostate and prostate with benign prostatic hyperplasia. (From Wilson, 1980.)

of BPH are present in the majority of men 60 years of age and older (Chute et al., 1993). In view of its high prevalence, BPH might be regarded as a normal physiological accompaniment of aging in men. Until recently, BPH was treated almost exclusively by surgery, most commonly by transurethral resection of the obstructing tissue (TURP). In 1992, BPH accounted for an estimated 1.7 million physician office visits and more than 300,000 prostatectomies annually in the United States (Guess, 1992). Although the pathogenesis of BPH is still incompletely understood at the molecular level, morphological features of its origin and evolution at both a gross anatomic level and a microscopic level have been described (McNeal, 1980; McNeal, 1988). Nodules of BPH originate exclusively in a region of the prostate (transition zone and periurethral gland region) located near the proximal urethra (Fig. 3). In the absence of BPH, this region accounts for less than 2% of the total prostate mass, which in the young adult male is about 20 g. With age, pathological processes involving nodule formation, diffuse transition zone enlargement, and nodule enlargement appear, to lead ultimately to the clinical manifestations of the disease (McNeal, 1980; McNeal, 1988).

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As BPH progresses, the excess tissue results in prostatic enlargement and causes urethral obstruction. This obstruction is typically associated with characteristic symptoms such as hesitancy in starting urination, diminished urine stream size and force, involuntary interruptions in stream, and a sensation of incomplete bladder emptying (Claridge, 1966). In men with BPH, the mean decrease in maximum urinary flow rate is about 0.2 ml/s/year (Ball et al., 1981). Because the expansile nodules of BPH originate in a highly localized periurethral region, it is possible for obstruction to occur without much overall increase in total prostate size. Alternatively, diffuse enlargement of the transition zone may produce considerable increase in prostate size without much obstruction. Hence, gross measures of total prostate size have not shown a strong correlation with either symptoms or other clinical measures of obstruction (Guess, 1992), despite the fact that the obstruction is a direct biological consequence of the abnormal growth. Obstruction and agerelated bladder dysfunction also contribute to lower urinary tract symptoms in men (McConnell, 1998). 3. Effects of Reducing DHT Levels in the Prostate In patients with BPH treated with finasteride (1–100 mg/day) for 7 to 10 days prior to prostatectomy, prostatic tissue removed at surgery contained approximately 80% less DHT and 10 times more testosterone, as compared with that for placebo-treated patients (McConnell et al., 1992). In the dog, the only known animal model of spontaneous BPH, treatment with finasteride resulted in a significant reduction in prostate volume, as determined by magnetic resonance imaging (MRI), which was accompanied by a marked decrease in intraprostatic DHT levels (Cohen et al., 1991). Testosterone and DHT normally act in the prostate to stimulate epithelial cell function and inhibit apoptosis (programmed cell death) in the epithelial cells (Wright et al., 1996). Dihydrotestosterone is more potent than testosterone at maintaining normal epithelial cell function. The two androgens are equipotent in preventing epithelial apoptosis. Finasteride reduces prostate volume by suppressing intraprostatic DHT levels, thereby reducing the stimulatory effect of DHT on epithelial cell function and decreasing its protective effect against apoptosis, thereby resulting in a progressive decrease in epithelial cell size and function and transiently increasing the rate of epithelial cell death (Rittmaster et al., 1996; Wright et al., 1996). The minimal increase in epithelial cell death associated with finasteride therapy is thought to be due to the increased

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intraprostatic testosterone levels resulting from inhibition of 5αreductase, which may partially offset the loss of the protective effect of DHT against apoptosis (Wright et al., 1996). The major reduction in prostate volume produced by finasteride occurs in the periurethral zone of the gland (Wilson, 1980; Tempany et al., 1993). Placebo-controlled urodynamic studies in men with BPH have unequivocally demonstrated that finasteride produces objective improvement in bladder outlet obstruction (Tammela and Kontturi, 1993; Kirby et al., 1992; Abrams et al., 1999; Schäffer et al., 1999). Singlecenter studies using detrusor pressure to measure outflow obstruction demonstrated that the majority of finasteride-treated patients were shifted out of the obstructed range as compared with placebo patients over a 3- to 6-month study period (Tammela and Kontturi, 1993; Kirby et al., 1992). Similar results were reported in a 1-year multicenter study in men with BPH (Abrams et al., 1999). Moreover, this study demonstrated that the magnitude of the improvement in bladder outlet obstruction and maximal urinary flow rate with finasteride over placebo increases with increasing prostate volume. Long-term (1-year), openlabel follow-up evaluation for this study demonstrated further reductions in bladder outlet obstruction with chronic finasteride therapy (Schäffer et al., 1999). 4. Phase III Studies The efficacy and safety of finasteride for the treatment of symptomatic BPH were assessed in two 12-month, randomized, doubleblind, placebo-controlled studies in men with BPH, these were a North American study (Gormley et al., 1992) and an international study (Stoner et al., 1993). In the North American study, men receiving finasteride 5 mg/day had a 19% decrease in prostate volume, compared with a 3% decrease in the placebo-treated men. In the international study, prostate volume was reduced by 23% in the finasteride 5 mg/day group, compared with a 5% decrease in the placebo group. Men treated with finasteride 5 mg/day had a significant increase of 1.6 ml/s in the maximum urinary flow rate in the North American study, compared with a slight increase of 0.2 ml/s in men receiving placebo. In the international study, there was a 1.7 ml/s increase in maximum urinary flow rate in the finasteride 5 mg/day group compared with 0.4 ml/s in the placebo group. In both studies, men treated with finasteride 5 mg/day had a significant decrease in total urinary symptoms as compared with men in the placebo group. Safety was evaluated extensively in the two phase III trials. The frequency of adverse effects observed in the 12-month controlled studies

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was remarkably similar in the placebo and finasteride groups, except for a slightly higher incidence (finasteride 5 mg versus placebo) of decreased libido (3.3% versus 1.6%), impotence (3.3% versus 1.6%), and decreased ejaculate volume (2.8% versus 0.9%). The incidence of new drug-related sexual adverse events decreased over time, and in many cases the sexual adverse events resolved while the patients were still receiving finasteride treatment. To examine the long-term efficacy and safety of finasteride 5 mg, eligible patients completing the 12-month placebo-controlled base studies of the North American and international BPH trials continued in 5-year open-label extensions. The results up to the fourth year of the extension for the North American study were recently reported (Hudson et al., 1999). In patients who had taken finasteride 5 mg/day during the entire 5-year period, the reduction in prostate volume demonstrated after the initial year of controlled treatment was maintained over the 4 years of open-label treatment with finasteride 5 mg/day. Moreover, the improvement in maximum urinary flow and urinary symptoms observed in these patients after 1 year continued to increase during the 4-year extension period. These results suggested that chronic treatment with finasteride continued to produce regression of BPH and halted the progression of the disease. Finasteride continued to be generally well tolerated during the 4-year extension. These findings, together with those of the Proscar Long-Term Efficacy and Safety Study (PLESS), confirmed the appropriate use and safety of finasteride for long-term maintenance therapy in men with BPH. 5. Proscar Long-Term Efficacy and Safety Study PLESS is the largest and longest randomized, placebo-controlled trial completed in men with BPH (McConnell et al., 1998). This study enrolled men with moderate to severe urinary symptoms who had an enlarged prostate as determined by digital rectal examination. In agreement with the results of the earlier placebo-controlled trials with finasteride in men with BPH, PLESS demonstrated that finasteride 5 mg/day produced a sustained reduction in prostate volume, improved urinary symptoms, and increased maximal urinary flow rate over a 4year treatment period (Fig. 4). Treatment with finasteride also resulted in a 50% decrease in the relative risk of need for TURP and other BPHrelated surgical procedures and of developing AUR over time, compared with placebo (Figs. 5 and 6). These beneficial effects were seen as early as 4 months into the study and continued throughout the 4-year period. No other medical therapies or minimally invasive surgeries have

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FIG. 4. Effect of finasteride 5 mg or placebo on (A) prostate volume, (B) symptom score, and (C) maximal urinary flow rate over time, stratified in tertiles by baseline prostate-specific antigen (PSA) level. Values are mean percent (panel A) or mean (panels B and C) (± SE) changes from baseline. (Panels A and B from Roehrborn et al., 1999a; panel C from Roehrborn et al., 2000.)

been demonstrated to reduce significantly the incidence of BPHrelated surgery or AUR in long-term studies. Although α-adrenoceptor blocking drugs are also effective in providing symptomatic relief of BPH, a significant reduction in the risk of developing AUR or of needing surgical intervention has not been demonstrated during long-term

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FIG. 5. Four-year incidences of AUR and/or BPH-related surgery in patients treated with placebo or finasteride, stratified in tertiles by baseline serum prostate-specific antigen (PSA) level and baseline prostate volume. (From Roehrborn et al., 1999b.)

treatment with these drugs (Roehrborn et al., 1996; Somers et al., 1996). A decrease in the need for surgery in men with BPH has major public health and economic implications, as TURP is the second most common operation performed in men over age 65 (Oesterling, 1995). The reduction in risk of spontaneous AUR is also important in terms of reducing the morbidity associated with BPH and in decreasing the pool of patients who are at highest risk of negative outcomes with subsequent surgery; AUR is a painful condition, which has been shown to

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FIG. 6. Risk of all AUR and/or BPH-related surgery for patients with placebo or finasteride stratified in tertiles by baseline serum prostate-specific antigen (PSA) level. (From Roehrborn et al., 1999b.)

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increase the morbidity of TURP and other BPH-related surgery (Powell et al., 1980; Malone et al., 1988; Higgins et al., 1991; Pickard et al., 1998). A recent study showed that men with AUR preceeding surgery had increased risk of death after surgery and increased risk of perioperative complications (Pickard et al., 1998). In addition to reducing the incidence of spontaneous AUR, finasteride also significantly decreased the incidence of episodes of AUR that resulted from precipitating factors, such as recent surgical procedures. For physicians treating men with symptomatic BPH in the clinic, the decision regarding appropriate treatment strategies can be a difficult one. As discussed below, recent analyses of data from PLESS and other clinical trial and communitybased studies indicate that prostate volume and serum prostate-specific antigen (PSA) are useful predictors of responsiveness to 5α-reductase inhibitor therapy. 6. Prostate Volume and Serum Prostate-Specific Antigen: Predictors of Responsiveness to 5α-Reductase Inhibitors in Men with Benign Prostatic Hyperplasia Further analysis of PLESS data demonstrated that while a statistically significant reduction in the risk of AUR and need for BPH-related surgery was found, the absolute differences between the finasterideand placebo-treated patients became more pronounced with increasing baseline prostate volume and serum PSA values (Roehrborn et al., 1999b) (Figs. 5 and 6). This was due to the fact that men with higher PSA levels and larger prostates were at increased risk of developing AUR or needing BPH-related surgery in the future and therefore showed the greatest benefit from the 50% reduction in relative risk for these outcomes produced by finasteride. A meta-analysis of all published studies with finasteride in men with BPH, available in 1996, demonstrated that the magnitude of improvement in urinary symptoms and maximal flow rate with finasteride over placebo was dependent on baseline prostate volume (Boyle et al., 1996). An extension of this analysis based on baseline serum PSA predictably demonstrated a similar relationship with an improved benefit of finasteride over placebo seen in men with higher PSA levels (Boyle et al., 1996). Data from PLESS independently confirmed and extended these observations over a 4year period (Roehrborn et al., 1999a, 2000) (Fig. 4). These findings indicate that men with little or no enlargement of their prostate (as indicated by a low serum PSA level) are less likely to experience symptomatic improvement with 5α-reductase inhibitor therapy. Thus, baseline prostate volume and serum PSA levels are of use to physicians in deciding on appropriate treatment options for men with

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symptomatic BPH. Among men with BPH who have been sufficiently screened for prostate cancer, those with larger prostate volumes and/or higher PSA values might be best advised to undertake active treatment rather than to follow a strategy of watchful waiting, since they are at greater risk of experiencing serious outcomes such as AUR or the need for BPH-related surgery. 7. Effect on the Predictive Value of Serum Prostate-Specific Antigen in the Early Detection of Prostate Cancer in Men with Benign Prostatic Hyperplasia In view of the significant reductions in serum PSA produced by finasteride (Gormley et al., 1992; Guess et al., 1993), the question arises as to whether finasteride treatment affects the usefulness of PSA as an early predictor of prostate cancer in men with BPH. The results from studies in such men demonstrated that treatment with finasteride 5 mg/day preserves the usefulness of serum PSA in the detection of prostate cancer (Andriole et al., 1998). Use of an upper limit for PSA of 2.0 ng/ml for finasteride and the customary upper limit of normal of 4.0 ng/ml for placebo yielded similar sensitivity (66% versus 70%) and higher specificity (82% versus 74%) for finasteride compared with placebo. Thus, in order to interpret serum PSA levels in men with BPH who have been treated with finasteride 5 mg/day for 6 months or more, the serum PSA level should be multiplied by 2 and compared with the normal range for serum PSA in untreated men with BPH (Guess et al., 1993; Andriole et al., 1998). 8. Summary The realization that DHT plays an important role in the control of prostate growth led to the discovery of the type 2 5α-reductase inhibitor finasteride as an important new treatment for men with BPH. Finasteride offers a therapeutic option of demonstrated efficacy, whose benefits and risks have been carefully quantified. The symptoms and signs of BPH are, at least in part, due to a slow, progressive increase in the volume of the prostate gland occurring after the fourth decade. Longterm clinical data in men with enlarged prostates show that finasteride produces a sustained reduction in prostate volume, which is associated with sustained improvements in symptoms, urinary flow, urodynamics, and reductions in the risk of serious outcomes such as developing AUR or the need for BPH-related surgery. The magnitude of finasteride’s beneficial effects in men with BPH have been demonstrated to increase with increasing prostate volume and serum PSA levels. For a given patient presenting with BPH in the clinic, the use of prostate volume and serum PSA as predictors enable the physician to more accurately

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assess whether this patient is among those most likely to benefit from treatment with finasteride. B. Prostate Cancer Prostate cancer is the most common form of cancer occurring in men, with approximately 184,500 new cases diagnosed in 1998 (Landis et al., 1998). The chance of developing prostate cancer among men in the United States has been estimated to be 5% to 10%, depending on race and ethnic background (Gaddipati et al., 1987). The prognosis and therapeutic options available depend to a great extent on the clinical stage of the disease at the time of diagnosis. Cancer confined to the prostate is often treated by radical prostatectomy, sometimes with adjuvant therapy (Smith and Armitage, 1987). In some cases, no treatment is recommended because many men with early-stage disease never require therapy (Smith and Armitage, 1987; George, 1988). Once the cancer has spread beyond the capsule of the prostate, radiation therapy, hormonal treatment, or both are often recommended. A landmark study reported by Huggins and Hodges in 1941 clearly established the suppressive effects of castration, and thereby of androgen deprivation, on prostate cancer. Androgen deprivation has been demonstrated to reduce significantly prostate tumor volume and to delay disease progression (Huggins and Hodges, 1941; Huggins et al., 1943; Melamed, 1987; Smith, 1987). However, many patients treated with pharmacologic or surgical castration eventually develop hormonally resistant, androgen-insensitive disease, which can be rapidly progressive and is usually fatal (Isaacs and Coffey, 1981). Because of the significant burden of prostate cancer on society, a strong rationale existed for the development of chemopreventive strategies. Such a strategy requires the exposure of a large number of men to a potential chemopreventive agent over many years in order to benefit the minority of men who potentially could develop prostate cancer. Thus, it is critically important that any pharmacologic intervention have minimal toxicity associated with its long-term use. Type 2 5α-reductase inhibitors produce long-term suppression of DHT formation, an important promoter of prostate growth, with minimal toxicity. In view of this, the National Cancer Institute in the United States has sponsored the Prostate Cancer Prevention Trial, a 9-year, randomized, placebo-controlled study in over 18,000 men, age 55 years and older, evaluating finasteride 5 mg/day as a chemopreventive agent in the development of prostate cancer, which is presently under way (Gormley et al., 1991; Gormley et al., 1995; Feigl et al., 1995; Thompson et al., 1997). It should

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be noted that the 80% decrease in intraprostatic DHT and the 10-fold increase in intraprostatic testosterone levels produced by finasteride therapy results in a net reduction in the overall intraprostatic androgen signal of approximately 50% (McConnell et al., 1992). Confirmation of the theoretical benefit of this reduction for the prevention of prostate cancer awaits completion of the Prostate Cancer Prevention Trial. C. Androgenetic Alopecia 1. The Role of DHT in Scalp Hair Growth Dihydrotestosterone has also been implicated in the pathogenesis of AGA (Imperato-McGinley et al., 1974; Walsh et al., 1974; Price, 1975; Kaufman, 1996; Imperato-McGinley, 1997). In men with AGA, the deleterious effect of DHT on scalp hair is due to a progressive shortening of the growth phase (anagen) duration and the resultant progressive miniaturization of terminal hairs to vellus-like follicles (Fig. 7A). Each hair follicle in the scalp undergoes cycles of activity. Anagen is followed by cessation of growth, termed catagen, during which there is partial involution of the follicle. Then follows a period of telogen, in which there is regrowth of the follicular germinal cells until a new hair starts to form, and the cycle repeats itself with a new period of anagen. The maximal length and thickness of a hair is determined by the rate of activity during anagen and the duration of that period. Androgens, by a poorly understood mechanism, produce a decrease in anagen duration, leading to follicular miniaturization and a reduction in the anagen to telogen ratio. As the number of miniaturized follicles increases, the ratio of terminal to miniaturized follicles decreases, as does the absolute number of follicles (Kligman, 1988; Whiting, 1990, 1993; Kaufman, 1996). The progressive loss of terminal scalp hair is readily perceived by the patient as thinning and eventually, baldness. On histologic biopsy, a perifollicular lymphocytic infiltrate surrounds follicles as they undergo miniaturization. “Streamers,” or remnants of the follicular connective tissue sheath, are seen that are indicative of AGA. In the late stage of AGA, fibrosis is detectable at sites of follicular dropout (Kligman, 1988; Whiting, 1990; Whiting, 1993). 2. Clinical Aspects of Androgenetic Alopecia and Pharmacologic Approaches to Treatment Androgenetic alopecia is a common, genetically determined, androgen-dependent disorder, affecting approximately 50% of men by age 40 (Olsen, 1994). The negative psychosocial impact of this condition, particularly in younger single men, can be significant, and considerable coping

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FIG. 7. Effects of finasteride in androgenetic alopecia. (A) 5α-Reductase–mediated conversion of testosterone (T) to dihydrotestosterone (DHT) leads to miniaturization of scalp hair follicles and eventual hair loss. (B) Finasteride inhibits continued miniaturization of scalp hair follicles and (C) converts miniaturized follicles back to terminal anagen hairs with time, leading to an increase of terminal scalp hair growth and slowing of further hair loss. (Panel A adapted from Randall et al., 1991; panels B and C from Kaufman and Dawber, 1999.)

efforts may be undertaken by the patient to maintain body image integrity (Cash, 1990; Cash et al., 1993; Dawber and Van Neste, 1995). Until recently, the only pharmacologic preparation approved for the treatment of AGA was minoxidil, which is applied topically as a solution

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on the scalp (U.S. product circular for Rogaine, 1996; Olsen et al., 1990). Minoxidil, a potent vasodilator, was originally developed as a systemic therapy for the treatment of severe hypertension and was found to cause hypertrichosis in some patients. Other potential therapies, such as estrogen, progesterone, and oral antiandrogens (spironolactone, flutamide, cyproterone acetate) have been used in female patients with AGA, but their use in men is unacceptable because of their side effects (Simpson and Barth, 1997). In view of the fact that DHT had been implicated in AGA and in light of the potential advancement in the treatment of this disorder that could be provided by a safe oral pharmacologic therapy, finasteride was developed for the treatment of men with AGA. 3. Effects of Reducing Dihydrotestosterone Levels in the Scalp In addition to markedly suppressing serum and intraprostatic DHT levels, finasteride also produces significant suppression (to about 65% below baseline) of scalp DHT (Dallob et al., 1994; Waldstreicher et al., 1994). The parallel decrease in serum and scalp DHT produced by finasteride is presumably facilitated by the dense vascularization of the scalp, providing a large reservoir of circulating androgens that can contribute to the total scalp DHT. Thus, it appears that the effect of finasteride in suppressing scalp DHT is due in part to the concomitant reduction in serum DHT, as well as to inhibition of type 2 5α-reductase located in the root sheaths of scalp hair follicles (Eicheler et al., 1995; Bayne et al., 1999). Complete reduction of DHT does not occur in the scalp owing to the residual level of DHT produced peripherally and locally by type 1 5α-reductase, which is unaffected by finasteride. In an animal model of AGA, the stumptail macaque monkey, finasteride prevented the decrease in scalp hair that occurs in both sexes of this species with puberty, increased scalp hair weight, and corrected the ratio of anagen to telogen follicles induced by pubertal androgens, such that anagen/telogen follicular balance in individual biopsy sections reverted to a more juvenile (prepubertal) pattern (Rhodes et al., 1994). Finasteride can improve androgen-induced scalp hair loss by several mechanisms. By reversing DHT-mediated shortening of anagen, this growing phase is prolonged toward normal, resulting in an increase in hair growth. This normalizing of anagen duration will also decrease hair follicle turnover from anagen to telogen, lessening the rate of shedding and hair loss. Continuous treatment with finasteride produces a sustained suppression of DHT levels, thereby reducing the deleterious effects of DHT on hair follicles. This results in reversal of the continued miniaturization and decline in the number of cosmetically

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important terminal hairs and leads to conversion of miniaturized hairs back to terminal anagen hairs with time, further increasing hair density and slowing additional hair loss (Fig. 7). 4. Phase III Studies The safety and efficacy of finasteride in men with AGA was definitively established in three large phase III multicenter studies. Two of these studies (Pivotal studies) were in men with predominantly vertex AGA and were identical in design, one being conducted in the United States and one conducted in 16 other countries (Pivotal studies; Kaufman et al., 1998). The third phase III study (frontal hair loss study) evaluated men with predominantly anterior mid-scalp hair loss (Leyden et al., 1999). Each of these studies demonstrated the benefit of treatment with finasteride 1 mg/day compared with placebo, based on four efficacy end points: hair counts obtained in a defined, representative area of hair loss; patient self-administrated assessment of hair growth; investigator clinical assessment of hair growth; and blinded assessment of standardized clinical scalp photographs by an expert panel of dermatologists. In the two phase III Pivotal studies, treatment with finasteride 1 mg/day produced clinical benefit compared with placebo based on all predefined end points. The assessment of standardized clinical photographs by the expert review panel demonstrated that 48% of men treated with finasteride had improvement in hair growth at 1 year, compared with 7% of men receiving placebo. The benefit of finasteride was increased further at 2 years: 66% of men experienced improvement in hair growth, compared with only 7% of men receiving placebo (Fig. 8). The improvements in hair growth in finasteride-treated patients demonstrated from the assessment of standardized clinical photographs were associated with improvements in hair density obtained from a 1inch diameter circular target area located at the anterior leading edge of the vertex bald spot (mean baseline hair count ±SE, 876 ± 6.9 hairs). For patients receiving finasteride, there was an 11% mean increase from baseline in hair count at 1 year, and this increase was maintained at 2 years (Fig. 9). In contrast, the placebo group experienced a steady decrease from baseline in hair count over the 2 years. Thus, there was a 14% improvement in hair count with finasteride over placebo at 1 year, and a 16% improvement at 2 years. Only 17% of patients receiving finasteride lost hair by hair count, compared with 72% of patients given placebo, which demonstrates that finasteride slowed the progression of further hair loss. The standardized clinical photographs demonstrated further improvement from baseline for finasteride-treated patients between year 1 and year 2 of treatment, despite hair count being stable

FIG. 8. Percentage of men with change in scalp hair growth at year 2 as determined by assessment of standardized clinical photographs by an expert panel of dermatologists. FIN 1 MG: finasteride 1 mg; PBO: placebo. Error bars are standard errors. (Adapted from Kaufman et al., 1998.)

FIG. 9. Hair count mean change from baseline (± SE) from combined U.S. and international studies for men with vertex pattern hair loss. FIN 1 MG: finasteride 1 mg; PBO: placebo. (From Kaufman et al., 1998.)

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FIG. 10. Percentage of men improved at year 2 based on a patient-assessed hair growth questionnaire. FIN 1 MG: finasteride 1 mg; PBO: placebo. Error bars are standard errors. (Adapted from Kaufman et al., 1998.)

during this period, indicating that finasteride produced a continuous improvement in the quality of hair over the 2 years of observation. The clinical relevance of the improvements observed both by clinical photography and by hair count was further supported by the patient selfadministered hair growth questionnaire, with the majority of patients reporting slowing of further hair loss, increased hair growth, improved appearance of hair, and increased satisfaction with the appearance of their hair at 2 years (Fig. 10). Investigator clinical assessment also confirmed the benefit of finasteride in improving hair growth, although a significant placebo effect was observed in this measure. Significant improvements in the Pivotal studies were seen in the finasteride group as early as 3 months after start of treatment and were generally maintained or improved with continued therapy. The phase III Frontal Hair Loss study was conducted in parallel with the phase III Pivotal studies in order to evaluate the efficacy of finasteride 1 mg primarily in the frontal scalp area (Leyden et al., 1999), as opposed to the vertex. The frontal hair loss study used end points similar to those used in the Pivotal studies and demonstrated significant improvements in all predefined efficacy measures with finasteride as compared with placebo. Treatment with finasteride significantly

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improved anterior midscalp hair counts and led to cosmetic improvement, as assessed by patients, investigators, and the expert review panel. Finasteride 1 mg/day demonstrated an excellent safety profile. The only drug-related adverse events resulting from treatment with finasteride 1 mg in 1% or more of patients were decreased libido (reported by 1.8% of men taking finasteride compared with 1.3% given placebo), erectile dysfunction (reported by 1.3% of men taking finasteride compared with 0.7% receiving placebo) and ejaculation disorder (1.2% of men receiving finasteride compared with 0.7% given placebo). These side effects disappeared in all men who discontinued therapy because of them and resolved in most men who continued taking finasteride. In addition to improving hair growth in younger men (mean age 32 years) with AGA (Kaufman et al., 1998; Leyden et al., 1999), finasteride was recently demonstrated to produce a significant improvement in scalp hair count compared with placebo in older men (mean age 65 years) with AGA (Brenner and Matz, 1999), which indicates that the key role of type 2 5α-reductase and DHT in mediating hair loss in men with AGA is maintained with advancing age. 5. Effect on the Predictive Value of Serum Prostate-Specific Antigen in the Early Detection of Prostate Cancer in Men with Androgenetic Alopecia As anticipated, treatment with finasteride 1 mg/day was demonstrated to cause a significant decrease in serum PSA levels in men with AGA (Kaufman et al., 1998). Therefore, as was the case for men with BPH receiving finasteride 5 mg/day (Guess et al., 1992; Andriole et al., 1998), in order to preserve the usefulness of serum PSA as a predictor in the early detection of prostate cancer in men with AGA treated with finasteride 1 mg/day for 6 months or more, the serum PSA level should be multiplied by 2 and compared with the normal range for serum PSA. 6. Summary Dihydrotestosterone production is a key mechanism producing follicular miniaturization and resultant hair loss in men with AGA. Finasteride represents an entirely new, rationally designed therapy for the treatment of men with AGA, whereby its mechanism of action (selective inhibition of the type 2 5α-reductase enzyme) is targeted at the underlying pathophysiology of the disorder. The oral route of administration enables delivery of drug to the entire scalp. Clinical end point studies show efficacy across a wide spectrum of AGA patients studied—men from 18 to 76 years old—with both anterior midscalp and vertex scalp responding to treatment. The safety and tolerability profile for finasteride 1 mg shows no significant clinical or laboratory adverse effects that would prevent long-term use of the drug.

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V. CLINICAL STUDIES IN WOMEN WITH ANDROGENIC DISORDERS A. Androgenetic Alopecia The underlying pathophysiology of AGA in women is thought to be similar to that in men, whereby DHT induces progressive miniaturization of scalp hair follicles and eventual hair thinning (Olsen, 1994). The hair thinning associated with AGA is generally more diffuse in women than in men and is usually greatest in the frontal-parietal region, with the frontal hairline being retained (Olsen, 1994; Dawber and Van Neste, 1995). This may be due, at least in part, to differences in the relative levels of 5α-reductase, aromatase, and androgen receptors in scalp hair follicles in women as compared with men (Sawaya and Price, 1997). In view of the proposed role of DHT in mediating AGA in women, a 1-year, placebo-controlled study was conducted to evaluate the efficacy of finasteride in slowing the progression of hair loss and increasing hair growth in women with AGA (Roberts et al., 1998). Because finasteride is contraindicated in women who are pregnant or who may become pregnant owing to the potential risk to a developing male fetus (U.S. product circulars for Propecia and Proscar, 1999), this study was limited to postmenopausal women with AGA. Efficacy was evaluated by scalp hair counts, patient and investigator assessments, review of photographs by an expert panel, and histological analysis of scalp biopsies. In contrast to its beneficial effects in men with AGA, finasteride produced no beneficial effects for any of the efficacy measures assessed in the postmenopausal women. However, finasteride produced a significant reduction in serum DHT and a marked reduction in serum levels of the DHT metabolite, androstanediol glucoronide, compared with placebo, indicating inhibition of type 2 5α-reductase. These findings suggest that DHT generated by type 2 5α-reductase does not play an important role in mediating hair loss in postmenopausal women with AGA. It would therefore appear that topical minoxidil is the only available pharmacologic treatment option for these patients. B. Hirsutism Hirsutism in women is characterized by an excess androgen-dependent hair growth distribution pattern, which is normally associated with men. Dihydrotestosterone has also been implicated in hirsutism in women. The activity of 5α-reductase in genital skin of hirsute women has been demonstrated to be higher than that of normal women (Serrafini et al., 1985; Miles et al., 1992). Moreover, a recent study in hirsute women demonstrated a significant correlation between 5α-reductase activity in the skin and hirsutism score (Miles et al., 1992).

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In view of the findings suggesting a role for DHT in hirsutism, a number of studies have examined the effects of finasteride in women with hirsutism (Fruzzetti et al., 1994; Moghetti et al., 1994; Wong et al., 1995; Ciotta et al., 1995; Castello et al., 1996; Tolino et al., 1996; Falsetti et al., 1997; Erenus et al., 1997; Sahin et al., 1998; Faloia et al., 1998; Fruzzetti et al., 1999; Venturoli et al., 1999). Despite the contraindication for finasteride in women who are pregnant or who may become pregnant, these studies were conducted in young, premenopausal women. All of the women participating in these studies were counseled and monitored as to the use of appropriate contraceptive methods during the trials. There were no pregnancies reported during any of the trials. These studies involved a variety of designs including double-blind placebo-controlled, open-label, active comparator, and single-arm studies. All of the studies used 5 to 7.5 mg/day doses of finasteride. The duration of the studies ranged from 3 to 12 months. All of the studies used the investigator-rated Ferriman-Gallwey (Ferriman and Gallway, 1961) or modified Ferriman-Gallwey (Hatch et al., 1981) hirsutism score to assess efficacy. Only four studies (Fruzzetti et al., 1994; Erenus et al., 1997; Sahin et al., 1998; Fruzzetti et al., 1999) stated that the clinician assessing for efficacy was blinded to treatment. Three of the studies (Wong et al., 1995; Falsetti et al., 1997; Venturoli et al., 1999) also used hair diameter measurements to quantitate differences between finasteride- and active comparator-treated patients. Patient self-administered assessment of hirsutism was also a measurement of efficacy in some trials (Wong et al., 1995; Tolino et al., 1996; Castello et al., 1996; Venturoli et al., 1999). In all of the studies, finasteride produce a modest improvement in investigator-rated hirsutism score, which was reported to occur as early as 3 months after the start of treatment. The magnitude of the improvement with finasteride was demonstrated to increase over a 1-year period (Ciotta et al., 1995) and was generally comparable with that produced by other antiandrogenic/estrogenic agents, including spironolactone, flutamide, ketoconazole, and cyproterone/estradiol. In the three studies assessing hair diameter and growth rate, the finasteride treatment groups demonstrated a similar reduction in hair diameter and growth rate as the comparator groups. An improvement with finasteride was also reported for the patient assessment of hirsutism, which was comparable with that for the active comparators. Notably, most of the studies reported no adverse effects in finasteride-treated patients. In the studies that did report adverse events with finasteride, the events were of mild intensity and were self-limited in nature. The lack of clinically significant adverse effects with finasteride was striking, as some of the active comparator drugs produced significant adverse effects, which in some cases prompted discontinuation of treatment.

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FIG. 11. 5α-Reductase inhibitors. (From Harris and Kozarich, 1997.)

VI. OTHER 5α-REDUCTASE INHIBITORS A. Other Type 2 5α-Reductase Inhibitors With the exception of finasteride, no other 5α-reductase inhibitors have yet been approved for clinical use. Several other 5α-reductase inhibitors, including specific inhibitors of the type 2 isozyme, have been synthesized (Fig. 11), some of which have been evaluated in clinical trials (Van Hecken et al., 1994; Levy et al., 1994; Nakayama et al., 1997). It therefore seems likely that other type 2 5α-reductase inhibitors will become available for the treatment of patients with androgen-dependent disorders. B. Type 1 5α-Reductase Inhibitors Several selective inhibitors of type 1 5α-reductase have been developed (Jones et al., 1993; Hirsch et al., 1993; Ellsworth et al., 1996; Schwartz et al., 1997). Early clinical studies with an orally active, selective type 1 5α-reductase inhibitor, MK-386, demonstrated less suppres-

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sion of serum DHT as compared with the type 2 inhibitor finasteride (22% versus 66%) but greater suppression of sebum DHT levels (49% versus 15%) (Schwartz et al., 1997). These findings are in keeping with the tissue localization of the two isozymes, with type 1 5α-reductase being predominant in sebaceous glands (Harris et al., 1992; Russell et al., 1994; Thigpen et al., 1993; Thiboutot et al., 1995). The significant suppression by MK-386 of sebum DHT suggested a possible involvement of type 1 5α-reductase in conditions involving excessive sebum secretion, such as acne. However, there are no published clinical data on the effects of type 1 5α-reductase inhibitors in patients with acne. C. Type 1/Type 2 (Dual) 5α-Reductase Inhibitors Dual inhibition of type 1 and type 2 5α-reductase offers the possibility of added efficacy in the treatment of conditions in which both isozymes contribute to the overall level of tissue DHT. One method to achieve such dual inhibition involves the combined use of selective type 1 and type 2 5α-reductase inhibitors. A study in men treated with a combination of the selective type 1 5α-reductase inhibitor MK-386 together with the type 2 inhibitor finasteride demonstrated nearly complete (90%) suppression of serum DHT (Schwartz et al., 1996). This study confirmed unequivocally that both the type 1 and type 2 isozymes of 5α-reductase contribute significantly to the pool of circulating DHT. In addition to the combined administration of selective inhibitors of type 1 and type 2 5α-reductase, another approach to achieving inhibition of both isozymes involves using compounds with inhibitory activity against both isozymes, so-called dual inhibitors (Bakshi et al., 1995; Kojo et al., 1995; Bramson et al., 1997; Hermann et al., 1996; Hobbs et al., 1998). Of these dual inhibitors, dutasteride (GG745) (Fig 11) appears to be at the most advanced stage of clinical development for the treatment of AGA and BPH (Bramson et al., 1997; Hermann et al., 1996; Hobbs et al., 1998). In a manner similar to that seen with the combination of a type 1 inhibitor and finasteride (Schwartz et al., 1996), dutasteride produced nearly complete (>90%) suppression of serum DHT in healthy male subjects (Bramson et al., 1997; Hermann et al., 1996; Hobbs et al., 1998). The clinical benefits and safety profiles of inhibitors that produce dual inhibition of the type 1 and type 2 5α-reductase isozymes are unclear at present, and their determination awaits completion of clinical studies with these drugs. D. Natural Products with 5α-Reductase Inhibitory Activity A number of natural products reportedly possessing 5α-reductase inhibitory activity have been advocated for use in the treatment of andro-

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genic disorders (Dreikorn et al., 1998). Permixon, a lipid-soluble extract from the fruit of Serenoa repens (also known as Sabal serruta and saw palmetto berry), is reported to have an inhibitory effect on 5α-reductase (Delos et al., 1994). This inhibitory effect is reportedly due to a saponifiable fraction of the extract, the main constituents of which are fatty acids (Weisser et al., 1996). However, a number of other studies with Permixon have produced data that do not support the suggestion that the extract inhibits 5α-reductase (Casarosa et al., 1988; Rhodes et al., 1993; Strauch et al., 1994; Braeckman, 1994). The clinical findings of randomized, double-blind, placebo-controlled studies with Permixon in men with BPH have also been mixed, with some studies demonstrating moderate improvements in urinary symptoms, flow rate, quality of life, and prostate size (Champault et al., 1984; Descotes et al., 1995), whereas other studies have shown no significant benefit over placebo (Reece Smith et al., 1986). Recently, a large, randomized, multicenter study comparing the effects of Permixon with those of finasteride over 6 months of treatment in men with BPH demonstrated a similar level of improvement in urinary symptoms and flow rate with the two treatments (Carraro et al., 1996). However, only the finasteride group demonstrated a significant reduction from baseline in serum PSA and prostate size; the Permixon group had no significant change from baseline in these parameters. These clinical data would appear to further argue against Permixon having a significant inhibitory effect on 5α-reductase. Cactus flower extracts have also been reputed to be of benefit in the treatment of men with BPH, although there are no published studies regarding their clinical effects. A recent study demonstrated such extracts to have 5α-reductase inhibitory activity in cultured foreskin fibroblasts, and also in human placental and prostatic homogenates (Jonas et al., 1998). Cernilton, a pollen extract, has also been reported to have an inhibitory effect on 5α-reductase activity (Tunn and Krieg, 1992) In summary, further studies are needed to fully characterize the effects of natural products reported to inhibit 5α-reductase activity, as well as to define the clinical usefulness of these products in the treatment of BPH and potentially of other androgenic disorders. VII. CONCLUSION The development of the new class of compounds known as 5α-reductase inhibitors has significantly advanced our understanding of androgen biology. Finasteride, a selective inhibitor of the human type 2 5α-reductase enzyme, was the first of this class of compounds in clinical development, and extensive clinical trials have established its usefulness

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in the treatment of BPH and AGA in men. The long-term safety of finasteride has been demonstrated by the absence of chemical or mechanism-based toxicity in preclinical studies and the lack of deleterious effects of chronic DHT suppression in adult men, based on observations in men with genetic deficiency of type 2 5α-reductase. Furthermore, long-term clinical trials and marketed experience with finasteride at the 5-mg dose in the treatment of men with BPH and at the 1-mg dose in the treatment of men with AGA confirm that chronic administration of the drug is generally well tolerated. A number of new 5α-reductase inhibitors presently in development will undoubtedly shed more light on the role of 5α-reductase and DHT in androgen biology and may also prove useful in the treatment of androgenic disorders. ACKNOWLEDGMENT The authors wish to thank Dr. Alan Meehan of Merck & Co., Inc. for his invaluable assistance in preparing this article for publication.

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PEROXISOME PROLIFERATOR-ACTIVATED RECEPTOR (PPAR)γ AGONISTS FOR DIABETES BY DAVID E. MOLLER AND DOUGLAS A. GREENE Departments of Metabolic Disorders and Clinical Development Merck Research Laboratories, Rahway, New Jersey 07065 and Division of Endocrinology and Metabolism, Department of Internal Medicine, University of Michigan Medical School, Ann Arbor, Michigan 48109

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Mechanism of Action of Peroxisome Proliferator–Activated Receptor (PPAR)γ Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Thiazolidinedione Insulin Sensitizers . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. PPARγ Structure and Function . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Spectrum of PPARγ Ligands. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Mechanisms of Insulin Sensitization by PPARγ Agonists . . . . . . . . . . . . . . . E. Additional Proposed Physiologic Functions, Therapeutic Indications, or Adverse Consequences Attributed to PPARγ Activation . . . . . . . . . . . . . III. Clinical Experience with PPARγ Agonists . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Benefits of Glycemic Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Antidiabetic Treatment in Type 2 Diabetes. . . . . . . . . . . . . . . . . . . . . . . . . . C. β-Cell Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Benefits of Treating Insulin Resistance . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Thiazolidinedione PPARγ Agonists Improve Insulin Sensitivity . . . . . . . . . F. Troglitazone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Rosiglitazone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . H. Pioglitazone. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions and Future Directions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Type 2 diabetes accounts for more than 80% of all diabetes. This disorder afflicts an estimated 6% of the adult U.S. population, and approximately 5.4 million cases are undiagnosed. The worldwide prevalence of type 2 diabetes is expected to continue to grow by 3% per annum, reaching an expected total of 210 million cases in 2010. Insulin resistance is a major component of the pathophysiology of type 2 diabetes. Since insulin resistance usually precedes the onset of diabetes, it also represents a well recognized susceptibility trait. Type 2 diabetes and insulin resistance per se are frequently associated with dyslipidemia (i.e., borderline elevation of LDL cholesterol, elevated triglycerides, and low HDL cholesterol), and a markedly increased incidence of atherosclerotic disease (i.e., coronary, cerebral, and peripheral artery disease). Atherosclerotic cardiovascular disease is 181 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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responsible for 80% of diabetic mortality and more than 75% of all hospitalizations for diabetic complications. Increased triglycerides and decreased HDL cholesterol levels are the most common lipid abnormalities in patients with insulin resistance, impaired glucose tolerance, and type 2 diabetes. Results from the U.K. Prospective Diabetes Study (involving 3,867 newly diagnosed type 2 diabetic subjects) have shown that aggressive control of hyperglycemia (with insulin or sulfonylureas) can attenuate complications such as nephropathy and retinopathy. These results provide a compelling rationale for the pursuit of new medicines for type 2 diabetes that will result in greater degrees of glucose control. Importantly, newer agents that also provide for improvements in the lipid profile and a net reduction in cardiovascular risk are also critically needed. II. MECHANISM OF ACTION OF PEROXISOME PROLIFERATOR–ACTIVATED RECEPTORΓ AGONISTS A. Thiazolidinedione Insulin Sensitizers 1. Discovery of the Thiazolidinediones Thiazolidinediones are a recently identified class of insulin-sensitizing antidiabetic drugs. Members of the thiazolidinedione class were first derived from efforts designed to improve the lipid-lowering and (weaker) glucose-lowering properties of the fibrates (1). In general, fibrates are amphipathic carboxylic acids with triglyceride- and cholesterol-lowering activity (2). Included in this class are clofibrate, ciprofibrate, gemfibrozil, bezafibrate, and fenofibrate. Further work on clofibrate gave the peroxisome proliferators their name (3), as these compounds were clearly associated with an increase in size and number of hepatic peroxisomes in rodents. More recent work has established that the mechanism of action of fibrates is largely explained by their activity as agonists for an orphan nuclear receptor referred to as peroxisome proliferator–activated receptor (PPAR) α (4, 5). A wide spectrum of thiazolidinedione compounds have been synthesized and characterized over the past 15 years. In general, these compounds were shown to be active in obese rodent models of type 2 diabetes but were not active in insulin-deficient diabetes, as in streptozotocin-treated rats. Thus, the compounds were shown to be insulin sensitizers with little or no potential to evoke hypoglycemia. In addition to potent insulin-sensitizing and glucose-lowering effects, the thiazolidinediones also showed substantial efficacy with respect to hypertriglyceridemia in animal models. A small number of thiazolidinediones have

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been characterized in more detail and were developed as novel agents for use in human type 2 diabetes. These include troglitazone, pioglitazone, and rosiglitazone (initially known as BRL 49653). 2. PPARγ Is Implicated as a Molecular Target for Thiazolidinediones In a landmark 1992 study, Kletzien et al. (6) discovered that pioglitazone could potently induce the differentiation of cultured 3T3L1 fibroblasts into adipocytes. This effect was common to many of the known thiazolidinediones. Shortly thereafter, Tontonoz et al. also found that the nuclear receptor PPARγ, which was closely related to PPARα, was an important component of an adipocyte transcription factor complex (ARF6) and was sufficient to drive adipocyte differentiation in cultured cells (7, 8). In 1995, Lehmann et al. (9) first reported that thiazolidinediones were high-affinity ligands for and agonists of PPARγ. This observation was also made by several other groups (10). As noted above, there were three key antecedent observations that set the stage for the discovery of PPARγ as a molecular target for thiazolidinediones: (1) thiazolidinediones were derived from fibrates that were known as PPARα activators; (2) thiazolidinediones were potent inducers of adipogenesis; and (3) PPARγ was a mediator of adipogenesis. B. PPARγ Structure and Function 1. PPAR Isoforms—Molecular Biology and Tissue Expression There are three related PPAR family members, namely PPARα, PPARγ, and PPARδ, which are subject to regulation by fatty acids and lipid metabolites. In 1990 Issemann et al. (11) cloned mouse PPARα and identified it as an orphan nuclear receptor, which was activated by peroxisome proliferators, including the fibrates. The DNA sequences for PPARα have now been obtained for several different species, including human (12). PPARα is expressed in liver and other tissues, including muscle and brown fat (13, 14). Its activation stimulates fatty acid oxidation in liver and possibly in muscle. Known PPARα target genes include the enzymes of peroxisome proliferation such as acyl-CoA oxidase, liver fatty acid binding protein, and carnitine palmitoyl transferase Following the cloning of mouse PPARα, three related nuclear receptors were cloned from a Xenopus cDNA library (15). Since all three receptors were capable of activating the acyl-CoA oxidase gene, these receptors were termed PPARα, β, and γ; PPARγ was subsequently cloned from mouse (16), hamster (17), and human (18) cells. There are two PPARγ isoforms, γ1 and γ2, in mouse (19) and human (20), which differ only in that γ2 has an additional 30 N-terminal amino acid units. For the murine gene, the

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two isoforms were shown to be products of alternative promoter use from a single gene located on chromosome 6 E3-F1 (19). The highest level of PPARγ expression is in adipose tissue and differentiated adipocytes (13, 14). Interestingly, differentiated adipocytes express high levels of PPARγ2 mRNA, whereas PPARγ1 is the only isoform expressed in other cell types (21). The mammalian PPAR family also includes a third isoform, referred to as PPARδ (22), which is also known as PPARβ or NUC1 and is expressed in many different cell types and tissues (13, 22). Specific target genes, natural ligands, and bona fide physiologic roles for PPARδ remain to be defined. 2. Role of PPARγ in Transcriptional Regulation Like other nuclear receptors (e.g., steroid hormone receptors, thyroid hormone receptors) the PPARs function as ligand-activated transcription factors. As illustrated in Fig. 1 (see color insert) individual PPARs function as dimers with members of the retinoid X receptor (RXR) family (23). Evidence for an interaction of PPARs with RXRs includes co-expression studies that were performed with yeast lacking endogenous nuclear receptors (24). The PPAR–RXR complex binds to specific DNA response elements (PPREs composed of two hexanucleotide direct repeats) in gene promoters and functions as a transcription factor, which can be activated by either RXR- or PPAR-specific ligands. The consensus site for PPAR-RXR binding is a direct repeat of two -AGGTCA- sequences with a single nucleotide spacer (a DR1 response element). However, DR1 elements may also bind other complexes, including RAR (retinoic acid receptor)/RXR heterodimers and RXR homodimers (25). Further specificity for binding of PPARs may be provided by sequences that flank the DR1 site (26). Following ligand binding, PPARs undergo specific conformational changes, which allow for the recruitment of one or more coactivator proteins such as steroid receptor coactivator 1 (SRC-1) or CREB binding protein (CBP). Coactivators are known to interact with nuclear receptors through a conserved LXXLL motif (where X denotes any amino acid) (27, 28). The receptor–coactivator complex then interacts with components of the basal transcriptional apparatus, allowing for the induction of RNA synthesis (Fig. 1). Recently, coactivators were also shown to mediate their effects via regulation of chromatin structure through histone modification (29). Specific ligand-induced in vitro interactions of PPARγ with SRC-1 (30, 31) and CBP (30) have been demonstrated. However, we have recently observed that PPARγ exhibits a clear preference for CBP over SRC-1 (30). In addition, PBP, for PPARγ-binding protein (a protein related to SRC-1), was cloned using a

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yeast two-hybrid system with Gal4-PPARγ as bait (32). More recently, a novel protein known as PPARγ Coactivator 1 (PGC-1) was cloned from a brown fat cDNA library and shown to interact with PPARγ as a positive regulator of PPARγ-mediated gene transcription (33). PGC-1 may play a key role in regulation of thermogenesis by PPARγ and other signaling pathways. Apart from direct activation via ligand binding, nuclear receptors are also subject to regulation by phosphorylation. Thus, transcriptional activity of PPARγ can be regulated by growth factor stimulation via the mitogen-activated protein (MAP) kinase pathway (34). 3. PPARγ Structural Biology and Mechanism of Receptor Activation Nuclear receptors have a modular structure consisting of six domains named A to F (35). The DNA binding domain (DBD) or C domain and the ligand binding domain (LBD) or E domain are highly conserved. The DBD is composed of two zinc fingers, which recognize specific DNA response elements in gene promoters (36). In PPARγ, as in other nuclear receptors, a critical “activation function domain,” termed AF-2 is located near the C-terminus of the E domain. The AF-2 is both necessary and sufficient for ligand-dependent transcriptional activation. The X-ray crystal structure for the isolated PPARγ LBD was recently determined (37, 38). As depicted in Fig. 2 (see color insert), the structure consists of 13 α-helices and a small, four-stranded β-sheet. Interestingly, the ligand binding pocket of apo-PPARγ is relatively large in comparison with other known nuclear receptor structures; this may account for the diversity of small molecules that appear to function as PPARγ ligands. Importantly, the AF-2 domain is located within helix 12; rosiglitazone was shown to bind with its thiazolidinedione head group, forming a direct contact with helix 12 (37). Moreover, a crystal structure of the ternary complex of rosiglitazone-PPARγ LBD and a fragment of the coactivator SRC-1 reveals that the agonist-bound LBD binds directly to an LXXLL motif of SRC-1 via close contacts between residues in helix 12 and helices 5 and 6 (37). A conformational change in PPARγ following binding of thiazolidinedione agonists could also be demonstrated via changes in the pattern of protease digestion, which are observed when comparing unliganded versus agonist-bound PPARγ LBD protein (10). C. Spectrum of PPARγ Ligands 1. Screening Assays Following the discovery of PPARγ as a probable molecular target for the thiazolidinediones, several approaches have been taken in order to rapidly screen for additional PPARγ ligands or agonists. Initial assays con-

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sisted of cell-based co-transfection of full-length PPARγ with a reporter gene driven by a PPRE-containing promoter (9). In such an assay, compounds with agonist activity will promote activation of the reporter gene (e.g., an increase in luciferase activity). A variation on this theme consists of using chimeric receptors composed of the PPARγ LBD coupled to a heterologous DBD derived from the yeast Gal4 transcription factor. When co-transfected with a Gal4-responsive reporter gene, PPARγ agonists will induce activation of the reporter gene (39). By using radiolabeled thiazolidinedione compounds, direct binding to the PPARγ LBD can also be assessed (9, 40). We also developed a scintillation proximity assay (SPA), which employs scintillant-containing beads to which PPAR LBDs can be coupled, allowing for more rapid and higher throughput detection of bound radioligands (41). Finally, we have also devised a novel high-throughput, cell-free method to detect ligand-induced interactions between nuclear receptor LBDs and selected coactivator proteins using fluorescence resonance energy transfer (30). The techniques described above have been used to search for new PPARγ ligands or agonists that possess greater potency, alternative selectivity profiles, and improved in vivo efficacy. 2. Chemistry of Thiazolidinedione and Nonthiazolidinedione PPARγ Ligands A number of naturally occurring fatty acids and eicosanoids have been implicated as ligands for, or activators of, PPARγ. Among these, the most prominent is 15-deoxy-∆12,14-prostaglandin J2 (15d-PGJ2), a prostaglandin metabolite, which was demonstrated to bind with moderate affinity (2.5 µM) to PPARγ (42, 43). Lower-affinity binding of related prostanoids, PGJ2 and ∆12-PGJ2, was also noted. As it is difficult to measure endogenous levels of 15d-PGJ2, the physiological relevance of this interaction is unclear. In addition to prostanoid metabolites, some evidence indicates that long-chain unsaturated fatty acids composed of more than 18 carbons are also PPARγ ligands. Thus, Forman et al. (5) used an indirect in vitro approach to show activation of mouse PPARγ, with unsaturated fatty acids, including linoleic, α-linolenic, γ-linolenic, and arachidonic acids. More recently, the 15-lipoxygenase-derived metabolites of linoleic acid, 13-HODE, and 9-HODE were also shown to be weak PPARγ agonists (44). In keeping with their high physiological concentrations, fatty acids have been tested at concentrations of 30 µM or above; although a relationship between binding and agonist activity was demonstrated for some fatty acids (5, 45), it has been difficult to demonstrate direct binding because of the apparent low affinity of these ligands. Taken together, current data do suggest

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that PPARγ is activated, as a “lipid sensor” by intracellular long-chain fatty acids. The existence of bona fide high-affinity natural ligands remains to be established. In addition to the thiazolidinediones, synthetic PPARγ agonists include the α-alkoxyphenylpropionates, a parallel chemical class derived from an original lead, AL-294 (40). Both of these classes have led to more potent drug candidates. Rosiglitazone (initially known as BRL-49653) is a potent is PPARγ selective ligand and agonist. In contrast, selected α-alkoxyphenylpropionate compounds, including SB 219994, have potent PPARγ agonist activity (EC50 0.070 µM) and possess additional PPARα agonist activity (EC50 2.5 µM). KRP-297 is a novel thiazolidinedione which activates both PPARγ and PPARα (46). Recently, we also reported the discovery of a novel class of phenylacetic acid derivatives, such as L-796449, which are potent PPARγ agonists with additional PPARδ activity (39). L-764406 is an alternative PPARγ-selective ligand with novel partial agonist activity (41). In addition to these potent synthetic PPARγ ligands, selected nonsteroidal anti-inflammatory drugs (NSAIDs) have been found to weakly activate human PPARγ (and PPARα) in transfection assays. Thus, indomethacin was shown to activate PPARγ at a 10 µM concentration (47). Table I shows the structures and PPARγ binding potency of selected thiazolidinedione and synthetic nonthiazolidinedione compounds. D. Mechanisms of Insulin Sensitization by PPARγ Agonists 1. Correlation Between PPARγ Activation and in Vivo Efficacy by Compounds Several lines of evidence implicate PPARγ activation as the predominant mechanism of action for the thiazolidinedione class of insulin sensitizers. Importantly, the in vivo efficacy of thiazolidinediones in rodents generally correlates with their in vitro PPARγ activity (10, 48). This correlation holds for a much broader range of nonthiazolidinedione PPARγ agonists of several structural types that also exert antihyperglycemic effects in rodent models (39, 49). We have also observed that, unlike compounds with potent PPARγ agonist activity, related compounds with PPARδ or PPARα selectivity were not effective as glucoselowering agents in db/db mice (39). Furthermore, structurally distinct compounds that function as selective RXR ligands can also activate PPARγ/RXR heterodimers and cause in vivo insulin sensitization in rodent models of type 2 diabetes (50, 51). The above findings have firmly established PPARγ activation as a prominent mechanism of insulin-sensitizing effects, which can be mediated by thiazolidinediones and a broader range of synthetic compounds.

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TABLE I Selected Synthetic PPARγ Ligands Structure

PPARγ binding

Troglitazone

7900 nM

Pioglitazone

5500 nM

Rosiglitazone

40–200 nM

L-796449

30 nM

L-764406

60 nM

GW-2570

20 nM

SB 219994

21 nM

2. Alterations in PPARγ Gene Expression or Gene Sequence In order to more firmly establish a role for PPARγ in the regulation of in vivo glucose homeostasis, insulin sensitivity, or adiposity, a number of recent studies have examined potential variation in PPARγ gene expression or the potential for disease-causing PPARγ mutations.

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Thus, subcutaneous adipose tissue PPARγ2, but not PPARγ1, mRNA may be modestly increased in obese versus lean American subjects (52). However, no association between altered PPARγ2 (or PPARγ1) mRNA expression and obesity or type 2 diabetes was evident in a similar study from France (53). Interestingly, one study reported that muscle PPARγ expression correlated with percent body fat and with in vivo insulinstimulated glucose disposal in obese subjects (54). Thus, a potential in vivo role for altered (increased) PPARγ expression as a mediator of increased adiposity and increased insulin sensitivity can be envisioned. Following the characterization of the human PPARγ gene (55), several investigators have reported single nucleotide sequence polymorphisms within its coding exons; however, to date, no significant genetic variants in PPARγ have been reported in nonhuman species. A silent polymorphism (C→T) in the sixth exon common to PPARγ1 and PPARγ2 (56, 57) has been implicated in association with altered circulating leptin levels (56). More importantly, a C→G substitution that encodes the substitution of Ala for Pro at amino acid 12 in PPARγ2 was found to exist with variable allele frequency (from 0.03 to 0.12) in several populations (57) and was associated with obesity in white persons (58). In contrast, no association of Ala12 with increased obesity (or type 2 diabetes) could be detected in other studies of white or Japanese subjects (59, 60). However, Deeb et al. recently reported that the Ala12 allele was associated with lower body mass index and improved insulin sensitivity (61). Although the role of the Pro→Ala12 polymorphism remains controversial, it was recently shown to impair thiazolidinedione-induced adipogenesis in cultured cells (62). A second PPARγ mutation in codon 115, which encodes a Pro→Gln substitution, was recently reported (63). In this case, the Glu115 allele was present in 4 of 121 obese German subjects but was absent in each of 237 normal weight control subjects. Interestingly, the Pro115→Gln substitution appears to result in increased activity of the recombinant expressed protein. Thus, it is possible that this change in PPARγ function (a gain of receptor activity) could result in a phenotype of increased adiposity even in patients bearing only one altered allele. Two independent kindreds with insulin-resistant type 2 diabetes in association with novel PPARγ mutations were recently described (64). In both cases, the mutations—Val→Met290 and Pro→Leu467—were shown to function as dominant-negative proteins when expressed in transfected cells. This compelling story provides further proof of the role of PPARγ as a potential regulator of in vivo insulin action. 3. In Vivo Physiologic Consequences of PPARγ Activation The mechanisms by which activation of PPARγ could promote a net increase in in vivo insulin sensitivity are likely to involve modulated

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TABLE II Summary of Potential Mechanisms for PPARγ-Mediated Insulin Sensitization Defined genes ↑ LPL ↑ PEPCK ↑ aP2, ↑ FATP ↑ acyl-CoA synthase ↑ stearoylCoA desaturase ↑ CD36 ↑ UCP 1 ↓ TNFα

Effects in cells ↑ Insulin stimulated glycogen synthase/glycogen synthesis (adipocyte) ↓ TNF α action-lipolysis (adipocyte) Differentiation of brown/white adipocytes ↑ Insulin-stimulated IRS-1 phosphorylation ↑ Insulin-stimulated Pl-3-kinase

Effects in tissues ↑ Glucose uptake, glycogen synthesis (adipose) ↑ Glucose uptake, glycogen synthase/ synthesis (muscle) ↓ Gluconeogenesis ↓ Glycogenolysis (liver) ↑ IRS Tyrosyl phosphorylation ↑ Pl-3-kinase ↓ Visceral white adipose tissue

Net in vivo effects ↓ FFA ↓ Triglyceride ↓ Hepatic glucose output

↑ Insulin-stimulated glucose disposal

expression of numerous genes and changes in a number of biochemical pathways. Table II provides a summary of some of these effects, which are described below. New insights into the physiologic functions of PPARγ were provided by recent studies conducted with knockout mice. Homozygous PPARγ null mice are embryonic lethal because of defects involving the placenta (65, 66). However, PPARγ null stem cells present in chimeric mice were used to demonstrate that intact expression of PPARγ was required for in vivo adipogenesis (65). Kubota et al. (66) recently described their results with mice heterozygous for a single PPARγ null allele. Although heterozygotes were indistinguishable from wild-type mice on a normal diet, these mice were resistant to high fat diet-induced obesity and insulin resistance (66). The latter finding is difficult to reconcile with overwhelming evidence that increased PPARγ activity promotes insulin sensitization and the fact that human subjects with reduced PPARγ activity (dominant-negative mutants) are insulin-resistant. A number of studies have shown that daily oral administration of PPARγ agonists to obese animal models of insulin resistance and type 2 diabetes (e.g. db/db, ob/ob mice and Zucker fatty rats) results in substantial correction of marked hyperglycemia and/or hyperinsulinemia (10, 67–69). In vivo effects in these models also include substantial lowering of high circulating triglyceride and nonesterified free fatty acid (FFA) levels. Chronic treatment with pioglitazone was also shown to lower glucose, insulin, and triglyceride levels in obese Rhesus monkeys

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(70). When studied under more controlled conditions using the “euglycemic hyperinsulinemic clamp” method, it is apparent that chronic treatment of insulin-resistant rats with PPARγ agonists can substantially improve peripheral insulin-stimulated glucose disposal and the ability of insulin to suppress hepatic glucose production (67, 71, 72). We have observed that suppression of elevated FFA levels is a very rapid (less than 12 hour) response to treatment of insulin-resistant rats with PPARγ agonists (T. Doebber, unpublished data). Since PPARγ agonists are known to promote adipose tissue uptake and storage of fatty acids, it is plausible that this effect constitutes a major mechanism of insulin sensitization, whereby elevated FFAs—a known cause of hepatic and muscle insulin resistance—can be alleviated. An additional effect of in vivo PPARγ activation, shown to occur in rats, was an increase in the number of small white adipocytes, along with a relative shift in the size of visceral (decreased) versus subcutaneous (increased) adipose depots (73). This has important implications because visceral adiposity and larger fat cells are both associated with insulin resistance. Importantly, chronic treatment of insulin-resistant rodents with PPARγ agonists also reverses discrete defects in tissue insulin action. Thus, several studies showed that in vivo treatment of mice or rats resulted in improved insulin-stimulated glucose uptake in adipocytes derived from the treated animals (68, 69, 74, 75). An increase in adipose tissue expression of the GLUT4 insulin-responsive glucose transporter isoform has also been observed (69,74). As skeletal muscle is a prominent site of in vivo glucose disposal, it is important to note that chronic treatment of insulin-resistant rats or mice also affects skeletal muscle insulin sensitivity. This has been shown by measuring improved in vivo glucose uptake into skeletal muscles under euglycemic hyperinsulinemic clamp conditions (71, 72), by improved insulin-mediated glucose uptake into perfused rat hindlimbs (76), and by enhanced in vitro insulin responsiveness of isolated muscles derived from chronically treated animals (72, 77). More specific signaling defects in insulin-resistant skeletal muscle such as reduced tyrosyl phosphorylation of the insulin receptor and its major substrate, IRS-1, as well as reduced phosphatidylinositol-3-kinase (PI-3-K) activity, can also be reversed by chronic treatment with PPARγ agonists (78). 4. Potential Direct Effects on Insulin Signaling and Insulin Action in Cells Although chronic in vivo PPARγ activation promotes insulin sensitization of both muscle and fat, we recently showed that, despite the presence of low levels of PPARγ protein in this tissue, direct in vitro incubation of skeletal muscles with PPARγ agonists does not enhance

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insulin sensitivity (72). In contrast, in vitro incubation of isolated rat adipose tissue with PPARγ agonists can clearly potentiate insulin-stimulated glucose incorporation into glycogen and activation of glycogen synthase within a 2 to 3-hour period (79). These results suggest that white adipose tissue is a primary target organ for PPARγ agonists, whereas effects in skeletal muscle may occur as a secondary consequence of the improved metabolic milieu (e.g., lowering of FFAs and other effects). In contrast, Burant et al. have reported that treatment of an insulin-resistant transgenic mouse model that lacks white adipose tissue with troglitazone was associated with metabolic improvement (80). Several studies have attempted to define specific effects of thiazolidinediones on aspects of insulin-mediated signal transduction in cultured cells. The most consistent results involve an increase in insulin-mediated PI-3-K activation (81, 82). A potential mechanism for enhanced insulin signaling at this level is an increase in insulin stimulation of IRS-1 phosphorylation, as demonstrated by Liu et al. (83). Since direct effects on insulin signaling have not been carefully correlated with in vivo effects or demonstrated with nonthiazolidinedione PPARγ agonists, it is not clear if such effects are PPARγ–mediated or if they really contribute to in vivo efficacy. An intriguing additional mechanism for in vivo insulin-sensitizing efficacy may involve the capacity of PPARγ agonists to attenuate the metabolic effects of tumor necrosis factor (TNF)-α, which is a potential systemic effector of insulin resistance; it is well described as a negative regulator of adipogenesis, and it can promote downregulation of insulin signaling and induce lipolysis in fat cells (84). Incubation of 3T3-L1 adipocytes with PPARγ agonists has been shown to completely prevent the effect of TNF-α to downregulate adipocyte gene expression or insulin-mediated glucose uptake (85). Similarly, PPARγ agonists can prevent the ability of TNF-α to induce lipolysis in 3T3-L1 adipocytes (86). Some in vivo results are consistent with this hypothesis in that pretreatment of normal lean rats with troglitazone prevented an increase in circulating FFAs and insulin resistance induced by TNFα infusion (87). 5. Identification of Defined PPARγTarget Genes At present, only a small number of genes have been described as being directly regulated by the PPARγ–RXR heterodimer in adipose tissue or cultured adipocytes. To date, there are no specific PPARγmediated gene expression changes that have been clearly shown to occur in vivo in tissues other than fat. Among the known genes, the adipocyte fatty acid binding protein, aP2, gene is clearly a direct target

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of PPARγ (7). Others include phosphoenolpyruvate carboxykinase (PEPCK) (88), lipoprotein lipase (LPL) (89), acyl-CoA synthetase (51), and two fatty acid transporters, FATP1 (51) and CD36 (44). One can speculate that a coordinated increase in the expression of genes that favor entry of lipids into white adipose tissue (LPL, CD36, FATP1, and others) would result in a relative decrease in circulating FFAs and thus a decrease in lipid flux into muscle or liver. Such a scenario, if true, would be predicted to result in insulin sensitization. Variable induction of GLUT4 expression (described above) may also contribute to insulin sensitization (69, 74). In addition to inhibition of the metabolic effects of TNF-α, suppression of TNF-α gene expression in white adipose tissue has been reported (90). Finally, we and others have shown that in brown adipose tissue, PPARγ agonist treatment can induce the expression of mitochondrial uncoupling proteins (91). This effect could act to attenuate a net increase in body adiposity, which might be favored by anabolic effects in white adipose cells. Although the list of PPARγ-regulated genes is still expanding, there is still no comprehensive model available to account for the insulin-sensitizing effects. Indeed, PPARγ-mediated induction of LPL gene expression is perhaps the only defined gene that can be clearly linked to a discrete in vivo metabolic effect—in this case, lowering of circulating triglyceride levels (Table II) (89). E. Additional Proposed Physiologic Functions, Therapeutic Indications, or Adverse Consequences Attributed to PPARγ Activation 1. Inflammation and Atheroclerosis Recently, studies have suggested that PPARγ may function as a negative regulator of macrophage activation (92) and inhibit the production of inflammatory cytokines in monocytes (93,94). Although these initial reports suggested that activation of PPARγ was able to inhibit the release of cytokines by activated macrophages, this conclusion was largely based on results obtained by using 15-deoxy-∆12,14-prostaglandin J2. In our own studies performed with a wider spectrum of thiazolidinedione and nonthiazolidinedione PPARγ ligands, we have failed to see effects of potent agonists to inhibit IL-6 or TNFα release from macrophages in vitro or in vivo (95). Despite conflicting data regarding anti-inflammatory effects in macrophages, it is now clear that PPARγ expression is induced during macrophage differentiation and that PPARγ is expressed in cells within atherosclerotic lesions (96). As noted above, CD36 is a probable PPARγ target gene. Since CD36 is a potential “scavenger receptor,” which

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could mediate cholesterol accumulation in foam cells, and since natural ligands for PPARγ may include 13-hydroxyoctadecadienoic acid (13-HODE) and 9-HODE, which exist within oxidized LDL particles, some have speculated that in vivo activation of PPARγ might produce an increase in atherosclerosis (44). This concept is counterbalanced by several lines of evidence suggesting that the net effects of PPARγ activation will favor a decrease in atherosclerosis: (1) PPARγ agonists have been shown to inhibit vascular smooth muscle cell proliferation and migration (97, 98); (2) PPARγ agonists inhibit vascular cell expression of matrix metalloproteinase 9, a protein implicated in plaque rupture (98); (3) PPARγ agonists inhibit adhesion molecule (ICAM-1 and VCAM-1) expression in vascular endothelial cells (99); (4) in vivo treatment of atherosclerosis-prone mice was associated with reduced “homing” of monocyte macrophages to vascular lesions (99); (5) chronic treatment of other atherosclerosis models (Wantanabe rabbits and cholesterol-fed hamsters) with troglitazone or pioglitazone produced a net reduction in vascular lesions (100). 2. Cancer Relatively high levels of PPARγ have been reported in the colon (101). Since cyclooxygenase inhibitors are known to reduce colon cancer risk (102), it is possible that the production of prostaglandins and other eicosanoids via cyclooxygenase might act to promote tumor formation by providing higher levels of natural PPARγ ligands. In attempting to address this hypothesis, Lefebvre et al. noted that chronic treatment of Min mice (a murine model of inherited polyposis) with high doses of two thiazolidinedione PPARγ agonists caused a modest increase in colon tumor number (103). In contrast, other studies have reportedly shown that growth of fully transformed human colon cancer cells can be inhibited by incubation with these same PPARγ-selective agonist compounds (101, 104). Thus, there is no firm evidence to support a pathophysiologic role for PPARγ in colon tumor formation. Thiazolidinedione PPARγ agonists have also been reported to induce terminal differentiation of malignant breast epithelial cells (105), which suggests that PPARγ activation could be used in the treatment of breast cancer. Recently, it was also shown that ligands for PPARγ are inhibitors of angiogenesis (106); however, the dose–response characteristics in this study do not agree with those for receptor binding and activation (9). 3. Additional Physiologic or Pathophysiologic Effects Administration of high dose of thiazolidinedione PPARγ agonists to animals has reportedly resulted in a number distinct toxic effects, such

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as cardiac enlargement, volume expansion, anemia, and proliferative and/or degenerative changes involving adipose tissue (107). With the exception of the effects in adipose tissue, none of these effects are clearly established consequences of PPARγ activation per se. Nevertheless, it remains possible that certain disease processes that affect these organ systems may ultimately be shown to be due, at least in part, to pathophysiologic mechanisms involving acquired or genetic changes in PPARγ expression or action. III. CLINICAL EXPERIENCE WITH PPARγ AGONISTS In contrast to type 1, or insulin-dependent, diabetes mellitus, in which insulin deficiency is the primary underlying cause of hyperglycemia, type 2 diabetes is pathophysiologically complex, multifactorial, and phenotypically heterogeneous (108, 109). Factors contributing to hyperglycemia include excess hepatic glucose production, impaired insulin secretion by pancreatic β cells, and insulin resistance in peripheral target tissue (e.g., liver, adipose, and muscle) (110). Overproduction of glucose by the liver is characteristic of type 2 diabetes and the primary cause of fasting hyperglycemia. Increased levels of glucagon and FFAs also contribute to increased hepatic glucose output in type 2 diabetes, as does hepatic insulin resistance (111). Clinically, insulin resistance is said to occur when normal circulating concentrations of plasma insulin produce a subnormal biologic response in an intact patient (112). Clinical studies point to insulin resistance in peripheral target tissue as the primary metabolic defect in type 2 diabetes (111). First, 80 to 90% of insulin-stimulated glucose uptake occurs in skeletal muscle, and patients with type 2 diabetes demonstrate a 60 to 80% deficiency in this action of insulin. Second, insulin resistance can be detected long before glucose tolerance deteriorates and often when insulin secretion is actually increased. Thus, insulin resistance and hyperinsulinemia often precede the development of type 2 diabetes and can be identified in most prediabetic individuals. Insulin resistance often worsens as the disease progresses because of the dysregulation of lipid and carbohydrate metabolism in type 2 diabetes (111). Importantly, epidemiologic and clinical studies point to a connection between insulin resistance and other important consequences or concomitants of hyperglycemia in type 2 diabetes, including dyslipidemia, hypertension, obesity, and atherosclerosis. These metabolic disorders are common comorbidities of type 2 diabetes; this cluster of disorders is known as the insulin resistance syndrome (113).

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A. Benefits of Glycemic Control There is little debate today regarding the benefits of tight glycemic control in type 2 diabetes. Results of the landmark United Kingdom Prospective Diabetes Study demonstrate conclusively that intensive glycemic control significantly influences the development of many of the destructive effects of type 2 diabetes. Over a 10-year period, for example, a reduction of 11% in the HbA1c of patients receiving intensive therapy was associated with a 25% reduction in microvascular complications (114). B. Antidiabetic Treatment in Type 2 Diabetes Life-style changes designed to diminish insulin resistance are a mainstay of antidiabetic therapy in type 2 diabetes, although most patients also require antidiabetic pharmacological therapy as well. Moderate exercise, weight loss, and a healthy diet low in simple sugars and saturated fats are generally recommended. Several different classes of antidiabetic pharmacological agents are available as adjuncts to lifestyle therapy to correct the metabolic abnormalities associated with type 2 diabetes. Although type 2 diabetes is marked by relative insulin resistance, insulin in pharmacological doses is effective in improving hyperglycemia. Insulin secretagogues such as the sulfonylureas augment β-cell insulin secretion by interacting with a specific sulfonylurea receptor and directly inhibiting the ATP-sensitive K+ channel, depolorizing the cell, and stimulating insulin release. However, these agents have little effect on increased hepatic glucose production and peripheral insulin resistance (110), and patients may lose responsiveness to insulin secretogogues as secondary β-cell failure occurs with advancing disease (see below). Metformin, a member of the biguanide class of agents, primarily suppresses hepatic glucose production and indirectly improves insulin sensitivity. Acarbose is an α-glucosidase inhibitor, which blocks hydrolysis of oligosaccharides and thus limits the rate of postprandial glucose absorption. C. β-Cell Failure β-Cells in the pancreas normally respond to peripheral insulin resistance by increasing basal and stimulated insulin secretion to compensate for the insulin-resistant state. Initially, this compensation maintains normal or impaired glucose tolerance in the prediabetic insulin-resistant state and delays deterioration of glucose homeostasis and type 2 diabetes. In patients destined to develop type 2 diabetes, β-cells can no longer com-

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pensate for insulin resistance by secreting increased amounts of insulin, and glucose-induced insulin secretion falls. This leads to fasting hyperglycemia and the development of frank diabetes, followed by resistance to the effects of insulin secretogogues (111). D. Benefits of Treating Insulin Resistance Many experts believe that treating insulin resistance in the prediabetic state or after type 2 diabetes has become manifest may prevent or delay the development and progression of type 2 diabetes (111). Because insulin resistance is an underlying factor in β-cell failure and in other concomitants of type 2 diabetes such as obesity, dyslipidemia, and hypertension, early resolution of insulin resistance in the at-risk population may also reduce critical cardiovascular risk factors and thus prevent the development of late arterial and renal complications (115). E. Thiazolidinedione PPARγ Agonists Improve Insulin Sensitivity Clinical experience with PPARγ agonists is largely confined to the thiazolidinedione class of compounds (Table I). Representative agents in this class include troglitazone, rosiglitazone, and pioglitazone (116). The thiazolidinediones improve insulin resistance by enhancing insulin sensitivity in skeletal muscle, liver, and adipose tissue, although as discussed above, the precise mechanism of action of these drugs is not yet known (113). As reviewed above, euglycemic-hyperinsulinemic clamp studies in various rodent models of insulin resistance have investigated the effects of thiazolidinediones on insulin sensitivity. The ability of insulin to suppress hepatic glucose output and increase glucose disposal in peripheral tissues was restored following treatment with thiazolidinediones. These metabolic effects were accompanied by a vast improvement in insulin sensitivity in isolated fat and muscle tissue, resulting in a normalization of glucose and insulin levels and thereby preventing the progression to diabetes (111). F. Troglitazone Troglitazone, the first clinically available thiazolidinedione, was approved for use in patients who have failed diet therapy and, in combination with insulin and/or sulfonylureas, in patients inadequately controlled with these agents alone (117). Although studies have documented the hypoglycemic efficacy of troglitazone in patients with type 2 diabetes, its use was associated with elevated liver enzymes, liver damage, and death

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secondary to liver failure (117). Troglitazone was subsequently withdrawn from the U.S. market because of associated hepatotoxicity. G. Rosiglitazone Rosiglitazone is currently the most widely prescribed thiazolidinedione compound and is considered a model for the class. Rosiglitazone exhibits insulin-sensitizing activity manyfold higher than that of troglitazone or pioglitazone (118), which is consistent with its significantly higher affinity for PPARγ (119) (Table I). The results of rosiglitazone in clinical trials have confirmed many preclinical findings and observations. Rosiglitazone is currently believed to be safe and effective in the treatment of type 2 diabetes and to have significant advantages over troglitazone with regard to hepatotoxicity. 1. Phase 2 Studies The first clinical efficacy study of rosiglitazone was a 12-week, double-blind, multicenter trial conducted in 380 patients with type 2 diabetes and a fasting plasma glucose (FPG) of 140 to 240 mg/dl (108, 120). This dose-ranging study explored total daily doses of rosiglitazone ranging from 0.1 to 4 mg. The drug was administered twice daily. Mean results are summarized in Table III (108). Rosiglitazone exhibited a clear dose–response relation. Although the top of the dose–response curve may not have been attained in this study, the minimally effective dose was 2 mg/day. This magnitude of response in patients receiving 4 mg/day was similar to the response observed with troglitazone 800 mg/day (108, 121). These results also demonstrate that rosiglitazone significantly improved glycemic control without an accompanying increase in fasting insulin. The treatTABLE III Effect of Four Rosiglitazone Doses on Glycemic Parameters (108, 120) Total daily dose (mg)

FPGa (mg/dl)

0.1 0.5 2 4

–2.4 1.1 –29.4b –40.1b

FPG, fasting plasma glucose. p <.001. c FPG <140 mg/dl.

a b

% Normalizedc

% Reduction >40 mg/dl

Insulin (mU/liter)

8.6 9.7 15.2 25.0

9.5 11.1 27.8 38.8

–1.6 –0.1 –0.5 –2.7

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ments were safe and well tolerated, with no significant differences between treatment groups in the adverse events reported (108). There was a small but clinically insignificant decrease in hemoglobin and hematocrit in rosiglitazone-treated patients (120). With regard to the reversible cardiac hypertrophy demonstrated in preclinical studies, echocardiography in these patients showed no evidence of increased left ventricular mass (120). Food had no demonstrable effect on the overall bioavailability of this agent (122). 2. Phase 3 Studies The safety and efficacy of rosiglitazone as monotherapy were assessed in a multicenter, placebo-controlled study in 493 patients with type 2 diabetes who had failed to achieve glycemic control with either diet or pharmacologic therapy (FPG between 140 and 300 mg/dl) (123). Patients were randomly assigned to receive either placebo or a total daily dose of rosiglitazone 4 or 8 mg administered in divided doses twice daily for 26 weeks. The results are summarized in Table IV. In this study, rosiglitazone had a clinically and highly statistically significant glucose-lowering effect compared with baseline and placebo. Overall, the proportion of patients reporting adverse experiences was comparable between treatment groups. There was a small dose-dependent decrease in hemoglobin and hematocrit, and there were no serious adverse experiences related to the liver or hypoglycemia. TABLE IV Efficacy of 4- and 8-mg/day Doses of Rosiglitazone (108, 123) Rosiglitazone Placebo (n = 158) FPG (mg/dl)a Baseline Mean ∆ from baseline (SE) Comparison with placebob 95% CIc Glycosylated hemoglobin (HbAlc) (%) Mean ∆ from baseline (SE) Comparison with placebob 95% CI FPG = fasting plasma glucose Adjusted mean difference. c 9.5% CI, 95% confidence interval d p <.0001. a b

228.8 18.9 (5.1) – – 9.04 0.92 (0.1) – –

4 mg (n = 166)

8 mg (n = 169)

226.9 –38.4 (4.1) –57.7d (–70.9, –44.6) 9.02 –0.28 (0.1) –1.21d (–1.52, –0.89)

219.7 –54.0 (3.9) –76d (–89.2, –62.9) 8.75 –0.56 (0.1) –1.54d (–1.85, –1.22)

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TABLE V Efficacy of 4- and 8-mg/day Doses of Rosiglitazone Combined with Sulfonylureas (108, 124) Sulfonylurea + Sulfonylurea + Sulfonylurea alone Rosiglitazone Rosiglitazone 4 (n = 192) 2 mg/day (n = 199) mg/day (n = 183) HbAlc (%)a Mean baseline Mean ∆ from baseline ± SD Difference from SU aloneb % with reduction ≥ 0.7% FPG (mg/dl)c Mean baseline Mean ∆ from baseline ± SD Difference from SU alone % with reduction ≥ 30 mg/dl

9.2 + 0.2 ± 1.11 NA 19%

9.2 –0.5d ± 1.05 –0.6d 39e

9.2 –0.9d ± 1.10 –1.0d 60%e

207.3 +5.8 ± 49.4 NA 21%

203.7 –17.1d±48.5 –24.3d 38%e

205.4 –37.7d±47.2 –43.9d 56%e

HbAlc, glycosylated hemoglobin. SU = sulfonylurea. c FPG, fatty plasma glucose. d p <.0001. e p <.0001 vs. placebo. a b

3. Combination Studies with Rosiglitazone Plus Sulfonylurea, Metformin or Insulin Stringent diabetic control mandated by the results of the United Kingdom Prospective Diabetes Study often requires therapy with multiple pharmacological agents in combination (114). The efficacy of low doses of rosiglitazone added to existing sulfonylurea therapy was assessed in 574 patients with type 2 diabetes in a randomized, doubleblind, placebo-controlled trial (108, 124). Patients had taken gliclazide, glibenclamide, or glipizide for 6 months or longer prior to entry in the study. They were randomized to receive rosiglitazone 2 or 4 mg or placebo daily in two divided doses or placebo in combination with their sulfonylurea. After 6 months of treatment, rosiglitazone produced clinically and statistically significant reductions in glycosylated hemoglobin (HbA1c) and FPG, with more pronounced effects at the 4 mg/day dose (Table V) (108). Improvements in HbA1c were the same for all three sulfonylureas. Decreases in FFAs and increases in high-density lipoprotein (HDL) and low-density lipoprotein (LDL) cholesterol were also observed with the 4 mg/day dose of rosiglitazone. The combination treatment was safe and well tolerated, with no significant hepatotoxicity or hypoglycemia reported (108).

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The effect of combination metformin and rosiglitazone treatment on glycemic control and lipid levels was assessed in a randomized, placebocontrolled, 26-week study (125). Only patients who were not well controlled despite maximum therapy with metformin (2.5g/day) (N = 348) were entered into the study. These subjects were randomly assigned to receive placebo or rosiglitazone 4 or 8 mg once daily in combination with metformin 2.5 g/day for 26 weeks. In both the 4 and 8 mg rosiglitazone groups, metformin plus rosiglitazone therapy produced clinically and statistically significant improvements in FPG and HbA1c without an increase in endogenous insulin production as compared with metformin plus placebo. In addition, 30% and 22% of patients receiving metformin plus rosiglitazone 8 and 4 mg, respectively, achieved the predetermined goal of FPG below 140 mg/dl, compared with 8% of those receiving metformin plus placebo. There was no change in the total cholesterol to HDL, cholesterol ratio in any group, whereas LDL cholesterol increased slightly in all treatment groups. Free fatty acids also decreased significantly in both rosiglitazone plus metformin treatment groups. No patients had alanine aminotransferase levels more than three times the upper limit of normal (108, 125). Although most patients with type 2 diabetes are expected to ultimately require insulin to control their disease, combination with oral medications may obviate the need for nonphysiologically large doses of insulin. This combination may improve glycemic control as well as minimize many of the undesirable side effects of insulin therapy, including weight gain and hyperinsulinemia (126). The safety and efficacy of combination therapy with rosiglitazone and insulin was investigated in a 26-week placebo-controlled trial in 319 patients with type 2 diabetes inadequately controlled on twice-daily insulin. Patients were randomly assigned to additional treatment with placebo or rosiglitazone 2 or 4 mg administered in divided doses twice daily for 26 weeks. Glycemic control improved significantly in patients receiving insulin plus rosiglitazone 2 or 4 mg, which allowed a reduction in insulin dose (Table VI) (108). More than 50% of patients receiving insulin plus rosiglitazone had a decrease in HbA1c of more than 1%. In the insulin plus rosiglitazone treatment groups, FFAs also decreased significantly. There was little change in total cholesterol to HDL cholesterol ratios. In general, the treatments were well tolerated, and no patients had alanine aminotransferase levels more than three times the upper limit of normal (127). 4. Safety Studies conducted to date indicate that rosiglitazone is safe and well tolerated in appropriately selected patients (120, 123). The effect of

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TABLE VI Efficacy of 4- and 8-mg/day Doses of Rosiglitazone in Combination with Insulin (108,127)

HbA1c (%) a Baseline Change from baseline Insulin dose (units) Baseline Change from baseline a b

Insulin + Placebo (n = 103)

Insulin + Rosiglitazone 2 mg bid (n = 106)

Insulin + Rosiglitazone 4 mg bid (n = 103)

8.9 ± 1.1 0.1 ± 1.0

9.1 ± 1.3 –0.6 ± 1.1b

9.0 ± 1.3 –1.2 ± 1.1b

70.1 ± 30.4 –0.4 ± 5.6

71.3 ± 43.8 –4.8 ± 14.6b

77.7 ± 36.4 –9.4 ± 16.7b

HbA1c = glycosylated hemoglobin. p <.006 vs. insulin + placebo.

rosiglitazone on liver function is being closely monitored because of the hepatotoxicity reported with the earlier thiazolidinedione troglitazone (117). In clinical trials conducted to date, however, there have been no reports of clinically significant elevations in liver enzymes or hepatic injury associated with administration of rosiglitazone (120, 123). Adverse experiences associated with rosiglitazone consist primarily of an increased frequency of mild lower extremity edema, mild weight gain, and mild reduction in hemoglobin and hematocrit. H. Pioglitazone Parallel results have been obtained in similarly designed studies with pioglitazone, the other thiazolidinedione approved in the United States. Pioglitazone in monotherapy exhibits a dose response between 7.5 and 45 mg, with the minimally effective dose appearing to be 15 mg daily (128). A 16-week randomized placebo-controlled monotherapy study with pioglitazone 30 mg once daily produced lowering of FPG and HbA1c in a range similar to that of the higher doses of rosiglitazone, with an additional benefit of reducing triglycerides (129). A similar pattern was demonstrated when pioglitazone 30 mg was used in combination with sulfonylurea (130, 131), metformin (132, 133), and insulin (134). Head-to-head comparisons between the two thiazolidinediones have not been reported. The safety profile of pioglitazone appears similar to that of rosiglitazone, with no reported cases of hepatotoxicity, and with mild adverse experiences of weight gain, edema, and falling hemoglobin and hematocrit.

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IV. CONCLUSIONS AND FUTURE DIRECTIONS The discovery of PPARγ as a major molecular target of the thiazolidinedione insulin sensitizers has sparked an all-out race to identify improved compounds that act through this mechanism to enhance insulin sensitivity and to treat type 2 diabetes and its complications. Since severe hepatotoxicity has been seen in humans with troglitazone, a desired goal of ongoing research is to derive more selective and potent PPARγ ligands that are structurally distinct from the thiazolidinedione platform. Given that PPARs function much like other nuclear receptors, it may also be possible to develop a wider spectrum of compounds that act as PPARγ ligands but exert alternative in vivo biological effects (e.g. tissue selectivity). This concept has been clearly validated in the estrogen receptor system, in which binding of partial agonists such as tamoxifen is associated with alternative receptor conformations, different profiles of receptor-associated cofactors, and selective biological responses versus estradiol (135). REFERENCES 1. Devchand, P. R., Keller, H., Peters, J. M., Vazquez, M., Gonzalez, F. J., and Wahli, W. (1996). The PPAR alpha-Leukotriene B4 Pathway to Inflammation Control. Nature 384, 39–43. 2. Thorp, J. M., and Waring, W. S. (1962). Modification of metabolism and distribution of lipids by ethylchlorophenoxyisobutyrate. Nature 194, 948–949. 3. Hess, R., Staubli, W., and Riess, W. (1965). Nature of the Hepatomegalic Effect Produced by Ethyl-Chlorophenoxy-Isobutyrate in the Rat. Nature 208, 856–858. 4. Lalwani, N. D., Alvares, K., Reddy, M. K., Reddy, M. N., Parikh, I., and Reddy, J. K. (1987). Peroxisome proliferator-Binding Protein: Identification and Partial Characterization of Nafenopin-Clofibric Acid-, and Ciprofibrate-Binding Proteins from Rat liver. Proc. Natl. Acad. Sci. U.S.A. 84, 5242–5246. 5. Forman, B. M., Chen, J., and Evans, R. M. (1997). Hypolipidemic Drugs, Polyunsaturated Fatty Acids, and Eicosanoids Are Ligands for Peroxisome Proliferator-Activated Receptors Alpha and Delta. Proc. Natl. Acad. Sci. U.S.A. 94, 4312–4317. 6. Kletzien, R. F., Clarke, S. D., and Ulrich, R. G. (1992). Enhancement of Adipocyte Differentiation by an Insulin-Sensitizing Agent. Mol. Pharmacol. 41, 393–398. 7. Tontonoz, P., Graves, R. A., Budavari, A. I., Erdjument-Bromage, H., Lui, M., Hu, E., Tempst, P., and Spiegelman, B. M. (1994). Adipocyte-Specific Transcription Factor ARF6 Is a Heterodimeric Complex of Two Nuclear Hormone Receptors, PPAR Gamma and RXR Alpha. Nucleic Acids Res. 22, 5628–5634. 8. Tontonoz, P., Hu, E., and Spiegelman, B. M. (1994). Stimulation of Adipogenesis in Fibroblasts by PPAR Gamma 2, a Lipid-Activated Transcription Factor. Cell 79, 1147–1156. 9. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A., Wilkison, W. O., Willson, T. M., and Kliewer, S. A. (1995). An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-Activated Receptor Gamma (PPAR Gamma). J. Biol. Chem. 270, 12953–12956.

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113. Opara, J. U., and Levine, J. H. (1997). The Deadly Quartet—the Insulin Resistance Syndrome. South. Med. J. 90, 1162–1168. 114. UK Prospective Diabetes Study (UKPDS) Group. (1998). Intensive Blood-Glucose Control with Sulphonylureas or Insulin Compared with Conventional Treatment and Risk of Complications in Patients with Type 2 Diabetes (UKPDS 33). Lancet 352, 837–853. 115. Buckingham, R. E., Al-Barazanji, K. A., Toseland, C. D. N. et al. (1998). Peroxisome Proliferator-Activated Receptor-γ Agonist, Rosiglitazone, Protects Against Nephropathy and Pancreatic Islet Abnormalities in Zucker Fatty Rrts. Diabetes 47, 1326–1334. 116. Whitcomb, R. W., and Saltiel, A. R. (1995). Thiazolidinediones. Expert Opin. Invest. Drugs. 4, 1299–1309. 117. Parke-Davis Pharmaceuticals. (1998). Rezulin package insert. 118. Cantello, B. C. C., Cawthorne, M. A., Haigh, D. et al. (1994). The Synthesis of BRL 49653—a Novel and Potent Antihyperglycemic Agent. Bioorg. Med. Chem. Lett. 4, 1181–1184. 119. Lehmann, J. M., Moore, L. B., Smith-Oliver, T. A. et al. (1995). An Antidiabetic Thiazolidinedione Is a High Affinity Ligand for Peroxisome Proliferator-Activated Receptor γ (PPARγ). J. Biol. Chem. 270, 12953–12956. 120. Patel, J., Anderson, R. J., and Rappaport, E. B. (1999). Rosiglitazone Monotherapy: Improves Glycemic Control in Patients with Type 2 Diabetes: A 12-Week Randomized, Placebo-Controlled Study. Diabetes Obesity Metab. 1, 165–172. 121. Kumar, S., Boulton, A. J. M., Beck-Nielsen H. et al. (1996). Troglitazone, an Insulin Action Enhancer, Improves Metabolic Control in NIDDM Patients. Diabetologia 39, 701–709. 122. Freed, M. I., Allen, A., Jorkasky, D. K. et al. (1999). The Bioavailability of Rosiglitazone Is Unaltered by Food. Eur. J. Clin. Pharmacol. 55, 53–56. 123. Patel, J., Miller, E., and Patwardhan, R. (1998). The Rosiglitazone 011 Study Group. Rosiglitazone (BRL 49653) Monotherapy Has Significant Glucose Lowering Effect in Type 2 Diabetic Patients (Abstract 0067). Diabetes 47, (Suppl. 1), A17. 124. Gomis, R, Jones, N. P., Vallance, S. E., and Patwardhan, R. (1999). Low Dose Rosiglitazone Provides Additional Glycaemic Control When Combined with Sulfonylureas in Type 2 Diabetes (Abstract). Diabetes 48, (Suppl. 1), A63. 125. Fonseca, V., Biswas, N., and Salzman, A. (1999). Once Daily Rosiglitazone in Combination with Metformin Effectively Reduces Hyperglycemia in Patients with Type 2 Diabetes. (Abstract), Diabetes 48, (Suppl. 1), A100. 126. Schwartz, S., Raskin, P., Fonseca, V. et al. (1998). Effect of Troglitazone in InsulinTreated Patients with Type II Diabetes Mellitus. N. Engl. J. Med. 338, 861–866. 127. Raskin, P., Dole, J. F., and Rappaport, E. B. (1999). Rosiglitazone Improves Glycemic Control in Poorly Controlled, Insulin-Treated Type 2 Diabetes (Abstract) Diabetes 48, (Suppl. 1), A94. 128. Schneider, R., Lessem, J., and Lekich, R. (1999). Pioglitazone is Effective in the Treatment of Patients with Type 2 Diabetes (Abstract 469). Diabetes 48, (Suppl, 1), A109. 129. Mathisen, A., Geerlof, J., Houser, V., and Pioglitazone 026 Study Group. (1999). The Effect of Pioglitazone on Glucose Control and Lipid Profile in Patients with Type 2 Diabetes (Abstract 441). Diabetes 48, (Suppl. 1), A102. 130. Mathisen, A., Egan, J., Schneider, R., and Pioglitazone 010 Study Group. (1999). The Effect of Combination Therapy with Pioglitazone and Sulfonylurea on the Lipid Profile in Patients with Type 2 Diabetes (Abstract 457). Diabetes 48, (Suppl. 1), A106.

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131. Schneider, R., Egan, J., Houser, V., and Pioglitazone 010 Study Group. (1999). Combination Therapy with Pioglitazone and Sulfonylurea in Patients with Type 2 Diabetes (Abstract 458). Diabetes 48, (Suppl. 1), A106. 132. Egan, J., Rubin, C., Mathisen, A., and Pioglitazone 027 Study Group. (1999). Adding Pioglitazone to Metformin Therapy Improves Lipid Profile in Patients with Type 2 Diabetes (Abstract 459). Diabetes 48, (Suppl. 1), 106. 133. Egan, J., Rubin, C., Mathisen, A., and Pioglitazone 027 Study Group. (1999). Combination Therapy With Pioglitazone and Metformin in Patients with Type 2 Diabetes (Abstract 504). Diabetes 48, (Suppl. 1), 117. 134. Rubin, C., Eagan, J., Schneider, R., and Pioglitazone 014 Study Group. (1999). Combination Therapy with Pioglitazone and Insulin in Patients with Type 2 Diabetes (Abstract 474). Diabetes 48, (Suppl. 1), A110. 135. McDonnell, D. P., Clemm, D. L., Hermann, T., Goldman, M. E., and Pike, J. W. (1995). Analysis of Estrogen Receptor Function in vitro Reveals Three Distinct Classes of Antiestrogens. Mol. Endocrinol. 9, 659–669.

DISCOVERY AND CLINICAL DEVELOPMENT OF HIV-1 PROTEASE INHIBITORS BY JOEL R. HUFF* AND JAMES KAHN† *Department of Medicinal Chemistry, Merck Research Laboratories, West Point, Pennsylvania 19486 and †University of California, San Francisco and Positive Health Program, San Francisco General Hospital, San Francisco, California 94110

I. II. III. IV. V. VI. VII. VIII. IX. X.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Selection and Validation of HIV-1 Protease as a Therapeutic Target. . . . . . . Development of HIV-1 Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . Structure-Based Design . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Inhibitor Identification through Broad-Based Screening. . . . . . . . . . . . . . . . Mechanism-Based Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Future Directions for Discovery . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . HIV-1 Protease Inhibitors: The Clinical Perspective . . . . . . . . . . . . . . . . . . . . Clinical Development Milestones . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Issues of Ongoing Concern for the Clinical Use of HIV-1 Protease Inhibitors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . XI. Rational Treatment Combinations That Include HIV-1 Protease Inhibitors . . . XII. Future Considerations for HIV-1 Protease Inhibitors . . . . . . . . . . . . . . . . . . . XIII. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

213 214 215 216 223 227 234 235 236 239 242 244 244 245

I. INTRODUCTION In June, 1981 the U.S. Centers for Disease Control (CDC) reported that five previously healthy young homosexual men in Los Angeles had developed an unexplained collapse of their immune systems leaving them vulnerable to a variety of opportunistic infections, including Pneumocystis pneumonia and a relatively rare skin tumor called Kaposi’s sarcoma (CDC, 1981). This devastating illness, subsequently defined as acquired immunodeficiency syndrome (AIDS), had been responsible for as many as 14 million deaths worldwide by the end of 1998 (UNAIDS, 1998). In the United States, almost 700,000 cases of AIDS had been reported to the CDC by the end of 1998; 60% of those patients had died (CDC, 1998). There are currently 800,000 U. S. residents estimated to be living with human immunodeficiency virus (HIV) infection (Karon, 1996). The global figure may be in excess of 33 million (UNAIDS, 1998). In the face of this global pandemic, a major effort was mobilized as early as the mid-1980s to find ways of controlling and treating this disease. While still no cure or even a treatment feasible for global imple213 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0065-3233/01 $35.00

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mentation is yet available, unprecedented progress has been made when viewed from a historical perspective. In less than 15 years a new disease was defined, the etiological agent was identified, several molecular targets were characterized, and effective therapeutic agents were developed, clinically characterized, and made available to many patients. This progress represents the enormous efforts by and high degree of synergy among academic scientists, pharmaceutical laboratories, clinicians, and the patient community. Almost within a decade of recognizing a new and complex disease, treatments were available that changed an almost uniformly fatal disease into a chronic condition for many individuals. While perhaps no longer unique, the development of HIV-1 protease inhibitors demonstrated a new paradigm for drug discovery and may be one of the first examples of this paradigm to come to fruition. In particular, the power of molecular and structural biology applied to the drug discovery effort was convincingly demonstrated. II. SELECTION AND VALIDATION OF HIV-1 PROTEASE AS A THERAPEUTIC TARGET Within 3 years of characterizing the new disease referred to as AIDS, two laboratories identified the responsible etiological agent as a retrovirus of the Lentiviridae family (Barre-Sinoussi et al., 1983; Gallo et al., 1984; Popovic et al., 1984). Originally referred to as LAV or HTLV-III, this enveloped, single-stranded RNA virus is now designated human immunodeficiency virus (HIV) (Coffin et al., 1986; Gallo and Montaigner, 1988). The virus selectively infects monocytes expressing CD4 and certain cytokine receptors and eventually depletes CD4+ T lymphocytes, producing profound defects in cell-mediated immunity (Bowen et al., 1985). The resulting immunodeficiency results in opportunistic infections, neurologic and neoplastic disease, and ultimately death. Ensuing studies of HIV elucidated the critical molecular events required for viral replication and identified several possible molecular targets for therapeutic intervention. Two years after HIV was identified as the agent responsible for AIDS, the genomic sequence of the virus was determined. Analysis of the genomic sequence led Ratner et al. (1985) to postulate that the second open reading frame of HIV-1 encoded a protease analogous to those found in other retroviruses. Furthermore, a highly characteristic amino acid triad, Asp-Thr(Ser)-Gly, found within the putative protease sequence suggested that the enzyme was a member of the aspartic acid protease family (Toh et al., 1985). However, despite the common, highly

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conserved catalytic triad, significant structural differences were apparent between retroviral and classical aspartic acid proteases. In fungal and mammalian aspartic acid proteases, the enzyme is generally composed of two homologous domains, with each domain containing the key catalytic triad (Tang et al., 1978). In contrast, the proposed HIV-1 protease contained about half the number of expected amino acid residues and only one catalytic triad. These observations led Pearl and Taylor (1987) to propose that the catalytically active form of retroviral proteases exists as a homodimer, with each monomer contributing one of the two key aspartic acid residues to the catalytic site. This proposal was confirmed by site-directed mutagenesis of the active site (Kohl et al., 1988), by inhibition by prototypical inhibitors of aspartic acid proteases (Krausslich et al., 1988; Nutt et al., 1988), and ultimately by single crystal X-ray crystallography (Lapatto et al., 1989; Navia et al., 1989; Wlodawer et al., 1989). Kramer et al. (1986) first proposed that inhibition of HIV-1 protease would serve as an effective strategy to block viral replication. Subsequently, Kohl and colleagues demonstrated that a conservative single amino acid substitution of asparagine for the active-site aspartic acid abolished enzymatic activity. Furthermore, when proviral DNA containing this same single mutation in the HIV-1 protease was transfected into human colon carcinoma cells, aberrant viral particles were produced, which contained unprocessed viral proteins and which were unable to infect susceptible lymphoid cells (Kohl et al., 1988) These results unambiguously demonstrated that the virally encoded protease was essential for replication and that its function could not be assumed by host cell enzymes. This and similar experiments (Gottlinger et al., 1989; Peng et al., 1989) validated HIV-1 protease as an important target for therapeutic intervention. III. DEVELOPMENT OF HIV-1 PROTEASE INHIBITORS In the quest to develop therapeutically useful inhibitors of HIV-1 protease, three fundemental strategies were employed. Each of these has produced potent inhibitors suitable for clinical evaluation. Two of these approaches employed relatively traditional strategies, involving either high-volume screening or incorporation of a transition state mimic into substrate analogues. The third strategy depended on insight gained from structural information obtained early in the quest for HIV-1 protease inhibitors. These approaches were not mutually exclusive. Indeed, knowledge gained from high-resolution crystal structures of the enzyme and

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enzyme inhibitor complexes influenced the progress of all three strategies (Wlodawer and Erickson, 1993; Wlodawer and Vondrasek, 1998). What is striking is the remarkable diversity of structural solutions to the problem of finding potent and selective HIV-1 protease inhibitors. IV. STRUCTURE-BASED DESIGN The impact of molecular biology on the identification and validation of important biochemical antiviral targets has been noted. In addition, the early success of cloning and expression of HIV-1 protease provided access to large quantities of pure protein for crystallography experiments (Hirel et al., 1990; Ido et al., 1991). This, in turn, resulted in the availability of high-resolution structural information very early in the search for inhibitors. The initial crystal structures confirmed the previously proposed architecture and mechanistic class (Lapatto et al., 1989; Navia et al., 1989; Wlodawer et al., 1989). The most striking feature of the protein was the C2 symmetry observed for the homodimer. In addition, residues 42 to 58 from each 99-amino acid monomer form a prominent hairpin turn, or flap, which projects over the substrate binding cleft. These flexible flaps collapse to enclose inhibitors and presumably substrates within a hydrophobic tunnel formed by the interface of the two monomers (Spinelli et al., 1991). The almost perfect crystallographic C2 symmetry of the homodimeric enzyme inspired a significant effort to develop inhibitors that embodied complementary symmetry. The rationale for developing symmetrical inhibitors encompassed an expectation for high selectivity with respect to other mammalian enzymes as well as the potential for greater metabolic durability than standard peptide mimetics. It was hoped that greater metabolic stability might provide greater bioavailability. Erickson et al. (1990) described the potent, pseudosymmetric inhibitor 1 (see Table I), which displayed excellent selectivity with respect to other aspartic acid proteases and demonstrated antiviral activity against HIV-1 in cell culture (IC50 = 0.4 µM). No oral bioavailability was observed in animal models, probably, at least partially, as a result of the very low aqueous solubility of the compound and its consequent poor absorption from the intestinal tract. Employing a slightly different symmetry analysis of substrate analogues, Kempf et al. (1991) reported the dihydroxy inhibitor 2 (Table I), which had been modified to improve solubility properties. A potent and selective inhibitor of HIV-1 protease, 2 (A77003) also showed poor oral bioavailability in animals. The solubility properties were significantly improved, however, allowing clinical

TABLE I Structure-Based Inhibitors Compound 1

Ki (IC50), nM

Antiviral activity IC50 (IC90), nM

Reference

4.5

400

Erickson et al. (1990)

(<1)

2

Kempf et al. (1991)

(A-77003) (<1)

3

220

Kempf (1994)

(A-80987) 4

217

(ritonavir)

(<0.5)

25

Kempf et al. (1995)

Kempf et al. (1998) (continues)

218

TABLE I Continued Compound

Ki (IC50), nM 0.0013

5

Antiviral Activity IC50 (IC90), nM

Reference

17

Sham et al. (1998)

0.27

(60)

Lam et al. (1994)

0.28

(126)

Hodge et al. (1996)

0.031

(62)

Rodgers et al. (1998)

(ABT-378, Lopinavir) 6

(DMP-323) 7

(DMP-450) 8

(DMP-850)

0.021

(56)

Rodgers et al. (1998)

10

0.65

(71)

De Lucca et al. (1999)

11

(1.0)

(20)

Kim et al. (1996)

9

(DMP-851)

219

220

JOEL R. HUFF AND JAMES KAHN

studies on this symmetrical inhibitor to be carried out via intravenous administration. A short plasma half-life and severe phlebitis at the injection site resulted in the early termination of those studies. No reduction in circulating virus was observed (Reedijk et al., 1995). Further investigation of structural features that affected solubility, oral bioavailability, and metabolic stability prompted the Abbott workers to depart from design strategies based on C2 symmetric inhibitors. A truncated, asymmetric inhibitor, 3 (Table I) (A-80987) (Kempf, 1994), demonstrated improved oral bioavailability in animal models and humans (Kempf et al., 1995). However, the compound possessed a short plasma half life as a result of extensive metabolism. Careful analysis of the sites for metabolism in 3 guided the chemical research to produce more stable molecules while retaining appropriate solubility properties. Replacement of both pyridine substituents with the thiazole nucleus resulted in compounds with much improved metabolic stability. Ritonavir, 4 (Table I) was the culmination of this effort (Kempf et al., 1995; Kempf et al., 1998). Administration of ritonavir to rats gave plasma levels more than 100-fold in excess of the antiviral EC50. Furthermore, plasma concentrations declined quite slowly. The extended pharmacokinetic profile of ritonavir corresponded to inhibition of cytochrome P450-3A4, the primary metabolic enzyme involved with its clearance. Ritonavir inhibits HIV-1 protease with an IC50 value of less than 0.5 nM and blocked the cytopathic effect of HIV-1 in cell culture with an EC50 = 25 nM. The in vitro potency of ritonavir is attenuated almost 20-fold by protein binding in the presence of human serum (Sham et al., 1998). The reduced antiviral potency may contribute to the emergence of resistant strains of HIV despite relatively high plasma levels in patients. Sequence analysis of ritonavir-resistant HIV indicates frequent mutation of valine82 to alanine, threonine, or phenylalanine (Molla et al., 1996). Analysis of the X-ray structure of ritonavir bound to HIV-1 protease suggested specific modifications of the inhibitor to minimize the effect of mutations at valine-82. Implementation of such modifications yielded ABT378 (lopinavir), 5 (Table I), a potent HIV protease inhibitor that retains improved antiviral activity against Val 82 mutant strains (Sham et al., 1998). The compound also proved to be less affected by serum protein binding. It inhibited wild-type HIV-1 protease with a Ki of 1.3 pM and blocked replication of the virus with an EC50 of 17 nM. Addition of human serum did reduce the antiviral potency of ABT-378 in cell culture; however, lopinavir retained an EC50 of 100 nM in the presence of 50% human serum. Against clinical isolates of HIV-1 that contained valine-82 mutations and were 20- to 40-fold resistant to ritonavir, lopinavir showed only a 6- to 10-fold shift in antiviral activity. Lopinavir

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221

exhibited poor pharmacokinetics when administered to rats, dogs, or monkeys. Coadministering the potent cytochrome P450 inhibitor ritonavir resulted in plasma concentrations of lopinavir that significantly exceeded the antiviral EC50 value for extended periods of time. This observation was reproduced in human volunteers, in whom coadministration of 50 mg of ritonavir produced a 77-fold increase in the total plasma exposure of lopinavir. Lopinavir in combination with rifonavir has been approved by the FDA for use in the United States. In addition to the C2 symmetry of the enzyme, a feature common to almost all inhibitor complexes of HIV-1 protease is a tightly bound water molecule, which bridges two carbonyl oxygens of a peptidomimetic inhibitor and two isoleucine amide hydrogens in the flaps of the enzyme through a network of hydrogen bonds (Miller et al., 1989; Wlodawer and Erickson, 1993). Incorporation of structural elements into inhibitors to mimic the function of this structural water should provide enhanced binding energy through favorable entropic changes. Additionally, such structures would potentially provide greater specificity for viral versus mammalian proteases (Swain et al., 1990). In an elegant example of inhibitor design, Lam et al., (1994) developed a series of cyclic ureas that incorporated both a water mimic and cyclic conformational constraint of the inhibitor side chains. Analysis of crystal structures of inhibitor–HIV-1 protease complexes had suggested the possibility of incorporating cyclic constraints within substrate analogues as a way of introducing the structural water mimic. Choosing the dihydroxy transition state mimic as a core resulted in the selection of a seven-membered ring, and the urea functionality was introduced to strengthen the hydrogen bonds to the flaps. Execution of this strategy resulted in 6 (Table I) (DMP323). This subnanomolar inhibitor of HIV1 protease blocked viral replication in cell culture by 90% at a concentration of 60 nM. Based on acceptable pharmacokintetics in animal models, DMP323 was taken into clinical trials; however, unacceptable oral bioavailability in humans forced early termination of the studies. Several design factors probably contribute to the excellent potency and selectivity of the cyclic ureas. The cyclic conformational constraint preorganizes the structure to complement the three-dimensional surface of the binding site. Thus, the entropic penalties typically associated with binding a linear, flexible inhibitor accrue during the synthesis of the molecule rather than during the binding event. Displacement of the bound water molecule is also thermodynamically favorable as a result of increased entropy. Finally, hydrophobic interactions between the inhibitor side chains and the binding subsites of the enzyme are optimized through constrained conformation and stereochemistry.

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Modifications to improve inhibitor solubility yielded 7 (DMP450) (Table I), which showed improved pharmacokinetics in humans. The compound, however, was much less effective against HIV that had acquired mutations resulting in resistance against other protease inhibitors (Hodge et al., 1996). Compounds 6 and 7 are C2-symmetric. Unsymmetrical compounds were also designed with the objective of optimizing enzyme and antiviral potency by modifying one of the nitrogen substituents and also improving physicochemical properties to enhance oral bioavailability by modifying the other (De Lucca et al., 1998; Wilkerson et al., 1997). Two interesting compounds emerged from this strategy. Previous compounds demonstrated that an increased number of hydrogen bonds due to the nitrogen substituents binding to the P2/P2′ subsites resulted in greatly increased potency against both wild-type and protease-resistant strains of HIV (Jadhav et al., 1997). Unfortunately, the increased polarity that necessarily accompanied an increased hydrogen bond network significantly attenuated the ability of a compound to penetrate cells, resulting in reduced antiviral activity in cell culture. Furthermore, these polar compounds had poor oral bioavailability. Simple lipophilic substituents were incorporated on one of the urea nitrogen atoms to balance the polarity of potency-enhancing substituents placed on the other urea nitrogen. Compounds 8 (DMP850) and 9 (DMP851) (Table I) demonstrated excellent potency, with Ki’s of 20–30 pM and antiviral IC90’s of 50–60 nM (Rodgers et al., 1998). These compounds showed much improved activity against protease-resistant strains of HIV as compared with DMP450. Both 8 and 9 exhibited good oral bioavailability (60%) and a sustained plasma halflife (3–8 hours). One additional advantage of the nonsymmetrical inhibitors was their reduced molecular weight compared with polar symmetrical inhibitors. Plasma levels of cyclic ureas dropped abruptly when the molecular weight of a compound exceeded 620 Da. Other variations of the design strategy that led to cyclic ureas have been described. Analysis of crystal structures of cyclic urea–HIV-1 protease complexes suggested that the unsymmetrical tetrahydropyrimidinone 10 (Table I) could achieve the proper conformation for binding when one of the benzyl substituents was lengthened to a phenethyl substituent (De Lucca et al., 1997). Optimization of these structures produced highly potent inhibitors with good antiviral activity in cell culture; however, the pharmacokinetic properties offered no improvement over the seven-membered ring cyclic ureas (De Lucca et al., 1999). The symmetrical seven-membered sulfone, 11 (Table I), has been reported by Kim to show high in vitro potency (IC50 = 1 nM), good antiviral activity in cell culture (EC90 = 20 nM), and excellent pharma-

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cokinetics in dogs (74% bioavailability; 4.7-hour plasma half-life). A single crystal X-ray structure confirmed the expected binding mode, with the sulfone oxygens performing the bridging function of the commonly observed bound water molecule (Kim et al., 1996). V. INHIBITOR IDENTIFICATION THROUGH BROAD-BASED SCREENING Broad-based screening for HIV-1 protease inhibitors provided a complementary approach to finding lead structures for optimization. A wide variety of structures have been identified that show inhibitory activity against the enzyme (Darke and Huff, 1994). Among those that have been developed further are the 4-hydroxypyranones. Wafarin, 12 (Table II), and several related compounds were identified as modestly potent inhibitors of HIV-1 protease independently in two laboratories (Thaisrivongs et al., 1994; Tummino et al., 1994a, 1994b). Crystal structures were obtained on several compounds in this series early in the development effort and used iteratively with structural modifications to optimize intrinsic affinity. The key features of the inhibitor–enzyme complex showed a hydrogen bond interaction between the enolic 4hydroxyl substituent and the catalytic aspartic acid residues of the enzyme. The pyranone carbonyl and ether oxygens formed hydrogen bonds with backbone amide hydrogens of the flaps, replacing the ubiquitous bound water molecule in other protease-inhibitor complexes (Prasad et al., 1996; Thaisrivongs et al., 1994; Vara Prasad et al., 1994). Further optimization yielded compounds with improved potency against HIV-1 protease in vitro. For example, compound 13 (U-96988) (Table II) had an IC50 of 38 nM as a mixture of four stereoisomers. An important feature of inhibitors in this class was their ability to comparably inhibit both HIV-1 and HIV-2 protease. This property suggested the potential for activity against protease-resistant strains of HIV-1. Compound 13 showed only modest antiviral activity in cell culture (IC50 = 3 µM); however, the pharmacokinetic properties of this compound in animal models were sufficiently promising to permit selection of the compound for clinical studies. One feature of the hydroxypyranones that presented a major challenge was the poor translation of high intrinsic inhibitory potency against the enzyme into antiviral activity in cell culture (Prasad et al., 1996). The acidic character of the enolic hydroxy group (pKa 4.2–6.5) as compared with aliphatic hydroxyl groups found in other classes of inhibitors did not appear to offer an explanation in as much as inhibitors of the pyranone class penetrate cells efficiently (Vara Prasad et al., 1995). Empirically,

224 TABLE II Inhibitors Originating from Screening Compound 12

Ki (IC50), nM

Antiviral activity IC50 (IC90), nM

18,000

—

38

3000

Reference Tummino et al. (1994)

(Wafarin) 13

(U-96988)

Thaisrivongs et al. (1994)

<1

14

1,000–2,000

Skulnick et al. (1995)

(PNU-103017) 0.008

15

(PNU-140690, tipranavir)

(100)

Turner et al. (1998)

225

226

JOEL R. HUFF AND JAMES KAHN

introduction of more polar substituents resulted in increased antiviral activity (Hagen et al., 1997). A probable explanation of the unexpectedly poor antiviral activity is likely related to protein binding. Warfarin and related coumarins are extensively bound to plasma proteins, which affects both pharmacological activity and pharmacokinetics (Yacobi and Levi, 1975). Representatives of the pyranone class of HIV-1 protease inhibitors exhibited high affinity and selectivity for the warfarin site IIA of human serum albumin (He and Carter, 1992). Thus, addition of increasing amounts of serum protein reduced the antiviral activity of compound 14 (PNU-103017) (Table II) by more than two orders of magnitude (Padbury et al., 1998). Inspection of crystal structures of enzyme-inhibitor complexes for this class suggested that a more flexible core ring would allow better accommodation of the side chains to the enzyme active site. As a result, the pyranone ring was partially reduced to give 5,6-dihydro-4-hydroxypyranones with greater intrinsic and antiviral potency (Tait et al., 1997; Thaisrivongs et al., 1996). Continued optimization yielded 15 (Table II) (PNU-140690, Tipranavir) (Turner et al., 1998). Compound 15 exhibited excellent inhibitory potency against HIV-1 protease, with Ki = 8 pM. In the absence of protein, 15 inhibited viral replication in cell culture by 90% at a concentration of 100 nM; however, addition of 10% fetal calf serum and 75% human plasma shifted the IC90 concentration to 1.4 µM. Nevertheless, pharmacokinetic evaluation in animals suggested that the required plasma concentrations for therapeutic efficacy could be achieved in humans. In rats, the plasma half-life exceeded 5 hours, and the oral bioavailability was approximately 30%. Mechanistic studies demonstrated that the modest oral bioavailability in both rat and dog was limited by absorption rather than by metabolism and was probably a result of the low solubility of the compound. Compound 15 was administered to healthy male volunteers in a single-dose safety and tolerability study, and after a 500-mg dose, plasma levels exceeded the target concentration of 1 µM for at least 8 hours (Borin et al., 1997). Although 15 exhibited low potency with respect to inhibiting other aspartic acid proteases such as human pepsin, cathepsin D, and cathepsin E in vitro (Ki values of 2, 15, and 9 µM, respectively), the high plasma concentrations required for antiviral efficacy raise the possibility of losing this selectivity in vivo (Turner et al., 1998). Compound 15 inhibits HIV-2 protease with a subnanomolar Ki value and displays good activity against a variety of HIV-1 protease variants that demonstrated a high level of resistance to other HIV-1 protease inhibitors (Poppe et al., 1997). Laboratory strains of HIV-1 selected for resistance to ritonavir are also highly resistant to saquinavir, indinavir, and nelfinavir, with 50-

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to more than 100-fold greater concentrations required for antiviral activity as compared with wild-type virus. These isolates remained sensitive to 15, however, with less than a 10-fold shift in the IC90 value. Additionally, a study of ritonavir-resistant clinical isolates (30- to 60-fold resistant) indicated only a two- to three-fold decrease in the sensitivity to 15 (Poppe et al., 1997). VI. MECHANISM-BASED STRATEGY Characterization of HIV-1 protease as a member of the aspartic acid protease family provided the rationale for most of the efforts to design inhibitors (Kohl et al., 1988; Krausslich et al., 1988; Navia et al., 1989; Pearl and Taylor, 1987). Previous efforts to develop therapeutically useful inhibitors of the mechanistically related enzyme renin had demonstrated that potent inhibitors could be prepared by replacing the scissile amide bond of a substrate analogue with a nonhydrolyzable isostere to mimic the tetrahedral intermediate or transition state involved in amide hydrolysis (Greenlee, 1990). Although several dipeptide isosteres have been used to successfully generate highly potent HIV-1 protease inhibitors, a relatively small number have resulted in compounds that reached clinical development. Discovery programs based on variations of the hydroxyethylamine dipeptide isostere have been the most productive avenue of research by a very large margin. Roberts et al. (1990) developed a design strategy based on hydroxyethylamine isosteres to mimic the unusual phenylalanine–proline cleavage site found in HIV-1 protease substrates. Since amide bonds N-terminal to proline are not commonly susceptible to hydrolysis by mammalian proteases, there was an expectation that such inhibitors should be highly selective for the viral enzyme. Early inhibitors such as compound 16 (Table III) showed modest potency; however, ring expansion and elaboration of the proline to the decahydroisoquinoline nucleus provided 17 (Table III) (Ro 31-8959, saquinavir) (Roberts et al., 1990). The decahydroisoquinoline nucleus may be regarded as a conformationally constrained hydroxyethylamine isostere of phenylalanine–cyclohexylalanine. Saquinavir inhibits both HIV-1 and HIV-2 PR with a Ki value of 0.1 nM or lower and blocks viral replication in susceptible lymphoid cells by 50% at 2 nM. The expectation of high selectivity versus mammalian aspartic acid proteases was realized with no inhibition of pepsin, renin, cathepsins D and E, or gastricsin at micromolar concetrations. The modest oral bioavailability observed in animal models (Martin, 1991; Roberts et al., 1992) was

228 TABLE III Inhibitors Based on Mechanism Compound

Ki (IC50), nM 140

16

17

Antiviral activity IC50 (IC90), nM

Reference

—

Roberts et al. (1990)

≤0.1

2

Roberts et al. (1990)

2.0

20

Kaldor et al. (1994)

(saquinavir) 18

19

3.0

970

Kaldor et al. (1997)

20

2.0

14

Kaldor et al. (1997)

0.03

7

(Nelfinavir) 21

Lamarre et al. (1997)

229

(Palinavir) (continues)

230

TABLE III Continued Ki (IC50), nM

Antiviral activity IC50 (IC90), nM

22

(0.3)

(200)

23

(<0.03)

Compound

24

25

(3)

(8)

0.3

Reference Lyle et al. (1991)

Ghosh et al. (1993)

Dorsey et al. (1994)

(50)

Vacca et al. (1994)

Dorsey et al. (1994)

(indinavir) (6)

26

21

Getman et al. (1993)

Vazquez et al. (1995)

(SC-52151) 27

1.0

5

28

0.6

(40)

Kim et al. (1995)

(3)

Bold et al. (1998)

(amprenavir) 29

(26)

231

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JOEL R. HUFF AND JAMES KAHN

reproduced in humans. The compound exhibits an oral bioavailability of approximately 4% when administered with food (Muirhead et al., 1992). Plasma levels of saquinavir were achieved, however, that reduced circulating virus levels in patients and increased the time to AIDS-defining events or death in HIV-infected individuals. Improvements in the formulation of saquinavir have led to significantly improved pharmacokinetic properties in humans (Perry and Noble, 1998). A fusion of mechanism-driven design informed by structural information resulted in the development of nelfinavir (20, Table III). Replacement of the hydroxyethylamine dipeptide isostere in saquinavir (17) with a carbon analogue generated an equally potent enzyme inhibitor, 18 (LY289612) (Table III); however, like most peptidomimetic inhibitors, the compound possessed poor pharmacokinetic properties (Kaldor et al., 1994). X-Ray crystallographic information guided the modification of the N-terminal portion that 18 still had in common with saquinavir. The resulting nonpeptide compound 19 potently inhibited HIV-1 protease with a Ki value of 3 nM; however, the antiviral activity in cell culture was disappointing, with an EC50 of approximately 1 µM (Kaldor et al., 1997). Compound 18 also had the disadvantage of poor aqueous solubility. Reintroduction of the decahydroisoquinoline found in saquinavir with its modestly basic amine produced 20 (nelfinavir) (Table III). Nelfinavir inhibits HIV-1 protease with a Ki value of 2.0 nM and blocks the cytopathic effect of HIV-1 in cell culture with an EC50 value of 14 nM. Compound 20 showed good pharmacokinetics in animal models. The oral bioavailability in fed animals ranged from 15 to 50% with a plasma half-life of 1 to 2 hours. Oral bioavailability was less in fasted dogs. An extended plasma half-life observed after oral dosing was likely due to slow dissolution and absorption (Kaldor et al., 1997). The compound effectively inhibited virus replication for a variety of laboratory and clinical isolates at concentrations ranging from 9 to 60 nM (Patick et al., 1996). Other close structural analogues of saquinavir (17) incorporating the hydroxyethylamine isostere have been developed to provide potent inhibitors of HIV-1 protease. For example, palinavir (21) (Table III) inhibits the enzyme with a Ki of 0.03 nM and blocks viral replication with an EC50 of 7 nM. This antiviral potency was not substantially affected by the addition of α1-acid glycoprotein at physiological concentrations; however, 21 did show reduced activity against HIV-1 variants with active site mutations, including valine-32 to isoleucine and isoleucine-84 to valine (Lamarre et al., 1997). Pharmacokinetics in rats have been reported for palinavir. The compound exhibited an oral bioavailability of 20 to 30%, with a plasma half-life of 30 to 50 minutes.

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A novel variation of the hydroxyethylamine transition state mimic was discovered in the development of indinavir, 25 (Table III). In the course of investigating HIV-1 protease inhibitors based on the hydroxyethylene isostere of the scissile amide bond, highly potent compounds were identified, for example, 22 (Table III) (Lyle et al., 1991) and 23 (Table III) (Ghosh et al., 1993). Although highly potent and selective, compounds 22 and 23 exhibited poor pharmacokinetics in animals. The extremely low aqueous solubility and metabolic lability of these compounds probably contributed to poor absorption and rapid clearance. Comparison of the structures of 22 with that of saquinavir, 17, emphasized two significant differences. Saquinavir, which showed at least modest oral bioavailability in animals, had a moderately basic amine incorporated into the inhibitor backbone. The polarity of the amine would be expected to improve aqueous solubility. In addition, the chiral configuration of the hydroxyl substituent in saquinavir was opposite to that required in compounds such as 22 and 23. Molecular modeling studies suggested that saquinavir and 22 could be aligned to allow superposition of the critical hydroxyl substituents if they were superimposed with one of the compounds aligned in the reverse orientation. Because the enzyme is C2-symmetric, inhibitors may bind in either of two orientations (Fitzgerald et al., 1990; Murthy et al., 1992). In this alignment, the C-terminal bicyclic decahydroisoquinoline of saquinavir would overlap the N-terminal portion of 22. This analysis suggested the hybrid compound 24 (Table III) as a target. This compound did prove to be a reasonable inhibitor of HIV-1 protease, with an IC50 value of 8 nM. More importantly, 24 showed modest bioavailability in animals. Further modification of the decahydroisoquinoline portion of 24 to increase polarity and solubility resulted in the development of indinavir, 25 (Dorsey et al., 1994; Vacca et al., 1994). Indinavir inhibited HIV-1 protease with a Ki value of 0.3 nM and blocked viral replication in cell culture by 95% at concentrations of 50 nM. Indinavir also exhibited good pharmacokinetic properties after oral administration to animals, justifying its development for human trials (Vacca et al., 1994). Other interesting hybrids between hydroxyethylene and hydroxyethylamine dipeptide isosteres have been reported. Getman et al. (1993) developed a series of hydroxyethylureas that potently inhibited HIV-1 protease. The concept of these urea isosteres, first introduced as renin inhibitors, may be envisioned as a modification of the hydroxyethylene isostere (e.g., compound 22), in which the P1′ chiral α-carbon is replaced with a trigonal nitrogen. One example of this class of inhibitors, SC-52151 (26) (Table III), inhibited HIV-1 protease with an IC50 value of 6 nM and blocked the cytopathic effect of HIV-1 in cell cul-

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JOEL R. HUFF AND JAMES KAHN

ture with an EC50 value of 21 nM. Although plasma concentrations of SC-52151 were achieved in patients that exceeded the in vitro IC90 value, no significant antiviral effect was observed (Fischl et al., 1997). The fact that SC-52151 is highly protein-bound in human plasma, particularly to α1-acid glycoprotein, was proposed as an explanation for the lack of antiviral activity in vivo. Additíon of physiologic concentrations of α1acid glycoprotein resulted in a 17-fold increase in the concentration of SC-52151 required to inhibit HIV-1 replication by 95% in cell culture (Bryant et al., 1995). Closely related analogues of 26, in which the urea functionality was replaced by a sulfonamide, have also been reported. Compound 27 (Table III) inhibits HIV-1 protease with a Ki value of 1.0 nM and blocks the cytopathic effect of HIV in cell culture with an EC50 of 5 nM (Vazquez et al., 1995). Employing the previously reported tetrahydrofuran (Ghosh et al., 1993) to replace the N-terminal quinolinylasparagine, Kim et al. (1995) were able to significantly reduce the molecular weight of inhibitors in this structural class without sacrificing potency. Amprenavir, 28 (Table III), inhibits HIV-1 protease with a Ki value of 0.6 nM and blocks viral replication in cell culture with an IC90 value of 40 nM (Kim et al., 1995). Aza-dipeptide isosteres have been also employed successfully as transition state mimics to generate potent HIV-1 protease inhibitors (Fassler et al., 1993; Grobelny et al., 1997; Sham et al., 1995). Compound 29 (BMS-232632; CGP-73547) (Table III) inhibited HIV-1 protease with an IC50 values of 26 nM and reduced the production of viral reverse transcriptase production by 90% at a concentrations of 3 nM (Bold et al., 1998; Rabasseda et al., 1999). The antiviral potency in cell culture was not significantly affected in the presence of 40% human serum. Furthermore, the antiviral activity of 29 was not diminished by mutations conferring resistance to nelfinavir, saquinavir, and amprenavir. However, cross-resistance was observed among 29, indinavir, and ritonavir (Gong et al., 1998, 1999). Plasma levels of 29 exceeded the antiviral EC50 for more than 24 hours after an oral dose of 300 mg to human volunteers (O’Mara et al., 1998, 1999). VII. FUTURE DIRECTIONS FOR DISCOVERY Future generations of HIV-1 protease inhibitors will focus primarily on two challenges. Improved pharmacokinetics are needed in new agents in order to reduce the complexity of current dosing regimens. Lack of patient compliance resulting from the complexity and life-style

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changes required by current dosing regimens undoubtedly reduce the effectiveness of therapy. The chief pharmacokinetic limitation of individual protease inhibitors appears to be rapid oxidative metabolism by cytochrome P450 enzymes. The propensity for elimination by this metabolic pathway is enhanced by the relatively high molecular weight of current protease inhibitors and their highly lipophilic character. It has been difficult to identify a general solution to this problem because the binding affinity of potent inhibitors relies significantly on extensive surface contact with the lipophilic protein environment. However, clinical data for some combinations of protease inhibitors already suggest that reduced frequency of dosing is a possibility, although relatively high doses are still required. In particular, combinations of protease inhibitors that inhibit these cytochrome P450 enzymes appear to provide effective therapeutic coverage with less frequent dosing (Farthing et al., 1997; Gallant et al., 1998; Hsu et al., 1997; Lal et al., 1998). Active transport of protease inhibitors out of the intestinal tract and central nervous system may also contribute to the poor pharmacokinetic profile and relative low drug levels in the brain (Kim et al., 1998a, 1998b); Lee et al., 1998; Washington et al., 1998). The second challenge has to do with resistance. Development of resistance to inhibitors is a complex process (Boden and Markowitz, 1998; Condra, 1998; Molla et al., 1998). Mutations occur within the enzyme active site, modifying residues that directly contact bound inhibitors. The role of these mutations in reducing the affinity of inhibitors for the enzyme is apparent. Numerous mutations are also observed that occur outside the active site and do not make contact with the inhibitor or significantly change the conformation of the protein (Chen et al., 1995). These changes were thought to provide compensatory enhancement to the reduced enzymatic activity of the mutant protease (Pazhanisamy et al., 1996; Schock et al., 1996). It is now clear that these distal mutations also directly affect the inhibitor binding (Olsen et al., 1999; Wilson et al., 1997). Developing HIV-1 protease inhibitors that do not show cross-resistance to current agents is clearly important; however, the demonstrated plasticity of the enzyme and substrates, as well as the high mutation rate of virus replication, make the durability of such a strategy uncertain in the clinical setting. VIII. HIV-1 PROTEASE INHIBITORS: THE CLINICAL PERSPECTIVE The pivotal clinical advance accounting for the remarkable decline in HIV-associated disease progression and mortality is the development

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and standard implementation of combination antiretroviral therapy (ART), which effectively suppresses viral replication to the lowest limits of available technology (Palella et al., 1998; Hogg et al., 1998; Mocroft et al., 1998; Vittinghoff et al., 1999). HIV-1 protease inhibitors represent the cornerstone of the active ART regimens. HIV-1 protease inhibitors in combination with nucleoside HIV-1 reverse transcriptase inhibitors appear to prevent ongoing immune destruction and contribute to partial reconstitution of selective immune responses (Powderly et al., 1998; Weverling et al., 1999). It has been hypothesized that the maintenance of immune responses and the restoration of other lost critical immune function are responsible for the observed reduction in disease progression (Vittinghoff et al., 1999). Seen from the perspective of recent events, the clinical development of protease inhibitors follows rather conventional programs for drug development. The most remarkable characteristic of the clinical development of HIV-1 protease inhibitors is their rapid clinical evaluation, regulatory approval, and subsequent incorporation into standard treatment regimens. Undoubtedly, the pressure to develop new and active compounds to treat persons with HIV infection contributed to the urgency of drug development. This section will review the clinical milestones for HIV-1 protease inhibitors and discuss some of the near future clinical directions for these compounds. Peer-reviewed, published manuscripts are the basis for this review. Data presented at meetings and published in abstracts were not used. IX. CLINICAL DEVELOPMENTAL MILESTONES HIV-1 protease inhibitors were initially tested to determine dosing and interval administration (Markowitz et al., 1995; Danner et al., 1995). These studies provided key insights into the pharmacologic properties for this class of compounds. A direct result of these studies was the early recognition of resistance (since nonfully suppressive doses and intervals were initially tested) to HIV-1 protease inhibitors. The rapid clinical assessment of these medications began with an evaluation of the addition of HIV-1 protease inhibitors for patients with advanced immune destruction as defined by less than 100 CD4 cells. This was followed by HIV-1 protease inhibitor evaluation for patients with modest immune suppression, defined as less than 200 CD4 cells, and then for patients with minimal immune destruction (200–500 CD4 cells), and even exploratory studies for patients with no immune destruction (more than 500 CD4 cells).

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The first randomized trial that demonstrated clinical activity of an HIV-1 protease was a randomized, placebo-controlled study of persons with advanced immune destruction (Cameron et al., 1998). The study involved 1090 patients, randomly assigned to ritonavir (n = 543) or placebo (n = 547). The primary objective was to determine if a protease inhibitor added to any concurrent licensed anti-HIV-1 therapy would reduce mortality or reduce the occurrence of AIDS-defining illnesses. Patients had advanced disease with a baseline median CD4 lymphoycte count of 18 and 22, respectively in each group. Ritonavir was provided as the 600-mg liquid oral solution twice daily, and a matched placebo was provided to the other group. During the blinded phase of the study, 119 patients (21.9%) assigned to ritonavir developed an AIDS-defined event or died, compared with 205 patients (37.5%) assigned to placebo. The hazard ratio was 0.53 (95% confidence interval [CI] 0.42–0.66; p .0001), indicating that among persons with advanced immune destruction, the addition of ritonavir reduced disease progression by 50%. The decline in disease progression paralleled the immunologic and virologic benefit of ritonavir treatment. Patients randomized to ritonavir sustained an increase of 31 CD4 cells from baseline, and in the subset assessing antiviral activity, plasma viral load fell, with a maximum mean reduction of 1.3 log10 copies per milliliter. Following the randomized portion, all patients were provided with ritonavir. A survival analysis was performed among all patients and demonstrated that 87 patients (16%) initially randomized to ritonavir, compared with 126 patients (23%) initially randomized to placebo, died during the study. The hazard ratio was .69 (95% CI .52–.91; log-rank p=.007) and indicated that the risk of dying was cut by almost one-third with the addition of the HIV-1 protease inhibitor. The improvement in clinical outcomes in this trial was the critical finding; it demonstrated not only that protease inhibitors contributed to reduced disease progression and reduced mortality but also that the improvement correlated with effective reduction of viral replication and improved CD4 cell count. The findings of this study were consistent with the surrogate marker studies of the protease inhibitor saquinavir (Markowitz et al., 1995; Danner et al., 1995; Collier et al., 1996) and subsequent studies of indinavir (Hammer et al., 1997; Hirsch et al., 1999; Gulick et al., 1997). This study was different from many others that will be cited, and the clinical situation requires some explanation. First, this study did not attempt to compare strategies of multiple ART combinations. Rather, it demonstrated the value of adding a protease inhibitor various ART regimens. In addition to the study design, the patient population was critical for the conclusions of the study. Patients with the most profound immune

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destruction represent the group most likely to experience disease progression and unfortunately die, and the study was powered based on the estimation that the annual rate of disease progression or death would be 40%. Studies that focused on patients with more CD4 cells and less immune destruction would not be able demonstrate mortality benefit in as much as the event rate would be markedly lower. Finally, the consistency of the surrogate marker data reduced the need for other studies to demonstrate a difference in mortality in order to convince clinicians of the value of HIV-1 protease inhibitors. The next clinical question involved whether the observed benefit demonstrated for patients with advanced immune suppression could also be extended to groups of patients with less immune destruction. This stage was undertaken with great confidence, because studies of shorter duration had already demonstrated that HIV-1 protease inhibitors combined with nucleosides had profound effects on surrogate markers as compared with changes observed with nucleoside therapy alone (Collier et al., 1996). The study demonstrating that the benefits of protease inhibitor could be extended into a healthier population involved 1156 patients with less than 200 CD4 cells (Hammer et al., 1997). Patients were randomized to dual nucleosides compared with the combination of nucleosides plus indinavir. The primary end point was the combination end point of time to the development of an initial AIDS-defining clinical end point or death. A total of 63 patients (11%) assigned to combination nucleosides as compared with 33 patients (6%) assigned to the indinavir-based regimen, developed an AIDS-defining clinical diagnosis or died (p .001; hazard ratio .5; 95% CI .33–.76). A reduction in mortality alone was also observed. There were 18 patients (3%) randomized to nucleosides who died compared with 8 patients (1%) randomized to indinavir plus combination nucleosides who subsequently died (p .04; hazard ratio 0.43; 95% CI 0.19–0.99). The profound effect of indinavir plus nucleosides observed in the entire cohort was also observed in the subset of patients with less than 50 CD4 cells. Conversely, when the subgroup of patients with 51 to 200 CD4 cells was analyzed, the rate of disease progression and death or death alone was not significantly different between the two treatment groups. The CD4 cell increases and the reduction in plasma HIV-1 RNA paralleled the key clinical observation that a protease inhibitor plus combination nucleosides was associated with a more profound reduction in clinical disease progression than combination nucleosides alone. The one nagging question that remained from this study was whether the optimal use of protease inhibitors should be restricted to those patients with advanced disease. The lack of a clinical effect in the substudy of patients with 50 to 200 CD4 cells did not spark a controversy in

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the matter of optimal timing of initial protease inhibitors with combination nucleosides. The reason was that the substudy was not powered to detect the anticipated difference, and thus the failure to demonstrate a difference among patients with 50 to 200 CD4 cells was an issue of trial design and not potency of the regimen. In addition, the clinical study demonstrated surrogate marker improvements that were consistent with the clinical improvement and continued to validate the use of these markers for interpreting clinical trials. Consensus panels evaluated the data from these and other clinical studies and determined that the profound activity of protease inhibitors validated their early and consistent use to maximally suppress viral replication (Carpenter et al., 1998; Gazzard and Moyle, 1998; Fauci et al., 1998). Furthermore, the changes observed with some of the initial smaller studies (Gulick et al., 1997) continued to demonstrate that HIV-1 protease inhibitor–based therapy combined with dual nucleosides would provide long-term immunologic improvement and sustained virologic suppression (Gulick et al., 1998). In addition, the observed surrogate markers in the clinical efficacy studies of ritonavir and indinavir were similar to the surrogate marker changes observed in smaller clinical trials for the other HIV-1 protease inhibitors, nelfinavir and amprenavir (Moyle et al., 1998; Markowitz et al., 1998; Murphy et al., 1999). Thus these new HIV1 protease inhibitors with a mechanism of action and function similar to those of the previously approved HIV-1 protease inhibitors, even without studies that demonstrated their clinical benefits, were approved for clinical use. X. ISSUES OF ONGOING CONCERN FOR THE CLINICAL USE OF HIV-1 PROTEASE INHIBITORS The effectiveness of HIV-1 protease inhibitors in reducing disease progression and mortality and the associated increase in CD4 cells along with the viral burden established protease inhibitors plus dual nucleosides as the paradigm for HIV-1 treatment. Several key questions remain: What is the optimal time to initiate therapy? What is the optimal treatment regimen? What is the most effective treatment after initial medications have failed? The rationale to initiate treatment with an HIV-1 protease inhibitor plus combination nucleosides for patients with HIV-1 infection and CD4 cells above 200 relies on interpretation of large randomized studies as-well as on investigations with small numbers of patients in trials with elegant laboratory-based evaluations (Table IV). This line of reasoning begins with the observation that ART delays disease progression

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TABLE IV HIV-1 Protease Inhibitors Reasons to Initiate Protease Inhibitors Prevent disease progression and death among patients with advanced disease Reduce death and disease progression among patients with CD4 <100 Death and disease progression among patient with CD4 <200 Reduce HIV-1 replication to the lowest limits of detection Preserve immunologic competence Allow for restoration of immune system Reasons to Delay Protease Inhibitors Unproven clinical benefit among patients with relatively intact immune system Concern with known short-term toxicities and unknown long-term complications High levels of triglycerides and lipodystrophy Drug-specific toxicities: ritonavir, gastrointestinal distress; saquinavir, hard gel capsules; indinavir, renal colic; nelfinavir, diarrhea; amprenavir, nausea and vomiting. Increased risk of poor adherence with long-term treatment, leading to resistance

better than no therapy for patients with CD4 cells below 500 cells/µl. The combination of two nucleoside agents also reduces disease progression more effectively than a single nucleoside analogue for persons with less than 500 CD4 cells/µl. As described above, HIV-1 protease inhibitors with combination nucleosides delays disease progression for persons with less than 200 CD4 cells/µl and has profound effects on surrogate markers compared with combination nucleoside agents alone. Various preliminary studies demonstrate that patients with more than 200 CD4 cells treated with HIV-1 protease inhibitors plus nucleosides experience potent viral suppression and sustained increases in CD4 cells. Therefore, for patients with more preserved immune systems, those with CD4 cells above 200 (and probably below 500), HIV-1 protease inhibitor–based combination therapy will likely preserve key immunologic responses while effectively suppressing viral replication, perhaps reduce virus-associated immune destruction, and ultimately reduce disease progression and mortality. The many consensus panels that support this early treatment paradigm can reassure the clinician faced with this type of reasoning. Tempering the data that urge early treatment with HIV-1 protease inhibitors is the reality of using these complicated medications, with complex administration regimens requiring daily administrations and consistent patient adherence to achieve the clinical and surrogate marker benefit (Carpenter et al., 1998; Gazzard and Moyle, 1998; Fauci et al., 1998; Lerner et al., 1998). The absolute necessity for the patient to commit to an intensive therapy regimen provides critical information

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to the clinicians; despite a variety of data to support the use of protease inhibitor and dual nucleoside therapy, the key to a treatment’s benefit will be patient commitment to the therapy. Patient adherence is a crucial element to long-term medication efficacy (Bassetti et al. 1999). The rationale for delaying the early use of protease inhibitors as a part of the initial anti-HIV therapy regimens relates to the risk benefit analysis for treatment. There have been no clinical trials involving an HIV-1 protease inhibitor combination testing the hypothesis that maximally suppressive combination therapy results in delayed disease progression for persons with CD4 cells above 200 cells/µl. The lack of randomized clinical trial data result from an impossibly large sample size for long-term clinical trials, ethical constraints to continue therapy among patients beyond virologic failure in order to determine clinical disease progression, and finally the difficulty in determining options and comparisons in a rapidly evolving arena of HIV medicine, especially when the present therapeutic dilemmas might not have much relevance for the future of HIV treatments. Therefore, studies proposed to answer the question of best initial treatment options and most advantageous time to initiate therapy will target treatment strategies rather than specific medications. The strategies include using early combinations of nucleosides plus either an HIV-1 protease inhibitor or a nonnucleoside reverse transcriptase inhibitor, or even delaying initial treatments until there has been some immunologic deterioration. Several studies have been launched and are actively enrolling that will attempt to answer this question, but for now, clinicians are left with interpreting clinical trials “between the lines” and their own clinical intuition. Ultimately, the risk/benefit analysis for persons with HIV infection fundamentally focuses on when to begin treatment with HIV-1 protease inhibitors. The benefits of early treatment tend to be laboratory-based and are reflected in CD4 cell count and immunologic function, as well as by measurement of HIV-1 replication and dormancy. But what are the risks of combination therapy? The first risk is that a person beginning therapy early will not have the same treatment options when it is most needed, namely, when the immune system has deteriorated. If resistance develops to an HIV-1 protease inhibitor, then the second or third treatment regimen is less effective. The reduced effectiveness is due to the increased chance that genotypic resistance specific to individual HIV-1 protease inhibitors may reduce viral susceptibility to other similar drugs. Therefore when medications fail because of resistance, the switch to other available, but similar, medications will confer more limited benefit than expected. The reduced benefits with ever increasingly complex regimens have already been observed in several small clinical studies. Thus, early treatment with HIV-1 protease inhibitors

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may induce resistance to the agent used, reducing subsequent options for treatment regimens, and ultimately may lead to less than optimal clinical outcomes. The unwanted resistance may also have significant complications in the arena of public health. Multidrug resistance can be transmitted (Hecht et al., 1998), and some investigators suggest that the incidence of multidrug-resistant HIV-1 transmission is increasing (Yerly et al., 1999; Little et al., 1999; Boden et al., 1999). The widespread appearance and transmission of multidrug-resistant HIV-1 may be a disastrous complication of decisions for early treatment undertaken prior to the patient’s full commitment to the complicated medications. This association has not been proved; however, poorly managed treatments leading to multiple drug resistance that can be transmitted will likely contribute to this potentially disastrous condition. Long-term toxicities are significant concerns with prolonged treatment with HIV-1 protease inhibitors alone and in combination with nucleoside analogues. Complications, including lipodystrophy, abnormal glucose metabolism, accelerated atherosclerosis, renal colic, gastrointestinal intolerance, sleep disturbances, and perhaps an increase in mutagenesis characterize combination antiretroviral therapies (Lo et al., 1998; Miller et al., 1998; Henry et al., 1998). These toxicities have been observed with a relatively short duration of therapy in a small number of patients. Longer-term treatments may increase the prevalence of these and other new clinical problems that may limit the treatment effectiveness and change the risk/benefit analysis. The problems associated with medication adherence become a dominant theme once a patient is committed to treatment with an HIV-1 protease inhibitor (Bassetti et al., 1999). The benefit of treatment is only achievable with consistent use of the medications, although the exact amount of adherence required for viral suppression remains unknown. This is a central problem for HIV-1 protease inhibitors and the nucleoside combinations—the treatments are difficult, requiring compromise of one’s life-style, and may induce toxicities without apparent improvement. After all, how can one improve what is an asymptomatic (and synonymous with a healthy) condition? The art of HIV therapy in the present form is to adequately assess the risks and benefits at every time point and to initiate therapy at the point of maximal benefit and minimal risk. XI. RATIONAL TREATMENT COMBINATIONS THAT INCLUDE HIV-1 PROTEASE INHIBITORS Once treatment is initiated, the goal for treatment is to reduce replicating virus to the lowest limits of quantitation. Consensus panels have

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TABLE V Combinations of HIV-1 Protease Inhibitors for Persons with HIV-1 Infection Two compatible reverse transcriptase agents plus a protease inhibitor: Zidovudine/lamivudine plus either Ritonavir 600 mg twice daily Didanosine/stavudine Saquinavir soft gel 1,200 mg three times daily Stavudine/lamivudine Indinavir at 800 mg every 8 hours Didanosine/zidovudine Nelfinavir at 750 mg three times daily Saquinavir 400 mg twice daily plus Ritonavir 400 mg twice daily Efavirenz 600 mg at night with a meal

suggested treatment options, and these are available for review by clinicians and patients. The most potent and clinically tolerable combinations are listed in Table V. The choice among various antiretroviral combinations should include the following considerations: potency, durability, ease of adherence, short- and long-term toxicities, patterns of resistant mutations, and subsequent rescue treatment options. The decision on the best strategy for treatment options awaits blinded and randomized controlled studies; however, the present standard is to use dual nucleosides and either a single HIV-1 protease inhibitor or a nonnucleoside reverse transcriptase inhibitor. (The nonnucleosides are not discussed in this review.) Table VI provides information regarding some practical recommendations when treatment for HIV infection with an HIV-1 protease inhibitor combined with dual nucleosides is initiated. To maximize adherence, treatment should not begin until the patient is committed to a long-term treatment strategy. Use of medication trays or vitamin pills to mimic the complexity of the treatment regimen so that the patient can realistically appraise the required commitment to treatment should be considered. TABLE VI Key Concerns When Beginning HIV-1 Protease Inhibitors 1. 2. 3. 4. 5. 6. 7. 8.

Begin therapy when the patient is ready for the complicated regimen. Consider checking baseline resistance patterns prior to initiating therapy. Always and only use maximally suppressive therapy. Establish a consistent baseline for CD4 cells and HIV-1 RNA. Prepare patient for toxicities and then monitor appropriately. Never initiate therapy and plan to reduce its intensity (induction maintenance). Never initiate therapy by adding one treatment at a time as a way to reduce toxicities. Stop all medications at once when toxicities develop rather than discontinuing treatments one at a time.

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Clinicians should anticipate toxicities, informing their patients of likely side effects. Alerting patients to possible toxicities enhances the likelihood that patients will be able to recognize common toxicities and initiate the proper response to mitigate their severity. XII. FUTURE CONSIDERATIONS FOR HIV-1 PROTEASE INHIBITORS One of the key areas of active research involves the idea of combining HIV-1 protease inhibitors to increase their activity (Cameron et al., 1999; Reiser et al., 1999; Deeks et al., 1999). From a mechanistic standpoint, combining agents with the same mechanism of action might have little value, but the pharmacokinetic profile of these agents is complex. These drugs have significant pharmacologic interactions. Combining these agents may offer dosing advantages and may influence viral mutations. Already some clinicians routinely use dual protease inhibitors with dual nucleosides for patients with extraordinarily high viral burdens and to recapture anti-HIV activity for those who have previously failed standard combination therapy. Another area of active research centers on second-generation HIV-1 protease inhibitors. The idea here is to develop agents that would demonstrate activity regardless of the genotypic resistance induced by the already approved and marketed agents. Although these newer agents are still relegated to the preclinical phase of development, the commitment to rapid development of agents that characterized the first generation of HIV-1 protease inhibitors is likely to contribute to the rapid exploration of suitable follow-up compounds. XIII. CONCLUSIONS Initiating HIV-1 protease inhibitors with dual nucleosides represents the most important treatment development for persons with HIV infection. These inhibitors can significantly reduce the risk for disease progression and death for persons with advanced immune suppression (less than 200 CD4 cells). They can profoundly and durably (more than 2 years in some small studies) suppress viral replication among patients with modest immune suppression (200 to 500 CD4 cells). Clinicians must educate patients regarding the risk and benefit for initiating therapy with this complex therapy and be responsible for selecting a combination that will maximally suppress viral replication. Consensus exists among international experts to individualize therapy and to offer only maximally suppressive

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combination therapy. More information is needed regarding the optimal agents and the most effective time, based on laboratory data, to initiate therapy. Therapy with HIV-1 protease inhibitor and dual nucleosides may represent the most active combination. Expert clinicians and patients must also balance toxicity, convenience, and adherence with the known beneficial activity associated with HIV-1 protease inhibitors.

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CALCINEURIN INHIBITORS AND THE GENERALIZATION OF THE PRESENTING PROTEIN STRATEGY BY KURT W. VOGEL,*1 ROGER BRIESEWITZ,*2 THOMAS J. WANDLESS,† AND GERALD R. CRABTREE‡ *Department of Pathology and ‡Department of Developmental Biology, Stanford University Medical School and †Department of Chemistry, Stanford University

I. Calcineurin, Calcineurin Inhibitors, and the Effects of Inhibition of Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Discovery and Development of Cyclosporin A and FK506 . . . . . . . . . . . . . . B. Identification of Calcineurin as the Common Target for Cyclosporin A and FK506 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Role of Calcineurin Outside of the Immune System . . . . . . . . . . . . . . . . . . 1. Role of Calcineurin and the Effects of CsA and FK506 in the Nervous System . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . a. FKBP and Release of Intracellular Ca2+ . . . . . . . . . . . . . . . . . . . . . . . . b. FKBP Ligands and Neuroregeneration . . . . . . . . . . . . . . . . . . . . . . . . 2. Calcineurin and Synaptic Plasticity . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Signaling through Calcineurin and NF/AT in the Developing Heart . . . . 4. Calcineurin and Cardiac Hypertrophy . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Inhibition of Calcineurin and Transforming Growth Factor-β Release . D. Specificity of Action of CsA and FK506 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Physiological Inhibitors of Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1. Calcineurin Autoinhibition . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2. Oxidative Inactivation of Calcineurin. . . . . . . . . . . . . . . . . . . . . . . . . . . . 3. Regulation by Localization: Bcl-2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4. Regulation by Localization: AKAP79 . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Inhibition by Cabin-1/Cain . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Inhibition by Disruption of Calcineurin/NF-AT Interaction . . . . . . . . . F. Other Inhibitors of Calcineurin . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Inhibition by Immunophilin/Immunosuppressant Complexes: The Presenting Protein Strategy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Structures of Immunophilin-Immunosuppressant Complexes . . . . . . . . . . B. Structure of the FKBP-FK506-Calcineurin Ternary Complex . . . . . . . . . . . C. Structure of the FKBP-Rapamycin-FRB Ternary Complex . . . . . . . . . . . . . . III. Generalization of the Presenting Protein Strategy. . . . . . . . . . . . . . . . . . . . . . . A. Using a Presenter Protein to Enhance the Affinity of a Small Molecule Ligand: Towards a New Approach to Enhance Drug-Target Interactions . . . . B. Properties of Good Presenter Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Enhancing the Affinity of an SH2 Domain Ligand with a Presenter Protein . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 2

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I. CALCINEURIN, CALCINEURIN INHIBITORS, AND THE EFFECTS OF INHIBITION OF CALCINEURIN The highly specific calcineurin inhibitors cyclosporin A (CsA) and FK506 are powerful drugs, which have made organ transplantation far more successful because they inhibit the rejection reaction that would otherwise be launched by the host immune system. In addition, the basis for their immunosuppressive activity has led to an understanding of the cytoplasmic signal transduction events in T-cell activation and in the development and function of specific tissues. The various components of the T-cell signaling pathway and their elucidation have been thoroughly reviewed (Weiss and Littman 1994, Crabtree 1989; Schreiber 1991; Schreiber & Crabtree 1997), and thus this chapter will seek to give a historical overview of the use of CsA and FK506 in teasing out the specific signaling mechanisms in these events. Although over the last decade CsA and FK506 have been used in the laboratory primarily to define the cytoplasmic events involved in lymphocyte activation, very recent results indicate that these compounds may be powerful tools in studying such diverse processes as heart valve development, cardiac hypertrophy, TGF-—induced oncogenesis, neuroregeneration, memory, and synaptic plasticity. We will discuss recent advances in these areas that have been brought about, in part, by taking advantage of the specificity of these drugs for calcineurin. In addition, we will examine the structures of the inhibited calcineurin complexes, as it is the unique mode of action of CsA and FK506 that give these drugs their exquisite specificity. Finally, a generalization of the mode of action of these drugs suggests a new approach for drug development, and these implications will be discussed at the end of the chapter. A. Discovery and Development of Cyclosporin A and FK506 In 1976, scientists at Sandoz reported the isolation and structural determination of cyclosporin A (CsA), a novel immunosuppressive compound isolated from Trichoderma polysporum, a fungus found in a Norwegian soil sample (Ruegger et al., 1976). This discovery put into motion over two decades of intensive research into immunosuppression, lymphocyte activation pathways, and the prevention of transplant rejection due to host-versus-graft immune response. These workers showed that CsA was a neutral 11-membered cyclic peptide containing several novel amino acids and several amino acids existing as D-isomers, as well as several N-methylated residues (Fig. 1). The fact that CsA proved to be orally bioavailable and had good pharmacokinetics led to its rapid adoption as the drug of choice for preventing rejection of kid-

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FIG. 1. Chemical structures of cyclosporin A and FK506.

ney transplants. It is not an overstatement to suggest that the introduction of CsA revolutionized transplant medicine. In the 20 years following its introduction, the 1-year graft survival rate for recipients of cadaveric kidneys increased from 45% to 90% (Morris, 1994), and by 1992 over 250,000 human renal transplant had been performed. In fact, the greatest hurdles in transplantation therapy are now sociological rather than technical, as demand for donor tissue far exceeds the supply of donors. A decade after the discovery of CsA, researchers at Fujisawa reported the isolation (Kino et al., 1987) and characterization (Tanaka et al., 1987) of another potent immunosuppressant, the macrolide polyketide FK506 (Fig. 1), which was isolated from Streptomyces tsukubeansis, a bacterism from the Tsukuba region in northern Japan. FK506 was shown to exhibit immunosuppressive properties nearly identical to those of CsA, with similarly good oral bioavailability and pharmacokinetics, but FK506 was found to be effective at suppressing immune responses both in vitro and in vivo at concentrations 10- to 100-fold lower than those required for CsA. Despite the lack of any apparent structural homology, the common actions of these compounds (inhibition of antigen-stimulated production of interleukin (IL)-2, IL-3, and interferon (IFN)-γ, suppression of mixed lymphocyte reaction, and other indicators of an immune response) suggested that they act through a common cellular target. The cellular target for CsA was identified and purified by Handschumacher et al. (1984) at Yale, using standard biochemical purification techniques and an assay in which the target protein was used to pull radiolabeled CsA through the void volume of a gel filtration column. The target protein, named cyclophilin owing to its high affinity for CsA, was a small (18-kDa) cytosolic protein, which bound CsA with a dissociation constant (Kd) of 200 nM. In addition, cyclophilin was shown to

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FIG. 2. Peptidylproline cis-trans isomerization reaction catalyzed by immunophilins.

bind natural and unnatural analogues of CsA with affinities proportional to the strength of the analogues as immunosuppressants. However, it was not until 5 years later that a biological activity for cyclophilin was found that provided a tantalizing (albeit ultimately misleading) clue as to how CsA might mediate its immunosuppressive effects. Researchers in Japan (Takahashi et al., 1989) and Germany (Fischer et al., 1989) simultaneously reported that a known enzyme, peptidylprolyl cis-trans-isomerase (PPIase), was identical to the cyclophilin identified by Handschumacher. This enzyme is a chaperone protein that accelerates the rate of isomerization of peptidylproline bonds in nascent proteins (Fig. 2), the rate-limiting step in the folding process of many proteins. Cyclosporin A was shown to strongly inhibit this isomerase (or “rotamase”) activity toward a model proline-containing oligopeptide substrate. Previous research suggested that cis-trans proline isomerization played an important role in signal transduction involving membrane receptors and membrane transport proteins in other systems. At the time these results strongly suggested that CsA acted by inhibiting a cytoplasmic peptidylprolyl cis-trans isomerization event that was crucial in the lymphocyte activation pathway. Prolyl isomerization is highly attractive as a signaling mechanism, as it is a reversible process resulting in allosteric changes in the conformation of a protein. As a result, many investigators were seduced into believing that inhibition of cyclophilin’s isomerase activity was central to the immunosuppressive action of CsA, and massive efforts were initiated toward identifying or developing inhibitors that acted against this isomerase activity. Support for the importance of a cytoplasmic peptidylprolyl cis-trans isomerization event in the lymphocyte activation pathway was bolstered by the subsequent identification of the protein target for FK506. Independently, researchers at Harvard (Harding et al., 1989) and at Merck (Siekierka et al., 1989) isolated a second small (12-kDa) cytoplasmic protein that bound FK506 with subnanomolar affinity. This target protein, called FKBP12 (for 12-kDa FK506 Binding Protein) constitutes approximately 0.4% of the total cytoplasmic protein in Jurkat T cells, an

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FIG. 3. Chemical structures of rapamycin and 506BD.

amount comparable with to that seen for cyclophilin. In addition, and most striking, FKBP was also shown to possess peptidylprolyl cis-trans isomerization activity. This rotamase activity was shown to be strongly inhibited by FK506 but not by CsA (likewise, the isomerization activity of cyclophilin was shown to be resistant to inhibition by FK506). Further evidence supporting the view that FK506 and CsA have similar modes of action was revealed when it was shown that the only lymphocyte activation pathways that were sensitive to inhibition by FK506 or CsA were those in which a measurable rise in intracellular Ca2+ could be detected and also that pathways that did not depend on a rise in intracellular Ca2+ were insensitive to inhibition by CsA or FK506 (Emmel et al., 1989; Mattila et al., 1990). Taken together, cyclophilin and FKBP are referred to as immunophilins in reference for their strong affinity for the immunosuppressants CsA and FK506. With the discovery of cyclophilin and FKBP, early efforts toward elucidating the mode of action of FK506 and CsA focused on the immediate targets of the drugs FKBP and cyclophilin and on the rotamase activity of these targets. Despite the tantalizing evidence that cis-trans peptidylprolyl isomerization was central to the effects of CsA and FK506 on the immune response, several lines of evidence suggested it was unlikely that inhibition of rotamase activity was directly responsible for the immunosuppressive effects of these drugs. For example, while FK506 exhibits immunosuppressive activity at a concentration of 1 nM, its immediate target, the highly abundant FKBP, is present at 5 µM in the cytosol of Jurkat cells. Rapamycin (Fig. 3) is another immunosuppressive drug with considerable chemical homology to FK506 that had also been shown to bind to FKBP and inhibit its rotamase activity at nanomolar concentrations (Bierer et al., 1990a). However, the effects of rapamycin on lymphocytes are distinct from those of FK506 or CsA, which prevent

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progression of the lymphocyte cell cycle from G0 to G1, which is a Ca2+dependent process. Rapamycin, on the other hand, prevents the transition from G1 to S phase, which is a Ca2+-independent process. Although FK506 can suppress the actions of rapamycin and rapamycin can suppress the effects of FK506, a more than 10-fold excess of either drug is required to suppress the effects of the other. Such observations suggested that the excess cytosolic FKBP acts as a buffer, and therefore that even at effective immunosuppressant concentrations, considerable immunophilin rotamase activity remains. Shortly after the identification of FKBP and the subsequent characterization of its rotamase activity, inhibition of this rotamase activity was shown to be insufficient for immunosuppression. Studies using the synthetic FKBP rotamase inhibitor 506BD (Fig. 3) (Bierer et al., 1990b, Somers et al., 1991), a derivative of FK506 and rapamycin that retains the common chemical functionality of these drugs necessary for binding to FKBP (Ki = 5nM) as an inhibitor of FKBP rotamase activity but lacks functionality not necessary for this binding, was shown to not inhibit T-cell receptor (TCR) or immunoglobulin E receptor-mediated signaling pathways. However, 506BD was shown to antagonize the effects of FK506 or rapamycin (Bierer et al., 1990b). Similar CsA-derived cyclophilin ligands were also shown to have comparable effects (Sigal et al., 1991). From these studies a model was beginning to emerge in which the inhibition of the peptidylprolyl isomerase activity of cyclophilin and FKBP was not responsible for their observed immunosuppressive actions (Bierer et al., 1990a; Somers et al., 1991). Rather, the immunosuppressants CsA, FK506, and rapamycin were revealing themselves to be molecules with two chemical domains, one an immunophilin-binding domain and the other an “effector” domain, which in concert with the bound immunophilin protein targeted a second protein or process. In this model, FK506 and rapamycin bind a common immunophilin, FKBP, but the resulting FKBPFK506 and FKBP-rapamycin binary complexes exert effects on different targets. Cyclosporin A, in turn, binds to its immunophilin partner, cyclophilin, and the resulting binary complex then exerts its effects on the same target as the FKBP-FK506 complex. B. Identification of Calcineurin as the Common Target for Cyclosporin A and FK506 In 1991 the focus of immunosuppression caused by FK506 or CsA shifted away from their immediate immunophilin targets and associated rotamase activities and toward a common target that was identified downstream of the initial immunosuppressant–immunophilin interaction. In a seminal report, Schreiber and colleagues showed that either immobi-

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lized FKBP in the presence of FK506 or immobilized cyclophilin in the presence of CsA could be used to isolate the same set of four proteins from calf thymus extracts (Liu et al., 1991). These interactions could be disrupted with soluble FKBP in the presence of FK506 or with soluble cyclophilin in the presence of CsA but not with free immunophilin or free FK506, CsA, or rapamycin. In addition, the formation of either complex was dependent on the presence of both Mg2+ and Ca2+, and the complexes could be disrupted with the Ca2+ chelator EGTA. The four isolated proteins were subsequently identified as calcineurin A, a proteolytic fragment of calcineurin A, calcineurin B, and the divalent metal-binding calcineurin-regulating protein calmodulin. Calcineurin (also known as protein phosphatase 2B or PP2B) is a Ca2+- and calmodulin-sensitive serine/threonine protein phosphatase heterodimer composed of a 61-kDa phosphatase-containing protein (calcineurin A) and a 19-kDa regulatory subunit (calcineurin B). Calmodulin associates with calcineurin B to regulate the activity of calcineurin A. It was subsequently shown that the immunophilin–immunosuppressant complexes interacted directly with calcineurin and did not exert their effects via interaction with calmodulin. Although it was possible to isolate this same set of proteins from a variety of tissues from different organisms, bovine brain was found to be the most reliable source, probably owing to the fact that calcineurin constitutes approximately 1% of total protein in the brain (Klee et al., 1979). With this report, the focus of immunosuppression by FK506 and CsA shifted away from their immunophilin partners, FKBP and cyclophilin, and toward the secondary targeting of calcineurin by the immunophilin–immunosuppressant complexes. The identification of calcineurin as the in vitro target of the CsA–cyclophilin and FK506–FKBP complexes raised several questions: What significance did calcineurin play in the immune response, and was the inhibition of calcineurin by the immunosuppressant–immunophilin complexes the crucial step in the T-cell activation that was being inhibited? Previous work had shown that the function of the NF-AT (nuclear factor of activated T cells) transcription complex was completely and selectively blocked by CsA and FK506 (Emmel et al., 1989). Furthermore, these drugs blocked the cytoplasmic-to-nuclear translocation of one component of the NF-AT transcription complex, NF-ATc (Flanagan et al. 1991). The NF-AT functions as both a signal transducer and a transcription factor in that one component (NF-ATc) resides in the cytoplasm in nonstimulated cells, whereas the other component (NFATn) can be contributed by various nuclear proteins, including AP-1. The NF-ATc component (the “c” refers to the fact that this subunit is cytoplasmic when transcriptionally inactive, is calcium-dependent, and is CsA/FK506-sensitive) encompasses a family of four transcription factors

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with distant homology to the rel/NF-κB proteins.2 In unstimulated lymphocytes, NF-ATc is phosphorylated and transcriptionally inactive, residing in the cytoplasm of the cell. On stimulation through the TCR, or through pharmacological agents that mimic TCR-mediated elevations in intracellular Ca2+ concentrations and activation of protein kinase-C (PKC), NF-ATc is dephosphorylated. Dephosphorylated NF-ATc then translocates to the nucleus and associates with nuclear partners (NFATn) to drive transcription of the IL-2 gene, the IL-2 protein being the primary growth factor for T lymphocytes (Shaw et al., 1988). FK506 and CsA inhibit NF-ATc activity by preventing its translocation from the cytoplasm to the nucleus, its site of action (Emmel et al. 1989). Calcineurin was demonstrated to be responsible for the nuclear translocation of NFATc because cells overexpressing calcineurin are resistant to the inhibition of NF-ATc translocation by CsA or FK506, and show augmented NF-ATc-mediated transcription (Clipstone and Crabtree, 1992). Furthermore, it has recently been shown that calcineurin translocates to the nucleus with dephosphorylated NF-AT (Wesselborg et al., 1996; Loh et al., 1996; Shibasaki et al., 1996), where it is required to maintain NF-AT transcriptional activity (Timmerman et al., 1996). While the NF-ATc transcription factor was originally identified and characterized in T lymphocytes, it has since been recognized that a family of NF-AT proteins exists and that these proteins play important transcriptional roles in a wide variety of processes outside of the immune system (Crabtree 1999). Regulation of the NF-ATc family proteins by calcineurin has been thoroughly reviewed recently (Rao et al., 1997). With the identification of calcineurin-mediated NF-ATc translocation in T cells, a model of the cytosolic events involved in T-cell activation has emerged, as outlined in Fig. 4. Stimulation of the TCR results in a series of membrane-associated events culminating in a rise in intracellular Ca2+, which binds to and activates calmodulin/calcineurin. Activated calcineurin then causes the cytoplasmic-to-nuclear translocation of NF-ATc, an event potentially mediated via the dephosphorylation of critical serine and/or threonine residues on the NF-ATc protein (Beals et al., 1997a, 1997b). Once nuclear, NF-ATc binds DNA with its nuclear partner, NF-ATn to drive production of IL-2 and other early genes, leading to lymphocyte activation and proliferation. 2 NF-AT nomenclature is varied and confusing in the literature. NF-ATc refers to the family of cytoplasmic, calcium-dependent, CsA/FK506-sensitive proteins that translocate to the nucleus to associate with nuclear partners, collectively referred to as NF-ATn. NF-ATn can vary. Human Genome Database nomenclature for the NF-ATc family members (which this chapter will follow) is as follows: NF-ATc1 = NF-ATc; NF-ATc2 = NF-ATp or NF-AT1; NF-ATc3 = NF-AT4 or NF-ATx; NF-ATc4 = NF-AT3.

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FIG. 4. Schematic model showing the role of calcineurin in T-cell activation.

C. Role of Calcineurin Outside of the Immune System The absolute requirement for NF-AT dephosphorylation by calcineurin (and subsequent translocation in association with calcineurin to the lymphocyte nucleus) for lymphocyte activation, coupled with the specificity of CsA and FK506 for inhibition of calcineurin in lymphocytes due to the low levels of calcineurin present in these cells relative to other tissues, has allowed these drugs to be used as critical tools in elucidating the cytoplasmic events involved in lymphocyte activation. However, because of the ubiquitous expression of the immunophilins and calcineurin, as well as the multitude of phosphoproteins that can be acted on by calcineurin, the effects of CsA and FK506 in cells outside of the immune system have prevented the use of these drugs in treating diseases of autoimmunity, such as lupus or arthritis. Nephrotoxicity and renal failure in transplant recipients are often complications of CsA- or FK506-based therapy, and as a result, transplant recipients receiving CsA or FK506 must be monitored closely. Because of the toxicity of these drugs, they are not used clinically except for preventing graft

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rejection after transplants. The side effects associated with these drugs are a result of the inhibition of calcineurin in tissues other than lymphocytes, indicating that calcineurin plays a role not only in the signaling pathway that leads to the activation of T cells but also in many processes outside of the immune system. In this section we will review the effects of calcineurin inhibition in nonlymphocyte cells in order to illustrate the need for inhibitors of events downstream from calcineurin or inhibitors that prevent dephosphorylation by calcineurin of more specific target proteins. Such inhibitors would be of great benefit in treating a broader spectrum of autoimmune diseases beyond transplant rejection and could potentially be used as tools to allow for a finer elucidation of signaling pathways in cells outside of lymphocytes. 1. Role of Calcineurin and the Effects of CsA and FK506 in the Nervous System The rotamase activity of the immunophilins cyclophilin and FKBP was initially thought to be the important physiological function of these proteins. Proper geometry of proline residues is crucial for proper protein folding and function, and cyclophilin and FKBP were assumed to act primarily as intermediates in this process. While both enzymes have been shown to interconvert cis and trans isomers of proline residues within a polypeptide chain, several other roles for immunophilins have been identified, with much of the recent work focused on roles that immunophilins, specifically FKBP, play in neuroregeneration and neuroprotection (Snyder et al., 1998a, 1998b). The effect that the calcineurin inhibitor FK506 has on these processes will be discussed below, regardless of calcineurin’s involvement in the process. a. FKBP and Release of Intracellular Ca2+. Calcium is a critical signaling molecule in neurons, and FKBP and calcineurin have been shown to play a role in mediating intracellular Ca2+ concentrations in neurons. FKBP12 is present in the brain at high levels, which suggests that it could play important roles in neuronal processes. FKBP12 has been found to associate with two endoplasmic reticulum-localized Ca2+ channels, the ryanodine receptor (RyR) (Collins, 1991; Jayaraman et al., 1992), and the inositol 1,4,5-trisphosphate (IP3) receptor (Cameron et al., 1995a). In each case, FKBP was identified by virtue of being a “contaminant” in highly purified preparations of the respective receptors. FKBP appears to play a similar role in the regulation of each of these channels. FKBP works by associating with and stabilizing the channels, reducing instances of spontaneous opening and passage of Ca2+ into the cytoplasm (“leakiness”) (Brillantes et al., 1994). Binding of IP3 or ryanodine to the receptor leads to an influx of Ca2+ into the cytoplasm, activating

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kinases such as PKC and CamKII. These kinases can in turn phosphorylate the IP3 or ryanodine receptor, which increases the sensitivity of the receptor for its ligand and therefore increases the efflux of Ca2+ from the endoplasmic reticulum to the cytoplasm. Phosphorylation of the receptor is opposed by calcineurin, which is also associated with the FKBP-receptor complexes (Cameron et al., 1995b) and which is activated by the raised local concentrations of Ca2+. Subsequent dephosphorylation of the receptor decreases its sensitivity to ligand and thus leads to a lower influx of Ca2+ into the cytoplasm. This interplay between PKC-mediated phosphorylation and calcineurin-mediated dephosphorylation of receptors acting as the gatekeepers for intracellular Ca2+ stores could play an important role in intracellular oscillations of Ca2+ concentrations. Such a hypothesis is supported by studies of mice lacking FKBP12, in which defects in Ca2+ release mediated by skeletal and cardiac ryanodine receptors led to embryonic lethality (Shou et al., 1998). It is interesting to note that in this scenario, FKBP acts as a “scaffold” or “adapter” protein to localize calcineurin to its site of action. The localization of both FKBP12 and calcineurin to the receptor can be disrupted by adding FK506 (Cameron et al., 1995a), suggesting that FKBP associates with the receptor through the FKBP binding pocket, possibly at a leucine-proline site. In fact, a leucine-proline dipeptide sequence in the IP3 receptor has been shown to be critical for association with FKBP (Cameron et al., 1997). Such a model also suggests that calcineurin binds to FKBP when FKBP is bound to the IP3 receptor. b. FKBP Ligands and Neuroregeneration. Perhaps the most therapeutically promising area for calcineurin inhibitors or FKBP ligands may lie in the area of neuroregeneration and neuroprotection. Interestingly, these effects appear to be regulated both through calcineurin-mediated pathways and through a calcineurin-independent, FKBP-dependent mechanism (Hamilton and Steiner 1998). Snyder and colleagues showed that in PC12 cells, FK506, rapamycin, and CsA, as well as nonimmunosuppressive analogues of these compounds (analogues that retained their immunophilin-binding domain but that were modified in their effector domain) all enhanced neurite outgrowth in response to nerve growth factor (NGF) (Lyons et al. 1994; Steiner et al. 1997a, 1997b). Interestingly, a nonimmunosuppressive CsA analogue showed a greater effect on neurite outgrowth than did CsA. FK506 and rapamycin showed EC50 values for neurite outgrowth of 500 pM each, while that for CsA was 50 nM. The fact that rapamycin augmented, rather than antagonized, the effects of FK506 suggested that the observed effects were mediated through FKBP rather than calcineurin. Parallel results were seen in animal studies, in which rats with lesioned

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sciatic nerves were seen to recover more quickly when treated with FK506 than control animals (Gold et al., 1994, 1995). Further testing with a wide variety of FKBP ligands that contained little chemical functionality outside of that which interacts with FKBP showed that the neurotrophic effects of these ligands resided fully within their interaction with FKBP (Hamilton et al., 1997). What was perhaps most interesting was the finding that ligand potency as a neurotrophic agent did not closely parallel the inhibition of FKBP rotamase activity, with several ligands possessing ED50 values for neurite outgrowth that were two orders of magnitude below their IC50 values for inhibition of rotamase activity. In addition, several FKBP ligands possessed ED50 values for neurite outgrowth in the low picomolar range, which is far below the concentration of FKBP in neurons. It is interesting to note that at extremely low doses, CsA and FK506 have both been shown to produce a small but reproducible enhancement of lymphocyte activation and NF-AT-dependent transcription (Emmel et al., 1989; Mattila et al., 1990). These results have led to the suggestion that perhaps subtle, as yet undetected, changes in FKBP structure on ligand binding can lead to a gain of function that is responsible for the observed results. A further possibility is that the FKBP ligands disrupt a preexisting complex between an FKBP and a protein partner, leading to the observed neurotrophic effects. Alternatively, an as yet unidentified FKBP isoform may involved (Hamilton and Steiner 1998). Gold has recently reported that the neurotrophic effects of FKBP ligands are mediated through FKBP52 and not FKBP12 and suggests that disruption of FKBP52 from the hsp-90 complex is responsible for the neurotrophic effects of FKBP ligands (Gold et al., 1999). However, this model will require further investigation, as neuroregeneration is observed at FKBP ligand concentrations far below those required to saturate the pool of known FKBPs in a cell. 2. Calcineurin and Synaptic Plasticity In recent years, calcineurin has revealed itself as a key player in the establishment in neurons of long-term potentiation (LTP) and longterm depression (LTD) which are two cellular mechanisms thought to underlie synaptic plasticity and some forms of memory (Silva et al., 1997; Stevens, 1998). Transgenic mice overexpressing a constitutively active form of calcineurin show defects in spatial long term memory, and isolated neurons from these mice show defects in the induction of several forms of LTP (Mansuy et al., 1998; Winder et al., 1998). In dayold chicks, CsA treatment was seen to impair memory formation (Bennett et al., 1996). In rat hippocampal slices, induction of LTD was shown to be inhibited by FK506 (Mulkey et al. 1994), but conflicting results

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have been reported with regard to induction of LTP, some investigators reporting inhibitory effects of CsA and FK506 on LTP induction (Lu et al. 1996a, 1996b) and others an enhancing effect (Torii et al. 1995; Wang and Kelly 1997; Yakel 1997). Some of the conflicting results in the literature could be an effect of isoform-specific roles of calcineurin. It was recently shown in mice lacking the calcineurin A-α gene that the induction of neither LTP nor LTD was affected, although depotentiation (the reversal of LTP) was abolished completely (Zhuo et al. 1999). These results suggest that the establishment of LTD and the reversal of LTP operate through different mechanisms. Alternatively, calcineurin A-β or -γ could play a redundant role in neurons. Neuronal stimulation either physiologically in vivo or through the application of an electric potential ex vivo leads to a rise in intercellular Ca2+, with a concomitant activation of Ca2+-dependent kinases and phosphatases, the interplay of which stimulates a cascade of events that ultimately contributes to the establishment of synaptic strength. While in lymphocytes the activation of NF-AT by calcineurin appears to commit a cell to activation via induction of early activation genes, including the IL-2 gene, in neurons a picture is emerging in which the cyclic AMP-responsive element binding protein (CREB) transcription factor is proving to have a pivotal role in the establishment of LTP or LTD (Bito et al., 1996; Mulkey et al., 1994). Unlike NF-AT, which lies immediately downstream of calcineurin and is activated by dephosphorylation, CREB lies several steps downstream of calcineurin and is active in its phosphorylated state. Synaptic activity leads to a rise in intracellular Ca2+ with activation of calmodulin. Calmodulin then activates both the phosphatase calcineurin and the CaM kinase-kinase. Activated calcineurin dephosphorylates phosphoinhibitor 1 (PI-1), which leads to active protein phosphatase 1 (PP1); PP1 can then dephosphorylate nuclear phospho-CREB, inactivating its transcriptional activity. CaM kinase-kinase, on the other hand, phosphorylates and there by activates CaM kinase-IV (CaMKIV), which can then phosphorylate and activate CREB. Prolonged activation of CREB leads to gene expression required for the establishment of LTP. Under this scenario, oxidative conditions generated by prolonged neuronal stimulation lead to inactivation of calcineurin, thus shifting the opposing phosphorylation/dephosphorylation processes to favor CREB phosphorylation and CREB-mediated transcription required for the formation of memory. 3. Signaling through Calcineurin and NFAT in the Developing Heart Recently, calcineurin and its effect on NF-AT nuclear localization has been shown to play roles in the proper development of the heart as well

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as in the pathway leading to cardiac hypertrophy. Mice with targeted disruptions of the NF-ATc1 gene were seen to die in utero at day 13.5 to 17.5 of gestation owing to improper formation of heart valves (de la Pompa et al. 1998; Ranger et al., 1998). In wild-type mice, treatment of embryonic cultures ex utero with CsA or FK506 was seen to prevent NFATc from entering the nucleus in endocardial cells that expressed NFATc1, whereas untreated cells from control cultures showed NF-ATc1 to be nuclear at this time point. Similar defects in heart valve formation have been observed in zebra fish treated with FK506 or CsA (S.-H. Park and G.R. Crabtree, unpublished observations). What is exciting about these results is that the heart valve defects observed in the NF-ATc1 knockout mice were physiologically similar to those that are observed in approximately 1% of live human births (Hoffman 1995), suggesting a role for calcineurin/NF-ATc1 in a debilitating human disease that affects a large population. 4. Calcineurin and Cardiac Hypertrophy Besides its role in heart valve development, the calcineurin/NF-AT pathway is also involved in cardiac hypertrophy. Hypertrophy refers to an increase in cell size without cell division. This is the healthy response of cardiac myocytes to increased stress and allows for increased circulation in response to such stress. This adaptive hypertrophy, however, can progress into a maladaptive hypertrophy in which the heart muscle cells become long and thin and then fail to contract properly. Under these conditions the heart must work harder to circulate the same amount of blood, which can ultimately lead to congestive heart failure. One in 500 individuals is affected by hypertrophic cardiomyopathy (Maron et al., 1995), but the biochemical pathways involved in the pathology of this disease are not yet clearly understood. It is known that the cardiac-specific zinc-finger transcription factor GATA4 is required for transcription of genes in response to hypertrophic stimuli (Molkentin and Olson, 1997), and Olson and colleagues searched for partners for this protein using a yeast two-hybrid screen (Molkentin et al., 1998). Among the proteins identified was NF-ATC4, and as it was known that many hypertrophic stimuli lead to increased intracellular Ca2+ concentrations, this finding led to an immediate inference of calcineurin involvement in the signaling pathway. In addition, it had been shown previously that overexpression of calmodulin in the hearts of transgenic mice led to cardiac hypertrophy (Gruver et al., 1993). In cultured myocytes, the Olson group demonstrated that CsA and FK506 were able to prevent angiotensin II stimulation from inducing hypertrophic genes. They then bred transgenic mice that expressed a consti-

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tutively active calcineurin under the control of the cardiac-specific αMHC promoter, and these mice were seen to develop cardiac hypertrophy and were susceptible to sudden death due to heart failure. Similar results were obtained with mice expressing a constitutively active NFATC4 under the control of the α-MHC promoter, suggesting that hypertrophy was mediated through the action of calcineurin on NFATC4. In the calcineurin transgenic mice, treatment with CsA was seen to prevent the development of hypertrophy, although similar experiments were not conducted on the NF-ATC4 transgenic mice. While the studies described above have clearly shown that inhibition of a constitutively active calcineurin can prevent the induction of hypertrophy in transgenic mice, the results of studies of whether calcineurin inhibition prevents hypertrophy in other models of induced hypertrophy have given much less clear results (Müller et al., 1998; Olson and Molkentin, 1999; Sugden 1999; Walsh 1999). Sussman and colleagues showed that CsA or FK506 could prevent induction of cardiac hypertrophy in mice predisposed to hypertrophy by cardiac-specific overexpression of tropomodulin, a mutant form of myosin light chain-2, or fetal β-tropomyosin. Although the above three model systems have a common defect in heart muscle contractility due to dysfunctions in sarcomeric proteins that predisposes the mice to hypertrophy, CsA treatment of mice overexpressing constitutively active retanoic acid receptor (which leads to hypertrophy without a defect in sarcomeric function) did not prevent the development of hypertrophy. The most controversial results from this study involve a rat pressure-overload model, in which CsA treatment was seen to prevent hypertrophy in abdominal aortic-banded rats. In studies by others, such an effect was not observed (Luo et al., 1998), and a reexamination of the data by the Sussman group showed that although CsA treatment of aortic-banded rats led to decreased heart body ratios after 6 days of drug treatment, such an effect was not seen at longer time periods (Molkentin, 1998). Other studies have similarly shown little or no effect of CsA treatment in preventing hypertrophy in pressure-overload model systems (Ding et al., 1999; Meguro et al., 1999). One critical issue in these studies is whether the dose of CsA used was sufficient to fully inhibit cardiac muscle calcineurin. Calcineurin is abundant in cardiac muscle and may not have been fully inhibited at the concentrations used. These investigators attempted to address this question by using assays of calcineurin activity in splenic lymphocytes. However, spleen cells contain far lower levels of calcineurin than are found in most other tissues. If calcineurin is involved in cardiac hypertrophy in humans, CsA and FK506 would not be the drugs of choice for therapeutic treatment. The

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expression level of calcineurin in heart is much higher than in lymphocytes, and so in order to inhibit cardiac calcineurin, large doses of FK506 or CsA would be required. Not only would patients be immunosuppressed, but they would probably suffer from hypertension and nephrotoxicity, effects that are observed when patients are treated with the comparatively low doses necessary for immunosuppression. A more viable drug target may be a downstream effector molecule of calcineurin such as NF-ATC4. Small-molecule drugs that can disrupt the interaction between calcineurin and NF-ATC4 or the interaction between GATA4 and NF-ATC4 would be more specific than general calcineurin inhibitors and would potentially cause less side effects. However, it remains to be seen if NF-ATC4 and the interaction of GATA4 and NF-ATC4 are viable drug targets for treating cardiac hypertrophy. 5. Inhibition of Calcineurin and Transforming Growth Factor-β Release A final example of a study that illustrates the need for inhibitors that directly affect the downstream targets of calcineurin and there by bypass the general inhibition of calcineurin phosphatase activity involves the potential role of calcineurin inhibition in an increased incidence of cancer. In humans, CsA treatment has been shown to give rise to an increased incidence of malignancy, primarily lymphomas. Hojo et al. (1999) have recently presented data raising the possibility that the ability of CsA to induce transforming growth factor (TGF)-β may play a role in this process. The Hojo group had previously shown that CsA treatment of a variety of cell types leads to induction of TGF-β (Khanna et al., 1994; Li et al., 1991) and therefore investigated the implications of this effect both in vitro and in vivo. Treatment of a variety of cancerous cell lines with CsA led to the development of morphological alterations that are characteristic of an invasive phenotype: the development of exploratory pseudopodia, an increase in cell motility, and the ability for anchorage-independent growth. These effects could be inhibited by cotreatment with anti-TGF-β antibodies. In addition, tumors implanted into immunodeficient SCID-beige mice (mice lacking functional B cells, T cells, and natural killer cells) showed increased metastasis when the mice were treated with CsA. This effect was also countered by the administration of anti-TGF-β antibodies. These results do not suggest that CsA directly causes cancer, but rather that it may play a role in augmenting the development of noninvasive transformed cells into invasive transformed cells. Whether or not calcineurin plays a role in this process remains to be investigated. Several lines of evidence argue against a role of TGF-β in CsAinduced cancers. The CsA doses used in the Hojo studies are 10 to 20 times as high as those used clinically for immune system suppression,

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and in CsA-treated patients, tumors develop and progress long after drug is withdrawn (in contrast to the immediate results seen in the Hojo study). In addition, TGF-β production has not been observed either in the malignant cells or in the cells of patients being treated. Finally, these studies were conducted on cells from an unrelated malignancy, not a CsA-induced malignancy. D. Specificity of Action of CsA and FK506 In clinical practice, FK506 and CsA show subtle differences in efficacy following organ transplantation, the details of which are beyond the scope of this chapter but have been reviewed recently elsewhere (Vanrenterghem, 1998). Although many of these differences are likely due to differences in tissue distribution, dosage, dosing regimens, pharmacokinetics, and formulations, there is evidence that FK506 may play a role in preventing acute allograft rejection by a mechanism independent of its inhibition of calcineurin (Kaibori et al., 1999). In cultured rat hepatocytes, stimulation with IL-1β was shown to lead to increased levels of inducible nitric oxide synthase (iNOS), which generates nitric oxide (NO), a mediator of cellular damage. Treatment of stimulated hepatocytes with FK506 but not CsA was shown to lead to decreased iNOS mRNA and protein levels. Calmodulin antagonists were seen to have no effect on IL-1β induced NO formation, and in addition, translocation of NF-κB (a transcription factor involved in iNOS expression) to the nucleus was shown to be inhibited by FK506 but not CsA. Because both FKBP and cyclophilin are abundant in the liver, these results suggest a calcineurin-independent, FK506-sensitive pathway for NF-κB translocation and iNOS gene expression in rat hepatocytes. To date, these results have not been repeated, and therefore further studies will be necessary to confirm them. E. Physiological Inhibitors of Calcineurin Although CsA and FK506 are extremely selective inhibitors of calcineurin, some of the studies above indicate that these drugs could possibly have calcineurin-independent effects. Moreover, calcineurin shows phosphatase activity towards a wide variety of phosphoprotein substrates, and inhibition of calcineurin by immunosuppressant–immunophilin complexes blocks phosphatase activity toward a broad spectrum of phosphoproteins. Inhibitors that blocked calcineurin mediated dephosphorylation of a specific substrate (such as NF-AT, or specific isoforms of NF-AT) without affecting the dephosphorylation of other substrates would be of great therapeutic as well as academic interest. The search for

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compounds that are inhibitory only with regard to specific substrates can be aided by an understanding of the mechanisms presently in use by nature to modulate calcineurin activity. 1. Calcineurin Autoinhibition Although much is known about the pathways leading to calcineurin activation following TCR engagement with major histocompatibility complex (MHC)/peptide, comparatively little is known about calcineurin downregulation or inactivation. As described earlier, in resting T cells and at low Ca2+ concentrations, the autoinhibitory domain of calcineurin is bound to the catalytic domain, suppressing enzyme activity (Hashimoto et al., 1990). At elevated Ca2+ concentrations, Ca2+ binds and activates calmodulin, which in turn binds to the calmodulin-binding domain of calcineurin. This action causes release of the calcineurin autoinhibitory domain from the calcineurin active site, thus unleashing the phosphatase activity toward substrates such as NF-AT. Removal of or mutations in the autoinhibitory domain of calcineurin have been shown to lead to a constitutively active form of the enzyme (Fruman et al., 1995; Hubbard and Klee 1989). Unlike the CsA-calcineurin or FK506-FKBP inhibition of calcineurin activity against NF-AT, which is noncompetitive and mediated by sterically blocking NF-AT access to the active site, inhibition by the autoinhibitory domain is competitive, as was shown with kinetic studies of a 25-residue peptide derived from this domain (Parsons et al., 1994). Mutation of a conserved aspartate, thought to mimic a substrate phosphoserine or phosphothreonine, in the middle of this peptide led to substantially reduced inhibition. Increasing the size of the inhibitory peptide used from 25 to 97 residues decreased the corresponding IC50 values eight-fold from 20 µM to 2.5 µM, suggesting that additional contacts at areas remote from the active site were important in mediating the affinity of the autoinhibitory domain for the calcineurin active site (Sagoo et al., 1996). 2. Oxidative Inactivation of Calcineurin Several proteins have been implicated in regulating the activity of calcineurin in vivo. Oxidative damage is known to lead to calcineurin inactivation, and studies have shown that superoxide dismutase can prevent such inactivation, possibly by preventing oxidative damage by superoxide anions (O2–) to the Fe-Zn active center of the enzyme (Wang et al., 1996). Based on these findings, it has been suggested that the regulation of calcineurin activity by superoxide dismutase could allow for Ca2+/calmodulin-dependent cellular processes to be modulated by the cellular redox potential via inactivation of calcineurin. Tsien’s group, in looking at the duration of stimulation required to maintain elevated

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phospho-CREB levels in hippocampal neurons, showed that calcineurin is involved in elevating the rate of decrease in nuclear phospho-CREB and that inactivation of calcineurin by inhibiting superoxide dismutase facilitated prolonged elevated nuclear phospho-CREB in response to electrical stimulation (Bito et al., 1996). In contrast, in the presence of the strong radical scavenger (and antioxidant) S-PBN, even prolonged neuronal stimulation was insufficient to give rise to nuclear phospho-CREB. These results provided further evidence for a physiologic role of cellular redox potential in mediating the activity of calcineurin and calcineurin-mediated processes. Recently, Carballo et al. (1999) have shown similar effects of reactive oxygen intermediates on calcineurin activity in human neutrophils. 3. Regulation by Localization: Bcl-2 The active site of calcineurin lies in a broad, shallow groove on the enzyme surface, which is consistent with the enzyme’s broad substrate specificity. For enzymes that lack strict substrate specificity, specificity can be narrowed by localization of the enzyme to its substrate by the use of adapter proteins. Conversely, enzymes can be functionally inactivated by “mislocalizing” or relocalizing them to areas of the cell where they cannot exert their effect on substrate(s). Overexpression of a constitutively active calcineurin has been shown to induce apoptosis in some cell types, likely through activation of NFAT, and apoptosis can be suppressed by the antiapoptotic protein Bcl-2 in a dose-dependent manner (Shibasaki and McKeon, 1995). This protein is known to be localized to the mitochondrial and endoplasmic reticulum membranes via a transmembrane domain. Bcl-2 was shown to associate with calcineurin in vivo, inhibiting Ca2+ ionophore-stimulated calcineurin phosphatase activity toward NF-AT, subsequent nuclear translocation of NF-AT, and reporter gene expression (Shibasaki et al., 1997). Immunofluorescent staining of cells overexpressing Bcl-2 and calcineurin were seen to have calcineurin colocalized at internal membranes with Bcl-2, in contrast to being diffuse throughout the cytoplasm in cells not overexpressing Bcl-2. Interestingly, the Bcl-2 was only seen to exert its inhibitory effects on calcineurin when localized to mitochondrial and endoplasmic reticulum membranes. Transfection of Bcl2 mutants lacking the membrane-localizing transmembrane domain was shown to have no effect on calcineurin-mediated NF-AT dephosphorylation, translocation, or reporter gene activity. The transmembrane-deleted mutant was seen to coprecipitate with calcineurin, demonstrating that deletion of the transmembrane domain did not abolish calcineurin/Bcl-2 association. In contrast, Bcl-2 was seen to have no effect on the activation of an NF-κB-sensitive reporter gene,

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although the NF-κB transcription factor is a substrate for calcineurin. The protein Bax, a proapoptotic member of the Bcl-2 family, was shown to disrupt Bcl-2 association with calcineurin, reestablishing calcineurin phosphatase activity toward NF-AT. Whether or not Bcl-2 and Bax play a physiological role in activation-induced, calcineurin-mediated apoptosis in T cells remains to be seen. 4. Regulation by Localization: AKAP79 Calcineurin has also been shown to associate with the membranebound A kinase anchor protein (AKAP79), which is found localized in dendritic spines in hippocampal pyramidal neurons and which targets protein kinase A (PKA) to this locus, where it plays a role in the modulation of glutamate receptor channels (Rosenmund et al., 1994). Calcineurin was identified as a second AKAP-binding protein using the yeast 2-hybrid system, and it was subsequently shown that AKAP79 was able to bind calcineurin and PKA simultaneously (Coghlan et al., 1995). In vitro it was seen that AKAP79 inhibited calcineurin phosphatase activity, even in the presence of calmodulin and Ca2+, suggesting that additional regulatory mechanisms are in place modulating the activity of AKAP79-associated calcineurin. In bovine brain lysates, approximately 5% of total calcineurin was associated with AKAP79, which argues against AKAP79 inhibition of calcineurin playing a regulatory role in dephosphorylation of cytoplasmic transcription factors but rather suggests a role for calcineurin in neuronal dendritic spines. The fact that AKAP79 simultaneously interacts with the phosphatase calcineurin and the kinase PKA suggests that it could play a role in switching between the phosphorylated and unphosphorylated state of a common substrate. In contrast to inhibition of calcineurin by Bcl-2, where membrane localization was required for inhibition of calcineurin-mediated NF-AT dephosphorylation, cytosolic mutant AKAP79, lacking the membrane targeting N-terminal domain, was seen to still bind and inhibit calcineurin’s activity toward NF-AT. 5. Inhibition by Cabin-1/Cain Very recently a new, physiologic inhibitor of calcineurin has been described by Liu and colleagues at MIT and OriGene (Sun et al., 1998). This protein, called cabin-1 (calcineurin binding protein) is a 2220residue nuclear protein, which was isolated from a murine T-cell cDNA library in a yeast two-hybrid system with a truncated, catalytically inactive calcineurin used as the bait. Cabin-1 was shown to be a phosphoprotein capable of binding to and inhibiting the phosphatase activity of calcineurin, and the interaction between cabin-1 and calcineurin was shown to be sensitive to disruption by FK506 or CsA. In a mammalian

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two-hybrid system, the interaction of cabin-1 and full-length calcineurin was seen to be dependent on both phorbol 12-myristate 13-acetate (PMA) and ionomycin. Although ionomycin leads to increased Ca2+ concentrations necessary for calcineurin activation by calmodulin, calcineurin activation is independent of treatment with PMA. Because PMA leads to activation of PKC, specific PKC inhibitors were used to define the role of PKC in the cabin-1/calcineurin association. Protein kinase C activation was shown to lead to cabin-1 hyperphosphorylation, with a corresponding increase in affinity for and inhibitory activity toward calcineurin. Overexpression of cabin-1 was shown to downregulate expression of reporter genes under the control of oligomerized AP1, ARRE, NF-AT, and NF-κB binding sites, all sites that are dependent on signaling through calcineurin and are sensitive (although to varying degrees) to treatment with FK506 or CsA. The fact that cabin-1 was shown to be localized in the nucleus further supports the recent findings that activated calcineurin translocates with NF-AT from the cytoplasm to the nucleus and that calcineurin is required in its activated form in the nucleus for NF-AT-mediated transcription to occur (Loh et al., 1996; Shibasaki et al., 1996; Zhu and McKeon 1999). Shortly after the report by the Liu group, Synder and colleagues at Johns Hopkins reported on the isolation and characterization of a nearly identical protein from a rat hippocampal cDNA library using the yeast two-hybrid system, naming it cain (for calcineurin inhibitor) (Lai et al., 1998). The Snyder group showed that cain expression patterns closely matched those of calcineurin in the rat brain. In addition, the inhibitory, calcineurin-binding domain of cain was further defined as residing in a 38residue region proximal to the C-terminus of the protein. Kinetic studies showed cain to be a potent noncompetitive inhibitor of calcineurin phosphatase activity, with a Ki of 440 nM. However, these kinetic studies were undertaken with purified cain of an unknown phosphorylation state. Given Liu’s results showing that hyperphosphorylated cabin-1 is a more potent inhibitor of calcineurin activity, it is possible that the in vivo physiological Ki is significantly lower. The localization of the inhibitory domain to a 38-residue region of a 2220residue protein suggests strongly the possibility of regulated inhibition of calcineurin by cabin-1/cain. The exact role of cabin-1/cain in the downregulation of calcineurin activity remains to be seen. 6. Inhibition by Disruption of Calcineurin/NF-AT Interaction Perhaps the most exciting progress in the development of calcineurin inhibitors that specifically interfere with calcineurin dephosphorylation of NF-AT while not affecting the dephosphorlyation of other phosphoprotein substrates has come from work by Rao and colleagues (Aramburu et

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al., 1998; Loh et al., 1996). Previous studies had shown that NF-AT is associated with calcineurin in a stable complex regardless of the activation state of calcineurin (Wesselborg et al., 1996) and that it is the N-terminal region of NF-AT, which is conserved across all known NF-AT family members, that is responsible for this interaction with calcineurin (Northrop et al., 1994). Cyclosporin A or FK506 does not abolish this interaction in cell lysates, which suggests that the interaction of this N-terminal region of NFAT with calcineurin is in a region distal to the contact of calcineurin with immunophilin–immunosuppressant complexes. Further work using NFATc1 mutants localized this interaction to the sequence SPRIEIT, which is conserved in NF-ATc1 and NF-ATc2 and has a similar motif in NF-ATc3 and NF-ATc4. Peptides containing this SPRIEIT motif were shown to prevent calcineurin-mediated dephosphorylation of NF-ATc1, NF-ATc2, and NF-ATc3 in vitro without affecting calcineurin-mediated dephosphorylation of other phosphoprotein or phosphopeptide substrates. Overexpression of a peptide containing the SPRIEIT motif in the CI.7W2 T-cell line prevented NF-ATc1 translocation to the nucleus on T-cell stimulation. These studies suggest a model in which calcineurin-mediated dephosphorylation of NF-AT is dependent on interactions between the enzyme and the substrate protein at sites remote from the enzyme active site and further suggest that by interrupting these interactions one can prevent NFAT dephosphorylation. If the disruptive activity of the SPRIEIT peptide could be mimicked by a small, cell-permeable organic molecule, such a molecule could prove to be superior to CsA or FK506 either in clinical applications in treating transplant recipients or in academic studies. F. Other Inhibitors of Calcineurin Cyclosporin A and FK506 are extremely specific for inhibiting calcineurin phosphatase activity as opposed to other phosphatases. This can be explained in part because they are not active site-directed and therefore would not be expected to bind to similar active-site structural motifs found in a wide variety of phosphatases. In addition, the absolute requirement for an immunophilin partner confers even greater target specificity. To date, no other small-molecule calcineurin-specific phosphatase inhibitors have been reported. While the type 2 pyrethroid insecticide cypermethrin is being marketed by Calbiochem as an inhibitor of calcineurin with an IC50 of 50 pM, recent reports have shown no effect of cypermethrin on calcineurin activity either in purified enzyme preparations or in cellular homogenates (Fakata et al., 1998). And although the original reference reporting cypermethrin as a calcineurin inhibitor is entitled “Specific inhibition of calcineurin by type II synthetic pyrethroid insecticides,” a reading of the paper reveals

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that the specificity refers to the fact that type I pyrethroid insecticides showed no effects on calcineurin in the assay system used by these investigators, whereas the type 2 compounds did show an effect (Enan and Matsumura, 1992). The fact that these compounds are known to interfere with Na+ channel regulation, coupled with their disputed efficacy and unknown specificity, argues against the use of such compounds as calcineurin inhibitors. II. INHIBITION BY IMMUNOPHILIN/IMMUNOSUPPRESSANT COMPLEXES: THE PRESENTING PROTEIN STRATEGY Detailed study of an enzyme can provide insights leading to better, more specific inhibitors, and by studying the ways that calcineurin acts on its substrates investigators are already developing such compounds. However, the unique mechanism by which the immunosuppressants CsA, FK506, and rapamycin exert their immunosuppressive effects—by binding to an immunophilin partner to form in binary complex, which then binds to calcineurin in the case of CsA or FK506 or to the fKBP rapamycin associated protein (FRAP) in the case of rapamycin—can also provide inspiration and insight leading to new-generation enzyme inhibitors or pharmaceuticals. The crystal structures for the ternary FKBP-FK506-calcineurin complexes (Griffith et al., 1995; Kissinger et al., 1995), as well as the FKBP-rapamycin-FRAP complex (Choi et al., 1996; Liang et al., 1999) have been determined, and the lessons learned from an examination of these structures has indeed provided guidance toward the desired goals (Briesewitz et al., 1999). Given the facts that neither cyclophilin nor FKBP binds to calcineurin with measurable affinity in the absence of its immunosuppressant ligand, nor do the immunosuppressants bind to calcineurin in the absence of their immunophilin protein partners, it was postulated early on that the gain of function seen on the formation of the immunosuppressantimmunophilin complex was due to conformational changes induced on binding in either the immunosuppressant or the immunophilin. Ligandinduced allosteric changes in a protein leading to a gain of function is a common phenomenon, as is seen on binding of steroids to hormone receptors. Cyclosporin A, FK506, and rapamycin are all macrocyclic compounds and are able to assume a variety of conformational isoforms. A. Structures of Immunophilin-Immunosuppressant Complexes In 1991, Schreiber and Clardy and associates reported the crystal structure of FK506 bound with FKBP (Van Duyne et al., 1991a). While

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little conformational change was observed in the protein, major conformational changes were observed in the FK506 ligand relative to the conformation of free FK506 in the unbound state (Tanaka et al., 1987). In free FK506, the pipecolinyl amide bond is predominantly in the cis conformation, but FK506 bound to FKBP shows this bond to be in the trans conformation. The trans conformation of the pipecolinyl amide bond in bound FK506 is the same as that seen for the analogous pipecolinyl amide functionality in rapamycin in either the free or bound forms (Findlay and Radics, 1980; Van Duyne et al., 1991b). In fact, the crystal structure of rapamycin bound to FKBP revealed that rapamycin itself undergoes very little conformational change on binding to FKBP, with only minor changes in the conformation of FKBP on binding as well. It was subsequently shown that the amino acids of FKBP that are perturbed on rapamycin binding are remote from the site of interaction with FRAP in the crystal structure of the ternary complex and thus not likely to be of importance in the formation of the ternary complex (Choi et al., 1996). The crystal structure of CsA bound to cyclophilin has been determined (Pflugl et al., 1993; Ke et al., 1994), but the structure of CsA in aqueous media has not yet been described. B. Structure of the FKBP-FK506-Calcineurin Ternary Complex In 1995, the crystal structure of the ternary FKBP-FK506-calcineurin inhibitory complex was elucidated by groups at Agouron Pharmaceuticals (Kissinger et al., 1995) and Vertex Pharmaceuticals (Griffith et al., 1995). Calcineurin A has an N-terminal catalytic domain of approximately 340 residues followed by a 40-residue calcineurin B binding domain, a shorter (25-residue) calmodulin binding domain, and finally a short C-terminal autoinhibitory domain, which interacts with the active site of the catalytic domain. The Vertex group determined with a 2.5 Å resolution the crystal structure of the ternary complex using calcineurin B and a proteolytic fragment of bovine calcineurin A that contained the catalytic and calcineurin B binding domains but lacked the C-terminal calmodulin-binding and autoinhibitory domains. The Agouron group resolved the crystal structure of the human calcineurin A-calcineurin B heterodimer to 2.1 Å resolution using full-length calcineurin A, as well as the inhibited ternary complex containing FKBP12 and FK506 with 3.5 Å resolution. In the Agouron structure of the calcineurin A-calcineurin B heterodimer unassociated with the FKBPFK506 complex, the majority of the C-terminal residues constituting the calmodulin-binding and autoinhibitory domains were found to be disordered and thus indistinguishable on the electron density map.

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However, an 18-residue segment of the autoinhibitory domain, composed of two short α-helical regions, was clearly visible in close contact with the catalytic domain, spanning the enzyme active site. This observation was consistent with previous studies showing competitive inhibition kinetics using a 25-residue peptide from the calcineurin A autoinhibitory domain (Parsons et al., 1994; Sagoo et al., 1996). The crystal structures of the ternary FKBP-FK506-calcineurin complex as described by both groups is remarkably similar, with the additional information from the Vertex group showing that binding of FKBP-FK506 displaces the autoinhibitory domain from contact with the catalytic domain (in contrast to the structure lacking FKBP-FK506, electron density for the autoinhibitory domain could not be discerned in the ternary complex). A diagram of the structures is shown in Fig. 5 (see color insert). The FKBP-FK506 binary complex interacts with calcineurin at a site removed from the active site, making its primary interactions with the exposed face of the calcineurin-B binding helix and with calcineurin B. Additional interactions are made with the catalytic domain, but remarkably, no part of the FKBP-FK506 protein comes within 10 Å of the enzyme active site. This finding immediately clarified a puzzling result that had been observed in previous studies: Whereas the FKBP-FK506 complex inhibits calcineurin activity toward large protein substrates such as NF-AT, dephosphorylation of smaller phosphopeptides or small-molecule organophosphates is not inhibited by formation of the ternary complex. In fact, binding of FKBP-FK506 to calcineurin actually increases calcineurin phosphatase activity toward the small organic molecule p-nitrophenyl phosphate (Liu et al., 1991). Although no structural changes could be observed in the calcineurin A catalytic domain at the resolution of these studies, it is possible that subtle alterations in active-site geometry brought about by the few contacts between FKBP and the calcineurin A catalytic domain might be responsible for this increase in activity toward certain substrates. However, the crystal structures suggest a model in which the FKBP-FK506 complex inhibits calcineurin activity toward phosphoprotein substrates by forming a complex with calcineurin that sterically blocks access of large phosphoproteins to the catalytic active site. The overall structure of the FKBP-FK506 binary complex is unperturbed on binding to calcineurin, and no gross structural perturbations could be detected in calcineurin at the resolution of these studies, aside from the displacement of the autoinhibitory domain seen in the study with full-length calcineurin. However, on binding, 400 to 550 Å2 of solvent-accessible surface area is buried for each component (Stoddard and Flick, 1996). The C15-C21 region of FK506 is seen to be inserted

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into an 8 Å long binding cleft formed between calcineurin B and the calcineurin B binding domain of the calcineurin A chain, with the C21 allyl functionality of FK506 inserted deep into a pocket within this cleft, making numerous hydrophobic interactions with the protein. The Vertex group noted that there were “subtle but significant changes scattered throughout FKBP12 at the calcineurin interface” and that “it is possible that some of this flexibility is necessary to optimize complementarity between the calcineurin and binary complex effector surfaces.” The fact that no component of the ternary complex undergoes gross conformational perturbations on complex formation beautifully illustrates the fact that it is a new composite surface formed by the immunosuppressant and immunophilin that is that actual inhibitory species in the complex. The expression “composite surface” refers to the chemical functionality that is displayed on the surface of the immunophilin protein once it binds to its immunosuppressant ligand. When the immunosuppressant-immunophilin complex is considered as a single species, it is as if the surface area of the immunosuppressant that can make contact with its ultimate target has been “enlarged” by annexing the chemical functionality of the immunophilin protein. FK506 serves to bring the FKBP and calcineurin protein surfaces together, and then by slight movements of residues at those interacting surfaces the most energetically favorable complementary conformations of those interacting residues can be achieved, leading to tight binding interactions where previously interactions were undetectably weak. In this way, FKBP acts as a “presenting protein” in that by presentation of the effector domain of FK506 to calcineurin along with the FKBP protein surface, not only are FK506-calcineurin contacts made, but FKBP-calcineurin contacts as well. It is this mode of action that is the basis for the high-affinity binding interaction that is achieved. C. Structure of the FKBP-Rapamycin-FRB Ternary Complex The crystal structure of the cyclophilin–CsA–calcineurin ternary structure has yet to be resolved but the ternary structure formed by rapamycin-mediated interactions between FKBP12 and the 12-kDa FKBP-rapamycin binding (FRB) domain of the 289-kDa FRAP protein has been determined with 2.7 Å resolution (Choi et al., 1996), which has more recently been refined to 2.2 Å resolution (Liang et al., 1999). The structure of this complex is shown in Fig. 6 (see color insert). Several similarities as well as differences in the overall mode of interaction can be seen relative to what is observed with the FKBP-FK506-calcineurin structure. As was seen with the FKBP-FK506 calcineurin ternary complex, there are no overall gross conformational changes

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observed for any member for the complex. Whereas in the FK506 was seen to not shift position relative to FKBP on formation of the FKBPFK506-calcineurin complex, rapamycin rotates by 9 degrees in the FKBP binding pocket on formation of the FKBP-rapamycin-FRP complex. This rotation translates into a 1.3 Å shift in the position of the C23 methyl group, the part of the rapamycin molecule most deeply buried in the FRB pocket (Van Duyne et al. 1991b). Also in contrast to the FKBP-FK506-calcineurin structure, in which numerous protein-protein interactions are observed between FKBP and calcineurin, relatively few interactions are observed between the FKBP and FRB binding partners in the rapamycin-mediated complex. Two loops of the FKBP protein, referred to as the 40s loop and the 80s loop (for the residues they contain) make the only contacts with FRB in the ternary complex. There is one hydrogen bond and one water-mediated salt bridge in the interaction of the 40s loop with FRB, and there are two hydrogen bonds and two water-mediated interactions in the 80s loop interaction with FRB. However, 400 Å2 of surface area is buried between the two proteins by the rapamycin-mediated interactions. III. GENERALIZATION OF THE PRESENTING PROTEIN STRATEGY The key to the remarkable specificity and high affinity of CsA and FK506 for their ultimate target, calcineurin, is the formation of a composite surface between these drugs and FKBP or cyclophilin, respectively. Cyclosporin A, FK506, and rapamycin are bifunctional molecules. One region of these molecules binds to an immunophilin protein, and the other region of the molecule, the effector domain, interacts with the drug target. Cyclophilin and FKBP therefore act as presenting proteins, binding to one half of the bifunctional molecule and displaying (presenting) the other half, the effector domain, along with the presenting protein surface. In this way a new, composite surface is formed by the immunophilin and the immunosuppressant. In essence, this composite surface represents a surface enlargement of the drug. When the drug target (i.e., calcineurin) binds the immunosuppressantimmunophilin complex, it establishes contacts with the effector domain of the immunosuppressant as well as with the presenter protein surface. By themselves, the contacts with presenter protein alone or with drug alone do not result in appreciable binding. However, the combination of effector domain–target and presenter protein–target interactions leads to high-affinity binding in the ternary complex. As a result of the immunophilin protein presenting the immunosuppressant effector domain in the context of its own protein surface, the unde-

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tectable affinity of the drugs for calcineurin is increased 104- to 105-fold. An affinity enhancement effect of such magnitude can only be the envy of any medicinal chemist wishing to boost the affinity of a lead compound to a point where the molecule becomes a viable drug candidate. The strategy of presenting a low-affinity ligand in the context of a presenting protein surface in order to boost the affinity of the ligand for its target is not unique to the immunosuppressants and immunophilins. The cell-surface major histocompatibility complex (MHC) presents small antigenic peptides to the T-cell receptor on T lymphocytes in order to elicit an immune response. The MHC or the antigenic peptide alone has insufficient affinity for the TCR to elicit such a response, but when the peptide is presented within the context of the MHC surface, interaction with the TCR is of sufficient strength to elicit such a response. In recent years we have gained insights into the characteristics of protein-protein interactions, and together with our understanding of the mode of action of CsA, FK506, and rapamycin, it may be time to consider if the lessons learned from nature can be applied to existing small-molecule ligands. The approach taken by nature is unlike any tool used in the development of drugs today, and it should give us an incentive to consider if the lessons learned can be applied to an alternative approach for drug development. A. Using a Presenter Protein to Enhance the Affinity of a Small Molecule Ligand: Toward a New Approach to Enhance Drug–Target Interactions The creation of high-affinity ligands for drug targets is an important challenge to the pharmaceutical industry. The process often involves the identification of lead compounds by screening large libraries of molecules. Once a candidate molecule is found, the tools of medicinal chemistry are used to optimize the molecule, modifying its functional groups and structural properties with the primary goal of improving affinity and specificity for the target. However, it is often not possible to create high-affinity ligands for a given protein domain because of the shape or size of the targeted binding pocket. In addition, it is particularly difficult to use a small-molecule drug to disrupt protein–protein interactions, since such interactions often take place over hundreds of square angstroms. Such interactions are often marked by a multitude of favorable interactions between the protein partners, which can be difficult to disrupt with a small-molecule drug that interacts favorably with only a few functional groups. In addition, the surfaces involved in mediating protein–protein interactions are often relatively flat, providing no pocket that could support a high-affinity small-molecule ligand.

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The mode of action of CsA, FK506, and rapamycin suggests a novel general approach to enhance the affinity of drugs for their targets. These drugs all achieve high affinity for their targets by binding to presenting proteins that enlarge their effective surfaces so that additional contacts can be established with their target. This observation suggests that a promising compound that lacks strong affinity for its target could potentially be improved by being presented in the context of a protein surface such as FKBP or cyclophilin. To achieve such presentation, the compound of interest would be chemically coupled to a ligand for the endogenous presenter protein so that a new bifunctional molecule is obtained. When the bifunctional molecule enters the cell, the presenter protein binds to the half of the new dimeric compound that contains its ligand moiety. As a result, the compound of interest, which now constitutes the effector domain of the bifunctional molecule, forms a composite surface with the surface of the presenter protein. When the target protein binds its ligand in the context of the presenter protein, it is brought into close proximity with the presenting protein surface so that additional protein-protein contacts may be established. If these interactions are energetically favorable, the binding to the target will occur with enhanced affinity relative to that of the unmodified ligand. Critical for the establishment of favorable protein–protein interactions is the linker that connects the ligand for the presenting protein and the drug of interest. First, the linker must have the right length. If it is too long, the protein surfaces will not interact, and if it is too short, steric hindrance will prevent the formation of the trimeric complex. Furthermore, the linker has to juxtapose the two protein surfaces in such a way that favorable protein–protein interactions are established. Not every juxtaposition of the presenting protein surface with the surface of the drug target protein will lead to favorable contacts. For some presenting proteins and drug targets a favorable juxtaposition may not exist at all. However, protein surfaces have revealed a certain degree of plasticity (Atwell et al., 1997; Garcia et al., 1998), which will support the mutual accommodation of two touching protein surfaces. By using a combinatorial approach for the generation of linkers, the spatial orientation of the two protein surfaces relative to one another can be systematically varied in the search for an orientation that leads to the formation of a stable trimeric complex. B. Properties of Good Presenter Proteins A good presenter protein should be expressed at high levels in the same cell as the drug target. It should have a high-affinity, cell-perme-

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able ligand, which can be linked to molecules of interest without affecting the affinity of the ligand for the presenter protein. Furthermore, the ligand should not cause an adverse biological effect on interacting with the presenting protein. FKBP, which is expressed at high concentrations in all cells and tissues, fulfills these criteria (Galat, 1993; Kay, 1996). The toxic effects caused by FK506 in humans have been linked to the inhibition of calcineurin and not to the inactivation of FKBP. In animal studies, ligands of FKBP that do not bind calcineurin have been shown to be nontoxic even at high concentrations (Dumont et al., 1992). The fact that a large number of FKBP molecules can be usurped for the purpose of serving as a presenter protein is further supported by the finding that the ablation of one FKBP allele, and hence the reduction of the protein levels by 50%, shows no phenotypic effects in mice (Shou et al., 1998). Moreover, there are many known high-affinity FKBP ligands in the literature that do not inhibit calcineurin. Just as different linkers between presenter protein ligands and drugs can be used to control the orientation of presenter protein and drug target surfaces, different FKBP ligands will also allow for linkages to drugs that present the drugs in different orientations relative to the FKBP surface. Perhaps the most convincing argument for using FKBP as a presenting protein to create a composite surface with a ligand of interest is the fact that this protein has been chosen twice by nature to fulfill this role. Both FK506 and rapamycin use FKBP to form a composite surface in order to bind tightly to their ultimate targets. The composite surfaces formed by FKBP with FK506 and by FKBP with rapamycin interact with two very different target proteins. This may reflect an inherent propensity of FKBP to interact with a wide variety of proteins. FKBP is a peptidylprolyl isomerase, as is cyclophilin. It may be that the role of these proteins in the folding of a wide range of protein substrates predisposes them to establish favorable contacts with a wide variety of protein surfaces. However, the choice of presenter proteins need not be limited to peptidylprolyl isomerases because all protein surfaces should show some degree of plasticity. Although the plasticity of some surfaces may be limited to the movement of amino acid side chains because of a rigid peptide backbone structure, other protein surfaces may be more accommodating because of the potential for large movements of relatively flexible structures such as loops. C. Enhancing the Affinity of an SH2 Domain Ligand with a Presenter Protein The important role of SH2 domains in many signaling pathways, makes them ideal candidates for therapeutic intervention. In order to

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test if the affinity of a small-molecule ligand for its target protein can be enhanced by presenting the ligand in the context of a protein surface, studies were undertaken using the Fyn SH2 domain as the target protein, a phosphopeptide tetramer (phosphotyrosine-glutamate-glutamate-isoleucine, pYEEI) as the small-molecule ligand, and two different FKBP family members, FKBP12 and FKBP52, as presenter proteins (Briesewitz et al., 1999). Aided by the availability of the cocrystal structures of Fyn-pYEEI (Mulhern et al., 1997), FKBP12-FK506 (Holt et al., 1993; Van Duyne et al., 1991a) and FKBP52-FK506 (Craescu et al., 1996), a linker was designed to chemically connect pYEEI to FK506 in such a way that the FKBP and Fyn SH2 domain surfaces were predicted to make contact on formation of the trimeric presenter protein – bifunctional molecule – target protein complex. In the resulting bifunctional molecule (referred to as FKpYEEI (Fig. 7), FK506 has lost its calcineurin binding activity but maintains its high affinity for FKBP. When the affinity of FKpYEEI binding to the Fyn SH2 domain was measured in the presence and in the absence of FKBP52, it was found that binding occurred with threefold higher affinity in the presence of FKBP52 relative to in its absence. These results support a model in which pYEEI is presented to the Fyn SH2 domain in the context of the FKBP52 surface (via association of the FK506 moiety with the FKBP52 protein). The formation of this trimeric complex allows favorable interactions to be established between the presenting protein and the target protein, leading to an enhancement of the affinity of the pYEEI moiety for its target. Binding of FKpYEEI by FKBP12 neither enhanced nor decreased the affinity of the pYEEI moiety for the Fyn SH2 domain, probably because no protein contacts are established or because the created protein contracts have a net neutral effect. These experiments demonstrated that the affinity of a small-molecule ligand for its target can be enhanced through additional contacts between the target protein and a presenter protein. IV. CONCLUSIONS The phosphatase calcineurin plays a critical role in many different physiological systems. After the initial elucidation of the central role this enzyme plays in the immune system, it has recently been implicated in synaptic plasticity and other neuronal processes, in cardiac valve development, and even in cardiac hypertrophy. The later is a disease process for which a calcineurin-regulated pathway may be a good site for therapeutic intervention. In all of these systems, the calcineurin

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FIG. 7. Structure of FKpYEEI and enhanced binding to Fyn SH2 domain in the presence of FKBP52.

inhibitors CsA and FK506 have played key roles in developing our understanding of the mechanisms involved. The ability of CsA and FK506 to prevent graft rejection by suppression of the immune response has made organ transplantation much more successful. However, because calcineurin plays central roles in many processes outside of the immune system, better calcineurin inhibitors may not make better pharmaceuticals. Instead, pathways that lie downstream of calcineurin are ripe for therapeutic targeting, and better immuno-

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suppressants or drugs that treat cardiac hypertrophy may be developed by targeting such pathways. The mode of action of the calcineurin inhibitors CsA and FK506 is remarkable. By themselves, these molecules have no measurable affinity for calcineurin, but by first binding to an endogenous presenter protein, they enlarge their effective surfaces so as to make presenter protein–target protein contacts in addition to drug–target protein interactions. As a result, binding takes place with very high affinity. This mode of action may be generalizable for enhancing the affinity of other small molecules for their targets. ACKNOWLEDGMENT Kurt Vogel is supported by PHS grant number 5T32CA09151, awarded by the National Cancer Institute, U.S. Department of Health and Human Services.

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PURE SELECTIVE ESTROGEN RECEPTOR MODULATORS, NEW MOLECULES HAVING ABSOLUTE CELL SPECIFICITY RANGING FROM PURE ANTIESTROGENIC TO COMPLETE ESTROGEN-LIKE ACTIVITIES BY FERNAND LABRIE, CLAUDE LABRIE, ALAIN BÉLANGER, VINCENT GIGUERE, JACQUES SIMARD, YVES MÉRAND, SYLVAIN GAUTHIER, VAN LUU-THE, BERNARD CANDAS, CÉLINE MARTEL, AND SHOUQI LUO Oncology and Molecular Endocrinology Research Center, Laval University Medical Center (CHUL), Québec, G1V 4G2, Canada

I. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. Women’s Health Needs. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Breast Cancer—Tamoxifen . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Menopause, Osteoporosis, and Cardiovascular Disease . . . . . . . . . . . . . . . . C. Intracrinology—Estrogens from Both Ovarian and Adrenal Origins . . . . . D. Limitations of Estrogen Replacement Therapy . . . . . . . . . . . . . . . . . . . . . . . E. Need of an Antiestrogen Having Pure Antiestrogenic Activity in the Mammary Gland and Endometrium . . . . . . . . . . . . . . . . . . . . . . . . . . III. The Estrogen Receptors and Their Multiple Gene Activation Mechanisms . . A. Nuclear Receptors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Estrogen Receptors ERα and ERβ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Activation Functions AF-1 and AF-2 of the Estrogen Receptors . . . . . . . . . . D. AP-1 Response Element . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . E. Coactivators and Corepressors . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. Classes of Antiestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Steroidal Antiestrogens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Triphenylethylenes. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Benzothiophenes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. The Benzopyrans EM-652 (SCH 57068) and EM-800 (SC H57050). . . . . . . V. Properties of EM-652 (SCH 57068) and EM-800 (SCH 57050) . . . . . . . . . . . . . A. Binding Characteristics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. EM-652 Blocks Both AF-1 and AF-2 Functions of ERα and ERβ . . . . . . . . . . C. EM-652 Blocks SRC-1–Induced Activity of Both ERα and ERβ. . . . . . . . . . . D. EM-652 Blocks the Recruitment of SRC-1 at the AF-1 of ERβ. . . . . . . . . . . . E. Inhibition of the Development and Growth of DMBA-Induced Mammary Tumors in the Rat. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . F. Inhibition of the Growth of Human Breast Cancer ZR-75-1, MCF-7, and T-47D Cell Lines in vitro . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . G. Comparison of the Effect of EM-800 and Tamoxifen on the Growth of Human Breast Cancer Xenografts in Nude Mice . . . . . . . . . . . . . . . . . . . H. Prevention of Bone Loss by EM-652 and Raloxifene . . . . . . . . . . . . . . . . . . . I. Inhibitory Effects of EM-652 on Serum Cholesterol and Triglyceride Levels References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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Copyright © 2001 by Academic Press. All rights of reproduction in any form reserved. 0065-3233/01 $35.00

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I. INTRODUCTION The progress being achieved in the field of antiestrogens is simply remarkable. Some new compounds possess characteristics that could never have been predicted by even the most optimistic scientist. Just a few years ago, the potential benefits of these new molecules for both the prevention and treatment of a series of diseases could not be foreseen. In fact, these new antiestrogens could well become the best example of the success achievable by biomedical and pharmaceutical research and of the efficient partnership between university and industry. The new chemically provided antiestrogens induce three-dimensional structural changes of the estrogen receptor (ER), which lead to a multitude of different activities of the ER–antiestrogen complex that are specific for each target cell type and gene. Such ligand-induced modifications of the three-dimensional structure of the ER, which are unique to each antiestrogen, lead at one extreme to a complete blockade of the normal action of estrogens in some tissues while in other tissues the same ER complex completely mimics or even surpasses this natural action. Knowing that such an absolute selective action of antiestrogens is possible, the objective of pharmaceutical research is to design compounds that will act in a beneficial way in all the tissues of special interest for women’s health. Since breast and uterine cancer are estimated to represent 35.5% of all new cancer cases and to have been responsible for 18.3% of all cancer deaths in women in the United States in 1999 (Landis et al., 1999), and since osteoporosis and cardiovascular disease are the main causes of morbidity and mortality after menopause, the ideal compound would be one having preventive as well as curative effects on all these diseases that most frequently affect women’s health. What could only be a dream a few years ago has become a reality: recent discoveries of pharmaceutical research offer women the hope of achieving a dramatic reduction in the incidence of breast and uterine cancer while protecting them against bone loss and fracture as well as reducing their risk of cardiovascular diseases. While of major importance for preventive and therapeutic medicine, these unexpected cell-specific properties of the new antiestrogens offer unique tools to investigate and further understand the detailed mechanisms of action of estrogens and antiestrogens, including the structural domains of ERs responsible for their delicate interactions with the numerous other intracellular regulatory proteins (coactivators and corepressors) that culminate in cell-specific stimulation or inhibition of the expression of each estrogen-sensitive gene. The availability of these new compounds should provide the means to make significant progress

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in the understanding of the detailed mechanisms involved in the numerous cellular functions regulated by estrogens under both normal and pathological conditions. II. WOMEN’S HEALTH NEEDS Estrogens, especially 17β-estradiol (E2), have long been recognized as playing a key role in the development, growth, and function of female sex organs, mammary glands, and female sex characteristics (Grese and Dodge, 1998; Henderson et al., 1988). An important role of estrogens has also been identified for the skeletal system, the cardiovascular system, and the central nervous system (Ciocca and Roig, 1995; Ciocca et al., 1995; Smith et al., 1994). Although estrogen replacement therapy at menopause can prevent bone loss and cardiovascular disease, there is evidence that estrogens are associated with an increased risk of breast cancer as well as endometrial cancer(Gambrell, 1994), which thus seriously limits the use of estrogen replacement therapy. The ideal compound for women’s health should be one able to decrease the risk of the most important causes of morbidity and mortality in women, namely breast cancer, uterine cancer, osteoporosis, bone fractures, and cardiovascular disease. Heart disease is, in fact, the leading cause of death in postmenopausal women (Lerner and Kannel, 1986). This ideal compound should also have an excellent safety profile to ensure compliance over 20 to 40 years of a woman’s life. A. Breast Cancer—Tamoxifen 1. General Breast cancer is the most frequently found cancer in women, with approximately 176,300 new cases and 43,700 deaths in the United States in 1999 (Landis et al., 1999). Present therapies for breast cancer achieve meaningful clinical results in only 30 to 40% of patients, with response duration usually limited to 12 to 18 months (Dickson and Lippman, 1987; Horwitz and McGuire, 1978; Mouridsen et al., 1978; Wakeling, 1993). Five-year survival in women with metastatic disease is still only 10 to 40%. Among all risk factors, estrogens are well recognized to play the predominant role in breast cancer development and growth (Asselin and Labrie, 1978; Davidson and Lippman, 1989; Dowsett et al., 1993; McGuire et al., 1975). However, existing surgical or medical ablative procedures do not result in complete elimination of estrogens in

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women (Miller, 1987) because of the contribution of the adrenal glands, which secrete high levels of dehydroepiandrosterone (DHEA) and its sulfate (DHEA-S), which are converted into estrogens and androgens in peripheral target tissues (Labrie, 1991; Labrie et al., 1995a; Labrie et al., 1996). Considerable attention has thus focused on the development of blockers of estrogen biosynthesis and action (Dauvois et al., 1991; de Launoit et al., 1991; Gronemeyer et al., 1992; Labrie et al., 1992a; Labrie et al., 1995b; Lévesque et al., 1991; Wakeling and Bowler, 1988a). Since the first step in the action of estrogens in target tissues is binding to the estrogen receptor (Green et al., 1986; Kumar and Chambon, 1988), a logical approach for the treatment of estrogen-sensitive breast cancer is the use of antiestrogens, or compounds that block the interaction of estrogens with their specific receptors. Until very recently, however, no agent with pure antiestrogenic activity in vivo has been available. 2. Tamoxifen Tamoxifen, the antiestrogen most widely used for the treatment of breast cancer, has shown clear clinical benefits in advanced breast cancer, its efficacy being comparable with that achieved by ablative and additive therapies (Furr and Jordan, 1984) (Fig. 1). In the first clinical studies, initiated in 1969, tamoxifen was found to achieve remissions in advanced breast carcinoma similar to those observed following estrogen therapy but with fewer side effects (Cole et al., 1971). Since then, because of its favorable profile and clinical efficacy, comparable with that of other endocrine therapies, including oophorectomy and estrogens, tamoxifen has become the treatment of choice for patients with advanced or metastatic breast cancer (Buchanan et al., 1986; Howell et al., 1990; Ingle et al., 1981). Tamoxifen, however, is known to possess mixed estrogenic and antiestrogenic activities (Furr and Jordan, 1984; Labrie et al., 1992a; Poulin et al., 1989b), which are species-, tissue-, cell-, and even gene-specific (Chalbos et al., 1993; Gottardis et al., 1988). In support of the clinical evidence for the estrogenic activity of tamoxifen on human breast cancer growth (Canney et al., 1987; Howell et al., 1992), tamoxifen and its active metabolite 4-hydroxytamoxifen have been found to stimulate the growth of human breast cancer cells in vitro and in vivo (Darbre et al., 1984; DeFriend et al., 1994b; Gottardis et al., 1988; Katzenellenbogen et al., 1987; Poulin et al., 1989a; Reddel and Sutherland, 1984; Roos et al., 1982; Simard et al., 1997a; Thompson et al., 1989; Wakeling et al., 1989). Tamoxifen probably acts as an estrogen agonist more frequently than is generally thought, and this may explain some of the apparent para-

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FIG. 1. Molecular structures of antiestrogens.

doxes of endocrine treatments, such as response to second endocrine therapy and withdrawal responses (Howell et al., 1990). Additionally, while benefits of tamoxifen on breast cancer are observed in up to 40% of patients, the long-term use of this compound has recently been recognized as being associated with a significant increase in the incidence of endometrial carcinoma (Andersson et al., 1991; Atlante et al., 1990; Boudouris et al., 1989; Cohen et al., 1993; Fornander et al., 1989, 1993; Gusberg, 1990; Hardell, 1988; Jordan, 1988; Killackey et al., 1985; Magriples et al., 1993; Malfetano, 1990; Mathew et al., 1990; Nayfield et al., 1991; Neven et al., 1989). This effect is likely caused by the intrinsic estrogenic activity of the compound, possibly because of its genotoxic action on DNA, by forming DNA adducts. All analogues of tamoxifen, including toremifene, droloxifene, and idoxifene, also possess estrogenic effects analogous to those of tamoxifen (Gauthier et al., 1997; Simard et al., 1997b).

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In fact, the most widely recognized adverse effects of tamoxifen are those related to the stimulatory effects on the endometrium. These effects include proliferation of the endometrium (Dijkhuizen et al., 1996; Hann et al., 1997; Lahti et al., 1993), formation of endometrial polyps (Cohen et al., 1997; Hann et al., 1997; Kedar et al., 1994a; Lahti et al., 1993), hyperplasia (Cohen, 1997; Lahti et al., 1993), and cancer (Fisher et al., 1994; Rutqvist et al., 1995; Stearns and Gelmann, 1998; Wilking et al., 1997). Other reproductive side effects of tamoxifen are worsening of endometriosis (Buckley, 1990; Hajjar et al., 1993; Ismail and Maulik, 1997), adenomyosis (Cohen et al., 1997), and proliferation of benign uterine tumors (Cohen, 1997; Kang et al., 1996). While tamoxifen was being used for the treatment or prevention of breast cancer, it was discovered by chance that this compound had positive effects on bone, although its antiestrogenic activity was originally expected to accelerate and not to reduce bone loss (Evans and Turner, 1995; Jordan, 1993). B. Menopause, Osteoporosis, and Cardiovascular Disease There is no medical problem related to women’s health that has a higher negative impact on morbidity (and frequently mortality) than menopause, a condition closely associated with declining sex steroid availability. The most important problems associated with menopause are osteoporosis, atherosclerosis, and cardiovascular diseases. It is estimated that 40 million women are menopausal or postmenopausal in the United States alone (Andrews, 1995; Curtis, 1999). Osteoporosis, an already serious problem, is compounded by increasing life expectancy. Such an incidence of osteoporosis results in approximately 300,000 hip fractures annually in the United States, with an annual increase of 10,000 to 20,000 due to aging of the population. More than 50% of hip fracture patients lose their social independence permanently. Moreover, between 12 and 20% of hip fracture patients die within 1 year from complications of such fractures. It is estimated that 1.7 million hip fractures occur annually worldwide as a complication of osteoporosis. The most widely recognized fact concerning menopause is that there is a progressive decrease and finally an arrest of estrogen secretion by the ovaries. The cessation of ovarian estrogen secretion is illustrated by the marked decline in circulating 17β-estradiol (E2) levels. This easily measurable change in circulating E2 levels, coupled with the demonstrated beneficial effects of estrogens on menopausal symptoms and bone resorption (Christiansen et al., 1982), has concentrated most of the efforts of hormone replacement therapy (HRT) on various forms of

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estrogens as well as on combinations of estrogen and progestin in order to avoid the potentially harmful stimulatory effects of estrogens used alone on the endometrium, which can result in endometrial hyperplasia and cancer. The action of estrogens in the prevention and treatment of osteoporosis is related to an inhibitory effect on bone resorption, which leads to decreased bone formation and therefore decreased bone turnover (Turner et al., 1994). It should be mentioned, however, that recent data suggest that progestins as well as estrogens have a negative impact on breast cancer (Clarke and Sutherland, 1990; Horwitz, 1992; Musgrove et al., 1991), with reports indicating an increased risk of this cancer (Colditz et al., 1995). Estrogen replacement therapy has been found to lower serum levels low-density lipoprotein (LDL) cholesterol, lipoprotein (a), and apoliprotein B, and to increase serum levels of high-density lipoprotein (HDL) cholesterol and apolipoprotein A-1 (PEPI Trial, 1995; Tikkanen, 1996; Ylikorkala et al., 1995). Estrogens have also been found to have direct effects on blood vessels, including increased synthesis of nitric oxide and increased vasodilation (Farhat et al., 1996; McCrohon et al., 1996). The observed decrease in the risk of cardiovascular disease and atherosclerosis by estrogens (Pines et al., 1997; Punnonen et al., 1995; Stampfer and Colditz, 1991) is thought to be due to the combined effects of estrogens on serum lipids and vascular reactivity. Despite the well-known beneficial effects of estrogen therapy on menopausal symptoms (Grady et al., 1992; Greendale and Judd, 1993; Lomax and Schonbaum, 1993) and their role in reducing bone loss and coronary heart disease (Barrett-Connor and Bush, 1991; Field et al., 1993; Harris et al., 1991; Lindsay, 1993; Lobo, 1991; Stampfer et al., 1991), compliance is low. Women decide not to take estrogens or stop treatment early because of the fear of breast and uterine cancer (Grady et al., 1992) and of symptoms associated with their therapy, namely, uterine bleeding, breast tenderness, and fluid retention. Menopause is thus the condition in most urgent need of tissue- or cell-targeted endocrine therapy. In fact, estrogen-like compounds are required for prevention of bone loss and cardiovascular disease. On the other hand, for the breast and endometrium, the ideal approach is a pure antiestrogen that would inhibit the development and growth of breast and endometrial carcinoma. The effects required in the skeletal and cardiovascular systems (estrogenic) are thus the opposite of those required in the breast and endometrium, for which antiestrogenic activity is recommended. The availability of such a compound would be a major step forward as compared with standard estrogen-based replacement therapy. Such a

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compound could be called a pure selective estrogen receptor modulator (SERM) or a pure breast and uterus SERM. The acronym SERM used alone has serious limitations, since it includes compounds having a wide range of ratios of antiestrogenic and estrogenic properties with no indication of any difference of activity in specific tissues. As an example, both tamoxifen and raloxifene are SERMs, but they are very different compounds, which indicates the need for a more explicit terminology. In fact, the acronym SERM does not identify the compounds that are pure antiestrogens in the mammary gland and endometrium and thus are the compounds most likely to have the highest efficacy in preventing breast and endometrial cancer. C. Intracrinology—Estrogens from Both Ovarian and Adrenal Origins Humans, along with some other primates, are unique among animal species in having adrenals that secrete large amounts of the inactive precursor steroid DHEA, especially as DHEA-S, which is converted into potent androgens and/or estrogens in peripheral tissues. Plasma DHEA-S levels in adult women are 5,000 to 25,000 times higher than those of estradiol, thus providing a large supply of substrate for the formation of androgens and/or estrogens. The local synthesis and action of sex steroids in peripheral target tissues has been called intracrinology (Labrie et al., 1988; Labrie, 1991). Recent and rapid progress in this area has been made possible by elucidation of the structure of most of the tissue-specific genes that encode the steroidogenic enzymes responsible for the transformation of DHEA-S and DHEA into androgens and/or estrogens locally in peripheral tissues (Labrie et al., 1997c; Labrie et al., 1992c; Labrie et al., 1996; Labrie et al., 1992d; Labrie et al., 1995c; Luu-The et al., 1995). The major importance of adrenal DHEA and DHEA-S in human sex steroid physiology is illustrated by the estimate of the intracrine formation of estrogens in peripheral tissues in women, which is of the order of 75% before menopause and close to 100% after menopause (Labrie, 1991). At menopause, the ovary ceases secreting estrogens, with the resulting well-known negative effects on the skeletal system, the cardiovascular system, and the central nervous system. As mentioned above, estrogen replacement therapy has provided beneficial effects in reducing the frequency and severity of these signs and symptoms of hypoestrogenism, while at the same time bringing new negative effects by increasing the risk of breast and endometrial cancer. The almost exclusive focus on the role of ovarian estrogens has removed attention from the dramatic 70% fall in circulating DHEA that

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already occurs between the ages of 20 to 30 and 40 to 50 years (Bélanger et al., 1994; Labrie et al., 1997a; Migeon et al., 1957; Orentreich et al., 1984; Vermeulen et al., 1982; Vermeulen and Verdonck, 1976). Since DHEA is transformed into both androgens and estrogens in peripheral tissues, such a fall in serum DHEA and DHEA-S explains why women at menopause are not only lacking estrogens but are also deprived of androgens. Consequently, in the interpretation of the signs and symptoms that occur at menopause, we have to take into account the fact that estrogens in women originate from two sources, namely, the ovaries, which cease functioning at menopause, and the adrenals, which secrete the inactive steroid precursor DHEA, which is transformed into estrogens and/or androgens in target tissues at a rate that depends on the level of expression of the various steroidogenic enzymes present in each cell type (Labrie, 1991). This new field of endocrinology, called intracrinology, is the formation of sex steroids from DHEA in the same cells where their action takes place without release significant amounts of the active sex steroids into the general circulation. D. Limitations of Estrogen Replacement Therapy Despite its well-recognized benefits on the skeletal and cardiovascular systems (Barrett-Connor and Bush, 1991; Stampfer et al., 1991; Wolf et al., 1991) and its possible advantages with respect to the risk and time of onset of Alzheimer’s disease (Kawas et al., 1997; Paganini-Hill and Henderson, 1994; Tang et al., 1996), HRT is practically limited to women with disabling vasomotor symptoms (Colditz et al., 1995; Collaborative Group on Hormonal Factors in Breast Cancer, 1997). Two important clinical problems associated with menopause are hot flashes and vaginal dryness. In fact, the hot flashes are the main reason why women consult their physicians and decide to take estrogen replacement therapy. It should be remembered that the benefits of HRT on bone and cardiovascular disease require continuous and long-term treatment. For example, 4 years after stopping HRT in ovariectomized women, the benefit of HRT on bone mass was practically lost (Lindsay et al., 1978). Other studies have confirmed an attenuation of the benefits of HRT on bone loss (Felson et al., 1993) and hip fractures (Kiel et al., 1987) after cessation of treatment. Among all the risk factors of breast cancer, age is the most important. In fact, about 77% of breast cancers are diagnosed after the age of 50 years, or the age that corresponds to menopause (Harris et al., 1992),

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the period in life at which hypoestrogenism is a significant factor in women’s health. A compound that could efficiently prevent breast cancer and at the same time prevent bone loss and cardiovascular disease would be a major breakthrough. In fact, most of the other factors leading to a higher risk of breast cancer are practically impossible or very difficult to control; these factors pertain to age at puberty, age at first full-term childbirth, age at menopause, obesity, diet, and family history of breast cancer (BRCA1 and BRCA2 and yet other susceptibility genes to be defined) (Brinton, 1997; Key and Pike, 1988). All these factors, except in hereditary cases, have in common longer and higher level of exposure to estrogen. Such risk factors clearly suggest the benefits of estrogen blockade to decrease the risk of breast cancer. The data summarized above are important to consider when designing approaches to counteract problems associated with menopause. E. Need of an Antiestrogen Having Pure Antiestrogenic Activity in the Mammary Gland and Endometrium Since clinical data suggest that long-term (5-year) tamoxifen adjuvant therapy is preferable to the short-term (2-year) use of the antiestrogen (Scottish Study, 1987; Tormey and Jordan, 1984) and studies have shown the benefits of long-term use of tamoxifen as a preventive for breast cancer (Fisher et al., 1998), it has become important to develop a pure antiestrogen to avoid the negative effects of the partial estrogenic activity of tamoxifen and thus make available a compound having activities limited to the desired therapeutic actions. The first class of pure antiestrogens obtained were 7α-substituted estradiol derivatives (de Launoit et al., 1991; Labrie et al., 1992a; Lévesque et al., 1991; Wakeling, 1993; Wakeling and Bowler, 1988a; Wakeling and Bowler, 1992; Wakeling et al., 1991), especially ICI 164,384, EM-139, and ICI 182,780 (Fig. 1). These compounds have been shown to possess pure and potent antiestrogenic activity in most well recognized in vitro and in vivo systems, including human breast cancer cells (de Launoit et al., 1991; Labrie et al., 1992a; Lévesque et al., 1991; Simard et al., 1990; Tremblay et al., 1997; Wakeling and Bowler, 1988a). The 7α-alkylestradiol derivative ICI 164384, however, has been found to possess some estrogenic agonistic activity in guinea pig uterine cells (Chetrite and Pasqualini, 1991; Giambiagi and Pasqualini, 1991). Furthermore, both hydroxytamoxifen and ICI 164384 can stimulate CAT activity in MCF-7 cells transfected with a pS2-tkCAT fusion gene (Weaver et al., 1988). Moreover, such 7α-alkylestradiol derivatives are difficult to synthesize, and their

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bioavailability by the oral route is very low, thus necessitating parenteral administration. Before describing recent progress in the design and development of orally active pure antiestrogens, it seems appropriate to briefly summarize data that provide some explanation for the unexpected activities of this new class of compounds, which act by highly specific ligand-induced three-dimensional conformations of ERs. III. THE ESTROGEN RECEPTORS AND THEIR MULTIPLE GENE ACTIVATION MECHANISMS A. Nuclear Receptors Approximately 70 nuclear receptors have been identified so far, of which about 50% have identified ligands (Gustafsson, 1999; Kliewer et al., 1999). The receptors with still unknown ligands are called orphan receptors. There are suggestions from the presence of 228 two–zinc finger structures in the Caenorhabditis elegans genome that the human genome may well contain homologues for all these nematode nuclear receptors (Gustafsson, 1999). As mentioned above, a large series of analogous receptors called orphan receptors have been isolated and sequenced (Kliewer et al., 1998; Resche-Rigon and Gronemeyer, 1998; Willson and Wahli, 1997). These receptors are known to bind prostaglandins, leukotrienes, and farnesoyl and steroid metabolites. There are obviously exciting possibilities that these nuclear receptors will become the elements that will permit elucidation of a large proportion of the control of cellular activity by hormones, environmental factors, and intracellularly made or intracrine (Labrie, 1991) factors, and at the same time provide unprecedented possibilities for well controlled pharmaceutical intervention. B. Estrogen Receptors ERα and ERβ Estrogen receptors constitute the only known members of the family of steroid receptors that have multiple subtypes. This diversity of ERs offers unique possibilities for the modulation of estrogen-responsive genes. The human ER was first cloned and its structure determined from human breast cancer MCF-7 cells (Green et al., 1986; Greene et al., 1986). ERα consists of 595 amino acids separated into six functional domains (Kumar et al., 1987). Over the past decade, all studies to elucidate the molecular events underlying the mode of ER action as well as antiestrogendesigned therapies have focused on the ERα identified and cloned several years ago (Green et al., 1986; Greene et al., 1986; White et al., 1987).

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Recently, a second estrogen receptor, designated ERβ, has been described and shown to share common structural and functional characteristics with ERα (Kuiper et al., 1996; Mosselman et al., 1996; Tremblay et al., 1997). Amino acid sequence comparison indicates that ERβ shares with ERα the same modular structure composed of six domains (A-F) (Krust et al., 1986). Domain C, which contains the two zinc fingers responsible for DNA binding, is the most conserved, followed by domain E, which is responsible for ligand binding, homodimerization, and nuclear localization. Domain E also contains a ligand-dependent activation function, AF2, involved in trans activation by the ERs. As mentioned above, a second activation function, AF-1, resides in the A/B domain and acts in a ligandindependent manner (Berry et al., 1990; Kumar et al., 1987; Metzger et al., 1995). Both ERs recognize a specific estrogen response element (ERE) composed of two AGGTCA motif half-sites configured as a palindrome spaced by three nucleotides (Tremblay et al., 1997). ERα has also been shown to interact with a number of coregulators via the AF-2 domain, and these protein–protein interactions promote transcriptional regulation of target genes (Cavaillès et al., 1995; Halachmi et al., 1994; Horwitz et al., 1996; Onate et al., 1995). A most important question is the distribution of ERα and ERβ in different tissues to explain the tissue-specific effects of estrogens and, especially, the new antiestrogens. ERβ has a high level of expression in bone, prostate, hypothalamus, and specific areas in the central nervous system (Kuiper et al., 1996, 1997; Laflamme et al., 1998). As illustrated in Fig. 2, the ERα-E2 and ERβ-E2 complexes can form homodimers (ERα–ERα and ERβ–ERβ) or heterodimers (ERα–ERβ). Such a diversity of ERα and ERβ interactions provides additional means of exerting gene-specific actions. C. Activation Functions AF-1 and AF-2 of Estrogen Receptors Estrogen receptors are members of a family of ligand-inducible transcriptional regulators, which include steroids, thyroid hormones, retinoids, and vitamin D. 1. Estrogen-Induced AF-2 Activation Classically, E2 binds to the ER, thus leading to conformational changes that result in dimerization of E2–ER complexes, with subsequent binding to specific EREs located within the promoter area of genes responsive to estrogen (Fig. 3). The classical ERE is an inverted hexanucleotide repeat sequence separated by three nucleotides (Beato et al., 1996). Such binding of the dimerized ER–E2 complex to ERE results in activation of transcription.

FIG. 2. Formation of the homodimers ERα-E2–ERα-E2 and ERβ-E2–ERβ-E2 as well as the heterodimer ERα-E2–ERβ-E2 in cells that express both ERα and ERβ.

FIG. 3. Schematic representation of the dual activation mechanisms of an ER by the AF-1 and AF-2 sites.

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The structural alignment of the ligand-binding domains of all known nuclear receptors has shown that all receptors show a similar fold and an analogous ligand binding pocket structure (Wurtz et al., 1996). Elucidation of the crystal structure of the ERα ligand binding domain in the presence of estradiol or of the antiestrogen raloxifene has shown that helix 12 takes different conformations when bound to the estrogen agonist estradiol and to the estrogen antagonist raloxifene (Brzozowski et al., 1997) (Fig. 4). The position of helix 12 (H12), the most carboxy-terminal α-helix, is critical for the agonist and antagonist activity of ER. First, binding of an agonist induces a major structural change in H12, which forms a lid over the ligand binding pocket while simultaneously providing a binding site for coactivator interaction. Steroid receptors are thus immediately induced to transactivate the expression of specific genes when their ligand binding domain (LBD) is bound by the cognate ligand. On the other hand, it is of great interest that the binding to ERα of the estrogen antagonist raloxifene causes a very different alteration of the position of the

FIG. 4. Positioning of helix H12 is shown in (a) for the ER LBD–E2 complex and in (b) for the ER LBD-raloxifene complex. H12 is drawn as cylinder (a = E2 complex), (b = raloxifene complex). Dotted lines indicate unmodeled regions of the structures. Hydrophobic residues located in the groove between H3 and H5 and Lys 382 (K362) are depicted in space-filling form. The locations of Asp 538, Glu 542, and Asp 545 are drawn as spheres, along with the helices that interact with H12 in the two complexes (Brzozowski et al., 1997).

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AF-2 core, in such a way that H12 covers the hydrophobic pocket in the LBD, thus precluding the binding of coactivators (Brzozowski et al., 1997; Shiau et al., 1998). Such exciting findings provide an explanation for the mechanism of action of antiestrogens, or at least raloxifene. 2. Kinase-Induced AF-1 Activation Estrogen receptors, in analogy with many other nuclear receptors, fill a double role, since they can be activated by kinase cascades independently of estrogens and of activation of the estrogen binding site by an estrogenic ligand (Fig. 3). Polypeptide growth factors have been proposed as autocrine or paracrine mediators of classically estrogen-regulated mitogenesis (Dickson and Lippman, 1987; Ignar-Trowbridge et al., 1992). For example, exogenous administration of epidermal growth factor (EGF) mimics the effect of estrogens on differentiation and proliferation of the reproductive tract in mice (Nelson et al., 1991). Much evidence has accumulated to indicate that such action of EGF is mediated by ERs (Ignar-Trowbridge et al., 1992; Nelson et al., 1991; Power et al., 1991). We will describe in more detail later the activation of AF-1 by Ras and EGF and, most interestingly, the complete inhibition of such action by the pure antiestrogen EM-652 (Fig. 1). In fact, AF-1 is thought to be responsible for the partial estrogenic activity of tamoxifen in cells that express ERα (McInerney and Katzenellenbogen, 1996). It should be mentioned, moreover, that tamoxifen, in contrast to EM-652, is unable to interfere with the activation of genes by factors acting through AF-1 (Berry et al., 1990), whereas such activation can be blocked by pure antiestrogens such as EM-652. As an example, the stimulatory effects of EGF and diethylstilbestrol on DNA synthesis and phosphatidylinositol turnover in the ovariectomized mouse uterus were blocked to the same extent by simultaneous administration of the pure antiestrogen ICI 164,384, which indicated that both EGF and diethylstilbestrol act through ER, the effects of the two very different compounds being completely blocked by a pure antiestrogen (Ignar-Trowbridge et al., 1992). It can be mentioned that EGF, in the absence of estrogen, caused a translocation of the ER to the nucleus in a way analogous to the effect of estrogen itself. Such data show that EGF can induce proliferative effects that are indistinguishable from the action of estrogens. D. AP-1 Response Element Not all genes responsive to estradiol possess an ERE in their promoter; instead, they possess other specific DNA sequences. The AP-1

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FIG. 5. Schematic representation of the activation of ER-regulated genes by classical interaction of dimerized ER-E2 with an ERE in the promoter (A) and by interaction of ERE2 in association with c-fos and c-jun with the AP-1 element of the promoter (B) of ER-regulated genes.

response element is such a specific DNA sequence that requires the protein transcription factors c-jun and c-fos for activity (Fig. 5). The tissue specificity of an estrogenic, antiestrogenic, or mixed estrogenic–antiestrogenic response can thus be explained by two different sites of DNA action of ERα and ERβ. The classical ERE (Fig. 5A) binds both ERα-E2 and ERβ-E2, with a positive response in both cases (Fig. 6). With ERα or ERβ and a reporter gene under the transcriptional control of an ERE, tamoxifen and raloxifene as well as ICI 164,384 inhibit the stimulatory effect of E2 (Paech et al., 1997). When the reporter gene was controlled by an AP-1 instead of an ERE element, E2, DES (diethylstilbestrol), tamoxifen, and raloxifene as well as ICI 164,384 all stimulated transcription with ERα. When ERβ was transfected instead of ERα, E2 and DES had an inhibitory effect, whereas the three antiestrogens exerted a stimulatory effect (Fig. 6). Most interestingly, E2 could reverse the stimulatory effect of raloxifene mediated by ERβ and the AP-1 element in a dose-dependent manner. Although these experiments were performed in HeLa cells, it is relevant to mention that in human breast cancer MCF-7 and uterine cancer Ishikawa cells (Paech et al., 1997), ERβ acting at an AP-1 element led to the same observations, namely stimulation by antiestrogens and inhibi-

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FIG. 6. Specificity of the effect of 17β-estradiol (E2), diethystilbestrol (DES), tamoxifen, raloxifene, and ICI 164,384 on transcription induced by the ERE and AP-1-element ER-regulated genes (from Paech et al., 1997).

tion by estrogens, these effects being opposite to the classical action of estrogens and antiestrogens on the transcription of genes controlled by an ERE. In agreement with these data, tamoxifen has been reported to inhibit ERE-mediated estrogen action, while stimulating transcription through the AP-1 element (Webb et al., 1995). The above-mentioned data indicate that variation in the ratio of ERα to ERβ (Fig. 2), as well as the ratio of ERE to AP-1 sites in the cell, can lead to markedly different and even opposite responses to the same agents, thus adding to cell- and even gene-specific types of response to any ER ligand. E. Coactivators and Corepressors Although the simple mechanisms of estrogen action illustrated schematically in Fig. 5 can offer a highly simplified explanation of the multiple effects mediated by estrogens, it has become clear that the stimulation or inhibition of transcription by ER-regulated genes is a highly complex phenomenon involving the interaction of dozens of proteins that enhance or inhibit the activity of the ER-E2 complex. Estrogen receptors are part of the family of nuclear receptors that share many common properties. In fact, in addition to ligand-induced specific changes of the ER structure (Brzozowski et al., 1997), target-cell specific factors play a major role in determining the final conformation of the ER–ligand complex and the response achieved. The protein–DNA complex known as chromatin loosens or tightens following tightly regulated modifications, thus permitting fine adjust-

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ments of cell functions (Hagmann, 1999). To open up the chromatin so that gene expression can occur, some transcription factors and coactivators attach different chemical tags, such as acetyl or methyl, groups to certain histone proteins. Similarly, growth factors, stress, and other external signals lead to histone phosphorylation, which activates the appropriate response genes. Histone phosphorylation is also a driving force behind the condensation of chromatin into the densely packed chromosomes needed for proper cell division (Hagmann, 1999). As described recently in an editorial entitled “How Chromatin Changes Its Shape” (Hagmann, 1999), many of the coactivators and proteins known to be involved in ER action are also key factors in the control of gene activity by a long series of nonhormonal cell modulators. Clearly, the mechanism of action of ER is central to the control of cell function in a long series of tissues that play essential roles in reproduction and thus permit the continuation of human life. Rapid progress is being made in the identification and understanding of the function of the large series of coactivators and corepressors that are expressed at various levels (from zero to high values) in a cellspecific fashion (O’Malley et al., 1999). Only when all these determinants of ERα and ERβ function are known for each ligand in each cell type will it be possible to predict with some confidence the agonist, antagonist, and mixed agonist–antagonist effects of each ligand on each gene in each cell type. 1. Coactivators The most widely studied coactivators of ER are SRC-1 and p300 or CBP (cAMP response element binding protein [CREB]) (Fig. 7). Since coactivators bind to the LBD of nuclear receptors, the LBD has been used to isolate coactivator proteins represented by the families SRC-1/NCoA-1, TIF-2, GRIP-1/NCoA-2 and pCIP/ACTR/AIBI (Freedman, 1999; Xu et al., 1999). These coactivators have been shown to have in common multiple LXXLL motifs, which interact with the LBD of nuclear receptors. As an example, in human hepatocellular carcinoma (Hep 62) cells, in which 4-hydroxytamoxifen is an agonist, the addition of SRC-1 (Smith et al., 1997) but not GRIP-1, a glucocorticoid receptor interacting protein, could enhance the 4-hydroxytamoxifen-induced transcription. As another example, AIB1, a member of the SRC-1 family, has been found to be elevated in 70% of breast cancers (Anzick et al., 1997). On the other hand, disruption of the SRC-1 gene in mice led to partial hormone resistance (Xu et al., 1998). CBP/p300 has been found to interact directly with nuclear receptors and with SRC-1, thus acting synergistically to stimulate transcription (Kamei et al., 1996; Yao et al., 1996). CBP/p300 and other coactivators

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FIG. 7. Model for activation by coactivators (A) and inhibition by corepressors (B) of transcription. Abbreviations: CBP/p300, cAMP response element binding protein; SRC-1, steroid receptor coactivator 1; TBP, TATA-binding protein; TAF, TBP-associated factor; pol II, RNA polymerase II; N-CoR, nuclear receptor corepressor; SMRT, silencing mediator of retinoic and thyroid hormone receptors.

possess histone acetyltransferase activity, which could induce remodeling of the DNA structure, thereby facilitating specific gene transcription. Also, CBP interacts with RNA polymerase II (Smith et al., 1996) as well as with TFIIB (Kwok et al., 1994). Thus, CBP could help to recruit polymerase II to the promoters of specific genes. Overexpression of CBP can increase estrogen-stimulated ER-mediated transcriptional activity approximately 10-fold (Smith et al., 1996). Recently, a complex called DRIP or TRAP has been identified as bind to many members of the nuclear receptor family. The single subunit DRIP-205/TRAP-220 binds to the LBD AF-2 core following binding of the receptor to it cognate ligand (Freedman, 1999; Rachez et al., 1998) before attracting the other 15 to 20 proteins that form the DRIP/TRAP complex. Formation of this complex is absolutely required for ligand-induced transcriptional activity of the receptor–ligand complex. In addition to the highly complex battery of protein coactivators, an RNA coactivator has recently been identified, and its interaction with other coactivators has been studied (Lanz et al., 1999). The RNA coacti-

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vator, termed SRA, interacts with the AF-1 domain of the progesterone receptor, and it has been suggested that SRA could serve as a ligand to recruit SRC-1 in the activation complexes of steroid receptors. 2. Corepressors On the other hand, transcription induced by nuclear receptors can be inhibited by protein molecules called corepressors. The silencing mediator of retinoic and thyroid hormone receptors (SMRT) (Chen and Evans, 1995) and the nuclear receptor corepressor (N-CoR) (Horlein et al., 1995) are among the best known of this class of proteins, which inhibit transcription (Fig. 7). In fact, in their inactive state ERs are thought to be bound by inactivating corepressors. On binding of the cognate ligand or activation by kinases (Fig. 3), the corepressors are released free while coactivators are recruited, thus potentiating the transcriptional capacity of ER. At least for some nuclear receptors, in the absence of agonists the corepressors N-CoR and SMRT interact with Sin3 and RPD3, a protein showing histone deacetylase activity, thereby tightening the chromatin structure and inhibiting access to the genes (Jackson et al., 1997; Smith et al., 1997; Wagner et al., 1998). The exact mechanisms by which ER AF-2 functions remains to be elucidated, but it is clear that the activation by estrogen of ER AF-2 involves multiple interactions of ER with a long series of coactivators and corepressors. It has also become clear that the interaction of a corepressor or a coactivator with the ER–ligand complex depends, not only on the ligand, but also on the cell type or intracellular environment, as well as on the element responsive to ER in the promoter of each gene. Different compounds will thus all have different agonist – antagonist activities, depending on the promoter and cell content of various coactivators and/or corepressors (Lefstin and Yamamoto, 1998). Binding of the ligand to the LBD of ER includes changes in the threedimensional conformation of ER that is well illustrated in the studies with E2 and raloxifene (Brzozowski et al., 1997) (Fig. 4). In addition, the interaction of ER with coactivators and corepressors specific for each cell type permits inducing a complete estrogenic effect in some tissues, whereas in other tissues pure antiestrogenic activity is obtained (Fig. 8). When referring to the endometrium, the full estrogenic activity is represented by E2 (Fig. 8A); tamoxifen presents relatively strong agonistic effects (Fig. 8B) and raloxifene possesses low estrogenic activity (Fig. 8C), an opposite effect being observed in terms of antiestrogenic activity. EM-652 (SCH 57068) and ICI 182,780, on the other hand, show pure antiestrogenic activity in the mammary gland and uterus (Fig. 8D).

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FIG. 8. Schematic representation of the ligand-induced changes in the confirmation of ER that lead to activities ranging from pure estrogenic (A) to pure antiestrogenic (D) effects. In addition to the ligand-induced changes in the three-dimensional structure of ER, the multiple interactions with cell-specific coactivators and corepressors determine the final activity of each ER-ligand complex in each cell type.

Despite such impressive progress, much remains to be done to understand the specific role of each coactivator and/or corepressor in the intact cell situation. It is clear, however, that such a long series of control elements permits signal-, cell-, and even gene-specific control under the influence of the multitude of physiological and pathological situations that occur in each cell type in each tissue of the organism in response to the large series of internal and external environmental stimuli to which it is subject. IV. CLASSES OF ANTIESTROGENS A. Steroidal Antiestrogens The steroidal antiestrogens ICI 164,384, ICI 182,780, and EM-139 are devoid of estrogenic activity in the mammary gland, uterus, and hypothalamic-pituitary-ovarian axis (Labrie et al., 1992b; Wakeling and Bowler, 1988b).

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These compounds, however, do not possess cell or tissue specificity and show antiestrogenic activity in all the tissues tested. For example, ICI 164,384 and ICI 182,780 do not lower serum cholesterol levels and do not prevent ovariectomy-induced bone loss (Dodds et al., 1993; Jordan, 1992; Wakeling, 1993), thus limiting our interest in them when there are other compounds that also exert pure antiestrogenic activity in the breast and uterus, while at the same time preventing bone loss and decreasing serum cholesterol (Labrie et al., 1999). B. Triphenylethylenes The finding that tamoxifen given as adjuvant to surgery improved survival (Fisher et al., 1996) and decreased bone loss (Evans and Turner, 1995; Jordan, 1993) has been a major stimulus to search for other and possibly more potent and specific compounds that would act as antiestrogens in the breast and uterus while having estrogen-like effects in the skeletal and cardiovascular systems. In the National Surgical Adjuvant Breast and Bowel Project (B-14), in which tamoxifen was administered for 5 years as adjuvant to surgery in patients with ER-positive and no axillary lymph node involvement, disease-free survival (relating to failure at distant sites p = .02) was increased from 57% to 69% (p < .0001), distant disease-free survival from 67% to 76% (p < .0001) and overall survival from 76% to 80% (Fisher et al., 1996). The incidence of contralateral breast cancer was reduced by 37% (p = .007). Somewhat surprisingly, however, for those who continued tamoxifen for 4 years after the initial 5 years, the advantages were reversed in favor of those who discontinued tamoxifen at 5 years. In fact, disease-free survival was 92% in the control group who received only 5 years of tamoxifen versus 86% in the group of women who continued tamoxifen for an additional 4 years (p = .003). Longerterm disease-free survival, on the other hand, was 96% in the control group versus 90% in the tamoxifen group (p = .01). Overall survival, on the other hand, was 96% in the control group versus 94% in the group of women who continued tamoxifen for an additional 4 years (p = .08). The conclusion was that the advantage of 5 years of tamoxifen therapy persists through 10 years of follow-up evaluations and no additional advantage is obtained from continuing tamoxifen therapy for more than 5 years. In fact, as mentioned above, continuation of tamoxifen after the initial 5 years had negative effects. In a breast cancer prevention study in high-risk women, treatment with a daily dose of 20 mg of tamoxifen for a median duration of 55 months decreased the risk of invasive breast cancer by 49% and the risk of ER-positive breast cancer by 69% (Fisher et al., 1998). However, in

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the same trial, which included 13,175 women, the relative risk of endometrial cancer was increased 2.53-fold (risk ratio, 2.53; 95% confidence interval, 1.35–4.97), which confirms previous data on the stimulatory effect of tamoxifen on the endometrium. The data showing that continuation of tamoxifen for 4 additional years gives results inferior to those obtained after only 5 years of treatment seriously limit the applicability of compounds of the class of tamoxifen for prevention of breast cancer. In fact, for efficient prevention of breast cancer, treatment should last as long as the risk of breast cancer exists. If such treatment starts at menopause or perimenopause, it should ideally continue for the remaining years of life. The clinical findings that led to the discontinuation of trial B-14 and to the suggestion of limiting tamoxifen use to 5 years likely result from the well-known resistance or loss of efficacy that usually develops under chronic administration of tamoxifen (Canney et al., 1987; Howell et al., 1992; Jordan, 1995; Osborne, 1993). The reasons for this time-limited beneficial effect of tamoxifen are likely to include the well recognized stimulatory estrogenic effects exerted by tamoxifen on breast cancer cell proliferation at the preclinical and clinical levels (Chalbos et al., 1993; Gottardis et al., 1988; Howell et al., 1990; Poulin et al., 1989c; Wakeling et al., 1989). The lack or loss of response to tamoxifen is also likely to be explained by the fact that tamoxifen does not block the action of growth factors that stimulate through the AF-1 of ERs (Fig. 3), whereas pure antiestrogens block activation of ERs by estrogens as well as by growth factors (Tremblay et al., 1998b, 1999). With tamoxifen, the stimulation of breast cancer proliferation by growth factors continues unhampered. With such knowledge, the clinical findings summarized above are not too surprising. Following observations going back to Beatson (1896), tamoxifen has clearly provided proof of the beneficial effect of estrogen blockade in breast cancer at all stages of the disease. The optimal success of estrogen blockade, however, is likely to be achieved with the new antiestrogens showing pure antiestrogenic activity in the mammary gland and uterus. In addition to its limited duration of beneficial effects in breast cancer, a serious problem associated with tamoxifen is its stimulatory effect on the endometrium, leading to endometrial hyperplasia and even cancer (Fisher et al., 1996; Kedar et al., 1994; Robinson et al., 1995). We thus have the example of a compound for which the benefits exerted on breast cancer and bone are accompanied by adverse effects in another tissue. In fact, as mentioned above, even the beneficial effects observed in breast cancer deserve some important comments because of the limit of 5 years of treatment, which raises practical questions about the use of this compound for prevention of breast cancer (Fisher et al., 1996) while, in fact, long-term use is required. Analogues of tamoxifen have

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been synthesized and developed to various stages, including clinical trials and commercialization. These compounds include toremifene, droloxifene, idoxifene, TAT-59, and GW5638. These compounds, however, exert a stimulatory effect on the uterus comparable with that of tamoxifen, as will be discussed in more detail later. C. Benzothiophenes Raloxifene, also known as keoxifene and LY 156758, was synthesized quite a few years ago and was originally observed to inhibit estrogen-induced proliferation of breast cancer cells in vitro (Anzano et al., 1996; Jones et al., 1984; Poulin et al., 1989c; Simard et al., 1997b; Thompson et al., 1988). Raloxifene was then found to protect against bone loss and to reduce serum cholesterol levels (Black et al., 1994; Ettinger et al., 1999; Evans et al., 1994; Kauffman et al., 1997). Clinical studies in postmenopausal women have confirmed the protective effect of raloxifene on bone (Delmas et al., 1997; Draper et al., 1996; Ettinger et al., 1999; Lufkin et al., 1997). In fact, although the protective effects of raloxifene on bone mineral density are generally inferior to those achieved by estrogens in both animal and human studies, at least at the doses used, the risk reduction of vertebral fractures observed at 3 years (Ettinger et al., 1999) is most encouraging. In fact, although 10.1% of placebo patients had at least one new vertebral fracture, in patients who took raloxifene this percentage decreased to 6.6% in the 60-mg group and to 5.4% in the 20-mg group. Such data correspond to a 50% reduction in vertebral fracture risk in the group of women with low bone mass and a 30% reduction among women with previous vertebral fractures. A most important observation made with raloxifene pertains to the data showing a marked reduction in the risk of breast cancer. In fact, in the Multiple Outcomes of Raloxifene Evaluation (MORE) trial, treatment for 3 years with raloxifene decreased the risk of invasive breast cancer by 76% (Cummings et al., 1999) (Fig. 9). These results were obtained in a group of 7705 postmenopausal osteoporotic women. The risk of ER-positive breast cancer was decreased by 90% (RR, 0.10; 95% confidence interval, 0.04–0.24) with no effect on ER-negative breast cancer (RR, 0.88, 95% confidence interval, 0.26–3.0). These results are impressive and indicate the much higher sensitivity of early breast cancer to estrogen blockade, an observation comparable with that already made on survival in localized prostate cancer (Labrie et al., 1997b). These results, obtained after a relatively short-term administration of raloxifene, certainly raise the hope that the use of a compound having

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pure antiestrogenic activity in the breast and uterus could dramatically reduce breast cancer incidence and mortality in women. A significant difference between the benzothiophenes (raloxifene and analogues) and the triphenylethylenes is the effect on the uterus, namely, whereas tamoxifen and its analogues exert relatively potent stimulatory effects on the uterus, raloxifene and its analogues have weak albeit statistically significant stimulatory effects (Ashby et al., 1997; Bryant, 1996; Sato et al., 1996). In contrast to tamoxifen, treatment with raloxifene for 3 years did not increase the risk of endometrial cancer although there was no significant inhibition (RR, 0.8; 95% confidence interval, 0.2–2.7). In the 1781 women who had transvaginal ultrasonography at baseline and had at least one follow-up examination, an increase in endometrial thickness of 0.01 mm was observed in the raloxifene group compared with a decrease of 0.27 mm in the placebo group (p < .01). In that study, 10.1% of women in the placebo group and 14.2% of women in the raloxifene group had an endometrial thickness evaluated at more than 5 mm at least once (p = .02). In the placebo group, endometrial thickness increased by more than 5 mm during the course of the study in 1.5% of women, compared with 3.3% in the raloxifene group (p = .03).

FIG. 9. Cumulative incidence of all confirmed breast cancers in the placebo and raloxifene groups (Cummings et al., 1999).

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Fluid was seen in the endometrial cavity in 5.7% of control subjects and 8.4% of raloxifene-treated subjects (p = .02). As will be discussed in more detail below, raloxifene exerts some weak but significant stimulatory effects on the proliferation of human breast cancer cells in vitro (Poulin et al., 1989c). Moreover, an increase in uterine weight and uterine epithelial thickness has been reported in ovariectomized rats (Ashby et al., 1997; Sato et al., 1996). As previously reported for raloxifene (Simard et al., 1997a), LY 353381, an analogue of raloxifene, which shows improved in vivo potency (Bryant et al., 1997), does possess comparable stimulatory effect on alkaline phosphatase in human uterine Ishikawa carcinoma cells (Martel et al., 1999). LY 353381 is thus unlikely to show superiority over raloxifene in terms of specificity of the effects or ratio of antiestrogenic to estrogenic activity in the mammary gland and uterus. As will be discussed later, this stimulatory effect of LY 353381, in analogy with that of raloxifene, can be completely prevented by simultaneous addition of EM-652, which supports the intrinsic estrogenic activity of the compounds. Because of the absence of protective effects of raloxifene on vasomotor symptoms or hot flashes, many authors suggest that conventional estrogen replacement therapy “is likely to remain the intervention of choice for the prevention of bone loss in symptomatic postmenopausal women” (Baynes and Compston, 1998). In fact, since the majority of women seek medical attention because of vasomotor symptoms (Gambrell, 1996) and the compounds of the SERM class, such as raloxifene (Draper et al., 1996), can lead to exacerbation (Draper et al., 1996) or have no influence (Delmas et al., 1997) on these symptoms, compliance is likely to be a problem. In fact, even tamoxifen has been shown to increase hot flashes (Love et al., 1991a). D. The Benzopyrans EM-652 (SCH 57068) and EM-800 (SCH 57050) Stimulated by the need for an improved therapy for breast cancer, considerable efforts have been devoted to the synthesis of compounds that would exert pure antiestrogenic activity in the mammary gland and uterus. As mentioned above, while tamoxifen has beneficial effects on breast cancer, it clearly acts as an estrogen agonist in the endometrium, leading to an increased rate of endometrial carcinoma in women taking tamoxifen under chronic conditions. Moreover, it is most likely that a pure antiestrogen will have beneficial effects superior to those of tamoxifen in breast cancer prevention and treatment. In order to meet the objective of a completely tissue-specific antiestrogen, a long series of benzopyran derivatives was synthesized with

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the objective of developing an orally active compound having pure antiestrogenic activity in the mammary gland and uterus. The compound EM-652 was selected for clinical development (Fig. 10). In order to facilitate large-scale purification, EM-800 (SCH 57050), the bipivalate derivative of EM-652, was synthesized. EM-800 is rapidly transformed into EM-652 in intact cells and following in vivo administration. The derivative currently used in our studies is EM-652. HCl (SCH 57068. HCl). As will be discussed later, the active compound EM-652 derived from EM-800 or EM-652. HCl behaves as a highly potent and pure antiestrogen in human breast and uterine cancer cells in vitro as well as in vivo in nude mice (Couillard et al., 1998a, 1998b; Gauthier et al., 1997; Luo et al., 1997a; Simard et al., 1997a). V. PROPERTIES OF EM-652 (SCH 57068) AND EM-800 (SCH 57050) A. Binding Characteristics The estrogen receptor affinity of EM-652, the active drug of EM-800, was first measured in human breast cancer and normal human uterine cytosol (Asselin et al., 1980). As measured by competition studies in human breast cancer tissue, the affinity of EM-652 (Ki = 0.047 ± 0.003 nM, RBA = 291, relative to 17β -estradiol set at 100), studied in the presence of

FIG. 10. Structure of EM-652 (SCH 57068).

320 TABLE I Comparison of the Estrogen Receptor Affinity of a Series of Antiestrogens and Related Compounds with Estradiol (E2) and Diethylstilbestrol (DES) in Human Breast Cancer and Normal Human Uterine Cytosol.a Breast Cancer Ethanol Compounds

Ki (nM) (max)

Uterus DMF

RBA

Ki (max)

Ethanol

RBA

Ki (max)

DMF

RBA

Ki (max)

RBA

E2

0.138

100

0.113

100

0.120

100

0.181

DES

0.126

110

–

–

0.128

93.5

–

100 –

(S)-6 (EM-652)

0.047

291

0.076

150

0.042

284

0.069

264

(R)-6 (EM-651)

2.09

6.62

–

–

1.89

6.34

–

–

(S)-1 (EM-800)

4.71

2.32

–

–

11.14

1.32

–

–

(R)-1 (EM-776)

>270

<0.04

–

–

–

–

–

–

ICI 164,384

4.60

3.00

1.53

7.46

2.33

5.15

1.76

10.3

ICI 182,780

7.63

1.81

0.755

15.1

–

–

0.668

27.2

(Z)-4-Hydroxytamoxifen Tamoxifen

0.249 11.9

43.8 0.92

– –

– –

0.346 34.4

43.8 0.92

– –

– –

aIncubations were performed at room temperature for 3 hours using 100 µl of cytosol, 100 µl of [3H]E (5 nM E , final) and 100 µl of the 2 2 indicated unlabeled compounds leading to final concentrations of 3.3% ethanol or 2.5% dimethylformamide (DMF). The apparent inhibition constant (Ki) and relative binding affinity (RBA) values were calculated as described (Asselin et al., 1980; Cheng and Prusoff, 1973). The apparent inhibition constant Ki values were calculated according to the equation Ki = IC50/(1+S/K), where S represents the concentration of labeled E2 and K is the KD value of E2 (0.14 nM) for the estrogen receptor. RBA values were calculated as follows: RBA = IC50 of E2/IC 50 of tested compound × 100.

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ethanol, was 2.9 higher than that of estradiol itself (RBA 6.62). Similar results were obtained on the human uterine ER. (Table I). It can be seen in this table that ICI 182,780 has about 10 times less affinity than EM-652 to displace [3H]E2 from the human estrogen receptor, whereas (Z)-4hydroxytamoxifen is about six times less potent under the experimental conditions used. The new antiestrogen EM-652 thus shows the highest affinity for the human ER of all the compounds tested (Gauthier et al., 1997) (Table I). It can be seen in Fig. 11 that EM-652 is seven to eight times as potent as E2 and ICI 182,780 in displacing [3H]E2 from the rat uterine estrogen receptor (IC50 values of 0.52 nM, 4.13 nM, and 3.59 nM for EM-652, E2, and ICI 182,780, respectively). ICI 164,384 and droloxifene are 21-fold less potent than EM-652, and toremifene is 400 times less potent than EM-652. Following cloning of mouse ERβ (mERβ; Tremblay et al., 1997), the activities of ERα and ERβ could be compared and the affinities of the

FIG. 11. Effect of increasing concentrations of EM-652, E2, ICI 182,780, droloxifene, ICI 164,384, and toremifene on [3H]17β-estradiol (E2) binding to the rat uterine estrogen receptor. The incubation was performed with 5 nM [3H]17β-estradiol for 2 hours at room temperature in the presence or absence of the indicated concentrations of unlabeled compounds (Martel et al., 1998a).

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two ERs for various ligands, especially antiestrogens, could be measured. We first tested the activity of both receptors in the presence of increasing E2 concentrations using the vitA2-ERE-TKLuc reporter in Cos-1 cells. Comparison of the dose–response curves of Fig. 12A shows an approximately fourfold shift of the E2 concentration required to achieve half of the maximal level of induction between the two receptors, the ERα being more sensitive to E2. The results indicated above already suggested that mERβ may have lower affinity for E2 than mERα. To verify if the difference in E2 responsiveness was due to a difference in ligand binding, we performed a binding analysis on both mERβ and mERα. [3H]-Estradiol was used to conduct binding studies with mERβ, and the results were plotted by the Scatchard method. As shown in Fig. 12B, this analysis yielded an average dissociation constant (Kd) of 0.5 nM for E2 when performed on ERβ prepared from rabbit reticulocyte lysates. This value is comparable with that obtained for the rat ERβ, which was reported to be 0.6 nM (Kuiper et al., 1996). On the other hand, we obtained an average Kd of 0.2 nM for mERα (Fig. 12C), which is well within the range of previously published determinations for the cloned human receptor (Tora et

FIG. 12. Dose-response and binding properties of mERα and mERβ. (A) Cos-1 cells were transfected with 500 ng mERβ (open circles) or mERα (closed circles) expression vectors and 1 µg vitA2-ERE-TKLuc and then incubated for 12 hours with increasing concentrations of E2 as indicated. (B) Specific binding of [2,4,6,7-3H]17β-estradiol ([3H]E2) to mERβ was determined by using receptors generated from rabbit reticulocyte lysates. Binding was determined over a concentration range of 0.01–3 nM [3H]E2 in the absence or presence of a 200-fold excess of unlabeled E2. The saturation plot is shown in the insert. The results were plotted by Scatchard’s method. Each point was determined in triplicate in each experiment, and the above results are representative of at least two separate experiments. (C) Specific binding to mERα using the conditions described in panel B (Tremblay et al., 1997).

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al., 1989). Therefore, this slightly reduced affinity of mERβ for E2 may provide an explanation for the shift in E2 responsiveness indicated by the dose–response curves (Fig. 12). B. EM-652 Blocks Both AF-1 and AF-2 Functions of ERα and ERβ The two ERs share many functional characteristics based on their well conserved modular structure. As summarized above, AF-2 is responsible for estrogen-dependent activation through recruitment of coactivator proteins including members of the steroid receptor coactivator (SRC) family (Anzick et al., 1997; Chen et al., 1997; Hong et al., 1996; Kamei et al., 1996; Li et al., 1997; Onate et al., 1995; Torchia et al., 1997; Voegel et al., 1996). On the other hand, AF-1 activity is constitutive and ligand-independent (Berry et al., 1990; Kumar et al., 1987; Metzger et al., 1995). In addition to the classical hormone activation pathway, a number of steroid receptors, including ERα and β, have thus been shown to be activated by nonsteroidal agents (Fig. 13), including dopamine, growth factors, and protein kinase A (PKA) activators (Aronica and Katzenellenbogen, 1993; Bunone et al., 1996; Ignar-Trowbridge et al., 1993; Power et al., 1991; Smith et al., 1993; Tremblay et al., 1997). Signal transduction pathways stimulated by growth factors have been well demonstrated to activate ER (Ignar-Trowbridge et al., 1992). Growth factors not only can activate an ER at the AF-1 site in the

FIG. 13. Schematic representation of the activation functions 1 and 2 of ERα and ERβ. AF-1 is ligand-independent but is activated by dopamine, growth factors, cyclic AMP, MAP kinase, PKA activators and Ras. AF-2, on the other hand, is activated by estrogenic compounds. EM-652 blocks both AF-1 and AF-2 completely while hydroxytamoxifen blocks AF-2 only (Labrie et al., 1999).

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absence of estrogens but can also enhance the stimulatory effect of estrogens (Katzenellenbogen et al., 1995) Potential phosphorylation of serine 118 in human ERα (Ali et al., 1993; Bunone et al., 1996; Kato et al., 1995) and serine 60 in mouse ERβ (Tremblay et al., 1997) through activation of the Ras-MAPK pathway has been shown to further maximize the E2 response of both ERs. To investigate whether EM-652 could efficiently block this effect, we used the wild-type H-Ras and its dominant active form H-RasV12 in our transfection experiments, as indicated in Fig. 14. As observed previously (Kato et al., 1995; Tremblay et al., 1997), the addition of H-Ras contributed to increase the activity of ERα in the presence of E2, with an even stronger response when H-RasV12 was used (Fig. 14A). These inductions by both Ras forms were completely abolished with the addition of EM-652 in the medium, as with ICI 182,780, which suggests that EM-652 is effective in blocking the AF-1 activity of ERα. The same experiment was conducted on ERβ, with which H-Ras and H-RasV12 augmented the E2 response in a similar manner (Fig. 14B). Here again, EM-652 and ICI 182,780 abolished the Ras effect in the presence of E2. Interestingly, we observed a ligand-independent effect of Ras on ERβ basal activity, in which a two- to three-fold induction occurred with H-RasV12 (Fig. 14B). On the other hand, no effect of Ras was seen on basal levels of ERα. The Ras induction of unliganded ERβ was blocked by EM-652 and ICI 182,780 (data not shown). We were also interested in testing whether EM-652 was efficient in blocking ER responsiveness on a natural promoter. The pS2 promoter has been extensively studied with respect to its ERα-mediated regulation (Berry et al., 1989). We previously showed that ERβ can also modulate transactivation of a reporter gene driven by the pS2 promoter in HeLa cells and also that the E2 response was potentiated by H-Ras (Tremblay et al., 1997). The effects of Ras on liganded ERα and β activities are completely abrogated by EM-652 (data not shown). Dose–response analyses were also performed to further evaluate the potency of EM-652 in inhibiting the effect of Ras on ER activities in the presence of E2. EM-652 was slightly more effective than ICI 182,780 in blocking H-RasV12 inductions of ERα and ERβ, especially at lower concentrations (Fig. 14C and D). C. EM-652 Blocks SRC-1 – Induced Activity of Both ERα and ERβ The coactivator SRC-1 has been shown to interact with and promote the transcriptional activity of a number of nuclear receptors, including ERα (McInerney et al., 1996; Onate et al., 1995). More recently, we have demonstrated that SRC-1 also stimulates ERβ activity through a direct interaction with its LBD, where the AF-2 domain resides (Tremblay et

FIG. 14. EM-652 blocks the Ras-induced ERα and ERβ transcriptional activity. (A) COS-1 cells were cotransfected with 1 µg vitA2ERETKLuc and 500 ng pCMX-ERα in the presence or absence of 1 µg Ha-Ras or Ha-RasV12 expression plasmids. The cells were then grown in the presence or absence of 10 nM E2 or 100 nM of EM-652 or ICI 182,780 (ICI). The basal activity of ERα in the absence of estradiol was set arbitrarily at 1.0. (B) Same as in (A), except that ERβ expression vector was used. (C). Dose responses of EM-652 (filled squares) and ICI 182,780 (open squares) in the presence of 10 nM E2 on ERα activity in COS-1 cells transfected with vitA2ERETKLuc reporter and Ha-RasV12 expression plasmid. The maximal induction by E2 alone was set arbitrarily at 100%. (D) Same as in (C) except that in ERβ expression vector was used (Tremblay et al., 1998a).

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al., 1997). We took advantage of this effect of SRC-1 to study whether EM-652 could block the E2-activated AF-2 of ERα and ERβ. We first generated glutathione-S-transferase (GST) fusion proteins with the E and F domains of mERα (GST-mERβEF) and domains D-F of mERα (GST-mERαDEF) for use in GST pulldown experiments (Fig. 15). GSTmERβEF and GST-mERαDEF were expressed in Escherichia coli, purified with GST-sepharose and incubated with [35S]methionine labeled SRC-1. As shown in Fig. 15A, the LBD of mERα interacted weakly with SRC-1 in the absence of E2 (lane 3), whereas addition of E2 caused an increase in interaction between the two proteins (lane 4). Both EM-652 (lane 5) and ICI 182,780 (lane 6) efficiently blocked the ligand-dependent SRC-1 interaction, with the stronger effect shown by EM-652. A similar inhibition of the E2-dependent interaction between SRC-1 and the LBD of ERβ was also observed, whereas ICI 182,780 was less efficient (Fig. 15, lanes 7–10). We also demonstrated that the stimulatory effect of SRC-1 on the E2 response of both ERs in COS-1 cells was completely abolished with the addition of EM-652 in the medium, as it was with ICI 182,780 at the concentration used (Fig. 15B and C). Furthermore, as observed with Ras (see above), SRC-1, under the present experimental conditions, enhanced the basal activity of ERβ but not that of ERα, in the absence of ligand. This ligandindependent effect of SRC-1 on ERβ was blocked by EM-652. Similar results were obtained on using HeLa cells transfected with a pS2Luc reporter construct (Fig. 15D and E). Dose–response analyses were also performed to further evaluate the potency of EM-652 in inhibiting the potentiating effect of SRC-1 on ER activities in the presence of E2. EM-652 was very effective in blocking SRC-1 potentiation of ligand-dependent ERα and ERβ transcriptional FIG. 15. EM-652 blocks the estrogen- and SRC-1-stimulated AF-2 activity of ERα and ERβ. (A) GST pull-down experiments. The purified fusion proteins were incubated with labeled SRC-1 in the absence (lanes 3 and 7) or presence of 5 nM E2 (lanes 4 to 6 and 8 to 10) in addition to a 100-fold excess of EM-652 (lanes 5 and 9) and ICI 182,780 (lanes 6 and 10). The input lane (lane 1) represents 20% of the total amount of labeled SRC-1 used in each binding reaction. An equivalent amount of protein was used in the sample containing only GST (lane 2). (B) COS-1 cells were cotransfected with 1 µg vitA2ERETKLuc and 500 ng pCMX-ERα in the presence or absence of 1 µg SRC-1 expression plasmid. Cells were incubated with or without 10 nM E2 or 100 nM antagonist as indicated. Results are expressed as the factor by which response exceeds basal levels set arbitrarily at 1.0. (C) Same as in (B), except that ERβ expression vector was used. (D) and (E) Same as in (B) and (C), respectively, except that pS2Luc reporter and HeLa cells were used in transfections. (F) Dose response of EM-652 (filled squares) and ICI 182,780 (open squares) in the presence of 10 nM E2 on ERα activity in COS-1 cells transfected with vitA2ERETKLuc reporter and SRC-1 expression plasmid. The maximal induction by E2 alone was set arbitrarily at 100%. (G) Same as in (F), except that ERβ expression vector was used (Tremblay et al., 1998a).

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activities with apparent IC50 values of 10–10M and 10–9M, respectively (Fig. 15F and G). ICI 182,780 was less potent in its ability to inhibit the SRC-1 induction of ERα and ERβ activities, with an IC50 value of 10–8M for both receptors. The present study describes the molecular action of EM-652, the active metabolite of EM-800, on ER transcriptional functions. We present evidence that EM-800 and its active metabolite EM-652 act as pure estrogen antagonists on ERα and ERβ transcriptional activities. This pure antiestrogenic profile is of primary importance in endocrinebased breast cancer therapy, since the objective, as mentioned earlier, is to develop a compound having both activities, because the widely available antiestrogen currently available, tamoxifen, acts as a mixed agonist–antagonist on ER function and does not inhibit AF-1. Despite the relatively good clinical record of tamoxifen in inducing remission of ER-positive breast cancer and in postsurgical adjuvant therapy, resistance to tamoxifen, a phenomenon likely due to its intrinsic agonist properties, does occur, and tumor progression ensues in most of the patients (Furr and Jordan, 1984). The potency of EM-652 to inhibit ER function was even more dramatic when the E2 response was maximized through activation of the AF-1 and AF-2 domains of ERs by Ras and SRC-1, respectively. Phosphorylation of Ser-118 triggered by the Ras-MAPK pathway has been described for ERα and shown to further increase its E2-stimulated transcriptional activity (Kato et al., 1995). Ras also activates liganded ERβ, presumably through phosphorylation of Ser-60 (Tremblay et al., 1997). Here we show that EM-652 strongly inhibited the E2-induced ERα and β activities triggered by either Ras or its dominant active form RasV12. We observed a similar pattern with SRC-1, which is well known as a general coactivator for steroid receptors and has been shown to up regulate ERα-stimulated transcription (McInerney and Katzenellenbogen, 1996; Onate et al., 1995). More recently, we demonstrated that SRC-1 interacts with ERβ and stimulates its transcriptional activity (Tremblay et al., 1997). This interaction occurred with the LBDs of both ERs (McInerney and Katzenellenbogen, 1996; Tremblay et al., 1997). Again, EM-652 was very potent in fully abolishing the E2 response of ERα and ERβ enhanced by SRC-1. These effects were not cell- or promoter-specific, as demonstrated with the pS2 promoter in HeLa cells. Hence, EM-652 can be regarded as a pure antagonist, which acts on both activation domains of the ERs. Interestingly, both Ras and RasV12 induced the activation of transcription of ERβ in the absence of E2. Such ligand-independent activation of Ras was not observed with ERα (Kato et al., 1995), although it was reported with EGF treatment (Bunone et al., 1996). A similar pattern of

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activation of ERβ but not ERα was also observed with SRC-1. Our previous work (Tremblay et al., 1997) has shown that the SRC-1-induced ligandindependent activation of ERβ was not blocked by 4-hydroxytamoxifen, which exerts an inhibiting effect on ER limited to AF-2 (Berry et al., 1989); this suggests that SRC-1 might interact with other regions of the receptor. A possible target region for such an interaction might be contained within the amino-terminal region of ERβ, as ICI 182,780 and EM-652 inhibit the ligand-independent effect of Ras and SRC-1. D. EM-652 Blocks the Recruitment of SRC-1 at the AF-1 of ERβ The ligand-independent activation of AF-1 is presumed to be closely related to phosphorylation of steroid receptors by cellular protein kinases (Weigel, 1996). Our previous observations that SRC-1 could stimulate ERα and β activity in absence of ligand prompted us to further investigate the mechanisms underlying this effect (Tremblay et al., 1998a). In the absence of E2 or other exogenously added stimulatory agents, SRC-1 increased in a dose-dependent manner the transcriptional activity of ERβ in transfected Cos-1 cells (Fig. 16A). This effect was not cell- or reporter-specific because a similar increase in ERβ activity was detected on various reporters such as pS2Luc and ERE3TKLuc and also in other cell lines including HeLa and 293T (data not shown). Interestingly, ERα was less sensitive than ERβ in terms of ligand-independent SRC-1 activation, a large excess of SRC-1 being required to reach an observable effect on ER activity (Fig. 16A). Thus, the balance between the cellular content of SRC-1 and ER isoforms may contribute to discrimination between activation of unliganded ERα and β. Based on the previous observation that SRC-1 could interact with steroid receptors in a ligand- and AF-2-independent manner (Henttu et al., 1997; Jeyakumar et al., 1997; Takeshita et al., 1996), we tested whether AF-2 was necessary to mediate the SRC-1 effect on basal ERα activation using the differential ability of mixed agonists-antagonists and pure antiestrogens to block AF-1 and AF-2 functions. EM-652, the active metabolite of EM-800, was previously identified as a very potent and pure antagonist of ERα and β transcriptional functions, whereas 4hydroxytamoxifen only blocked the AF-2 activity of both ERs (Tremblay et al., 1997). As shown in Fig. 16B, EM-652 strongly impaired the SRC-1mediated basal ERβ activity in Cos-1 cells, whereas 4-hydroxytamoxifen treatment minimally decreased ligand-independent activation of ERβ. The same pattern of inhibition was obtained in HeLa cells with the pS2Luc reporter (data not shown). These data suggest that AF-2 is not required for the E2-independent activation of ERβ by SRC-1. To test this

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FIG. 16. Basal ERβ transcriptional activation by SRC-1 is AF-2 independent. (A) Dosedependent activation of ERα and ERβ by SRC-1 in absence of E2. Cos-1 cells were transfected with ERETKLuc reporter along with ERα or ERβ and increasing amounts of SRC-1 expression plasmids. Luciferase activities were normalized with β-Gal expression and results are expressed as factor by which response exceeds basal levels (–) and represent the mean ± SEM of three independent experiments. (B) Pure antiestrogen EM-652 but not the mixed antagonist 4-hydroxytamoxifen (OHT) inhibits basal ERβ transcriptional activation by SRC-1. Cos-1 cells were transfected with ERETKLuc along with equivalent amounts of ERβ and SRC-1 expression plasmids and incubated with increasing amounts of OHT or EM-652 prior to being assayed for luciferase activity. The maximal induction by SRC-1 alone (solid bar) was defined as 100%. Basal level in absence of SRC-1 is indicated by an open bar. (C) Basal activity of an ERβ AF-2 mutant is induced by SRC-1. Cos-1 cells were transfected with ERETKLuc reporter and equivalent amounts of ERβ or ERβ L509A AF-2 mutant and SRC-1 (solid bars) expression plasmids. Cells were then treated with 10 nM E2 (striated bars) or left untreated (open and solid bars) for 16 hours prior to harvest. Results are plotted as factor by which induction exceeds basal levels (Tremblay et al., 1999).

possibility further, we used an ERβ AF-2 deficient mutant (ERβL509A) that is transcriptionally inactive in the presence of E2 (Fig. 16C). SRC-1 could still activate ERβL509A in the absence of ligand (Fig. 16C), thus demonstrating that the observed transcriptional effect of SRC-1 on unliganded ERβ occurred in an AF-2–independent fashion. We also assessed the effect of phosphorylation of the ERβ AF-1 in regulating interaction with SRC-1 in vivo using transfection experiments. Treatment of transfected cells with PD98059, a selective inhibitor of MAPK activation, completely abrogated the SRC-1-mediated activation of unliganded ERβ, whereas use of staurosporin, which inhibits protein kinase C, had no significant effect (Tremblay et al., 1999). The interaction between ERβ and SRC-1 in the absence of hormone was also demonstrated in vivo and shown to be influenced by factors known to change the phosphorylation status of nuclear receptors.

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Our observations suggest that Ser-106 and Ser-124 are both required in vivo to fully recruit SRC-1. In addition, when cells were treated with factors known to activate Ras, such as EGF or IGF-1 (data not shown), the in vivo interaction between SRC-1 and ERβ was also enhanced, thus mimicking the results obtained in the presence of activated Ras. This study demonstrates for the first time that phosphorylation of the AF-1 domain of a member of the nuclear receptor superfamily enhances the recruitment of a steroid receptor coactivator (SRC-1) and provides a molecular basis for ligand-independent activation of ERβ via the MAPK cascade. SRC-1 has been described as a coactivator that interacts and enhances the transcriptional activity of a number of nuclear receptors in a ligand- and AF2-dependent manner (Onate et al., 1995). On the basis of three different approaches, enhancement of ERβ activation by SRC-1 in the absence of ligand was found to be independent of AF-2. Very importantly, the partial antiestrogen hydroxytamoxifen had no appreciable effect on SRC-1-induced unliganded ERβ activity, whereas the pure antiestrogen EM-652 completely abolished this effect. This observation strengthens the need for pure antiestrogens in breast cancer therapy, in which all aspects of ER-regulated gene expression, including coactivator-mediated hormone-dependent as well as hormone-independent activation pathways, must be regarded as direct targets for antiestrogen action. In fact, the absence of blockade of AF-2 by hydroxytamoxifen could explain why the benefits of tamoxifen observed for up to 5 years become negative at longer treatment times and why resistance develops to tamoxifen. E. Inhibition of the Development and Growth of DMBA-Induced Mammary Tumors in the Rat 1. Prevention of Estrone-Stimulated Development of Dimethylbenz(a)anthracene-Induced Mammary Carcinoma in the Rat 7,12-Dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat is a widely used animal model to study the factors that control hormone-sensitive breast cancer in women. In fact, the development and growth of these tumors are particularly sensitive to the stimulatory action of estrogen and prolactin (Asselin et al., 1977; Asselin and Labrie, 1978; Dauvois and Labrie, 1990; Dauvois et al., 1989b; Huggins et al., 1961; Jordan and Allen, 1980; Kelly et al., 1977, 1979; Labrie et al., 1976, 1993; Leung et al., 1975; Li et al., 1993, 1995; Welsch, 1985). An ideal antiestrogen should exert a highly potent inhibitory effect on breast cancer without showing any adverse effects on the endometrium or serum lipids and bone metabolism. We have thus investigated the

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FIG. 17. Effect of daily oral administration of 25 µg, 75 µg, or 250 µg EM-800 on the number of animals that developed palpable mammary carcinoma induced by dimethylbenz(a)asthracene throughout the 279-day observation period. Data are expressed as percentage of the total number of animals in each group *,p < .05;**, p < .01 vs control (Luo et al., 1998a).

effect of the new pure antiestrogen, EM-800 (Gauthier et al., 1997; Luo et al., 1997b, 1997c, 1998c); Simard et al., 1997a; Tremblay et al., 1997) on the development of mammary carcinoma induced by DMBA and the effect of such treatment on serum lipid profile as well as bone mass in the female rat. As illustrated in Fig. 17, 9 months after DMBA administration, 95% of control animals had developed palpable mammary carcinoma. In contrast, treatment with 25, 75, or 250 mg of EM-800 caused a progressive inhibition of tumor development (p < .0001, for both Fisher’s exact test and the logistic regression), the incidence being reduced to 60%, 38%, and 28%, respectively. However, the difference between EM-800 doses is not statistically significant. It is of interest to see in Fig. 18A that mean tumor number per animal was markedly decreased from 4.5 ± 0.5 tumors in the control group to 0.9 ± 0.2 (p < .0001), 0.5 ± 0.2 (p < .0001),

FIG. 18. Effect of daily oral administration of 25 µg, 75 µg, or 250 µg EM-800 on average tumor number per rat (A) and average tumor size per rat (B) throughout the 279-day observation period. Data are presented as means ± SEM (Luo et al., 1998a).

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and 0.3 ± 0.1 (p < .0001) tumors in the groups of rats treated with the antiestrogen. There is no statistically significant difference between the three groups treated with EM-800. In addition, the mean tumor area per animal was reduced from 12.2 ± 1.33 cm2 to 3.76 ± 0.78 cm2 (p < .0001), 2.94 ± 1.07 cm2 (p < .0001), and 2.58 ± 1.21 cm2 (p < .0001) following the same treatments (Fig. 18B). Antiestrogens have been found to suppress tumorigenesis induced by chemical carcinogenic agents in the rat (Jordan, 1974, 1976; Kawamura et al., 1991; Kelly et al., 1977; Labrie et al., 1976; Li et al., 1995). The present data clearly show that EM-800 not only significantly reduces the percentage of rats bearing DMBA-induced tumors but also decreases tumor number per animal that has developed tumors during treatment with EM-800. Such data indicate the potential chemopreventive action of this compound in breast cancer. It is also of interest to note that the tumor size reached in the rats treated with EM-800 was smaller than that of control animals, a finding apparently in contrast with the data obtained with tamoxifen in the same animal model. In fact, Fendl and Zimniski (1992) reported that the tumors that developed in rats treated with tamoxifen displayed a higher growth rate than the tumors in the control group. A strict comparison would, however, require a parallel evaluation of the two compounds in the same study. The effects of EM-800 on serum lipids will be described later. 2. Inhibition by EM-800 of Estrone-Stimulated Growth of DMBA-Induced Mammary Carcinoma in the Rat—Combination with DHEA Since antiestrogens (Dauvois et al., 1991; Jordan, 1976, 1978; Kawamura et al., 1991; Labrie et al., 1995b) as well as DHEA (Li et al., 1993) can independently inhibit the development of DMBA-induced mammary carcinoma, we have studied the potential benefits of combining the new antiestrogen EM-800 with DHEA on the development of mammary carcinoma induced by DMBA in the rat. As illustrated in Fig. 19, 95% of control animals developed palpable mammary tumors by 279 days after DMBA administration. Treatment with DHEA or EM-800 partially prevented the development of DMBAinduced mammary carcinoma, the incidence being thus reduced to 57% (p < .01) or 38% (p < .01), respectively. Interestingly, combination of the two compounds led to a significantly higher inhibitory effect than those achieved by each compound alone (p < .01 versus DHEA or EM-800 alone). In fact, the only two tumors that developed in the group of animals treated with both compounds disappeared before the end of the experiment.

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FIG. 19. Effect of treatment with dehydroepiandrosterone (DHEA) (10 mg, percutaneously, once daily) or EM-800 (75 µg, orally, once daily) alone or in combination for 9 months on the incidence of dimethylbenz(a)anthracene (DMBA)-induced mammary carcinoma in the rat throughout the 279-day observation period. Data are expressed as percentage of the total number of animals in each group (Luo et al., 1997c).

It has been observed that androgens exert a direct antiproliferative activity on the growth of ZR-75-1 human breast cancer cells in vitro and that such an inhibitory effect of androgens is additive to that of an antiestrogen (Poulin et al., 1988; Poulin and Labrie, 1986). Similar inhibitory effects have been observed in vivo on ZR-75-1 xenografts in nude mice (Couillard et al., 1998a; Dauvois et al., 1991). Androgens have also been shown to inhibit the growth of DMBAinduced mammary carcinoma in the rat, this inhibition being reversed by the simultaneous administration of the pure antiandrogen flutamide (Dauvois et al., 1989a). Taken together, the present data indicate the involvement of the androgen receptor in the chemopreventive action of DHEA. Since antiestrogens and DHEA exert chemopreventive effects on breast cancer via different mechanisms, it is reasonable to expect that the combination of EM-800 and DHEA exerts more potent inhibitory effects than each compound alone on the development of DMBA-induced rat mammary carcinoma, as is well illustrated by the present data.

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F. Inhibition of the Growth of Human Breast Cancer ZR-75-1, MCF-7, and T-47D Cell Lines In Vitro This section describes the effects of EM-652 and other antiestrogens on basal and E2-induced cell proliferation in three well characterized estrogen receptor–positive human breast cancer cell lines. Our results show that EM-652 and its precursor EM-800 are the most potent known antiestrogens in vitro in human breast cancer cells and are, most importantly, devoid of any intrinsic estrogenic activity. 1. Comparison of the Effects of EM-652 and EM-800 with Those of ICI 164,384, ICI 182,780, Hydroxytamoxifen, Tamoxifen, Droloxifene, Toremifene, and Hydroxytoremifene on Basal and E2-Induced Cell Proliferation in the T-47D Human Breast Cancer Cell Line Since EM-800 is rapidly metabolized into the active compound EM-652 in intact cells, we compared the effect of increasing concentrations of the nonsteroidal antiestrogens EM-652 and EM-800 with those of hydroxytamoxifen and tamoxifen and of the steroidal antiestrogen ICI 164384 on basal and E2-induced cell proliferation in T-47D, ZR-751 and MCF-7 cells (Simard et al., 1997a). As illustrated in Fig. 20, a 10day exposure to 0.1 nM E2 increased the proliferation of T-47D cells 4.77-fold. This E2-induced stimulation of cell proliferation was competitively blocked by simultaneous incubation with EM-800, EM-652, hydroxytamoxifen, ICI 164,384, and tamoxifen at respective IC50 values of 0.148, 0.146, 0.522, 2.41, and about 100 nM. It can also be seen in Fig. 20 that none of these compounds affected basal T47-D cell proliferation when incubated alone. Droloxifene, toremifene, and ICI 182780 are being developed for the treatment of breast cancer (Loser et al., 1985; Valavaara, 1990; Wakeling and Bowler, 1992). It was thus of interest to compare the effects of these compounds on breast cancer cell proliferation with that of EM-800. As illustrated in Fig. 21, a 9-day exposure to 0.1 nM E2 resulted in a 3.8-fold increase in the proliferation of T-47D cells (Simard et al., 1997a). This E2induced stimulation of cell proliferation was competitively blocked by simultaneous incubation with EM-800, hydroxytamoxifen, ICI 182780, droloxifene, or toremifene at respective IC50 values of 0.158, 0.400, 0.434, 7.30, and more than 100 nM (Fig. 21). In fact, we observed in another independent experiment that after a 9-day incubation with increasing concentrations of EM-800, hydroxytoremifene or toremifene in T-47D cells, the 2.47-fold increase in cell proliferation induced by 0.1 nM E2 was reversed at respective IC50 values of 0.112, 0.430, and 179 nM (Simard et al., 1997a, data not shown).

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FIG. 20. Effect of increasing concentrations of EM-652, EM-800, ICI 164,384, 4 hydroxy-trans-tamoxifen (OH-TAM), or tamoxifen (TAM) on basal and E2-induced cell proliferation in T-47D human breast cancer cells. Three days after plating, cells were exposed for 10 days to the indicated concentrations of compounds in the presence or absence of 0.1 nM E2. Media were changed at 2- or 3-day intervals. At the end of the incubation period, cell number was determined by measurement of DNA content. Data are expressed as the means ± SEM of triplicate dishes. When the SEM overlaps with the symbol used, only the symbol is illustrated (Simard et al., 1997a).

2. Comparison of the Effect of EM-652, EM-800 and Tamoxifen on the Proportion of Cycling MCF-7 Cells To assess the percentage of MCF-7 cells that progressed through the S-phase of the cycle during incubation with EM-652, EM-800, or tamoxifen in the presence or absence of E2, the continous BrdUrd exposure technique was used. As measured after a 48-hour exposure to BrdUrd, 72-hour pretreatment with 1 nM EM-652, EM-800, or hydroxytamoxifen alone decreased the percentage of BrdUrd-positive cells from 43.6% to 20.2%, 21.5%, and 30.9%, respectively (p < .01) (Fig. 22A). On the other

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FIG. 21. Effect of increasing concentrations of EM-800, ICI 182780, or 4-hydroxytrans-tamoxifen (OH-TAM), droloxifene, or toremifene on basal and E2-induced cell proliferation in T-47D human breast cancer cells. Three days after plating, cells were exposed for 9 days to the indicated concentrations of compounds in the presence or absence of 0.1 nM E2. Media were changed at 2- or 3-day intervals. Data are expressed as described in the legend of Fig. 20 (Simard et al., 1997a).

hand, incubation with 0.1 nM E2 increased the percentage of BrdUrdpositive cells to 77.9% (p < .01). Addition of increasing concentrations of EM-652, EM-800, or OH-tamoxifen completely blocked the stimulatory effect of E2 on this parameter and caused a further decrease below the control value to levels similar to those obtained with these compounds in the absence of E2 (Fig. 22B). The inhibitory effect of EM-652, EM-800, and hydroxytamoxifen on the percentage of BrdUrd-positive cells was observed at respective IC50 values of 0.60, 1.26, and 3.8 nM. It can also be seen in Fig. 22 that treatment with tamoxifen was approximately 1000fold less effective in decreasing the proportion of cycling MCF-7 cells. The results described above show that none of the compound tested exerts a more potent antagonistic effect than the novel nonsteroidal compound EM-652 on E2-induced proliferation in T-47D, ZR-75-1, and MCF-7 human breast cancer cells in vitro. Most importantly, EM-652 and EM-800 have no estrogenic activity in the three breast cancer cell lines studied, whereas raloxifene, hydroxytamoxifen, toremifene, and droloxifene cause a significant stimulation of

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FIG. 22. Effect of increasing concentrations of EM-652, EM-800, hydroxytamoxifen (OH-TAM) or tamoxifen (TAM) on the proportion of cycling MCF-7 cells after exposure to BrdUrd. Three days after plating at an initial density of 0.85 × 105 per 10-cm2 well, cells were pretreated for 3 days with the indicated concentrations of compounds in the presence (B) or absence (A) of 0.1 nM E2 before changing to fresh medium containing the same compounds and 10 µM BrdUrd. Cells were then harvested after 2 days, fixed, and stained with the dye Hoechst 33358. The percentage of BrdUrd-positive cells was calculated as described by Simard et al., (1997a). Data obtained with control medium alone in the presence or absence of 0.1 nM E2 are indicated on the Y axis. Data are expressed as described in the legend of Fig. 20 (Simard et al., 1997a).

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ZR-75-1 and MCF-7 human breast cancer cell proliferation in the absence of estrogens. As mentioned earlier, the estrogenic activity of tamoxifen on breast cancer cell proliferation is well illustrated by other preclinical as well as clinical studies (Canney et al., 1987; Gottardis et al., 1988; Howell et al., 1992). The clinical data are well supported by the tamoxifen-induced stimulation of breast cancer cells cultured directly from patients (DeFriend et al., 1994b). Such data suggest that progression of the disease observed in patients on tamoxifen therapy is related to the stimulation of the cancer by the intrinsic estrogenic activity of the compound. This study is also in agreement with the partial agonistic activity of raloxifene on basal ZR-75-1 cell proliferation, which was reported 10 years ago (Poulin et al., 1989b). This study also shows that EM-652 and EM-800 decrease the proportion of MCF-7 cells that advance through the S phase, and completely block the stimulatory effect of E2 on this parameter. In fact, EM-652 and EM-800 were at least 1000-fold more effective than tamoxifen in reducing the proportion of BrdUrd-positive cells in the presence or absence of E2, and EM-652 was six times as potent as hydroxytamoxifen in the presence of E2. G. Comparison of the Effect of EM-800 and Tamoxifen on the Growth of Human Breast Cancer Xenografts in Nude Mice Tamoxifen has shown important benefits in breast cancer and has become the standard therapy at all stages of the disease. Although 30 to 50% of the patients with advanced breast cancer show a positive response to tamoxifen, the duration of response is usually limited to 12 to 18 months, with the development of resistance to further treatment with this antiestrogen (Dickson and Lippman, 1987; Howell et al., 1995; Mouridsen et al., 1978). As mentioned above and demonstrated in a series of studies with human breast cancer cell lines in vitro and in vivo (Gottardis et al., 1988; Katzenellenbogen et al., 1987; Lykkesfeldt and Sorensen, 1992; Osborne et al., 1995; Poulin et al., 1989b; Wakeling et al., 1989) and supported by clinical observations (Canney et al., 1987; Hoogstraten et al., 1984; Howell et al., 1990, 1992; Pritchard et al., 1980; Wiebe et al., 1993), it seems reasonable to suggest that the loss of positive response to tamoxifen treatment in breast cancer patients could be, at least in part, due to the intrinsic estrogenic activity of the compound. This explanation is supported by the finding that human breast cancer cell lines showing resistance to tamoxifen retain their sensitivity to specific antiestrogens in vitro (Brunner et al., 1993; Coopman et al., 1994; Lykkesfeldt et al., 1994; Lykkesfeldt and Sorensen, 1992), as well as in vivo in nude mice (Gottardis et al., 1989; Osborne et al., 1991, 1995).

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FIG. 23. Time course of the effect of treatment with the pure antiestrogen EM-800 or tamoxifen at the daily oral dose of 50 µg, 150 µg, or 400 µg for 4 months on the average size of ZR-75-1 human breast cancer xenografts in ovariectomized nude mice supplemented with an implant of estrone. The size of tumors at start of treatment was 31.1 ± 0.8 mm2. Ovariectomized mice receiving the vehicle alone were used as additional controls. Results are expressed as percentage of pretreatment values (means ± SEM of 28 to 37 tumors per group) (Couillard et al., 1998a).

Since human breast carcinoma xenografts in nude mice provide the closest available model of human breast cancer, we have compared the effect of EM-800 and tamoxifen alone and in combination on the growth of ZR-75-1 breast cancer xenografts in nude mice. While estrone caused a 365% increase in ZR-75 tumor size during the 4-month treatment period, administration of the oral daily 50-µg, 150µg, or 400-µg dose of the antiestrogen EM-800 completely prevented tumor growth (Fig.23). In fact, at the 400-µg dose, average tumor size was reduced by 25% (p < .001) at 4 months as compared with the initial size at start of treatment. When the same doses of tamoxifen alone were administered, it can be seen in Fig. 23 that average tumor sizes after 4 months were 289%, 217%, and 220% of pretreatment values at the 50 µg, 150 µg, and 400 µg doses, respectively (p < .0001 for all doses).

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FIG. 24. Time course of the effect of (A) daily oral doses of 150 µg of EM-800, 150 µg of tamoxifen, or the combination of both drugs; or (B) daily oral doses of 150 µg of EM-800, 400 µg of tamoxifen, or the combination of both drugs for 4 months on the average size of ZR-75-1 human breast cancer xenografts in ovariectomized nude mice supplemented with an implant of estrone. Ovariectomized nude mice receiving the vehicle alone or supplemented with estrone implants were added as controls. The size of tumors at start of treatment was 31.1 ± 0.8 mm2. Results are expressed as percentage of pretreatment values (means ± SEM of 25 to 37 tumors per group) (Couillard et al., 1998a).

When EM-800 at the daily oral dose of 150 µg was combined with the same dose of tamoxifen, average tumor size decreased from 217% of pretreatment values for tamoxifen alone to 112% for tamoxifen plus EM-800 (p < .001) (Fig. 24). When the higher dose of tamoxifen was used, namely 400 µg daily, addition of 150 µg of EM-800 decreased tumor size from 220% of average initial size for tamoxifen alone to 138% for tamoxifen plus EM-800 (p < .01, Fig. 24B). In the presence of EM-800, average tumor size was not significantly different from the pretreatment values. These data clearly show that under in vivo conditions in nude mice, tamoxifen has a direct stimulatory effect on the growth of human breast cancer xenografts, whereas the novel antiestrogen EM-800 has no stimulatory effect. In fact, 73% of tumors progressed when tamoxifen alone was administered to ovariectomized animals, but no tumor progressed with EM-800. Moreover, in ovariectomized animals supplemented with estrone, EM-800 (150 µg daily) could completely neutralize the increase

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in average tumor size observed with tamoxifen at both the 150 µg (Fig. 24A) and 400 µg (Fig. 24B) doses. This demonstration of a stimulatory effect of tamoxifen on human breast cancer growth is in agreement with previous data obtained in human breast cancer cell lines in vitro (Katzenellenbogen et al., 1987; Lykkesfeldt and Sorensen, 1992; Poulin et al., 1989b; Wakeling et al., 1989) as well as in vivo in nude mice (Gottardis et al., 1988; Osborne et al., 1995). These experimental data are also in agreement with clinical observations suggesting the stimulatory effect of tamoxifen on breast cancer in women (Canney et al., 1987; Hoogstraten et al., 1984; Howell et al., 1990, 1992; Pritchard et al., 1980; Wiebe et al., 1993). Particularly convincing evidence of the estrogenic activity of tamoxifen is also provided by the finding that human breast cancer cell lines showing resistance to tamoxifen retain their sensitivity to specific or pure antiestrogens in vitro (Brunner et al., 1993; Coopman et al., 1994; Lykkesfeldt et al., 1994; Lykkesfeldt and Sorensen, 1992) as well as in vivo in nude mice (Gottardis et al., 1989; Osborne et al., 1991, 1995). Although adjuvant treatment with tamoxifen delays breast cancer recurrence, improves survival in early breast cancer, and induces remission in patients with advanced disease, its benefits are ultimately limited by the development of tamoxifen resistance (Osborne et al., 1994). Similarly, in the in vivo model using nude mice, tamoxifen inhibited MCF-7 tumor growth for 4 to 6 months, but tumor growth then continued despite tamoxifen treatment (Osborne et al., 1987, 1991). In analogy with the present data, Gottardis et al., (1988) have observed the acquired ability of tamoxifen to stimulate rather than to inhibit tumor growth. Since, as mentioned above, pure antiestrogens can block the stimulatory effect of tamoxifen (Couillard et al., 1998a; Gottardis et al., 1989; Osborne et al., 1994, 1995; Osborne et al., 1994) such data suggest that the stimulatory effect of tamoxifen on long-term treatment is due to the intrinsic estrogenic activity of the compound or its metabolites (Osborne et al., 1992). Treatment of nude mice bearing MCF-7 xenografts with 10 mg ICI 182,780 once per week led to a transient decrease of tumor size followed by a plateau of no change for about 200 days followed by progression (Osborne et al., 1995). In mice treated with ICI 182,780, regrowth of tumors or resistance to ICI 182,780 occurred in most tumors (Osborne et al., 1995). The stimulatory effect of tamoxifen or hydroxytamoxifen on human breast cancer cell growth has been reported previously by many laboratories under in vitro (Berthois et al., 1986; Cormier and Jordan, 1989; Darbre et al., 1984; DeFriend et al., 1994a; Katzenellenbogen et al., 1987; Nomura et al., 1990; Osborne et al., 1985; Poulin et

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al., 1989b; Reddel and Sutherland, 1984; Roos et al., 1982; Wakeling, 1989; Weaver et al., 1988) as well as in vivo (Gottardis et al., 1988) conditions. Such intrinsic estrogenic activity of tamoxifen probably limits its success in the treatment of breast cancer in women (Furr and Jordan, 1984). In addition to the data mentioned earlier, the estrogenic action of tamoxifen in breast cancer is supported clinically by the tumor flare observed at the start of therapy (Clarysse, 1985; McIntosh and Thynne, 1977; Plotkin et al., 1978). This early stimulatory effect of tamoxifen is analogous to the present data showing a stimulatory effect of the same drug on the growth of ZR-75-1 xenografts. The withdrawal response observed following arrest of tamoxifen in patients who progress under tamoxifen therapy (Canney et al., 1987; Howell et al., 1992) can also be explained as a result of withdrawing the estrogenic activity of tamoxifen. Since data suggest that continuous long-term tamoxifen therapy is preferable to its usual short-term use (Powles, 1997) and studies are already in progress on long-term administration of tamoxifen or raloxifene to prevent breast cancer (Fisher et al., 1998; Nayfield et al., 1991), it becomes important to make available a pure antiestrogen, which, owing to its lack of estrogenic activity, should theoretically be more efficient than tamoxifen in treating breast cancer while simultaneously eliminating the risk of uterine carcinoma development during its longterm use. We have thus compared the effect of EM-800 or its active metabolite EM-652 with those of hydroxytamoxifen, hydroxytoremifene, droloxifene and raloxifene on estrogen-sensitive alkaline phosphatase activity in human endometrial carcinoma Ishikawa cells. Alkaline phosphatase activity is well known to be stimulated by estrogens, whereas the other steroids, namely androgens, progestins, mineralocorticoids, and glucocorticoids, have no effect on this parameter (Littlefield et al., 1990). We have previously reported that the marked stimulatory effect induced by 1 nM E2 was competitively and completely reversed by EM-800, EM-652, and ICI 182,780, at IC50 values of 1.98 ± 0.23 nM, 1.01 ± 0.16 nM and 5.64 ± 0.59 nM, respectively (data not shown) (Simard et al., 1997b). On the other hand, incubation of Ishikawa cells for 5 days with increasing concentrations of hydroxytamoxifen caused a maximal 3.7-fold increase in AP activity, a half-maximal effect being achieved at 0.15 ± 0.02 nM, while hydroxytoremifene caused a similar stimulatory effect on this estrogen-sensitive parameter at an EC50 of 0.30 ± 0.05 nM (Fig. 25). It can also be seen in Fig. 25 that exposure to 0.1, 1, 10, and 100 nM raloxifene increased alkaline phosphatase activity 3.0–, 2.5–, 2.3–, and 2.1-fold, respectively. This figure also shows that the marked stimulatory effect exerted by 1 nM E2 was competitively, but not completely, reversed by

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FIG. 25. Effect of increasing concentrations of EM-800, hydroxytamoxifen, hydroxytoremifene, and raloxifene on alkaline phosphatase activity in human Ishikawa cells. Alkaline phosphatase activity was measured after a 5-day exposure to increasing concentrations of the indicated compounds in the presence or absence of 1.0 nM E2. The data are expressed as the means ± SEM of four wells. When SEM overlaps with the symbol used, only the symbol is shown (Simard et al., 1997b).

hydroxytamoxifen, hydroxytoremifene, and raloxifene, their partial inhibitory action being exerted at IC50 values of 13.5 ± 3.8 nM, 41.0 ± 7.2 nM, and 3.74 ± 0.43 nM, respectively, while the E2-induced alkaline phosphatase activity was completely blocked by simultaneous exposure to EM-800 at an IC50 value of 1.73 ± 0.19 nM (Simard et al., 1997b). The present data clearly demonstrate that the novel nonsteroidal antiestrogen EM-800 and its active metabolite EM-652 exert pure antagonistic effects while being the most potent of the compounds tested on E2-induced alkaline phosphatase activity in human Ishikawa endometrial adenocarcinoma cells. Hydroxytamoxifen and hydroxytoremifene, in contrast to EM-652, exert a stimulatory effect on this estrogen-sensitive parameter, an effect that can be competitively blocked by simultaneous exposure to the antiestrogen EM-652, EM-652-HCl, or EM-800, thus supporting the suggestion that the stimulatory effect of these

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antiestrogens is mediated through activation of the estrogen receptor (Simard et al., 1997b). The appearance of uterine carcinoma in women treated with tamoxifen (Carlson, 1997; MacMahon, 1997) is not surprising, since tamoxifen has been shown to stimulate the growth of two human endometrial tumors implanted in nude mice (Clarke and Satyaswaroop, 1985; Gottardis et al., 1988; Satyaswaroop et al., 1984) as well as in vitro (Anzai et al., 1989; Croxtall et al., 1990; Jamil et al., 1991). Furthermore, hydroxytamoxifen has been shown to be potent and sometimes even more potent than E2 itself in stimulating progesterone receptors in the human Ishikawa endometrial cell line (Jamil et al., 1991). It should be added that the relationship between estrogens and endometrial carcinoma is well known (Hoover et al., 1976; Smith et al., 1975; Ziel and Finkle, 1975). As further support to the data obtained in human endometrial carcinoma, the potent stimulatory effect of tamoxifen on estrogen-sensitive parameters in the normal uterus is also well known in the mouse, rat, and hamster (Harper and Walpole, 1966, 1967; Labrie et al., 1992a). It thus appears that the estrogenic activity of tamoxifen in the uterus is common to all estrogen-sensitive parameters and species studied so far. The consequence of the partial agonistic activity of tamoxifen is that “complete blockade of the action of estrogens cannot be achieved with tamoxifen” (Wakeling, 1993) or the other compounds demonstrated to exert stimulatory effects, reversible with EM-652 on the proliferation of human breast cancer cells and alkaline phosphatase in human endometrial carcinoma cells. It is thus reasonable to expect that the availability of a pure antiestrogen, in addition to avoiding the risk of inducing endometrial carcinoma, should provide significant benefits over tamoxifen in the treatment of breast cancer. H. Prevention of Bone Loss by EM-652 and Raloxifene Osteoporosis is a disease characterized by a generalized loss of bone mass with the associated increased risk of fracture (Riggs, 1991). The reduction in circulating ovarian estrogen levels at menopause is thought to be largely responsible for the accelerated bone loss in women (Stevenson et al., 1989) and is also associated with the higher risk of coronary heart disease, which is at least partially related to an increase in serum lipids (Gordon et al., 1978; Matthews et al., 1989). Tamoxifen, an antiestrogen with partial agonistic properties, has been shown to maintain bone mass and lower serum cholesterol levels in postmenopausal women (Love et al., 1991b, 1992). As indicated above, the uterotrophic activity of tamoxifen, however, limits its acceptability for the prevention and treatment of osteoporosis. In a previous

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study in rats, we observed that addition of EM-800 to dehydroepiandrosterone (DHEA) treatment showed an additive effect on many parameters of bone physiology, thus suggesting a positive action of EM-800 on bone loss (Martel et al., 1998b). This report describes the ability of EM-800 to prevent bone loss and lower serum cholesterol levels in ovariectomized (OVX) rats and compares its effects with those of raloxifene (Black et al., 1994). The OVX rat is a well-recognized animal model, which mimics the development of estrogen deficiency–induced osteopenia in humans. It is also a useful model for studying the lipid profile of compounds (Lundeen et al., 1997), a close parallel being found between the effect of selective estrogen receptor modulators (SERMs) as inhibitors of serum cholesterol and prevention of bone resorption (Black et al., 1994; Grese et al., 1996). Increasing doses of EM-800 and raloxifene were thus administered orally for 37 weeks to OVX animals, and the effect of these two compounds as well as that of 17β-estradiol (E2) on parameters of bone physiology and serum lipids were examined. Despite its pure antiestrogenic activity in the mammary gland and endometrium as summarized above, EM-652 exerts potent protective effects in bones. However, it is unique in having pure antiestrogenic activity in both human breast and uterine cells (Couillard et al., 1998a; Gauthier et al., 1997; Simard et al., 1997a, 1997b), while being the most potent among SERMs studied so far in preventing loss of bone mineral density (BMD) and lowering serum cholesterol in the rat. It is of interest that studies with estrogens have shown that the inhibition of bone turnover found in short-term studies translates into increased BMD and decreased fracture rate in long-term studies (Ettinger et al., 1985; Field et al., 1993; Lufkin et al., 1992; Weiss et al., 1980). The “estrogen-like” action of SERMs in the bone should thus lead to a decrease in bone fractures as confirmed recently in the MORE trial (Cummings et al., 1999). The mean pretreatment values of bone mineral density (BMD) measured in vivo by DEXA during the acclimation period at the lumbar spine, total body skeleton, and femoral site were 0.148 ± 0.003g/cm2, 0.118 ± 0.001 g/cm2, and 0.245 ± 0.004 g/cm2, respectively. The BMD of the lumbar spine was 19% lower in OVX control rats than in intact controls (p < .01) (Labrie et al., 1999). The animals given EM-800 or raloxifene at doses of 0.01–1 mg/kg had 90 to 93% and 85 to 90%, respectively, of the BMD observed in intact rats, the BMD values being significantly higher than those of OVX control rats (p < .01), with the exception of the lowest dose of raloxifene (0.01 mg/kg), which did not have a statistically significant effect on this parameter. The lumbar spine BMD of rats treated with E2 was 92% (p < .01) of that observed in the

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intact controls. This stimulatory effect of E2 is not statistically different from that of EM-800 at all doses studied. It is of interest to mention that EM-800 already had a maximal stimulatory effect on lumbar spine BMD at the lowest dose used (0.01 mg per kilogram of body weight, p < .01), whereas a statistically significant effect of raloxifene was first observed at the 0.03 mg per kilogram of body weight (p < .01); thus EM-800 is at least three times more potent than raloxifene on lumbar spine BMD. Thirty-seven weeks after ovariectomy, marked decreases of 73% (p < .01) and 77% (p < .01) in trabecular bone volume and trabecular bone number (data not shown), respectively, were observed at 1 to 5 mm of the growth plate metaphyseal junction of the proximal tibia. Simultaneously, a marked increase in trabecular bone separation from a control value in intact rats of 262 ± 19 to 1486 ± 236 µm (p < .01) was observed in OVX animals. Treatment with 1 mg/kg of EM-800 and raloxifene resulted in 68% (p < .01) and 64% (p < .01) reversals, respectively, of the decrease in trabecular bone volume caused by ovariectomy. In fact, treatment with EM-800 and raloxifene at the daily 1 mg/kg dose increased trabecular bone volume of the proximal tibia from a control value of 5.8 ± 0.9% in OVX animals to 16.4 ± 0.4% and 15.8 ± 1.0%, respectively. These stimulatory effects are not statistically different from the 53% reversal achieved with E2. At the lowest dose used (0.01 mg/kg), EM-800 already reversed by 34% (p < .01) the effect of OVX whereas raloxifene had no detectable effect. The administration of 0.1 mg/kg of EM-800 and raloxifene, on the other hand, resulted in 40% (p < .01) and 24% (p < .05) reversals, respectively, of the decrease in trabecular bone volume caused by OVX. Even the 0.01 mg/kg dose of EM-800 caused a 66% (p < .01) reversal of the effect of OVX on trabecular bone separation, and a 76% (p < .01) reversal of this parameter was observed at the 0.1 mg/kg dose (Fig. 26). Raloxifene, on the other hand, had no detectable effect at the lowest dose used (0.01 mg/kg), but a 63% (p < .01) reversal of the effect of OVX was observed at the 0.1 mg/kg dose. At the 1 mg/kg dose, EM-800 and raloxifene caused 85% (p < .01) and 88% (p < .01) decrease in trabecular bone separation, as compared with OVX controls. Estradiol, on the other hand, reversed by 85% (p < .01) the effect of OVX, a value similar to that achieved with the 1 mg/kg dose of EM-800 or raloxifene. Figure 27 (see color insert) illustrates the prevention of trabecular bone volume in the proximal tibial metaphysis induced by EM-800 and raloxifene in ovariectomized treated animals compared with OVX controls (Fig. 27B). The administration of 0.01 mg/kg of EM-800 (Fig. 27D) already prevented by 52% the OVX-induced osteopenia, whereas raloxifene had no detectable effect at the same dose (Fig. 27F). Treatment

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FIG. 26. Effect of 37-week treatment with increasing daily oral doses of EM-800 or raloxifene on trabecular separation in ovariectomized (OVX) rats. Comparison is made with intact rats. **,p < .01, experimental versus OVX control rats (Labrie et al., 1999).

with 1 mg/kg of EM-800 or raloxifene (Figs. 27E & G) resulted in prevention of approximately 75% of the ovariectomy-induced osteopenia. I. Inhibitory Effects of EM-652 on Serum Cholesterol and Triglyceride Levels As can be seen in Fig. 28, a 36% reduction of serum cholesterol from 2.9 ± 0.18 mmol/liter to 1.8 ± 0.09 mmol/liter was observed even with the lowest dose (25 µg) of EM-800 used (p < .01). A daily 75-µg dose of EM-800 further decreased serum cholesterol to 1.6 ± 0.12 mmol/liter (p < .01), and a 250-µg dose of EM-800 caused a maximal 52% inhibition to a value of 1.4 ± 0.06 mmol/liter (p < .01). The 250-µg dose had an inhibitory effect significantly (p < .01) more important than that of the 25-µg dose of EM-800, whereas the 75-µg dose had an intermediate inhibitory effect, not significantly different from that of the 25-µg and 250-µg doses. A similar inhibitory effect of EM-800 was observed on serum triglyceride levels. The daily administration of 25 µg of EM-800 for 9 months

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FIG. 28. Effect of daily oral administration of 25 µg, 75 µg, or 250 µg EM-800 for 9 months on serum cholesterol (A) and triglyceride (B) levels in the rat. The number of animals per group was 9, 14, 16, and 20, respectively. Data are expressed as mean ± SEM. **,p < .01 vs control (Luo et al., 1998a).

induced a near maximal inhibition (69%) of serum triglyceride levels, which were measured at 1.4 ± 0.21 mmol/liter (p < .01), while the value in control animals was 4.3 ± 0.62 mmol/liter. Daily oral administration of 75 µg of EM-800 caused a maximal inhibition (72%) of serum triglyceride levels to 1.2 ± 0.15 mmol/liter (p < .01), while the 250-µg dose of EM-800 decreased serum triglyceride levels to 1.3 ± 0.12 mmol/l (p < .01). There was no statistically significant difference between the inhibitory effect of the three doses of EM-800. We also compared the effect of increasing doses of EM-800 and raloxifene on serum cholesterol levels (Fig. 29). Thirty-seven weeks after ovariectomy, a 35% increase (p < .01) in serum cholesterol was observed in OVX control rats compared with intact controls. The daily oral administration of even 0.01 and 0.03 mg/kg of EM-800 to OVX animals caused respective 54% (p < .01) and 56% (p < .01) reductions of serum cholesterol levels relative to OVX controls, raloxifene administered at the same doses caused respective 24% (p < .01) and 41% (p < .01) decreases in the same parameter. When administered in daily doses of 0.1, 0.3, and 1 mg/kg, EM-800 caused respective 58%, 58%, and 66% inhibitions (all p < .01 versus OVX control rats) of serum cholesterol levels, and raloxifene caused respective 60%, 62%, and 65% decreases of this parameter at the same doses (all p < .01). The E2 implant, on the other hand, only reduced serum cholesterol by 20% (p < .01) compared with OVX control rats.

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FIG. 29. Effect of 37-week treatment with increasing daily oral doses of EM-800 or raloxifene on total serum cholesterol levels in ovariectomized (OVX) rats. Comparison is made with intact rats and with OVX animals bearing an implant of 17β-estradiol (E2). **, p < .01, experimental versus OVX control rats (Labrie et al., 1999).

The present data show that EM-800 produces marked hypocholesterolemic and hypotriglyceridemic effects in the rat, thus suggesting the possibility of additional beneficial effects in women. It has been repeatedly reported that estrogens and some antiestrogen compounds lower serum cholesterol levels in the rat (Black et al., 1994; Chao et al., 1979; Davis and Roheim, 1978; Dipippo et al., 1995; Ke et al., 1995; Russell et al., 1993; Weinstein et al., 1986; Wilcox et al., 1981; Windler et al., 1980) as well as in the human (Barrett-Connor, 1993; Bruning et al., 1988; Love et al., 1990, 1991b; Matthews et al., 1989; Walsh et al., 1991). The effect of estrogens on human and rat high-density lipoproteins (HDL) have been found to be opposite, with an usual increase in serum HDL levels in the human (Barrett-Connor, 1993; Walsh et al., 1991), but a decrease in the rat (Chao et al., 1979; Staels et al., 1989; Windler et al., 1980). On the other hand, estrogens are known to elevate serum triglyceride levels in both the rat (Dipippo et al., 1995; Russell et al., 1993) and the human (Barrett-Connor, 1993; Love et al., 1990, 1991b; Matthews et al., 1989; Walsh et al., 1991), thus demonstrating a potential adverse effect on lipid metabolism. It is thus of particular interest to see that EM-800 reduces both serum cholesterol and triglyceride levels, which indicates a potentially more global beneficial effect of EM-800 on lipid metabolism. The

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other antiestrogens, such as tamoxifen (Brunner et al., 1993; Dipippo et al., 1995; Love et al., 1990, 1991b), droloxifene (Ke et al., 1995), and raloxifene (Black et al., 1994), have been reported to elicit beneficial effects on the serum lipid profile, but they have not demonstrated such an effect on serum triglycerides in the rat or in the human. The ability to lower both serum cholesterol and triglyceride levels seems to be unique to EM-800. The similar inhibitory effect achieved with the 25 µg, 75 µg and 250 µg doses of EM-800 on serum triglyceride levels suggests a higher sensitivity of this parameter to the action of EM-800 as compared with serum cholesterol. An ideal therapy at menopause should prevent bone loss and simultaneously reduce cardiovascular risks without producing significant estrogenic effects on the endometrium and mammary gland, which seriously limit the acceptance of the current estrogen replacement therapy. EM-800 lacks a stimulatory effect on the endometrium, as shown at histopathological examination in this study as well as in previous studies (Couillard et al., 1998b, Sourla et al., 1997; Luo et al., 1998b). Similarly, EM-800 shows pure antiestrogenic activity in human endometrial Ishikawa carcinoma cells (Simard et al., 1997b). Raloxifene, on the other hand, was shown to have no stimulatory effect on the endometrium in a short-term study in the rat (Black et al., 1994). However, in the present long-term study, raloxifene has been shown to cause a significant stimulation of the endometrial epithelium at doses that are effective in preventing bone loss (0.1 to 1 mg/kg). It should be mentioned also that raloxifene, droloxifene, and tamoxifen stimulate, to various degrees, the estrogen-sensitive parameter alkaline phosphatase in human endometrial Ishikawa carcinoma cells, the stimulatory effect of these compounds being fully reversible by EM-800 (Gauthier et al., 1997; Simard et al., 1997b). The present data clearly demonstrate that in the rat, low doses of EM-800 prevent bone loss and lower serum cholesterol levels without stimulatory effect on the endometrium, and previous studies have described the pure antiestrogenic activity of this compound in the mammary gland. Such data are encouraging and suggest that the antiestrogen EM-652 and its precursor EM-800 have the potential for exerting simultaneous beneficial effects on four important aspects of women’s health, namely, prevention and/or treatment of breast and uterine cancer, osteoporosis, and coronary heart disease. Although pure steroidal antiestrogens such as ICI 164,384, ICI 182,780, and EM-139 could also be more effective than tamoxifen in controlling estrogen-sensitive breast cancer, they cannot prevent bone loss and might even have harmful effects on the skeletal and

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cardiovascular systems (Favoni and de Cupis, 1998; Nicholson et al., 1995; Nique et al., 1994; Wakeling and Bowler, 1988b). In order to test the hypothesis that a more specific and more potent antiestrogen devoid of estrogenic activity in human breast (Gauthier et al., 1997; Simard et al., 1997a; Tremblay et al., 1997) and endometrial (Simard et al., 1997b) cancer cells could show increased clinical efficacy, we have administered the novel antiestrogen EM-800 to women who had failed tamoxifen therapy. This approach is supported by the finding that human breast cancer cell lines showing resistance to tamoxifen retain their sensitivity to pure antiestrogens under in vitro conditions (Brunner et al., 1993; Coopman et al., 1994; Lykkesfeldt et al., 1994; Lykkesfeldt and Sorensen, 1992) and as xenografts in nude mice (Couillard et al., 1998a; Gottardis et al., 1989; Osborne et al., 1991, 1995). Forty-three (43) postmenopausal or ovariectomized women of median age 67 years (43 to 86 years) with breast cancer resistant to tamoxifen were treated with daily oral doses of 20 mg or 40 mg of EM-800 (SCH 57050). The patients had progressive metastatic or locally advanced inoperable breast cancer as proven by biopsy or fine-needle aspiration, which had responded to tamoxifen (completely or partially) or which had been stable for at least 6 months but was in progression. Patients originally treated with tamoxifen as an adjuvant to surgery for at least 1 year who were in progression on tamoxifen or who were progressing after cessation of tamoxifen were also candidates. Tamoxifen had to be stopped for at least 1 month unless the investigator judged that the disease was progressing rapidly, where as 2 weeks were sufficient before starting treatment with EM-800. The study was approved by the institutional review board of each hospital or university. The predominant sites of failure with tamoxifen at the start of EM-800 administration were in decreasing order of incidence rate: bone (28), nodes (15), liver (11), skin (8), lung (7), breast (2), and parotid gland (1). Progression was present at one site in 20 patients, at two sites in 17 patients, and at three sites in 6 patients at the start of treatment with the antiestrogen. As shown in Table II, one patient had a complete response and is still responding at 27 months, and five patients had a partial response. Complete and partial responses have been observed so far in six patients (13.9%), and no change for at least 3 months has been observed in 13 patients (30.2%), for a total of 19 positive responses among 43 evaluable patients (44.2%). When stable response for at least 6 months is considered, 10 patients meet this criterion for a total of 16 positive objective responses, or 37.2% of the 43 patients. Four patients are still responding, namely, one completely at 27 months, one partially at 21

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TABLE II Best Response to EM-800 and Response Durations by Dose 20 mg (n = 21) Best a response No. (%) Response duration, months b

No. (%) Response duration, monthsb

CR PR SD PD

1 (4) 2 (9) 8 (36) 11 (50)

a b

0 (0) 3 (14) 5 (24) 13 (62)

— 9,15,21+ 4.5,6,9,9,11 —

40 mg (n = 22)

27+ 8, 9 4,4.5,6,9,16,17,23+,24+ —

CR, complete response; SD, stable disease; PR, partial response; PD, progressive disease. +, response still ongoing.

months, and two at 23 and 24 months of stable disease, respectively (Table II). A complete response was observed after 3 months of treatment in a patient who had shown progression in a node of the right axilla while being treated with tamoxifen as adjuvant for 42 months. Of the five partial responders, one patient had received tamoxifen for advanced disease for 8 months and four had received adjuvant therapy for 5, 61, 64, and 108 months, respectively. In the no change category, two had received adjuvant tamoxifen for only 13 and 14 months while two had received adjuvant tamoxifen for 37 and 70 months. In the same category of response, six had received palliative treatment for advanced disease for 10, 25, 26, 34, 64, and 92 months, respectively, while three had received tamoxifen for both adjuvant therapy and advanced disease. In the group of patients who showed no positive response to EM-800, 12 had received adjuvant tamoxifen only for 13 to 115 months, 3 had adjuvant followed by palliative tamoxifen, and 9 had received tamoxifen for advanced disease for 10 to 38 months. The present findings suggest that the progression of breast cancer that occurs with tamoxifen treatment can be due to the intrinsic estrogenic stimulatory activity of tamoxifen on breast cancer proliferation (Brunner et al., 1993; Canney et al., 1987; Couillard et al., 1998b; Gottardis et al., 1988, 1989; Howell et al., 1990, 1992; Katzenellenbogen et al., 1987; Lykkesfeldt et al., 1994; Lykkesfeldt and Sorensen, 1992; Osborne et al., 1995; Poulin et al., 1989b; Wakeling et al., 1989). As mentioned above, clinical evidence supports the suggestion that tamoxifen-stimulated tumor growth is a mechanism responsible for tamoxifen resistance or no response in an unknown proportion of breast cancer patients (Canney et al., 1987; Hoogstraten et al., 1984; Howell et al., 1990, 1992), Pritchard et al. 1980; Wiebe et al., 1993). The possibility also exists that changes in the intracellular metabolism or distribution of tamoxifen could explain the loss of response to this antiestrogen (Osborne et al., 1991; Pavlik et al.,

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1992). In a small proportion of cases, the resistance to tamoxifen could possibly be explained by loss of ER expression or by ER mutation (Encarnacion et al., 1993; Osborne et al., 1995). When LY156758 (raloxifene) was used as second-line treatment in a group of 14 patients, no complete or partial response was found, with 5 patients (36%) showing no change (Buzdar et al., 1988). It is clear, however, that large-scale and randomized studies are required to truly assess the benefits of a new drug in such a heterogeneous and difficult to treat population of patients in whom tamoxifen therapy has failed. Owing to its specific antiestrogenic activity and its particularly high potency, it is reasonable to expect that EM-800 should not only be more efficient than tamoxifen in treating breast cancer but that its use should also decrease the estrogen-related risk of carcinogenicity (Vancutsem et al., 1994) and induction of uterine carcinoma (Moyer et al., 1993) during long-term use. In fact, EM-800 is the only nonsteroidal antiestrogen showing no estrogenic activity in human Ishikawa endometrial carcinoma cells, as assessed by changes in alkaline phosphatase activity, a well known estrogen-sensitive parameter (Simard et al., 1997b). In long-term studies in the rat, mouse, and monkey, EM-800 shows a potent inhibitory effect on the uterus (Luo et al., 1997b; Sourla et al., 1997, Labrie et al., unpublished data). Moreover, the new antiestrogen EM-800 has been shown to have good oral bioavailability in the mouse, rat, monkey, and human, thus providing an important advantage over the steroidal antiestrogens, which possess poor oral bioavailability (Howell et al., 1995; Labrie et al., 1992a). In toxicology studies using up to 25 mg per kg body weight, a dose approximately 35-fold higher than the highest dose used in the present study, no toxic effects other than the endocrine changes expected from a pure antiestrogen have been observed in female rats and monkeys treated daily for 6 months. In phase 1 studies in which 145 normal postmenopausal women received daily doses of EM-800 of up to 40 mg for up to 14 days, as well as in this phase 2 study, in which breast cancer patients received the daily 40 mg dose for up to more than 2 years, no serious adverse effect related to the drug has been observed. The present data, strongly supported by a long series of preclinical studies, suggest that EM-800 (SCH 57050) may improve the rate, quality, and duration of response of advanced breast cancer to endocrine therapy. Further studies are required to obtain a more precise assessment of the response rate and its duration and to determine the longterm effects on bone, lipids, and the endometrium. It is of interest that this compound has been shown to prevent bone loss in the ovariectomized rat (Figs. 26 and 27) and to decrease serum cholesterol and triglycerides in the rat (Fig. 29) (Luo et al., 1998a) and in post-

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FIG. 30. Schematic representation of the estrogenic and/or antiestrogenic action of estradiol, tamoxifen, raloxifene, and EM-652 on the main parameters important for women’s health, namely the breast, endometrium, bone, and serum lipids.

menopausal women (Labrie et al., unpublished data). Coupled with the pure antiestrogenic activity of EM 800 in human breast and endometrial cancer cells and the lack of toxicity seen in any phase I or II study in women, as well as in all the toxicology studies performed in the rat and monkey, the present data suggest the reason for the interest in studying the effect of EM-800 (SCH 57050) or its active compound EM652 (SCH 57068) in the neoadjuvant and adjuvant settings and, most importantly, for prevention of breast cancer. As predicted by detailed preclinical studies, the present clinical data obtained in tamoxifen failure patients suggest that EM-800 (SCH 57050) is a promising new drug for the prevention and treatment of breast and endometrial cancer while also exerting beneficial effects on bone and lipids, which are major parameters of women’s good health at menopause. Figure 30 summarizes the characteristic effects of 17β-estradiol and of the three classes of antiestrogens so far available, namely tamoxifen (first-generation SERM), raloxifene (second-generation SERM) and EM-652 (pure SERM), on the best known parameters of women’s

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MONOCLONAL ANTIBODY THERAPY BY JOHN W. PARK* AND JOSEF SMOLEN† *Division of Hematology and Oncology, Department of Medicine, University of California, San Franciso, Medical Center, San Franciscos, California 94115, and †Division of Rheumatology, Internal Medicine III, University of Vienna General Hospital, and Second Department of Medicine, Center for Rheumatic Diseases, Lainz Hospital, Vienna, Austria

I. Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. General Aspects of Monoclonal Antibody Therapy . . . . . . . . . . . . . . . . . . . . A. Generation of Monoclonal Antibodies. . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Monoclonal Antibody Engineering . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Monoclonal Antibody Therapy in Organ Transplantation. . . . . . . . . . . . . . . A. Monoclonal Antibodies to T-Cell Differentiation and Function-Associated Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. mAb to Activation-Associated T-Cell Antigens . . . . . . . . . . . . . . . . . . . . . . IV. Monoclonal Antibody Therapy in Cardiac Disease. . . . . . . . . . . . . . . . . . . . . A. Coronery Artery Disease . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cardiomyopathy and Heart Failure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Monoclonal Antibody Therapy in Infections Diseases . . . . . . . . . . . . . . . . . . A. Septic Shock . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Monoclonal Antibodies to Viral Antigens . . . . . . . . . . . . . . . . . . . . . . . . . VI. Monoclonal Antibody Therapy in Rheumatologic and Autoimmune Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Rheumatoid Arthritis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Systemic Lupus Erythematosus . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Other Rheumatologic Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Crohn’s Diseases. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Monoclonal Antibody Therapy of Cancer . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. General Considerations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Lymphocyte Differentiation Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Oncogene-Product Antigens . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VIII. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION Defense mechanisms employing the innate and the adaptive immune response evolved primarily to protect the host against the attack of infectious agents (1, 2). However, the host is also threatened by a variety of insults that are not necessarily infectious in origin, such as malignancy, autoimmunity, or an otherwise dysregulated physiology. To combat such insults by counterbalancing an inappropriate, inadequate, or nonexistent immune response is the idea behind all immunotherapy. 369 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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Antibodies constitute a key component of the adaptive immune response. Each immunoglobulin (Ig) molecule consists of heavy (H) and light (L) chains, the genes for which are drawn from a vast number of distinct DNA segments that are present in the germline and that subsequently recombine during B-cell maturation (3, 4). During a specific immune response and depending on the type and location of the response, antibody genes undergo somatic recombination, isotype switching, and affinity maturation in order to generate a highly functional Ig as a defense protein (3–6). Immunogloblin classes include the IgA, IgD, IgE, IgG, and IgM isotypes. Of these, the IgG molecule has been particularly important in antibody therapy. The IgG molecule is a dimer of heavy (H) and light (L) chains organized into two structurally distinct components: a C-terminal crystallizable fragment (Fc portion), containing two of the three constant regions of the H chains, bound to each other in part by disulfide bonds; and two N-terminal Fab regions (fragment for antigen binding), each consisting of constant and variable regions of the L chain and the N-terminal half of the H chain, with L and H chains interconnected. The portion of the Fab region consisting solely of variableregion sequences is the Fv. The IgG molecule can be enzymatically split into its single Fc and two Fab fragments by using papain. When pepsin is employed, the two Fab portions remain bound together as a single F(ab’)2 fragment, leaving a smaller Fc portion. The variable (V) segments of both the H and the L chains (VH and VL, respectively) contain the complementarity-determining regions (CDRs) as well as the framework regions. The CDRs in turn consist of three hypervariable domains, which mediate binding to a cognate structure on the antigen, the epitope. Individual B cells usually produce antibodies of a single specificity or antigenic reactivity, and the fact that many different B cells react to a single antigen (and sometimes even to a single epitope) allows the generation of a polyclonal immune response. A single antibody molecule usually reacts with a specific antigenic epitope to which it possesses a specific binding site, the idiotype. The Fc region determines the antibody isotype or class, and, depending on the isotype, is involved in different functions such as complement fixation (with subsequent lysis or enhanced phagocytosis), Fc-receptor binding (with subsequent antibody-dependent cellular cytotoxicity (ADCC) or enhanced phagocytosis), or other effector mechanisms (7). Given the common and sometimes fatal weakness of the adaptive immune response in its interaction with infectious agents, various means to enhance the activity of the immune system have been developed. One, and the oldest, of these approaches is vaccination, which started in 1796 with Jenner’s cowpox vaccine (8) and is of course ongoing today.

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This approach has led to prevention, eradication, or at least amelioration of a number of bacterial and viral infectious diseases (9, 10). Another therapeutic approach is serotherapy, which exploits the presence of antibodies to antigen(s) in individuals who have previously undergone a specific immune response, either by natural exposure or by active immunization (11). Such serum or more recently, purified Ig preparations can be homologous or heterologous. These injectable proteins have been used to prevent the onset of certain diseases or to provide treatment acutely, as with infectious diseases, exposure to biological toxins, and rejection of transplanted organs. These serotherapeutic approaches can be regarded as predecessors of monoclonal antibody therapy. II. GENERAL ASPECTS OF MONOCLONAL ANTIBODY THERAPY A. Generation of Monoclonal Antibodies Monoclonal antibody therapy (MAT) makes use of all the major features of the immune response. It involves vaccination/ immunization, albeit in experimental animals, to induce the desired specific immune response. It exploits the high specificity, selectivity, and affinity of the antibody CDR toward the target antigen to be recognized, highlighted, inactivated, or eliminated, using the characteristics of the Fc portion of an immunoglobulin to facilitate the means for such inactivation or elimination and for selection of appropriate effector mechanisms. Finally, MAT represents a modern form of serotherapy, in which parenteral administration of whole serum or Ig preparations has been replaced by recombinant antibody molecules of a defined specificity. The single major breakthrough leading to MAT came from the finding of Köhler and `Milstein that homogeneous or monoclonal antibodies (mAbs) of a given specificity could be obtained by fusion of a normal cell producing an antibody against a defined antigen (usually a B cell derived from an immunized mouse) with immortalized myeloma cells (12). These hybridomas can produce specific antibodies in virtually unlimited amounts. Since these seminal observations a quarter of a century ago, this technique has revolutionized the biological sciences, as mAbs have provided a research tool of enormous power and versatility. In medicine, mAbs were initially used mostly for in vitro diagnostic or pathologic applications, but they have now entered clinical use as in vivo diagnostics or imaging agents and, particularly, for targeted therapy. It is especially the potential for highly specific targeting of selected structures that has led to this new era of mAb technology, a potential that has arisen from advances in mAb technology itself as well as from a better understanding of pathogenetic mechanisms in various disorders.

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B. Monoclonal Antibody Engineering One of the major obstacles to successful MAT was the limitation of the applicability of murine (or other xenogeneic) mAbs. Their biologic activity in the human environment is limited, since the host’s immune response to these antibodies, namely production of human antimouse antibodies (HAMAs), is not only potentially associated with undesirable and sometimes life-threatening clinical side effects, but also with neutralization or enhanced elimination of the therapeutic mAb. This could be partly prevented by concomitant immunosuppression, including the use of immunosuppressive mAbs as in the case of organ transplantation. Several approaches have been undertaken to reduce or eliminate the immunogenicity of mAbs. In some cases, limited administration of mAb fragments devoid of the Fc portion may reduce immunogenicity, although it does not eliminate it. Thus, strategies to reengineer mAbs have emerged, and the first such approach was the development of chimeric antibodies. For chimerization, cloned antibody genes are first generated in which the variable regions from the original murine mAb are combined with human antibody-constant regions (13). Examples of such chimeric antibodies (cAbs) are abciximab, infliximab, and rituximab. The biologic activity and half-life of such mouse/human chimeric antibodies correspond more closely to those of human immunoglobulins (hIg). Since the antigen recognition domain is still of foreign origin, immune responses to this domain of the cAb can still occur; such human antichimeric antibodies (HACA) may reduce clinical efficacy or elicit side effects, although this tends to be much lower in frequency and extent than with fully murine mAbs. The immunogenicity of mAbs could be further reduced by combining a primate-derived variable region with human constant regions (primatized antibodies) (14). A still further step was the development of humanized mAbs (15). For humanization, all portions of the mAb not required for antigen binding, including framework residues in the variable region, are replaced with human sequences, usually resulting in less than 10% of the CDR being retained from the parental murine mAb. Examples of humanized mAbs include dacliximab, anti-CD33, anti-RSV (respiratory syncytial virus) F protein, and trastuzumab. These humanized antibodies have half-lives that are very similar to that of endogenous IgG. In addition to these advances in mAb engineering, in which xenogeneic mAbs can be converted to increasingly more human-like mAbs, techniques to produce fully human mAbs have been developed in which mAbs never pass through a xenogeneic stage and are in fact devoid of any xenogeneic sequences whatsoever. For example,

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two such approaches are being increasingly used, one employing human antibody genes in phage display libraries and the other using human antibody genes in transgenic mice. In the human antibody gene–phage display technique, genes encoding the VH and VL chains are generated by polymerase chain reaction (PCR) cloning from “naive” human lymphocytes; these genes are then assembled into a library from which they can be expressed either as disulfide-linked Fab fragments or as single-chain Fv (scFv) fragments; the Fab- or scFv-encoding genes are also fused to a surface coat protein of filamentous bacteriophage (16, 154). Fab or scFv of the desired specificity can then be identified by screening the library with purified antigen or other antigen source, with reactive phage clones subject to further iterations of the screening process. Molecular evolution or affinity maturation procedures can be employed to enhance the affinity of the Fab or scFv fragment. An example of a fully human mAb with high affinity is the anti-TNF (tumor necrosis factor) antibody D2E7. The transgenic mouse technique uses animals in which the endogenous murine Ig gene loci have been replaced by homologous recombination with their human homologues. Thus, immunization of the hIg-transgenic mouse followed by conventional hybridoma technology yields fully human mAb. Transgenic mice with different antibody repertoires have been derived (17, 18). An example of a human mAb produced via the transgenic mouse is anti-IL-8. In addition to conventional mAbs based on the IgG (or occasionally IgM) structure, hybrid constructs containing constant-region mAb sequences fused to receptor sequences rather than an antigen-binding region have been created (19). Such so-called immunoadhesins provide the ability to use endogenous receptors, which may be directed against potentially pathogenic mediators, in the context of a potentially long circulating antibody-like molecule. Immunoadhesins also may be less likely to elicit anti-idiotypic immune responses than conventional mAbs, as the host is likely to be made tolerant to the receptor component. Nevertheless, immune responses to the hinge region connecting the receptor and antibody sequences can ensue (20). While these advances in mAb technology have progressively overcome the obstacle of host immune responses to mAbs, other advances have made it possible to overcome the previously limiting obstacle of large-scale production. To this end, a number of strategies have been successfully developed, including highly efficient expression of cloned antibody genes in prokaryotic and eukaryotic cell culture systems. Other recent innovations have included production of mAbs in the milk of transgenic cows or goats (21).

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III. MONOCLONAL ANTIBODY THERAPY IN ORGAN TRANSPLANTATION A. Monoclonal Antibodies to T-Cell Differentiation and Function-Associated Antigens The rejection of allografts is an imunological response mediated primarily by T cells (22). Consequently, targeting T cells by pharmacological and biological means has been a major aim in the development of therapeutic strategies for organ transplantion. Heterologous, polyclonal antilymphocyte globulin (ALG) and, later, antithymocyte globulin (ATG) were the first biological compounds to be used in transplant rejection; these preparations induced rapid elimination of lymphocytes and effective immunosuppression (23). However, these preparations also had a broad spectrum of reactivities, including cross-reactivities with surface antigens in normal tissues other than T cells. Aside from constitutional symptoms, such as nausea, fever, arthralgias, diarrhea, and the risk of infections by virtue of their pharmacological effect, ALG and ATG also induced thrombocytopenia and increased the risk of lymphoid malignancy. Other side effects included occasional serum sickness and shock. As xenoproteins, they elicited strong immune responses, which in turned to rapid elimination and precluded repeat administration. With the emergence of mAb technology and the approval of the first pan–T cell mAb, OKT3 (24), a new era was introduced. OKT3 is a murine mAb directed against the ε chain of the CD3 molecule; CD3 is a differentiation antigen present on all T cells, is associated with the Tcell antigen receptor (TCR), and functions as a signal transduction complex for the TCR (25). OKT3 is administered systemically for prophylaxis of graft rejection, for induction therapy (26), and particularly for the salvage of grafts in the course of steroid nonresponsive rejection; in the latter setting OKT3 has a success rate of more than 90% (27). OKT3 leads to profound T-cell depletion, partly by complement-mediated cytotoxicity, and CD3(+) cell depletion is prolonged by the emergence of CD3(–) cells during repopulation. Consequently, infections, particularly opportunistic viral infections, occur frequently, and the incidence of B-cell non-Hodgkin’s lymphomas or posttransplantation lymphoproliferative disorders (PTLD), usually associated with activation of endogenous Epstein-Barr virus (EBV), is increased. Other adverse effects include a cytokine release syndrome that is commonly seen. The frequent generation of HAMA reduces the half-life of OKT3, can lead to inactivation of the pharmacological effect, and increases the risk of anaphylactic reactions. Monoclonal antibodies directed against other T-cell antigens, such as CD4, CD7, and CD11a

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(28), which were theoretically promising, have been found to be less successful in clinical studies. Other promising targets are adhesion and costimulatory molecules such as LFA-1 and CD28. B. Monoclonal Antibodies to Activation-Associated T-Cell Antigens An mAb which is already approved for renal transplant patients is anti-CD25 or anti-Tac (29). CD25 is the 55-kDa α-chain of the IL-2 receptor (IL-2R), which is expressed only on activated T cells. The 75kDa β-chain of the IL-2R is also part of the IL-15R, and the 64-kDa γchain is common to the IL-4R, IL-7R, IL-9R, and IL-13R receptors. The α chain: (1) is specific for the IL-2R; (2) by virtue of its short intracytoplasmic tail lacks signaling ability; (3) as a single chain binds IL-2 only very weakly; and (4) together with the β and γ chains forms the highaffinity IL-2R. Anti-CD25, therefore, inhibits IL-2 activation of the high-affinity IL-2R. It does not prevent IL-2 from binding to the intermediate-affinity IL-2R consisting of the β and γ chains and present on cells such as nonactivated T cells and natural killer cells. However, activation of the intermediate affinity IL-2R requires higher IL-2 production than that required to activate the high-affinity receptor. In the context of renal transplantation, IL-2 production is largely suppressed by agents such as cyclosporin A or FK 506; hence, typical IL-2 levels are usually insufficient to activate the intermediate IL-2R. Two anti-CD25 mAbs are currently available for therapeutic purposes: these are basiliximab, a chimeric anti-CD25 mAb, and daclizumab (or dacliximab), a humanized mAb. The mAbs mainly differ in their half-life, which is four to five fold longer for dacliximab (Table I), and in their affinity, which is ten fold higher for basiliximab; the change in framework residues around the CDRs in the course of humanization of dacliximab appears to have led to reduced affinity. Although basiliximab is chimeric rather than humanized or human, HACA are generally a minor problem in renal transplant patients, since they are immunosuppressed and the anti-CD25 mAb itself may further support a state of (transitory) tolerance to the chimeric mAb. When administered on the day of renal transplantation, both antiCD25 mAbs were highly effective in reducing the frequencies of first rejection episodes or graft loss (by approximately 20 to 30%) (29–32). Moreover, the number of rejection episodes subjected to antibody therapy was reduced by about 50% as compared with placebo. The safety profile of both mAbs was similar to that of placebo, at least within the first 6 to 12 months after administration. In particular, neither infections nor malignancies were found to be increased in incidence, and

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TABLE I Anti-IL-2R Monoclonal Antibodies: Daclizumab and Basiliximab Daclizumab Monoclonal antibody Source Isotype Half-life Affinity (Kd) Dose

Anti-IL-2Rα (CD25) Humanized mAb IgG1κ 29 days 1.0 nM 5 × 1 mg/kg i.v.

Basiliximab Anti-IL-2Rα (CD25) Chimeric mouse/human mAb 1gG1κ 6 days 0.1 nM 2 × 0.3 mg/kg i.v.

the treatment itself was very well tolerated. For basiliximab, a serum concentration of 0.2µg/ml was necessary to achieve sufficient IL-2R blockade on activated T cells. Anti-CD25 mAbs are probably not helpful in the therapy of acute episodes of renal transplant rejection, given that acute rejections occurred even during periods of high circulating mAb levels (31). This is not too surprising, since after initiation of the rejection event, different mechanisms involving cytokines other than IL-2 and several different cell populations appear to be operative (33). Anti-CD25 mAbs are also useful in the therapy of other allografts, such as liver transplantation and bone marrow transplantation (34, 35) and may also prove beneficial in other disorders, such as rheumatoid arthritis and systemic lupus erythematosus (see Section VI.) IV. MONOCLONAL ANTIBODY THERAPY IN CARDIAC DISEASES A. Coronary Artery Disease Coronary artery disease is usually atherosclerotic in origin. The atherosclerotic lesions, particularly rupture of plaques, lead to vascular injury by alteration of the endothelial surface and exposure of components of the subendothelial matrix (such as von Willebrand factor [vWF], fibronectins, and collagens) to platelet receptors. The sequence of receptor changes and platelet aggregation leads to activation of the coagulation cascade and thrombus formation (37–39). One of the platelet receptors involved in platelet activation is the integrin αIIbβ3 or platelet glycoprotein (GP) IIb/IIIa. This receptor functions in the final common pathway for platelet aggregation and thus bears a central role in thrombogenic events (36–38). Platelet aggregation appears to be of pivotal importance in several acute or subacute coronary syndromes: acute myocardial infarction (MI), unstable angina (UA), postangioplasty ischemia, and poststent-

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ing ischemia (39). Since both aspirin and heparin have only limited efficacy in preventing platelet aggregation and exert their inhibitory capacity upstream of GPIIb/IIIa activation (36, 37, 39, 40), blockade of GPIIb/IIIa has been attempted in the above clinical settings. Particularly impressive results were obtained with abciximab, a Fab fragment derived from a chimeric mAb to GPIIb/IIIa. Abciximab originates from a murine monoclonal antibody, 7E3, which specifically binds human GPIIb/IIIa (41). In vitro, this mAb inhibited platelet aggregation, and in experimental models of angioplasty injury in vivo, it inhibited thrombosis (42). Since Fab fragments were similarly active in vitro and could reduce the risk of sensitization and other potentially undesirable effects associated with the Fc portion, 7E3-derived Fab fragments were assessed in clinical trials and proved effective (43). To further reduce immunogenicity, a chimeric molecule, c7E3 Fab, was constructed, consisting of the murine variable regions linked to human IgG constant-region sequences. This molecule again was highly effective (44), and essentially did not elicit serologic reactions (45), thus allowing successful retreatment (46). The major results of abciximab therapy are summarized in Table II. The placebo-controlled, randomized abciximab trials have all involved percutaneous coronary interventions. In the EPIC trial, in which highrisk patients underwent percutaneous transluminal coronary angioplasty (PTCA) with either abciximab or placebo, there was an approximately 35% reduction in a composite end point consisting of death, nonfatal MI, or urgent repeat revascularization at 30 days after bolus abciximab plus 12-hour abciximab infusion (bolus alone was not sufficiently effective) (45). Follow-up evaluation at both 6 months (47) and 3 years (48) revealed continuing benefit; in fact, 3 years after therapy there was a 60% reduction in late death. However, abciximab therapy in the EPIC trial was accompanied by an increased incidence of bleeding. In the EPILOG trial, in which PTCA with or without abciximab was investigated irrespective of patients’ risk, with weight-adjusted low-dose heparinization, the previously observed increase in bleeding was shown to have been due to high heparin dose rather than to abciximab. The EPILOG trial was terminated after the first interim analysis because the events (as in EPIC) were reduced by 56% as compared with placebo (49). The effects of abciximab were again also seen in the long term (50). When the effects of abciximab on the benefit of stent implantation was investigated in the EPISTENT trial, a complementary effect was seen, with a reduction of death or MI by 53% as compared with placebo (51). In the CAPTURE trial, patients with refractory unstable angina were treated with abciximab or placebo infusion for 18 to 24 hours prior to

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TABLE II Abciximab Studies in Patients Receiving Angioplasty in the Course of Unstable Angin Composite end points (%) (usually death or myocardial infarction) Study EPICa 30 days 6 months 3 years EPILOGb 30 days 6 months CAPTUREc 30 days Elevated Troponin T Normal Troponin T EPISTENT 6 months

Placebo

Abciximab

p<

12.8 35.1 47.2

8.3 27.0 41.1

.003 .001 .009

11.7 25.8

5.3 22.5

.001 ~.05

15.9 23.9 9.5

11.3 7.5 9.4

.012 .001 n.s.

11.4

7.8d 5.6e

.01 .001

a High-risk patients undergoing angioplasty with abciximab given as bolus injection plus infusion together with standard, not weight-adjusted doses of heparin. b Abciximab given as bolus injection plus infusion together with low-dose heparin. c Patients with unstable angina undergoing angioplasty with or without abciximab. d Abciximab and balloon angioplasty. e Abciximab and stent implantation.

PTCA and 1 hour afterward. Reduction in primary outcome variables was 30% in favor of abciximab and was again maintained over 6 months (52). Patients with elevated serum troponin T levels showed a particular benefit (53). Finally, patients with acute MI benefited from abciximab given as an adjunct to primary PTCA in the RAPPORT trial (54). Other GPIIb/IIIa inhibitors have also shown some clinical benefits, but these are usually of lesser extent than those of abciximab (50, 55). The particular efficacy of abciximab is likely due to the fact that the antibody binds not only to the integrin αIIbβ3, but also to the related vitronectin receptor, αVβ3, which contains the same β3 subunit as GPIIb/IIIa. The αVβ3 receptor is expressed by platelets, endothelial cells, and smooth muscle cells; migration and thrombospondininduced proliferation of the latter is inhibited by abciximab (56, 57). In addition, abciximab binds to an activation-induced neoepitope on the leukocyte integrin Mac-1 (αMβ2), which mediates binding of neutrophils and monocytes to the injured endothelium. In fact, abciximab

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inhibits monocyte adhesion, inhibits binding of fibrinogen to monocytes (58, 59), and inhibits upregulation of Mac-1 during cell activation (60). Finally, abciximab binds also to other adhesion molecules, such as ICAM-1, which is expressed on endothelial cells (58). These results taken together show that abciximab is an effective agent in preventing acute coronary syndromes based on its inhibition of platelet GP IIb/IIIa and probably also of αvβ3 and Mac-1, which thereby induces interference with several thrombogenic and inflammatory pathways. B. Cardiomyopathy and Heart Failure Patients with advanced heart failure have increased levels of circulating TNFα. The biological effects of TNFα may explain several clinical features of heart failure, such as ventricular dysfunction and pulmonary edema (61, 62). These data are also supported by experimental evidence (63, 64). Thus, the efficacy of TNFα blockade is currently under investigation in cardiomyopathy and congestive heart failure, and in vitro studies support the concept (65). V. MONOCLONAL ANTIBODY THERAPY IN INFECTIOUS DISEASES A. Septic Shock Sepsis and septic shock remain a major complication of infections with gram-negative and gram-positive bacteria and fungi (66). Mortality rates due to septic shock are estimated to be 40 to 70% (67). Currently, strategies to improve clinical outcome are not sufficiently successful. Therefore, approaches that interfere with the pathogens or pathogenetic steps of the septic process have been actively sought. 1. Tumor Necrosis Factor–Blocking Agents Since TNFα appears to play an important role in the pathogenesis of septic shock (68–70), one of the approaches to treating sepsis has been the application of TNF blockade with mAb or receptor constructs. Several anti-TNFα mAbs have been tested in clinical trials, including the murine mAbs CB 0006 and MAK 195F and an unnamed anti-TNFα mAb (71–73), as well as the chimeric mAb cA2. Taken together (71–78), studies of anti-TNFα mAbs have shown no compelling differences in survival in patients treated with there TNFα mAbs as compared with control subjects. Although subgroup analyses revealed that patients with high (over 1,000 pg/ml) versus low IL-6 levels (74) and patients with high (over 50 pg/ml) versus low TNFα levels (71) may have minor survival benefits, these data are as yet unconfirmed. How-

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ever, given this subgroup data and the efficacy of anti-TNFα mAb in a baboon model of group A streptococcal sepsis, in which 50% improvement of survival was achieved (78), further studies may be needed to answer the question of whether anti-TNFα mAb may be beneficial in certain patient subgroups with sepsis syndrome. As with the anti-TNFα mAbs, neither an immunoadhesin containing type 1 TNF receptor sequences (TNF-RI-Fc fusion protein) (79) nor type II receptor sequences (TNF-RII-Fc fusion protein, etanercept) reduced mortality in patients with septic shock (80). However, unlike the case of the mAb therapy, there appeared to be a significant, dosedependent increase in mortality with the TNF-RII-Fc construct (doses tested were 0.15 mg/kg, 0.45 mg/kg and 1.5 mg/kg as a single infusion). The reasons for this result and the discrepancy with the mAb data are not clear. It may be related to the lymphotoxin-binding capacity of the TNF-R fusion protein, to the capacity of some mAbs to lyse membrane-associated TNFα-expressing monoytes, or to chance observation. 2. Antiendotoxin Monoclonal Antibodies The lipopolysaccharide (LPS) component of the cell wall of gramnegative bacteria is considered one of the most important factors in the induction of septic shock (81). As early as the early 1980s, a survival benefit was demonstrated for an antiserum against Escherichia coli in a controlled clinical trial (83), but this was not reproduced in meningococcal sepsis (83) More recently, antiendotoxin mAbs, including the murine mAb E5 and the humanized HA-1A, both of the IgM class, have been evaluated. Therapy with E5 initially seemed to lead to an increased survival rate in patients with gram-negative sepsis who did not suffer from a shock syndrome (84), but in a second study this improved survival was not evident (85). Treatment with HA-1A, likewise, did not increase overall survival, although the subgroup of patients with gramnegative septic shock had a reduction in mortality (86). In a second trial, there was again no overall survival benefit in HA-1A-treated patients, and there was even a nonsignificant trend toward decreased survival among patients without gram-negative bacteremia (87). Finally, HA-1A treatment of children with meningococcal septic shock led to a mortality reduction of 33%, but this was not significant (p = .11) compared with placebo (88). Thus, attempts to improve outcome in patients with sepsis and septic shock using an antiendotoxin antibody strategy have not been shown unequivocally to be of benefit. 3. Other Approaches It should be stated here that other immunotherapeutic strategies for sepsis, such as interleukin-1 receptor antagonist (IL-1ra), also did not

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increase survival significantly (89). Among other possible reasons, the failure to obtain benefit in septic shock with the above strategies may be due to the heterogeneity of the patient populations, to the use of mAbs that interfere with the disease process insufficiently (90), or to selection of the wrong antigenic targets. B. Monoclonal Antibodies to Viral Antigens In contrast to antibacterial antibiotic therapy, inhibition of viral replication is usually difficult to achieve. Therefore preventive strategies, such as vaccination, are frequently more successful and clinically important. However, vaccines are not available for all viruses; furthermore, some viruses, such as Epstein-Barr virus (EBV) and cytomegalovirus (CMV), are ubiquitously present and usually not very pathogenic unless in an immunocompromised host. One strategy to combat viral infections in the immunocompromised host is the application of neutralizing mAbs. One such mAb is directed to the F protein of the respiratory syncytial virus (RSV), which afflicts premature newborns with often severe pulmonary infections; this mAb appears to be useful in such situations (91). Other mAbs to viral antigens are in development. VI. MONOCLONAL ANTIBODY THERAPY IN RHEUMATOLOGIC AND AUTOIMMUNE DISEASES A. Rheumatoid Arthritis 1. Tumor Necrosis Factor-α Blockade Rheumatoid arthritis (RA) is a chronic joint disease with autoimmune features. Its hallmarks include a destructive activity of the inflamed synovium, the pannus, which erodes cartilage and bone, ultimately leading to loss of function, morbidity, and premature death (92). Although diseasemodifying antirheumatic drugs (DMARDs) can retard clinical and radiographic disease progression, particularly if applied early in the disease course, most DMARDs have to be stopped within a few weeks to years because of either toxicity or insufficient efficacy (92, 93). Therefore, the search for new therapeutic modalities is ongoing. Although the etiology of RA is unknown, important steps in its pathogenesis have been elucidated in recent years. It is clear that in addition to T cells, cells of the monocyte/macrophage and fibroblast lineages (of which the hyperplastic synovial lining layer consists) and their cytokines, particularly IL-1 and TNFα, play decisive roles (94–98), TNFα has a variety of proinflammatory effects, such as induction of IL-

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1, acute-phase responses, adhesion molecules, and metalloproteinases; IL-1 itself has similar activities (96, 97, 99). In experimental models of arthritis, anti-TNFα mAb therapy was shown to be effective in reducing and preventing joint damage (100). This chimeric antibody, infliximab, was then taken into open and controlled clinical trials (101–104) and shown to be highly effective in reducing signs and symptoms of RA if given in single or repeated doses. Within days, acute-phase responses, particularly C-reactive protein (CRP), decreased by more than 60%; within one to a few weeks, joint counts were significantly reduced, leading to more than 50% improvement of Paulus response criteria in almost 60% of the patients at a dose of 10 mg/kg within 4 weeks (102). In patients refractory to the hitherto most active drug, methotrexate (MTX), the combination of low-dose MTX with infliximab was much more effective than MTX combined with placebo during a 4-month period (104). Since this was the first trial of combination therapy of anti-TNF blockade with MTX, the MTX dose chosen was low (7.5 mg/week). Moreover, at the time of the design of this study, much lower doses of MTX were generally used than today. Since the combination not only was efficacious and safe but also led to a significantly lower frequency of HACA as compared with infliximab without MTX, a trial of combination therapy using higher MTX doses (the ATTRACT trial) was performed and confirmed the high degree of efficacy of infliximab in patients failing to obtain sufficient responses with MTX within 6 months (105). Intravenous administration of 3 mg/kg every 8 weeks was effective, although higher doses (up to 10 mg/kg every 4 weeks) tended to be more effective in the longer term. Finally, a significant reduction in and even arrest of new bone erosions was seen with infliximab as compared with placebo in patients with baseline MTX therapy (106), indicating that TNF blockade may have to be seriously considered as a baseline medication in RA patients, at least in those with an aggressive course. Some details regarding its efficacy can be seen in Table III. Infliximab, which is already approved for Crohn’s disease, has also been approved for RA treatment in the United States and probably will shortly also be approved in Europe. The effects of infliximab provided proof of the concept that TNFα has a pivotal role in RA pathogenesis. This proof came not only from the observed clinical efficacy but also from the following observations, which are consistent with an effect on TNF activation: infliximab led to a reduction in IL-6 levels (99) adhesion molecule expression (107, 108), and synovial cellularity (109). Other monoclonal antibodies have also been employed. One of these, a humanized mAb, CDP 571, reduces acute-phase proteins to a similar extent as infliximab but appears to be less effective in reduction

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TABLE III Effects of TNF-Blocking Therapy in Patients with Rheumatoid Arthritis as Defined According to the ACR 20 Criteria (103–107, 116–119). Responders (%) Trial

Control

TNF-blocker

p<

44 79

.01 .0001

46 57

.01 .004

50 53 52 58

.001 .001 .001 .001

75

.001

51 59

.001

71

.001

Infliximab Trial 1 (4 weeks) Placebo Infliximab 1 mg/kg Infliximab 10 mg/kg Trial 2 (26 weeks) MTX in all patients plusa Placebo Infliximab 3 mg/kg Infliximab 10 mg/kg Trial 3 “ATTRACT” (30 weeks)b MTX in all patients plus Placebo Infliximab 3 mg/kg q 8 weeks Infliximab 3 mg/kg q 4 weeks Infliximab 10 mg/kg q 8 weeks Infliximab 10 mg/kg q 4 weeks

8

17

20

Etanerceptc Trial 1 (3 months) Placebo 32 mg/m2/week Trial 2 (6 months) Placebo Etanercept 20 mg/week Etanercept 50 mg/week Trial 3 (6 months) MTX in all patients plus Placebo Etanercept 50 mg/week

14

11

27

Last infusion at week 14 (12 weeks before final evaluation). Interim analysis of 54-week trial. c Indicated doses are total doses; these doses were divided into two subcutaneous injections. a b

of joint pain and swelling (110). On the other hand, a human monoclonal anti-TNFα antibody, D2E7, appears equally as effective as infliximab (111), and is currently undergoing phase III studies. Anti-TNFα mAbs, particularly infliximab, bind soluble as well as membrane-bound

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TNFα. In fact, infliximab may lead to lysis of TNFα-expressing cells (112), an event that may contribute to the therapeutic effects of the mAb. In vivo, TNFα activity is partly regulated by natural TNF receptors, and the p55 TNF-RI and the p75 TNF-RII IgG-Fc fusion proteins of these receptors have been studied in RA. The first such study involved a TNF-RI fusion protein, lenercept, which initially appeared efficacious (113) but did not show a clear-cut dose response in larger studies and may not be further pursued. Another TNF-RI protein, which was PEGylated to produce a divalent TNF-binding protein, has revealed efficacy (114) and is currently undergoing phase IIb/III trials. Finally, the TNF-RII fusion protein, etanercept, has proved to be clinically efficacious to a similar extent as infliximab, both as a single agent and in combination with MTX (115–118). Among patients receiving MTX plus etanercept, 39% had an ACR 50% response, versus 3% on placebo plus MTX. Etanercept is administered subcutaneously at 10 to about 25 mg twice weekly. This product is already approved for the therapy of RA in the United States and is awaiting licensing in Europe. Tumor necrosis factor blocking agents appear to be relatively safe. Antibody formation has been observed during both infliximab and etanercept therapy but does not appear to reduce efficacy. In the course of TNF blockade, antinuclear antibodies (ANA) may be induced, and although there has been an occasional report of a lupus-like syndrome (104), it is not very likely that TNF blockade induces severe systemic autoimmune disease for a variety of reasons: (1) TNF and TNF-R are increased in systemic lupus erythematosus and correlate with disease activity (119, 120); (2) a number of agents employed in RA, including gold salts, D-penicillamine, sulfasalazine, and minocycline, lead to druginduced lupus erythematasus (121), and it does not appear that this is more frequent for TNF blocking agents than for those drugs; (3) thousands of patients have been treated with infliximab and etanercept for several months to several years, and a drug- induced lupus-like syndrome does not appear to be a noticeable side effect. Nevertheless, with the occurrence of ANA in ANA-negative patients, this issue must be kept in mind until more experience is gained. Malignancy has been another concern, but malignancies do not appear to be more frequent in patients treated with TNF blocking agents than would be expected in the general population or in RA patients. Finally, infections are a special issue. Although bacterial and viral infections do not appear to be increased over placebo rates, at least at lower doses of TNF blocking agents, infections with intracellular bacteria may be exacerbated. Thus, special attention has to be devoted to possible tuberculosis, listeriosis, or fungal infection before and during

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TNFα blockade. A provisional consensus statement on the use of TNF blocking agents has been formulated by an international group of rheumatologists (122). In summary, TNF blockade appears to be a new and successful approach to the therapy of RA and may be considered the major breakthrough of RA treatment in the late 1990s. Despite its efficacy, up to 30% of patients still do not respond, remissions are rare, and cure is not yet in sight. Thus, the search for even better remedies for RA must go on. 2. Targeting Other Cytokines Other cytokines and their pathways are also attractive candidates for therapeutic intervention. In particular IL-1, a proinflammatory cytokine, which has several effects in common with TNFα, is an important target, and it can be assumed that the application of mAbs to the IL-1 receptor (123) will have efficacy similar to that of the IL-1 receptor antagonist, which retards radiographic disease progression (124). In fact, some authors have suggested that IL-1 may have an important role in cartilage degradation (125), and it is conceivable that the combination of TNFα and IL-1 blockade may be even more efficacious than the individual agents. Interestingly, anti-IL-6 mAb therapy has been suggested to be efficacious in RA in an open-label study (126). However, a controlled study has not been published as yet. Support for the potential of interfering with IL-6 activities also stems from a study of anti-IL-6 receptor mAbs in experimental arthritis (127). 3. Targeting T Cells Although the role of T cells in RA has been the focus of intensive debate during the past decade, there is now unequivocal evidence that T cells and their products are active in this disease (128). Initial studies using a humanized anti-CDw52 mAb, CAMPATH-1H, revealed only transient effects (responses for about 2 to 4 weeks in 55 to 65% of the patients) but significant side effects (129–131). Aside from prolonged lymphocyte depletion, mainly CD4(+) and CD8(+) cells, there were significant immediate infusion-associated toxicities, possibly due to TNF release (132), and infections. Thus, nonspecific T-cell depletion using CAMPATH-1H does not appear to lead to sustained improvement of RA and entails significant side effects. An important contribution to the pathogenesis of RA may come from CD4(+)cells. Thus, the approach of targeting this cell population has aroused significant interest. However, although initial open-label studies have indicated efficacy (133), use of anti-CD4 mAbs has not been as successful in controlled trials (134). The reasons for this failure are not clear:

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they may be involve a wrong target or wrong dose, but higher doses were precluded by the depleting activities of several of the anti-CD4 mAbs. The application of a fully humanized anti-CD4 mAb, which is supposedly nonimmunogenic and nondepleting, led only to brief symptomatic relief (135), and larger, double-blind trials would be needed to learn about true efficacy. Likewise, a primatized anti-CD4 antibody, while promising initially (136), is not being pursued at present, possibly because of undesired effects. Another humanized anti-CD4 mAb led to good clinical responses (137), but further data are lacking at present. Thus, anti-CD4 therapy does not appear to constitute a breakthrough in RA therapy. Nevertheless, a combination of anti-CD4 and TNFα blockade appears to be even more effective in experimental arthritis (138) as well as in human RA, at least in open-label trials (139). The apparent failure of anti-CD4 monotherapy does not imply that targeting T cells is inappropriate in RA, since other antigens may be more fruitful for achieving clinically meaningful results. One such candidate antigen could be the IL-2 receptor, and since mAbs against IL-2R are already in use in renal transplant patients (as mentioned above), it is conceivable that studies in RA will soon follow. 4. Targeting Adhesion and Costimulatory Molecules Since adhesion molecules are of pivotal importance in cell trafficking and thus inflammation, they could constitute good therapeutic targets in RA. In fact, a murine mAb to intercellular adhesion molecule (ICAM)-1 proved to lead to clinical improvement, (140) but repeated administration may have less effects, and the side-effect profile was also of major concern (141). Thus, anti-ICAM-1 may not be the strategy of choice. In this context it should be mentioned that TNFα blockade leads not only to clinical benefit but also to reduction of adhesion molecule expression. Another potential target of interest in RA is CD40 ligand (see below). B. Systemic Lupus Erythematosus Systemic lupus erythematosus (SLE), one of the prototypic autoimmune diseases, has constituted a focus of immunotherapeutic interest since the early report of successful anti-CD4 therapy in a murine SLE model (141). This therapy is also somewhat efficacious in human SLE, but to a lesser extent (142). On the other hand, interference with CD40-ligand (L) appears to be quite promising. CD40L (or gp39) is a cell surface molecule present particularly on activated CD4(+) cells, interacts with the CD40 molecule expressed by B cells, and is important in the development of the

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humoral immune response (143). However, CD40 is also expressed on a variety of other cells, including various antigen-presenting cells (144). When CD40L-CD40 interactions are blocked, tolerance is induced (145). In experimental models, anti-CD40L could block collageninduced arthritis as well as the induction of anti-collagen antibodies in these animals (146), and inhibited lupus in several lupus-prone mouse strains (147, 148). Among the latter, lupus nephritis was inhibited and survival significantly prolonged. Since human SLE is a disease characterized by the production of large amounts of autoantibodies and often severe systemic disease manifestations which may be refractory to current therapy, the effects of anti-CD40L therapy are awaited with significant interest. Nevertheless, the expression of CD40 on platelets may entail a risk of thromboembolic complications. Anti-CD40L, in experimental models, also inhibits graft-versus-host disease (149) and experimental allergic encephalitis (150) and may find use also in the human counterparts of those models. C. Other Rheumatologic Diseases Similar MAT may enter clinical trials in a variety of other rheumatologic conditions, including scleroderma, for which the success of current treatments is extremely limited. Another autoimmune disease that has already been studied, though not extensively and only in early phases, is psoriatic arthritis, and it appears as if this disease will respond to similar measures as rheumatoid arthritis. D. Crohn’s Disease Crohn’s disease was one of the first disorders for which MAT and in particular anticytokine therapy have been approved both in the United States (in 1998) and in Europe (1999). The chimeric anti-TNFα mAb infliximab has shown efficacy in several clinical trials in Crohn’s disease, including reduced fistula formation (151–153). The effects may last for several months even after a single infusion. VII. MONOCLONAL ANTIBODY THERAPY OF CANCER A. General Considerations Of all of the potential applications of mAb therapy, cancer treatment has been the most intensively studied. A broad array of antigenic targets and antibody constructs have been evaluated since the advent of mAb technology in 1975, including two decades of clinical trials. Despite

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TABLE IV Monoclonal Antibody Therapy of Cancer: General Considerations Antigen-related obstacles Tumor specificity Heterogeneity of expression Shed antigen Immunogenicity Tumor penetration Biological activity and potency

Strategies Oncogene-product antigen Tissue-specific antigen Functionally important antigen Repeat administration Chimerization/humanization Human mAbs Long circulation Repeat administration Antiproliferative mAbs Proapoptotic mAbs Bispecific mAbs Immunotoxins Radioimmunoconjugates Drug immunoconjugates Immunoliposomes

this, mAb research has only very recently yielded agents of proven utility in cancer treatment: in 1998, rituximab and trastuzumab became the first mAbs approved by the U.S. Food and Drug Administration (FDA) for cancer therapy. Obstacles that have impeded the development of mAb therapy for cancer treatment have included those relating to selection of clinically useful antigenic targets as well as limitations of mAb technology itself (Table IV). Progress in mAb research has resulted in an improved understanding of these obstacles and has generated strategies to overcome them. Many previous antigenic targets have proved inadequate in several key aspects: (1) Tumor specificity. Properties of an ideal target antigen would include absolute tumor specificity (i.e., expression restricted to tumor cells), high-level expression, and universal prevalence in specific cancers. Although an antigen fulfilling all of these criteria has yet to be identified, tumor-associated antigens useful for mAb therapy have emerged, and identification and characterization of promising new antigenic targets is ongoing. One strategy, now validated, has been the use of highly overexpressed or mutant oncogene products such as HER2. Another is the use of differentiation antigens in nonessential cells or tissues, such as the B-cell antigen CD20. (2) Heterogeneity of antigen expression. Related to specificity of expression is the question of heterogeneity, as many antigens vary widely in their expression within a population of tumor cells. Furthermore, many antigens can undergo downmodulation as a result of mAb therapy, leading to resis-

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tant antigen-negative variants. Again, antigenic targets such as oncogene products or regulatory molecules are much more likely to be homogeneously expressed owing to their critical functions and are less likely to undergo downmodulation without compromising oncogenicity. Indeed, some mAb strategies exploit antigen internalization for efficacy, either for downregulation of prooncogenic signal transduction or for delivery of intracellular cytotoxins. (3) Shed or secreted antigen. High levels of circulating soluble forms of tumor-associated antigens can adversely affect mAb pharmacokinetics and theoretically lead to immune complex–related toxicities. Sometimes, pharmacokinetic limitations can be overcome with dose and schedule modifications. Limitations of many anticancer mAbs have included: (1) Immunogenicity. As previously discussed, rodent mAbs suffer severe limitations with regard to pharmacokinetics and immunogenicity and are generally unsuitable for repeated administration owing to the induction of human antimouse antibodies (HAMA). The advent of technologies for chimeric, humanized, or human mAb has revolutionized this area. (2) Tumor penetration. The treatment of solid tumors poses unique problems for macromolecular agents such as mAbs and mAb-derived constructs. Theoretical barriers to mAb penetration of solid tumors include heterogeneous vascular supply, high interstitial pressure in central necrotic zones, and the relative slowness of macromolecular diffusion over large intratumoral distances (155). On the other hand, it has become increasingly clear that a countervailing tendency toward tumor accumulation also exists in many solid tumors. An enhanced permeability and retention (EPR) effect has been demonstrated for long circulating macromolecular agents, which undergo preferential extravasation at sites of abnormal tumor vessel microanatomy associated with tumor angiogenesis and which, combined with deficient lymphatic drainage in tumors, results in enhanced tumor levels (156). Furthermore, improvements in mAb technology have made possible the administration of increasingly higher doses of long circulating mAb given repeatedly, which also facilitates mAb penetration and accumulation. (3) Biological activity and potency. Obviously, mAb binding is not necessarily sufficient to induce a therapeutic response. Monoclonal antibodies containing human constant-region sequences are theoretically capable of mediating antibody-dependent cytotoxicity (ADCC) and possibly complement-dependent cytotoxicity (CDC). However, the efficiency of these processes within the setting of active tumor mechanisms in resisting immunologic rejection is clearly variable. Strategies to boost therapeutic potency include mAbs that inhibit critical oncogenic or tissue-specific mediators. In addition to these “naked” mAb approaches, immunoconjugate strategies such as immunotoxins,

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radioimmunoconjugates, drug immunoconjugates, and immunoliposomes, which can provide markedly greater potency, have emerged and appear promising. In this section, we will review selected examples of mAb therapy of cancer, particularly the successful targeting of the lymphocyte differentiation antigen CD20 by rituximab and the oncogene product HER2 by trastuzumab. Preclinical and clinical data demonstrating the efficacy of these mAbs has not only validated these two antigens as targets but has provided long awaited proof of the concept for mAb therapy of cancer. B. Lymphocyte Differentiation Antigens 1. Idiotype One of the earliest attempts to exploit hybridoma technology for cancer treatment was the work of Levy and co-workers, who produced mAbs reactive with the idiotype expressed on malignant B lymphocytes obtained from individual patients (157–158). Although the need to produce “custom-made” mAbs for each patient has severely limited the feasibility of this strategy, these pioneering studies did provide early and convincing evidence that mAb therapy could elicit durable antitumor responses. 2. CD20 as a Target for mAb-Based Therapies CD20, a 35-kDa cell-surface protein expressed during B-cell ontogeny, plays a critical role in that process by regulating cell cycle initiation and differentiation as a component of a signal transduction complex, which may function as a calcium channel (159–161). CD20 is expressed by pre-B cells and B cells in the thymus, splenic white pulp, lymph nodes, and peripheral blood (162); but is not generally present in hematopoietic stem cells, early B cell progenitors, plasma cells, or nonlymphoid cells. In B-cell neoplasms, CD20 expression is retained in more than 90% of B-cell non-Hodgkin’s lymphoma (NHL) and chronic lymphocytic leukemia (CLL), as well as frequently in pre-B-cell acute lymphoblastic leukemia (ALL) (162–163). CD20 possesses a number of advantages as a target for immunotherapy. Although clearly not a tumor-specific antigen, CD20 is highly specific for normal or malignant cells of B-cell lineage. Hence, the normal tissue expression of CD20 is restricted to cells that are nonessential to survival; that is, short-term B-cell depletion is not necessarily associated with major host toxicity. As an important regulator of B-cell growth, CD20 tends to be consistently expressed in the target cell population. CD20 is accessible as a cell-surface protein but does not appear to be shed or secreted into the circulation (164–165). Also, CD20 does not appear to undergo internalization following mAb binding. This may be

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advantageous for mAb strategies in which prolonged surface display is paramount, such as recruitment of immune effectors via Fc. 3. Rituximab Rituximab (IDEC-C2B8, Rituxan) is a chimeric anti-CD20 mAb in which variable regions from the parental murine mAb, IDEC-2B8, were joined via recombinant DNA methods to human IgG1 and k constantregion sequences (166). The resulting chimeric mAb was shown to retain the binding specificity and affinity (Kd = 8.0 nM) of the murine mAb. Preclinical studies demonstrated that rituximab binding to CD20(+) B cells induced cell lysis, both directly and indirectly via host immune effector mechanisms. The direct effects of rituximab in vitro included growth inhibition and induction of apoptosis in B-cell lymphoma lines (167). Immune-related effects included mediation of complementdependent cytotoxicity via binding of rituximab to C1q and mediation of ADCC via binding to Fc receptors on immune effector cells (166). As predicted, the human IgG1-containing rituximab was significantly more potent (about 1000-fold) in mediating lysis in the presence of human effectors than was the parental murine mAb. Rituximab has been evaluated in a series of clinical trials. An initial phase I study in 15 patients with relapsed B-cell NHL showed that single-dose intravenous administration of 10–500 mg/m2 could produce a rapid, dose-dependent depletion of normal and malignant B cells which persisted for as long as 3 months (168). Two partial responses (PRs) were observed, and the treatment was generally well tolerated. A phase I/II study employed an initial multiple-dose, dose-escalation design in 20 low-grade/indolent NHL patients, who were given rituximab intravenously, 125–375 mg/m2 weekly for four doses (169); the second part of the study used 375 mg/m2 in 27 additional NHL patients (170). The response rate in 37 patients receiving rituximab at 375 mg/m2 was 46%, and included 3 complete (CR) (8%) and 14 partial responses (PRs) (38%). One of 37 patients displayed human antichimeric antibodies (HACA) at low titer. A large multicenter pivotal phase II study was performed to evaluate the efficacy of rituximab in the treatment of relapsed low-grade or follicular CD20(+) B-cell NHL (171). A total of 166 patients with small lymphocytic (n = 33), follicular small cleaved (n = 67), follicular mixed (n = 53), follicular large-cell (n = 10), or low-grade variant (n = 3) NHL were enrolled at 31 centers. Patients with bulky disease (≥10 cm), pleural or peritoneal involvement, CNS lymphoma, AIDS-related lymphoma, CLL or leukemic component (more than 5,000 lymphocytes/µl) were excluded. Rituximab was again administered intravenously at 375 mg/m2 weekly for four doses. The overall response rate, which was the primary study end point, was

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48% in the intent-to-treat population of 166 patients. This included 6% CRs and 42% PRs. The median duration of response was 11.6 months, HACA were observed in one patient. Toxicities observed throughout the rituximab clinical studies included an infusion reaction of fever, chills/rigors, nausea, asthenia, headache, and occasionally other hypersensitivity-type symptoms (168–171). These reactions occurred particularly with the first infusion, and were usually self-limited and resolved following slowing or interruption of the infusion along with supportive care. Serious infusion-related toxicities were uncommon, although grade 3–4 hypotension, bronchospasm, and urticaria were occasionally observed. Other serious adverse events included occasional grade 3–4 neutropenia and thrombocytopenia. Based on these data, rituximab was approved by the FDA in 1998 for the treatment of relapsed or refractory low-grade or follicular, CD20(+), B-cell NHL. Rituximab was the first mAb to receive approval for the treatment of cancer. The combination of rituximab and chemotherapy was evaluated in an open-label phase II study in 40 patients with relapsed low-grade or follicular CD20(+) B-cell NHL (172). The chemotherapy regimen consisted of the standard cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) regimen, given every 3 weeks for six cycles; rituximab was administered at 375 mg/m2 for six doses over 20 weeks. Of 40 patients, 38(95%) responded, including 22 CRs (55%) and 16 PRs (40%). This combination was not associated with any novel toxicities. Another phase II study has tested rituximab monotherapy in the treatment of relapsed or refractory intermediate- and high-grade CD20(+) B-cell NHL (173). A total of 54 enrolled patients were randomized to receive intravenous rituximab at 375 mg/m2 weekly for eight doses versus 375 mg/m2 initially followed by 500 mg/m2 weekly for seven doses. In the entire intent-to-treat population of 54, the overall response rate was 31%, with CRs in 9% and PRs in 22%. No difference in response was noted between the two regimens. 4. Anti-CD20 Radioimmunotherapy Anti-CD20 mAbs have also been developed for use in the radioimmunotherapy (RIT) of B-cell neoplasms. Compared with unlabeled or “naked” mAb strategies, RIT has the theoretical advantage of increased potency via radionuclide emission that does not depend on host immune mechanisms, and so-called bystander toxicity in which cancer cells not bound by mAbs may still be lethally irradiated if located sufficiently close to bound mAb on other cells. An additional rationale for RIT in the treatment of NHL and other hematologic malignancies is the exquisite sensitivity of many of these diseases to radiation therapy.

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131I-anti-B1

(Bexxar) consists of a murine anti-B1 (anti-CD20) IgG2a, tositumomab, labeled via the iodogen method to 131I (174). It is administered via two intravenous infusion sessions, an initial dosimetric dose followed 1 week later by a therapeutic dose. Phase I results demonstrated the feasibility of this regimen, which was generally well tolerated but with a moderate degree of hematologic toxicity (175). Of 28 patients who received the therapeutic dose; 14 achieved CR (50%) and 8 PR (29%); 34 patients overall were enrolled. 131 I-anti-B1 is currently undergoing phase II–III evaluation in relapsed or refractory low-grade and transformed NHL. Since myelosuppression is the usual dose-limiting toxicity of RIT, 131Ianti-B1 therapy has also been evaluated in the setting of high-dose, myeloblative doses followed by autologous stem cell rescue (176–178). Another anti-CD20 radioimmunoconjugate, IDEC-Y2B8, is a 90Ytlabeled version of the murine mAb parent of rituximab. In a phase I–II study, B-cell NHL patients received an initial dose of unlabeled rituximab to clear peripheral B cells and improve biodistribution of the RIT mAb, followed by escalating doses of IDEC-Y2B8, (179). Recently, randomized studies comparing unlabeled anti-CD20 mAb therapy with anti-CD20 RIT have been initiated. These studies should help clarify the relative risks and benefits of these two CD20-targeted strategies. 5. Other Lymphocyte Differentiation Antigens Monoclonal antibodies against CD 19, another cell-surface B-cell differentiation antigen, have also been developed for MAT of B-cell neoplasms. A murine IgG2a mAb was evaluated in a phase I study and produced some clinical responses (180). Because CD19, unlike CD20, can undergo internalization following mAb binding, anti-CD19 immunotoxins have been developed, including a chemical conjugate of murine mAb HD37 with deglycosylated ricin A chain (181). CD22 is also a cell-surface B-cell differentiation antigen capable of internalization and thus also an attractive target for immunotoxin strategies. These include an immunotoxin consisting of anti-CD22 mAb conjugated to ricin A chain (182), and another immunotoxin in which anti-CD22 mAb was conjugated to whole ricin containing a blocked B chain (183). BL22 is a recombinant anti-CD22 immunotoxin in which Fv sequences have been fused to those encoding the ricin A chain (184). Anti-CD22 RIT has also been pursued using 131I-LL2 (185). As previously discussed, CD25 is a component of the IL-2 receptor on activated T cells. A recombinant anti-CD25 immunotoxin, LMB-2, contains fused Fv and Pseudomonas exotoxin A sequences and is currently in clinical trials (186).

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CDw52, a 21- to 28-kDa phosphatidylinositol-linked glycoprotein of unclear function, is widely expressed on the cell surface of both B and T lymphocytes (187). Anti-CDw52 mAbs include CAMPATH-1 and its humanized version CAMPATH-1H (187–188). CAMPATH-IH has been evaluated in multiple clinical trials, in which it has shown anticancer efficacy against a variety of lymphoid neoplasms (188–190). However, CAMPATH-1H also induced rapid depletion of both B cells and T cells, resulting in potentially profound immunosuppression (191). C. Oncogene-Product Antigens 1. HER2 as a Target for mAb-Based Therapies The neu oncogene was first identified in rat neuroblastomas that arose following gestational exposure to ethylnitrosourea (192). The transforming gene, designated neu because of its association with neuroblastoma, was found to encode a 185-kDa membrane-bound glycoprotein closely related to the epidermal growth factor receptor (EGFR) (193). This initial oncogene was subsequently appreciated to be a mutant allele, which contained a point mutation in the transmembrane domain associated with constitutive tyrosine kinase activity (194). The c-erbB-2 or HER2 gene was identified by screening human genomic and cDNA libraries for receptors related to EGFR; using this strategy, several groups simultaneously isolated the human homologue of neu (195–197). This gene was designated c-erbB-2 or HER2 to reflect its relation to EGFR, also known as c-erbB-1 or HER1, and has also itself been referred to as neu or HER2/neu. Unlike the wild-type rat neu proto-oncogene, from which an activated tyrosine kinase can arise via mutation, the HER2 gene does not appear to undergo mutation in human cancer. Indeed, multiple studies have failed to demonstrate an analogous activating mutation of HER2 in human cancers (198). However, the HER2 gene was observed to be amplified in certain human cancer cell lines (195–196, 199). These studies suggested that HER2 gene amplification might be an alternative oncogenic mechanism to mutational activation. The direct role of HER2 in breast tumorigenesis has been elucidated by multiple laboratory studies. For example, when overexpressed, HER2 has proven to be a potent oncogene, which transforms cells in vitro and confers tumorigenicity in vivo (200–201). Transgenic mice overexpressing the mutant or wild-type rat neu gene or the mutant or wild-type human HER2 gene developed cancers at high frequency, including mammary cancer (202–206). Finally, certain mAbs directed against HER2 or rodent neu were able to inhibit cancer cell growth in

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vitro and/or in vivo; these mAbs include trastuzumab as well as a number of independently derived mAbs (207–210). The clinical significance of HER2 amplification was first demonstrated by Slamon and co-workers, who found HER2 amplification in approximately 25% of primary breast tumors and noted a particularly poor prognosis when present in axillary lymph node–positive patients (212). A subsequent study demonstrated that overexpression of HER2 at the RNA and protein levels was similarly prognostic for poor outcome (213). It has since been widely confirmed that HER2 overexpression occurs in 20 to 30% of breast cancers and is associated with poor prognosis in node-positive and probably also node-negative patients (214). In addition to its prognostic significance, HER2 overexpression is also predictive of differential responses to specific therapies. In studies using clinical samples, HER2 overexpression correlated with increased responsiveness to anthracycline chemotherapy in the adjuvant setting (215–217) and possibly with resistance to other chemotherapies (218). In experimental models, HER2 overexpression has induced resistance to tamoxifen therapy (219, 220). HER2-overexpression has also been observed in a variety of other cancers, including ovarian cancer (213), endometrial cancer (221, 222), salivary gland adenocarcinoma (223), non–small cell lung cancer (224), gastric cancer (225), pancreatic cancer (226), colorectal cancer (227–228), bladder cancer (229–230), and prostate cancer (231–232). With the exception of ovarian cancer and possibly some others, it remains unclear whether HER2 gene amplification is the predominant mechanism for overexpression and whether amplification or overexpression is strongly correlated with prognosis, as in breast cancer (219). HER2 provides an attractive target for mAb therapy. It is accessible as a cell-surface receptor, and when overexpressed is present at up to 106 receptors per cancer cell, or 100-fold higher concentration than in normal cells. In normal tissues, its expression is detected only in certain predominantly epithelial cell types (233). As an oncogene, its continued expression appears to remain important throughout malignant progression, including metastasis (234). 2. Trastuzumab Fendly and co-workers generated a panel of mAbs reactive with the extracellular domain (ECD) of HER2 by immunizing mice with HER2transfected NIH 3T3 cells (235). Characterization of these mAbs revealed variable effects on the growth of HER2-overexpressing cell lines in vitro; growth effects included cytostatic inhibition, no effect, and even growth stimulation depending on the mAb and cell line

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(236). Monoclonal antibody 4D5 was noted to have the most consistently potent inhibitory activity against a series of HER2-overexpressing breast cancer cell lines. For example, continuous incubation of SKBR3 cells for 5 days with 4D5 at 10 µg/ml resulted in 67% reduction of monolayer growth. Further in vitro studies of 4D5 showed that continuous incubation with HER2-overexpressing cells similarly inhibited anchorage-independent growth in soft agar. The precise mechanism by which 4D5 exerts an antiproliferative effect in vitro has remained unclear. The effect is clearly cytostatic rather than cytotoxic or cytocidal, as removal of 4D5 from the medium results in immediate resumption of growth; in addition, 4D5 treatment does not produce apoptosis or observable cytopathic effect (236). An early hypothesis was that 4D5 binding might competitively inhibit ligand activation of HER2. However, following the identification of the heregulins as activators of HER2 (237–238), subsequent studies indicated that 4D5 binding exerts at most a partial blockade of heregulin-mediated stimulation of HER2 (239). Studies of 4D5 itself on HER2-associated signal transduction pathways revealed that certain downstream events occur following 4D5 treatment, including receptor phosphorylation and internalization (240), although not shc phosphorylation or mitogen-activated kinase activation (241). Thus, 4D5 may function as a partial agonist of HER2, possibly via an effect on HER2 homodimerization, resulting in altered signaling and/or receptor downmodulation. Because of its promising preclinical activity against HER2-overexpressing breast cancers, 4D5 was evaluated in phase I clinical studies (see below). However, in order to circumvent the problems associated with rodent mAbs as therapeutics, a humanized version of murine 4D5 mAb was constructed by Carter and co-workers using gene conversion mutagenesis of cloned 4D5 sequences (242). The antigen-binding or complementarity determining regions (CDRs) of murine 4D5 were fused to consensus human variable-region framework sequences and IgG1 constant-region domains. The most humanized construct, in which only the CDRs were retained from 4D5, showed impaired binding affinity, and thus additional variants were generated in which selected framework residues in 4D5 were also retained. The best of these variants, rhuMAb HER2 or trastuzumab (Herceptin), bound to recombinant HER2 ECD with an affinity (Kd = 0.1 nM) superior to that of 4D5 and showed comparable antiproliferative activity in vitro. In addition, trastuzumab mediated ADCC against HER2-overexpressing cells in the presence of human effector cells in vitro (236, 242). Preclinical efficacy studies were performed with both 4D5 and trastuzumab in HER2-overexpressing breast cancer xenograft models. Anti-HER2 mAb treatment produced cytostatic growth inhibition of

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HER2-overexpressing breast cancer xenografts in vivo (243). Furthermore, trastuzumab enhanced the antitumor efficacy of multiple chemotherapeutic agents, including cisplatin, paclitaxel, and doxorubicin, in animal models (244–246). Initial phase I testing was performed with murine mAb 4D5 in patients with advanced breast or ovarian cancer. In this and subsequent studies, patients were required to have HER2-overexpressing tumors as evidenced by immunohistochemistry (IHC) assay, in which membrane staining was scored semiquantitatively. 4D5 was found to be safe, although 4 of 20 patients did develop evidence of an antibody response to the murine mAb (HAMA). Following construction, characterization, and scale-up of trastuzumab, phase I testing of the humanized mAb was carried out in patients with HER2-overexpressing metastatic breast cancer. The initial phase I study evaluated the safety and pharmacokinetics of a single, escalating (10–500 mg) intravenous dose of trastuzumab. A subsequent phase I study evaluated the safety and pharmacokinetics of multiple-dose administration, with weekly intravenous doses and similar dose escalation by cohort (10–500 mg). Both studies of 32 patients overall demonstrated that trastuzumab monotherapy was very well tolerated, with no serious adverse events attributable to mAb treatment. A third phase I study included trastuzumab, again via weekly intravenous administration of 10–500 mg, in combination with cisplatin chemotherapy at 50 or 100 mg/m2 per 4week cycle. Toxicities in this trial were those commonly seen with cisplatin. In these phase I studies, trastuzumab displayed dose-dependent pharmacokinetics, with a serum half-life ranging from 1 day at the 10-mg dose level to 14 days at the 500-mg dose level. Importantly, when administered at the higher dose levels, consistent serum trough concentrations of trastuzumab above 10 µg/ml were achieved, which was the target concentration based on the in vitro antiproliferative activity. Soluble HER ECD was detected in 24 of 28 patients and was correlated with altered trastuzumab pharmacokinetics. Also, in contrast to 4D5 therapy, no human antihuman antibodies (HAHA) were observed following trastuzumab administration. Phase II trials of trastuzumab demonstrated anticancer activity both as a single agent and in combination with cisplatin in metastatic and refractory breast cancer patients with HER2 overexpression. For monotherapy, trastuzumab was administered intravenously at an initial loading dose of 250 mg, followed by 100 mg weekly (247). Objective responses were observed in 5 out of 46 patients (11%). As in the phase I studies, trastuzumab was well tolerated, and no HAHA was observed. With this regimen, 91% of patients achieved mAb trough levels of 10 µg/ml or higher, although pharmacokinetics were impacted in patients with high

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circulating HER2 ECD concentrations. The second phase II study combined trastuzumab at the same dose and schedule with cisplatin at 75 mg/m2 per 4-week cycle (248). Of 37 evaluable patients, 9 (24%) achieved partial responses, with no complete responses observed. This response rate was deemed somewhat higher than would have been expected for cisplatin chemotherapy alone, based on historical data. Based on these initial phase II results, an expanded worldwide phase II pivotal study was performed to assess the efficacy and safety of trastuzumab monotherapy (249). This study enrolled 222 metastatic breast cancer patients whose tumors had HER2 overexpression and who had progressed after one or two prior chemotherapy regimens. As with previous trastuzumab studies, HER2 overexpression was determined by a centralized IHC assay, in which HER2 staining was required to be 2+ or 3+ on a 0 to 3+ scale. Patients received trastuzumab intravenously as a 4 mg/kg loading dose followed by 2 mg/kg weekly maintenance. Although 213 patients actually received at least one dose of trastuzumab, results were based on the intent-to-treat population of 222. Eight complete (4%) and 26 partial responses (11%) were observed, for an objective response rate of 15%. Median time to disease progression was 3.0 months. Time to disease progression was longer for patients with HER2 overexpression at the 3+ level than for those at 2+ (3.3 vs. 1.9 months, p = .0034). Toxicities included an infusion reaction consisting of fever, chills, pain, asthenia, nausea, and vomiting. This syndrome was typically observed after the initial infusion, was self-limited, and frequently did not recur with subsequent infusions. The most serious toxicity was cardiac dysfunction, defined as clinical findings of congestive heart failure and/or subclinical declines in cardiac ejection fraction, which was seen in 5% of patients. Cardiotoxicity was unanticipated, as it had not been detected in previous studies. The mechanism for this effect is unclear, but is likely related to trastuzumab’s antiproferative effect on HER2-mediated homeostasis and response to injury. Another phase II study has evaluated trastuzumab monotherapy in HER2-overexpressing metastatic breast cancer patients who had not yet received chemotherapy for advanced disease (283). In this more favorable patient population, trastuzmab treatment was associated with a somewhat higher response rate, 23%. Also, no difference in response rate was observed between two trastuzumab doses, 4 mg/kg followed by 2 mg/kg versus 8 mg/kg followed by 4 mg/kg. Phase II studies of trastuzumab monotherapy are summarized in Table V. A phase III pivotal study was performed to directly compare chemotherapy plus trastuzmab with chemotherapy alone in patients with HER2-overexpressing (2+ or 3+) breast cancers as first-line therapy for metastatic

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TABLE V Phase II Studies of Trastuzumab Monotherapy for Metastatic Breast Cancer

Trastuzumab regimen 250 mg, then 100 mg weekly (247) 4 mg/kg, then 2 mg/kg weekly (248) 4 or 8 mg/kg, then 2 or 4 mg/kg weekly (283)

Time to progression (median, months)

Response rate (%)

Response duration (median, months)

5.1

11

6.6

222

Any (median = 2) 1–2

3.0

14

9.1

113

None

3.4

23

16.6

N 46

Prior chemoRx for stage IV (# regimens)

disease (250). This was a worldwide, open-label, randomized study in which over 150 centers and 469 patients participated. The chemotherapy regimen for most patients consisted of doxorubicin (Adriamycin®) 60 mg/m2 or epirubicin 75 mg/m2 plus cyclophosphamide (Cytoxan®) 600 mg/m2 [Adriamycin/Cytoxan (AC) or Epirubicin/Cytoxan (EC)]). However, for patients who had previously received an anthracycline-based chemotherapy regimen in the adjuvant treatment of their initial breast cancer, the regimen was paclitaxel 175 mg/m2. All chemotherapy was administered every 3 weeks for six cycles, with additional cycles at the investigator’s discretion. Trastuzumab was administered intravenously as a 4 mg/kg loading dose on the same day as chemotherapy and continued weekly at 2 mg/kg until disease progression, which represented the primary end point of the study. Trastuzumab plus chemotherapy was associated with significant improvements in time to disease progression, response rate, and 1-year survival as compared with chemotherapy alone (Table VI). Time to progression was increased from 4.6 months with chemotherapy alone to 7.4 months with trastuzumab plus chemotherapy (p < .0001). The response rate to trastuzumab plus chemotherapy was also significantly higher than that to chemotherapy alone, 50% vs. 32% (p < .0001). Results were particularly notable in the paclitaxel subgroups (n=188), where trastuzumab plus paclitaxel was associated with a time to progression of 6.7 versus 2.5 months for paclitaxel alone (p < .0001) and a response rate of 38% versus 15% for paclitaxel alone (p < .001). Updated results continue to show increased survival for the trastuzumab plus chemotherapy group, with a median survival of 25.4 months versus 20.3 months for chemotherapy alone (p = .025) (251). Toxicities in the phase III trial attributable to trastuzumab were generally similar to those seen previously. However, the incidence of car-

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TABLE VI Phase III Trial of First-Line Chemotherapy versus Chemotherapy plus Trastuzumab for Metastatic Breast Cancer a

Treatment Doxorubicin/ cyclophosphamide (AC) AC + trastuzumab Paclitaxel (T) T + trastuzumab All Chemotherapy (CT) CT + Trastuzumab

N

Time to progression (median, months)

p value

Response rate (%)

p value

138

6.1

.0004

42

.02

143 96 92 234 235

7.8 2.5 6.7 4.6 7.4

.0001 .0001

56 15 38 32 50

.001 .0001

a All patients had stage IV breast cancer receiving first-line chemotherapy, either AC (doxorubicin or epirubicin plus cyclophosphamide) or T (paclitaxel). Data from Slamon et al. (250).

diotoxicity was significantly higher in the trastuzumab groups and was particularly notable in patients receiving trastuzumab plus AC chemotherapy: 28% versus 7% for AC alone. The incidence of cardiotoxicity was also markedly increased in patients receiving trastuzumab plus paclitaxel (11%) versus paclitaxel alone (1%). Following the results of these two pivotal studies, trastuzumab was approved by the FDA in 1998, the first mAb approved for solid tumor treatment. Its indicated use is in patients with metastatic breast cancer whose tumors overexpress HER2, either as a single agent for patients previously treated with chemotherapy or in combination with paclitaxel as first-line therapy. Thorough baseline cardiac assessment and extreme caution in patient with preexisting cardiac dysfunction is recommended. Controversy exists as to the most useful assay for HER2-overexpression, particularly for identification of candidates for trastuzumab therapy. Following the approval of trastuzumab, an IHC-based assay (Herceptest), similar but not identical to the trastuzumab clinical trials assay, was developed for this use. Fluorescence in situ hybridization (FISH)-based assays have also been developed to determine HER2 gene amplification. The concordance of these assays with each other and with the trastuzumab clinical trials assay has been questioned (252). Since the probability of response to trastuzumab therapy appears to correlate with the level of HER2 overexpression, further studies will be required to establish standardized HER2 testing procedures. Ongoing and future studies have been designed to evaluate the efficacy of trastuzumab in combination with other chemotherapy regimens, including single agents and combinations. A phase II trial has

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used weekly trastuzumab plus weekly paclitaxel at 90 mg/m2 in place of the paclitaxel regimen (175 mg/m2 every 3 weeks) used in the phase III study; preliminary results indicate a response rate of 62% in patients with HER2-overexpressing tumors, as compared with 44% in patients without HER2 overexpression who were also enrolled (253). A multicenter phase II study is evaluating trastuzumab plus docetaxel, while a multicenter randomized phase III study is treating patients with trastuzumab plus paclitaxel with or without carboplatin. Trastuzumab in combination with hormonal therapies will also be tested. This strategy is based on the rationale that, as discussed, HER2 overexpression may be associated with tamoxifen resistance, and that trastuzumab therapy may alter or reverse this. Finally, studies using trastuzumab in earlier stages of breast cancer have been initiated. Three large cooperative group trials are currently evaluating the use of trastuzumab with chemotherapy in the adjuvant setting for HER2-overexpressing breast cancers with positive axillary lymph nodes. A multicenter trial is evaluating trastuzumab plus chemotherapy in the neoadjuvant setting for HER2-overexpressing, locally advanced breast cancers In addition to these breast cancer trials, trastuzumab is under evaluation for the treatment of other cancers in which HER2 overexpression can occur, including ovarian cancer, non–small cell lung cancer, colorectal cancer, and prostate cancer. 3. Other Anti-HER2 Monoclonal Antibody–Based Therapies a. Anti-HER2 Bispecific Antibodies. Bispecific antibodies (BsAbs) consist of two mAb fragments, which recognize distinct antigenic targets linked in one hybrid construct. They can be generated by chemical conjugation or cross-linking, cell fusion of hybridomas to produce quadromas, or recombinant DNA methods using cloned antibody genes. The most common BsAb strategy involves combining an mAb fragment directed against a tumor-associated antigen with a fragment directed against an antigen present on immune effector cells, in order to facilitate immune-mediated killing of target cells (directed killing). The BsAbs have theoretical advantages over naked mAbs relying on ADCC, as BsAbs exploit specific and potentially high-affinitiy Ag-Ab binding rather than the relatively nonspecific and low affinity binding between the mAb Fc and immune cell Fc receptors. The bispecific mAb MDX-210 recognizes both HER2 and CD64, the high-affinity type I Fc receptor (FcγRI) expressed by mononuclear phagocytes and activated neutrophils. MDX-210 was evaluated in a phase I clinical trial in advanced breast and ovarian cancer and observed to produce immunologic effects, including transient monocytopenia and cytokine release; 1 of 10 assessable patients achieved a partial response (254). The

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BsAb 2B1 recognizes both HER2 and CD16, the low-affinity type III Fc receptor (FcγRIII) expressed by mononuclear phagocytes and natural killer cells. In a phase I clinical study, 2B1 was shown to induce cytokine release, and several minor antitumor responses were observed (255). b. Anti-HER2 Immunotoxins. Immunotoxins, in which an mAb or an mAb fragment is conjugated or fused to a toxin molecule, represent an attractive strategy to greatly increase the anticancer effect following mAb binding. The anti-HER2 immunotoxin erb-38, containing sequences derived from Pseudomonas exotoxin A (PE), was evaluated in a phase I clinical trial in six patients (256). All six experienced hepatotoxicity, with significant transaminase elevations despite the low level of HER2 expression in hepatocytes and the low doses of erb-38 administered. This result suggests that immunotoxin strategies can sometimes be too potent, since normal tissues with very low levels of antigen expression (such as HER expression in hepatocytes) can still be targeted by these exquisitely active and otherwise nonspecific toxins. c. Anti-HER2 Immunoliposomes. Immunoliposomes represent a strategy for tumor-targeted drug delivery by conjugation of antibody fragments to liposomes (257). Anti-HER2 immunoliposomes have been developed which consist of Fab or scFv fragments linked to long-circulating sterically stabilized liposomes (104). Anti-HER2 immunoliposomes bound efficiently to and internalized in HER2-overexpressing cells in vitro and in vivo, resulting in intracellular drug delivery (258–260). In HER2-overexpressing tumor xenograft models, anti-HER2 immunoliposomes loaded with doxorubicin displayed potent and selective anticancer activity, including significantly superior efficacy versus all other treatment conditions tested: free doxorubicin, liposomal doxorubicin, free mAb (trastuzumab), and combinations of doxorubicin or liposomal doxorubicin with mAb (261). Anti-HER2 immunoliposomes are currently undergoing scale-up for clinical studies. There are a number of potential advantages of the immunoliposome approach versus other mAb-based strategies directed against HER2. For example, anti-HER2 immunoliposome delivery of doxorubicin may circumvent the prohibitive cardiotoxicity associated with combined trastuzumab and doxorubicin treatment. These immunoliposomes can be constructed with alternative anti-HER2 scFv that lack antiproliferative activity, are incapable of ADCC, and require threshold levels of HER2 expression for delivery (261); these properties enable targeted drug delivery but are likely to avoid the cardiotoxicity associated with steady-state exposure to trastuzumab. In contrast to most immunotoxins, drug-loaded immunoliposomes provide an additional level of specificity through the use of chemotherapeutic

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agents with their own therapeutic index, can provide bystander toxicity by diffusion of drugs to proximal cells, and can be constructed so as to be nonimmunogenic. 4. Other Oncoprotein Antigens Monoclonal antibodies directed against other oncogene products have been developed. Epidermal growth factor race (EGFR) is the prototypic member of the EGFR receptor tyrosine kinase family, which also includes the previously discussion HER2, as well as HER3 (ErbB3) and HER4 (ErbB4). Overexpression of EGFR occurs in a number of cancers, including malignant gliomas (262), breast cancer (263,264), lung cancer (265), bladder cancer (266), and head and neck cancer (267). In most of these cancers, EGFR gene amplification is infrequent, although it is the predominant mechanism for overexpression in malignant gliomas (268–270). Furthermore, a variety of mutations have also been described in gliomas (271–275). Mendelsohn and co-workers developed a murine anti-EGFR mAb, mAb 225, which competitively inhibits endogenous ligands such as EGF and TGF-α from binding and activating EGFR (276–277). In preclinical studies, mAb 225 inhibited the growth of nontransformed cells and cancer cells in vitro and the growth of human tumor xenografts in nude mice (277,278). This mAb also enhanced the antitumor activity of chemotherapeutic agents, including doxorubicin (279) and cisplatin (280). C225, a chimeric version of mAb 225, retains the ability to block EGFR-mediated signaling and to inhibit cancer cell growth (281,282). In clinical trials, C225 has displayed activity against several types of solid tumors, both alone and in combination with chemotherapeutic agents or with radiotherapy. Phase III evaluation of C225 in combination with radiotherapy in the treatment of advanced head and neck cancer is currently underway. VIII. CONCLUSION Twenty-five years after the revolutionary description of mAb production via hybridoma technology, mAbs have fully entered the clinical arena as new, unique, and important components of the medical armamentarium for the treatment of a variety of diseases. In fact, some of these mAbs can be regarded as major breakthroughs in the treatment of such disorders as transplant rejection, coronary artery disease, rheumatoid arthritis, non-Hodgkin’s lymphoma, and breast cancer. It is virtually certain that this initial set of agents is but the first wave of what will become a broad array of mAb therapeutics for disease targets in all areas of medicine.

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GLUCAN SYNTHASE INHIBITORS AS ANTIFUNGAL AGENTS BY MYRA B. KURTZ* AND JOHN H. REX† *Merck Research Laboratories, R80Y-220, Infectious Diseases, P.O. Box 2000, Rahway, NJ 07065, and †Division of Infectious Diseases, Department of Internal Medicine, Center for the Study of Emerging and Re-emerging Pathogens, University of Texas Medical School, 1728 JFB, Houston, Texas 770302

I. Introduction and Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Clinical Need for Antifungal Agents. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Antifungal Resistance Has Accentuated the Need for a New Class of Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . II. The Fungal Cell Wall Is an Attractive Target . . . . . . . . . . . . . . . . . . . . . . . . . . . III. Early Research On Cell Wall Active Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Echinocandins and Papulacandins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. Cilofungin: Limited Spectrum and Solubility Problems Ended Development, Leading to Reduced Interest in the Entire Class. . . . . . . . . C. Inhibition of Cell Wall Synthesis Remained Attractive—Cell Wall Screens—Overview. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . IV. The Pneumocandins: Mycology and Parasitology Collide . . . . . . . . . . . . . . . . A. Activity Against Pneumocystis carinii Revives Interest in the Class . . . . . . . . B. Early Work Focused on Solubility Issues and Fermentation . . . . . . . . . . . . C. A Phosphate Ester Prodrug Emerges . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . V. Development of Amino Compounds . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . A. Total Synthesis, Developing SAR, Discovery of Amino Compounds . . . . . B. Biological Spectrum and Potency Significantly Enhanced – In Vivo Studies. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . C. Mode of Action: Genetic Insights . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VI. Current Compounds in Clinical Development . . . . . . . . . . . . . . . . . . . . . . . . . A. MK-0911 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . B. LY303366, a Lipid-Modified Echinocandin . . . . . . . . . . . . . . . . . . . . . . . . . C. FK463 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . D. Related Agents . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . VII. Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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I. INTRODUCTION AND BACKGROUND A. Clinical Need for Antifungal Agents Fungal infections can range from superficial, noninvasive diseases of normal children and adults to life-threatening systemic diseases of immunocompromised individuals. The incidence of disseminated infections has grown in the past decade, at least in part as a consequence of changing clinical practices. With the use of effective broad423 ADVANCES IN PROTEIN CHEMISTRY, Vol. 56

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spectrum antibacterial agents for treatment and prophylaxis, fungal infections have now become an important cause of morbidity and mortality in bone marrow transplant patients, cancer patients, and other severely immunocompromised individuals. This has been reflected in the steady rise in number of infections due to Candida species during the 1980s (Beck-Sague and Jarvis, 1993) and more recently in increasing rates of invasive aspergillosis (Groll et al., 1996; Wald et al., 1997). The increase in Aspergillus infections is particularly troublesome, since even with the best treatments, first-line therapy fails in at least 50% of patients and mortality approaches 100% in the absence of effective therapy (Denning, 1998). Although the diversity of the fungal organisms that infect humans is as great as the affected patient populations (Kwon-Chung and Bennett, 1992), three groups of organisms cause the majority of invasive mycoses. Candida species are rapidly growing ascomycete yeasts, which are the most common cause of invasive mycoses, but they are also frequent normal commensals of the gut and genital tract. The most important pathogen of this diverse genus is Candida albicans. This species is unusual in its ability to assume several morphological forms (Kwon-Chung and Bennett, 1992); under standard laboratory conditions it grows as a typical budding yeast, an elipsoid form, which replicates by producing single daughter cells as a small bud from one pole. Under the appropriate conditions, the yeast form extends a long narrow tube, which becomes a branched multinuclear structure similar to the hyphae of true filamentous fungi. Sometimes buds remain with the mother cell, forming long chains of elongated cells, which differ from true hyphae by their width and by the constrictions found at the septa that separate each cell. The ability to undergo this dimorphic transition may contribute to the greater virulence of Candida albicans as compared with the other Candida species. The Candida species can cause a wide range of infections, including mucocutaneous candidiasis, candidemia, and visceral infections of almost any organ (Rex et al., 1997). The second major yeast infection of humans is due to Cryptococcus neoformans, a basidiomycete yeast with a thick polysaccharide capsule, which belongs to a separate phylum (Basidiomycota) from the rest of human pathogenic fungi such as Aspergillus, Candida, Coccidioides, Histoplasma, and Blastomyces (Ascomycota) (Perfect et al., 1998; Taylor, 1995). This organism is normally found in avian droppings and is spread by inhalation. Following inhalation, it may disseminate to any organ but has a strong propensity to produce meningitis. The third major group of pathogens contains the various species of Aspergillus (Denning, 1998). These true filamentous ascomycete fungi are soil organisms and

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are generally of low virulence. As part of their life cycle, they produce airborne asexual conidial spores, which may be inhaled. In patients with defective immunity, especially patients with defects in either function or number of neutrophils, these conidia may germinate and produce invasive disease. A large number of other filamentous fungi are also occasional pathogens of humans (Rex et al., 1997). Ideally, clinicians would like to treat or prevent these wide-ranging fungal infections with a single, nontoxic antifungal agent. Unfortunately, this requires eradication of a very diverse set of eukaryotic microbes from a eukaryotic host. Three groups of agents have been used to date, namely, the polyenes, the azoles, and flucytosine. Amphotericin B is the prototypical polyene antifungal agent and achieves a modest degree of specificity by preferentially targeting the ergosterolcontaining membranes of fungi rather than the cholesterol-containg membranes of mammals (Gallis et al., 1990). However, an effective dose of this broad-spectrum and fungicidal agent results in severe fevers and chills and more importantly, dose-limiting nephrotoxicity. Liposomal formulations reduce but do not completely eliminate these side effects and are quite costly (Hiemenz and Walsh, 1998). The second class of agents, the azoles, has far fewer side effects than amphotericin B and members of this class have proved useful as systemic therapy for invasive mycoses (Rodriguez et al., 1996). Members of this drug class inhibit fungal lanosterol demethylase (Marriott and Richardson, 1987), a cytochrome P450–containing enzyme, which catalyzes a key step in ergosterol biosynthesis. Recently, evidence has been obtained that the azoles may inhibit a second cytochrome P450 enzyme, sterol ∆22-desaturase, the enzyme that catalyzes the last step in ergosterol biosynthesis (Kelly et al., 1997). Three azoles are now in common use as therapy of systemic function infections. Fluconazole, a hydrophilic triazole, is available for oral and parenteral administration and is active against Candida spp. and C. neoformans, but has no meaningful activity against Aspergillus species or the other mold fungi. The lipophilic azole itraconazole has long been available for oral use and possesses potent activity against both Candida and Aspergillus. Because of its limited solubility, a parenteral preparation was not licensed until 1999. Finally, ketoconazole is an older agent, which is structurally related to itraconazole. As itraconazole generally has superior activity to ketoconazole, use of the latter is now quite limited. All three of these agents are well tolerated and have been widely used. Their principal drawbacks are that they are not fungicidal and that prolonged use is associated with a significant risk of developing antifungal resistance (Rex et al., 1995).

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The last of the three groups of current systemic antifungal agents is a class that contains one drug, the fluorinated cytosine analogue known as flucytosine. On conversion to 5-fluorodeoxyuridylic acid monophosphate, this compound interferes with fungal DNA synthesis (Polak and Scholer, 1975). Although it is a potent agent, resistance to this drug develops readily. Because it also has a significant risk of hematologic and gut toxicity, it is now little used. Thus, all three classes of current antifungal agents possess significant drawbacks, and new agents would be welcomed. This review will focus on the development history of one new class of antifungal agents, the lipopeptides, which are broadly known as echinocandins. Agents of this class that are currently under development include MK-0911 (previously L-743,872, and now known as caspofungin acetate and Cancidas/Merck), LY303366, and FK463. As one of us (MBK) has been closely involved with the discovery and development of MK-0911, a historical perspective on its development will be used both as the framework for this review and to convey the interaction between changing medical needs, growing scientific databases, and serendipity in drug development. B. Antifungal Resistance Has Accentuated the Need for a New Class of Agents All growing cell populations, from bacteria to human, have some capacity to develop resistance mechanisms to toxic substances. The frequency of observing resistance to a particular cytotoxic compound depends on the target cell as well as on compound-specific characteristics. For fast-growing pathogenic bacteria that carry episomal DNA encoding antibiotic-inactivating enzymes, resistance to widely used antibiotics can develop quickly and spread rapidly. Resistance to antifungal agents has been much slower to develop and spread, in part because of lower levels of antifungal use but also because fungi lack vectorial transmission systems and are opportunists that do not cause disease in most individuals. Standardized methods for surveillance of antifungal drug susceptibility have been a recent development (Rex et al., 1993). The M27 protocol of the National Committee for Clinical Laboratory Standards (NCCLS) for testing of yeasts focused on laboratory to laboratory reproducibility and became an approved standard in 1997 (National Committee for Clinical Laboratory Standards, 1997). A modification of M27 for testing of molds has recently been proposed as NCCLS document M38-P (National Committee for Clinical Laboratory Standards, 1998). With these tools, collaborative studies to validate the predictive power of these results have been possible. Interpretive breakpoints for

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the azoles and for flucytosine have been proposed for Candida (National Committee for Clinical Laboratory Standards, 1997; Rex et al., 1997). Technical issues have limited the interpretability of tests of C. neoformans (Ghannoum et al., 1992), but recent work suggests that reliable correlations will be possible (Witt et al., 1996), at least for the azoles. Methods for identifying azole-resistant isolates of Aspergillus have also been demonstrated (Denning et al., 1997). Unfortunately, reliable determination of the susceptibility of both yeasts and molds to amphotericin B has proved difficult (Verweij et al., 1998; Rex et al., 1995; Lozano-Chiu et al., 1997a, 1997b). Great attention to choice of media, test format, and type of end point appear required for meaningful results (Wanger et al., 1995; Nguyen et al., 1998). As we will see, these problems are paralleled by difficulties with the glucan synthase inhibitors, for which in vitro methodologies play an important, complex role in predicting in vivo efficacy. These difficulties aside, banks of strains have now been collected and studied to determine the mechanisms of resistance. Elegant studies have been done on clinical and laboratory strains to elucidate the molecular mechanisms of resistance to the azole antifungal agents (White et al., 1998; Vanden Bossche et al., 1998; Sanglard, 1998). Mechanisms described to date include (1) increased level of the target enzyme; (2) mutations in the target enzyme that reduce affinity for azoles; (3) increased passive or active efflux of drug, manifest either as a stable or transient trait; and (4) altered sterol biosynthesis to bypass the accumulation of toxic intermediates. Each of these mechanisms can contribute stepwise to the overall loss of susceptibility to growth inhibition by azoles (White et al., 1998). In addition to these genetic alterations to produce resistance, there is evidence that a transient, reversible adaptation to sterol inhibitors can occur by derepression of the sterol biosynthetic pathway. Resistance to amphotericin B most often appears to be due to decreases in membrane ergosterol (Pierce et al., 1978). Other forms of resistance have been described but not extensively characterized (Lozano-Chiu and Rex, 1998). Finally, resistance to flucytosine may be due to loss of the permease, deaminase, or to uridine monophosphate pyrophosphorylase activity required for conversion of flucytosine to its active form, or to an increase pyrimidine synthesis (Jund and Lacroute, 1970, 1974). One obvious way to treat pathogens that have accumulated multiple resistance mechanisms or are intrinsically resistant to a given therapy is to use a drug with an entirely different mode of action. Since the major antifungal therapies target sterol biosynthesis or composition, a large variety of additional metabolic pathways are theoretically available. In reality,

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however, useful therapeutic choices are not completely obvious. One would like to target a process or enzyme that is common to the diverse pathogenic fungi and yet is sufficiently different (or absent) in the eukaryotic host to prevent mechanism-based toxicity. It is logical that killing the organism is preferable to merely inhibiting it, so the process should be essential to the fungi for growth and preferably one that is required to maintain cell viability. As genetic resistance emerges at a low but finite rate, which is a function of the total number of organisms, it is also possible that a fungicidal agent could reduce the organism load to a low level that would delay the appearance of drug-resistance. II. THE FUNGAL CELL WALL IS AN ATTRACTIVE TARGET The criteria we have established for a good broad-spectrum target for an antifungal agent are fulfilled by the enzymes of cell wall biosynthesis. The cell wall both is a universal feature of the fungi and is unique to the fungi. The enzymes required for the synthesis and maintenance of this essential structure are unlikely to have closely related counterparts in mammals. Finally, the clinical success of antibacterial agents that target the bacterial cell wall suggests that the functional equivalent of “penicillin for fungi” would have great potential. Figure 1 shows a working and undoubtedly oversimplified model for the structure of the yeast cell wall based on genetic and biochemical studies in Saccharomyces cerevisiae and C. albicans. There are four major components of the wall: 1,3-β-D-glucan, 1,6-β-D-glucan, chitin (a polymer of N-acetylglucosamine), and cell wall proteins (Lipke and Ovalle, 1998). As suggested in Fig. 1, these components are organized in two layers, which can be visualized by electron microscopy (Osumi, 1998) and detected by biochemical analysis (Klis, 1994). The outermost layer is composed of more than 40 heterogeneous cell wall mannoproteins covalently linked to 1,3-β-D-glucan fibrils, which form the inner layer (Vandervaart et al., 1996a, 1996b; Kapteyn et al., 1995, 1996). 1,3-β-DGlucans are the most abundant carbohydrate polymers in the ascomycete fungi and are shown as the stippled interwoven fibers in Fig. 1. They constitute as much as 60% of the cell wall in S. cerevisiae and are responsible for the rigidity of this structure. These 1,3-β-D homopolymer fibers are relatively long and unbranched and contain approximately 1500 glucose residues. The third component, the 1,6-β-D glucans, are only 5% of the cell wall and form an intermediate linkage of the cell wall proteins to 1,3-β-D-glucan (Kollar et al., 1997). 1,6-β-DGlucan can be found in highly branched chains covalently bound to 1,3-β-D-glucan (Kollar et al., 1997; Kapteyn et al., 1997). Chitin is the

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FIG. 1. Fungal cell wall components. Reproduced with permission from Kurtz (1998).

fourth and least abundant component (1 to 2%) but plays a key structural role. It is found in a ring at the neck of a budding yeast, in the septum between cells, and in the lateral walls. The amount of chitin and the degree of chitin–glucan cross-linking determine the alkali solubility of 1,3-β-D-glucan fractions of cell wall and the rigidity of the structure. The mesh work formed by the glucan fibrils protect the cells from the

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wide range of osmotic conditions encountered in the hostile environment, either as a pathogen or within its natural ecological niche. It provides the scaffold for numerous enzymes and adherence factors and is also a barrier for large molecules. Cell wall architecture determines cell shape throughout the cell cycle. Although it is shown as a fixed structure, cell wall composition can change in response to a variety of internal and external stimuli (Osumi, 1998). Genetic studies have shown that mutations that prevent synthesis or disturb the regulation of some cell wall components can be lethal, and therefore these processes represent good fungal-specific targets for antifungal drugs (Ram et al., 1994). It should be noted that the simplified model shown for S. cerevisiae appears to be applicable to many fungi and especially to Candida species. Although all the pathogenic fungi possess some 1,3-β-D glucan, there are differences among the fungi that may be significant for development of broad-spectrum agents. Pathogens such as Aspergillus fumigatus and Histoplasma capsulatum have galactomannan in their cell walls (Sakaguchi et al., 1968; Reiss et al., 1974) and have a greater proportion of chitin (Hector, 1993). Cryptococcus neoformans has a large portion of its glucan in α linkages (Bacon et al., 1968; James et al., 1990). Despite the interest by pharmaceutical companies in cell wall synthesis as an antifungal target, the high degree of genetic and biochemical complexity of the cell wall has limited our understanding of the genes and enzymes essential for building the wall. Fungi have developed mechanisms to compensate for alterations in cell wall composition and integrity, further complicating the task of elucidating what is undoubtedly not a simple linear pathway for synthesis. For example, chitin synthesis is upregulated under a variety of genetic and environmental alterations in cell wall synthesis (Choi et al., 1994) and is catalyzed by multiple isozymes, which differ in number and enzymatic properties among various fungal species. In addition, construction and remodeling of the cell wall during cell growth, morphogenesis, and cell division pose regulatory constraints both temporally and spatially. Most of these considerations are outside the scope of this review. However, we have learned much about the genetic and biochemical steps in glucan synthesis from the study of natural products that inhibit steps of cell wall synthesis, and we will focus on these results. III. EARLY RESEARCH ON CELL-WALL ACTIVE AGENTS A. Echinocandins and Papulacandins In the 1970s two structural classes of fungal fermentation products were discovered in screening programs for antifungal agents: (1) the

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lipopeptide class, referred to as echinocandins and (2) the glycolipid class, known as papulacandins. However, early data on their mode of action were limited by technology—no purified enzyme, no genetics. Since their discovery, additional members of each class have been described (Table I). The naturally occurring papulacandins include papulacandin B (Traxler et al., 1977, 1987), chaetiacandin (Komori et al., 1985; Komori and Itoh, 1985), L-687,781 (VanMiddlesworth et al., 1991), BU-4794 (Aoki et al., 1993), saricandin (Chen et al., 1996), fusacandins A and B (Jackson et al., 1995), BE-29602 (Okada et al., 1996), and PF-1042; there are also numerous lipopeptide compounds such as echinocandins B, C, and D (Nyfeler and Keller, 1974; Traber et al., 1979), aculeacin A (Mizuno et al., 1977; Satoi et al., 1977), as well as mulundocandin (Mukhopadhyay et al., 1987, 1992; Roy et al., 1987) and more than a half dozen pneumocandins (Hensens et al., 1992) and FR901379 (Iwamoto et al., 1993). Several reviews have been published describing these agents (Debono and Gordee, 1994; Balkovec et al., 1997; Georgopapadakou and Tkacz, 1995; Current et al., 1995; Kurtz and Douglas, 1997). At the time of the discovery of these natural products, the cell wall biosynthetic enzymes had not been purified or defined by genetic analyses. The echinocandins are amphiphilic cyclic hexapeptides with an Nlinked acyl side chain. The first member of the class to be discovered was echinocandin B, a fermentation product of Aspergillus nidulans (Nyfeler and Keller, 1974). The hexapeptide nucleus is derived from several unusual amino acids: dihydroxyornithine, 4-hydroxyproline, dihydroxyhomotyrosine, and 3-hydroxy-4-methylproline, as well as two threonines. The lipid tail of the compound is formed from a linoleoyl group on the N terminus of the cyclic peptide, and the ring is completed by an aminal group that connects the 3-hydroxy-4-methylproline to the δ amino group of dihydroxyornithine. Nomenclature of these lipopeptides can be confusing. The term echinocandin has been used to refer to the family of compounds that have the same hexapeptide nucleus as echinocandin B with variations in the hydroxylation patterns and the lipid tail, but it has also been used it as a more inclusive term encompassing the pneumocandins, despite the different hexapeptide nucleus and lipid tail of the latter, as discussed below. All of the lipopeptides (echinocandins, aculeacins, pneumocandins, mulundocandins, sporiofungins) discussed in this chapter are compounds that have similar biological activity and have glucan synthase as their enzymatic target. Because papulacandin B had good antifungal activity against the model organisms S. cerevisiae and Schizosaccharomyces pombe, scientists in academic and pharmaceutical laboratories have used papulacandin B or related compounds in genetic, physiological, and biochemical analy-

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TABLE I

Naturally Occurring Glucan Synthase Inhibitorsa

Year 1974 1977 1979 1979 1982 1987 1989 1993

Name Echinocandin B Aculeacin Echinocandin C Echinocandin D S31794 Sporiofungin A Mulundocandin L-671,329 Pneumocandin A0 FR901379d

Company Sandoz Ciba-Geigy Lilly Toyo Jozo Sandoz Sandoz Sandoz Hoechst Merck Fujisawa

R1

R2

R3

OH OH OH H OH OH OH OH OH

OH OH OH H OH OH OH OH OH

OH OH H H OH H OH OH OH

R4 CH3 CH3 CH3 CH3 CH3 H CH3 CH3 CH3

R5 CH3 CH3 CH3 CH3 CH2CONH2 CH2CONH2 H CH2CONH2 CH2CONH2

Fatty acid Linoleic acid Palmitic acid Linoleic acid Linoleic acid Myristic acid DMMb MMMc DMMb Palmitic acid

a Note that the term echinocandin is used (a) in a general sense to refer to entire the entire lipopeptide class (including the pneumocandins and all compounds in this table except for papulacandin B; and (b) in a specific sense to refer only to compounds with the same hexapeptide core structure as echinocandin B. b Dimethyl myristate. c Monomethyl myristate. d Has OSO H ortho to OH on homotyrosine. 3

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ses of mode of action (Ribas et al., 1991a, 1991b). Mutants of S. pombe resistant to papulacandin B were isolated in the hope that they would facilitate the discovery of the genes encoding the components of glucan synthesis and aid in the purification and characterization of the enzyme. Throughout the 1970s and 1980s it remained difficult to demonstrate that genes conferring resistance were part of the glucan synthesis machinery. It turns out that purifying glucan synthase to homogeneity has been even more difficult and has not been achieved for any fungus to date, despite considerable effort since the pioneering work by Kang and Cabib (1986). Another reason that papulacandin B became the natural product of choice for study was that it was possible to demonstrate inhibition of an in vitro enzyme reaction with fungal membrane preparations at levels close to the minimum inhibitory concentration (MIC) of the whole cells (Baguley et al., 1979; Kang et al., 1986). This was in contrast to the early observations that the IC50 of echinocandins for crude membrane preparations of glucan synthase were considerably higher than the concentration needed to inhibit growth. Highly enriched membrane preparations with glucan synthase activity were first obtained from the filamentous fungus Neurospora crassa (Awald et al., 1993) by a novel method called product entrapment (Cabib and Kang, 1987), which had been developed to purify chitin synthase. By allowing the crude membranes to synthesize fibrous glucan in an in vitro reaction, the enzyme complex remains associated (entrapped) with the product selectively and can be partially purified by low-speed centrifugation. This remained the state of the art throughout the period of development of the early echinocandins. More recently, highly enriched soluble preparations from S. cerevisiae (Inoue et al., 1995), A. nidulans (Kelly et al., 1996), and C. albicans (Frost et al., 1997) have been reported; these will be discussed below in conjunction with the discussion of the cloning of the S. cerevisiae genes for the putative catalytic subunit of the enzyme (Inoue et al., 1995; Mio et al., 1997). B. Cilofungin: Limited Spectrum and Solubility Problems Ended Development, Leading to Reduced Interest in the Entire Class In the 1970s scientists at Eli Lilly & Co. determined that echinocandin B had potent antifungal activity against C. albicans and Candida tropicalis and in vivo activity in animal models of candidiasis. Its limitations for human therapeutics were lack of oral bioavailability and poor water solubility. There was also concern because it produced in vitro lysis of red blood cells. Early chemical modifications focused on reducing the lytic potential of echinocandin B and showed that the fatty acyl tail

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could be modified to produce nonlytic compounds (Debono et al., 1988, 1989). Methods were developed to enzymatically cleave echinocandin B selectively to yield the cyclic peptide and the fatty acid, neither of which had antifungal activity by itself (Debono et al., 1989). Echinocandin B analogues with modified fatty acids were synthesized by chemically reacylating the cyclic peptide nucleus (Debono et al., 1989). To retain antifungal activity, the fatty acid had to be at least C12 in length; C18 unbranched fatty acids were optimal. Activity required a lipophilic tail, and highly polar or short-chain acyl groups did not restore antifungal activity to the echinocandin B nucleus. These observations suggest the possibility that the tail must be sufficiently long and lipophilic to tether the compound to the membrane (the site of glucan synthesis) for inhibition to occur. Improved potency was obtained with side chain modifications. Cilofungin (LY121019) (Fig. 2), (echinocandin B with a p-octyloxybenzoyl side chain), was selected for clinical development as a parenteral agent. Since the modifications did not decrease the intrinsic water insolubility of the natural product, the drug was administered with the cosolvent polyethylene glycol. The solvent system itself proved toxic and the trials were ended before the efficacy of the compound could be fully assessed (Doebbeling et al., 1990). However, initial studies indicated promising results for Candida esophagitis and disseminated candidiasis (CopleyMerriman et al., 1990a, 1990b). C. Inhibition of Cell Wall Synthesis Remained Attractive— Cell Wall Screens—Overview The clinical experience in the late 1980s with cilofungin did not change the view of cell wall synthesis as an attractive target for an antifungal agent, and pharmaceutical companies continue to search for broad-spectrum inhibitors of cell wall integrity to this day. However, clinical needs were changing at that time, and it was becoming clear that an agent with a spectrum limited to Candida would not satisfy the needs for broad-spectrum empirical therapy of the growing immunocompromised patient population. At the same time, the secondary infections of patients with the acquired immunodeficiency syndrome (AIDS) required therapy for infectious agents that had been rarely seen before. Thus, the requirements for a useful new antifungal agent were evolving. The primary search for a lead compound for further medicinal chemistry was, however, still the same. With little in the way of mechanistic information on the synthetic process, discovery of antifungal agents affecting cell wall synthesis has been an empirical process. Although pharmaceuti-

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FIG. 2. Structures of the clinical candidates.

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cal companies screen through their in-house collection of chemical compounds, natural products have often been the richest source of antimicrobial agents. Traditionally, this search entails testing hundreds of thousands of fermentation broths with an assay that can distinguish nonselective poisons from cell wall inhibitors in the complex mixture. This screening process can be as sophisticated as a robotic enzymatic assay or as basic as detecting a zone of growth inhibition around a drop of fermentation broth on an agar plate. Generally, large pharmaceutical companies and biotechnology companies alike guard their proprietary screening technologies, and therefore the details of highly successful paradigms are not likely to be revealed in a timely way. Despite these limitations, we can glean some information from the literature. The representatives of the lipopeptide classes were originally found as potent anti-Candida agents or as compounds that interfered with the cell’s ability to exclude a nonpermeant dye (Schwartz et al., 1992; Nyfeler and Keller, 1974). As our understanding of the properties of glucan synthase improved, high-throughput in vitro enzyme assays were developed. These included enzyme preparations from N. crassa, C. albicans, and A. fumigatus based on crude cell lysates and microsomal fractions that could be harvested in bulk (Wood et al., 1998; Taft et al., 1994; Frost et al., 1994; Shedletzky et al., 1997). Since it was not known whether these membrane preparations contained all the accessory components needed for glucan synthesis, strategies were developed for whole-cell assays that specifically detected changes in glucan synthesis. In S. cerevisiae, strains were genetically manipulated to funnel galactose preferentially into cell wall glucan (Coen et al., 1994; Tkacz, 1984). Specific inhibitors could be detected as those compounds that change the ratio of glucan synthesis to protein synthesis. Thus, different whole-cell assays were developed to detect inhibition of glucan synthesis. In one, toluene was used to make cells permeable to the nonpermeant uridine diphosphate (UDP)–glucose substrate, following which glucan synthesis could be measured (Frost et al., 1994). In another system, differential whole-cell labeling with glucose, glucosamine, and an amino acid was used as to assess inhibition of glucan synthesis (Onishi, J., Merck Research Laboratories, personal communication). Genetic techniques were also used to create strains that were specifically more sensitive or more resistant to inhibitors of glucan synthase than the parent strain (Onishi, J., Merck Research Laboratories, personal communication); (Frost et al., 1997). It was reasoned that only inhibitors of glucan synthesis would produce a differential response between the wild-type and the resistant strain. By using methods based

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on these tools, new echinocandin and papulacandin inhibitors of glucan synthase were sought and found. IV. THE PNEUMOCANDINS: MYCOLOGY AND PARASITOLOGY COLLIDE A. Activity Against Pneumocystis carinii Revives Interest in the Class At Merck, a novel class of lipopeptides was discovered in 1985 in a broad screening for cell wall agents, which detected compounds that allowed the accumulation of nonpenetrant dyes (Schwartz et al., 1992). The pneumocandin-producing culture was isolated from pond water obtained in the valley of the Lozoya River in Spain. The major fermentation product of the original culture is pneumocandin A0 (present at 330 mg/liter). Pneumocandin B0 (26 mg/liter) and pneumocandin D0 (4 mg/liter) are the most abundant of the additional minor components (Masurekar et al., 1992). As the major active component, pneumocandin A0 was studied first. It differs in structure from the echinocandin B class by replacement of threonine with hydroxyglutamine, along with a modified proline in place of the 3-hydroxy-4methylproline in the cyclic peptide nucleus. The lipid tail has 10,12-dimethylmyristate in place of linoleate. Like many members of this class, pneumocandin A0 has excellent in vitro activity against C. albicans and C. tropicalis and good in vivo activity in animal models of C. albicans infection. What made this compound more than routine was a somewhat broader anti-Candida spectrum than observed with echinocandins, including activity against Candida krusei and Candida glabrata (Bartizal et al., 1992). It was also not lytic against red blood cells. This prompted the evaluation of pneumocandin A0 for additional antifungal activities and a search among the minor components for products with an even better spectrum (e.g., anti-Cryptococcus activity), but no such products were discovered. In 1989, a Merck scientist (D. Schmatz) attended a parasitology meeting at which it was reported that Pneumocystis carinii “cysts” contain 1,3β-D-glucan (Matsumoto et al., 1989)). Interest in this organism was increasing because of the many AIDS patients with life-threatening P. carinii pneumonia (PCP). The most robust rat model for PCP at the time was quite crude. In brief, PCP was induced in otherwise normal rats by immunosuppressing them for 6 weeks. This endogenously arising disease can be monitored by counting cysts in the lung and observing mortality. In one bold experiment that used the remaining purified supplies of pneumocandin A0, Schmatz et al. (1990) showed that pneumocandin A0 prolonged survival and reduced mortality in the rat PCP

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model. This exciting discovery initiated a diverse collaborative effort between the antifungal and antiparasite discovery groups to identify an agent that could treat Candida infections and pneumocystosis. B. Early Work Focused on Solubility Issues and Fermentation It was recognized that the original fermentation product pneumocandin A0 had some limitations and that improvements in potency, spectrum, water solubility, and chemical stability were needed. A number of assays were used to evaluate the biological properties of synthetic analogues, semisynthetic analogues, and fermentation components. Initially, the in vitro antifungal spectrum was assessed by measuring each compound’s MIC against a panel of targeted Candida, Aspergillus, and Cryptococcus strains. This MIC correlated well with the minimum fungicidal concentration (MFC) for Candida, defined as the concentration required to reduce the number of colony-forming units (CFUs) 100fold. Both MIC and MFC were highly correlated with inhibition of in vitro glucan synthase. At the beginning of the program, a rapid, compound-sparing animal model of candidiasis was developed to evaluate in vivo efficacy. The model was given the acronym TOKA for “target organ kidney assay,” because efficacy was determined by quantitating the number of Candida CFUs that could be recovered from the kidney of infected mice a fixed period after intravenous infection. Use of complement-deficient rather than immune-competent mice, results in fewer infectious organisms being required for a lethal infection. The potential for rapid and large (up to 10,000-fold) increases in CFUs in the kidneys provided a sensitive quantitative measure of a compound’s efficacy (Bartizal et al., 1992). Mice could also be sampled well before death, making this approach both faster and more humane. If representative samples were taken at various time points, it was also possible to determine the pace and magnitude of the response. After analyzing as many of the minor components as was feasible, pneumocandin B0 was chosen as the basis for further modification. This analogue has very potent anti-Candida activity, excellent anti-Pneumocystis activity, and little ability to lyse red blood cells. The bioprocess engineers then developed media and conditions to favor pneumocandin B0 production over pneumocandin A0, while the microbiologists isolated strains which produced more pneumocandin B0 relative to unwanted products. Starting with a culture that produced only 4 mg/liter of pneumocandin A0, the combination of efforts resulted in 20-fold improvements (Masurekar et al., 1992). Later, when a semisynthetic analogue was selected for clinical trials, further titer improvements and reduction

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in by-products of the fermentation were achieved. One of the important achievements was the development of a defined medium that allowed fermentations at the production level (N.C. Connors, Merck Research Laboratories, personal communication). C. A Phosphate Ester Prodrug Emerges Unlike cilofungin, pneumocandin B0 was not hemolytic, even at concentrations up to 400 µg/ml. Therefore, the aim of the early medicinal chemistry work was to improve solubility. One strategy was to develop a prodrug that would be rapidly converted to the active form (Balkovec et al., 1992). The physical properties of pneumocandin B0 presented a difficult challenge for selective modification. The natural product was unstable in both acidic and basic solutions and had limited solubility in the solvents useful for chemical synthesis. The homotyrosine ring provided an electron-rich ring and an opportunity for selective acylation. The best candidate among a number of homotyrosine derivatives was a phosphate ester prodrug, L-693,989 (Balkovec et al., 1992). The compound was soluble in water to a concentration of over 50 mg/ml, a major improvement as compared to less than 0.1 mg/ml for its parent. The phenolic hydroxyl group is essential for antifungal activity and thus the prodrug is not active as an antifungal agent until it is dephosphorylated by the serum and tissue phosphatases of the host. This cleavage occurs rapidly and the in vivo activity of L-693,989 in animal models of PCP and candidiasis was indistinguishable from that of the parent (Balkovec et al., 1992). V. DEVELOPMENT OF AMINO COMPOUNDS A. Total Synthesis, Developing SAR, Discovery of Amino Compounds As L-693,989 was on its way to becoming a clinical candidate for the treatment of candidiasis and pneumocystosis, medicinal chemists were learning to modify additional positions on the cyclic peptide nucleus. Their work was guided, in part, by the successful total synthesis of a defunctionalized echinocandin by solid-phase synthesis (Zambias et al., 1992). The information on the minimum requirements for antifungal activity and glucan synthase inhibition suggested that the acid-labile hemiaminal group might be an opportunity to prepare additional analogues (Balkovec and Black, 1992). Studies of a series of ether derivatives at the hemiaminal group showed that the greater the lipophilicity of the group, the less inhibitory the activity. The aminoethyl ether ana-

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logue (L-705,589) showed a 10-fold improvement in activity against glucan synthase and had anti-Candida activity, in vitro and in vivo (Bouffard et al., 1994). The corresponding β isomer was 45-fold less potent against the enzyme (Bouffard et al., 1994). L-705,589 was also more stable than the parent compound and intrinsically water-soluble. Surprisingly, this compound and other ether analogues also had improved antiAspergillus activity in an in vivo model (Bouffard et al., 1994) (Table II). This observation had a significant impact on subsequent work. To this point, development had been spurred by the potential utility of a new agent with activity against Candida and P. carinii, both of which were major causes of AIDS-related opportunistic infections. However, the emergence of generally effective treatment and prophylaxis regimes for these infections (fluconazole and trimethoprim–sulfamethoxazole, respectively) had made these research goals somewhat less compelling. Furthermore, confusion existed at that time about the in vitro activity of the echinocandins against Aspergillus. The antifungal spectra of all the natural product glucan synthase inhibitors are similar: all are clearly potent fungicidal inhibitors of C. albicans and C. tropicalis, with differing activities against other Candida species and no activity against Cryptococcus spp. However, debate remained about their antiAspergillus activity. The natural products do alter growth of Aspergillus mycelia to the extent that a zone of inhibition can be seen around a disk containing the compounds on agar plates seeded with the organTABLE II Improved In Vivo Efficacy with Stepwise Amino Modifications of Pneumocandin B0a,b Activity (mg/kg) Compound name: pneumocandin B0 modification Pneumocandin B0 L-705,589 CH2CH2NH2 at hemiaminal (R1) L-731,373 Reduced glutamine at R5 (CH2NH2) L-733,560 Both of the above modifications at R1 and R5

ED99.9

Aspergillusd ED50

PCP ED90

6.0 0.78 0.375 0.09

>20 0.06 >20 0.03

0.15 0.037 0.037 0.019

TOKAc

Reproduced with permission from (Bouffard et al. (1994). R1 and R5 are as defined in Table I. c TOKA ED 99.9, minimum dose reducing renal CFUs per gram of C. albicans by 99.9% vs. untreated controls. d Aspergillus ED , minimum dose administered on days 0 to 4 that increases the 28-day sur50 vival rate by 50%. e PCP ED , minimum administered for 4 days that decreases the lung cyst count by 90% 90 over untreated controls. a

b

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ism. In fact, during the development of cilofungin and in the early development of pneumocandin derivatives, the potency of semisynthetic analogues was monitored by a quantitative bioassay using zones of inhibition on Aspergillus montevidensis (Gordee et al., 1984). However, sparse growth may occur with the zone of inhibition on agar (Arikan et al., 1999), and the synthase inhibitors do not produce a conventional completely clear (no growth) MIC against Aspergillus species in brothbased testing (Hanson and Stevens, 1989). Furthermore, cilofungin showed only limited activity in a stringent model of Aspergillus infection, requiring 62.5 mg/kg for 80 to 83% protection in a survival model (Denning and Stevens, 1991), significantly more than the ED50 of about 7.0 mg/kg needed for treating Candida infections (Gordee et al., 1984). When compared with the sharp growth end point that could be obtained for amphotericin B, these data led to the idea that these compounds had little activity against Aspergillus. Microscopic observation of pneumocandin-treated Aspergillus cultures showed that the lipopeptides caused aberrant filamentous growth; hyphae were short and thick, with abundant abnormal branches. They also had unusual bulbous tips, consistent with expansion of a weakened cell wall under osmotic pressure (Kurtz et al., 1994). In electron microscopic studies, the cell wall changes were even more dramatic. Whereas the untreated controls had cell wall structures of uniform thickness throughout the apical and subapical tip regions, the treated cultures had much thicker walls overall and were thickest in the subapical regions. These abnormal structures were dark and intensely stained. The septa were also thicker and more frequent in treated hyphae. Despite the alterations in the wall, the internal organelles appeared unchanged, and there were no signs of overt damage to the cytosolic contents. The concentration of drug required to produce this effect (0.03 to 1 µg/ml) was at or below that needed to kill C. albicans (1–2 µg/ml), indicating that it is unlikely to be a nonspecific detergent effect. Detection of the morphological effect of these compounds does not require microscopic examination; it can be seen with the naked eye in broth dilution assays as significant growth inhibition and is especially easy to detect in microtiter dishes. We have called the concentration of drug required to produce the morphological effect minimum effective concentration (MEC). This morphological effect accompanies significant growth impairment, and as we expected, the MEC correlates well with an MIC that used a cut-off of 50 or 80% growth reduction compared with the untreated control (Kurtz et al., 1994). Similar gross morphological effects on hyphae with other glucan synthase inhibitors have been observed for the filamentous fungi N. crassa (Taft and Selitren-

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nikoff, 1988) and Paracoccidioides brasiliensis (Davila et al., 1986). More recently, the MEC nomenclature has been used to describe the same effect of LY303366 (Oakley et al., 1998). With this array of observations of anti-Aspergillus activity in mind, L693,989 and several related compounds were tested in a immunocompromised rat model of pulmonary infection (Kurtz et al., 1995; Kurtz et al., 1994). In this model L-693,989 prolonged survival compared with untreated animals in a dose-dependent manner. At doses as low as 5 mg/kg, 90% of animals survived compared with 10% of the infected control animals. Although it was difficult to find mycelia in light micrographs of A. fumigatus-infected rat lung tissue in drug-treated animals, the few examples that were obtained showed the characteristic abnormal morphology of pneumocandin-treated cultures (Fig. 3). These strong demonstrations of good anti-Aspergillus activity in vivo with less evident in vitro activity led to the need for a high-throughout screening animal model. Again, the complement-deficient mouse turned out to be sufficiently compromised to develop a lethal disseminated infection when injected intravenously with a spore suspension of Aspergillus. This model was also more stringent than the rat model, as L-693,989 was protective only at doses of more than 20 mg/kg. This was actually useful, as compounds 10-fold more potent than L-693,989 were needed, and it seemed likely that this would be easily detected. In this mouse model, ether analogues of pneumocandin B0 were remarkably improved (Abruzzo et al., 1995). The methyl ether derivative had an ED50 of 1.8 mg/kg for Aspergillus but lost some Candida activity, whereas the aminoethyl ether analogue had an ED50 of 0.06 (over 100-fold improvement) for Aspergillus and an ED90 of 0.3 (a 10-fold improvement) in the candidiasis model (Abruzzo et al., 1995; Bouffard et al., 1994). B. Biological Spectrum and Potency Significantly Enhanced—In Vivo Studies Medicinal chemists developed methods for the selective modification of the 3-hydroxyglutamine of pneumocandin B0 at the primary carboximide to yield the 3-hydroxyornithine analogue L-731,373. This derivative had the anti-Candida activity of L-705,589 but did not have detectable activity against Aspergillus (Bouffard et al., 1994; Abruzzo et al., 1995). When the pharmacophore of L-731,373 was combined with that of L-705,589 to produce L-733,560, the improvements in anti-Candida activity were additive. Furthermore, the excellent anti-Aspergillus activity of the aminoethyl ether modification was retained in L-733,560. The results with this doubly modified compound were obtained just as the phosphate prodrug was in preclinical safety trials. When it became

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FIG. 3. Morphological effects of pneumocandin treatment on Aspergillus and Candida. Panel 1: Light micrographs of drug-induced morphologic changes in A. fumigatus showing (A) control fungus and (B) effect of treatment of 2 µg/ml of L-733,560 (reproduced with permission from Kurtz et al., 1994).

apparent that SAR was developing, which made profound spectrum improvements and also conferred intrinsic water solubility, emphasis was shifted to the amino-containing analogues. Although MK-0911 (L743,872, the heminaminal aza analogue of L-733,560) was later found to have pharmacokinetic and safety advantages over L-733,560 and was chosen for clinical trials, most of the work on mode of action was done with L-733,560.

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FIG. 3. Panel 2: Light micrographs of A. fumigatus-infected rat lung tissue stained with toluidine blue-O from (A and B) sham-treated controls and (C and D) animals treated with 0.625 mg/kg of L-733,560 (reproduced with permission from Kurtz et al., 1995).

C. Mode of Action: Genetic Insights Before proceeding, the unprecedented 100-fold improvement in the antifungal activity required confirmation that the original fungal-specific mode of action had not changed. Since the echinocandins were known to be inhibitors of cell wall synthesis in C. albicans, it seemed likely that the amino analogues of pneumocandin B0 were more potent antifungal agents because increased potency as inhibitors of glucan synthesis. The IC50 of the hemiaminal-modified compound L-705,589 against the particulate enzyme preparation was seven-fold improved over pneumocandin B0 (11 nM vs. 70 nM) (Abruzzo et al., 1995; Bouffard et al., 1994). The 3-hydroxyornithine-modified compound, L731,373, was also seven fold more potent in this assay. The combination of these modifications yielded L-733,560 and was synergistic for inhibiting the glucan synthase reaction with an the IC50 of 1 nM, a 70-fold improvement in enzyme inhibition. This increased potency as a glucan synthase inhibitor was sufficient to account for the reduced MIC. In

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FIG. 3. Panel 3: Light micrographs showing exclusion of viability dye by C. albicans under different conditions: (A) exclusion of dye by untreated controls; (B) exclusion of dye by 0.8 M sorbitol-treated controls; (C) uptake of dye by essentially all cells treated with 0.2 µg/ml of MK-0911 without sorbitol; and (D) exclusion of dye by at least some cells treated with 0.2 µg/ml MK-0911 in the presence of 0.8. M sorbitol.

fact, the improvement correlated quite well with the observed activity in the candidiasis animal model: the ED99.9 values were 6.0, 0.78, 0.375, and 0.06 mg/kg for pneumocandin B0, L-705,589, L-731,373, and L733,560, respectively. Surprisingly, the MICs for Candida of a variety of cationic structural analogues were only roughly correlated with the enzyme inhibition and animal efficacy data. The most potent compounds had lower MICs than the less effective ones, but the relative ranking could not predict in vivo efficacy for all compounds. As more data accumulated from the medicinal chemistry effort, it became clear that the in vitro enzyme data for the Candida enzyme constituted the best predictor of in vivo activity.

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Thus, all compounds with good activity against the Candida enzyme, regardless of their MIC, were scaled up for animal testing. 1. Development of a Model in S. cerevisiae To pinpoint the mode of action of the potent analogues more precisely and to generate the biochemical and molecular tools for designing better inhibitors, a genetic study in S. cerevisiae was also initiated. Traditionally, the targets of antimicrobial agents have been identified and characterized by isolating mutants resistant to the drug. This approach was difficult for the echinocandins because the natural products had such weak activity (MIC 32 µg/ml) against the model genetic organism, S. cerevisiae (Douglas et al., 1994). Furthermore, inhibition of the glucan synthase enzyme in crude membrane preparations occurred at concentrations higher than needed for whole-cell activity, raising the question as to whether the mechanism in S. cerevisiae was the same as in C. albicans. With the synthesis of much more potent analogues that had good activity against S. cerevisiae, this concern was put to rest. L-733,560 was eight fold more potent against whole cells of S. cerevisiae and had a corresponding eight fold greater IC50 against the glucan synthase of that organism (Douglas et al., 1994). We were then able to isolate mutants resistant to L-733,560. This was certainly not the first attempt to clone the glucan synthase enzyme(s) by resistance, but the mutant searches conducted in many other laboratories had been sparsely documented in the literature (Mehta et al., 1982; DeMora et al., 1991; Ribas et al., 1991b). As it turned out, the FKS1 gene encoding the putative catalytic subunit of glucan synthase was independently isolated by six different laboratories at about the same time, including two laboratories at Merck (Eng et al., 1994; Parent et al., 1993; Garrett-Engele et al., 1995; Castro et al., 1995; Dixon and Ma, 1995; Ram et al., 1994, 1995; Inoue et al., 1995). As mentioned earlier, papulacandin-resistant and osmotically sensitive S. pombe strains had been isolated, and ultimately one of these (cps1+) would also prove to be an FKS1 homologue (Ishiguro et al., 1997). Remarkably, not all of these researchers were seeking to clone the glucan synthase genes. Rather, the repeated cloning of FKS1 has to do with its unique regulation in S. cerevisiae. In our work, a series of L733,560-resistant mutants, initially referred to as echinocandin target genes (ETGs), were isolated, which had decreased susceptibility to inhibition of glucan synthase in vitro (Douglas et al., 1994). The resistance was specific to echinocandins, and susceptibility to antifungal agents with different modes of action was unaffected. In heterozygous diploids, whole-cell resistance was intermediate (MIC value of 0.78

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µg/ml) compared with the wild-type susceptible parent (0.13 µg/ml) and the original mutant (6.25 µg/ml) (Douglas et al., 1994). This semidominant feature of the mutation allowed cloning of the wild-type gene by screening a library for plasmids that conferred reduced resistance in the mutant (Douglas et al., 1994). The large (> 5.0 kb) gene isolated by this procedure was called ETG1 and was found on chromosome 12 (Douglas et al., 1994). Since these studies were done before the complete sequencing of the yeast genome, we first asked whether disruption of the gene affected glucan synthase. To our surprise (and disappointment), the ETG1 null mutant was viable but had reduced viability and grew slowly (Mazur et al., 1995). The strain was highly susceptible to L-733,560 and other glucan synthase inhibitors. It had reduced levels of glucan synthase (approximately 20% that of the wild-type strain) and the residual activity was more than five-fold more susceptible to L-733,560 than the parent. The loss of ETG1 function did not alter drug susceptibility in a generalized way, since the defective mutant was not more susceptible to unrelated antifungal compounds, with one notable exception. The etg null strain was exquisitely sensitive to the immunosuppressive agent FK506 as compared with either the wild-type or resistant parent. Independently, colleagues in adjacent laboratories at Merck & Co. had cloned a gene known as FKS1 based on hypersensitivity to FK506, which inhibits the Ca2+-dependent phosphatase calcineurin (Parent et al., 1993). By comparing chromosomal location, restriction mapping, and gene knockout phenotypes, it became apparent during one extraordinary lunch in the Merck cafeteria that the ETG1 gene was identical to the FKS1 gene (Douglas et al., 1994). 2. A Working Model for the Glucan Synthase Genes The surprising relationship between cell wall glucan synthase and calcineurin was explained by the discovery that there is a second homologue of FKS1 in S. cerevisiae called FKS2 (88% identity at the amino acid level) (Mazur et al., 1995; Kurtz and Douglas, 1997) (Fig. 4). Our current model for the related roles of FKS1 and FKS2 comes from studies of the transcriptional regulation of the two genes as well as the phenotypes of the disruption strains. In this model, both Fks1p and Fks2p can function as the catalytic subunit of glucan synthase but are regulated differently. Whereas FKS1 is the predominant form during vegetative cell growth and is cell cycle–regulated, FKS2 is expressed during sporulation or in response to specific nutritional or environment signals. Disruption of FKS2 does not affect normal vegetative growth but does impair sporulation. Levels of glucan synthase activity in the FKS2 null

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FIG. 4. Model of Fks1p and Fks2p regulation. Reproduced with permission from Kurtz and Douglas (1997).

strains are not appreciably different from those in the wild-type strain. However, loss of FKS1 has severe consequences for the cell, since it must rely on the residual activity from the gene for growth. The fks1 null strain grows poorly and has reduced glucan in its cell wall. The doubly disrupted strain is not viable because no glucan synthase can be made. The FKS2 gene differs from FKS1 primarily by its regulation, not by the biochemical characteristics of its gene product. In fact, the only detectable difference between the Fks1p enzyme and the Fks2p form is that the latter enzyme is more susceptible to echinocandins in vitro, which explains the susceptibility of S. cerevisiae to echinocandins (Mazur et al., 1995). FKS2 is subject to regulation by a calcineurin-requiring step, which does not affect FKS1 transcription. Therefore, in the presence of FK506, calcineurin is inhibited, leading to reduced FKS2 transcription and severe growth inhibition of fks null strains. It can now be seen that the connection between FK506 and glucan synthase is at a step quite removed from the enzyme activity itself (Fig. 4). A third homologue, FKS3 has been discovered, but its function remains obscure. Loss of function produces no recognizable phenotype, either alone or in conjunction with FKS1 or FKS2 deletions. No mRNA can be detected under various conditions surveyed (A. Ram, personal communication). In view of the partial functional redundancy of FKS gene products and their complex transcriptional regulation, it is not surprising that the FKS1 gene was discovered in a diverse set of genetic and biochemical screens. These include papulacandin B resistance (PBR1) (Castro et al., 1995); resistance to the echinocandin B analogue LY280949 (PBR1)

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FIG. 5. Fks transmembrane domains. Reproduced with permission from Kurtz and Douglas (1997).

(Dixon and Ma, 1995); enrichment during partial purification of glucan synthase (Inoue et al., 1995)); hypersensitivity to calcofluor white, an agent that impairs cell wall assembly (CWH53) (Ram et al., 1994, 1995); synthetic lethality with calcineurin-defective mutants (CND1) (GarrettEngele et al., 1995); and hypersensitivity to FK506 (FKS1) (Eng et al., 1994). The FKS1 DNA sequence encodes a protein that is predicted to be 215 Kda, with 16 potential transmembrane domains. The putative topology places a large loop (582 amino acids) on the cytoplasmic side of the protein, as well as two smaller loops of 78 and 89 amino acids (Fig. 5). Although there are no known UDP-glucose binding motifs, one of the cytoplasmic domains does have limited homology to the bcsA gene from Acetobacter xylinium, which encodes the catalytic subunit of cellulose synthase. The FKS1 gene also has structural homology to the ChvA/NdvA genes from Agrobacterium and Rhizobium, which are involved in transport of cyclic β-1,2-glucan. This large integral membrane protein has sufficient complexity to catalyze the polymerization of glucan and also to form a pore to extrude the growing fibril. Over the past few years, FKS1 homologues have been cloned and sequenced from A. nidulans, A. fumigatus, S. pombe, C. neoformans, C. albicans, and N. crassa (Mio et al., 1997; Thompson et al., 1999; Douglas et al., 1997; Ishiguro et al., 1997). A single FKS1 homologue was found in the aspergilli (Kelly et al., 1996; Latge, unpublished data) and in Cryptococcus, whereas S. pombe (Ishiguro et al., 1997) and C. albicans (Mio et al., 1997; Douglas et al., 1997) have two full-length homologues. The Candida genes are discussed in detail below. The genes are highly conserved at the sequence and structural level, but it is the internal large loop that is most conserved among the different fungi (Thompson et al., 1999). In addition to an integral membrane component specified by FKS1, it has long been known that the glucan synthase complex requires a readily dissociable subunit with guanosine triphosphate (GTP)ase activity

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(Notario et al., 1982; Szaniszlo et al., 1985; Cabib et al., 1998). This GTPase has been identified as Rholp, a small, geranylgeranylated protein with homology to Ras, which is essential for viability (Mazur and Baginsky, 1996; Qadota et al., 1996; Drgonova et al., 1996). Interestingly, Rholp has two roles in cell wall assembly, in which it is both a necessary subunit of glucan synthase and a regulator of phosphokinase C (PKC) function (Nonaka et al., 1995; Kamada et al., 1995, 1996). Conditional mutants of RHO1 have a temperature-sensitive phenotype, and the level of glucan synthase that can be measured in extracts prepared from strains grown at the restrictive temperature is reduced (Qadota et al., 1996). The activity of these preparations can be restored by the addition of recombinant Rholp-containing fusion protein. Since RHO1 is an essential gene and is found in both complexes of Fks1p and Fks2p, it is likely common to both the vegetative enzyme and the highly regulated enzyme complex. Thus, glucan synthase is composed of at least two subunits. Mutant searches for additional factors involved in glucan synthase have uncovered a number of other genes that affect the levels of glucan made by the cells. One of these, GNS1, originally isolated for low-level resistance to L-733,560, has been shown to be identical to FEN1/ELO3 (El-Sherbeini and Clemas, 1995; Oh et al., 1997). It now appears that the reduced glucan synthase activity in GNS1 null mutants may be an indirect result of altered membrane composition. ELO3/GNS1 belongs to a family of genes that the encode enzymes that are responsible for fatty acid elongation. Other genes such as KNR4 and HKR1, which are also implicated in regulation of glucan levels, may affect chitin or other regulatory aspects of cell wall synthesis (Hong et al., 1994; Kasahara et al., 1994; Yabe et al., 1996; Martin et al., 1999). Direct, specific interaction between the echinocandins and the FKS1 product has not yet been demonstrated, in part because of the complex nature of the protein and the propensity for these large lipophilic compounds to stick to many surfaces. LY303366, a lipid-modified echinocandin B, was derivatized to produce a photoactivable cross-linking echinocandin analogue with antifungal activity (Radding et al., 1998). The photoaffinity probe specifically labeled proteins of 40 and 18 kDa in membrane preparations. Sequences from internal peptides of the 40-kDa protein identified an unknown protein not previously described as being involved in glucan synthesis (Radding et al., 1998). In contrast, work at Merck with a pneumocandin B cross-linking analogue did not find specific binding in whole cells, spheroplasts, or partially purified enzyme preparations (C.M. Douglas, J. Balkovec and J. Nielsen, unpublished). In addition, attempts to demonstrate specific binding with the 89-amino-acid loop that contains the FKS1 mutations that alter susceptibility to the echinocandins have not been convincing

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(C.M. Douglas, unpublished). Demonstration of specific physical binding of MK-0911 to a fungal target remains a technical hurdle. 3. Glucan Synthase Genes of C. albicans The studies described above with S. cerevisiae were conducted to serve as a model for the clinically relevant fungi, especially C. albicans. The activity of Candida glucan synthase is quite similar to that of S. cerevisiae glucan synthase in terms of Km, Vmax, subunit composition, regulation, and difficulty in purifying to homogeneity (Frost et al., 1997; Mazur and Baginsky, 1996; Mio et al., 1997). The Candida enzyme can also be partially purified from plasma membrane preparations and enriched by product entrapment. One significant difference is that the Candida enzyme is much more susceptible to echinocandins than the S. cerevisiae enzyme (IC50s of 0.001 and 1.3 µM, respectively, for L-733,560). The Candida FKS1 homologue has been cloned by scientists at Merck (Douglas et al., 1997) and at Nippon Roche as GSC1 (Mio et al., 1997). This gene has 73% identity to S. cerevisiae FKS1 and shares the same topological features (Mio et al., 1997). Of particular note are the highly conserved large internal loop and the first smaller internal loop. The greatest divergence lies in the N-terminal end of the deduced protein. From the inability to disrupt all alleles in the wild type, it is likely that the gene specifies an essential function in C. albicans. It may be the functional equivalent of the vegetative FKS1 in S. cerevisiae. Two additional CaFKS1 homologues have been identified by Nippon Roche scientists: (1) GSL2 has 53% homology to CaFKS1 and ScFKS1 and (2) GSL1 is a truncated form (130 kDa compared with 210 kDa) with 54% homology to CaFKS1. The fact that it has been impossible to disrupt the FKS1 gene suggests that neither of these two homologues can readily serve as an alternate source of Fks protein (Mio et al., 1997; Douglas et al., 1997). The gene for the second known component of glucan synthase, RHO1, has been cloned by crosshybridization and polymerase chain reaction to the S. cerevisiae gene (Kondoh et al., 1997). The deduced amino acid sequence is 89% identical to the S. cerevisiae counterpart, and the gene can complement RHO1deficient mutants of the model yeast. CaRho1p copurifies with product-entrapped glucan synthase and interacts directly with Fks1p in a ligand overlay assay (Kondoh et al., 1997). Further confirmation that the model developed for S. cerevisiae is an accurate one for Candida comes from studies of a small set of spontaneous L-733,560-resistant mutants (CAI4R560, NR1-4) and strains selected for resistance by mutagenesis (CA-2 and M-2) (Kurtz et al., 1995, 1996; Douglas et al., 1997). These mutants have enzyme activity that was 5 to 40 times as resistant to inhibition by L-733,560 as the parent strain (Douglas et al., 1997). They are cross-resistant to other glucan

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synthase inhibitors (echinocandins and papulacandins) but are just as susceptible to antifungal agents with different modes of action as their parent strains. Like the S. cerevisiae FKS1 mutants that show intermediate resistance in diploids, these Candida mutants show semidominance in constructed hybrids. This point has potential clinical significance. Since most Candida species are likely to be diploid, mutation to resistance must be dominant, semidominant, or more rarely, due to two independent mutational events leading to recessive resistance. Separate events can also produce a recessive mutation by gene conversion, a process by which a single mutation serves as the template for the second allele during DNA replication and generates two identical mutant alleles. The low resistance to L-733,560 in the laboratory (1 in 108 per generation) suggests that there are few mechanisms to bypass this essential step (Kurtz and Douglas, 1997). 4. Glucan Synthase Genes of Aspergillus With the development of a model of glucan synthase in S. cerevisiae, it was possible to determine whether this concept applied to Aspergillus and could explain the observations with the more potent glucan synthase inhibitors. Earlier studies had demonstrated that crude glucan synthase could be prepared from A. fumigatus and that echinocandins would inhibit the formation of trichloroacetic acid (TCA)-insoluble product from UDP-glucose (Beaulieu et al., 1993, 1994; Beauvais et al., 1993; Wood et al., 1998; Tang et al., 1992). The discovery that the polyoxyethylene ether detergent W-1 could solubilize the enzyme facilitated further purification of the enzyme (Beaulieu et al., 1994). The enzyme was characterized from the true pathogen A. fumigatus and purified from the closely related genetic model organism A. nidulans (Kelly et al., 1996). The reaction was inhibited by pneumocandins in a time- and dose-dependent fashion. As with the kinetics for the Candida enzyme, inhibition was noncompetitive with substrate. A similar noncompetitive inhibition of the C. albicans and Aspergillus enzymes was found for cilofungin, a semisynthetic echinocandin B with a lipid side-chain modification (Beaulieu et al., 1994). For A. fumigatus, the IC50’s were 50 nM for pneumocandin B0 and 8 nM for L-733,560, potent enough to account for the growth inhibition demonstrated by these compounds (Kelly et al., 1996). Although there is a rough correlation of inhibition of glucan synthesis with in vivo activity and the morphological effect concentration (MEC), there are enough additional pharmacokinetic parameters that also affect in vivo efficacy that this measure was not relied on to evaluate compounds (Beaulieu et al., 1993; Kurtz et al., 1994). Whereas most compounds with good in vivo activity had good MECs and inhibited the in vitro enzyme, the glucan synthase IC50 could

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not provide an accurate ranking of compounds. Therefore, all potentially interesting compounds were tested in vivo for Aspergillus activity. The A. nidulans enzyme was characterized in detail because it offered the opportunity to clone the gene(s) encoding the catalytic subunit of glucan synthase and to disrupt the gene to test for function. The morphological effect of treatment was identical, producing growth-inhibited hyphae that were short and stubby with distended tips. The concentrations needed to produce the morphological effect (4.0 µg/ml or 3.8 nM/ml for pneumocandin B0) and inhibition of the glucan synthase assay (2.0 µg/ml or 1.7 nM/ml) were the same as those required to produce these effects in the strain used for the mouse survival model (Kelly et al., 1996). The enzyme was purified about 300-fold by product entrapment and found to be inhibited by echinocandins at a concentration equal to that which inhibited growth of whole cells. The enzyme activity required a GTP-regulated component that was inactivated by C-3 toxin, presumably a functional equivalent of Rholp. A 20-kDa polypeptide was highly enriched in the partially purified enzyme and is a good candidate for the Aspergillus Rho1p. The A. nidulans FKS1 homologue (fksA) was cloned by crosshybridization with S. cerevisiae FKS1 (Kelly et al., 1996). The gene is 5716 nucleotides long and encodes a predicted protein of 229 kDa. It is highly conserved with respect to size, charge, amino acid sequence, and predicted membrane topology. The putative FksAp is 64% identical to S. cerevisiae Fks1p and has 79% similarity. The most highly conserved region is in the large cytoplasmic domain that is the same size as and 86% identical to the S. cerevisiae and C. albicans domains. Antibodies raised against a peptide derived from the FksAp sequence immunodepleted nearly all of the glucan synthase activity in crude or purified extracts and recognized an about 200-kDa band in Western blots of Aspergillus crude membrane preparations. No additional fksA homologues have been detected by cross-hybridization in Aspergillus. Recent attempts by Latge et al. to disrupt the A. fumigatus, fksA gene have repeatedly failed to isolate a strain without an intact fksA gene (J. P. Latge, personal communication). Thus, the fksA gene product plays an important role in glucan synthase activity and is most likely the essential target of echinocandins for this fungus. 5. Mechanisms of Resistance and Meaningful In Vitro Testing Mutant analysis in S. cerevisiae has helped in understanding the mode of action of the echinocandins and in defining the components of the enzyme in this organism and in other fungi. It also raised the issue of the significance of resistance for the clinic. A careful study of mutants in Candida was therefore conducted (Douglas et al., 1997). The spontaneous

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mutants from a genetically marked strain of C. albicans were mapped by gene cloning and disruption experiments on the FKS1 homologue, just as for their S. cerevisiae counterparts. The restriction site polymorphism of the FKS1 locus was used to distinguish which allele of the resistant strain had been disrupted and thus identify the chromosome carrying the resistant allele. This study confirmed that for three of the four strains analyzed, only one of the alleles had been mutated, and the mutation must therefore be dominant or semidominant. The fourth strain may have undergone a gene conversion event to produce two resistant alleles. The amino acid changes that confer resistance to echinocandin have been identified in several high-level resistant mutants of S. cerevisiae (Thompson et al., 1999). Almost all the mutations are in a highly conserved region of the 89 amino acid loop (Fig. 5). With this growing database defining a consensus “sensitive” loop, it was possible to examine the sequence of additional fungi, including those with intrinsic resistance to echinocandins (e.g., C. neoformans), because an amino acid sequence that would predict resistance (Fig. 6) shows the multiple sequence alignment of the region of known echinocandin-resistant mutants as well as those of the unique sequences of Aspergillus and Cryptococcus. The Cryptococcus sequence is identical for seven of eight amino acids in the consensus sequence. The only alteration, a change of Leu to Phe at position 6, is also found in A. fumigatus, which has a glucan synthase that is highly sensitive to lipopeptide inhibitors (Thompson et al., 1999). The lack of potency against the cryptococcal enzymes thus must be due to some other unidentified factor.

FIG. 6. Multiple alignment in region of known echinocandin mutations. Wild-type deviations from the consensus sequence are shown in bold type. The sequences of three known echinocandin-resistant mutations are listed at the bottom with the substituted residues underlined. Reproduced with permission from Thompson et al. (1999).

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The other confirmed component of the glucan synthase complex is Rho1p. Given our current understanding of the dual role of this GTPase, it is not entirely surprising that thus far no mutants that have been isolated for resistance to echinocandins have been mapped to changes in this protein. However, overexpression of the rho1+ gene in S. pombe results in hypersensitivity to papulacandin (Arellano et al., 1996, 1997). Other drug resistance mechanisms that might confer cross-resistance to existing therapies have not been observed. Strains of S. cerevisiae that have defective alleles of PDR3 and PDR5, genes that specify proteins in the multidrug resistance ABC transport family and MDR regulator, respectively, are not highly susceptible to pneumocandins (M. B. Kurtz and S. Dreikorn, unpublished). More importantly, disruptions in the C. albicans CDR1, CDR2, and CaMDR1/BENr genes do not affect MK-0911 susceptibility (M. B. Kurtz and S. Dreikorn, unpublished). These genes encode proteins important for efflux of fluconazole and itraconazole. CDR1 and CDR2 are members of the ATP-binding cassette (ABC) drug efflux superfamily, while CaMDR1/BENr is in the class of major facilitators that have been shown to be responsible for the low level of accumulation of azole antifungal agents (Vanden Bossche et al., 1998; White, 1997; White et al., 1998; Prasad et al., 1995; Hernaez et al., 1998; Sanglard et al., 1995, 1996). CDR1 and the major facilitator (gene, CaMDR1 (BEN), were shown to be overexpressed in resistant C. albicans isolates. MK-0911 is just as potent against a panel of fluconazole- and amphotericin B-resistant clinical isolates as against susceptible isolates (Marco et al., 1998; Vazquez et al., 1996, 1997; Nelson et al., 1997). An echinocandin B derivative LY303366 was also effective against similar drug-resistant strains, which suggests that the lipopeptide class of antifungals are not likely to be affected by resistance mechanisms common to marketed drugs (Marco et al., 1998; Pfaller et al., 1999; Karlowsky et al., 1997b). A serial transfer study was designed to search for resistant isolates in a setting that would more closely mimic clinical situations than the genetic mutant searches (Bartizal et al., 1997). In the experiment, a culture of C. albicans was treated with two-fold dilutions of drug as for a standard measurement of MIC. The cells from the highest concentration that did not inhibit growth were used as inoculum for the next MIC. After 40 serial transfers, the MIC increased no more than twofold. This increase is within the error of such measurements. Even with the assumption that resistance is a rare phenomenon, it is of critical importance that future studies determine (1) whether the observed increase in MIC in vitro correlates with clinical outcome and (2) whether there is cross-resistance to other clinically relevant therapies. Although definitive conclusions can only be made with sufficient clinical experience, a few studies with the spontaneous mutants have

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provided some interesting observations (Kurtz et al., 1996). First, all three spontaneously resistant mutants of the marked C. albicans strain were just as susceptible as the parent strain to fluconazole, amphotericin B, and itraconazole, again demonstrating the specificity of the mutations needed for resistance to this novel agent. Second, there is a general trend, but not a precise correlation between the ED90 for MK0911 and the MIC of these spontaneous mutants of C. albicans. All the strains required more MK-0911 to clear the infections in immunocompromised mice, but the increase was not as large as would be predicted by the increase in MIC, and the relative ranking could not be predicted by the in vitro test. For example, mutant CAI4R1 had an MIC that was more than 64 times as high as its parent strain but had an ED99 only 8 times as high to clear the infection in mice (Kurtz et al., 1995a). Although a variety of media and conditions were tested to obtain a better correlation, none gave a better prediction of in vivo outcome. As clinical data accumulate, we expect to better evaluate in vitro testing procedures for Candida strains. As indicated in Section VA, the MEC for Aspergillus strains is that concentration of drug that produces characteristic morphological changes and a significant reduction in growth. This correlates well with MIC determined by the NCCLS M-38 protocol in RPMI for over 80 strains if the MIC is defined as the concentration required to reduce growth (either visually or spectrophotometrically) by 50 or 80% as compared with the untreated control (Arikan et al., 1999). This partial inhibition definition is analogous to the partial inhibition definition shown relevant for susceptibility testing of the azole antifungal agents (Rex et al., 1993, 1997). This cutoff point is also the same as the concentration at which the pH indicator Alamar Blue is affected, thus further simplifying readout (Flattery, unpublished). There was little difference in the MIC value when either antibiotic medium 3 (AM3) with 2% glucose or RPMI with 2% glucose was used in place of standard RPMI medium, which contains 0.2% glucose (Arikan et al., 1999). The relative lack of medium effects contrasts with the significant impact a rich medium has on MIC for Candida. Using AM3, for example, can lower the MIC 10- to 100-fold for echinocandins as compared with a defined medium such as YNBG or RPMI (Nelson et al., 1997). One explanation for this phenomenon is that the faster growth of Candida on rich medium increases susceptibility to lysis by cells with fragile and defective cell walls. On the other hand, aeration rather than medium composition may be the limiting factor for growth of Aspergillus on microtiter plates. Another alternative to liquid microtiter protocols could be the E-test. This is a commercial test strip with a calibrated gradient of drug that can diffuse

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onto a seeded agar plate, which is used for antibacterial susceptibility testing and is being evaluated for various antifungals (Linares et al., 1998; Lozano-Chiu et al., 1998; Szekely et al., 1999; Clancy and Nguyen, 1999; Sewell et al., 1994; Colombo et al., 1995). Since the zone of inhibition is directly related to the concentration of drug on the strip, the intersection of the tear drop–shaped zone with the test strip is read as the MIC. If the physical properties of MK-0911 are compatible with the proprietary solid phase of the E-test, this may prove to be another valid method of susceptibility testing. VI. CURRENT COMPOUNDS IN CLINICAL DEVELOPMENT Three echinocandin derivatives are now in clinical development. These include MK-0911 (formerly L-743,872 and now named caspofungin), LY303366, and FK463 (Fig. 2). A. MK-0911 When the early work on the amino-containing compounds showed that they had significant spectrum improvements and intrinsic water solubility, additional analogues were screened for a variety of biological and chemical properties, and especially for once per day dosing. MK0911 (L-743,872, the heminaminal aza analogue of L-733,560, (Fig. 2) was found to have pharmacokinetic and safety advantages over other derivatives and was therefore selected as the clinical candidate. 1. In Vitro Activity During the search that led to the selection of MK-0911 as a clinical candidate, the chemically defined yeast medium YNBG (yeast nitrogen base with glucose) was used because it allowed good growth of all the fungal pathogens being studied and had few problems with trailing end points. More recently, the spectrum and potency of MK-0911 has been evaluated against a wide variety of clinical isolates of Candida spp. with use of the NCCLS M27 RPMI-based medium and methodology (Marco et al., 1998; Bartizal et al., 1997; Del Poeta et al., 1997; Krishnarao and Galgiani, 1997; Vazquez et al., 1996, 1997; Espinel-Ingroff, 1996, 1998; Fothergill et al., 1996; Pfaller et al., 1997a, 1998); Nelson et al., 1997). MK-0911 has MICs of 0.05 to 1.0 µg/ml for essentially all Candida isolates. This MIC range does not change for isolates that are intrinsically resistant to the azole antifungal agents (C. krusei and C. glabrata), as well as those C. albicans and C. glabrata strains that have acquired resistance

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(Nelson et al., 1997; Marco et al., 1998; Vazquez et al., 1997). Within this MIC range, there is a reproducible pattern of MIC by species that shows an overall rank order of C. albicans = C. tropicalis = C. glabrata ≤ C. parapsilosis ≤ C. krusei = C. lusitaniae (Marco et al., 1998; Vazquez et al., 1997). Recent clinical isolates demonstrate similar susceptibility as the defined type cultures (Pfaller et al., 1998; Rex et al., 1998a). MK-0911, like compounds in the echinocandin class, also has significantly lower MICs with AM3 against Candida species (Nelson et al., 1997). MK-0911 is fungicidal for C. albicans in RPMI (i.e., a 99% reduction in viability) within 6 to 8 hours at concentrations ranging from 0.06 to 0.25 µg/ml (0.25 to 2 times the MIC) (Bartizal et al., 1997a). A similar 99% reduction in viable colonies of C. tropicalis was observed with 2 to 4 hours at these same concentrations. Loss of viability can be partially prevented by providing an isotonic medium, such as 0.8 M sorbitol (Fig. 3). Although these 2-log reductions in CFU just miss the traditional 3log reduction used to defined microbicidal activity, detection of fungicidal activity of the echinocandins is likely heavily dependent on medium, strains, and methodology (Turner and Current, 1997; Klepser et al., 1998; Ernst et al., 1996, 1999; Klepser, 1999). The echinocandins are often able to completely clear infections of selected organs (Abruzzo et al., 1997), thus supporting the idea that they are indeed fungicidal for Candida spp. MK-0911 has an MIC90 of 32 µg/ml against C. neoformans (Franzot and Casadevall, 1997; Bartizal et al., 1997a). Of interest, however, is the observation that the combination of MK-0911 with either fluconazole or amphotericin B may be synergistic (Franzot and Casadevall, 1997; Bartizal et al., 1997a). Subinhibitory concentrations of MK-0911 reduced the apparent MIC of amphotericin B up to 16-fold, and the interaction was synergistic for all of 18 isolates in a recent survey (Franzot and Casadevall, 1997). The effect of the combination with fluconazole was less striking, with an up to fourfold reduction in MIC and synergy for 4 of the 18 tested isolates. These results are similar to the synergistic interaction previously noted between L-733,560 and amphotericin B for C. neoformans and A. fumigatus (Bartizal et al., 1995), as well as the indifferent or additive interactions noted for cilofungin in combination with several antifungal agents (Smith et al., 1991). A previous demonstration of in vivo synergy between cilofungin and amphotericin B murine models of invasive candidiasis (Hanson et al., 1991; Sugar et al., 1991) suggests that these observations may be more than in vitro artifacts. However, the effects of a combination with an echinocandin are not consistent. Cilofungin antagonized amphotericin B in normal CD-1 mice with disseminated aspergillosis (Denning and Stevens, 1991), MK-0911 and

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amphotericin B had an indifferent interaction in pancytopenic ICR mice with disseminated aspergillosis (Flattery et al., 1998), and neither fluconazole nor amphotericin B had a synergistic interaction with MK0911 in DBA/2N mice with cryptococcosis (Flattery et al., 1998). Thus, this area needs to be approached with care. MK-0911 also has potent activity against Aspergillus strains (MIC90 0.03 to 0.12 µg/ml) (Pfaller et al., 1998). The MIC (based on 80% reduction of growth from control) may be as much as 10-fold higher than the MEC for A. fumigatus but is similar to the MEC for other species (Arikan et al., 1999). The geometric mean MEC is 0.27–0.5 µg/ml and is the same for both RPMI and AM3 broth (Arikan et al., 1999). Isolates of Blastomyces dermatitidis, Histoplasma capsulatum, Acremonium strictum, Bipolaris spp., Cladophialophora bantiana, Curvularia lunata, Exophiala jeanselmei, Fonsecaea pedrosoi, Pseudallescheria boydii, and Scedosporium spp. had MK-0911 MICs of 0.125 to 8 µg/ml (Espinel-Ingroff, 1998; Del Poeta et al., 1997). Less consistent evidence of activity was seen with isolates of Sporothrix schenckii (1 to ≥16 µg/ml), Trichosporon beigelii (16 to >16 µg/ml), Fusarium spp. (≥16 µg/ml), Phialophora spp. (1 to 16 µg/ml), and Rhizopus arrhizus (> 16 µg/ml) (Del Poeta et al., 1997; Espinel-Ingroff, 1998; Chin et al., 1996). The significance of these results has not been determined in vivo. 2. In Vivo Activity In support of human trials, multiple animal models have been used to demonstrate the potent dose-dependent activity of MK-0911 in clearing C. albicans and A. fumigatus infections and prolonging survival in the face of lethal infections (Smith et al., 1996; Flattery et al., 1996; Abruzzo et al., 1997; Graybill et al., 1997a, 1997b; Bartizal et al., 1997b). In genetically immunodeficient mice (DBA/2N) and in immunocompetent outbred mice (CD-1), the ED50’s for prolongation of survival in a disseminated C. albicans model were 0.04 and 0.10 mg/kg per dose with twice daily dosing, respectively (Abruzzo et al., 1997). MK-0991 was also effective at low doses when therapy was delayed by as much as 24 hours after intravenous infection. This may be more relevant to the clinical situation, in which therapy is often initiated after infection has become well established. Similar results were seen for all species tested, including C. tropicalis, C. glabrata, C. lusitaniae, C. parapsilosis, and C. krusei, as well as for C. albicans in a pancytopenic mouse model (Bartizal et al., 1997b). These results have been confirmed by other workers (Graybill et al., 1997a, 1997b). In the most extensive study, the rank order for in vivo susceptibility in the DBA/2N mouse was C. albicans = C. tropicalis = C. glabrata ≤ C. lusitaniae ≤ C. parapsilosis ≤ C. krusei (Abruzzo et al., 1997).

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MK-0911 has been effective in both prevention and treatment of disseminated aspergillosis in mice (Bernard et al., 1996; Abruzzo et al., 1997; Bartizal et al., 1997b). When administered in twice daily doses of 0.02 mg/kg or more MK-0911 significantly prolonged the survival of DBA/2N mice, with estimated ED50 and ED90 of 0.03 and 0.12, respectively (Abruzzo et al., 1997). These values approximately doubled if treatment was initiated 24 hours after the infection was initiated. In steroid-treated rats, MK-0911 prolonged survival both when given as a single dose prior to infection and as therapy following of infection (Bernard et al., 1996). To evaluate the therapeutic potential of MK-0911 in situations that mimic the clinical syndromes more closely, the compound was tested in several animal models of more severe immunosuppression. These included a mouse survival model of disseminated candidiasis and aspergillosis in animals made neutropenic via cyclophosphamide treatment or via granulocyte depletion by administration of a monoclonal antibody specific for Gr-1 receptor on mouse granulocytes (Smith et al., 1996). In both instances, MK-911 showed activity comparable with that of amphotericin B in prolonging survival after 5 or 10 days of therapy. Immunosuppression was maintained throughout the experiment by repeated injections of cyclophosphamide and confirmed by differential blood counts. Antifungal treatments were initiated 24 hours after challenge and continued intraperitoneally daily for 14 days. Figure 7 shows a separate test in which the survival after dosing with MK-0911 was compared with that achieved with amphotericin B at doses titrated down from the equivalent to those given to humans in the clinical setting. The ED50 values for MK-0911 from days 14 to 28 ranged between 0.192 and 0.245 mg/kg per dose. These MK-0911 ED50 values were slightly lower than those obtained with amphotericin B (0.257 to 0.264 mg/kg per dose) and approximately sixfold lower than those for Amphotericin B Lipid Complex (ABLC) (1.225 to 1.438 mg/kg per dose). MK-0911 prolonged survival and reduced colony counts in spleen and liver at a dose as low as 0.05 mg/kg of body weight/day in immunocompetent Balb/c Nu/+ mice infected with H. capsulatum (Graybill et al., 1998). In their athymyic Balb/c nu/nu counterparts, MK-0911 was effective but clearly less potent. Doses of 5 mg/kg per day were required. Finally, MK-0911 was active in a murine model of P. carinii pneumonia (Powles et al., 1998). 3. Pharmacology MK-0911 has shown activity in animal models following intravenous, intraperitoneal, and oral administration. However, the oral route requires doses higher by as much as 300-fold than the parenteral routes

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FIG. 7. Efficacy of MK991 against a disseminated C. albicans infection. Mice were infected with 7.4 × 104 colony-forming units per mouse. Therapy was initiated within 30 minutes after challenge, and mice were treated intraperitoneally twice per day for 4 days (total of eight doses). Data are for five mice per time point. ■, 0.001 mg/kg; ▲, 0.02 mg/kg; ■, 0.005 mg/kg; ●, sham treatment; •, 0.09mg/kg; ▲, 0.375 mg/kg. Reproduced with permission from Abruzzo et al. (1997).

(Abruzzo et al., 1997), suggesting a bioavailability of 0.3 to 0.4%. Useful oral activity thus seems unlikely. MK-0911 is strongly bound to serum protein, with a free fraction of less than 4% (Hajdu et al., 1997). As is consistent with this, addition of 50% serum to in vitro assays reduces the antifungal activity approximately fourfold (Bartizal et al., 1997a). The pharmacokinetics of intravenous MK-0911 in healthy volunteers have been described (Stone et al., 1998). Following doses of 35 to 70 mg, dose-proportional increases in serum concentrations were seen. The beta half-life was 9 to 10 hours for all doses. With daily dosing at 70 mg/day for 2 weeks, the trough serum concentration rose from 1.4 to 2.7 µg/ml. This demonstrates some accumulation of MK-0911 on repeat dosing but also demonstrates that this dosing regimen produces serum concentrations that exceed the MIC of essentially all Candida and Aspergillus isolates during the entire dosing interval.

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4. Therapeutic Efficacy in Humans MK-0911 is the only echinocandin for which results of clinical trials of therapy for human mycoses have been reported. In a randomized, double-blind trial of esophageal candidiasis therapy in adults, 74 patients were treated with MK-0911 at 50 mg/day (46 patients) or 70 mg/day (28 patients), and 54 patients were treated with 0.5 mg/kg/day amphotericin B (Sable et al., 1997). Clinical response occurred in 84% of the MK0911–treated patients and 67% of the amphotericin B–treated group (Fig. 8, see color insert). MK-0911 was generally well tolerated and was not associated with any serious adverse events. A rise in creatinine to more than 2 mg/dl was seen in 15% of the amphotericin B–treated group and 1% of the MK-0911–treated patients. These results were confirmed in a second randomized, double-blind trial of therapy of esophageal and oropharyngeal candidiasis in adults, in which 95 evaluable patients received 35 to 70 mg/day MK-0911 (Arathoon et al., 1998). Higher doses of MK-0911 trended toward higher response rates, and MK0911 was better tolerated than amphotericin B. B. LY303366, a Lipid-Modified Echinocandin An alternative approach to improving the tolerability of the echinocandins was being pursued at Eli Lilly at the same time as the phosphate prodrug and MK-0911 were being developed at Merck. In view of their clinical experience with cilofungin, Lilly scientists focused on improving the tolerability and potency by different modifications of the lipid side chain. They learned that the structure and physical properties of the side chain, especially the lipophilicity and rigidity, were key for the antifungal potency (Debono et al., 1995). By incorporating additional aromatic rings into the side chain, they were able to improve potency and unexpectedly began to detect some oral efficacy. Optimization of the oral activity led to the identification of LY303366 (Fig. 2), a compound that is composed of the echinocandin B nucleus with a terphenyl head group with a C5 tail that had an oral ED50 of less than 10 mg/kg (Turner and Current, 1997). The oral bioavailability was 4.3% to 9% in dogs and less than 2% in rats, and thus the low doses required for in vivo efficacy represents a significant improvement in potency (Zornes et al., 1993; Zornes and Stratford, 1997). LY303366 has recently been licensed to Versicor (Fremont, CA) for development as a parenteral agent. 1. In Vitro Activity Like MK-0911, LY303366 is active against essentially all isolates of Candida (Marco et al., 1998; Krishnarao and Galgiani, 1997; Pfaller et al.,

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1997b; Uzun et al., 1997). The MICs for most isolates by the NCCLS M27 methodology are 0.06 to 2 µg/ml. The observed MIC is no different for either intrinsically azole-resistant Candida species or C. albicans strains that have become azole-resistant by mutation (Turner and Current, 1997). There are some differences in MIC by species. In one large survey, the MIC50 was 0.12 to 0.5 µg/ml for C. albicans, C. glabrata, C. tropicalis, C. krusei, and C. lusitaniae but 2 µg/ml for C. parapsilosis and 4 µg/ml for C. guilliermondii (Pfaller et al., 1997b). Testing in antibiotic medium 3 lowers the observed MIC approximately 30-fold (Pfaller et al., 1997b; Klepser et al., 1997). LY303366 produced a 3 log10 reduction in CFU of one of two isolates each of C. albicans and C. glabrata and thus met the traditional definition for having fungicidal activity (Ernst et al., 1996). In these same studies, LY303366 did not appear fungicidal for either of two tested isolates of C. tropicalis. Choice of medium and MIC end point may significantly improve the apparent in vivo activity of LY303366. Unlike MK-0911, LY303366 often produces partial inhibition over several twofold dilutions before producing a clear well (Ernst et al., 1996; Klepser et al., 1998). Most work with LY303366 has used the first clear well to define the end point, but Klepser et al., found that reading the MIC at the concentration that produced 80% growth reduction improved the correlation between time versus kill curves and minimum fungicidal concentrations (Klepser et al., 1998). Such behavior is well known from studies of fluconazole (Rex et al., 1998b), but use of a partial inhibition end point seems most rational for static drugs such as fluconazole, and further work is needed in this area. The effects of LY30366 are rapid. Candida albicans cells exposed to LY303366 for 5 minutes, washed, and then incubated for 3 hours showed the same percent reduction in CFUs as did a culture continuously exposed to the drug (Green et al., 1999). However, cells exposed for 5 minutes and then plated directly for CFUs were viable (Turner, 1997). Like MK-0911, LY303366 does not have significant activity against C. neoformans (Espinel-Ingroff, 1998; Zhanel et al., 1997). Combinations of LY30336 with other drugs have not been reported for C. neoformans but were generally indifferent for Candida (Karlowsky et al., 1997a). The MEC end point was used to determine susceptibility for Aspergillus spp. in AM3 and Casitone medium, both of which gave similar results, with a range from 0.0018 to over 0.5 µg/ml. At tenfold higher concentrations, LY303366 was fungicidal for 87% of species in AM3 medium (Oakley et al., 1998). The NCCLS RPMI-based test gave higher MICs, 0.03 to 0.12 µg/ml (Pfaller et al., 1998). In a pattern very similar to that seen for MK-0911, isolates of Histoplasma capsulatum, Bipolaris spp., Cladophialophora bantiana, Pseudallescheria boydii,

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and Scedosporium prolificans had LY303366 MICs of 1 to 8 µg/ml (EspinelIngroff, 1998). Less activity was seen with isolates of Blastomyces dermatitidis (2 to ≥ 16 µg/ml). Sporothrix schenckii (0.25 to > 16 µg/ml), Trichosporon beigelii (>16 µg/ml), Acremonium strictum (> 16 µg/ml), Fusarium spp. (> 16 µg/ml), Phialophora spp. (1 to >16 µg/ml), and Rhizopus arrhizus (> 16 µg/ml) (Zhanel et al., 1997; Uzun et al., 1997; Espinel-Ingroff, 1998). 2. In Vivo Activity The improved in vitro potency of LY303366 was correlated with efficacy in a mouse model of disseminated candidiasis (Turner and Current, 1997). There was a 2 log reduction in CFUs in the kidneys of animals treated with oral doses of the drug as compared with infected control mice. LY303366 has also been active in models of invasive candidiasis reported by others (Petraitis et al., 1998a), including a model of fluconazole-resistant esophageal candidiasis (Petraitiene et al., 1998). LY303366 was active in several animal models of aspergillosis (Petraitis et al., 1998b; Turner and Current, 1997; Verweij et al., 1998), including both a mouse model, which allowed conidial germination and hyphal growth in target organs before treatment was initiated (Turner and Current, 1997) and a severely neutropenic rabbit model (Petraitis et al., 1998b). The detailed data provided for the neutropenic rabbit model provide further insight into the relative activity of the echinocandins against Aspergillus. Although LY303366 prolonged survival and decreased overall pulmonary injury, there was no effect on number of CFUs of fungi recovered from the lung, and fungi were clearly visible in tissue sections (Petraitis et al., 1998). These data strongly suggest that LY303366 acts in a fungistatic fashion in this model. 3. Pharmacology Like MK-0911, LY303366 is extensively bound to serum proteins, and the addition of serum to in vitro assay systems increases the observed MIC four- to eightfold for Candida (Hoban et al., 1998). Following intravenous doses of 35 to 100 mg in humans, dose-proportional Cmax values of 1.8 to 3.8 mg/ml and areas under the curve of 37 to 104 µg/ml/h were observed (Rajman et al., 1997). The volume of distribution was 0.8 to 0.9 liters/kg, and the half-life was 39 to 46 hours. Oral dosing of LY303366 in humans has also been studied. Dose-proportional increases in Cmax and area under the curve were seen for oral doses of 100 to 500 mg in healthy volunteers (Lucas et al., 1996). Ingestion with a high-fat meal reduced Cmax and area under the curve by 75% (Lucas et al., 1996). Following a single 500-mg dose, a Cmax of 0.36 to 0.37 µg/ml and an area under the curve of 12.7 to 18.7 µg/ml/h was observed in a second study,

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which included both healthy and HIV-infected volunteers (Ni et al., 1998). These data suggest that oral LY303366 has a bioavailability of approximately 3%. Results of therapy trials in humans have not yet been reported for LY303366. C. FK463 FK463 is the most recent echinocandin to enter clinical trials (Fig. 2). Published data on it are limited, but the available information suggests that FK463 is very similar to MK-0911 and LY303366. For Candida spp., MICs are 0.0039 to 2 µg/ml, and for Aspergillus spp. they are 0.0039 to 0.0313 µg/ml (Maki et al., 1998). Using once daily dosing, FK463 improved survival in immunosuppressed mice infected with a variety of Candida spp., with an ED50 for C. albicans of 0.28 to 0.45 mg/kg (Matsunoto et al., 1998). Likewise, the ED50 of FK463 was 0.26 to 0.51 mg/kg in a neutropenic mouse model of invasive aspergillosis (Wakai et al., 1998). Protein binding is more than 99% (Suzuki et al., 1998). FK463 had doseproportional pharmacokinetics in humans at doses up to 50 mg (Azuma et al., 1998). The average half-life was 14.7 hours. Results of therapy trials in humans have not yet been reported for FK463. D. Related Agents Mulundoncandin, an echinocandin-like lipopeptide isolated from Aspergillus sydowii, was reported to have activity against Candida spp. but appears relatively inactive against Aspergillus spp. (Hawser et al., 1999). A-175800.0 is a cyclic hexapeptide, which has been reported to have activity in vivo against C. albicans with an ED50 of 3.62 mg/kg following a single dose and with overall activity comparable with that of amphotericin B on repeated dosing (Meulbroek et al., 1997). A group of lipopeptides, WF11899A, B, and C, were described in 1994 and reported to inhibit C. albicans at 0.004 to 0.03 µg/ml (Iwamoto et al., 1994a, 1994b). Chemists at Abbott explored the minimum structural versus functional requirements of the hexapeptide nucleus using solution-phase total synthesis. This approach circumvented the stability problems associated with preparing derivatives containing a hemiaminal. They found that many of the unusual amino acids could be replaced by more conventional ones. Aminoproline hexapeptides with increased water solubility and in vitro antifungal activity were among the most promising compounds (Klein and Li, 1999).

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VII. OUTLOOK The search for new treatment options for life-threatening disseminated fungal infections that have a novel, fungal-specific mode of action has been more difficult than expected. Long after the discovery of the first member of the echinocandin class, the semisynthetic lipopeptides are now entering the last stages of testing and development as therapeutic agents for human use. The novel, fungal-specific mode of action suggests that these compounds will not produce mechanism-based toxicity and can act on fungi that have intrinsic or acquired resistance to currently marketed drugs. Although a linear path from discovery to clinical application is never truly expected, the detours experienced in this journey illustrate the multiple influences that affect the drug discovery process, including changing clinical needs, enabling medicinal chemistry, and a better understanding of the molecular biology of human pathogens. And of course, there is also the powerful, but unreliable factor of serendipity, which can change the road traveled. We look forward to the development of new compounds as powerful new tools for treating important fungal infections. ACKNOWLEDGMENTS We wish to thank our colleagues Carole Sable, George Abruzzo, Milton Hammond, Jim Balkovec, and Ken Bartizal for all their help in preparing this chapter.

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AUTHOR INDEX

A

Akagi, T., 290 Akanuma, Y., 206, 207, 208 Akita, R. W., 418 Akiyama, T., 416 Akl, S., 108 Ala, P., 247 Alabaster, V. A., 21, 65 Alaoui-Jamali, M. A., 416 Alary, M., 404 Alaupovic, P., 108 Albanell, J., 419 Al-Barazanji, K. A., 211 Alberg, D. G., 288 Albers, 108 Alberti, D., 47, 53, 72 Alberts, A. W., 82, 108, 110 Alberts, J., 404 Albertsen, P., 177 Albini, A., 367 Albrecht, U., 363 Albus, U., 46, 48, 55, 71 Aldape, R. A., 286 Alden, C. L., 139 Alder, J. D., 471 Alderman, E. L., 110 Alderman, M. H., 51, 55, 58, 73, 74, 75 Aldigier, J. C., 44, 53, 71, 74 Aldrich, P. E., 246, 247 Alegre, C., 138 Alessio, P., 51, 73 Alexander, R. W., 114 Alexandre, J. M., 16, 39, 44, 63, 68, 71 Algren, H., 248 Alhenc-Gelas, F., 18, 19, 40, 41, 56, 61, 64, 69, 75 Ali, N., 359 Ali, S., 324, 357, 365 Ali, S. M., 286 Allegrini, J., 19, 40, 64, 69 Allen, A., 211 Allen, H. L., 110

Aaronson, S. A., 415, 416 Abadi, N., 418 Abbey, M., 364 Abbondanza, C., 360 Abbott, B. J., 468 Abel, P. D., 420 Ablan, F., 179 Abraham, E., 379, 380, 407, 408 Abrams, P., 154, 174, 178 Abrams, P. H., 174 Abrams, W. B., 30, 66 Abruzzo, G., 467, 470, 475 Abruzzo, G. K., 441, 444, 458, 459, 460, 461, 466, 467, 468, 469, 471, 474 Acchiardo, S. R., 22, 65 Acemoglu, F., 245 Acevedo, E., 139 Actor, P., 471 Adachi, M., 469 Adachishimizu, M., 471 Adam, M., 140 Adams, A., 206 Adams, D. F., 17, 20, 63 Adams, H. P. Jr., 109 Adams, M. R., 364 Adcock, S., 141 Adgey, J., 406 Admiraal, P. J. J., 42, 70 Agard, D. A., 366 Aghdasi, B., 290 Agosti, J. M., 408 Agostini, M., 207 Ahmann, D. L., 361 Ahmed, T., 176 Aikawa, M., 473 Aisen, P. S., 134, 140 Aitchison, R., 411 Aizawa, S., 208, 416 Ajayi, A. A., 42, 70 477

478

AUTHOR INDEX

Allen, K. E., 331, 361 Allred, C., 417 Allred, D. C., 365 Alpaugh, R. K., 420 Alper, S. L., 52, 73 Alt, F. W., 404 Altaras, M. M., 358 Alteri, E., 246 Altieri, D. C., 379, 407 Altman, D. G., 8, 12 Altuna, R., 137 Alvares, K., 203, 204 Alvarez, J. G. A., 206 Alvaro-Garcia, J. M., 411 Alzari, P. M., 250 Amant, C., 53, 74 Amara, I. A., 178 Amberson, J. B., 2, 11 Ambros, R. A., 418 Ambudkar, S. V., 248 Amidon, G. L., 30, 33, 66, 67 Amlot, P., 405, 414 Amlot, P. L., 375, 405 Amorosa, L. F., 111 Amouyel, P., 114, 207 Amplatz, K., 108 Amy, R. M., 366 Anagnostopoulos, A., 415 Anaissie, E. A., 473 Anaissie, E. J., 473 Andelinovic, S., 417 Ander, M. W., 245 Andersen, J. T., 174, 178 Anderson, C. F., 93, 111 Anderson, D., 404 Anderson, D. R., 413 Anderson, E., 359, 361 Anderson, H., 361 Anderson, J., 246, 473 Anderson, J. J., 359, 362 Anderson, K., 108 Anderson, K. C., 390, 413 Anderson, K. M., 145, 174, 175, 405, 406 Anderson, P. A., 248 Anderson, P. C., 248 Anderson, P. S., 246, 248, 249, 250 Anderson, R., 246 Anderson, R. E., 207 Anderson, R. J., 211 Anderson, R. M., 404 Anderson, S., 46, 53, 71, 72

Andersson, J., 408 Andersson, K. E., 175 Andersson, M., 297, 357 Andersson, S., 146, 174, 176 Andoh, T., 466 Andrade, J., 466 Andres, R., 207 Andrews, H., 367 Andrews, K. M., 208, 209 Andrews, M. J., 138 Andrews, W. C., 298, 357 Andriole, G., 177, 178 Andriole, G. L., 160, 168, 174, 176 Angel, J. B., 245 Angelin, B., 108, 113, 114 Angers, M., 16, 63 Anraku, Y., 470, 473 Ansetti, C., 376, 405 Antes, L., 137 Antman, E. M., 8, 12 Anton, E. D., 247 Antonaccio, M. J., 26, 65 Antoni, C., 409 Antony, I., 68 Antos, C. L., 289 Anzai, Y., 346, 357 Anzano, M. A., 316, 357 Anzick, S. L., 310, 323, 357 Aoki, M., 431, 466 Aoyagi, T., 27, 33, 66 Apaja-Sarkkinen, M., 363 Aperlo, C., 183, 204 Apostoloff, E., 412 Appelbaum, F., 413 Appelbaum, F. R., 413, 414 Appelt, K., 247, 249 Applegate, W. B., 109 Appleton, D. R., 420 Arad, Y., 89, 108 Arai, I., 138 Arai, M., 114 Aramburu, J., 285 Arathon, E., 473 Arathoon, A., 462, 466 Arbegast, P. T., 30, 66 Arboleda, J., 417 Archer, G. E., 421 Archibald, D. G., 49, 73 Arellano, M., 455, 466 Arend, W. P., 381, 382, 409 Argos, P., 360, 362

479

AUTHOR INDEX

Argueta, R., 364 Arikan, S., 441, 456, 459, 466, 474 Arisawa, M., 469, 470, 471, 473, 475 Aristoff, P. A., 250 Arizmendi, A., 473 Armanini, D., 177 Armayor, G. M., 33, 67 Armitage, J. M., 110 Armitage, P., 43, 70 Armitage, T. G., 161, 179 Armstead, R., 246 Armstrong, D., 467, 471 Armstrong, D. L., 290 Armstrong, P. W., 48, 72 Armstrong, R. L., 138 Arnadottir, M., 94, 108 Arnal, J. F., 40, 69 Arnaud, C., 359 Arnold, J. M. O., 113 Arnolda, L., 26, 66 Aronica, S. M., 323, 357 Arroyo, V., 137 Aruffo, A., 412 Arveiler, D., 40, 69, 114 Arvizu, C., 419 Asai, K., 289 Asano, K., 473 Asano, T., 287 Asgari, H., 420 Ash, M., 419, 421 Ashby, J., 317, 318, 357 Ashcroft, T., 420 Ashley, S. E., 362 Ashton, C. H., 96, 110 Ashwell, G., 470 Ashworth, A., 359 Assan, C. J., 37, 68 Asselin, J., 295, 319, 331, 357, 362, 363 Aster, S. D., 27, 28, 29, 32, 66 Athens, J. W., 361 Atkins, D., 107, 108 Atkins, J., 360 Atkinson, A. B., 27, 66 Atlante, G., 297, 357 Atlas, S. A., 38, 40, 68, 69 Atwal, K. S., 35, 67 Atwell, S., 281, 285 Auboeuf, D., 189, 207 Auden, J. A., 474 Audette-Arruda, J., 178 Audia, J. E., 176

Auerbach, S., 178 August, J. T., 245 Aum¸muller, G., 175 Aurell, M., 46, 71 Austin, D. R., 287 Auwerx, J., 204, 206, 207, 209, 210 Auwerx, L., 366 Auzan, C., 46, 53, 71, 74 Averna, M., 112 Awald, P., 433, 466 Axel, M. G., 246 Axler-Blin, C., 245 Ayala, J., 174 Azen, S. P., 108 Azizi, M., 41, 42, 44, 69, 70 Azorsa, D. O., 357 Azuma, J., 465, 467 Azzolina, B., 174, 175, 176

B Babaian, R. J., 418 Bacal, C., 362 Bach, A., 419 Bach, A. M., 360 Bach, M., 174, 178 Bach, M. E., 289 Bacheler, L. T., 245, 247, 248, 249 Bacher, J., 472 Bachinger, H. P., 362 Bachmann, K. A., 141 Bachvarov, D., 363 Backlund, E. P., 28, 30, 66 Bacon, J. S., 430, 467 Bacon, S., 91, 110 Bacon, S. P., 110, 111 Bacon, S. R., 110 Bacotti, D., 419 Bacus, S. S., 418 Badger, C. C., 414 Badia, M. C., 34, 67 Baer, L., 39, 50, 68, 73 Baert, F., 412 Bagchi, M. K., 361 Baginsky, W., 175, 176, 450, 451, 468, 471 Bagnoli, A., 179 Baguley, B., 433, 467 Bailey, P., 204 Bain, D. L., 361 Bain, R. P., 3, 12, 47, 53, 72 Bainbridge, A. D., 39, 68

480

AUTHOR INDEX

Bajamonde, A., 419 Baker, A., 360 Baker, A. C., 246 Baker, C. T., 247 Baker, D., 365 Baker, V. V., 417 Bakhle, Y. S., 20, 21, 65, 141 Bakker, J., 407 Bakshi, R. K., 172, 174, 175 Bal, E. T., 111 Balasubramanian, S., 89, 108 Baldwin, B. C., 470 Baldwin, E., 251 Baldwin, E. T., 250 Baldwin, H., 177 Baldwin, J. R., 245 Balk, R. A., 408 Balko, T., 469, 470 Balko, T. V., 470, 475 Balkovec, J., 450, 467 Balkovec, J. M., 431, 439, 466, 467, 473 Ball, A. J., 153, 174 Ball, S. G., 47, 72 Ballantyne, C. M., 94, 108 Ballard, J. H., 367 Baly, D., 419 Balzi, E., 473 Band, G., 363 Bander, N. H., 421 Bankhurst, A. D., 410 Bao, G., 17, 63 Bao, S., 290 Barahm, P., 55, 74 Barakat, R. R., 360 Barbagallo, C. M., 112 Barbir, M., 94, 108, 111 Barchiesi, F., 467 Barchiesi, F. J., 469 Barcos, M., 417 Barden, H. S., 364 Barder, H., 53, 74 Barger, A. C., 48, 72 Bargmann, C. I., 394, 415 Barlett, M. S., 473 Barnes, D. M., 415 Barnes, M. J., 419 Barnett, J., 140 Barnhart, M., 468 Barr, D., 246 Barr, L. A., 406 Barratt, P., 204

Barre-Sinoussi, F., 214, 245 Barret, D. M., 174, 178 Barrett-Connor, E., 299, 301, 351, 357 Barroso, I., 189, 207 Barry, A. L., 473 Barstad, D., 366 Bartal, A. D., 420 Bartelt, D. C., 20, 65 Barth, J. H., 164, 179 Bartizal, K., 437, 438, 455, 457, 458, 459, 460, 461, 466, 467, 468, 469, 470, 474, 475 Bartizal, K. F., 467 Bartlett, J., 246 Baselga, J., 397, 403, 419, 421 Baselga, J. M., 419 Bass, E. R., 2, 11 Bassetti, S., 241, 242, 245 Bast, R. C., 417 Basta, L., 50, 73 Bastien, L., 140 Bastion, Y., 415 Batchelor, K. W., 174 Bates, E. R., 377, 406 Bates, M. P., 413 Bath, R., 139 Battegay, M., 245 Battersby, L., 365 Battle, T., 40, 55, 69, 74 Bauer, S., 112 Baughman, S., 419 Baughman, S. A., 419 Baum, H. J., 360 Baum, M., 359 Baum, S. T., 208 Baumeister, K., 249 Baumelou, E., 138 Baumgartner, S. W., 404, 410 Bauriedl, G., 378, 406 Bauters, C., 53, 74 Bautier, P., 43, 70 Baxi, L., 361 Baylink, D. J., 361 Bayly, C., 137, 138, 139, 140, 141 Bayly, C. I., 118, 125, 137 Bayne, E. K., 146, 164, 174 Baynes, K. C., 318, 357 Bayram, F., 178 Bays, H., 113 Beals, C. R., 260, 285 Beamer, B. A., 189, 207

AUTHOR INDEX

Bean, J. S., 362 Bearn, A. G., 18, 64 Beato, M., 205, 304, 357 Beatson, G., 315, 357 Beaulieu, A., 139 Beaulieu, D., 452, 467, 474 Beauvais, A., 452, 467 Beck, K. D., 206 Beck, W., 245 Becker, J., 417 Becker, R. H., 60, 75 Beck-Nielsen, H., 211 Beck-Sague, C., 424, 467 Beere, P. A., 109 Beevers, D. G., 16, 63 BÈgaud, B., 138 Behn, C., 16, 63 Behnke, B., 180 Bei, M., 358 Bekele, A., 358 Belanger, A., 293–357, 359, 360, 362, 363, 364, 366 Belanger, P., 471 Belder, R., 113 Bell, A., 472 Bell, A. R., 204 Bell, D. R., 204 Bell, G. D., 114 Bell, J., 359 Bell, P. D., 413 Bell, R. C., 3, 12 Bell, S., 472 Bellet, M., 39, 43, 68, 70 Belley, M., 137, 139 Belloti, G., 177 Belshaw, P. J., 288 Bence-Bruckler, I., 413 Bendele, R., 358 Benetos, A., 17, 63 Benfield, P., 68 Benfield, T. L., 248 Benhamou, B., 360 Bennett, C. D., 248 Bennett, J. E., 424, 471 Bennett, M. K., 420 Bennett, P. C., 264, 285 Bensch, W. R., 358, 362 Benz, C. C., 395, 417, 419, 420 Berchuck, A., 395, 417 Berek, C., 370, 404 Berg, J., 246

481

Berg, K., 112 Berger, G. D., 205 Berger, J., 183, 185, 186, 187, 190, 192, 204, 205, 206, 208, 209, 210, 406 Berger, P. B., 53, 74 Bergeson, S. E., 289 Bergfeld, W., 177, 178 Berglund, L., 113, 114 Bergman, M., 114 Bergman, P. G., 14, 62 Bergner, D., 178 Bergsma, D. J., 177 Bergstrom, J. D., 82, 89, 108 Berinstein, N. L., 415 Berkenstam, A., 205 Berkoben, J., 49, 73 Berlin, J. A., 405 Berman, C., 174, 178 Berman, D. M., 174 Berman, R., 473 Berman, R. N., 208 Bernard, E. M., 460, 467, 471 Bernheim, J., 358 Berns, P. M. J. J., 420 Bernstein, E. A., 19, 64 Bernstein, K. E., 19, 64 Bernstein, L., 361 Bernstein, L. O., 414 Berrut, G., 68 Berry, D. A., 417 Berry, M., 304, 307, 323, 324, 329, 357, 360, 362 Berry, S. J., 151, 174 Bersi, C., 175 Bertel, O., 39, 68 Berthois, Y., 343, 357, 362 Bertilsson, G., 205 Beryt, M., 419 Besnard, P., 206 Best, S., 177 Betebenner, D., 247, 249 Betebenner, D. A., 249 Beyth, Y., 358 Bhardwaj, B., 362 Bhat, R. G., 472 Bhat, T. N., 250 Bhattacharyya, D. K., 123, 137 Bidwell, M. C., 361 Biemann, K., 20, 64 Bierer, B. E., 257, 258, 285, 287, 290, 291 Biernat, L., 417

482

AUTHOR INDEX

Bigner, D. D., 420, 421 Bigner, S. H., 403, 420, 421 Bijl, H., 409 Bild, D. E., 289 Biles, C., 250 Bilheimer, D. W., 82, 108, 111 Billaud, E., 56, 75 Bille, J., 473 Bills, G., 474 Bills, J., 55, 74 Binder, C., 410 Binkowitz, B., 176, 177 Biollaz, J., 30, 32, 33, 41, 66, 67, 69 Birch, C., 246 Birch, T., 55, 74 Birenhager, K. W. H., 18, 64 Birkenmeier, T. M., 141 Biron, P., 16, 63 Birtwell, J., 114 Bischofberger, N., 247 Bischoff, E. D., 206 Bisel, H. F., 361 Bissett, J. K., 108 Biswas, C., 204, 205, 209 Biswas, N., 211 Bito, H., 265, 271, 285 Bitterman, W., 176 Bjarnason, I., 123, 131, 137 Bjarnason, N. H., 359 Bjerkvig, R., 421 Bjorkhem, I., 113 Black, D., 110, 359, 360 Black, D. M., 3, 12, 112 Black, L. J., 316, 347, 351, 352, 358, 361 Black, R. M., 439, 467 Black, W. C., 126, 137, 139, 140 Blackshaw, S., 288 Blackwell, C. F., 22, 65 Blahey, O. M., 250 Blamey, R., 361 Blamey, R. W., 358, 359 Blanco, G., 363 Blankenhorn, D. H., 100, 101, 105, 108 Blaschke, T. F., 251 Bl‰ttler, W. A., 414 Blick, M., 420 Block, G., 109, 112 Block, G. A., 109 Blomley, M., 245 Bloss, J. D., 366 Bluhmki, E., 138, 139

Blumbach, J., 472 Blumberg, B., 204, 205 Blumenstein, B., 175 Blundell, T., 248 Blundell, T. L., 250 Boake, R., 176 Bocanegra, R., 469 Boccuzzi, S. J., 114 Bocquel, M. T., 360 Bodart, J. C., 53, 74 Boddaert, M., 112 Boden, D., 235, 242, 245 Bodkin, D. J., 413 Boehm, M. F., 206 Boekstegers, P., 379, 408 Boerma, G. J. M., 111 Boermann, O. C., 411 Boger, J., 39, 68 Bohanon, M. J., 250 Bohle, R. M., 53, 74 Bohn, D. L., 28, 30, 66 Boikov, D., 474 Boily, M. C., 370, 404 Boiziau, J., 247 Bokelman, D. L., 110 Bold, G., 231, 234, 245, 246 Bolden, S., 177, 363 Bolli, P., 39, 68 Bolognese, J., 137, 139, 141 Bolognese, J. A., 43, 70, 110, 114, 139 Bondjers, G., 114 Bone, R. C., 379, 407, 408 Bonfrer, J. M., 358 Bonilla, J., 178 Bonn, T., 358 Bonnard, A., 175 Boomsma, F., 42, 70 Booth, R. N., 406 Borenstein, H. B., 15, 63 Borg, T. K., 286 Borge, M., 110 Borgonovi, M., 469 Borin, M. T., 226, 245 Borkowski, A., 175 Bornert, J. M., 357, 360, 365 Bornert, J.-M., 362 Borst, M. P., 395, 417 Bosch, J., 47, 72 Bosch, R. J., 179 Bosch-MarcÈ, M., 133, 137 Bosman, F. T., 47, 72

AUTHOR INDEX

Boss, E., 245 Bossert, N. L., 365 Bostedor, R. G., 108 Bostian, K. A., 472 Bottari, S. P., 47, 72 Botteri, F., 44, 71 Botting, R. M., 141 Bouchard, L., 394, 416 Boucher, C. A. B., 248, 249 Boucher, R., 16, 63 Boudouris, O., 297, 358 Bouffard, A., 467 Bouffard, A. F., 466 Bouffard, F. A., 440, 444, 467, 469, 473 Bouhour, J. B., 113 Boulay, R., 247 Boulton, A. J. M., 211 Boulukos, K. E., 204 Bourgon, L., 248 Bourguet, W., 368 Bourne, T. H., 362 Boustead, C. M., 40, 69 Bowden, R. A., 474, 475 Bowen, B., 139 Bowen, D. L., 214, 245 Bowen, L., 190, 191, 208 Bowker, T. J., 100, 108 Bowler, J., 296, 302, 313, 336, 353, 367 Bowley, E. R., 358 Bowman, D. M., 360 Boyce, S., 138, 140 Boyd, A. W., 413 Boyd, J., 366 Boyer, F. E., 247 Boylan, C., 468, 475 Boyle, P., 159, 174, 178 Boyll, B., 475 Bozzette, S., 246 Braakman, T., 412 Bracken, B., 178 Bracken, B. R., 176 Bradford, R. H., 86, 92, 93, 95, 108 Bradwin, G., 208 Brady, S. F., 248 Braeckman, J., 173, 174, 175 Braissant, O., 183, 184, 204 Braithwaite, S. S., 209 Bram, R. J., 287 Bramson, H. N., 172, 174 Brancati, F. L., 54, 74 Branden, G. A., 406

Brandt, K., 469, 470 Brandt, M., 177 Brannon, E. S., 48, 72 Brant, K. D., 467 Brassard, J. A., 139 Brater, D. C., 141 Braunwald, E., 50, 73, 113 Bravo, E. L., 40, 69 Brawley, O., 176 Brawley, O. W., 175 Brecher, P., 48, 72 Breedveld, F., 137, 409 Breedveld, F. C., 409, 410, 411 Brekelmans, S., 473 Bremaud, J., 365 Brener, S., 378, 406 Brennan, F. M., 381, 382, 400, 409 Brennan, R. G., 362 Brenner, B. M., 46, 53, 54, 72, 74 Brenner, M., 46, 53, 71 Brenner, S., 168, 174 Brenton, R., 363 Bresnihan, B., 385, 411 Brewer, B. K., 110 Brewer, J. E., 245 Briand, P., 364 Briand, P. A., 358 Brideau, C., 118, 120, 137, 138, 140 Briesewitz, R., 253–285, 275, 283 Briggs, J. G., 16, 63 Briggs, M., 209, 210 Brill, C., 406 Brillantes, A. M., 288 Brillantes, A.-M. B., 262, 286 Brillantes, F. P., 361 Brind, J. L., 365 Brinton, L. A., 302, 358 Britt, P. M., 30, 66 Broadhurst, A. V., 249 Brockman, J. A., 194, 210 Brogan, D., 57, 75 Brogden, R. N., 68, 123, 138 Broijersen, A., 102, 108 Brolmann, H. A., 359 Bron, A. J., 110 Bronning, P. J., 53, 74 Brookhaven Protein Databank, 124 Brookmeyer, R., 362 Brooks, J. R., 149, 174, 177 Brooks, P., 119, 137 Brooks, S., 471, 473

483

484

AUTHOR INDEX

Brouillet, J., 16, 63 Browler, J., 367 Brown, C. C., 357 Brown, D., 56, 75 Brown, E. J. Jr., 50, 73 Brown, J., 416 Brown, J. J., 16, 17, 63 Brown, J. P., 413 Brown, K. A., 207 Brown, L., 113 Brown, M., 360 Brown, M. S., 89, 108, 109, 110, 111, 368 Brown, N. A. P., 110 Brown, N. H., 469 Brown, P. J., 206 Brown, W. V., 112 Browner, M, F., 140 Bruce, H., 359 Bruchovsky, N., 145, 174 Brun, R. P., 206 Brune, K., 141 Brunel, P., 39, 68, 123, 137 Bruneval, P., 45, 53, 71 Bruning, P. F., 351, 358 Brunner, D., 44, 71 Brunner, D. B., 30, 41, 66, 69 Brunner, H. R., 14, 20, 22, 26, 30, 32, 33, 37, 39, 41, 42, 44, 49, 50, 62, 64, 65, 66, 67, 68, 69, 70, 71, 73, 141 Brunner, N., 340, 343, 352, 353, 354, 358 Brunt, J. N. H., 114 Brun-Vezinet, F., 245 Bruschke, A. V. G., 111 Bruskewitz, R., 174, 177, 178 Bruskewitz, R. C., 176 Bryan, J., 177 Bryant, A. E., 408 Bryant, E., 317, 318, 358 Bryant, H. U., 358, 359, 360, 362, 366, 367 Bryant, J., 360, 417 Bryant, M., 234, 245 Bryant, M. L., 246, 250 Bryant, P., 247 Brzozowski, A. M., 306, 307, 309, 312, 358 Bubien, J. K., 390, 413 Buchanan, L., 246 Buchanan, R. B., 296, 358 Bucher, H. C., 107, 108 Buchwald, H., 97, 101, 108 Buchwalder-Csajka, C., 41, 69 Buckingham, R. E., 197, 208, 211

Buckle, D. R., 187, 206 Buckley, C. H., 298, 358 Buckner, A., 365 Buclin, T., 41, 69 Budavari, A. I., 203 Budde, K., 112 Budman, D. R., 417 Buechel, K. C., 141 Buechi, M., 113 B¸hler, F. R., 39, 68 Buhlmann, J. E., 387, 412 Bukasa, A., 141 Bull, H., 175 Bull, H. G., 32, 66 Bulovas, K., 417 Bulpitt, K. J., 410, 411 Bultpitt, C. S., 43, 70 Bumpus, F. M., 45, 46, 71 Bunch, R. T., 139 Bundred, N. J., 359 Bunjes, D., 415 Bunkenburg, B., 55, 74 Bunone, G., 323, 324, 328, 358 Buntinx, A., 114 Burakoff, S. J., 285, 287, 291 Burakov, D., 366 Burant, C. F., 192, 209 Burbaum, J. J., 289 Burchenal, J., 406 Burger, A. G., 205 Burger, P. C., 420 Burgess, J. A., 247 Burgess, J. M., 414 Burkhardt, F. J., 468 Burmester, G. R., 411 Burnett, P. E., 288, 290 Burnier, M., 30, 41, 66, 69, 141 Burns, C. M., 412 Burns, D. K., 207 Burrows, N., 411 Burton, J. A., 48, 72 Bush, S. A., 414 Bush, T. L., 299, 301, 357 Busse, R., 60, 75 Bustos, C., 46, 53, 72 Butchko, G., 414 Butchko, G. M., 414 Butler, T., 408, 475 Buurman, W., 407 Buzdar, A. U., 355, 358 Byers, L. D., 22, 27, 65

AUTHOR INDEX

Byington, R. P., 109 Byzova, T. V., 406

C Cabanillas, F., 413 Cabib, E., 433, 450, 467, 468, 470, 472, 474 Cabot, A. T., 151, 175 Cabot, C. F., 406 Cafferkey, R., 468 Caggiula, A. W., 364 Cagliola, A., 139 Cai, L. Q., 176 Cai, L.-Q., 177 Cakir, N., 410 Cala, K. M., 177 Calabrese, J., 246 Calandra, C., 205 Calandra, G. B., 473 Caldwell, J., 137, 410 Califf, R. M., 3, 12, 406 Callahan, L. F., 409 Callender, D. P., 472 Calogero, A. E., 175 Cambien, F., 40, 69 Cambou, B., 247 Cambou, J. P., 40, 69 Cameron, A. M., 262, 263, 286 Cameron, D. W., 237, 244, 245 Cameron, L., 364 Cameron, W., 246 Camilla, T., 53, 74 Campana, C., 113 Campanale, K. M., 247 Campbell, D. J., 42, 70 Campbell, G. S., 108 Campbell, I. D., 289 Campbell, S., 362 Campodonico, S., 112 Campos, C. T., 108 Candas, B., 293–357, 359, 362, 363 Candelario, M., 472 Candelore, M. R., 209 Cann, C. E., 359 Canney, P. A., 296, 315, 340, 343, 344, 354, 358 Cannon, A., 174, 178 Cannon, G., 129, 137 Cannon, J. G., 379, 407 Cant, J. R., 48, 72 Cantello, B. C. C., 198, 206, 211

Cao, G., 247 Capdeville, R., 414 Capizzi, T. P., 178 Caplan, R. H., 364 Capobianco, J., 469 Capobianco, J. O., 467 Capone, J. P., 204 Cappucio, F. P., 43, 71 Capraro, H. G., 245 Car, B. D., 138 Carabello, B. A., 289 Carballo, M., 271, 286 Carbin, L., 210 Carbone, P. P., 364 Carcangiu, M. L., 364 Carderelli, C. O., 248 Cardiff, R. D., 416 Cardona, G. R., 367 Carere, R. G., 52, 73 Carew, J., 288 Carey, R. M., 17, 64 Cark, S. J., 43, 70 Carlet, J., 379, 408 Carlin, J. R., 150, 175 Carlquist, M., 358 Carlsen, J. E., 43, 70 Carlson, G. F., 245 Carlson, R. W., 346, 358 Carlsson, A., 114 Carlsson, B., 362 Carner, K., 413 Carnina, E., 177 Caro, J. F., 206 Caron, B., 360 Caron, M. G., 357, 362 Carothers, L., 247 Carpenter, C. C. J., 239, 240, 245 Carper, B., 112 Carr, A., 245 Carr, D. W., 289 Carraro, J. C., 173, 175 Carretero, O. A., 15, 17, 53, 63, 74 Carrington, P. R., 179 Carroll, M. C., 369, 404 Carter, A. J., 53, 74 Carter, C. A., 248 Carter, D. C., 226, 247 Carter, I. R., 109 Carter, P., 396, 418, 420 Cartwright, T., 247

485

486 Carvalho, A. C. A., 102, 108 Carver, J. M., 364 Carver, J. R., 57, 75 Carver, M. E., 418 Casadevall, A., 458, 469 Casal, M., 471 Casarosa, C., 173, 175 Case, D. B., 22, 38, 40, 65, 68, 69 Casey, M. I., 179 Cash, T. F., 163, 175 Cashin-Hemphill, L., 108 Casper, K. A., 410 Cassidenti, D. I., 177 Castaigne, A., 48, 72 Castaigne, J. P., 405 Castaneda-Zuniga, W. R., 108 Castaner, J., 249 Castellanos, R., 178 Castelli, W. P., 77, 108 Castello, R., 170, 175, 177 Castro, B., 39, 68 Castro, C., 446, 448, 467 Catella-Lawson, F., 132, 137, 140 Catovsky, D., 415 Cattin, L., 109 Catzeflis, F., 205 Cauley, J. A., 359 Cauli, A., 411 Cauquil, J., 175 Caux, C., 387, 412 CavaillËs, V., 304, 358 Cawthorne, M. A., 206, 208, 211 Cedarholm, J. C., 114 Cedermark, B., 360 Celermajer, D. S., 113, 364 Cenedella, R. J., 96, 112 Cerami, A., 288 Cerpolini, E., 286 Cesano, L., 416 Cesario, R. M., 206 Cetinkaya, Y., 474 Chabon, A. B., 364 Chadalavada, V. S. R., 176 Chakravarti, D., 358 Chalbos, D., 296, 315, 358 Challah, M., 40, 53, 69 Chalmers, D., 56, 75, 412 Chalmers, I., 8, 12 Chalmers, J., 43, 71 Chalmers, J. P., 33, 57, 59, 67, 75 Chalmers, T. C., 108

AUTHOR INDEX

Chamaret, S., 245 Chambers, K. S., 413 Chambon, P., 205, 296, 357, 360, 361, 362, 365, 367, 368 Champagne, M. A., 110 Champagne, P., 362 Champault, G., 173, 175 Chan, B., 381, 408 Chan, C., 139, 468 Chan, C.-C., 119, 120, 137, 138, 139, 140, 141 Chan, K. S., 110 Chan, L., 366 Chandler, L., 108 Chang, A., 408 Chang, B., 174, 175 Chang, C. H., 249 Chang, C.-H., 246, 247, 248 Chang, C. P., 366 Chang, L., 180 Chang, N. T., 249 Chang, S., 246 Chang, T. W., 249 Chang, Y. C., 472 Chanmugan, P., 139 Chantry, D., 409 Chao, Y. S., 108, 114, 351, 358, 368 Chapman, J. W., 474 Charboneau, F., 110 Charbonneau, R., 363 Charles, P., 409 Charleson, S., 137, 138, 139, 140 Charpenet, G., 363 Charpentier, B., 375, 405 Chatelain, C., 175 Chatellier, G., 41, 42, 44, 46, 54, 55, 56, 69, 70, 71, 74, 75 Chatterjee, V. K. K., 207 Chauvet, M. T., 19, 41, 64, 69 Chavez, C. M., 179 Chavez, S., 357 Cheatham, W. W., 251 Chen, C.-M., 249 Chen, E., 245 Chen, G. C., 358, 468 Chen, H., 206, 323, 358 Chen, I.-W., 246, 250 Chen, J., 108, 203, 206, 286, 370, 404 Chen, J. C., 246 Chen, J. D., 312, 358, 363 Chen, L., 210, 289 Chen, L. B., 210

AUTHOR INDEX

Chen, M. S., 247 Chen, Q., 246 Chen, R. H., 431, 467 Chen, X., 249 Chen, X. N., 204 Chen, Y., 204, 206 Chen, Z., 105, 108, 235, 245 Cheng, H, F., 139 Cheng, L., 366 Cheng, Y., 358 Chenitz, W. R., 17, 20, 63 Chermann, J.-C., 245 Cherniak, R., 470 Chernyavskiy, T., 248 Cherrington, J. M., 247 Cheskin, L. J., 207 Chetrite, G., 302, 358 Cheung, A. H., 177 Cheung, H. S., 22, 23, 24, 25, 26, 27, 29, 65 Chevallier, J. C., 113 Chevillard, C., 39, 68 Chi, G. H. Y., 44, 71 Chida, N., 177 Chin, D. J., 109, 111 Chin, M. M., 111 Chin, N. X., 459, 467 Chin, W. W., 367 Chinn, P. C., 413 Chio, L. C., 469 Chipman, J. G., 141 Chirgadze, N. Y., 247 Chisholm, D. J., 208 Chisholm, G. D., 175 Chiu, S. H. L., 176 Chizzonite, R., 411 Chobanian, A. V., 48, 72 Chodakewitz, J., 473 Choi, J., 275, 276, 278, 286, 288, 358 Choi, W., 467 Choi, W.-J., 430, 467 Choi, W. S., 470 Chong, K. T., 249, 250 Chong, K.-T., 250 Chopin, D. K., 175 Choudat, L., 53, 74 Chow, J., 140 Chow, T. W., 407 Choy, E. H. S., 386, 410, 411 Chrebet, G., 472 Chremos, A. N., 108, 111, 114 Christen, A., 138

Christensen, M. S., 358 Christians, U., 112 Christiansen, C., 298, 358, 359 Christmas, T. J., 177 Christofalo, P., 175 Christophersen, B., 112 Chrivia, J. C., 362 Chromlish, W., 140 Chrysant, S. G., 33, 67 Chu, C., 409 Chulada, P. C., 139 Chute, C. G., 152, 175 Chylack, L. T. Jr., 96, 109 Cianci, A., 175 Ciccarone, T. M., 248 Cilla, D. D., 86, 109 Cimis, G., 175, 176, 179 Ciocca, D. R., 359, 365 Ciocca, R. G., 295, 358 Ciotta, L., 170, 175 Cipollone, F., 140 Cirillo, D., 179 Cirillo, V. J., 32, 33, 43, 67, 70 Cirrincione, C., 417 Cirrincione, C. T., 417 Clancy, C. J., 457, 467, 472 Clardy, J., 275, 286, 288, 291 Clare, M., 245, 246, 250 Claria, J., 137 Claridge, M., 153, 175 Clark, A. C., 363 Clark, D. A., 206 Clark, E. A., 413 Clark, G. M., 359, 416 Clark, J. I., 420 Clark, M., 404 Clark, M. G., 208 Clark, P., 414 Clark, R. Y., 176 Clark, S., 41, 69 Clarke, C. L., 299, 346, 358 Clarke, R., 358 Clarke, S. D., 203 Clarke-Pearson, D. L., 417 Clarysse, A., 344, 358 Claus, C., 359 Clausen, E., 19, 64 Clauser, E., 41, 69 Clawson, D. K., 247 Clawson, L., 248, 251 Clayton, T. C., 108

487

488 Clearfield, M., 109 Clemas, J. A., 450, 468, 471 Clemens, J. A., 361 Clement, J. J., 247, 467 Clemm, D. L., 212 Clemons, K. V., 469 Clipstone, N. A., 260, 285, 286, 291 Clouette, C., 248 Cloutier, J., 360 Clozel, J. P., 45, 53, 71, 73 Coats, A. J. S., 43, 70 Cobb, F., 49, 73 Cobb, J. E., 205, 206 Cobbe, S. M., 113 Cobbold, S. P., 411 Cobbs, C. G., 474 Cobby, M., 411 Cobleigh, M. A., 398, 419, 421 Cocanougher, B., 108 Cochet, C., 421 Cockett, A. T. K., 175 Codacovi, L., 247 Codina, J., 366 Cody, R., 48, 72 Cody, R. J., 40, 69 Coebergh, J. W., 359 Coen, M. L., 436, 467 Coffee, K., 37, 68 Coffey, D. S., 161, 174, 176 Coffield, S., 178 Coffin, J., 214, 245 Coffman, S., 472 Coghlan, V. M., 272, 286 Cohan, H., 58, 75 Cohen, A., 209 Cohen, C., 245, 246 Cohen, C. J., 298, 358 Cohen, E., 248 Cohen, E. A., 406 Cohen, H., 51, 73 Cohen, I., 297, 358 Cohen, J., 407, 408 Cohen, R. A., 141, 153 Cohen, R. M., 366 Cohn, J. N., 49, 73 Coiffier, B., 392, 414 Colajanni, E., 53, 74 Colca, J. R., 208, 209 Colditz, G. A., 299, 301, 358, 366 Coldman, R., 469 Cole, G., 179

AUTHOR INDEX

Cole, H. W., 360, 362 Cole, M. P., 296, 358 Cole, S. L., 250 Cole, T. G., 113 Coleman, D. C., 472 Coleman, T. G., 16, 63 Coll, J., 205 Coller, B. S., 376, 377, 405 Colley, C., 111 Collier, A. C., 237, 238, 245 Collier, J. G., 22, 41, 65, 69 Collins, J. H., 262, 286 Collins, R., 54, 56, 74, 75, 108, 110, 414 Collins, R. H., 414 Collins, R. J., 138 Collins, V. P., 421 Collins, W. P., 362 Colman, R. W., 108 Colombo, A. L., 457, 467 Colombo, F. M., 179 Colonno, M., 246 Colquhoun, E. Q., 208 Coltart, D. J., 114 Coltman, C. A., 175, 179 Colton, C. D., 248 Colucci, W. S., 49, 73 Compston, J. E., 318, 357 Conant, M., 248 Conde, M., 286 Condra, J. H., 235, 245, 250 Conneely, O. M., 366 Conneley, O. M., 366 Connell, B. L., 364 Connelly, P. W., 109 Conner, M. W., 209 Connolly, M. A., 287, 290 Connor, W. E., 109, 111 Connors, N. C., 439 Conolly, D. J. A., 411 Considine, R. V., 206 Constanzer, M. L., 179 Contel, N. R., 138 Contributors, N., 360 Conway, J., 43, 70 Cook, A., 177 Cook, D. J., 59, 75 Cook, T. J., 112, 174, 178 Coombs, D., 419 Coombs, R. W., 245 Cooper, C. R. Jr., 473 Cooper, J. A., 51, 73

AUTHOR INDEX

Cooper, W. D., 56, 75 Coopman, P., 340, 343, 353, 358 Cope, R., 110 Copeland, N. G., 204, 367 Coplan, K., 421 Copley-Merriman, C. R., 434, 467, 468 Copping, L. G., 468 Coquet, A., 363 Coral, F., 414 Corbett, I. P., 418 Cordes, E. H., 28, 32, 66 Cordes, M. H. J., 32, 66 Cordoba, M., 358 Cordon-Cardo, C., 418 Cordova, B., 245, 246, 249 Cormier, E. M., 343, 358 Cornu, Ph., 56, 75 Coronado, E., 417 Coronado, E. B., 365 Coronary Drug Project, 97, 109 Corr, L. A., 90, 109 Corrada, M., 362 Corran, A. J., 470 Correa, I. D., 206 Corsetti, L., 112 Corstens, F. H., 411 Corthesy, B., 287 Corvol, P., 18, 19, 39, 40, 41, 43, 46, 53, 56, 61, 64, 68, 69, 70, 71, 74, 75 Corvol, P. L., 40, 68 Coscio di Coscio, M., 175 Cosgrove, D. O., 362 Costa, A., 359 Costantino, C., 360 Costantino, J., 360 Costantino, J. P., 360 Costanzi, J. J., 361 Costerousse, O., 18, 40, 64, 69 Cottel, D., 207 Cotton, J., 19, 41, 64, 69 Couillard, S., 318, 335, 341, 342, 347, 353, 354, 359 Coukell, A., 36, 67 Coussens, L., 415 Covington, M. B., 138 Cowan, D. J., 206 Cowley, A. W., 16, 63 Cowley, A. W. Jr., 15, 63 Cox, S., 246 Coyle, P. J., 206, 208 Cozens, R., 245

489

Crabtree, G. R., 253–285, 286, 287, 290 Craescu, C. T., 283, 286 Craig, J. C., 249 Crane, L. R., 468 Crane, R., 137 Craven, T., 114 Crawford, E. D., 174 Crawford, S. M., 414 Crawford, T. D., 362 Creager, M. A., 51, 73 Crean, P. A., 52, 73 Creminon, C., 140 Criddle, R. S., 469 Crine, P., 62, 75 Crissman, J. D., 417 Crombie, D., 206 Crombie, D. L., 206 Cromlish, W., 138, 139, 140 Cromlish, W. A., 139 Cronin, W., 360 Cronin, W. M., 360 Cross, A. S., 380, 408 Croteau, G., 248 Crouch, T. H., 288 Crouse, J. R. 3rd., 88, 107, 109 Crowell, T. A., 247 Crowley, J., 175, 179 Crowley, V. E. F., 207 Croxtall, J. D., 346, 359, 361 Cryer, B., 128, 138 Cuddy, T. E., 50, 73 Culbertson, J., 178 Cullinan, A. B., 249 Cullinan, C. A., 204, 205 Cullinan, G. J., 358, 362 Culotta, V. C., 291 Culp, J. S., 248 Culp, S. A., 139 Cumming, R. G., 96, 109 Cummings, R., 205 Cummings, S., 179 Cummings, S. R., 316, 317, 347, 359, 360 Cundy, K. C., 247 Cuniasse, P., 19, 64 Cunningham, D., 414 Curfman, G., 49, 73 Curnie, W. J. C., 56, 75 Current, W., 474 Current, W. L., 431, 462, 463, 464, 468, 469, 475 Currie, S., 108

490

AUTHOR INDEX

Currie, V., 419 Curtis, M. G., 298, 359 Curtis, S., 359 Curtis, S. W., 361 Cusan, L., 357, 362, 363 Cushman, D. W., 14, 18, 20, 21, 22, 23, 24, 25, 26, 27, 29, 34, 35, 42, 45, 62, 64, 65, 67, 70 Cust, M. P., 367 Cutler, J., 54, 74 Cutler, J. A., 42, 60, 70 Cwudzinski, D., 109 Cyert, M. S., 469 Cylc, D., 467 Cyran, J., 33, 67 Czerniak, P. M., 138 Czerwinski, D. K., 413 Czuczman, M. S., 391, 392, 413, 414

D D, I. J., 468 Daar, E. S., 248 Da Col, P., 91, 109 Daemen, M. J. A. P., 47, 72 Daenen, W., 114 Daenzer, C. L., 245 Dagenais, G., 47, 72 Dagkessamanskaia, A., 471 Daha, M. R., 410, 411 Dahl, A. M., 468, 472 Dahl, K. J., 110 Dahlˆf, B., 46, 71 Dahod, S. K., 470 Dalen, J. E., 104, 109 Dalga, R. J., 250 Daling, J. R., 367 Dallaire, B., 414 Dallaire, B. K., 413, 414 Dallies, N., 471 Dallob, A., 138, 179 Dallob, A. L., 137, 164, 175 Dallongeville, J., 207 Dalton, W. S., 104, 109 Daly, M., 360 Dana, S., 207 Danchin, N., 53, 74 Daniels, B., 137, 141 Danilov, S., 40, 69 Danley, D. E., 248 Danne, S., 245

Danner, S. A., 236, 237, 245, 249 Dantis, L., 419 D’Aquila, R. T., 248 Darbre, P. D., 296, 343, 359 Dargie, H. J., 49, 73 Darke, P. L., 223, 245, 246, 248, 250 Das, M., 18, 64 Da Silva, F. C., 175 Dastidar, P. S., 366 Datta, S. K., 412 Dauca, M., 204 Dauda, G., 16, 63 Dauguet, C., 245 Daures, J. P., 54, 74 Dauter, Z., 358 Dauvois, S., 296, 331, 334, 335, 358, 359, 363, 366 Davey, M., 420 Davey Smith, G., 97, 109 Davi, G., 112 Davidson, M., 110, 113 Davidson, M. H., 111, 112, 113, 141 Davidson, N. E., 295, 359 Davidson, N. O., 209 Davie, A. P., 49, 73 Davies, D. I., 16, 63 Davies, D. R., 370, 404 Davies, J. F. 2nd., 247 Davies, M. J., 102, 109 Davies, P. J., 206 Davies, R., 47, 72 Davignon, J., 91, 109, 110 Davila, T., 441, 468 Davis, B. R., 42, 60, 70, 113 Davis, D., 409 Davis, I. M., 176 Davis, J. O., 16, 48, 63, 72 Davis, L. J., 248 Davis, L. S., 412 Davis, R., 36, 67, 138 Davis, R. A., 351, 359 Davis, T., 413 Davis, T. A., 390, 413 Dawber, R., 179 Dawber, R. P., 177 Dawber, R. P. R., 163, 169, 175 Dawson, T. M., 288, 290 Dax, S., 251 Day, A. J., 289 Day, M., 56, 75 Day, R., 137

AUTHOR INDEX

Dayer, J. M., 409, 410 Dean, D. C., 176 Dean, D. K., 206 Deangelo, D. J., 210 De Backer, G., 113 DeBoer, L. W. V., 108 deBold, A. J., 15, 63 Debono, M., 431, 434, 462, 468 Debouck, C., 248 DeCillis, A., 360 Decker, S. J., 415 DeCrescenzo, G., 245 DeCrescenzo, G. A., 246, 250 de Cupis, A., 353, 359 Deeb, S., 207, 209 Deeb, S. S., 189, 207 Deeks, S. G., 244, 245 deFaire, U., 46, 71 DeFillo-Ricart, M., 177 DeForrest, J. M., 34, 35, 42, 67, 70 DeFriend, D., 361 DeFriend, D. J., 296, 340, 343, 359 De-Fusco, D., 175 Degano, M., 287 De Geest, H., 114 Degoey, D., 471 DeGregorio, M., 365 DeGregorio, M. W., 365, 368 DeGrutola, V., 248 Deisseroth, K., 285 Dejneka, T., 34, 67 deJong, P. E., 54, 74 de Jong Bakkar, M., 358 de la Brousse, F. C., 289 Delacruz, J., 470 De La Iglesia, F. A., 210 Delaney, C., 36, 67 Delaney, K. M., 249 Delaney, M., 246 de la Pompa, J. L., 266, 286 De La Torre, D., 33, 67 de Launoit, Y., 296, 302, 359, 363 deLeeuw, P. W., 68 De Lepeleire, I., 138 De-Lepeleire, I., 179 de Lignieres, D., 365 Della-Latta, P., 467 Dellavalle, A., 53, 74 Delmas, P. D., 316, 318, 359 Delmer, D. P., 474 Delong, E. R., 178

491

de Lorenzo, D., 175 Delos, S., 173, 175 Del Poeta, M., 457, 459, 468 del Rey, F., 467 De Lucca, G. V., 219, 222, 245, 246 De Lucca, I., 246 de Luna, F. A., 29, 66 DeMayo, F., 287 DeMayo, F. J., 368 Demer, L., 210 DeMets, D. L., 9, 12, 364 deMetz, D. L., 57, 75 D’Emilia, J., 395, 417 Deming, Q. B., 48, 72 Deminie, C., 246 DeMora, J., 446, 468 DeMuylder, X., 365 den Broeder, A., 410 Denis, D., 137, 139 Denis, L., 175 Denissen, J. F., 247 Dennehy, P. H., 409 Denning, D. W., 424, 441, 458, 468, 472, 474 Dennis, M., 359 Dennis, S., 247 Densmore, V., 287 Denton, C., 419 Deo, Y., 419 Depre, M., 179 DePriest, P. D., 365 Dequeker, J., 122, 138 D’Ercole, K., 417 Dergham, S. T., 417 Derkx, B., 380, 387, 408, 412 Derkx, F. H. M., 42, 70 Derocq, D., 358 Desager, J. P., 110 Desai, K., 404 Desante, K., 471, 473 Deschaseaux, P., 175 DeschÍnes, D., 138 DeschÍnes, L., 360, 362 De Schepper, P., 138 De Schepper, P. J., 179 De-Schepper, P. J., 179 Descotes, J. L., 173, 175 Desikan, K. R., 473 Deslypene, J. P., 367 Desreumaux, P., 210 Desvergne, B., 205

492 Detmers, P. A., 210 Devanarayan, V., 245 Devaux, C., 16, 63 Devchand, P., 206 Devchand, P. R., 182, 203 Devenport, M., 367 Devereux, R., 46, 71 Devereux, R. B., 46, 51, 71 deVereWhite, R., 178 DeVillez, R., 177 DeVincenzo, J., 409 Devine, J., 209 De Vos, A. M, 285 Dewbury, K., 178 De Wergifosse, P., 473 DeWitt, D. L., 123, 138, 139, 140, 141 Dey, S., 248 deZeeuw, D., 54, 74 D’haens, G., 387, 412 Dhainaut, J. F., 407, 408 Diamond, P., 357, 362 Dianzumba, S., 56, 75 Diaquin, M., 467 Diaz, E., 209 Diaz, M., 473 Dibbs, Z., 379, 407 DiBianco, R., 57, 75 Dickson, C., 415 Dickson, R., 367 Dickson, R. B., 295, 307, 340, 359 DiDomenico, B., 469 Diehl, V., 414 Dietz, R., 125, 138 Di Fiore, P. P., 394, 416 Dignam, J., 360 Dijkema, R., 365 Dijkgraaf, G., 470 Dijkhuizen, F. P., 298, 359 Dillard, R., 245 Dilzer, S. C., 141 Di-Marco, S., 175 DiMasi, J. A., 7, 12 Dimitrov, N., 360 Dimitrov, N. V., 360 Dinchuck, J. E., 136, 138, 140 Ding, B., 267, 286 Dinh, J. T., 177 Dintaman, J. M., 247 Dipippo, V. A., 351, 352, 359 Di Silverio, F., 175 Dismukes, W. E., 474

AUTHOR INDEX

Distel, M., 138, 139, 140 Distler, A., 207 Ditschumeit, H., 140 Dive, V., 19, 41, 64, 69 Dixon, C., 468 Dixon, C. K., 446, 468 Dixon, D., 417 Dixon, F. K., 468 Dixon, R. A., 248 Dixon, R. A. F., 246, 248 Dobratz, D., 141 Dobrinska, M., 110 Dobs, A., 113 Dodds, R., 314, 359 Dodge, J. A., 295, 360 Dodge, R. K., 417 Dodwell, D. J., 361 Doebbeling, B. N., 467, 468 Doebber, T., 208 Doebber, T. W., 204, 205, 209 Doern, G., 472 Doherty, E. M., 250 Doherty, M., 133, 138, 411 Dolak, L. A., 249, 250 Dole, J. F., 211 Doll, R., 2, 11, 112 Dolphin, P. J., 366 Domagala, J., 250 Domagala, J. M., 247, 249, 250 Dombey, S. L., 56, 75 Domljan, Z., 411 Donnely, R., 42, 56, 70 Doran, E. R., 249 Dorer, F. E., 14, 62 Dorfman, R. I., 144, 175 Dorr, F. A., 365 Dorsey, B. D., 230, 231, 233, 246, 250 Dory, Y. L., 360 Dougados, M., 137 Douglas, C., 471 Douglas, C. M., 431, 446, 447, 448, 449, 450, 451, 452, 453, 468, 470, 471, 474 Douglas, C. M. A., 468 Douglas, R. C., 180 Doussau, M. P., 46, 71 Douwes, J. E., 470 Downs, J. R., 92, 97, 98, 99, 103, 104, 109 Downton, M., 108 Dowsett, M., 295, 359 Doyle, A. E., 18, 64 Doyle, M. J., 118, 123, 138

493

AUTHOR INDEX

Draelos, Z., 177 Drake, L., 179 Drake, R., 467 Draper, M., 359 Draper, M. W., 316, 318, 359 Drayer, J. I., 22, 65 Drebin, J. A., 415, 416 Dreher, M., 175 Dreikorn, K., 173, 175 Dreikorn, S., 455, 471 Dreser, H., 116, 138 Dreslinski, G. R., 26, 66 Dressman, B. A., 247 Drew, R. H., 469 Drexler, H., 40, 48, 69, 72 Dreyer, C., 183, 204 Drgon, T., 467, 468 Drgonova, J., 450, 467, 468, 470 Drigues, P., 365 Driver, C. L., 357 Drolet, G., 363 Drolet, Y., 362 Dromer, F., 474 Dropinski, J., 471 Dropinski, J. F., 466, 467, 469, 473 Dropski, J. F., 467 Drucker, M., 364 Drugge, R. J., 287 Du, W., 417 Duan, L., 365 DubÈ, D., 126, 138 Dubler, N. N., 248 DuBois, R. N., 134, 138, 210 Du Cailar, G., 54, 74 Ducas, J., 406 Duchin, K. L., 36, 67 Duell, P. B., 91, 109 Dueweke, T. J., 249 Dufloux, M. A., 46, 71 Dufour, J. M., 362, 363, 366 Dufour, M., 359 Dufrene, J., 362 Dugan, M. C., 395, 417 Duggan, M., 35, 67 Duggan, M. E., 34, 67 Dugue, A., 247 Dujovne, C., 108, 110, 113 Dujovne, C. A., 110, 111, 113 Dukes, M., 367 Dumont, F., 290 Dumont, F. J., 282, 286

Dumont, M., 363, 366 Dunbar, J. B. Jr., 250 Duncan, A. E., 29, 66 Duncan, I. B., 249 Dunlap, F., 177 Dunn, B. M., 251 Dunn, B. R., 46, 53, 72 Duong, T. T., 246 Dupont, A., 357 Durack, L. D., 414 Duran, A., 466, 467, 469, 473 Duran, G. E., 251 Durand, D. B., 286, 290 Durcan, M. J., 207 Durie, F. H., 387, 412 Durmusoglu, F., 175 Durocher, F., 363 Durrant, K. R., 358 Durrette, P., 290 Dusleag, J., 52, 73 Dussaule, J. C., 46, 53, 71, 74 Dworkin, L. D., 53, 54, 74 Dwyer, M. D., 247 Dyer, M. J., 394, 415 Dykoski, D., 418 Dzau, V. J., 45, 46, 47, 49, 51, 53, 71, 72, 73, 74

E Eardley, I., 177 Early, G. C., 387, 412 Eary, J. F., 414 Ebel, D. L., 179 Eber, B., 52, 73 Eberl, G., 409 Eberlein, K. A., 56, 75 Eckart, J., 407 Eckel, J., 209 Eckernas, S. A., 96, 109 Eckert, S., 359 Edgerton, S., 417 Edgington, T. S., 407 Edmiston, W. A., 108 Edmond, J., 108 Edmondson, R., 177 Edmonson, J. H., 361 Edwards, A., 110 Edwards, A. S., 19, 64 Edwards, C. K., 410 Edwards, F. F., 471

494

AUTHOR INDEX

Edwards, J. E., 469 Edwards, J. E. Jr., 475 Edwards, P. A., 108 Edwards, W. D., 53, 74 Egan, J., 202, 212 Egli, P., 26, 65 Ehlers, M. R., 20, 64 Ehrich, E., 137, 141 Ehrich, E. W., 128, 129, 137, 138 Ehrlich, P., 371, 404 Eicheler, W., 146, 164, 175 Eichenwald, K., 9, 12 Eiermann, W., 419 Einarsson, K., 113 Einfeld, D. A., 390, 413 Einstein, M., 174 Eisbruch, A., 403, 420 Eisenberg, D., 112 Eisenhauer, T., 112 Ekstrand, A. J., 403, 421 Elahi, D., 207 Elbrecht, A., 183, 186, 204, 205, 206 Elder, M. G., 359 Eldershaw, T. P. D., 191, 208 Eleftheriou, G., 175 El Esper, N., 53, 74 el-Harith, E. A., 138 Elhilali, M., 176 Elia, A. J., 286 Eling, T. E., 141 Eliseo, L., 414 Ellen, R. L., 91, 109 Elliott, H. L., 42, 54, 56, 70 Elliott, M., 410, 411 Elliott, M. J., 382, 383, 409 Ellis, L. F., 469 Ellis, S. G., 377, 406 Ellsworth, E. L., 247, 250 Ellsworth, K., 171, 174, 175, 176 Elo, O., 109 El-Sherbeini, M., 450, 468, 471 El-Shourbagy, T., 247, 248 Elson, D. F., 208 Emberton, M., 178 Emerson, T., 408 Emery, P., 137, 409, 411, 412 Emini, E. A., 246, 248, 250 Emmanouilides, C., 414 Emmel, E. A., 257, 259, 260, 264, 286, 289, 290 Emmett, E., 415

Emmrich, F., 411, 412 Enan, E., 275, 286 Encarnacion, C. A., 355, 359 Enderlin, C. S., 474 Endo, A., 81, 109, 111, 114 Endo, N., 204 Endo, S., 289 Endoh, H., 361 Endou, Y., 417 Eng, V. M., 138 Eng, W. K., 446, 449, 468 Engel, P., 413 Engel, S. L., 21, 45, 65, 71 Engeli, S., 207 Engert, A., 414 Englert, M., 33, 67 Engstrom, O., 358 Enmark, E., 362 Eppenberger, U., 363, 366 Epstein, C. L., 414 Epstein, E. S., 179 Epstein, J., 176 Epstein, J. I., 174 Epstein, S., 364 Erdjument-Bromage, H., 203, 288, 366 Erenus, M., 170, 175 Erickson, J., 216, 217, 246, 247 Erickson, J. W., 216, 221, 247, 249, 250, 251 Erickson Viitanen, S., 245 Erickson-Viitanen, S., 247, 248, 249 Erikson, R. L., 141 Eriksson, L. O., 108, 109 Eriksson, L.-O., 175 Eriksson, M., 108 Erkelens, D. W., 110 Erne, P., 39, 68 Ernst, E. J., 458, 468, 470 Ernst, M. E., 458, 463, 468, 470 Ernster, V. L., 360 Eschbach, M., 245 Esche, G. C., 364 Eschenbach, A. C., 418 Escobar, J., 112 Espeland, M. A., 109 Espinel-Ingroff, A., 457, 459, 463, 464, 468, 473 Estacio, R. O., 60, 75 Estes, J., 414 Esteva, F., 419 Estill, C., 469 Ethier, D., 138, 139, 140

AUTHOR INDEX

Eto, K., 208 Ettinger, B., 3, 12, 316, 347, 359, 360 Evans, A., 40, 69 Evans, G., 316, 359 Evans, G. L., 298, 314, 359 Evans, J., 137, 138, 140, 360 Evans, J. F., 139, 140 Evans, R. M., 203, 204, 206, 210, 312, 358 Everett, D. W., 36, 67 Everson, R., 361 Evin, G., 39, 68 Ewerth, S., 113 Ewing, L. L., 174 Exley, A. R., 379, 407 Eydelloth, R. S., 113 Eyermann, C. J., 247, 248 Ezaki, M., 470 Ezaki, O., 208 Ezan, E., 41, 44, 69

F Faergeman, O., 112, 113 Fago, A., 48, 72 Fair, J. M., 110 Fajas, L., 207 Fakata, K. L., 274, 286 Falcone, J. F., 361 Falgueyret, J. P., 138, 139, 140 Faller, A., 206 Faloia, E., 170, 175 Falsetti, L., 170, 175 Fan, Z., 403, 421 Fand, R., 26, 66 Fang, S., 145, 175 Fanger, M. W., 419 Farhat, M. Y., 299, 359 Farhy, R. D., 53, 74 Farmer, J. A., 86, 108, 109 Farmer, J. D. Jr., 288 Farmer, V. C., 467 Farndon, J. R., 420 Farquhar, J. W., 110 Farrar, W., 360 Farthing, C., 235, 245, 246 Fassas, A. S., 415 Fassler, A., 234, 245, 246 Faucette, L., 468 Fauci, A., 239, 240, 246 Fauci, A. S., 245 Faure, G., 175

495

Faust, J. R., 109 Fava, R. A., 412 Favez, T., 205 Favoni, R. E., 353, 359 Favre, A., 360 Faxon, D. P., 49, 51, 73 Fay, J., 414 Fay, J. W., 414 Fears, R., 81, 109 Fehlhaber, H. W., 472 Fehrenbacher, L., 419, 421 Feigl, P., 161, 175 Fein, A., 408 Feldman, E. B., 110 Feldman, H. I., 405 Feldman, M., 421 Feldman, M. I., 360 Feldmann, M., 409, 410, 412 Felson, D. T., 301, 359, 362 Fendl, K. C., 334, 359 Fendly, B., 418 Fendly, B. M., 395, 418 Feneley, R. C. L., 174, 178 Fenner, H., 137 Fenner, M. C., 414 Fenyk-Melody, J. E., 210 Ferber, F., 30, 66 Ferencic, Z., 417 Ferguson, D., 247, 249, 250 Ferguson, E. A., 250 Ferguson, J. J., 406 Ferguson, R. K., 26, 41, 65, 69 Ferrand, S., 358 Ferrari, P., 175 Ferreira, S. H., 20, 21, 37, 64, 65, 68, 117, 138 Ferres, H., 109 Ferriman, D., 170, 175 Feuring-Buske, M., 414 Feyzi, J., 364 Fiebach, N. H., 56, 75 Fiedler, V., 178, 179 Field, C. S., 299, 347, 359 Fields, C., 246 Fiel-Ridley, A., 472 Fierer, J., 408 Fiering, S., 289 Figari, I., 418 Figg, W. D., 414 Filipponi, S., 175 Findlay, J. A., 276, 287 Fine, B. D. Jr., 468

496

AUTHOR INDEX

Finkle, W. D., 346, 368 Finn, P., 49, 73 Finn, R. S., 419 Fino, L., 247 Finucane, F. F., 368 Finzel, B. C., 250 Firestein, G. S., 409 Firth, J. C., 111 Fischer, G., 256, 287 Fischer, H., 47, 72 Fischl, M. A., 234, 245, 246 Fish, R. D., 114 Fishbein, M. C., 210 Fisher, B., 3, 12, 298, 302, 314, 315, 344, 360, 417 Fisher, C., 141 Fisher, C. J., 379, 380, 381, 407, 408 Fisher, C. J. Jr., 408 Fisher, D., 360, 414 Fisher, D. R., 414 Fisher, E. R., 360, 417 Fisher, H. A., 418 Fisher, J. L., 419 Fisher, S., 414 Fishman, A., 358 Fisicaro, M., 109 Fitch, L. L., 108 Fitton, A., 68 FitzGerald, D. J., 415 FitzGerald, G. A., 137, 140 Fitzgerald, P. M. D., 233, 246, 248 Flack, J. M., 289 Flamigni, C., 179 Flamm, R. K., 470 Flanagan, J., 174 Flanagan, W. M., 259, 287 Flather, M., 50, 73 Flattery, A., 468, 470 Flattery, A. M., 456, 459, 466, 467, 468, 469, 471, 474 Fleischer, S., 288 Fleischmann, R. M., 404, 410 Fleisher, D., 30, 66 Fleming, T., 419 Fleming, T. R., 57, 75 Flentge, C. A., 247 Fletcher, B. S., 139 Fletcher, C., 210 Fletcher, D. S., 120, 138 Fletcher, W. S., 361 Flexner, C., 246

Flick, K. E., 277, 290 Flier, J. S., 204, 206 Flower, R. J., 117, 138 Flowers, D. E., 359 Fluit, A., 472 Flury, G., 56, 75 Focht, R. J., 138 Foekens, J. A., 420 Follansbee, S., 245, 246 Fonda, M., 109 Fong, N. T., 87, 114 Fong, Y., 379, 407 Fonseca, V., 201, 211 Foon, K. A., 413 Foor, F., 468, 471, 472 Forchielli, E., 144, 175 Ford, C. E., 54, 74 Ford, G. F., 175 Ford, I., 113 Ford, J., 114 Ford, L., 360 Ford, L. G., 365 Ford, R. A., 468 Ford-Hutchinson, A. W., 138, 140 Forman, B. M., 182, 186, 203, 206 Fornander, T., 297, 360, 366 Fornier, M., 400, 419 Forrest, M., 140, 209 Forrest, M. J., 138 Fortin, R., 138 Foster, J. R., 357 Foster, P., 246 Fothergill, A., 469 Fothergill, A. W., 457, 467, 469 Fouad, F. M., 40, 69 Foufelle, F., 204 Foulkes, M., 361 Fournier, A., 53, 74 Fournier, M., 363, 364 Fournie-Zaluski, M. C., 62, 75 Foutoulakis, J. M., 471 Fox, A. A., 68 Fox, C. S., 360 Fox, J. A., 418 Fox, K. M., 52, 73 Fox, R. I., 410 Foy, T. M., 412 Frampton, J. E., 68 Franciosa, M. D., 209 Francis, A. B., 365 Francis, D. A., 138, 139

497

AUTHOR INDEX

Francis, G., 49, 73 Francis, I. R., 414 Francke, U., 415 Francois, J., 471 Frandsen, T. L., 358 Frank, G. J., 68 Frank, G. R., 366 Franklin, F. A., 108 Frantz, M., 36, 67 Franzot, S. P., 458, 469 Fraser, R., 17, 47, 63, 72 Fratta, M., 175 Frattola, A., 43, 70 Fraumeni, J. F., 361 Fray, J. C. S., 40, 61, 69 Frazier, O. H., 108 Frederic, J., 359 Freed, M. I., 199, 201, 202, 211 Freedman, A. S., 413 Freedman, L. P., 310, 311, 360, 366 Freeman, R. H., 16, 63 Freis, E. D., 45, 71 French, M. E., 176 Frering, V., 207 Freskos, J. N., 250 Freslon, J. L., 46, 71 Fressinaud, P., 68 Frick, M. H., 97, 109 Friedland, M., 176 Friedman, B. I., 22, 65 Friedman, D. I., 30, 33, 67 Friedman, H. S., 420, 421 Friedman, J., 288 Friedman, L., 9, 12 Friehe, H., 68 Friend, J., 109 Friesen, M., 475 Friesen, R. W., 138 Fritsche, L., 112 Fritz, J. E., 247 Frizzell, R. A., 413 Frohlich, I., 110 Frohlich, J., 109 Frolik, C. A., 358 Fromell, G. J., 111 Fromm, M. F., 248 Frommer, F. R., 468 Fromtling, R., 474 Fromtling, R. A., 473 Frost, D., 466, 467 Frost, D. J., 436, 469, 470

Frost, D. N., 433, 436, 451, 469 Frost, P., 113 Frost, P. H., 110 Fruchart, J. C., 206 Fruchart, J.-C., 210 Fruman, D. A., 270, 287, 290, 291 Fruzzetti, F., 170, 175 Frye, S. V., 174 Fu, J. Y., 117, 138 Fu, Y., 469 Fujie, A., 469 Fujii, Y., 469 Fujimoto, N., 362 Fujimoto, W., 207 Fujioka, H., 473 Fujita, T., 208 Fujiwara, F., 474 Fujiwara, T., 190, 191, 208, 472 Fukuda, D. S., 468 Fukunaga, Y., 114 Fukushige, S., 394, 416 Fuller, G. N., 421 Funck-Brentano, C., 44, 71 Fung, E. T., 286 Fung, V. P., 404 Fung, W. C., 42, 70 Fuqua, S. A., 359, 368 Furberg, C. D., 49, 57, 73, 75, 101, 107, 109, 110 Furchgott, R. F., 15, 63 Furr, B. J., 296, 328, 344, 360 Furr, B. S. A., 178 Furrer, H., 245 Furst, D., 409 Furst, D. E., 385, 411 Furuichi, Y., 469, 470, 475 Furuta, T., 470 Furuta, Y., 416 Fusco, O., 140 Fuselier, H. A., 178 Fushida, S., 417 Fuster, V., 102, 109 Futaki, N., 120, 138 Fyhrquist, F., 46, 71

G Gabay, C., 384, 410 Gaber, A., 405 Gabriel, M., 178, 179 Gabriel, T., 176

498 Gaddipati, J., 161, 176 Gad el Mawla, N., 361 Gagne, C., 109, 110 Gailliot, P., 473 Gaito, G., 178 Gajda, C., 247, 250 Gal, A., 409 Galas, D. J., 10, 12 Galat, A., 282, 287 Galgiani, J. N., 457, 462, 470, 473 Gallagher, J. C., 361 Gallant, J. E., 235, 246 Gallati, H., 410 Gallion, H. H., 365 Gallis, H., 467 Gallis, H. A., 425, 469 Gallo, R. C., 214, 246, 249 Gallois, H., 68 Gallois, Y., 41, 48, 69 Gallwey, J. D., 170, 175 Galpin, S. A., 249 Galtier, F., 358 Gambrell, R. D. Jr., 295, 318, 360 Gambrell, R. J., 360 Ganci, A., 112, 140 Gangarosa, L. M., 421 Ganger, K. F., 367 Ganguli, B. N., 472, 473 Ganguly, K., 248 Ganten, D., 18, 47, 64, 72 Ganz, P., 110, 114 Gao, Q., 248 Garavito, M., 137 Garavito, R. M., 140 Garber, S., 245, 249 Garber, S. S., 247 Garbino, J., 408 Garcia, K. C., 281, 287 Garcia, M., 358 Garcia, T., 360 Garcia-Cozar, F., 285 Garcia de Palazzo, I., 420 Gardener, P., 285 Gardes, J., 39, 68 Gardin, J. M., 289 Gardner, K., 110 Gardner, S. F., 90, 109 Garg, R., 3, 12, 49, 73 Garini, G., 47, 53, 72 Garland, W. T., 36, 67 Garnes, D., 174

AUTHOR INDEX

Garrett-Engele, P., 446, 449, 469 Garrison, J. C., 36, 67 Garrison, L. M., 177 Garsky, V. M., 249 Garwood, J., 363 Gaudet, D., 110 Gauer, O. H., 16, 63 Gaul, S. L., 30, 66 Gauthier, J., 418 Gauthier, J. Y., 137, 138, 139, 140 Gauthier, S., 293–357, 360, 363, 364, 366 Gautier, T., 176 Gavazzi, A., 113 Gavras, H., 17, 22, 26, 30, 37, 41, 49, 51, 63, 65, 66, 68, 69, 73 Gavras, I., 17, 22, 37, 63, 65 Gavrilova, O., 207 Gaziano, J. M., 110 Gazzard, G., 239, 240, 246 Geale, E. G., 209 Geboes, K., 210, 412 Gee, J. M. W., 365 Gee, S., 472 Geerlof, J., 211 Geissler, L., 179 Geissler, L. A., 175, 179 Geissler, W. M., 108 Geisslinger, G., 141 Geisterfer, A. A. J., 47, 72 Gelfand, J. A., 407 Geller, J., 176, 177, 178 Gelmann, E. P., 298, 366 Gelmont, D., 408 Genant, H. K., 359, 361 Genazzani, A. R., 175 Gencheff, C. A., 179 Genereux, P. E., 206, 208, 209 Genest, J., 16, 63 Geng, C. S., 359, 363 Gengo, F., 96, 109 Gentzabein, E., 177 Geoghegan, K. F., 248 George, B. S., 406 George, E. B., 288 George, F. W., 177 George, N. J. R., 161, 176 Georgopapadakou, N. H., 431, 469 Gerber, G., 175 Gerineau, V., 41, 44, 69 Germain, R. N., 369, 404 Germershausen, J. I., 108, 113

AUTHOR INDEX

Gerritse, K., 387, 412 Gerritsen, M. E., 210 Gerson, R. J., 95, 110 Gertez, B. J., 138 Gertz, B., 138 Gertz, B. J., 137, 141, 179 Getman, D., 245 Getman, D. P., 231, 233, 246, 250 Ghanayem, B. I., 139 Ghannoum, M. A., 427, 469, 471, 473, 475 Gherardi, G., 47, 53, 72 Ghetie, V., 414 Gholem, M. H., 178 Ghosh, A. K., 230, 233, 234, 246 Ghrayeb, J., 249, 409, 410, 412 Giambiagi, N., 302, 360 Giammarresi, C., 112 Giardiello, F. M., 138 Giatras, I., 54, 74 Gibbons, G. H., 47, 72 Gibbs, E. M., 206, 208, 209 Gibbs, J. S. R., 52, 73 Gibelin, B., 178, 179 Gibson, D. M., 109 Gibson, G. J., 420 Gidding, S. S., 289 Gierse, J. K., 139 Giese, K. P., 290 Giess, C. S., 360 Giguere, V., 293–357, 363, 367 Gijon-Banos, J., 137 Gil, C., 469 Gil, G., 111 Gil, R., 468 Gilbert, D. J., 204, 367 Gilboa, S., 358 Gildehaus, D., 139 Gilewski, T., 419 Gill, C. J., 466, 467, 468, 469, 474 Gill, G. N., 403, 421 Gill, M. W., 177 Gilliland, K., 179 Gillis, C. R., 62, 75 Gilman, A., 27, 66 Gilman, A. G., 114 Gilna, P., 360 Giltinan, D., 174 Ginocchio, G., 40, 69 Ginsberg, H. N., 108 Giorgio, N. A., 421 Girard, Y., 138

499

Girardi, L. S., 3, 12 Giraudeau, B., 40, 53, 69 Girman, C. J., 175 Gittleman, M., 178 Giudicelli, J. F., 46, 71 Giuliacci, C., 247 Giuliano, F., 141 Givel, F., 204 Glack, W. D., 22, 65 Gladstone, P., 114 Glas, U., 360 Glasebrook, A. L., 362 Glaspy, J. A., 419 Glass, C. K., 205, 209, 361, 367, 368 Glasson, S., 46, 71 Glaus, C., 359 Glauser, M. P., 408 Gleason, C., 473 Gledhill, J., 359 Glenn, S., 414 Glenn, S. D., 414 Gloss, B., 361 Glusman, J. E., 359 Godfrey, J. D., 35, 67 Godin, M., 53, 74 Godolphin, W., 416 Goei, T. H., 122, 138 Goetze, S., 210 Goffeau, A., 473 Gofflo, D., 360 Gohlke, P., 17, 63, 64 Goklen, K. A., 473 Gold, B. G., 264, 287 Gold, B. I., 21, 65 Goldani, L. Z., 474 Goldberg, M. R., 44, 71 Goldberg, R., 93, 110 Goldberg, R. B., 113 Goldblatt, H., 17, 64 Goldenberg, D. M., 393, 415 Goldenberg, H., 415 Goldfard, B., 53, 74 Goldhirsch, A., 361 Goldmacher, V. S., 414 Goldman, M., 472 Goldman, M. E., 212 Goldman, R., 469 Goldman, R. C., 467, 469 Goldmann, B., 406 Goldstein, J. L., 89, 108, 109, 110, 111, 179, 368

500

AUTHOR INDEX

Goldstein, M. A., 287 Goldstein, N. I., 421 Gomes, P. J., 363 Gomez, H. J., 30, 32, 33, 41, 43, 66, 67, 69, 70 Gomez, J. L., 357, 362 Gomis, R., 200, 211 Gong, Y., 234, 246 Gong, Y.-F., 234, 246 Gonzales, M. F., 41, 69 Gonzalez, F. J., 203, 204 Goodman, L. S., 27, 66 Goodman, R. H., 362 Goodwin, F. T., 50, 73 Goodwin, R. G., 404 Gooley, T., 414 Gooley, T. A., 414 Goorno, W. E., 53, 74 Goosby, E., 246 Gordee, R. S., 431, 441, 468, 469 Gordin, F., 246 Gordon, D. J., 42, 60, 70, 106, 110 Gordon, H. S., 287 Gordon, I. R., 174 Gordon, L., 414 Gordon, L. I., 414 Gordon, R., 137, 138, 139, 140 Gordon, T., 54, 74, 346, 360 Gorman, C., 418 Gorman, C. M., 417, 418 Gorman, R. R. 3rd, 249 Gormley, C. J., 176 Gormley, G., 179 Gormley, G. J., 149, 150, 154, 160, 161, 174, 176, 177, 178 Gorry, S. A., 138 Gotlib, L., 250 Goto, T., 288, 471, 474 Goto, Y., 113 Gotoh, Y., 361 Gottardis, M. M., 296, 315, 340, 343, 344, 346, 353, 354, 360 Gottesdiener, K., 138 Gottesman, M. M., 248 Gottlinger, H. G., 215, 246 Gotto, A. M. Jr., 108, 109 Gotuzzo, E., 466, 473 Gould, A. L., 103, 106, 108, 110, 111, 174 Goulding, E. H., 139 Gown, A. M., 419 Grabowski, B., 249

Gracheck, S. J., 247, 250 Gradman, A. H., 33, 67 Grady, D., 299, 359, 360 Graf, J., 367 Graf, P., 42, 44, 70 Graham, A. M., 358 Graham, I., 113 Graham, N., 250 Graham, P. I., 250 Graham, R. M., 56, 75 Graham, S., 366 Granberry, M. C., 109 Grand, C. B., 210 Grand, L. C., 361 Grandien, K., 362 Grandits, G. A., 56, 75 Granger, P., 18, 64 Granneman, G. R., 247, 248, 249 Granneman, R., 245, 249 Grant, R. M., 245, 247 Grappel, S. F., 471 Grasing, K., 141 Gravallese, E. M., 289 Graves, R., 205 Graves, R. A., 203, 209 Gray, T., 178 Gray, T. J., 204 Graybill, J., 467 Graybill, J. R., 459, 460, 469 Graziano, R., 419 Greaves, W., 246 Greco, A., 140 Green, B. E., 247 Green, D., 112 Green, E. M., 361 Green, J., 250 Green, L. J., 463, 469 Green, L. L., 404 Green, M. E., 183, 204 Green, M. R., 362 Green, S., 204, 296, 303, 360, 362 Green, S. J., 361 Greenberg, A. S., 209 Greenberg, L., 367 Greendale, G. A., 299, 360 Greene, D. A., 181–203, 210 Greene, G., 204 Greene, G. L., 303, 358, 360, 366 Greene, L. J., 20, 21, 65 Greene, L. L. J., 37, 68 Greene, M. I., 416, 417

501

AUTHOR INDEX

Greene, M. L., 415 Greenlee, W. J., 27, 28, 29, 32, 33, 66, 227, 246 Greenman, R. L., 380, 408 Gregg, H., 176 Gregoire, S. L., 175 Gregorini, L., 51, 73 Greig, G., 140 Greig, G. M., 123, 138, 139 Grese, T. A., 295, 347, 360 Gresser, M., 115–137, 138, 140 Griffin, C. A., 207 Griffin, L., 247 Griffin, P., 206 Griffith, J. P., 275, 276, 287 Griffith, L. E., 108 Griffiths, K., 175 Griffiths, T., 358 Grillo-Lopez, A., 413 Grillo-Lopez, A. J., 413, 414 Grimminger, F., 407 Grobelny, D., 234, 246 Groel, J. T., 26, 66 Grognet, J. M., 41, 69 Groll, A. H., 424, 469, 472 Grollier, G., 53, 74 Gronemeyer, H., 296, 303, 360, 366, 367, 368 Groos, E. M., 470 Gross, D. M., 28, 30, 32, 66 Gross, F., 16, 39, 63 Gross, T. P., 112 Grossbard, M. L., 393, 414 Grossman, S. J., 113 Grouix, B., 411 Grubb, M. F., 247 Gruber, W., 409 Gruer, P., 93, 94, 110 Gruest, J., 245 Grundy, S. M., 108 Gruner, J., 467, 474 Grusby, M. J., 289 Grutter, M., 246 Gruver, C. L., 266, 287 Gu, K., 395, 418 Gualberto, A., 288 Guan, X. Y., 357 Guare, J. P., 246, 248 Guay, D., 137, 138, 139, 140 Guay, J., 138, 140 Guerin, D. M. A., 250

Guerrero, L., 176 Guess, H. A., 152, 153, 160, 168, 174, 175, 176 Guileyardo, J. M., 179 Guillet, J. L., 358 Gulick, R. M., 237, 239, 246, 247, 248 Gullick, W. J., 415 Gulnik, S., 247 Gulnik, S. V., 250, 251 Gumbs, C. P., 141 Guo, H., 287, 290 Guo, L. Y., 420 Guo, Q., 290 Gupta, R. A., 134, 138, 210 Gurbuz, O., 175 Gurland, B., 367 Gurnell, M., 207 Gurpide, E., 357, 363 Gusberg, S. B., 297, 360 Gustafsson, J. A., 303, 358, 360, 362, 365 Gutheil, J., 414 Guthrie, R., 36, 67 Gutman, M., 359 Guttendorf, R. J., 250 Gutterman, J., 420 Guvre, P. M., 419 Guvre, V., 419 Guy, C. T., 394, 416 Guyatt, G. H., 2, 11, 15, 59, 62, 75, 108 Guyene, T. T., 39, 41, 42, 43, 44, 53, 68, 69, 70, 74, 137 Guyton, A. C., 16, 63

H Haag, B., 405 Haagensen, D. E., 366 Haapa, K., 109 Haas, E., 17, 64 Haas, T. A., 376, 405 Haba, T., 111 Haber, E., 48, 72 Haber, H. E., 112 Haber, R. S., 208 Hackett, S. P., 408 Hadjadj, S., 41, 48, 69 Hafkin, B., 179 Hagari, S., 179 Hagen, S., 250 Hagen, S. E., 226, 247 Haggbald, J., 362

502 Haghfelt, T., 112 Hagmann, M., 41, 69, 310, 360 Haigh, D., 206, 211 Haigh, J., 112 Haigh, J. R., 109 Haines, A., 15, 62 Haioun, C., 414 Hajdu, R., 461, 469 Hajjar, L. R., 298, 360 Hakes, T. B., 362 Halachmi, S., 304, 360 Hald, T., 174, 178 Hale, G., 393, 411, 412, 415 Hall, A., 50, 73 Hall, D., 245 Hall, L., 468 Hall, P. W., 16, 63 Hall, R. R., 420 Hall, T. C., 415 Hall, W. D., 57, 75 Hallada, T. C., 471 Halloran, P. F., 286 Halperin, J. L., 51, 73 Ham, J., 368 Hamada, K., 178 Hamann, A., 204 Hamann, L. G., 206 Hamasaka, Y., 138 Hamberg, M., 117, 138 Hamdy, F. C., 175 Hamel, P., 139 Hamilton, G. S., 263, 264, 287, 290 Hamilton, H. W., 247, 250 Hamilton, J. B., 147, 176 Hamilton, L. C., 118, 123, 139 Hamilton, S. R., 420 Hamm, C. W., 378, 406 Hammer, S. M., 237, 238, 245, 247 Hammerstone, S., 34, 67 Hammond, M., 247, 467 Hammond, M. L., 467, 475 Han, H. P., 247 Hanauer, S. B., 412 Hancock, B., 140 Hand, E., 32, 33, 67 Handa, B. K., 249 Handschmacher, R. E., 255, 287 Hankinson, S. E., 358 Hann, L. E., 298, 360 Hanna, N., 413 Hannan, M. T., 359

AUTHOR INDEX

Hannedouche, T., 53, 74 Hanni, C., 205 Hansen, B. J., 108 Hansen, J. A., 405 Hanson, L. H., 441, 458, 469 Hansson, L., 14, 44, 46, 62, 71 Hansson, V., 363 Hanus, M., 175 Happle, R., 175 Haq, I. U., 55, 74 Harada, Y., 247 Hardell, L., 297, 360 Harding, G. A., 470 Harding, G. A. J., 475 Harding, M. W., 256, 287 Hardman, J. G., 36, 67, 114 Hardy, M., 365 Hardy, M. C., 404 Hardy, R., 413 Hare, R. S., 469 Harper, J., 178 Harper, K. D., 359 Harper, M. J., 346, 360 Harper, S., 139 Harper, S. E., 139 Harriman, G., 409 Harrington, M., 246 Harris, A. L., 420 Harris, E., 27, 28, 29, 32, 66, 108 Harris, E. F., 27, 33, 66 Harris, G., 146, 147, 149, 172, 174, 176, 179 Harris, G. S., 171, 174, 175, 176 Harris, J. R., 361 Harris, L., 421 Harris, M. L., 96, 110 Harris, R. C., 131, 139 Harris, S. T., 299, 301, 361 Harris, W. J., 404 Harris, W. S., 110 Harrison, L. H., 178 Harrison, R. W., 96, 110 Harrod, M. J., 179 Harry, K., 359 Hart, A. A., 358 Hart, D. M., 363 Hart, S., 139 Hartmann, A., 110 Hartwell, T., 109 Harvengt, C., 110 Harvey, C., 34, 67 Harvey, C. M., 42, 70

AUTHOR INDEX

Harwood, B., 247 Hasain, A., 45, 71 Hasegawa, G., 208 Haseltine, W., 249 Haseltine, W. A., 246 Hasenclever, H. F., 473 Hashimoto, M., 288 Hashimoto, S., 469, 470 Hashimoto, Y., 270, 287 Haskell, W. L., 101, 110 Hasse, A., 245 Hata, T., 474 Hata, Y., 113 Hatanaka, H., 288, 291 Hatano, K., 474 Hatch, F. E., 22, 65 Hatch, K. D., 417 Hatch, R., 170, 176 Hatch, S. D., 247 Hatcher, B., 471, 472, 473 Hatinoglou, S., 17, 63 Haubrich, R., 246 Haubrich, R. H., 475 Haudenschild, C. C., 48, 72 Hauri, D., 175 Havakawa, T., 191, 208 Havel, R. J., 82, 84, 110, 358, 368 Haves, N. S., 205 Havlier, D., 246, 247 Hawkey, C., 131, 138, 139 Hawkey, C. J., 137 Hawkins, A. L., 207 Hawrylik, S. J., 248 Hawser, S., 465, 469 Hayano, T., 290 Hayashi, M., 471 Hayashi, Y., 288 Hayden, M. R., 109 Hayes, D. F., 395, 417, 419 Hayes, N., 204, 209 Hayes, N. S., 204 Hayes, R. J., 8, 12 Hayllar, J., 137 Hayner-Buchan, A. M., 418 Haynetags, B. F., 246 Hayward, R. S. A., 2, 11 Hazelman, B. L., 411 He, J., 2, 12 He, W., 290 He, X. M., 226, 247 Headlee, D., 414

Health-Chiozzi, M. G., 245 Healy, B., 45, 71 Heath, I. B., 471 Heath, K. V., 247 Heath-Chiozzi, M., 246 Hebert, P., 56, 75 Hebert, P. R., 107, 110 Hecht, F. M., 242, 247 Heck, J. V., 467, 475 Heck, L. W. Jr., 410 Hecker, D., 177 Hector, R. F., 469 Hedges, J., 141 Hedlund, H., 174, 178 Heery, D. M., 184, 205 Heeschen, C., 406 Hefu, F., 53, 73 Hegele, R., 109 Heidler, S. A., 473 Heikas, J. E., 34, 67 Heimbach, J. C., 246, 248 Heimberg, M., 367, 368 Heine, M. J. S., 367 Heinonen, O. P., 109 Heinonen, T., 84, 88, 110 Heinsalmi, P., 109 Heinsohn, E. B., 365 Heintz, A. P., 359 Heintz, R., 245 Heintz, R. M., 246 Heinzel, T., 361 Heiss, C. E., 177 Hekman, A., 393, 414 Helbecque, N., 207 Held, J., 110 Helfrich, R., 246 Helftenbein, G., 204 Heller, D., 361 Hellmann, N. S., 245 Hellstrˆm, A. C., 360 Helmchen, U., 53, 74 Helo, P., 109 Hemingway, J., 473 Hemler, M., 117, 139 Hemmings, A., 248 Henderson, B. E., 295, 361 Henderson, I. C., 417, 419 Henderson, V. W., 301, 365 Heningway, J., 471 Hennekens, C., 358 Hennekens, C. H., 110, 366

503

504 Henner, D., 418 Henning, J., 246 Hennuyer, N., 206 Henry, G. C., 51, 73 Henry, J. P., 16, 63 Henry, K., 242, 247 Henry, R. R., 195, 196, 210 Hensens, O., 108 Hensens, O. D., 431, 469 Henttu, P. M., 329, 361 Henzel, W. J., 418 Herber, W. K., 246, 248 Herbst, J. J., 208 Herczeg, S. A., 26, 66 Herlitz, H., 46, 71 Herman, J. R., 195, 210 Hermann, D., 174 Hermann, D. J., 172, 176 Hermann, R. A., 247 Hermann, T., 212 Hermes, J. D., 205 Hernaez, M. L., 455, 469 Hernandez-Presa, M., 46, 53, 72 Herrera, C., 177 Herrero, E., 468 Herrlich, P., 205 Herrmann, R. R., 359 Herrmann, W. L., 366 Herschman, H. R., 139 Herzenberg, L. A., 285 Hesney, M., 108 Hess, O. M., 113 Hess, R., 182, 203 Heubsch, J., 247 Heudes, D., 45, 53, 55, 71, 74 Hewitt, K., 415 Heyderman, R., 408 Heyman, M. R., 413 Heyman, R., 210 Heyman, R. A., 206, 209, 361 Heyse, J. F., 110, 176 Hichens, M., 32, 33, 67 Hicklin, D. J., 421 Hicks, J. B., 286 Hiemenz, J. W., 425, 469 Hiepe, F., 386, 412 Hiesse-Provost, O., 41, 69 Higgins, J., 108 Higgins, K. M., 286 Higgins, M., 368 Higgins, P. M., 159, 176

AUTHOR INDEX

Higginson, L., 114 Higgs, 248 Higo, K., 209 Higuchi, S., 138 Hilfiker, A., 51, 73 Hilfiker-Kleiner, D., 51, 73 Hill, R. G., 138 Hills, M., 43, 70 Hilsenbeck, S. G., 365 Hindley, R. M., 206 Hinshaw, L., 408 Hinshaw, R. R., 249, 250 Hirano, H., 472 Hirano, K.-I., 209 Hirel, P. H., 216, 247, 250 Hirosumi, J., 177 Hiroyoshi, H., 210 Hiroyuki, K., 46, 53, 72 Hirsch, K. S., 171, 176 Hirsch, L. J., 108 Hirsch, M., 237, 246, 247 Hirschman, R. J., 364 Hirschmann, R., 27, 28, 29, 32, 66 Hirshfield, J., 108 Hirvatari, M., 26, 66 Hisamatsu, K., 365 Hitzenberger, G., 114 Hjemdahl, P., 108 Hjortland, M. C., 360 Hla, T., 117, 139 Ho, A. D., 413 Ho, B. K., 249 Ho, D., 245, 246, 249 Ho, D. D., 248, 249 Ho, K. K. L., 58, 75 Ho, K. L., 53, 74 Ho, S. N., 285, 289, 291 Ho, Y. K., 111 Hoagland, V. L., 474 Hoare, S., 205, 358 Hoban, D., 464, 469 Hoban, D. J., 470, 475 Hobart, P. M., 248 Hobbs, S., 172, 176 Hobson, H. L., 108 Hochberg, R. B., 363 Hockings, N., 42, 70 Hockwin, O., 113 Hodge, C. N., 218, 222, 247, 248 Hodge, M. R., 289 Hodges, C. V., 161, 176

AUTHOR INDEX

Hodgson, J., 102, 110, 114 Hodgson, S. F., 364 Hodis, H. N., 108 Hodsman, G. P., 26, 65 Hoen, H. M., 109 Hofbauer, K., 16, 63 Hoffman, C., 108 Hoffman, J. I., 266, 287 Hoffmann, R., 175 Hoffsommer, R. D., 27, 28, 29, 32, 66 Hofmann, C., 191, 193, 208, 209 Hogan, P. G., 285, 289 Hogg, R. S., 236, 247 Hohlfeld, T., 111 Hojo, M., 268, 287 Hokfelt, B., 22, 65 Holand, A., 208 Holdaas, H., 94, 110 Holden, S. A., 210 Holden-Wiltse, J., 246 Holder, J. C., 208 Hole, D. J., 62, 75 Holford, N. H. G., 42, 70 Holinka, C. F., 357 Holland, S. D., 474 Hollenberg, N. K., 17, 20, 49, 63, 73 Hollis, R. J., 472 Hollis, V. W., 177 Hollomon, D. W., 468 Holloway, M. K., 246, 250 Holman, G. D., 208 Holmes, D. R., 53, 74 Holmes, F., 358 Holmes, I. B., 114 Holmes, S. A., 177 Holmes, W. E., 396, 418 Holst-Hansen, C., 358 Holt, D. A., 177, 283, 287 Holt, J. A., 416 Holt, P., 137 Holtgrewe, H. L., 177 Holtz, J., 40, 69 Holtzman, M. J., 141 Homsy, E., 16, 63 Hong, C., 289 Hong, H., 323, 361 Hong, K., 420 Hong, Z., 450, 469 Honjo, T., 404 Honn, K. V., 139 Honselaar, A., 414

505

Hood, W. B. Jr., 51, 73 Hoogsteen, K., 108 Hoogstraten, B., 340, 343, 354, 361 Hoover, M., 137 Hoover, M. E., 139 Hoover, R., 346, 361 Hope, P., 48, 72 Hopf, R., 43, 70 Hopkins, R. J., 3, 12 Hopper, S., 110 Hordinsky, M., 177, 178, 179 Horikoshi, H., 208 Horiuchi, M., 47, 72 Horlein, A. J., 312, 361 Horne, C. H., 418 Horneff, G., 385, 411 Horng, M. M., 250 Horng, M.-M., 249, 250 Hornych, A., 137 Horowitz, J. A., 415 Horsmans, Y., 91, 110 Hort, Y., 360 Hortobagyi, G., 358, 419 Hortsmann, P. M., 144, 179 Horwitz, K. B., 295, 299, 304, 361 Hosang, M., 53, 73 Hosenpud, J., 112 Hosie, J., 122, 139 Hostetter, T. H., 53, 54, 74 Hotaling, T. E., 418 Hotamisligil, G. S., 192, 209 Houseman, K., 245 Houseman, K. A., 246, 250 Houser, V., 211, 212 Houston, A., 472 Houston, A. C., 248 Houtzager, V., 125, 139 Howald, H., 39, 68 Howard, G. M., 250 Howard, L. C., 468, 469 Howard, M., 286 Howe, B., 250 Howe, W. J., 249, 250 Howell, A., 296, 297, 315, 340, 343, 344, 354, 355, 358, 359, 361 Howie, C. A., 42, 70 Hricik, D. E., 53, 74 Hruby, V., 34, 67 Hsieh, L., 204 Hsu, A., 235, 245, 247, 248, 249 Hsu, I. N., 250

506

AUTHOR INDEX

Hsu, M. J., 472 Hsu, M.-J., 470 Hsu, S., 419 Hsueh, W. A., 210 Hu, E., 185, 203, 204, 205, 209 Hu, N., 416 Hu, Z., 406 Huang, J. T., 209 Huang, M., 287 Huang, Q., 204 Huang, W., 287 Hubbard, M. J., 270, 288 Hubbard, R. E., 358 Huber, P., 366 Hubert, C., 18, 19, 40, 64, 69 Hudis, C., 419 Hudis, C. A., 419 Hudson, P., 174 Hudson, P. B., 155, 176, 178 Hudziak, R. M., 394, 416, 418 Huebner, K., 469 Huff, J., 108 Huff, J. R., 213–245, 246, 248, 250 Huff, J. W., 113 Huff, M., 112 Hug, V., 358 Huggins, C., 161, 176, 331, 361 Huggins, E. M. Jr., 140 Hughes, B. J., 407 Hughes, D., 405 Hughes, M. D., 247 Huguenin, R., 289 Huinink, W. W., 414 Hulin, B., 186, 187, 206 Hulley, S. B., 104, 105, 112 Hulthen, L., 22, 65 Humblet, C., 250 Humphrey, P. A., 403, 420, 421 Humphrey, P. E., 467, 470 Hung, H. M. J., 44, 71 Hung, M. C., 415 Hung, M.-C., 418 Hung, S. H. Y., 290 Hunninghake, D., 110 Hunninghake, D. B., 89, 90, 109, 110, 111, 113 Hunsicker, L. C., 47, 53, 72 Hunsicker, L. G., 3, 12 Hunt, B. J., 111 Hunt, M., 410 Hunt, R., 131, 139

Hunt, T. L., 474 Hunt, V., 108 Hunter, D. J., 358 Hunter, D. W., 108 Hupe, D., 247, 249, 250 Hupe, L., 250 Hurley, A., 245, 248 Hurley, D. P., 108, 111 Hurlimann, T., 245 Hurwitz, E., 416 Huskey, S. W., 150, 176 Huster, W. J., 359 Hutchesson, A. C., 110 Hutchinson, H., 47, 72 Hutter, R., 406 Huttunen, J. K., 109 Hwang, D., 139 Hyams, D., 417 Hyslop, D. L., 467, 468

I Iacobelli, S., 140 Iacona, I., 113 Iba, Y., 373, 404 Ibrahim, A. S., 469 Ichikawa, I., 48, 72 Ichiki, T., 17, 64 Ichishima, E., 470, 475 Ide, T., 206 Ido, E., 216, 247 IehlÈ, C., 175 Ignar-Trowbridge, D. M., 307, 323, 361 Iguchi, Y., 471 Ikawa, Y., 416 Ikeda, F., 471, 474 Ikeda, H., 208 Ikeler, T. J., 27, 28, 29, 32, 66 Ikoma, M., 48, 72 Ikram, H., 14, 20, 62, 64 Iles, V., 179 Illingworth, D., 110 Illingworth, D. R., 77–107, 109, 110, 111, 112, 113 Imaizumi, T., 114 Imhloz, B., 43, 70 Imperato-McGinley, J., 145, 146, 147, 148, 162, 176, 177 Improvisi, I., 474 Inagami, T., 17, 64 Ingham, J., 108

AUTHOR INDEX

Ingle, J. N., 296, 361 Inglessis, N., 41, 69 Inostroza, J., 205, 367 Inoue, A., 15, 63 Inoue, S. B., 433, 446, 449, 469, 471, 473 Inoue, T., 288 Insuasty, J., 41, 42, 69, 70 Irvin, D. J., 56, 75 Irwin, M. W., 379, 407 Isaacs, J. D., 385, 386, 411, 412 Isaacs, J. T., 161, 176 Isaacsohn, J., 110 Isaacsohn, J. L., 111, 113 Isakson, P. C., 139 Isaksson, E., 368 Ischer, F., 473 Isenberg, D. A., 410 Isert, D., 469 Ishiguro, J., 446, 449, 469 Ishii, C., 208 Ishii, S., 209 Ishikawa, T., 15, 63 Ishimaru, T., 467 Isitt, V. L., 56, 75 Isles, C. G., 113 Ismail, S. M., 298, 361 Isom‰ki, H., 138 Isreal, L., 363 Issemann, I., 183, 204 Itabe, H., 114 Ito, S., 288 Ito, T., 114, 210 Itoh, H., 47, 72 Itoh, Y., 431, 470 Ivanhoe, R. J., 57, 75 Ivanoff, L., 249 Iwamoto, K., 208 Iwamoto, T., 431, 465, 469, 470 Iwasaki, H., 51, 73 Izon, D. O., 415 Izumo, S., 47, 52, 72, 73

J Jackson, A., 409 Jackson, C. L., 174 Jackson, D. A., 247, 248 Jackson, E. K., 36, 67 Jackson, J. B., 246 Jackson, M., 431, 470 Jackson, P. R., 55, 74

Jackson, R., 55, 74 Jackson, S., 210 Jackson, T. A., 312, 361 Jacob, B., 289 Jacob, S., 359 Jacobs, D., 367 Jacobs, T. W., 399, 419 Jacobsen, C. A., 174, 176 Jacobsen, S. J., 175 Jacobson, C., 179 Jacobson, C. A., 178 Jacobson, T. A., 91, 111 Jadhav, P. K., 222, 247, 248 Jaffe, E. S., 414 Jaffe, H., 418 Jaffee, B, D., 138 Jain, S., 205 Jain, V., 413, 414 Jaki, M., 205 Jakobovits, A., 373, 404 Jamali, M. A., 416 James, C. D., 421 James, E., 248 James, I., 114 James, M. K., 174 James, M. N. G., 250 James, P. G., 430, 470 Jamil, A., 346, 361 Janakiraman, M. N., 249, 250 Janakiraman, N., 413, 414 Janeway, C. A., 404 Janeway, C. A. Jr., 374, 405 Janiak, P., 40, 53, 69 Janin, G., 47, 53, 72 Jansanius, J. N., 289 Jansen, H., 111 Japour, A., 246, 247, 248 Japour, A. J., 245, 249 Jaramillo, J. J., 111 Jarman, M., 365 Jarvis, W. R., 424, 467 Jaskolski, M., 251 Jaspard, E., 18, 64 Jayanti, V., 249 Jayaraman, T., 262, 288 Jeang, K. T., 248 Jefcott, L. J., 206 Jeffers, B. W. M. R. H., 60, 75 Jeffrey, P., 250 Jendeberg, L., 205 Jeng, M. H., 360

507

508 Jenkins, A. B., 208 Jenkins, A. C., 26, 66 Jenkins, E. P., 174 Jenkins, J. A., 250 Jenkins, N. A., 204, 367 Jenkinson, M., 114 Jennette, J. C., 140 Jensen, D. M., 3, 12, 139 Jenster, G., 416 Jeunemaitre, X., 41, 69 Jevnikar, M. G., 361 Jeyakumar, M., 329, 361 Ji, C., 139 Jiang, C., 193, 209 Jiang, S. Y., 360 JimÈnez, W., 137 Jimenez-Linan, M., 204, 206 Jin, D., 45, 51, 71 Jin, J. R., 362 Jiracek, J., 19, 41, 64, 69 Johannesson, M., 57, 75 Johansen, P., 43, 70 Johanson, J., 139 Johansson, H., 366 Johansson, U., 366 John, M., 19, 64 John, V., 40, 69 Johne, A., 112 Johnson, B. L., 249 Johnson, D. L., 361 Johnson, E. M., 468, 474 Johnson, G., 49, 73, 110 Johnson, J. G., 22, 65 Johnson, M., 248 Johnson, P., 414 Johnson, P. D., 249, 250 Johnson, R. K., 468 Johnson, R. L., 108 Johnson, R. M., 417, 418 Johnston, C. I., 26, 48, 66, 72 Johnston, J. J., 138 Johnston, J. M., 411 Johnston, P., 418 Johnstone, I. M., 110 Jokela, H. A., 366 Jokubaitis, L. A., 111 Jolicoeur, P., 416 Jolivet, J., 362 Joly, S., 472 Jonas, A., 173, 176 Jonas, C., 414

AUTHOR INDEX

Jones, A., 133, 138 Jones, A. F., 110 Jones, C. D., 171, 176, 316, 361 Jones, C. T., 358 Jones, D., 210, 467 Jones, J. C., 37, 68 Jones, K. H., 30, 66, 114 Jones, L. A., 416 Jones, N. P., 211 Jones, P. H., 108, 109, 112 Jones, P. J., 86, 111 Jones, P. S., 183, 184, 204 Jones, R., 472 Jones, R. G., 470 Jones, R. L., 139 Jones, R. N., 471, 472 Jones, S. A., 206, 362 Jones, T. M., 141 Jordan, V. C., 296, 297, 298, 302, 314, 315, 328, 331, 334, 343, 344, 358, 359, 360, 361, 364, 367 Jorkasky, D. K., 211 Jose, R., 359 Josephs, S. F., 249 Joshua, H., 27, 28, 29, 32, 66, 108, 473 Jow, L., 206, 207 Ju, W. D., 474 Judd, H. L., 299, 359, 360, 364 Jue, C. K., 474 Juge-Aubry, C., 184, 205 Juillerat, L., 42, 44, 70 Jukema, J. W., 100, 105, 111 Julien, J., 46, 71 Julien, J. A., 250 Jund, R., 427, 470 Jung, G., 247 Jung, U. S., 470 Jung, W. E., 365 Junot, C., 41, 69 Jurriaans, S., 249 Just, H., 40, 69 Justice, S. J., 179 JustNuebling, G., 469 Jynn, J. C., 250

K Kaartinen, M., 51, 73 Kabakow, B., 364 Kabani, A., 469, 470, 475 Kabani, A. M., 470

AUTHOR INDEX

Kabbinavar, F., 419 Kaczmarek, D., 102, 111 Kadam, S., 470 Kadam, S. K., 467 Kadmon, D., 174 Kadowaki, H., 207 Kadowaki, T., 206, 207, 208 Kageyama, H., 467 Kahan, A., 138 Kahn, C. R., 207 Kahn, J., 213–245 Kahn, J. R., 14, 17, 18, 62, 63, 64 Kaibori, M., 269, 288 Kaiser, L., 251 Kaitaniemi, P., 109 Kakinuma, Y., 48, 72 Kakuk, T. J., 250 Kalabalikis, P., 408 Kalden, J., 409 Kalden, J. R., 409, 411 Kaldor, S., 249 Kaldor, S. W., 228, 229, 232, 247 Kalinteris, A., 175 Kalish, V., 249 Kalish, V. J., 247 Kalkhoven, E., 205, 361 Kallakury, B. V., 418 Kallen, J., 289 Kallioniemi, O. P., 357 Kamada, Y., 450, 470 Kamata, N., 415 Kamegis, J. N., 108 Kamei, Y., 205, 310, 323, 361, 367 Kaminski, M. S., 392, 393, 414 Kamishita, T., 291 Kamiyama, Y., 288 Kandel, E. R., 289, 291 Kang, J., 298, 361 Kang, M. S., 433, 467, 470, 474 Kang, S., 177 Kannel, W. B., 54, 58, 74, 75, 295, 360, 363 Kapadia, S., 379, 407 Kapitanovic, M., 417 Kapitanovic, S., 395, 417 Kaplan, L., 471 Kaplan, M., 246 Kaplan, S., 178 Kaplan, S. A., 178 Kaplin, A. I., 286 Kapoor, S., 137, 140 Kapteyn, J. C., 428, 470

509

Kaptyn, J. C., 470 Karam, H., 45, 53, 71 Karanewsky, D. S., 34, 35, 67 Kargman, S., 118, 137, 138, 139, 140 Kargman, S. L., 140 Kari, P. H., 113 Karim, A., 246 Karkas, J. D., 108 Karkhanis, Y., 473 Karlowsky, J., 469 Karlowsky, J. A., 463, 470, 475 Karon, J. M., 213, 247 Karp, J. E., 365 Karp, S. K., 361 Karplus, P. A., 291 Karwowski, J. P., 467, 470 Kasahara, S., 450, 470, 475 Kasprzyk, P. G., 395, 416 Kassimos, D., 410 Kastelein, J., 112 Kastner, P., 205 Kasuga, M., 207 Katakura, T., 207 Kataoka, T., 370, 404 Kathryn, K., 359 Kati, W., 249 Kati, W. M., 247 Kato, H., 20, 65, 114 Kato, S., 324, 328, 361 Katoh, K., 287 Katsikis, P., 409 Katz, D., 367 Katz, I., 177 Katz, M. D., 146, 177 Katz, T. K., 246 Katzenellenbogen, B. S., 296, 307, 323, 324, 328, 340, 343, 354, 357, 362, 364, 365, 367 Katzenellenbogen, J. A., 357 Kauffman, R. F., 316, 358, 362 Kaufman, K. D., 145, 162, 165, 166, 167, 168, 177 Kaufman, P. A., 419 Kaufman, R. P. Jr., 418 Kaul, M., 407 Kaul, S., 249 Kauppila, A., 363 Kavanah, M., 360 Kavanaugh, A. F., 386, 412 Kawabata, K., 471 Kawai, H., 472

510

AUTHOR INDEX

Kawakami, T., 404 Kawamoto, T., 421 Kawamura, I., 334, 362 Kawamura, T., 48, 72 Kawano, Y., 210 Kawas, C., 301, 362 Kawashima, H., 361 Kay, J., 249, 251 Kay, J. E., 282, 288 Kazazis, D. M., 113 Ke, H., 276, 288 Ke, H. Z., 351, 362 Keates, E. U., 111 Keavney, B., 41, 69 Kedar, R. P., 315, 362 Keech, A. C., 110, 364 Keenan, R., 37, 68 Kehely, A., 91, 111 Keilson, L., 110 Keim, H. J., 22, 65 Keith, D. E., 416 Kelestimur, F., 178 Keller, A. R., 365 Keller, G. A., 420 Keller, H., 203, 204 Keller, I., 44, 71 Keller, S. W., 431, 436, 472 Keller, U., 110 Keller-Juslen, C., 474 Kelly, C. I., 209 Kelly, D. E., 470 Kelly, J. G., 36, 68 Kelly, L. J., 193, 209 Kelly, P. A., 331, 334, 357, 362, 363 Kelly, P. T., 265, 291 Kelly, R., 433, 449, 452, 453, 470 Kelly, S. L., 425, 470 Kelly, T., 177 Kelly, W., 16, 63 Kelsey, S. F., 364 Kelsey, S. M., 415 Kemnitz, M. W., 191, 208 Kempf, A. J., 473 Kempf, D. J., 216, 217, 220, 245, 246, 247, 248, 249 Kenady, D. E., 365 Kenagy, R., 368 Kendra, K. L., 362 Kenemans, P., 365 Kennedy, B., 138, 139, 140 Kennedy, B. P., 117, 139, 140

Kennedy, C. J., 208 Kenny, A. J., 40, 69 Kent, S. B. H., 248, 250, 251 Keogan, M. T., 411 Kereiakes, D. J., 406 Kerkerling, T. M, 468 Kern, J. A., 395, 417 Kern, M., 110, 114 Ketterer, N., 414 Key, T. J., 302, 362 Khairallah, P. A., 46, 71 Khalil, D. A., 247 Khan, K. N. M., 133, 139 Khanna, A., 268, 288 Khoury, S., 175 Khurmi, N. S., 52, 73 Khyne, A. T., 473 Kiang, D. T., 418 Kidd, P., 413 Kiefhaber, T., 287 Kiel, D. P., 301, 359, 362 Killackey, M. A., 297, 362 Kim, A. E., 235, 247 Kim, C. U., 219, 223, 247 Kim, E. E., 231, 234, 247, 287 Kim, H. S., 139 Kim, J. B., 205 Kim, J. L., 287 Kim, J. R., 360 Kim, M. H., 176 Kim, R. B., 235, 248 Kim, S., 51, 73 Kim, U. T., 245 Kim, W., 360 Kim, Y.-M., 419 Kim, Y. S., 466 Kimball, B., 114 Kimber, G. R., 56, 75 Kim-Motoyama, H., 207 Kimura, H., 417 Kimura, S., 208 Kinchington, D., 249 Kindl, S., 416 Kindred, L., 112 King, C. R., 394, 415, 416 King, F. J., 210 King, K. L., 418 King, R. J., 359 Kingsley, G. H., 410, 411 Kinkel, P., 109 Kinney, R. B., 417

AUTHOR INDEX

Kino, T., 255, 288, 291 Kinsella, J., 414 Kinzler, K. W., 420 Kiowski, W., 39, 68 Kirby, R., 174 Kirby, R. S., 154, 177 Kirby, T. J., 78, 96, 111 Kirchertz, E. J., 53, 74 Kirkham, D. M., 208 Kirkman, R., 405 Kirpotin, D., 402, 420 Kirpotin, D. B., 420 Kirschman, J. D., 37, 68 Kishihara, K., 407 Kishimoto, T., 411 Kissinger, C. R., 275, 276, 288 Kitamoto, T., 361 Kitamura, T., 208 Kitano, N., 114 Kjekshus, J., 99, 111, 112, 113 Kjekshus, J. T. M., 112 Klabe, R. M., 245, 246, 249 Klag, M. J., 54, 74 Klahr, S., 46, 53, 72 Klapper, L. N., 395, 416 Klatt, T., 206 Klee, C. B., 259, 270, 288, 291 Kleiman, N. S., 406, 407 Klein, J., 114 Klein, J. L., 110, 114 Klein, L. L., 465, 470, 471 Klein, N., 408 Klein, W. W., 52, 73 Kleinman, J. C., 368 Klepser, M. E., 458, 463, 468, 470 Kletzien, R. F., 183, 203 Kliem, V., 112 Kliewer, S. A., 186, 203, 206, 303, 362 Kligman, A. M., 162, 177 Klijn, G. G. M., 403, 420 Klimkait, T., 245 Klippenstein, D., 414 Klis, F. M., 428, 470, 473, 474 Kluckman, K. D., 139, 140 Kluin, P. M., 410, 411 Knapp, L. E., 68 Knapp, M., 469 Kneissler, U., 53, 74 Knigge, M., 246 Knigge, M. F., 247 Knight, D. M., 410

Knighton, D. R., 288 Knoll, G. A., 3, 12 Knopp, R., 110 Knopp, R. H., 111, 113 Knotts, T. A., 367 Ko, A., 176 Ko, S. S., 245, 249 Kober, L., 43, 50, 70, 73 Koble, C. S., 206 Kobrinsky, E., 286 Kocagoz, S., 474 Kocan, G. P., 250 Koch, G., 175 Kocy, O., 21, 65 Koeplinger, K. A., 249, 250 Koepsell, T. D., 108 Koerner, F., 417 Kogler, H., 472 Kohl, N. E., 215, 227, 248 Kohlbrenner, W. E., 246, 247 Kohli, K., 361 Kohno, H., 472 Kohno, K., 114 Kohsaka, M., 362, 469, 470 Koike, T., 207 Koizumi, J., 111 Kojiri, K., 472 Kojo, H., 172, 177 Koki, A. T., 139 Kolata, G., 9, 12 Kollar, R., 428, 468, 470 Koltin, Y., 468 Komeda, K., 208 Komiyama, T., 27, 33, 66 Komori, T., 431, 470 Kondo, E., 290 Kondo, N., 362 Kondoh, O., 451, 470 Kong, L., 466, 467, 469, 474 Kong, X.-P., 247 Konialian, A. L., 287 Kononen, J., 357 Kontturi, M., 154, 179 Koostra, J., 406 Kopelman, R., 53, 74 Koprak, S. L., 286 Korach, K. S., 361, 366 Korant, B., 247 Korenberg, J. R., 204 Kornbrust, D. J., 110 Korneyeva, M., 248, 249

511

512 Korngold, R., 405 Korngold, S., 406 Korw, E., 16, 63 Kosa, M. B., 247 Kosanke, S., 408 Koshinen, P., 109 Kosinski, A. S., 114 Koski, R. A., 418 Kostis, J. B., 36, 67, 96, 111 Kostuk, W. J., 112 Kotey, P., 178 Kothe, C., 412 Kotowicz, M. A., 364 Kotts, C., 418, 420 Kovanen, P. T., 89, 111, 368 Kovanen, W., 51, 73 Kowalski, J., 210 Kozarich, J. W., 171, 176 Kozka, I. J., 208 Kraegen, E. W., 208 Kraemer, S. A., 138 Krakoff, L. R., 50, 73 Kramar, M., 33, 67 Kramer, B. S., 175, 365 Kramer, J. H., 406 Kramer, R. A., 215, 248 Kramsch, D. M., 108 Krapcho, J., 35, 67 Krapf, D., 176 Kraszewski, A., 363 Kraus, M. H., 415, 416 Kraus, S., 177 Kraus, S. J., 179 Kraus, W. L., 362 Krauss, R. M., 110 Krausslich, H.-G., 215, 227, 248 Krawczyk, S. H., 247 Krayenbuehl, H. P., 113 Krebs, S., 141 Kreft-Jaos, C., 56, 75 Kreitman, R. J., 393, 415 Krekler, M., 112 Kremer, J. M., 410 Kreutter, D. K., 208, 209 Krey, G., 204 Krieg, M., 173, 179, 180 Krieger, E. M., 37, 68 Krinks, M. H., 288 Kripalani, K. J., 26, 65 Krishmarao, T. V., 457, 462, 470 Kristine, E., 359

AUTHOR INDEX

Krohn, A., 249 Krone, W., 207 Krˆnke, M., 407 Kronquist, K., 204 Kropp, H., 466, 467, 468, 469, 474 Krueger, K. A., 359 Krupa, D., 466, 467 Krust, A., 304, 362 Krust, V., 360 Kruszynska, Y. T., 189, 207 Kruyer, W., 109 Ku, G., 368 Kubota, N., 190, 208 Kuchler, K., 473 Kucway, R., 417 Kuehner, D., 473 Kuhlmann, J., 112 Kuhn, H., 53, 73 Kuhn, M., 289, 474 Kuiper, G. G. J. M., 304, 322, 362, 365 Kujubu, D., 137 Kujubu, D. A., 117, 139 Kukovetz, W. R., 114 Kuller, L. H., 364 Kulmacz, R. J., 141 Kumar, G. N., 249 Kumar, S., 198, 211 Kumar, V., 296, 303, 304, 323, 360, 362 Kunaver, M., 409 Kuo, L. C., 245, 249 Kuramoto, H., 357 Kurata, S., 178 Kuroda, A., 291 Kuroda, M., 81, 109, 111, 114 Kurokawa, R., 205, 361 Kuron, G., 108 Kurosaki, T. T., 289 Kurosawa, Y., 404 Kurrelmeyer, K., 407 Kurth, K. H., 179 Kurtz, M., 429, 470, 474 Kurtz, M. B., 423–466, 467, 468, 470, 471 Kurumbail, R. G., 123, 139 Kushner, P. J., 358, 365, 366, 367 Kushwaha, S., 108 Kute, T., 417 Kuulasmaa, K., 114 Kuusisto, J., 207 Kuwahara, S., 471, 474 Kvidal, P., 109 Kvien, T., 137

AUTHOR INDEX

Kvols, L. K., 361 Kwan, K., 204 Kwatra, M. M., 421 Kwok, R. P., 311, 362 Kwon-Chung, K. J., 424, 471, 472 Kwong, E., 140

L Laakso, M., 207 Laatikainen, T., 363 Lablanche, J. M., 53, 74 Labrie, C., 293–357, 359, 360, 362, 363, 364, 366, 367 Labrie, F., 293–357, 359, 360, 362, 363, 364, 365, 366, 367 Labrie, Y., 300, 363 Lachance, Y., 363 Lachno, R., 471, 473 Lacroute, F., 427, 470 Lacy, E., 286 Laerum, O. D., 421 Lafavette, R. A., 46, 53, 71 Laflamme, N., 304, 363 Lagace, L., 366 LaGrandeur, L., 468 Lahti, E., 298, 363 Lai, M. M., 273, 288, 290 Laidlaw, I., 361 Laine, L., 3, 12, 131, 139 Laippala, P. J., 366 Lakkis, N. M., 407 Lal, R., 235, 248, 249 Lalwani, N. D., 182, 203 Lam, G. N., 245 Lam, P. Y., 218, 221, 248, 249 Lam, P. Y. S., 247 Laman, J. D., 412 Lamarre, D., 229, 232, 248 Lamarre, L., 416 Lamas, G. A., 50, 73 Lamb, D. C., 470 Lambert, J. M., 414 Lambert, M. H., 205 Lambert, R. W., 249 Lambrecht, L. J., 141 Lamgfrom, H., 17, 64 Lamkin, G. E., 110 Lan, S.-J., 26, 65 Lancaster, M., 471, 473 Lancaster, S. G., 33, 67

513

Lanchbury, J. S., 411 Landais, P., 53, 74 Landis, S. H., 161, 177, 294, 295, 363 Landrum, D. P., 177 Lands, W. E. M., 139 Landy, A., 249 Lane, A. W., 364 Lane, H. C., 245 Lane, M., 114 Lane, W. S., 288 Laneuville, O., 139 Lang, K., 287 Lang, M., 245, 246 Langeberg, L. K., 286 Langenbach, R., 136, 139, 140 Langendorfer, A., 108, 109 Langman, M. J., 3, 12, 132, 139 Langton-Webster, B. C., 416 Lank, K. M., 474 Lankas, G. R., 110 Lansky, D., 245 Lanyon, L., 359 Lanz, R. B., 311, 363 Lanza, F., 123, 130, 139 Lapatto, R., 215, 216, 248 La Placa, F. P., 112 Laragh, J. H., 16, 22, 37, 38, 39, 40, 46, 50, 51, 57, 59, 63, 65, 68, 69, 71, 73, 75 Laroche, B., 17, 63 LaRosa, J. C., 2, 12 Larouche, D., 360 Larsen, N. E., 358 Larsen, R. A., 475 Larson, E. B., 108 Larson, M. G., 58, 75 Larson, P. J., 141 Lartey, P., 471 Laser, M., 289 Laskarzewski, P., 113, 114 Laskarzewski, P. M., 110, 111 Lasnitzki, I., 145, 180 Lasseter, K. C., 137, 176, 179 Lasseter, K. L., 141 Laterre, P. F., 408 LatgÈ, J. P., 449, 453, 467 Latief, T. N., 358 Laties, A. M., 96, 111 Latreille, J., 362 Lau, C. K., 126, 137, 139 Lau, C.-P., 111 Lau, J., 54, 74

514 Laudet, V., 185, 205 Laupacis, A., 15, 47, 48, 62, 72 Lautenberger, J. A., 249 Laverdiëre, J., 362 Lavigne, M. C., 359 Laville, M., 207 Law, M. R., 106, 111 Law, R. E., 194, 210 Law, W., 248 Lawen, A., 285 Lawhorn, D. E., 176 Lawrence, B., 365 Lawrence Wickerman, D., 360 Lawson, D. H., 56, 75 Lawson, J. A., 140 Laybutt, D. R., 208 Lazar, M. A., 210 Lazarus, P., 367 Lazdins, J., 245 Lazier, C. B., 180 Le, A., 421 Lea, A. P., 86, 111 Leake, B., 248 Leal, M. A. E., 475 Lebeau, M. C., 286 Lebel, P., 207 Leblanc, G., 360, 363, 364 Leblanc, H., 53, 74 Leblanc, Y., 137, 138, 139, 140 Lebwohl, M., 177 Lecerf, L., 40, 69 Lechner, J. J., 365 Leckie, B. J., 47, 72 Lecomte, M., 125, 137, 139 Ledbetter, J. A., 412, 413 Leder, P., 416 Ledergerber, B., 251 Lederman, M. M., 249 Ledford, A., 140 Lee, A., 247 Lee, C. A., 139, 140 Lee, C. G., 235, 248 Lee, C. S., 365 Lee, F. W., 174 Lee, J., 418 Lee, J. S., 420 Lee, R. E., 415 Lee, S. E., 248 Lee, S. H., 117, 139 Leeb, B. F., 409 Lees, A. M., 110

AUTHOR INDEX

Lees, B., 367 Lees, J. A., 368 Lees, K. R., 39, 68 Lees, R. S., 108, 110 Lefebvre, A. M., 209 Lefebvre, A.-M., 194, 210 Lefkowitz, D. S., 109 Lefstin, J. A., 312, 363 Leger, S., 137, 138, 140 Le Goff, P., 362 Le Grand, C. B., 210 Lehmann, J. M., 183, 186, 187, 194, 198, 203, 206, 211, 362 Lehmann, K., 43, 70 Leibowitz, M., 204 Leibowitz, M. D., 204, 205 Leighton, C., 467, 469 Leighton, C. E., 467 Leijd, B., 108 Leisenring, W., 474 Leiter, L., 110 Lekich, R., 211 Lelievre, Y., 247 Le Marec, H., 113 Lemay, A., 178 Le May, M., 114 Lemoine, N. R., 394, 415 Lenhard, J. M., 206 Lentz, K. E., 14, 18, 62, 64 Leon, A. S., 33, 67, 108, 110 Leonard, J., 246, 247 Leonard, J. E., 413 Leonard, J. M., 245, 247, 248, 249 Leonard, S. B., 110 Lepage, M., 363 Lerebours, G., 68 Lerner, B. H., 240, 248 Lerner, C. G., 467 Lerner, D. J., 295, 363 Lerouge, T., 360 Lesi, C., 113 Lesperance, J., 114 Lessem, J., 211 Leu, C. T., 246, 248 Leung, B. S., 331, 363 Leung, J. S., 363 Leung, L., 376, 377, 405 Leung, W.-H., 102, 111 Leventhal, H., 364 Lever, A. F., 16, 17, 62, 63, 75 Levesque, C., 296, 302, 359, 362, 363, 366

AUTHOR INDEX

Levey, A. S., 54, 74 Levi, G., 226, 251 Levin, D. E., 470, 473 Levin, M., 408 Levin, R., 175 Levin, R. B., 246, 250 Levin, W. J., 416 Levin, Y., 17, 18, 63 Levine, J. H., 211 Levine, M., 14, 62 Levine, W. G., 27, 66 Levy, D., 58, 75, 108 Levy, F. O., 363 Levy, H., 407 Levy, J. A., 245 Levy, M. A., 171, 177 Levy, R., 411, 413 Levy, R. B., 418 Lewis, B., 114 Lewis, C. T., 288 Lewis, D. M., 206 Lewis, E. J., 3, 12, 47, 53, 72 Lewis, E. S., 114 Lewis, G., 419 Lewis, G. D., 395, 396, 418 Lewis, G. R. J., 33, 67 Lewis, H., 207 Lewis, L. D., 419 Lewis, R. E., 470 Lewis, R. J., 475 Leyden, J., 165, 167, 168, 177 Leyland-Jones, B., 419 L’Horset, F., 358 Li, A. C., 209 Li, B., 288 Li, C. S., 138 Li, D., 365 Li, H., 245, 323, 363 Li, L., 465, 470, 471 Li, S., 331, 334, 359, 363, 364 Li, W., 108, 468, 470, 471 Li, W. L., 420, 474 Li, W. S., 420 Li, X., 364 Li, Y., 205, 245 Li, Z., 208 Liang, C. S., 51, 73 Liang, J., 245, 246, 275, 278, 288 Liang, T., 148, 149, 177 Liao, S., 145, 174, 175 Liao, W.-C., 36, 67

515

Libby, P., 210 Liberator, P., 473 Libermann, T. A., 402, 420 Lichtenstein, D. R., 141 Lichti, H., 289 Lickley, H. L., 360 Lie, K. I., 111 Lieber, M., 177, 178 Lieber, M. M., 175, 176 Lieberman, G., 419 Lieberman, S., 16, 63 Liebson, P. R., 56, 75 Lien, P., 209 Liesch, J., 108 Liesch, J. M., 469 Lievre, M., 41, 48, 69 Light, S., 405 Liles, T. M., 413 Lim, R. W., 139 Limas, C., 46, 71 Limbird, L. E., 36, 67, 114 Lin, C. S., 286, 290 Lin, J. H., 246, 250 Lin, P.-F., 246 Lin, R. J., 358 Lin, S., 249 Lin, S. C., 361 Lin, S. X., 363 Lin, Y., 475 Linares, M. J., 457, 471 Lincoff, A. M., 3, 12, 377, 378, 406 Linden, B., 122, 139 Linden, T., 114 Linders, D., 358 Lindgren, B., 56, 75 Lindholm, L. H., 44, 71 Lindsay, R., 299, 301, 359, 363 Lines, C., 109 Lingle, D. D., 362 Link, B. K., 413 Linz, W., 46, 47, 48, 55, 71, 72 Liou, S., 139 Lipani, J., 411 Lipari, M. T., 418 Lipert, S., 175 Lipicky, R. J., 44, 71 Lipid Research Clinics Program, 97, 111 Lipke, P. N., 470, 471 Lippa, E. A., 111 Lippman, M. E., 295, 307, 340, 358, 359, 361, 367, 417

516 Lipsky, P., 409 Lipsky, P. E., 382, 383, 409, 412 Lipton, A., 419 Liscum, L., 89, 111 Lister, A., 414 Lister, C. A., 206, 208 Lister, J., 414 Litin, S. C., 93, 111 Little, S. J., 242, 248 Littlefield, B. A., 344, 363 Littlejohn, G., 138 Littman, D. R., 254, 291 Liu, B., 250 Liu, E., 420 Liu, E. T., 417 Liu, J., 259, 277, 288 Liu, J. O., 290 Liu, L. S., 192, 209 Liu, Q. Z., 250 Liu, S., 137, 177 Liu, S. Y., 393, 414 Liu, Y., 367 Livak, K. J., 249 Livi, G. P., 468 Livingston, D. J., 249, 286 Livingston, D. M., 368 Llobell, A., 470 Lloyd, L. K., 178 Lo, D., 405 Lo, J. C., 242, 248 Lobo, R. A., 177, 179, 180, 299, 363 LoBuglio, A. F., 414 Locatelli, F., 47, 53, 72 Locke, C., 247 Lofgren, J. A., 418 Loftin, C. D., 139, 140 Logan, R. L., 15, 62 Logsdon, D. L., 357 Logue, K. A., 245 Loh, C., 260, 273, 274, 288, 290 Lohmiller, J., 209 Lohr, N. S., 27, 28, 29, 32, 66 Loll, P. J., 140 Lomans, J., 416 Lomax, P., 299, 363 Lombardi, D. M., 47, 72 Lombardi, G., 179 London, G. M., 40, 68 Long, J. M., 108 Long-Fox, A., 409 Longstreth, W. T. Jr., 108

AUTHOR INDEX

Loning, T., 417 Lonn, E., 47, 52, 72 Loosli, H., 289 Loosli, H. R., 474 Loots, M. J., 34, 67 Lopez, G., 358 Lopez, G. N., 367 Lopez, L. M., 33, 67 Lopez, M., 108 Lorell, B. H., 286 Lorenz, K., 208, 209 Lorenz, R., 56, 75 Loria, P. M., 366 Lorimer, A. R., 113 Loser, R., 335, 363 Lotsch, J., 141 Louie, D. M., 404 Louis, V., 359 Lovasz, K. D., 250 Love, R. R., 318, 346, 351, 352, 364 Lowe, F., 175 Lowe, N. J., 179 Lowell, B. B., 204 Lowry, S. F., 407, 408 Lozano-Chiu, M., 427, 457, 466, 471, 472, 473, 474 Lu, H. S., 418 Lu, J., 108 Lu, J. R., 289 Lu, Y. F., 265, 288 Lubahn, D. B., 366 Lubbehusen, P. P., 247 Lucas, L. M., 111 Lucas, R., 464, 471 Lucchini, F., 394, 416 Lucky, A., 178 Ludden, C. T., 30, 66 Luell, S., 138 Luengo, J. I., 287 Luetscher, J. R., 48, 72 Lufkin, E. G., 316, 347, 364 Lui, M., 203 Lukens, J., 209 Lund, B., 138 Lundblad, J. R., 362 Lundeen, S. G., 347, 364 Lund-Johansen, M., 421 Lunney, E. A., 247, 249, 250 Luo, C., 288, 289 Luo, S., 293–357, 363, 364, 366 Luo, Z. Y., 267, 288

AUTHOR INDEX

Luong, C., 123, 140 Lupien, P. J., 109, 110 Lupien, P.-J., 112 L¸scher, T. F., 52, 73 Lush, R. M., 414 Luskey, K. L., 109, 111 Luther, M., 469 Luther, M. F., 469 Luu-The, V., 293–357, 362, 363, 364 Lykkesfeldt, A. E., 340, 343, 353, 354, 364 Lyle, T. A., 230, 233, 246, 248 Lyman, C. A., 472 Lynch, L., 467 Lynch, M. E., 474 Lynn, J. C., 249, 250 Lynn, M., 139 Lyons, A., 248 Lyons, W. E., 263, 288

M Ma, D., 414, 446, 468 Ma, P. T., 89, 111 MAAS invetigators, 101, 105 Mabuchi, H., 82, 111 Macaulay, V., 359 Macaya, C., 51, 73 MacCarthy, E. P., 33, 67 MacDonald, B., 411 MacDonald, J. S., 110, 113 Macdonald, P. C., 179 Macfarlane, J. D., 409 MacFarlane, P. W., 113 MacFayden, R. J., 39, 42, 54, 68, 70 MacGregor, G. A., 37, 43, 68, 71 Machin, P. J., 249 Machold, K. P., 381, 409 Mack, W. J., 108 MacKay, H., 360 Mackay, H. J., 415 Mackenzie, J. W., 48, 72 Mackey, S. F., 110 Mackinnon, P. L., 62, 75 MacLean, A., 363 Maclouf, J., 140 MacMahon, B., 346, 364 MacMahon, M., 111 MacMahon, S., 54, 56, 74, 75, 108, 110 Macpherson, A., 137 Madani, H., 404 Madans, J. H., 368

517

Madden, J. D., 179 Maddison, P. J., 411 Madelenat, P., 358 Madhavan, S., 51, 58, 73, 75 Madias, N. E., 53, 74 Madigan, M. J., 248 Madsen, M. W., 364 Maeda, H., 413 Maenpaa, H., 109 Magarian, G. J., 93, 111 Magee, D., 359 Magee, D. E., 358, 360 Magnani, C. M., 177 Magriples, U., 297, 364 Mahendroo, M. S., 147, 148, 177 Mahler, J. F., 139, 140 Mahon, D. M., 246 Mahrer, P. R., 108 Mai, I., 112 Mailhot, J., 360 Maillard, M., 141 Maini, R., 382, 383, 409 Maini, R. N., 382, 383, 384, 402, 409, 410, 412 Mainwaring, W. I. P., 145, 177 Majerus, P. W., 141 Majka, J. A., 110 Mak, S., 407 Mak, T. W., 407 Maki, K., 465, 471, 474 Malatesta, P. F., 174 Malbecq, W., 179 Malcolm, A. J., 420 Maldonado-Cocco, J., 139 Malek, A. M., 52, 73 Malek, G. H., 176, 178 Malenka, R. C., 289 Malfetano, J. H., 297, 364 Malice, M. P., 179 Malice, M.-P., 174, 178 Malkonen, M., 109 Malley, R., 381, 409 Malmberg, L. H., 467, 470 Malone, P. R., 159, 177 Maloney, D. G., 391, 392, 413, 414 Maloney, T. R., 361 Maluccio, M., 287 Man, M. C., 251 Mancia, G., 43, 70 Mancini, G. B., 406 Mancini, J., 51, 73, 137, 138, 139, 140 Mancini, J. A., 123, 125, 138, 140, 141

518 Mancini, V., 175 Mandala, S., 472 Mandala, S. M., 468 Mandel, K. G., 138 Maneval, D., 418 Maneval, D. A., 419 Mangelsdorf, D. J., 184, 185, 204, 205 Mangiarini, L., 416 Mangold, B., 112 Manhem, P. J., 47, 72 Mani, S. K., 366 Man in’t veld, A. J., 42, 70 Mann, D. L., 407 Mann, J. F., 44, 71 Mann, J. F. E., 54, 74 Mann, J. I., 114 Mann, L. L., 469 Mann, P., 469 Manninen, V., 109 Manning, D. L., 365 Manning, L., 248 Manning, R. D., 16, 63 Mansel, R. E., 359 Manson, J. B., 366 Manson, J. E., 358 Mansuy, I., 291 Mansuy, I. M., 264, 289, 291 Mantell, G., 109 Mantell, G. E., 114 Mantero, F., 175 Manttari, M., 109 Manuck, S. B., 112 Marais, A. D., 89, 111 Maranhao, M. F. L., 33, 67 Marcecaux, G., 407 Marchioro, T. L., 405 Marco, F., 455, 457, 458, 462, 471, 472 Marcrelli, S., 179 Marcus, C., 293–357, 358 Marcus, S. L., 204 Marden, E., 360 Marder, K., 367 Marder, P., 469 Mardini, I. A., 140 Marencak, J., 175 Marescalchi, O., 179 Margolese, R. G., 360 Margolies, G., 410 Margolin, N., 249 Mark, J., 421 Mark, M., 205

AUTHOR INDEX

Markandu, N. D., 37, 43, 68, 71 Markham, B., 289 Markham, P. D., 246 Markiewicz, L., 363 Markoski, L. J., 247 Markou, M., 177 Markowitz, M., 235, 236, 237, 239, 245, 246, 248, 249 Marks, C., 412 Marks, J., 420 Marks, J. D., 412 Markus, A., 469 Marletta, E., 175 Marlink, R., 246 Marnett, L. J., 139, 141 Maron, B. J., 266, 289 Maron, D. J., 110 Marquez, G., 286 Marquis, R., 205 Marr, J., 245 Marr, J. J., 246 Marr, K. A., 475 Marre, M., 41, 48, 53, 69, 74 Marrinan, J., 471 Marrinan, J. A., 467, 468, 470, 471 Marriott, M. S., 425, 471 Marsh, C., 420 Marsh, K. C., 247, 249 Marsh, W. A., 3, 12 Marsh, W. H., 18, 64 Marshall, G. R., 248 Martel, C., 318, 321, 347, 359, 360, 362, 363, 364 Martin, B. M., 19, 64 Martin, C. E., 472 Martin, G., 187, 193, 206, 360 Martin, G. R., 367 Martin, H., 450, 471 Martin, J. A., 227, 248, 249 Martin, K. J., 210 Martin, M. A., 408 Martin, P. J., 413, 414 Martin, P. M., 175 Martinez, F. A., 44, 48, 71 Martin-Nieto, J., 286 Marusawa, H., 291 Marx, N., 193, 194, 210 Masashi, S., 210 Maschio, G., 47, 53, 72 Masferrer, J. L., 117, 137, 138, 140 Maslen, G., 207

AUTHOR INDEX

Mason, L. J., 409 Mason, M. F., 179 Mason, M. W., 114 Massacrier, C., 412 Masse, B. R., 404 Masso, E., 245 Masteller, M. J., 108 Masuda, H., 210 Masugi, J., 189, 207 Masuhiro, Y., 361 Masui, H., 421 Masurekar, P. S., 437, 438, 471 Masushige, S., 361 Masuyoshi, S., 466 Materson, B. J., 26, 66 Mathew, A., 297, 364 Mathews, M., 45, 71 Mathisen, A., 202, 211, 212 Matos-Ferreira, A., 174, 178 Matsubara, K., 416 Matsuda, S., 471 Matsui, H., 288 Matsumoto, A. M., 178 Matsumoto, F., 471, 474 Matsumoto, S., 474 Matsumoto, Y., 437, 471 Matsumura, F., 275, 286 Matsumura, Y., 389, 413 Matsunoto, S., 465, 471 Matsuoka, H., 114 Matsuyama, T., 407 Matthews, B. W., 20, 64 Matthews, D. C., 414 Matthews, K. A., 112, 346, 351, 364 Mattila, P. S., 257, 264, 285, 289 Matts, J. P., 108 Mattsson, A., 360 Matullo, G., 53, 74 Matz, H., 168, 174 Matzuk, M. M., 287 Mau, J., 138 Maulik, T. G., 298, 361 Maurin, M. B., 247 Maxwell, C., 246 May, R., 414 Mayaux, J.-F., 247 Maycock, A. L, 27, 28, 29, 32, 66 Mayer, G., 46, 53, 71 Mayer, L., 412 Mayeux, R., 367 Mayford, M., 289

519

Maynard-Currie, C. E., 404 Mayrose, D., 288 Mazees, R. B., 364 Maziasz, T. J., 139 Mazina, K. E., 474 Mazur, P., 447, 448, 450, 451, 468, 471 McAdam, B., 137 McAdam, B. F., 129, 140 McAlister, F. A., 47, 48, 72 Mcalpine, J. B., 467, 470 McBride, K. J., 141 McBride, O. W., 204 McCabe, D., 384, 410, 411 McCague, R., 365 McCall, C. E., 140 McCallum, I. R., 62, 75 McCarthy, P., 475 McCaw, S. E., 204 McClellan, K. J., 86, 111 McClellan, W. M., 57, 75 McClelland, R. A., 365 McCloskey, R., 408 McCloskey, R. V., 380, 408 McConnell, J., 175, 178 McConnell, J. D., 3, 12, 143–174, 153, 155, 162, 176, 177, 179 McConnell, M. A., 361 McCormick, L. S., 112 McCracken, R., 46, 53, 72 McCrary, C., 208 McCrea, P., 37, 68 McCredie, R. J., 364 McCrohon, J. A., 299, 364 McCue, B. L., 365 McCutchan, J. A., 408 McCutcheon, M., 289 McDaniel, S. L., 246, 250 McDonald, E., 247, 249 McDonald, J. J., 139 McDonnell, D. P., 203, 212, 367 McFadden, D., 473 McFadden, D. C., 467 McGee, L. R., 247 McGeer, E. G., 140 McGeer, P. L., 135, 140 McGiff, J. C., 15, 63 McGillem, M. J., 406 McGough, D. A., 467 McGrath, J. P., 249, 250 Mcguire, E. J., 210 McGuire, J. S., 144, 177

520

AUTHOR INDEX

McGuire, W. L., 295, 359, 361, 364, 416 McInerney, E. M., 307, 324, 328, 364 McInnes, G. T., 62, 75 McIntosh, I. H., 344, 364 McIntyre, M., 42, 70 McKanna, J. A., 139 McKay, J., 141 McKee, D. D., 362 McKee, S. P., 246 McKeen, M. L., 364 McKeever, B. M., 123, 140, 246, 248 McKenna, N. J., 363 McKenney, J., 110 McKenzie, C., 41, 69 McKeon, F., 271, 273, 290, 291 McKillop, J. H., 113 McKinley, B., 363 McKinna, J. A., 359 McKinsey, T. A., 289 McKinstry, D., 41, 69 McKinstry, D. N., 26, 40, 65, 69 McLachlan, J. A., 361, 365 McLain, R., 110 Mclaughlin, M. M., 468 McLaughlin, P., 391, 392, 413 McLean, A. G., 376, 405 McLean, B., 137 McLenachan, J. M., 114 McLennan, I., 55, 74 McLennan, N. R., 108 McLeod, C., 421 McMahon, B. T., 2, 11 McMahon, D., 245 McMahon, F., 43, 70 McMahon, S., 55, 74 McMillan, C., 472 McMillian, C. L., 472 McMurray, J. J. V., 49, 73 McMurtrey, A. E., 418 McNamara, P. M., 360 McNeal, J. E., 152, 177 McPherson, R., 91, 109 McPherson, R. K., 208 McQuaid, L., 176 McQuaid, L. A., 176 McQueen, M., 110 McTavish, D., 36, 67, 68, 86, 111, 112 Meade, T. W., 51, 73 Meakin, J. W., 366 Means, A. R., 287 Meehan, P. M., 246

Meehan, W. P., 210 Meek, J. L., 247, 248 Meggs, L., 49, 73 Meglasson, M. D., 208 Meguro, T., 267, 289 Mehlisch, D. R., 138 Mehta, R. J., 446, 471 Meier, C. A., 205 Meilahn, E., 364 Meinz, M., 468 Meirhaeghe, A., 189, 207 Meisel, S., 68 Meissner, H. C., 409 Melamed, A., 161, 177 Melancon, R., 357 Melief, C. J., 414 Melino, M., 113 Melino, M. R., 113 Mellies, M. J., 110 Mellon, K., 418 Mellors, J., 245, 246 Mellors, J. W., 246, 247 Mellstedt, H., 415 Melman, A., 178 Melroe, H., 247 Meltzer, P. S., 357 Memoli, V., 419 Memon, A., 178 Menard, D., 46, 71 Menard, J., 13–62, 63, 68, 69, 70, 71, 74, 75, 137 Menard, J. E., 40, 68 Mendelsohn, J., 403, 419, 421 Menick, D. R., 289 Menke, J., 206 Mentzel, C., 469 Merand, Y., 293–357, 359, 360, 362, 363, 364, 366 Mercuri, M., 109, 110, 112 Meredith, P. A., 42, 54, 56, 70 Merrett, J. H., 249 Merrill, A. J., 48, 72 Merson, J. R., 248 Merz, W. G., 473 Mes-Masson, A. M., 418 Messer, S. A., 468, 470, 471, 472 Mestan, J., 245 Metcalf, B. W., 177 Metter, E., 362 Metz, A. L., 210 Metzger, D., 304, 323, 357, 360, 361, 365, 367

AUTHOR INDEX

Metzger, H., 404 Metzger, R., 47, 72 Meulbroek, J. A., 465, 471 Meurer, R., 209 Meyer, L., 419 Meyer, M. E., 360 Meyer, O., 410, 420 Meyer, P., 16, 63 Meyer, Ph., 16, 63 Meyer, T. W., 46, 53, 71, 72, 74 Michalska, M., 411 Michaud, A., 18, 19, 41, 64, 69 Michel, J. B., 46, 53, 71, 74 Michelet, S., 41, 69 Mickelson, J. K., 379, 406, 407 Miettinen, T., 112, 114 Miettinen, T. A., 99, 112 Migdaloff, B. H., 26, 36, 65, 67 Migeon, C. J., 301, 365 Miguel, C. M., 406 Mihara, M., 411 Miichell, W. O., 473 Mijatovic, V., 365 Miki, H., 208 Miksche, U., 16, 63 Miksicek, R. J., 358 Milam, D. E., 178 Milan, D., 290 Milburn, M. V., 205 Milefchik, E. N., 475 Miles, C., 57, 75 Miles, P. D. G., 192, 209 Miles, R. A., 169, 177 Milestone, D. S., 208 Milik, A. W., 414 Miller, A., 140 Miller, B., 177 Miller, D. M., 417 Miller, E., 211 Miller, K. D., 242, 248 Miller, M., 221, 248, 251 Miller, M. M., 250 Miller, P., 174, 178 Miller, R. A., 413 Miller, R. R., 176 Miller, T. K., 475 Miller, V. T., 110, 111, 113 Miller, W. H. Jr., 421 Miller, W. R., 296, 365 Milliez, P., 16, 63 Milliez, P. L., 40, 68

Milligan, J., 470 Milligan, J. A., 471 Mills, J. S., 249, 251 Mills, K., 475 Milo, T., 470 Milstein, C., 404 Mimran, A., 17, 54, 63, 74 Minnich, J. L., 18, 64 Minnich, M. D., 248 Mino, A., 472 Mio, T., 433, 449, 451, 469, 471 Misiti, S., 367 Mitch, S., 35, 67 Mitchel, Y., 112, 113, 114 Mitchel, Y. B., 86, 112, 113 Mitchell, A., 108, 468 Mitchell, A. M., 248 Mitchell, J., 96, 112 Mitchell, J. A., 139, 141 Mitchell, M. S., 395, 417 Mitchell, P., 96, 109 Mitlak, B. H., 359 Mitra, S., 175, 205 Mivata, K. S., 184, 204 Miyamoto, C., 469, 470 Miyamoto, S., 111 Miyashiro, J. M., 139 Miyata, T., 250 Miyazaki, I., 417 Miyazaki, M., 45, 51, 52, 71, 73 Mizota, T., 362 Mizuno, K., 431, 471, 473 Mo, H., 249 Mo, H.-M., 248 Moachon, G., 357 Moallem, T. M., 291 Moasser, M., 419 Mobbs, B. G., 366 Moberger, B., 360 Mochizuki, M., 114 Mochizuki, T., 206 Mockus, L., 52, 73 Mocroft, A., 236, 248, 251 Moghetti, P., 170, 175, 177 Mohan, C., 387, 412 Mohler, K. M., 373, 404 Mohring, J., 16, 63 Moilanen, B., 469 Molinoff, P. B., 36, 67, 114 Moliterno, D. J., 406 Molkentin, J. D., 266, 267, 289

521

522

AUTHOR INDEX

Molla, A., 220, 235, 247, 248, 249 Moller, D. E., 181–203, 204, 205, 206, 208, 209, 210 Monaghan, R., 108 Moncada, S., 15, 63, 138 Moncloa, F., 32, 33, 67 Mondon, C. E., 206 Monks, C., 466 Monod, M., 473 Monroe, A., 362 Montaigner, L., 214, 245, 246 Montalbo, E. M., 469 Montano, M. M., 362 MonteseirÌn, J., 286 Montigny, M., 109, 110 Montijn, R. C., 470 Moon, J. B., 250 Moon, S. D., 414 Moore, C. B., 472 Moore, E., 178, 179 Moore, E. C., 175 Moore, J., 419 Moore, J. T., 362 Moore, K., 208 Moore, L. B., 203, 206, 211 Moore, M. A., 410 Moore, R., 405 Moore, T., 17, 63 Mooren, A. T. A., 474 Moorman, A. C., 249 Mooser, V., 42, 44, 70 Mora, M. J., 179 Moral, F., 410 Moran, A., 110 Moras, D., 368 Moreland, L., 410 Moreland, L. W., 373, 383, 384, 404, 410 Morgan, A. W., 386, 412 Morge, R. R., 250 Morham, S. G., 136, 139, 140 Mori, H., 207 Mori, Y., 189, 207, 208 Morikawa, H., 178 Morimoto, K., 287 Morimoto, T., 208, 287 Morin, N., 468, 471, 472 Morishita, R., 47, 72 Morishita, Y., 471 Morissey, J. J., 46, 53, 72 Moritz, C., 141 Moriwaki, A., 288

Moriya, Y., 411 Morley, T. L., 419 Morrill, R., 176 Morris, A. A., 28, 30, 66 Morris, A. J., 472 Morris, D. C., 206 Morris, D. L., 139 Morris, J. K., 250 Morris, P. J., 255, 289 Morris, R. A., 468 Morris, R. S., 180 Morrison, A., 362 Morrison, B. W., 137 Morrison, J. L., 48, 72 Morrison, R. A., 36, 67 Morrison, S., 372, 404 Morrissey, G., 474 Morrissey, J., 474 Morrow, M., 359 Mortel, R., 366 Mortensen, E., 139 Mortensen, R. M., 208 Morton, C. J., 289 Morton, J. J., 17, 37, 47, 63, 68, 72 Moser, M., 56, 75 Moskowitz, M. A., 362 Mosley, R., 205 Mosley, R. T., 174 Mosselman, S., 304, 365 Mouridsen, H., 295, 340, 365 Mouridsen, H. T., 357 Movnahan, M., 419 Mowery, D. C., 2, 11 Moye, L. A., 50, 73, 113 Moyer, D. L., 355, 365 Moyle, G., 246 Moyle, G. J., 239, 240, 248 Mrozek-Orlowski, M., 419 Muck, W., 93, 112 Muderris, I., 178 Mudra, H., 52, 73 Mueller, E., 194, 210 Mueller, R., 245 Mueller, R. A., 246, 250 Muesing, M. A., 247 Muggeo, M., 175, 177 Muhlbaier, L. H., 420 Muirhead, E. E., 22, 65 Muirhead, G. J., 232, 248 Mukai, T., 467 Mukherjee, R., 187, 206, 207

523

AUTHOR INDEX

Mukhopadhyay, S., 141 Mukhopadhyay, T., 431, 472, 473 Mukoyama, M., 47, 72 Muldoon, M. F., 97, 112 Mulhern, T. D., 283, 289 Mulh-Selbach, U., 141 Mulichak, A. M., 250 Mulkey, R. M., 264, 265, 289 Mullen, L. T., 357 Muller, B., 40, 69 Muller, D., 207 Muller, D. W. M., 406 Muller, J. G., 267, 289 Muller, R. K. M., 53, 73 Muller, W. J., 394, 416 Muller-Wieland, D., 207 Mullick, A., 367 Mulligan, K., 248 Mullins, K., 365 Mumford, R. A., 174 Munoz, J. F., 471 Munson, J., 112 Muraca, P. J., 418 Murad, R., 245 Murakami, K., 187, 206, 208 Murcko, M. A., 247 Murdoch, D., 36, 67 Murieta-Geoffroy, D., 42, 44, 70 Murphy, C., 210 Murphy, G., 175 Murphy, R. L., 239, 248 Murray, G., 50, 73 Murray, G. D., 47, 72 Murray, M., 415 Murray, T., 177, 363 Murthy, K. H. M., 233, 248 Murthy, S., 415 Murtomaki, E., 112 Musgrove, E. S., 299, 365 Musick, L., 249 Musikic, P., 411 Musliner, T. A., 112 Muss, H. B., 395, 417 Mustonen, S., 174, 178 Mutschler, E., 43, 70 Mutti, E., 43, 70 Myburgh, D. P., 33, 67 Myers, J. N., 395, 416 Myers, M. H., 361 Myers, R. E., 366 Mykkanen, L., 207

N Naar, A. M., 361 Nabel, E. G., 114 Nachman, R., 405 Nadaud, S., 18, 64 Nadler, L. M., 413 Nadzan, A. M., 206 Naftolin, F., 364 Nagai, R., 208 Nagashima, M., 472 Nagy, L., 186, 193, 194, 206, 358 Naito, M., 208 Najib, J., 210 Najvar, L. K., 469 Nakada, M. T., 382, 410 Nakahara, K., 467 Nakai, T., 471, 474 Nakajima, M., 47, 72 Nakajima, S., 472 Nakajima, T., 470, 475 Nakano, M., 407 Nakano, R., 208 Nakatani, Y., 358 Nakaya, N., 113 Nakayama, O., 171, 177 Nakshatri, H., 184, 205 Nangia, S., 473 Naoumova, R. P., 111 Napier, M. A., 418 Nappi, C., 179 Nappi, R. E., 363 Narayan, P., 176 Nash, A., 358 Nash, C. H., 471 Nash, D., 246 Nash, D. T., 108, 113 Nashan, B., 375, 376, 405 Nasraway, S., 407 Nastainczyk, W., 138 Natanson, C., 407 Natarajan, S., 35, 67 Natoli, C., 140 Navetta, F. I., 406 Navia, M. A., 215, 216, 227, 247, 248 Navis, G., 54, 74 Nawaz, Z., 366 Nawrocki, J. W., 88, 112 Nayfield, S. G., 297, 344, 365 Neafus, R., 112 Neafus, R. P., 109

524

AUTHOR INDEX

Neal, D. E., 178, 403, 418, 420 Neaton, J., 54, 74 Neaton, J. D., 54, 74 Needham, M., 368 Needleman, P., 138, 140 Neeman, I., 176 Negri, C., 175, 177, 207 Neidhart, D. J., 246 Neidhart, J. A., 413 Neild, J. A., 359 Neilson, K., 117, 139 Nell, V., 409 Nelp, W. B., 414 Nelson, E. B., 33, 67 Nelson, K., 365 Nelson, K. G., 307, 361, 365 Nelson, P. W., 455, 456, 457, 458, 471, 472, 473, 475 Nemeth, P. R., 138 Nemoto, M., 207 Nemoto, S., 289 Nessim, S. A., 108 Neubauer, B. L., 176 Neubeck, R., 35, 67 Neubek, M., 43, 70 Neumayer, H. H., 112 Neuwirth, C. K., 111 Nevala, M., 366 Neven, P., 297, 365 Newboult, E., 367 Newcomb, P. A., 364 Newland, A. C., 415 Newman, J., 414 Newman, R., 372, 404 Newman, R. A., 413 Newman, T. B., 104, 105, 112 Newton, L. S., 206 Newton, M. A., 50, 73 Ney, P., 68 Ng, J., 176, 179 Ng, K., 467 Ng, K. K., 20, 65 Ng, K. T., 285 Ng, T. H., 44, 71 Nguyen, H. H., 175, 179 Nguyen, M. H., 427, 457, 467, 472 Ni, L., 465, 472 Nicholls, J., 359 Nicholls, M. G., 14, 20, 62, 64 Nichols, K. K., 468 Nicholson, R. I., 353, 359, 365

Nickel, J. C., 177 Nickelson, T., 359, 364 Nickerson, C., 48, 72 Nie, Z. R., 416 Niehans, G. A., 395, 418 Nielsen, J., 450, 470 Nielsen, J. B., 468, 471, 472 Nies, A. S., 115–137 Nieves, J., 246 Nigam, S., 139 Nightingale, P., 407 Nikkila, E. A., 109 Nilaver, G., 289 Nilsson, S., 362, 365 Nimer, S. D., 204 Ninomiya, I., 417 Nique, F., 353, 365 Nir, P., 248, 249 Nishida, E., 361 Nishimotot, N., 411 Nishiyama, M., 288 Niskanen, L., 44, 71 Nissen, J. S., 468 Nitta, K., 469 Noble, F., 62, 75 Noble, S., 68, 232, 249 Nochy, D., 53, 74 Noelle, R. J., 412 Nolan, G. H., 360 Nollstadt, K., 467, 470, 471 Nollstadt, K. H., 467, 473 Nollstadt, K. M., 475 Nolte, R. T., 185, 205 Nombela, C., 469 Nomura, Y., 343, 365 Nonaka, H., 450, 472 Nooyen, W. J., 358 Norbeck, D. W., 246, 247, 248, 249 Nordberg, J. E., 417 Nordling, J., 174, 178 Noriega, L., 466 Norman, D. J., 94, 112 Norman, M. J., 362 Norman, R., 176 Norman, R. A., 16, 63 Norman, R. W., 178 Norola, S., 109 Norris, J. D., 367 Norris, P., 411 Northrop, J. P., 274, 289, 291 Norton, J., 36, 67

AUTHOR INDEX

Norton, L., 359, 398, 417, 419, 421 Nossal, G. J., 370, 404 Notarbartolo, A., 102, 112 Notario, V., 450, 470, 472 Notsu, Y., 177 Nouhan, C., 247, 250 Novilla, M., 475 Novotny, W., 421 Nowaczynski, W., 16, 63 Noya, D., 204 Nriega, L., 473 Nucifora, F. C. Jr., 286 Nugeyre, M. T., 245 Nuki, G., 411 Nunez, A. M., 357 Nussberger, J., 41, 42, 44, 56, 69, 70, 71, 75, 141 Nusse, R., 416 Nutt, E. M., 248 Nutt, R. F., 215, 248 Nyfeler, R., 431, 436, 472 Nyyssonen, K., 113

O Oakes, N. D., 191, 208 Oakley, K. L., 441, 463, 468, 472, 474 O’Banion, M. K., 117, 140 Obeirne, M. J., 467 O’Boyle, E., 208 Obrda, O., 141 Ochmann, K., 112 Oda, M., 288 Oddens, B. J., 359 Oddou-Stock, P., 44, 71 Odds, F. C., 473 Odman, B., 114 Theodoulou, M., 419 O’Driscoll, G., 102, 112 Odum, J., 357 Oehlen, L., 473 Oehlschlager, A. C., 472 Oelkers, W., 17, 63 Oesterling, J. E., 157, 175, 176, 178 Oeze, L., 366 Offen, W. W., 44, 71 Ogden, S. G., 418 O’Grady, P., 112 Ogura, M., 467 Oh, C. S., 450, 472 Oh, H.-J., 287

525

Ohashi, M., 206 Ohashi, P., 407 Ohki, H., 471 Ohman, E. M., 53, 74 Ohman, L., 358 Ohmori, Y., 114 Ohoyama, S., 417 Ohtawa, M., 150, 178 Ohya, Y., 470, 473 Oka, T., 114 Okada, H., 431, 472 Okamoto, M., 114 Okamura, H., 285 Oki, T., 466 Okuhara, M., 177, 469, 470 Okumura, T., 288 Okuno, A., 191, 208 Olbricht, C., 93, 112 Olefsky, J. M., 195, 207, 209, 210 Oleksijew, A., 471 Oleske, J., 246 Oliver, B. B., 206, 362 Oliver, M. F., 3, 12, 90, 97, 109, 112 Olofsson, S. O., 114 Olsen, D. B., 235, 249 Olsen, E., 178 Olsen, E. A., 162, 164, 169, 177, 178 Olsen, S. J., 249 Olson, E. N., 266, 267, 289 Olson, S. C., 68 Olsson, A. G., 112, 113 O’Malley, B. W., 363, 364, 365, 366, 368 O’Malley, B. W. Jr., 310, 365 O’Malley, K., 36, 68 O’Malley, P., 251 O’Mara, E. M., 234, 249 Omstead, M. N., 474 Onate, S. A., 304, 323, 324, 328, 331, 363, 365, 366 Ondetti, M. A., 14, 18, 20, 21, 22, 23, 24, 25, 26, 27, 29, 34, 45, 62, 64, 65, 67 Ondeyka, D. L., 27, 28, 29, 32, 66 Ondrias, K., 286 Ondriasova, E., 286 O’Neil, B. J., 52, 73 O’Neill, G., 140 O’Neill, G. P., 138, 139, 140 Onishi, J., 436, 468, 471 Ono, M., 250 Ooi, T., 110 Ooi, W. L., 51, 73

526

AUTHOR INDEX

Opal, S. M., 407, 408 Opara, J. U., 195, 197, 211 Opie, L. H., 26, 36, 66 O’Rahilly, S., 207 Oral, H., 379, 407 O’Reilly, B., 34, 67 Orentreich, N., 301, 365 Orevillo, C., 138 Orlic, S., 176 Oroszlan, S., 245 Orpana, A., 368 Ortego, M., 46, 53, 72 Orth, H., 16, 63 Ortlepp, S., 407 Ortwine, D. F., 250 Ory, S. J., 359 Osborne, C. K., 315, 340, 343, 353, 354, 355, 359, 365, 368, 417 Ose, L., 84, 86, 88, 93, 109, 112 O’Shea, J., 405 Oshima, H., 140 Oshima, M., 134, 140 Oskalns, R., 112 Osman, M., 291 Osterborg, A., 394, 415 Ostovic, D., 246, 250 O’Sullivan, M. G., 117, 140 Osumi, M., 428, 430, 472 Otomo, K., 471, 474 Otomo, S., 138 Otsu, Y., 291 Otterbein, E. S., 33, 67 Otto, J. C., 123, 140 Otto, M. J., 247, 248 Ouellet, C., 360 Ouellet, M., 119, 137, 138, 139, 140 Ouimet, N., 137 Ounishi, H., 45, 71 Outerovitch, D., 247 Ovalle, R., 471 Overington, J., 248 Owens, G. K., 47, 72 Oyen, W. J., 411

P Pacholok, C., 467 Packard, C. J., 113 Packer, M., 48, 57, 72, 75 Padbury, G. E., 226, 249, 250 Padhy, L. C., 415

Padley, R. J., 178, 179 Paech, K., 308, 309, 365 Paetznick, V., 466, 473 Paetznick, V. L., 471, 473 Pagani, J. L., 473 Paganini-Hill, A., 301, 365 Pagano, P. J., 249, 250 Page, I. H., 14, 15, 62, 63 Pahor, M. R. P. B., 60, 75 Pai, S.-Y., 287 Paik, S., 395, 417 Pairet, M., 116, 117, 120, 122, 140, 141 Pai-Scherf, L. H., 402, 420 Pajak, A., 114 Pak, J. Y., 139 Palatini, P., 43, 70 Palazuk, B. J., 209 Palazzo, I., 420 Palella, F. J., 3, 12, 236, 249 Paleolog, E., 409 Paleolog, E. W., 382, 383, 410 Palker, T. J., 246 Palmer, A., 41, 69 Palshof, T., 365 Palumbo, G., 175 Palumbo, M. A., 175 Panagides, J., 139 Panara, M. R., 140 Panayi, G. S., 381, 385, 409, 410, 411 Pandiella, A., 421 Pandya, S., 140 Pangalis, G. A., 415 Panneton, M., 138 Panser, L. A., 175 Papahadjopoulos, D., 420 Papanicolaou, N., 16, 63 Papas, T. S., 249 Papazoglou, S., 138 Pappas, F., 176, 177, 178 Pappas, P. G., 468, 473 Pappu, A. S., 113 Paquet, N., 363 Para, K. S., 249, 250 Para, M. F., 246 Paradisi, R., 179 Parati, G., 43, 70 Pardoll, D. M., 365 Parent, S. A., 446, 447, 472 Parge, H. E., 288 Pargellis, C., 248 Parikh, I., 203

AUTHOR INDEX

Pariser, D., 177 Pariser, D. M., 179 Parish, S., 41, 69 Parisi, A. F., 50, 73 Park, C., 249, 417 Park, C. H., 247 Park, C. W., 205 Park, I., 367 Park, J. S., 113 Park, J. W., 369–403, 418, 420 Park, S. K., 46, 53, 71 Parker, E., 414 Parker, F., 247 Parker, M., 368 Parker, M. G., 205, 358, 361, 417 Parker, M. M., 407 Parkes, K. E. B., 249 Parr, T. J., 467 Parr, T. R., 467, 474 Parrillo, J. E., 379, 407 Parrini, D., 175 Parsons, J. N., 270, 277, 289 Parsons, T. J., 416 Partin, A. W., 179 Partinen, M., 96, 112 Partridge, J. B., 210 Parving, H. H., 54, 74 Pasceri, V., 194, 210 Pasinetti, G. M., 127, 134, 140 Pasqualini, J. G., 302, 358 Pasqualini, J. R., 302, 360 Passa, P., 53, 74 Passamani, E., 8, 12 Passmore, L. A., 139 Pastan, I., 248, 393, 415, 420 Pasternack, A., 109 Pasty, B. M., 57, 75 Patarca, R., 249 Patchett, A., 108 Patchett, A. A., 13–62, 66 Patel, G. F., 174 Patel, I., 206 Patel, J., 198, 199, 201, 202, 211 Patel, J. C., 175 Paterniti, J. R., 207 Paterniti, J. R. Jr., 206 Paterson, A. G., 358 Paterson, A. H. G, 360 Patick, A. K., 232, 247, 249 Paton, V., 419 Patrick, D., 138, 140

527

Patrignani, P., 118, 127, 128, 140 Patrono, C., 112, 137, 140 Pattengale, P. K., 416 Patterson, J., 247, 365 Patterson, L., 174, 176 Patwardhan, R., 211 Paul, D. A., 246, 247 Paul, L., 359 Paul, M., 47, 72 Paulus, U., 113 Pavelic, K., 417 Pavelka, K., 411 Pavlik, E. J., 354, 365 Pavlovsky, A., 250 Payne, L. G., 27, 28, 29, 32, 66 Payne, L. S., 39, 68 Payton, 108 Pazhanisamy, S., 235, 249 Peach, M. J., 47, 72 Pead, M., 359 Pearce, M. B., 108 Pearl, L. H., 215, 227, 249 Pearson, D., 420 Pearson, M. L., 249 Pearson, T. A., 100, 112 Peart, W. S., 51, 73 Pease, L. R., 287 Pedersen, T., 112 Pedersen, T. R., 8, 12, 85, 87, 92, 93, 95, 96, 97, 105, 107, 111, 112, 113 Peer, C. W., 357 Peer, G., 408 Pees, C., 17, 64 Pegram, M., 397, 419 Pegram, M. D., 397, 417, 419 Peinado-Onsurbe, J., 209 Pekkanen, J., 97, 109 Pelak, B. A., 469 Peles, E., 396, 418 Pelletier, G., 363, 364 Pelletier, L. A., 288 Peltiers, C., 247 Pelto-Huikko, M., 362 Peng, C., 215, 249 Penning, T. D., 139 Pennington, P., 176 Pennington, P. A., 176 Penny, C., 111 Penzo, M., 43, 70 Perani, G., 113 Percival, D., 137, 139

528

AUTHOR INDEX

Percival, M. D., 119, 137, 138, 139, 140 Perez, A., 48, 72 Perez, P., 466, 473 Perfect, J. R., 424, 468, 472, 474 Perier, A., 175 Perl, T. M., 407 Perles, P., 179 Perlman, A. M., 469 Perlmann, T., 362 Perlow, D. S., 39, 68 Perna, A., 47, 53, 72 Pernin, A., 205 Perondi, R., 51, 73 Perone, G., 175 Perreault, J.-P., 176 Perri, M. G., 34, 67 Perrier, H., 138, 140 Perrin, P., 175 Perrino, B. A., 286, 287 Perris, T. F., 16, 63 Perry, C. M., 232, 249 Perryman, S., 246 Persson, L. M., 208 Pessina, A. C., 43, 70 Petera, P., 410 Peters, C. A., 151, 178 Peters, D. C., 68 Peters, D. H., 68 Peters, J. M., 203 Peters, M. K., 361 Peters, S. W., 367 Peters, T. D., 100, 112 Petersdorf, S. H., 414 Peterson, A. E., 36, 67 Peterson, A. V. Jr., 114 Peterson, E. R., 27, 28, 29, 32, 33, 66 Peterson, L., 290 Peterson, P. A., 287 Peterson, R. E., 176 Petitclerc, L., 366 Petitti, D. B., 8, 12, 360 Peto, R., 54, 56, 74, 75, 108 Petraitiene, R., 464, 472 Petraitis, V., 464, 472 Petrakova, E., 470 Petrillo, E. W., 22, 26, 34, 35, 65, 67 Petrillo, E. W. Jr., 34, 35, 67 Petrone, A., 179 Petropoulos, C. J., 247 Petry, N. A., 414 Petteway, S. R. Jr., 249

Pez, J. P., 365 Pfaller, M. A., 455, 457, 458, 459, 462, 463, 468, 470, 471, 472, 473 Pfeffer, J. M., 49, 73 Pfeffer, K., 379, 407 Pfeffer, M., 50, 73 Pfeffer, M. A., 49, 50, 73 Pfeiffer, A., 207 Pfister, P., 110 Pflugfelder, P. W., 94, 112 Pflugl, G., 276, 289 Pheffer, M. A., 113 Phelps, M. C., 68 Philibert, D., 365 Philippe, M., 40, 53, 69 Philips, A., 358 Phillips, D. L., 362 Phillipson, B. E., 111 Phylip, L. H., 251 Picard, D., 358 Picard, M., 474 Picard, S., 364 Pickard, R., 159, 178 Pickard, W. W., 469 Pickering, G., 54, 74 Picot, D., 123, 140 Pierce, A. M., 427, 472 Pierce, J. H., 416 Pierce, J. H. D., 472 Pierce, L. R., 91, 112 Pierce, V. K., 362 Pierre, D., 359 Pierre-Malice, M., 178 Pietras, R., 419 Pietras, R. J., 395, 397, 417, 419 Pihl, S., 112 Pihlajamaki, J., 207 Pike, A., 364 Pike, A. C., 358 Pike, A. J., 176, 361 Pike, J. W., 212 Pike, M. C., 302, 362 Pikkarainen, J., 109 Pikounis, B., 178 Pikounis, V. B., 466, 469 Pilcher, G. J., 113 Pillsbury, H. D., 45, 71 Pincus, T., 381, 409 Pines, A., 299, 365 Pinkas-Kramarski, R., 416 Pinkus, G. S., 413

AUTHOR INDEX

Pinner, M. A., 2, 11 Pinto, I. L., 206 Pinto, M. B., 141 Piotrowski, J., 470 Piper, R. C., 250 Pirie, C. M., 362 Pitt, B., 44, 48, 71, 72, 88, 100, 112, 406 Pittarelli, L. A., 473 Plattner, J. J., 246, 247, 249 Plosker, G. L., 68, 86, 112 Plotkin, D., 114, 344, 365 Plouin, P. F., 43, 46, 53, 56, 70, 71, 74, 75 Plow, E. F., 405, 406 Pluscec, J., 21, 65 Plutsky, J., 210 Plymate, S. R., 179 Pocetti, D. A., 36, 67 Pochi, P., 176 Poe, M., 290 Pognonec, P., 204 Pogson, G., 91, 112 Pogue, J., 47, 72 Poirier, D., 364, 365, 366 Poirier, H., 206 Poirier, O., 40, 69 Polak, A., 426, 473 Poljak, R. J., 250 Pollak, M., 362 Polman, J., 365 Pomidossi, G., 51, 73 Pomponi, S., 475 Poncioni, B., 245 Ponglikitmongkol, M., 367 Ponticelli, C., 44, 71 Poo, J. L., 137 Pool, J., 33, 67 Pool, J. L., 108 Poole-Wilson, P., 113 Poole-Wilson, P. A., 44, 48, 71 Poole-Wilson, Ph., 48, 72 Popescu, A., 286 Popovic, M., 214, 246, 249 Poppe, S. M., 226, 227, 249 Porchet, M., 30, 41, 66, 69 Poretz, D., 245, 246 Pories, W. J., 206 Porkkala, E., 113 Porras, A., 138 Porter, A., 177 Porter, B., 414 Porter, D. A., 405

Possert, P. L., 250 Posvar, E. L., 109 Pottage, J. C., 468 Potvin, M., 362 Poulin, R., 296, 315, 316, 318, 335, 340, 343, 354, 363, 365, 366 Pouliot, F., 359 Poulter, N. R., 43, 70 Powderly, W. G., 236, 248, 249 Powell, D. E., 365 Powell, J. R., 34, 35, 67 Powell, J. S., 53, 73 Powell, P. H., 159, 178 Power, R. F., 307, 323, 366 Powers, C. A., 359 Powles, M. A., 460, 467, 473 Powles, T., 359 Powles, T. J., 344, 362, 366 Pozzi, M., 357 Prakash, S. R., 206 Pramanik, B., 474 Prasad, J. V., 223, 247, 249, 250 Prasad, R., 455, 473 Prasit, P., 126, 137, 138, 139, 140, 141 Prasquier, R., 46, 71 Pratt, R. A., 45, 47, 71 Pratt, R. E., 47, 53, 72, 74 Preclik, G., 118, 123, 140 Prentice, R., 366 Prentice, R. L., 114 Prescott, R. J., 111 Present, D. H., 387, 412 Press, M. F., 395, 417, 418 Press, O. W., 390, 393, 413, 414 Presta, L., 418 Preston, R. A., 26, 66 Prewett, M., 403, 421 Price, E. J., 384, 410 Price, E. R., 290 Price, R. L., 286 Price, V. H., 162, 169, 175, 177, 178 Priestman, T. J., 358 Primka, R., 178 Primka, R. L., 174 Primorac, D., 417 Pritchard, K. I., 340, 343, 354, 366 Privalsky, M. L., 358 Probstfield, J., 109 Provencher, L., 362, 364 Prusoff, W. H., 358 Pruzanski, W., 385, 411

529

530

AUTHOR INDEX

Pryor, J. S., 114 Psaty, B. M., 3, 12, 108 Pucillo, A. L., 52, 73 Puigserver, P., 185, 205 Pujol, C., 472 Punnonen, R. H., 299, 366 Puolakka, J., 368 Pye, R. J., 411 Pyke, S. D., 108 Python, C. P., 470, 473

Q Qadota, H., 450, 470, 473 Qadri, F., 17, 63 Qi, C., 204, 205 Qiu, Y., 368 Quade, M. M., 68 Quan, H., 137, 139 Quillen, E. W. Jr., 15, 63 Quintero, J. C., 246, 248, 250 Quiter, E., 110

R Raab, H., 418 Raab, R., 413 Raal, F. J., 89, 113 Rabasseda, X., 234, 249 Rabinowe, S., 17, 63 Racadot, E., 411 Race, E., 251 Rachez, C., 311, 366 Rachubinski, R. A., 204 Racioppa, A., 43, 70 Rack, M. F., 139 Radaelli, A., 43, 70 Raddato, V., 113 Radding, J. A., 450, 467, 468, 473 Radema, S., 412 Radford, J. A., 414 Radford, S. A., 468 Radics, L., 276, 287 Radosevic, S., 417 Radovancevic, B., 108 Raetz, C. R., 176 Rafaat, K., 209 Rafalski, J. A., 249 Ragan, D., 45, 71 Raghaven, A., 285 Raimondeau, J., 91, 113

Rain, D. L., 361 Raines, C., 246 Rajakangas, A. M., 114 Rajman, I., 464, 472, 473 Raju, U. R., 360 Rakugi, H., 45, 47, 71 Ram, A., 430, 446, 449, 473 Ram, A. F. J., 470 Ramachandra, M., 248 Ramadan, N., 472 Ramakrishnan, R., 108 Rambeaud, J. J., 175 Ramesha, C., 140 Rami, H. K., 206 Ramilo, O., 409 Ramos, L., 417 Ramsay, L. E., 55, 56, 74, 75 Ramwell, P. W., 359 Randall, B. L., 54, 74 Randall, V. A., 163, 178 Ranger, A. M., 266, 289 Rankin, E. C. C., 383, 410 Ransburg, N. J., 468 Rao, A., 260, 285, 289 Rao, B. G., 247, 249 Rao, M. S., 204, 205 Rapaport, M., 177 Rapp, J. H., 111 Rappaport, E. B., 211 Rapson, N., 411 Rascati, K. L., 3, 12 Rasetti, C., 40, 69 Raskin, P., 201, 202, 211 Rasmussen, R. R., 470 Rasmusson, G. H., 149, 174, 176, 177, 178 Rasmusson, G. R., 175 Rasori, R., 138 Ratner, L., 214, 249 Rattigan, S., 208 Rau, R., 410, 411 Raub, T. J., 249 Raubitschek, A., 414 Raule, G., 43, 70 Ravaioli, B., 179 Ravnikar, V., 367 Ravoux, A. C., 359 Ray, G. T., 285 Raynaud, J. P., 175, 362, 363 Rayner, M. M., 246, 247, 248 Raz, A., 117, 138, 140, 141 Razon, N., 420

AUTHOR INDEX

Re, R., 46, 51, 72 Read, E., 249 Rebello, P. R., 411 Reddel, R. R., 296, 344, 366 Reddy, E. P., 248 Reddy, G. C., 473 Reddy, J. K., 203, 204, 205 Reddy, M. K., 203 Reddy, M. N., 203 Redfield, R., 246 Redford, J., 361 Redgrave, J., 17, 63 Redmond, C., 360 Redmond, C. K., 360 Redshaw, S., 249 Reece Smith, H., 173, 178 Reed, K., 245 Reed, K. L., 246 Reedijk, M., 220, 249 Reese, D. M., 417 Reff, M. E., 390, 413 Regan, D., 414 Regazzi, M. B., 93, 113 Register, E., 470 Reglier, J. C., 41, 48, 69 Reiber, J. H. C., 111 Reich, R., 367 Reich, S., 249 Reich, S. H., 247 Reid, C., 249 Reid, J., 41, 69 Reid, J. L., 39, 42, 54, 56, 68, 70 Reihner, E., 89, 113 Reinecke, H., 40, 69 Reines, S., 112 Reinhart, K., 379, 407 Reinherz, E. L., 374, 405 Reinhold, B. B., 470 Reis, M. D., 415 Reisch, C., 54, 74 Reiser, M., 244, 249 Reiss, E., 430, 470, 473 Reitman, M., 207 Remme, W. J., 48, 72 Renaud, J. P., 368 Renedo, G., 46, 53, 72 Rennke, H. G., 46, 53, 54, 71, 72, 74 Reny, J. C., 41, 44, 69 Resche-Rigon, M., 303, 366 Resnick, M., 178 Resnick, S., 362

531

Reuter, V., 419 Rew, D. J., 108 Rex, J. H., 423–466, 471, 472, 473, 474, 475 Rey, F., 245 Reyes, A., 46, 53, 72 Reyes, F., 414 Reyner, E. L., 250 Reynolds, G. F., 174, 177 Rheaume, E., 363 Rhodes, L., 164, 173, 178 Rhymer, P. A., 110 Ribadeau-Dumas, A., 40, 53, 69 Ribas, J. C., 433, 446, 467, 469, 473 Ribichini, F., 53, 74 Ribstein, J., 54, 74 Ricci, C., 175 Rice, J., 287 Rich, D. H., 34, 67, 250 Richard, S., 416 Richard, V., 363 Richards, D. H., 109 Richards, J. P., 362 Richards, J. S., 117, 141 Richardson, J., 289 Richardson, K., 425, 471 Richer, C., 46, 71 Richer, J. K., 361 Richie, J. A., 475 Richman, D. D., 246 Richter, W. F., 384, 410 Rico, H., 473 Ricote, M., 193, 209 Ridgway, J. B. B., 418 Riechmann, L., 372, 404 Rieger, M. M., 68 Rieke, C. J., 137 Therien, M., 138, 140, 141 Riendeau, D., 118, 126, 138, 139, 140, 141 Riess, W., 203 Rietschel, R., 177 Rieusset, J., 207 Rifkin, M. D., 418 Riggs, B. L., 346, 359, 364, 366 Riley, W. A., 109 Rinaldi, M. G., 467, 469, 472, 473 Rinaldi, M. J., 469 Rindi, G., 416 Ring, D. M., 420 Ringel, J., 189, 207 Ringold, G. M., 206

532

AUTHOR INDEX

Riordan, J. F., 20, 64 Riou, J. P., 207 Rippy, M. K., 366 Riser, L., 473 Rissanen, V., 43, 70 Ristow, M., 189, 207 Ritchie, A. J., 249 Ritsema Van Eck, H. J., 42, 70 Rittenhouse, J. W., 246 Rittmaster, R. S., 148, 149, 151, 153, 176, 178, 180 Ritz, E., 54, 74 Rivera, C., 473 Rivera, F., 137 Rivest, S., 363 Riviere, A., 395, 417 Rizer, R. L., 365 Ro, J. Y., 418 Robb, C. A., 48, 72 Robert, J., 362 Robert, N. J., 419 Roberts, A. B., 357 Roberts, J., 169, 178 Roberts, J. L., 177 Roberts, N. A., 227, 228, 249 Roberts, R. S., 15, 62 Roberts, S. G., 362 Robertson, D. L., 141 Robertson, J., 361 Robertson, J. F., 359 Robertson, J. I., 26, 65 Robertson, J. I. S., 16, 17, 27, 63, 66 Robidoux, A., 360 Robins, T., 247, 249 Robinson, B., 246 Robinson, B. F., 22, 41, 65, 69 Robinson, D. C., 315, 366 Robinson, F. M., 27, 28, 29, 32, 66 Robinson, J., 364 Robinson, J. P., 365 Robinson, R. A., 417 Robinson, S. P., 360 Rochefort, H., 358 Rochold, F. W., 44, 71 Rockwell, P., 421 Rockwell, R. F., 421 Rodan, G. A., 204 Rode, R., 245, 246 Rodeheffer, R. J., 57, 75 Roden, D. M., 248 Rodenhuis, S., 414

RodÈs, J., 137 Rodger, I., 138 Rodger, I. W., 137, 138, 140, 141 Rodgers, J. D., 218, 219, 222, 249 Rodier, M., 41, 69 Rodkey, J. A., 248 Rodriguez, F. C., 471 Rodriguez, G., 417 Rodriguez, L. J., 425, 473 Rodriguez, M., 468 Rodriguez, M. J., 468 Roecker, E. B., 208 Roederer, G., 109 Roehrborn, C. G., 156, 157, 158, 159, 174, 177, 178 Rohde, G., 112 Rohde, R. D., 3, 12, 47, 53, 72 Roheim, P. S., 351, 359 Roig, L., 358 Rojas, E., 473 Rokosz, L. L., 289 Rollema, H. J., 174, 178 Romancheck, M. A., 473 Romas, N. A., 174, 176 Romeo, O. M., 209 Romero, D. L., 250 Romines, K. R., 250 Rommele, G., 467 Romo, M., 109 Ronald, K., 359 Roncero, C., 473 Ronnett, G. V., 286 Ronsini, S., 179 Roos, B. E., 109 Roos, W., 296, 344, 363, 366 Roots, I., 112 Roques, B. P., 62, 75 Rose, D. W., 205, 361, 367 Rose, M., 43, 70, 108 Rose, R., 246 Rosel, J., 245, 246 Rosen, D. J., 358 Rosen, E. D., 190, 208 Rosen, G. D., 117, 141 Rosen, H., 466, 468, 469, 474 Rosen, P. J., 365 Rosen, P. P., 419 Rosen, R. C., 111 Rosenberg, J., 413 Rosenblat, G., 176 Rosenblatt, S., 176, 178

533

AUTHOR INDEX

Rosenfeld, M. G., 205, 361, 367, 368 Rosenfield, R. L., 176 Rosenmund, C., 289 Rosenthal, T., 68 Roses, A. D., 11, 12 Rosina, B., 175 Roskams, A. J., 286 Rosner, B., 109, 358, 366, 367 Ross, C., 108 Ross, C. W., 414 Ross, D. T., 287, 290 Ross, J. S., 395, 418 Ross, K. A., 361 Ross, R., 109, 361 Ross, S., 209 Rossat, J., 132, 141 Rosseneu, M., 366 Rossouw, J. E., 110 Roth, D., 93, 110 Roth, E., 250 Roth, J., 207 Roth, R. J., 117, 141 Rothberg, A. D., 56, 75 Rothman, M., 421 Rothrock, J., 108 Rothrock, J. W., 27, 28, 29, 32, 66 Rotman, H., 137 Roudebush, R. E., 362 Rouleau, J. L., 113 Roulston, J. E., 37, 68 Roumen, R. M., 359 Round, E., 177 Rousseau, A., 41, 69 Roussel, S., 245 Rouvtiere, N., 286 Roux-Lombard, P., 410 Rouzioux, C., 245 Rovnyak, G., 35, 67 Rovnyak, G. C., 34, 67 Rowden, G., 178 Rowland, A. M., 418 Rowley, E. R., 360 Roy, J., 178, 362 Roy, J. B., 176 Roy, K., 431, 472, 473 Roy, M., 412 Roy, P., 137, 138, 139, 140 Royston, I., 413 Rozenbaum, W., 245 Rozman, B., 411 Ru, Y., 248

Rubens, R., 367 Rubin, B., 21, 22, 24, 25, 26, 35, 45, 65, 67, 71 Rubin, C., 202, 212 Rubin, J., 250 Rubin, J. R., 250 Rubin, S. M., 360 Rudas, L., 112 Ruddon, R. W., 36, 67, 114 Rude, T. H., 474 Rudling, M., 113 Ruegger, A., 254, 289 Ruf, H. H., 138 Ruggenenti, P., 47, 53, 72 Ruilope, L. M., 44, 71 Ruiz, L., 247 Ruiz-Ortega, M., 46, 53, 72 Rumberger, J. M., 207 Rumke, P., 414 Rummukainen, J., 113 Rupniak, N., 140 Rush, B. D., 250 Rush, J. E., 33, 67 Russell, D. W., 146, 172, 174, 176, 177, 178, 179 Russell, J. C., 351, 366 Rutgeerts, P., 412 Rutherford, J. D., 113 Rutishauser, G., 179 Rutkowski, C. A., 249 Rutledge, S. J., 204 Rutqvist, L. E., 298, 360, 366 Rutsch, W., 406 Ruwart, M. J., 250 Ruyle, W. V., 27, 28, 29, 32, 66 Thervet, E., 405 Ryan, A., 361 Ryan, C. F., 141 Ryan, T. J., 49, 51, 73 Ryde, C., 359 Ryder, J. W., 208 Ryono, D. E., 35, 67

S Saad, F., 418 Saag, K., 129, 141 Saag, M., 248 Sabatini, D. M., 286, 290 Sable, C. A., 462, 466, 473 Sabo, E. F., 21, 22, 23, 24, 25, 26, 27, 29, 65

534

AUTHOR INDEX

Sacco, M. G., 416 Sack, M., 410 Sackett, D. L., 15, 47, 48, 62, 72 Sacks, F., 367 Sacks, F. M., 92, 97, 98, 99, 100, 102, 103, 104, 106, 107, 113 Sacks, P. G., 420 Sader, H., 472 Sadick, N. S., 175 Sadoff, J. C., 408 Sadoshima, J. U., 47, 72 Sadowski, H. B., 140 Saeki, M., 114 Safai, B., 246 Safar, M. E., 40, 68 Sagnella, G. A., 43, 71 Sagoo, J. K., 270, 277, 290, 291 Sahin, Y., 170, 178 Sahoo, S. P., 205 Saigo, K., 250 Saino, A., 51, 73 Sainsbury, J. R., 403, 420 Saito, Y., 86, 113 Saitoh, K., 416 Saitou, A., 469 Sajid, M., 406 Sakaguchi, M., 45, 51, 71 Sakaguchi, O., 430, 473 Sakai, T., 111 Sakai, Y., 111 Sakamoto, K., 469 Sakamoto, N., 470 Sakane, K., 471 Sakonjo, H., 52, 73 Saladin, R., 204 Salahuddin, S. Z., 246 Saldivar, A., 247, 249 Saleh, M., 414 Salgado, H. C., 37, 68 Salgo, M., 246 Salgo, M. P., 245 Salom, I. L., 139 Salomen, J., 43, 70 Salomen, R., 43, 70 Salonen, J. T., 113 Salonen, R., 101, 107, 113 Salowe, S., 289 Saltiel, A. R., 195, 197, 210, 211 Saltzman, B., 178 Salvadori, M., 47, 53, 72 Salvetti, A., 36, 67

Salzberger, B., 249 Salzman, A., 211 Samson, B., 362 Samuelsson, B., 138 San, B. F., 468 San, B. G., 468 Sanchez, R., 366 Sanda, M., 139 Sander, O., 410 Sanders, C., 408 Sangiorgi, F. A., 53, 74 Sanglard, D., 427, 455, 473, 474 Sanmarco, M. E., 108 Santanello, N. C., 110 Santini, C., 205 Santos, B., 467 Saperstein, R., 204 Sapp, S. K., 406 Sardana, V. V., 250 Sarkar, F. H., 417 Sarnacchiaro, F., 179 Sarngadharan, M. G., 249 Sarraf, P., 194, 205, 208, 210 Sarup, J. C., 396, 417, 418 Sasaki, G. H., 363 Sasaki, H., 361 Sasaki, M., 416 Sasaki, T., 417 Sassano, P., 39, 43, 56, 68, 70, 75 Satchanska, G., 471 Sathyanarayana, B. K., 248, 251 Sato, J. D., 421 Sato, M., 317, 318, 358, 359, 360, 366, 367 Satoh, S., 208 Satoi, S., 431, 471, 473 Satyaswaroop, P. G., 346, 358, 360, 366 Saunders, C., 359 Saunders, D., 469 Saunders, F. J., 145, 178 Saunders, J. M., 247 Sausville, E. A., 414 Sauter, G., 357 Savin, R., 177, 178 Savin, R. C., 175 Savoie, C., 138 Savory, R., 204 Sawada, K., 177 Sawant, S. N., 472 Sawaya, M. E., 169, 178 Sawin, H. S., 108 Sawyer, T. K., 250

AUTHOR INDEX

Scalese, B., 35, 67 Scalese, R., 34, 67 Scallon, B. J., 384, 410 Scanlan, T. S., 365 Schaber, M. D., 248 Schaefer, E., 96, 113 Schaefer, M., 405 Schaeffer, J. M., 205 Schaeffer, T. R., 21, 45, 65, 71 Schafer, A. J., 207 Sch‰ffer, W., 154, 174, 178 Schaible, T., 412 Schalekamp, M. A., 16, 63 Schalekamp, M. A. D. H., 18, 64 Schalekamp-Kuyken, M. P. A., 18, 64 Schaller, M., 416 Schambelan, M., 248 Schaper, J., 46, 48, 55, 71 Schaufele, R. L., 472 Schechter, A. L., 394, 415 Scheel, K. W., 16, 63 Scheer, S., 251 Schein, R. M. H., 408 Scheld, K. G., 248 Schelfhout, W., 367 Schell, W. A., 468 Schellhammer, P. F., 178 Schelling, J. L., 32, 33, 67 Schelling, P., 47, 72 Scher, H. I., 421 Scherr, M. H., 470 Schiano, M. A., 366 Schieffer, B., 51, 73 Schieffer, E., 51, 73 Schiff, I., 367 Schiff, M., 411 Schiff, M. H., 404, 410 Schilder, R. J., 413, 414 Schiller, C. D., 410 Schindler, J., 414 Schipper, P. J., 248 Schirmer, T., 289 Schlabach, A. J., 250 Schleif, W. A., 246, 248, 250 Schlessinger, J., 416, 420 Schlitt, H. J., 405 Schlitz, R. L., 358 Schlossman, S., 405 Schlossman, S. F., 413 Schluter, G., 95, 114 Schmatz, D., 437, 467, 473, 474

535

Schmatz, D. M., 437, 467, 473, 475 Schmid, F. X., 287 Schmidt, A., 184, 204 Schmidt, J., 96, 113 Schmitt, C., 113 Schmitt, H., 39, 68 Schmitz, P. I. M., 420 Schnaper, H., 108 Schneider, E. F., 109 Schneider, H., 248 Schneider, J., 248, 250, 251 Schneider, J. J., 144, 179 Schneider, M., 469 Schneider, R., 202, 211, 212 Schnell, C., 55, 74 Schnitt, S. J., 419 Schnitzer, T. J., 385, 411 Schock, H. B., 235, 245, 249 Schodin, D. J., 362 Schoeller, D. A., 86, 111 Schoenfeld, D. A., 245 Schofield, B., 108 Schofield, P., 367 Scholer, J. H., 426, 473 Scholkens, B. A., 46, 47, 48, 55, 60, 71, 72, 75 Scholl, S., 419 Schonbaum, E., 299, 363 Schonbeck, U., 210 Schonberg, G., 108 Schooley, R., 246 Schoonjans, K., 193, 209 Schotborgh, R. H., 141 Schreiber, S. L., 254, 275, 285, 286, 287, 288, 290, 291 Schroder, F. H., 151, 179 Schror, K., 111 Schrott, H., 110, 113 Schubiger, G., 56, 75 Schulz, K. F., 8, 12 Schulze-Koops, H., 412 Schulzer, M., 140 Schupp, J., 137 Schuster, A. E., 249 Schutz, G., 205 Schwabe, J. W., 207 Schwart, M., 474 Schwartz, D. A., 417 Schwartz, E., 178 Schwartz, H., 139 Schwartz, J., 34, 35, 67

536

AUTHOR INDEX

Schwartz, J. I., 130, 141, 171, 172, 179 Schwartz, K., 46, 71 Schwartz, L., 112 Schwartz, M. S., 113 Schwartz, P. E., 364 Schwartz, R. E., 436, 437, 467, 469, 473 Schwartz, R. S., 53, 74 Schwartz, S., 201, 211 Schwartz, S. L., 112 Schwartz, S. M., 47, 72 Schwende, F. J., 249, 250 Schwerdtfeger, A., 360 Schwinzer, R., 405 Scicli, A. G., 15, 17, 53, 63, 74 Sciuto, A., 175 Scolnick, E. M., 1–11, 248 Scorsone, K., 418 Scott, A., 286 Scott, G. K., 417 Scott, J. D., 289 Scott, P. J., 15, 62 Scott, P. M., 467 Scott, R., 112 Scott, S., 113 Scott, T., 467 Scotto, C., 204 Scully, R., 368 Sealey, J. E., 22, 37, 38, 40, 51, 65, 68, 69, 73 Seaman, W. E., 412 Seard, M. E., 364 Sebire, K., 246 Sedman, A. J., 68, 109 Seed, B., 209 Seest, E. P., 250 Segal, R., 44, 48, 71 Segatto, O., 416 Sehajpal, P. K., 288 Seibel, K., 363 Seibert, K., 138, 139, 140 Seibold, J. R., 138 Seidenberg, B., 137, 141 Seidman, A. D., 419 Seiler, C., 102, 113 Sein, J. J., 414 Sein, T., 472 Seitz, S. P., 249 Sekihara, H., 208 Sela, M., 416 Selitrennikoff, C. P., 441, 466, 474, 475 Selk, L., 248 Selk, L. M., 251

Selkurt, E. E., 16, 63 Selwyn, A. P., 114 Semba, K., 394, 415, 416 Sen, S., 46, 71 Senderowicz, A., 414 Senge, T., 175 Sentandreu, R., 468 Sereni, D., 249 Serrafini, P., 169, 179 Serrurier, D., 43, 70 Sesin, D. F., 473 Sessa, F., 416 Sever, P., 43, 70 Sewell, D. L., 457, 473 Sexton, G. J., 82, 111 Shackelton, C. H. L., 177 Shackleton, C., 176 Shade, R. E., 22, 65 Shadron, A., 469 Shafiq, M. C., 469 Shah, A. S., 359 Shah, P. M., 469 Shah, S., 138 Shahane, A., 112, 139 Shahinian, A., 407 Shak, S., 419, 421 Shalaby, R., 420 Shalita, A., 177 Sham, H. L., 218, 220, 234, 247, 249 Shamblen, E. C., 179 Shamiss, A., 68 Shanmuganathan, K., 177 Shao, Y., 420 Shapira, J., 358 Shapiro, D., 113 Shapiro, D. R., 109 Shapiro, J., 177 Share, L., 22, 65 Sharkey, R. M., 415 Sharma, A. M., 207 Sharpe, T. R., 247, 248 Shaw, G. L., 289 Shaw, J.-P., 260, 290 Shaw, K. J., 469 Shaw, K. T., 288 Shaw, T., 248 Shaw, W. C., 33, 67 Shear, C. L., 108, 111, 114 Shearer, G. M., 246 Shearer, J., 177 Shedletzky, E., 436, 474

AUTHOR INDEX

Sheedy, K. M., 177 Sheehan, C. E., 418 Sheet, C. T., 468 Shei, G. J., 468 Sheiner, L. B., 42, 70 Shelby, M. D., 104, 113 Sheldon, D., 56, 75 Sheldon, T. A., 15, 62 Shelton, B. A., 210 Shen, D., 414 Shenolikar, S., 289 Shenouda, A. N., 22, 65 Shepard, H. M., 417, 418 Shepart, II, T. H., 365 Shepherd, J., 91, 92, 97, 98, 99, 101, 103, 104, 113 Sher, T., 183, 204 Sherer, R., 246 Sheridan, C. M., 285 Shetty, B. V., 247 Shi, Y., 412 Shiau, A. K., 307, 366 Shibasaki, F., 260, 271, 273, 290 Shibata, H., 360 Shieh, H.-S., 246, 250 Shigematsu, N., 469 Shih, C., 394, 415 Shih, W., 114 Shima, T. B., 367 Shimazaki, J., 178 Shimizu, Y., 114 Shimoi, H., 470 Shimokawa, T., 123, 141 Shimomura, K., 362 Shinar, D., 204 Shine, J., 360 Shingleton, H. M., 417 Shiota, K., 208 Shiota, N., 51, 73 Shiou, L., 250 Shiraki, T., 208 Shiratori, Y., 470 Shore, A. C., 43, 71 Shou, W., 263, 282, 287, 290 Shown, T., 178 Shrader, M. W., 357 Shu, K., 208 Shuldiner, A. R., 207 Shulman, G. I., 208 Shum, L., 247 Shumway, N. P., 17, 18, 63, 64

537

Shurzinske, L., 112 Shuster, P., 33, 67 Shvu, K. G., 288 Sialer, S., 33, 67 Siegel, J. A., 415 Siegel, N., 140 Siekierka, J. J., 256, 286, 290 Sigal, I. S., 248 Sigal, N. H., 258, 290 Signomklao, T., 366 Sigthorsson, G., 137 Siiteri, P. K., 145, 179 Sikic, B. I., 251 Silva, A. J., 264, 290 Silver, K., 207 Silver, R. I., 179 Silverman, D. H. S., 414 Silverman, H., 407 Silverman, J. A., 247 Silverstein, A. M., 370, 404 Silvestre, J., 249 Simard, J., 293–357, 359, 360, 362, 363, 366 Simmer, R., 246 Simmons, D., 139 Simmons, D. L., 141 Simmons, H. A., 362 Simon, D. I., 379, 407 Simon, H., 33, 67 Simon, L. S., 3, 12 Simon, T., 137, 139 Simon, T. J., 139 Simons, J., 113 Simons, L. A., 102, 113 Simoons, M. L., 406 Simpson, N. B., 164, 179 Sing, K., 411 Singer, D. R. J., 43, 71 Singer, I. I., 82, 113, 174 Singer, S., 210 Singh, G., 141 Singh, K., 417 Singh, S. M., 363, 366 Singhvi, S. M., 26, 36, 65, 67 Singleton, T. P., 418 Sinn, E., 416 Sintchak, M. D., 287 Sioufi, A., 137 Siragy, H. M., 17, 64 Sirois, J., 117, 141 Sjoblom, T., 109

538

AUTHOR INDEX

Skalka, A. M., 248 Skeggs, L. T., 14, 17, 18, 62, 63, 64 Skeggs, L. T. Jr., 18, 64 Skelton, M. M, 15, 63 Skidgel, R. A., 19, 64 Sklarin, N., 419 Sklarin, N. T., 419 Skoback, D., 17, 63 Skoglund, M. L., 138 Skoog, L., 360 Skorey, K., 137, 139 Skulnick, H. I., 225, 249, 250 Slade, D. E., 249 Slamon, D., 398, 419, 421 Slamon, D. J., 395, 416, 417, 418, 419 Slater, E. E., 1–11 Slaughenhoupt, B. L., 413 Slayter, H. S., 47, 72 Sleight, P., 43, 47, 70, 72 Sliwkowski, M., 419 Sliwkowski, M. X., 396, 417, 418 Sluka, J. P., 360 Smalley, W., 210 Smalley, W. E., 138 Smart, C. J., 178 Smeets, T. J., 410, 411 Smidt, M., 245 Smink, R. D., 108 Smith, A. M., 290 Smith, B., 413, 472 Smith, C., 406 Smith, C. A., 404 Smith, C. L., 310, 311, 312, 323, 366 Smith, C. R., 408 Smith, C. W., 407 Smith, D. C., 346, 358, 366 Smith, D. G., 206 Smith, E. P., 295, 366 Smith, F. M., 48, 72 Smith, I. E., 359 Smith, J., 161, 179, 249 Smith, J. B., 117, 141 Smith, J. G., 459, 460, 466, 467, 468, 469, 474 Smith, J. L., 469 Smith, J. M., 357 Smith, K. R., 458, 474 Smith, L. D. R., 114 Smith, M., 246 Smith, P. F., 93, 113 Smith, P. H., 161, 179 Smith, P. J. B., 178

Smith, R., 209 Smith, R. G., 204, 205, 206 Smith, S. A., 206, 208 Smith, S. M., 408 Smith, W. L., 125, 137, 138, 139, 140, 141 Smithies, O., 139, 140 Smith-Oliver, T. A., 203, 211 Smolen, J., 137, 369–403, 409 Smolen, J. S., 409, 410, 411 Snibson, G., 110 Sniderman, A., 110 Snyder, S. H., 262, 286, 288, 290 Snydman, D. R., 472 Sobel, J. D., 474 Sobel, R. A., 291 Soderling, T. R., 287 Soderstrom, M., 361 Sodroski, J. G., 246 Soffer, R. L., 18, 20, 21, 22, 64 Solinger, A., 411 Solis, F., 471 Soll, D. R., 472 Solomon, D., 414 Somell, A., 360 Somers, E. P., 210 Somers, P. K., 258, 285, 290 Somers, R., 414 Somers, W. J., 157, 179 Son, H., 291 Song, K., 51, 73 Song, S. U., 416 Sonicki, Z., 417 Sonnenberg, H., 15, 63 Soos, M. A., 207 Soper, J. T., 417 Sopwith, A. M., 410 Sorensen, E. K., 340, 343, 353, 354, 364 Soreq, H., 420 Sorkin, E. M., 68 Sorlic, P., 54, 74 Sorlie, P., 54, 74 Sosa, M. S., 471 Soubrier, F., 18, 19, 40, 64, 69 Sourla, A., 352, 355, 364, 366 Souza, S. C., 192, 209 Soyoola, E., 139 Spahn, M. A., 180 Spaventi, R., 417 Spaventi, S., 417 Specker, B., 366 Spector, N. L., 414

AUTHOR INDEX

Spector, S., 246 Speizer, F. E., 358, 366 Spencer, M. P., 16, 63 Spencer-Green, G., 123, 141 Spicer, T., 246 Spiegelman, B., 204 Spiegelman, B. M., 203, 205, 206, 208, 209, 210 Spies, S., 414 Spinelli, P. A., 250 Spinelli, S., 216, 250 Spinola, P. G., 359 Spitzer, J., 208 Spitzmiller, E. R., 34, 35, 67 Sporn, M. B., 357 Sprecher, D. L., 112 Sprent, J., 374, 405 Springer, J., 108 Springer, J. P., 246, 248 Springer, P. A., 367 Sprung, C. L., 408 Squires, K. E., 247 Sreenan, S., 209 Sridhar, S., 139 Srinivasan, S., 365 Sromovsky, J. A., 33, 67 St. Clair, W. E., 409 Stack, G., 362 Staddon, S., 415 Staels, B., 207, 209, 351, 366 Staessen, J. A., 40, 69 Stagg, R., 418 Stahlberg, D., 113 Stahlhut, M. W., 249 Stallcup, M. R., 361 Stallings, W. C., 139, 246, 250 Stamm, N. B., 176 Stampfer, M. J., 299, 301, 358, 366 Stanczyk, F. Z., 180 Standaert, R. F., 285, 291 Stanford, N., 141 Stange, E. F., 140 Stapley, E., 108 Starcich, B., 249 Startzl, T. E., 374, 405 Staruch, M. J., 286 Stashenko, P., 390, 413 Staszewski, 247 Staubli, W., 203 Staudinger, J. L., 362 Stauffer, L., 109

539

Stearns, V., 298, 366 Steckelings, U. M., 47, 72 Steele, G. Jr., 417 Steffenino, G., 53, 74 Stegeman, R. A., 246, 250 Stegman, R. A., 139 Stehelin, D., 205 Steigbigel, R., 247 Stein, E., 110, 112 Stein, E. A., 84, 85, 87, 88, 90, 91, 109, 110, 111, 113, 114, 176 Stein, P. P., 208 Stein, R., 415 Steinbaugh, B. A., 247 Steinberg, C., 49, 73 Steinbr¸ck, K., 138 Steiner, G., 110, 381, 409, 410 Steiner, J. P., 263, 264, 286, 287, 288, 290 Stemmer, P. M., 286 Stenstrom, J., 110 Stenzel, K. H., 288 Stepanavage, M., 113 Stephen, E., 359 Stephens, J. D., 33, 67 Stephens, S., 407 Stephenson, W. P., 111 Stern, D. F., 415 Stern, S., 139 Stern, Y., 367 Stetler-Stevenson, M., 414 Steven, R., 359 Stevens, A. M., 139 Stevens, C. F., 264, 290 Stevens, D. A., 441, 458, 468, 469 Stevens, D. L., 379, 380, 408 Stevens, R. E., 176 Stevenson, J. C., 346, 367 Stevenson, J. W., 108 Stevenson, R., 208 Stevenson, R. W., 191, 192, 208, 209 Stewart, B. H., 250 Stewart, J. M., 17, 20, 21, 63, 65 Stewart, K., 249 Stewart, K. D., 247 Stewart, R. W., 45, 71 Stieglitz, S., 179 Stiepel, A., 249 Stillabower, M. E., 114 Stimpel, M., 68 Stoddard, B. L., 277, 290 Stojanovic, M., 364

540

AUTHOR INDEX

Stokes, J. B., 468 Stoll, K., 245 Stoll, M., 47, 72 Stoll, R. W., 361 Stolow, M., 290 Stolzenbach, J., 245 Stone, C. A., 28, 30, 66 Stone, E., 116, 141 Stone, J., 467 Stone, J. A., 461, 474 Stone, M., 414 Stone, M. J., 393, 414 Stone, S. H., 473 Stoner, E., 143–174, 176, 177, 179 Storm, H. H., 357 Storm-Dickerson, T., 287 Stortz, C. A., 470 Stouffer, G. A., 378, 406 Stover, D., 245 St-Pierre, A., 364 Strandberg, T., 112 Stratford, R., 462, 475 Stratford, R. E., 475 Straube, R. C., 408 Strauch, G., 173, 179 Stremler, K. E., 404 Strohbach, J. W., 250 Stronkhorst, A., 412 Stuart, S. G., 416 Stubbs, R. J., 110, 113 Studer, R., 40, 48, 69, 72 Studnicka-Benke, A., 383, 384, 409, 410 Sturla, A., 176 Sturrock, E. D., 20, 64 Stuver, C. M., 249 Styanczyk, F. Z., 177 Su, K. S., 247 Suarez, L., 53, 74 Suda, H., 27, 33, 66, 472 Suda, Y., 394, 416 Sudhof, T. C., 111 Suematsu, H., 467 Sugar, A. M., 458, 474 Sugawa, N., 403, 421 Sugawara, K., 466 Sugden, P. H., 267, 290 Sugimoto, Y., 363 Sugiyama, T., 208 Sukhova, G., 210 Suldan, Z., 366 Sullivan, D., 113

Sullivan, D. J., 472 Sullivan, P. A., 38, 68 Summerhayes, I. C., 417 Sun, E., 245, 246, 248, 249 Sun, L., 272, 290 Sun, W., 139 Sundblad, K. J., 141 Sundelof, J. G., 469 Sundseth, S. S., 206 Superko, H. R., 110 Surawicz, T. S., 364 Surbone, A., 419 Sussman, H. A., 51, 73 Sutcliffe, F., 359 Suter, T. M., 113 Suthanthiran, M., 288 Sutherland, D. J., 366 Sutherland, R. L., 296, 299, 344, 358, 365, 366 Sutton, D. A., 469, 472 Sutton, G. C., 52, 73 Suvorov, L. I., 250 Suzdak, P. D., 290 Suzuki, H., 472 Suzuki, M., 290, 473 Suzuki, S., 465, 474 Suzuki, T., 20, 65, 416 Svenson, J., 138 Svensson, C., 205 Swain, A. L., 221, 250 Swales, J. D., 38, 68 Swaminathan, S., 247 Swan, A. V., 114 Swan, S. K., 132, 141 Swanson, S. A., 286 Sweet, C. S., 14, 20, 28, 30, 32, 62, 64, 66 Sweny, P., 405 Swerlick, R. A., 410 Swick, A. G., 208 Swinehart, J. M., 179 Sybert, E. G., 18, 64 Szalkowski, D., 192, 209 Szaniszlo, P. J., 450, 470, 474 Szatrowski, T. P., 105, 114 Szczech, L. A., 374, 405 Szekely, A., 457, 474

T Tabuchi, H., 471 Tachedjian, G., 246

AUTHOR INDEX

Tachibana, Y., 470, 471 Tadros, S. S., 26, 66 Taft, C. S., 436, 441, 474, 475 Tagari, P., 137, 138, 139, 140 Tai, T.-A., 209 Tai, V. W., 248 Tait, B. D., 226, 247, 250 Tak, P. P., 382, 385, 410, 411 Takada, M., 471 Takagi, G., 289 Takagi, N., 385, 411 Takahashi, H., 366 Takahashi, N., 256, 290, 404 Takahashi, S., 138, 177 Takahashi, T., 365 Takai, S., 45, 51, 52, 71, 73 Takai, Y., 468, 472 Takashima, H., 51, 73 Takasugi, H., 471 Takasuka, T., 469 Takaya, T., 467 Takeda, R., 111 Takeshita, A., 329, 367 Taketo, M. M., 140 Takeuchi, T., 27, 33, 66 Takewaki, N., 469 Takimoto, G. S., 361 Takimoto, H., 286 Talley, J., 245 Talley, J. D., 114, 406 Talley, J. J., 246, 250 Tam, S. H., 410 Tamai, O., 102, 114 Tamamoto, H., 208 Taminiau, J., 412 Tammela, T., 154, 179 Tammela, T. L. J., 174, 178 Tamori, Y., 207 Tan, M. H., 109 Tanaka, H., 209, 255, 276, 291, 471 Tanaka, K., 468, 472 Tanaka, M., 417 Tanaka, S., 208 Tanaka, S. K., 471 Tanaka, W. K., 175, 179 Tan-Chiu, E., 360, 417 Tanen, M., 205 Tanen, M. R., 361 Tang, J., 215, 247, 250, 452, 467, 468, 474 Tang, M. X., 301, 367 Tang, S. C., 394, 415

Tanner, M. M., 357 Tanner, T., 249 Tanzawa, K., 109, 111, 114 Tao, Y., 360 Tarazi, R. C., 40, 46, 69, 71 Targan, S., 412 Targhetta, R., 17, 63 Tarpley, W. G., 249 Tashiro, H., 365 Tasken, K., 363 Taskinene, P. J., 363 Tatami, R., 111 Tate, A., 112 Tate, A. C., 111 Tatlock, J. H., 247 Tatti, P., 60, 75 Taub, D., 27, 28, 29, 32, 66 Tavassoli, M., 417 Tawara, S., 471, 474 Taylor, A., 176, 178 Taylor, A. M., 110, 138, 175, 177 Taylor, D. L., 249 Taylor, H. R., 111 Taylor, J. A., 414 Taylor, J. W., 424, 474 Taylor, P. C., 410 Taylor, R. R., 112 Taylor, S., 43, 71 Taylor, V., 473 Taylor, W. R., 215, 227, 249 Tcheng, J. E., 377, 406 Tchobroutsy, C., 56, 75 Tebiesebeke, R., 474 Tedder, T. F., 390, 413 Teece, D. J., 2, 11 Tegeder, I., 128, 141 Tegoshi, T., 471 Teich, N., 245 Teillac, P., 175 Temin, H., 245 Tempany, C. M. C., 154, 179 Temple, R., 10, 12, 42, 57, 70, 75 Tempst, P., 203, 366 ten Bokkel Huinink, W. W., 414 ten Broeke, J., 27, 28, 29, 32, 66 Teng, C. T., 361 Tenhoopen, R., 473 Tennant, S., 467 Tenover, J. S., 151, 176, 179 Tepper, R., 358 Terakawa, M., 474

541

542

AUTHOR INDEX

Teratani, N., 471 Terauchi, Y., 208 Termine, J. D., 358 Terranella, L., 177 Terz, J., 360 Tesser, J. R. P., 386, 411 Testut, P., 18, 64 Teutsch, G., 365 Thaisrivongs, S., 223, 224, 226, 249, 250 Thakkar, A. L., 469 ThÈriault, C., 363 Thibeault, D., 248 Thibonnier, M., 53, 74 Thiboutot, D., 147, 172, 174, 177, 179 Thieringer, R., 193, 210 Thigpen, A. E., 146, 172, 179 Thistlewaite, J., 374, 405 Thomas, D. J., 289 Thomas, E. D., 413 Thomas, G. J., 249 Thomas, L. G., 2, 12 Thomas, L. N., 178, 180 Thomas, N., 359 Thompson, A. R., 361 Thompson, D. D., 362 Thompson, D. J., 366 Thompson, D. L., 176 Thompson, E. W., 296, 316, 358, 367 Thompson, G., 110 Thompson, G. M., 209 Thompson, G. R., 82, 111, 114 Thompson, I., 175, 176 Thompson, I. M., 161, 179 Thompson, J. R., 449, 453, 474 Thompson, R., 469 Thompson, S. G., 111 Thompson, W. J., 246, 248 Thomson, D. B., 366 Thomson, J. A., 287 Thor, A. D., 395, 417 Thorgeirsson, G., 112, 113 Thornberry, N. A., 32, 66 Thornby, J., 407 Thornton, J. M., 178 Thornton, P., 415 Thorp, J. M., 182, 203 Thorsett, E. D., 27, 28, 29, 32, 33, 66 Thummel, C., 205 Thurston, H., 38, 68 Thynne, G. S., 344, 364 Thysell, H., 108

Tiano, H. F., 139, 140 Tigerstedt, R., 14, 62 Tighe, H. P., 415 Tikkanen, M. J., 110, 299, 367 Till, A. E., 32, 33, 67 Till, M., 414 Tilly, H., 414 Timerman, A. P., 288 Timmerman, L. A., 260, 286, 289, 291 Tindall, E. A., 404, 410 Ting, A. T., 209 Tintelnot-Blomley, M., 245, 246 Tio, R. A., 51, 73 Title, L. M., 112 Titos, E., 137 Tjandramaga, T. B., 179 Tkacz, J. S., 431, 436, 469, 474 Tobe, K., 206, 208 Tobert, J., 110 Tobert, J. A., 77–107, 109, 110, 111, 112, 113, 114 Tobian, I., 37, 68 Tocco, D. J., 29, 66 Toda, N., 45, 71 Todd, I. D., 358 Todd, P. A., 33, 67, 68 Toffaletti, D. L., 474 Toh, H., 214, 250 Tohidast-Akrad, M., 409 Toke, D. A., 472 Tokuda, M., 288 Tolino, A., 170, 179 Tolman, R. L., 174, 175, 176, 205 Tomich, P. K., 249, 250 Tomishima, M., 471 Tomizawa, K., 288 Tomkins, G. M., 144, 177, 179 Tomlinson, A. M., 139 Tommasi, R. A., 250 Tompkins, R. G., 407 Tonegawa, S., 370, 404 Toney, J., 175 Tonizzo, M., 109 Tontonoz, P., 183, 193, 203, 206, 209, 210 Toole, J. J., 290 Toomey, R. E., 176 Topel, E. J., 109 Topol, E. J., 3, 12, 376, 377, 378, 405, 406 Topol, I. A., 250 Tora, L., 322, 360, 367 Torchia, J., 184, 205, 323, 361, 367

543

AUTHOR INDEX

Torii, N., 265, 291 Tormey, D. C., 302, 367 Torney, L., 417 Torp-Pedersen, C., 43, 70 Torrance, D. S., 404 Torre-Amione, G., 407 Torres, G., 246 Tosch, W., 474 Toseland, C. D. N., 211 Tosi, F., 175, 177 Toth, L. N., 250 Toth, M. V., 248 Tovcimak, A., 471 Toyoshima, K., 245, 415, 416 Toyoshima, Y., 208 Traber, R., 431, 474 Trachtenberg, J., 176 Trainor, C., 467, 475 Trainor, G. L., 245, 249 Tran, L. E., 469 Transbol, I. B., 358 Tranum, B. L., 361 Traxler, P., 431, 474 Trayner, I., 114 Trazzi, S., 43, 70 Treacher, D., 407 Treasure, C., 114 Treasure, C. B., 102, 110, 114 Tredway, D., 176 Tregear, G., 19, 64 Trejecer, G., 40, 69 Tremblay, A., 325, 326, 329, 363, 367 Tremblay, G., 110, 363 Tremblay, G. B., 302, 304, 315, 321, 322, 323, 324, 328, 329, 330, 332, 353, 367 Tremblay, P. J., 416 Trent, J. M., 357 Trinh, H., 410 Tripathy, D., 417, 419, 421 Tristani, F., 49, 73 Tristram, E. W., 27, 28, 29, 32, 66 Trivedi, A., 361 Troy, A. E., 208 Trump, B. F., 139 Truss, M., 357 Trzaskos, J. M., 138, 140 Tsai, A., 141 Tsai, M. J., 363, 364, 366, 368 Tsai, M.-J., 365 Tsai, S. Y., 363, 365, 368 Tsien, R. W., 285

Tsubamoto, Y., 208 Tsuda, H., 404 Tsujita, Y., 81, 109, 111, 114, 194, 210 Tsumoto, T., 291 Tsurumi, Y., 469, 470 Tuck, M., 414 Tummino, P. J., 223, 224, 247, 249, 250 Tun, P., 110 Tuna, N., 108 Tung, L., 361 Tung, R. D., 247 Tunis, S. R., 2, 11 Tunn, S., 173, 179, 180 Tunnino, P. J., 250 Tunon, J., 46, 53, 72 Tunstall Pedoe, H., 105, 114 Tupy Visich, M. A., 111 Turcato, G., 473 Turck, C. W., 285 Turgeon, C., 363 Turgeon, F., 363 Turik, M., 470, 471, 472, 473, 475 Turini, G. A., 26, 30, 41, 65, 66, 69 Turk, C., 35, 67 Turner, C. H., 299, 367 Turner, D., 475 Turner, J. R., 468 Turner, M. J., 473 Turner, R. T., 298, 314, 359 Turner, S., 360 Turner, S. R., 225, 226, 250 Turner, W., 468, 475 Turner, W. W., 458, 462, 463, 464, 468, 473, 474 Turon, M., 246 Turri, M., 137 Twaddell, T., 419 Tyssen, D., 246 Tytgat, G., 412 Tytgat, G. N. J., 141 Tzeng, T. B., 249

U Ucci Stoll, K., 245 Uchiyama, S., 361 Udove, J., 416 Uehling, D. E., 287 Ueki, K., 208 Ueki, T., 466 Uhr, J. W., 414

544

AUTHOR INDEX

Uht, R. M., 367 Uip, D., 473 Ullman, K. S., 289 Ullrich, A., 415, 416, 418 Ulm, E. H., 27, 28, 29, 30, 32, 33, 66, 67 Ulrich, R. G., 203 Ultsch, M., 285 Umesono, K., 205, 208 Umezawa, H., 27, 33, 66 Umikawa, M., 472 Unal, S., 474 Unger, C., 474 Unger, S., 112 Unger, T., 17, 47, 63, 64, 72 Unger, W., 175, 179 Uno, H., 178 Unrau, A. M., 472 Uppenberg, J., 185, 205 Urata, H., 45, 71 Ure, C. L., 367 Urowitz, M. B., 411 Ushitani, T., 471 Ushiyama, I., 208 Utz, P. J., 290 Uzun, O., 463, 464, 474

V Vacca, J. P., 230, 233, 246, 250 Vadas, P., 411 Vagelos, P. R., 59, 75 Vahanian, A., 406 Vahlensieck, W., 179 Vaidyanathan, L., 415 Vailas, L. I., 108 Vaillancourt, M., 248 Vaisman, N., 416 Vaitkevicius, V. K., 417 Valavaara, R., 336, 367 Valdivieso, H., 467 Vale, J. A., 177 Valenti, M., 109 Valentine, H. L., 290 Valentine, M. A., 413 Valero, A., 471 Vallance, S. E., 211 Vallier, P., 207 VanBelle, Y., 365 Vanbervliet, B., 412 Van Bommel, T., 249

van Boven, A. J., 111, 112 vanBurik, J. A., 474 Van Cleemput, J., 114 Vancutsem, P. M., 355, 367 van Deemter, L., 416 Van den Berg, W. B., 385, 411 Vanden Bossche, H., 427, 455, 474 Van den Eertwegh, A. J. M., 386, 412 Vandenende, H., 470 Van Den Meiracker, A. H., 42, 70 Van de Putte, L. B. A., 383, 410 Van der Laken, C. J., 385, 411 van der Lubbe, P. A., 411 van der Meer, J. W., 411 van der Mooren, M. J., 365 VanderRoest, S., 247 Vandervaart, J. M., 428, 474 Van de Velde, P., 365 van de Ven, M. T., 411 van Deventer, S., 412 van de Vijver, M. J., 416 Vandrie, J., 246 Van Duyne, G. D., 275, 276, 279, 283, 291 Vane, J. R., 15, 18, 20, 21, 22, 41, 63, 64, 65, 69, 116, 117, 138, 141 Van Eeden, A., 123, 141 Vanhaecke, J., 94, 114 Vanhanen, H., 112 Van Hecken, A., 138, 171, 179 van Hogezand, R., 412 van Hogezand, R. A., 412 van Leeuwen, F., 394, 416 Van Lierde, J., 114 van Loon, J., 358 VanMiddlesworth, F., 431, 467, 474 VanMiddlesworth, F. L., 473 Vanmiddleswoth, J. F., 246 van Nagell, J. R. Jr., 365 Van Neste, D., 163, 169, 175, 177 van Olden, A. L., 249 van Rappard, F. M., 111 Vanrenterghem, Y., 269, 291 van Riel, P., 410 van Rijswijk, M. H., 137 van Ryn, J., 116, 117, 140, 141 Vanschagen, F. S., 474 Vara Prasad, J. V. N., 223, 250 Varco, R. L., 108 Varmus, H., 245 Varns, C., 413, 414

AUTHOR INDEX

Varnum, B. C., 139 Varona, R., 467 Vasan, R. S., 58, 75 Vasavanonda, S., 247, 248, 249 Vasmant, D., 41, 48, 69 Vassil, T. C., 27, 28, 29, 30, 32, 33, 66, 67 Vassiliou, S., 19, 41, 64, 69 Vastag, K., 250 Vaughan, D. E., 50, 73 Vaughan, E. D., 39, 68 Vaughn, C. B., 361 Vaughn, E. D., 176 Vavrek, R. J., 17, 63 Vazeux, G., 19, 64 Vazques, M., 203 Vazquez, J. A., 455, 457, 458, 474 Vazquez, M., 245 Vazquez, M. L., 231, 234, 246, 250 Theve, T., 360 Veale, D., 403, 420 Veber, D. F., 248 Vedin, A., 33, 67 Vega, J., 110 Veilleux, R., 363 Vekshtein, V. I., 114 Velander, W., 404 Vella, S., 248 Venables, P. J., 410 VÈniant, M., 45, 53, 71 Ventre, J., 205, 208, 209 Venturini, C. M., 139 Venturoli, S., 170, 179 Verdonck, L., 301, 367 Vereis, A. T., 15, 63 Vergezac, A., 360 Vergult, G., 178, 179 Verhoeff, J., 472 Verhoeven, G., 366 Vermeulen, A., 301, 367 Veronesi, U., 361 Verrips, C. T., 474 Vertsey, L., 472 Verweij, C. L., 286 Verweij, P. E., 427, 464, 474 Vesely, M., 409 Vessels, J. M., 467 Veyssier, P., 249 Viau, J. M., 56, 75 Vicario, P., 209 Vicaut, E., 40, 69

Vickers, P., 137, 138, 139, 140 Vickers, P. J., 140 Vidal, H., 207 Vidal-Puig, A., 184, 204 Vidal-Puig, A. J., 189, 206 Vigano, M., 113 Viinikka, L., 368 Viitanen, S., 245 Villa, A., 416 Villa, J., 420 Villanueva, A., 473 Villard, E., 40, 53, 69 Vincent, J. L., 379, 407 Vincenti, F., 375, 376, 405 Vincenzoni, C., 357 Vink, E., 470 Viola, J. P., 288 Virmani, R., 53, 74 Visco, D., 138, 140 Vita, J., 102, 110 Vita, J. A., 102, 114 Vitetta, E. S., 393, 414 Vittinghoff, E., 236, 251 Vivat, V., 368 Vlassara, H., 288 Vlasses, P. H., 176 Vocaturo, G., 357 Voegel, J. J., 323, 367 Vogel, C. L., 403, 419, 421 Vogel, K. W., 253–285 Vogel, R., 204 Vogel, V., 360 Vogelman, J. H., 365 Vogelstein, B., 420, 421 Vogt, P., 245 Vojnovic, I., 141 Volberding, P., 246 Volk, H. D., 412 Vollmer, E. P., 364 Volone, F. H., 401, 419 von Baehr, R., 412 von Bergmann, K., 113 Von B¸low, B., 48, 72 Vondrasek, J., 216, 251 von Hoff, D. D., 365 von Keutz, E., 95, 114 von Schoultz, E., 368 von Tol, A., 366 von Warburg, A., 474 Vorce, R. L., 286

545

546

AUTHOR INDEX

Vouloumanos, N., 140 Vuist, W. M., 414 Vukovich, R. A., 22, 37, 65 Vupputuri, S., 2, 12 Vyas, K. P., 113

W Wada, T., 416 Wada, Y., 114 Waddell, D. S., 247 Wade, L., 362 Wadworth, A. N., 68 Waeber, B., 39, 41, 42, 44, 68, 69, 70 Wagner, B. L., 312, 367 Wagstaff, A. J., 36, 67 Wahl, R. L., 414 Wahli, W., 203, 204, 205, 206, 303, 368 Wahner, H. W., 359, 364 Wakai, Y., 465, 471, 474 Wakasugi, T., 111 Wakeham, A., 407 Wakeling, A. E., 295, 296, 302, 313, 314, 315, 336, 340, 343, 344, 346, 353, 354, 358, 359, 365, 367 Waksal, H., 421 Wald, A., 424, 474 Wald, N. J., 111 Waldichuk, C., 413 Waldman, S., 474 Waldman, T. A., 415 Waldmann, H., 404, 411, 412, 415 Waldmann, T. A., 375, 405 Waldron, T. L., 34, 35, 67 Waldstreicher, J., 164, 174, 177, 178, 179 Waldstreicher, W., 178 Walensky, L. D., 286 Walker, J., 409 Walker, J. F., 14, 20, 62, 64 Walker, R. L., 357 Walkinshaw, M. D., 289 Wallace, J. M., 22, 65 Wallace, R., 416 Wallendszus, K. R., 110 Walley, V. M., 53, 74 Walmer, D. K., 365 Walpole, A. L., 346, 360 Walsh, B. W., 351, 367 Walsh, C. T., 290 Walsh, K., 288 Walsh, P., 177, 178

Walsh, P. C., 145, 146, 151, 162, 174, 176, 178, 179 Walsh, R. A., 267, 291 Walsh, T. J., 425, 469, 472, 473 Walter, P., 360, 362 Walton, P., 359, 361 Wambach, G., 111 Wandel, C., 248 Wandless, T. J., 253–285, 290 Wang, D., 178 Wang, D. S., 45, 47, 71 Wang, D. Z., 177, 179 Wang, F. L., 42, 70 Wang, H., 249 Wang, J. G., 40, 69 Wang, J. H., 265, 291 Wang, J. L., 139 Wang, N.-Y., 34, 67 Wang, Q. C., 415 Wang, X., 270, 291 Wang, X. C., 246, 247 Wang, Y., 245 Wang, Z., 138, 140, 141 Wanger, A., 427, 475 Wanner, C., 112 Ward, J., 366 Wardrop, E., 248 Wardwell, K., 419 Ware, J. H., 8, 12 Wargon, M., 42, 44, 70 Wargovitch, T. J., 52, 73 Waring, W. S., 203 Warner, T., 137 Warner, T. D., 118, 120, 122, 139, 141 Warnica, J. W., 113 Warnke, R., 413 Warnock, D. W., 468, 474 Warren, D. J., 16, 63 Washburn, T. F., 361 Washington, C. B., 235, 251 Washington, L. C., 109 Watabe, E., 471, 474 Watanabe, A., 111 Watanabe, T., 469, 470, 471, 473 Watanabe, Y., 81, 114, 471, 474 Watenpaugh, K. D., 249, 250 Waterfield, M., 360 Waterman, H. L., 141 Waters, D., 100, 101, 105, 112, 114 Watkins, L. J., 48, 72 Watkins, V. S., 472

AUTHOR INDEX

Watson, D. J., 139 Watson, M. A., 362 Watson, P., 468 Watson, R. D. S., 33, 67 Watson, T., 420 Watt, I., 411 Watts, G. F., 101, 114 Watts, R. A., 411 Waugh, M. H., 21, 45, 65, 71 Waxman, L. H., 248 Wdzieczak-Bakala, J., 41, 44, 69 Weakeling, A. E., 178 Weaver, A. L., 404, 410 Weaver, C. A., 302, 344, 367 Weaver, E. R., 21, 65 Webb, J., 138 Webb, J. A., 177 Webb, J. K., 141 Webb, P., 309, 365, 367 Webber, S., 249 Weber, B. L., 419 Weber, F. J., 108 Weber, I. T., 251 Weber, M. A., 22, 57, 59, 65, 75 Weber, P. C., 248 Webley, D. M., 467 Webster, G., 177 Webster, M. A., 416 Wedel, H., 112 Wehrli, W., 467 Wei, L., 18, 19, 41, 64, 69 Weidenhˆfer, S., 408 Weigel, N. L., 329, 367 Weil, B., 16, 63 Weinberg, E. O., 286 Weinberg, R. A., 415 Weinblatt, M. E., 383, 384, 385, 410, 411 Weiner, D. B., 417 Weiner, L. M., 401, 420 Weiner, M. S., 178 Weinstein, I., 351, 367, 368 Weinstein, S. P., 191, 208 Weintraub, W. S., 114 Weis, S., 109 Weisman, H. F., 405, 406 Weisman, M., 410 Weismann, H. F., 406 Weismann, M., 409 Weiss, A., 254, 291 Weiss, D., 179 Weiss, N. S., 57, 75, 347, 367

Weiss, R., 245 Weiss, S., 113 Weiss, S. R., 112 Weiss, Y. A., 40, 68 Weisser, H., 173, 180 Weisser, K., 43, 70 Weissman, I., 288 Weitz, D., 28, 30, 66 Weitzman, I., 467 Welch, J. S., 209 Wells, G. A., 47, 48, 72 Wells, J. A., 285 Welsch, C. W., 331, 367 Welsh, K. I., 405 Wen, D., 418 Wendling, D., 385, 411 Wenger, H. C., 28, 30, 66 Wenzel, L. M., 366 Wenzel, R. P., 380, 408, 468 Werman, A., 206 Werner, M. H., 420 Werns, S., 110, 114 Wesselborg, S., 290 Wesselborg, S. A., 260, 274, 291 West, C. M., 359 West, M. J., 33, 67 West, P., 467 Westbrook, G. L., 289 Westerhof, M., 179 Westin, S., 205, 367 Westrum, B., 46, 71 Weverling, G. J., 236, 251 Wey, K., 413, 414 Wheat, L. J., 472 Whelton, P. K., 54, 74 Whitaker, M., 364 Whitcomb, R. W., 197, 211 White, B. G., 407 White, C. A., 413, 414 White, C, M., 36, 68 White, G., 246 White, J. O., 359, 361 White, J. W., 151, 180 White, R., 303, 368, 473 White, R. P., 22, 65 White, T. C., 427, 455, 475 White, W. B., 68 White-Carrington, S., 209 Whitehorn, E. A., 249 Whitfield, L. R., 109 Whiting, D., 177, 178

547

548 Whiting, D. A., 162, 174, 180 Whitney, E., 109 Whittle, P. J., 248 Wichmann, C. F., 469 Wickerham, D. L., 360, 417 Wickerhan, L., 360 Wideburg, N., 246, 249 Wideburg, N. E., 247 Widenes, J., 411 Widmer, M. B., 404 Widmer, W. R., 138 Wieand, S., 360 Wiebe, D. A., 364 Wiebe, V., 365 Wiebe, V. J., 340, 343, 354, 365, 368 Wiederrecht, G. J., 289 Wiegmann, K., 407 Wiemer, G., 46, 48, 55, 60, 71, 75 Wiesenhutter, C., 411 Wiethe, R. W., 206 Wiklund, O., 91, 114 Wilber, J. A., 57, 75 Wilcox, H. G., 351, 367, 368 Wilderspin, A., 248 Wilhelmsen, L., 112 Wilkerson, W. W., 222, 251 Wilking, N., 298, 360, 366, 368 Wilkins, T. D., 373, 404 Wilkinson, G. R., 248 Wilkinson, H. A., 205 Wilkinson, K. F., 250 Wilkison, W. O., 203 Wilks, D. P., 359 Willard, D. A., 36, 67 Willard, K. E., 468 Willerson, J. T., 210 Willett, W., 361 Willett, W. C., 358, 366 Willey, J., 419 Williams, A. R., 367 Williams, B., 411 Williams, C. J., 358 Williams, C. S., 194, 210 Williams, D. C., 358 Williams, G. H., 17, 20, 49, 63, 73 Williams, G. M., 367 Williams, G. W., 1–11 Williams, M. E., 413 Williams, M. G., 250 Williams, N. J., 21, 65 Williams, P. E. O., 248

AUTHOR INDEX

Williams, P. T., 110 Williams, R. O., 382, 386, 409, 412 Williams, T. A., 18, 19, 64 Williams, T. C., 303, 366 Williams, T. D. M., 207 Williamson, P. R., 472 Willingham, M. C., 420 Willis, A. L., 117, 141 Willson, T. M., 187, 203, 205, 206, 209, 362, 368 Wilson, A. C., 111 Wilson, B., 176 Wilson, F. R., 139 Wilson, J. D., 145, 146, 152, 154, 174, 177, 178, 179, 180 Wilson, K. E., 473 Wilson, M. C., 2, 11 Wilson, P. W., 108, 359, 362 Wilson, R. G., 358 Wilson, S. I., 235, 251 Wilson, T. H., 176 Wimmer, E., 248 Winborne, E. L., 248 Winch, S., 178 Winchell, G. A., 179 Winder, D. G., 264, 291 Windisch, B., 409 Windler, E. E., 351, 358, 368 Wing, L., 43, 71 Wing, M., 411 Wing, R. R., 364 Winget, M., 418 Wingo, P. A., 177, 363 Winn, V., 140 Winneker, R. C., 364 Winniford, M., 110, 114 Winslow, D. L., 247 Winter, G., 373, 404 Winters, P., 177 Wirth, C., 420 Wiscount, C. M., 248 Wise, H., 174 Wisely, G. B., 205, 206 Wiseman, G. A., 414 Wiseman, L. R., 68, 111 Withington, S., 407 Witt, M. D., 427, 475 Witte, K., 43, 70 Wittes, J., 408 Wittmann-Liebold, B., 287 Wittreich, J., 138

549

AUTHOR INDEX

Witzig, T. E., 393, 414 Witzmann, G., 409 Witztum, J. L., 95, 114 Wlodawer, A., 215, 216, 221, 248, 250, 251 WM, O. F., 364 Woerner, F. J., 247 Wofsy, D., 386, 412 Wolf, B., 417 Wolf, J., 139 Wolf, M., 175 Wolf, P. H., 301, 368 Wolfe, E. J., 468 Wolfe, J. K., 109 Wolfe, M. M., 115, 141 Wolfenden, R., 22, 27, 65 Wollert, K. C., 48, 72 Wolmark, N., 360, 417 Wolosker, H., 288 Wolter, J., 419 Wolter, J. M., 419 Wolters, A., 473 Wong, A. J., 403, 420, 421 Wong, B., 472 Wong, C.-K., 111 Wong, E., 123, 124, 137, 138, 139, 140, 141 Wong, G. A., 108 Wong, I. L., 170, 180 Wong, J., 363 Wong, P., 138 Wong, P. C., 18, 64 Wong, P. Y., 139 Wong, S. F., 248 Wong, S. G., 416 Wong, W. L., 418 Wong, W. L. T., 418 Wong, Y. N., 248 Wong-Staal, F., 249 Wongvipat, N., 417 Woo, S. L., 365 Wood, A. J., 248 Wood, D., 113 Wood, D. A., 108 Wood, J. M., 55, 74 Wood, R. L., 452, 475 Wood, S., 248 Wood, W. C., 417 Wood, W. I., 420 Woods, J., 208 Woods, K. R., 18, 64 Woody, J., 410 Woody, J. N., 409

Word, R. A., 177 Wortel, C., 412 Woulfe, D. S., 410 Wray, R., 111 Wright, A., 475 Wright, A. S., 153, 154, 180 Wright, C., 52, 73, 395, 418 Wright, M., 205 Wright, M. R., 245, 249 Wright, S. D., 108, 210 Wu, B., 249 Wu, H. D., 210 Wu, M., 208, 209 Wu, M. T., 27, 28, 29, 32, 66 Wu, W. S., 205 Wu, Z., 20, 64, 205 Wun, C.-C., 113 Wunderink, R., 407 Wurtz, J. M., 306, 368 Wyche, A., 140 Wysowski, D. K., 112 Wyss, D., 245 Wyvratt, M. J., 27, 28, 29, 32, 66

X Xia, M. Q., 415 Xiao-Ping, X., 210 Xie, W., 117, 141 Xin, X., 194, 210 Xiong, S. P., 247 Xu, H., 407 Xu, J., 310, 368 Xu, L., 137, 141, 310, 361, 368 Xu, L.-J., 138, 140 Xu, Y., 47, 72, 245, 246

Y Yabe, T., 450, 470, 471, 475 Yacobi, A., 226, 251 Yacoub, M., 108 Yagi, A., 471, 473 Yagi, T., 416 Yakel, J. L., 265, 291 Yamada, A., 471 Yamada, H., 470 Yamada, Y., 416 Yamadaokabe, H., 471, 475 Yamadaokabe, T., 475 Yamaguchi, A., 417

550 Yamamoto, I., 467 Yamamoto, K. R., 312, 363 Yamamoto, M. T., 209 Yamamoto, T., 415, 416 Yamashita, D. S., 287 Yamashita, M., 469, 470 Yamauchi, T., 208 Yamodaokabe, T., 471 Yan, S. B., 467 Yan, X., 363 Yanagisawa, M., 15, 63 Yang, C. P., 250 Yang, H. Y. T., 17, 18, 63 Yang, S., 210 Yang-Feng, T. L., 415 Yansura, D., 418 Yao, E. F., 138 Yao, S. L., 141 Yao, T. P., 310, 368 Yarden, Y., 416, 418, 420 Yarnall, D. P., 207 Yasaki, Y., 207 Yasuda, K., 207 Yazaki, Y., 206, 208 Yaziji, H., 419 Yee, B., 176 Yee, H. S., 87, 114 Yeh, E. T. H., 210 Yeh, H. J. C., 470 Yellin, A. E., 108 Yen, C. J., 207 Yen, C.-J., 189, 207 Yen, L., 395, 416 Yen, P. M., 367 Yeo, W. N., 55, 74 Yeo, W. W., 56, 75 Yeramian, P., 246 Yergey, J. A., 139 Yerly, S., 242, 251 Yernell, D. P., 207 Yeung, A., 114 Yeung, A. C., 110, 114 Yi, H. F., 204 Yigael, D., 358 Yiotakis, A., 19, 41, 64, 69 Yip, B., 247 Ylikorkala, O., 299, 368 Yoakim, C., 248 Yocum, D. E., 411 Yokobayashi, F., 474 Yokota, K., 473

AUTHOR INDEX

Yokoyama, M., 138 Yokoyama, T., 407 Yonemura, Y., 395, 417 Yoshida, H., 286 Yoshida, K., 467 Yoshida, M., 416 Yoshida, O., 175 Yoshida, S., 113 Yoshimura, A., 111 Yoshimura, N., 94, 114 Yoshioka, S., 208 Yoshioka, T., 208 Yoshizaki, K., 411 You, X. L., 416 Youle, M., 248 Youn, H. D., 290 Young, D. A., 140 Young, J. B., 108 Young, P. R., 468 Young, P. W., 190, 191, 193, 208 Young, R. N., 138, 140, 141 Youngman, L., 41, 69 Yu, B., 247 Yu, V. L., 472 Yu, X. C., 20, 64 Yu, Y. V., 472 Yuan, A., 467 Yuan, W., 138 Yuan, X. L., 474 Yuasa, H., 30, 66 Yucelten, D., 175 Yusuf, S., 3, 12, 47, 49, 50, 52, 72, 73 Yyanovski, J. A., 248

Z Zaino, R. J., 366 Zak, O., 474 Zalipsky, S., 420 Zalutsky, M. R., 421 Zambias, R. A., 439, 467, 475 Zamboni, R., 138, 140, 141 Zaninotto, M., 43, 70 Zannad, F., 48, 72 Zarling, J., 413 Zasadny, K. R., 414 Zatz, R., 46, 53, 72 Zavoral, J., 110 Zawadski, J. V., 15, 63 Zaya, R. M., 250 Zechel, C., 367

AUTHOR INDEX

Zechkner, D., 468 Zeckner, D., 475 Zeckner, D. J., 467, 468, 469 Zeidler, H., 137 Zeitner, M. E., 179 Zeldis, S. M., 57, 75 Zelenitsky, S. A., 470, 475 Zell, R., 408 Zeng, Q., 137 Zenz, P., 409 Zerhouni, E. A., 179 Zetterstrom, R. H., 362 Zhanel, G., 469 Zhanel, G. G., 463, 464, 470, 475 Zhang, B., 192, 205, 206, 208, 209 Zhang, J., 114 Zhang, L., 47, 72, 245 Zhang, M., 210 Zhang, M. Z., 139 Zhang, W., 291 Zhang, X., 418 Zhang, Y., 359, 364 Zhang, Z., 474 Zhao, C., 247, 249 Zhao, P.-L., 139, 141 Zhao, W., 285, 412 Zhao, Z., 249, 250 Zhau, H. E., 395, 418 Zheng, Y., 473 Zhong, H., 139 Zhong, W.-Z., 250 Zhou, G., 184, 186, 205, 206

Zhou, L. J., 413 Zhou, N., 368 Zhu, J., 273, 291 Zhu, Y., 183, 184, 185, 204, 205 Zhu, Y.-S., 177 Zhuo, M., 265, 291 Ziegler, E. J., 380, 408 Ziel, H. K., 346, 368 Zierath, J. R., 191, 192, 208 Ziesche, S., 49, 73 Zimmerman, B. G., 18, 64 Zimmermann, J. L., 407 Zimniski, S. J., 334, 359 Zink, D. L., 469 Zinreich, S. J., 179 Zipp, G. L., 249, 250 Zoccali, C., 47, 53, 72 Zonderman, A., 362 Zornes, L. L., 462, 475 Zugay, J., 250 Zugay, J. A., 246, 248 Zugel, M., 466 Zurinum, G., 289 Zusman, R. M., 17, 63 Zvaifler, N. J., 381, 409 Zweifel, B., 140 Zweifel, B. S., 140 Zweifel, M. J., 468 Zwicker, H., 178 Zwinderman, A. H., 111 Zybarth, G., 248

551

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SUBJECT INDEX

A

aldosterone, production, angiotensin II role, 16 alendronate, osteoporosis treatment, 5 alopecia, see androgenetic alopecia α-alkoxyphenylpropionates, ligand chemistry, 187 5α-reductase inhibitors, 143–174 clinical studies, 151–168 benign prostatic hyperplasia, 6, 151–161 dihydrotestosterone role, 151, 153–154, 160 finasteride, 6, 144, 153–158 inhibitor responsiveness predictors, 159–160 natural history assessment, 144, 151–153 phase III studies, 154–155 proscar efficacy and safety, 155–159 prostate gland growth, 151 serum prostate-specific antigen role, 159–161 female androgenetic alopecia, 169 female hirsutism, 169–170 male androgenetic alopecia, 162–168 clinical aspects, 162–164 dihydrotestosterone role, 148, 162, 164–165, 168 phase III studies, 165–168 serum prostate-specific antigen role, 168 treatment approaches, 162–164 prostate cancer, 161–162, 168 development, 148–151 chemistry, 148–149 pharmacodynamics, 150–151 pharmacokinetics, 150, 174 rationale, 148 identification and characterization, 144–148

A-80987, structure, 217 A-770033, structure, 217 abciximab angina treatment, 4 coronary artery disease treatment, 377–379 ABT-378, structure, 218 acetaminophen, gastropathy incidence, 131 acetyl salicylate gastropathy incidence, 130 historical background, 116–117 myocardial infarction treatment, 4 acquired immune deficiency syndrome disease course, 213–214 treatment amprenavir, 239 clinical issues, 6, 8, 235–243 didanosine, 243 drug resistance, 235, 241–242 efavirenz, 243 HIV-1 protease inhibitors, see HIV-1 protease inhibitors indinavir, 6, 237–239, 243 lamivudine, 243 nelfinavir, 239, 243 ritonavir, 6, 237, 239, 243 saquinavir, 243 stavudine, 243 zidovudine, 243 acute myocardial infarction, treatment angiotensin-converting enzyme inhibitors, 5, 41, 49–50 clinical trials role, 4–5 ACE Inhibitor Myocardial Infarction Collaboration Group, 50, 73 adhesion molecules, monoclonal antibody therapy target, 386 AKAP79, calcineurin regulation by localization, 272 553

554

SUBJECT INDEX

5α-reductase inhibitors (continued) discovery in humans, 145–146 historical perspectives, 144–145 isozymes, 146 physiology and pathophysiology, 146–148 natural inhibitors, 172–173 overview, 143–144, 173–174 type 1 inhibitors, 171–172 type 2 inhibitors, 172 alteplase, myocardial infarction treatment, 4 Alzheimer’s disease, treatment cyclooxygenase-2 inhibitors, 134–135 nonsteroidal anti-inflammatory drugs, 134–135 amphotericin B, antifugal properties, 425, 427 amprenavir acquired immune deficiency syndrome treatment, 239 mechanism-based design, 231, 234 analgesics, see specific types androgenetic alopecia, treatment female form, 169 male form, 162–168 clinical aspects, 162–164 dihydrotestosterone role, 148, 162, 164–165, 168 phase III studies, 165–168 serum prostate-specific antigen role, 168 treatment approaches, 162–164 angina, clinical trials role in management, 4 angiotensin II aldosterone production link, 16 hypertension link, 16–17 synthesis, 14, 16 angiotensin-converting enzyme inhibitors, 14–62 captopril, see captopril cardiac disease treatment atherosis, 50–52 congestive heart failure, 5, 33, 48–49 coronary heart disease, 50–52 myocardial infarction, 5, 41, 49–50 renin-angiotensin system, 46–49 clinical aspects availability, 36–37 historical perspectives, 15–18 knowledge-base advances

drug development process advances, 40–41 physiological and pathophysiological contributions, 37–40 surrogate end point dilemma, 55–61 diabetic nephropathy treatment, 5 enalapril, see enalapril fosinopril, 34–37 hypertension treatment, 15, 41–45, 54–55 lisinopril, 30–33, 48 overview, 14–20, 61–62 peptide inhibitors, 20–22 bradykinin-potentiating peptides, 17, 20–24, 54 teprotide studies, 21–22, 27, 37 renal disease treatment normotension versus hypertension concepts, 54–55 renal insufficiency, 53–54 renin-angiotensin system, 45–48 restenosis treatment, 52–53 antiestrogens, 293–357 activity endometrium, 302–303 mammary glands, 302–303 benzopyrans, 318–357 AF-1 and AF-2 blocking mechanisms, 323–324, 329–331 binding characteristics, 318–323 bone loss prevention, 346–357 DMBA-induced mammary tumor development inhibition, 331–335 human breast cancer inhibition, 336–346 properties, 297, 318–319 SCR-1 blocking mechanisms, 324–331 benzothiophenes, 297, 316–318 estrogen receptor activation mechanisms, 303–313 AP-1 response element, 307–309 β-receptors, 303–304 coactivators, 309–312 corepressors, 309–310, 312–313 estrogen-induced AF-2-receptors, 304–307 kinase-induced AF-1-receptors, 307 modulators, 300, 318 nuclear-receptors, 303 α-receptors, 303–304 overview, 294–295 steroidal antiestrogens, 313–314

SUBJECT INDEX

tamoxifen action mechanisms, 296–297, 314–316 breast cancer treatment, 6, 295–298, 302, 314 triphenylethylenes, 297, 314–316 Antiplatelet Trialists’ Collaboration, 102, 108, 116, 137 arthritis, treatment celecoxib, 6, 129 clinical trials role, 6 cyclooxygenase-2 inhibitors, 123, 129 infliximab, 382–384 monoclonal antibody therapy, 381–386 interleukin-1 targeting, 385 T-cell targeting, 385–386 tumor necrosis factor-α blockade, 381–385 nonsteroidal anti-inflammatory drugs, 129, 133 rofecoxib, 6, 123, 129 Aspergillus species biology, 424–425 glucan synthase inhibitor antifugal properties, amino compound development, 440–441, 452–453 aspirin gastropathy incidence, 130 historical background, 116–117 myocardial infarction treatment, 4 atherosclerosis, see also hypertension diabetes link, 181–182, 193–194 treatment clinical trials role, 4, 48 monoclonal antibody therapy, 376–379 pioglitazone, 194 statins, 105–107 thiazolidinediones, 193–194 trandolapril, 48 troglitazone, 194 atherosis, treatment, angiotensinconverting enzyme inhibitors, 50–52 atorvastatin cholesterol-lowering effects, 83, 86, 88 diabetes treatment, 106

B basiliximab, organ transplant monoclonal antibody therapy, 375–376 β-cells, peroxisome proliferator-activated receptor γ agonist effects, 196–197

555

Bcl-2, calcineurin regulation by localization, 271–272 benign prostatic hyperplasia, treatment, 6, 151–161 dihydrotestosterone role, 151, 153–154, 160 finasteride, 6, 144, 153–158 inhibitor responsiveness predictors, 159–160 natural history assessment, 144, 151–153 phase III studies, 154–155 proscar efficacy and safety, 155–159 prostate gland growth, 151 serum prostate-specific antigen role, 159–161 benzopyrans, antiestrogen activity, 318–357 AF-1 and AF-2 blocking mechanisms, 323–324, 329–331 binding characteristics, 318–323 bone loss prevention, 346–357 DMBA-induced mammary tumor development inhibition, 331–335 human breast cancer inhibition, 336–346 properties, 297, 318–319 SCR-1 blocking mechanisms, 324–331 benzothiophenes antiestrogen activity, 316–318 structure, 297 bile acid sequestrants, cholesterol-lowering effects, 82, 90–91 bone loss, see osteoporosis bradykinin-potentiating peptides, properties, 17, 20–24, 54 breast cancer, treatment benzopyrans DMBA-induced mammary tumor development inhibition, 331–335 human breast cancer inhibition, 336–346 MCF-7 cancer cell cycling comparison, 337–340 clinical trials role, 6 estrogen replacement therapy, 295, 299–302 raloxifene, 316–318 statins, 103–105 tamoxifen, 6, 295–298, 302, 314 trastuzumab, 395–401

556

SUBJECT INDEX

C C225, cancer radiotherapy, 403 cabin-1, calcineurin inhibition, 272–273 cain, calcineurin inhibition, 272–273 calcineurin, role outside the immune system, 261–269 heart development, 265–268 nervous system function, 262–265 calcium ion release, 262–263 neuroregeneration, 263–264 synaptic plasticity, 264–265 NF-AT nuclear localization, 265–268 transforming growth factor-β release, 268–269 calcineurin inhibitors, 253–285 autoinhibition, 270 cabin-1, 272–273 cain, 272–273 calcineurin/NF-AT interaction disruption, 273–274 cyclosporin action specificity, 269 discovery and development, 254–258 heart development effects cardiac hypertrophy, 266–268 myopathy risk, 93 NF-AT signaling, 265–268 nervous system effects neuroregeneration, 263–264 synaptic plasticity, 264–265 structure, 255 target identification mechanisms, 258–261 transforming growth factor-β release, 268–269 cypermethrin, 274–275 FK506 action specificity, 269 discovery and development, 254–258 heart development effects cardiac hypertrophy, 266–268 NF-AT signaling, 265–268 nervous system effects intracellular calcium ion release, 263–263 neuroregeneration, 263–264 synaptic plasticity, 264–265 structure, 255 target identification mechanisms, 258–261

immunophilin/immunosuppressant complex inhibition, 275–279 complex structure, 275–276 FKBP-FK506-calcineurin ternary complex structure, 276–278 FKBP-rapamycin-FRB ternary complex structure, 278–279 overview, 283–285 oxidative inactivation, 270–271 regulation by localization AKAP79, 272 Bcl-2, 271–272 CAMPATH-1, cancer monoclonal antibody therapy, 394 cancer, treatment, see also specific types benzopyrans DMBA-induced mammary tumor development inhibition, 331–335 human breast cancer inhibition, 336–346 MCF-7 cancer cell cycling comparison, 337–340 celecoxib, 134 clinical trials role, 6 cyclooxygenase-2 inhibitors, 134 dihydrotestosterone, 161–162, 168 estrogen replacement therapy, 295, 299–302 HMG co-A reductase inhibitors, 103–105 monoclonal antibody therapy, 387–403 considerations, 387–390 lymphocyte differentiation antigens, 390–394 CAMPATH-1, 394 CD19 targeting, 393 CD20 targeting, 390–393 CD22 targeting, 393 CD25 targeting, 393 idiotype, 390 radioimmunotherapy, 392–393 rituximab, 391–392 oncogene-product antigens, 394–403 bispecific antibodies, 401–402 CD64 targeting, 401 C225 radiotherapy, 403 epidermal growth factor race, 403 HER2 targeting, 394–403 immunoliposomes, 402 immunotoxins, 402 trastuzumab, 395–401

SUBJECT INDEX

nonsteroidal anti-inflammatory drugs, 133–134 raloxifene, 316–318 rofecoxib, 134 simvastatin, 103–105 statins, 103–105 tamoxifen, 6, 295–298, 302, 314 thiazolidinediones, 194 trastuzumab, 395–401 Candida species biology, 424 glucan synthase inhibitor antifugal properties amino compound development, 451–452 cell wall targeting, 428–430 glucan synthase genes, 451–452 captopril adverse effects, 43 analogues, 24 carboxyalkanoylproline derivatives, 22–24 cardiovascular disease treatment, 5, 26–27, 43–44 characterization, 24–26, 43 clinical approvals, 26–27, 36 diabetic nephropathy treatment, 5 hypertension treatment, 5, 26, 43–44 pharmacokinetics, 26, 43–44 properties, 24, 37, 43 cardiovascular disease cholesterol effects, see cholesterol hypertension, see hypertension menopause relationship, 298–300 treatment angiotensin-converting enzyme inhibitors atherosis, 50–52 captopril, 5, 26–27, 43–44 congestive heart failure, 5, 33, 48–49 coronary heart disease, 50–52 enalapril, 5, 30, 49 fosinopril, 36 lisinopril, 33, 48 myocardial infarction, 5, 41, 49–50 ramipril, 5, 38, 48 renin-angiotensin system, 46–49 clinical trials role, 3–5 monoclonal antibody therapy, 376–379

557

CD19, monoclonal antibody therapy target, 393 CD20, monoclonal antibody therapy target, 390–393 CD22, monoclonal antibody therapy target, 393 CD25, monoclonal antibody therapy target, 393 CD40, monoclonal antibody therapy target, 386–387 CD64, monoclonal antibody therapy target, 401 celecoxib arthritis treatment, 6, 129 colon cancer treatment, 134 gastropathy incidence, 130–132 properties, 121–123 cerivastatin, cholesterol-lowering effects, 83, 88, 93 cholesterol biosynthesis, 77–81 controversy over effects, 97–98 EM-652 and EM-800 effects, 349–357 historical perspectives, 77–84 HMG co-A reductase inhibitors effects, 89–91 action mechanisms, 89 atorvastatin, 83, 86, 88 cerivastatin, 83, 88, 93 cholestyramine, 83 colestipol, 83 combination therapy, 89–91 bile acid sequestrants, 82, 90–91 diet, 83, 89–90 fibrate drugs, 84, 91 niacin, 83, 90–91, 93 statins, 90–91 compactin, 81–82 lipoprotein reaction, 84–88 chronopharmacology, 86 comparative effectiveness, 87–88 dose-response relationship, 86 outcome studies, 98–100 lovastatin, 4, 82–85, 88, 99–102 simvastatin, 4, 82–92, 99–102 randomized controlled trial, 2–3 cholestyramine, cholesterol-lowering effects, 83 cilofungin antifugal properties, 433–434 structure, 435

558

SUBJECT INDEX

clinical trials, see also specific drugs and proteins natural history determination by randomized controlled trials, 1–11 genomics role, 10–11 new chemical entity development, 3–8 overview, 1–2 patient safety, 9–10 pharmaceutical industry role, 2, 9 presenting protein strategy, see presenting protein strategy surrogate end point dilemma, 55–61 colestipol, cholesterol-lowering effects, 83 colon cancer, treatment celecoxib, 134 cyclooxygenase-2 inhibitors, 134 nonsteroidal anti-inflammatory drugs, 133–134 rofecoxib, 134 thiazolidinediones, 194 compactin cholesterol-lowering effects, 81–82 toxicity, 82 coronary artery disease, treatment angiotensin-converting enzyme inhibitors, 50–52 HMG co-A reductase inhibitors, 100–102, 107 monoclonal antibody therapy, 376–379 Crohn’s disease, monoclonal antibody therapy, 387 Cryptococcus species, biology, 424–425 cyclooxygenase-2 inhibitors, 115–137 assays, 118–120 preclinical in vitro assays, 119–120 in vitro assays, 118–119 clinical development, 127–133 efficacy, 6, 129–130 future research directions, 133–135 Alzheimer’s disease research, 134–135 colon cancer research, 133–134 safety, 130–133 articular cartilage damage, 133 gastrointestinal problems, 115–116, 127, 130–132 renal problems, 132–133 selectivity in humans, 127–129 enzymology, 124–127 historical perspectives, 116–117

medicinal chemistry, 124–127 overview, 115–116, 135–137 selectivity, 120–124, 127–129 cyclosporin action specificity, 269 discovery and development, 254–258 heart development effects cardiac hypertrophy, 266–268 NF-AT signaling, 265–268 nervous system effects neuroregeneration, 263–264 synaptic plasticity, 264–265 structure, 255 target identification mechanisms, 258–261 transforming growth factor-β release, 268–269 cypermethrin, calcineurin inhibition, 274–275

D daclizumab, organ transplant monoclonal antibody therapy, 375–376 dehydroepiandrosterone bone loss treatment, 347 conversion to estrogen, 296, 300–301 DFU, properties, 121–122 diabetes atherosclerosis link, 181–182, 193–194 insulin resistance, 181, 191, 195–197 treatment atorvastatin, 106 clinical trials role, 5 lifestyle changes, 196 metformin, 200–201 peroxisome proliferator-activated receptor γ agonists, see peroxisome proliferatoractivated receptor γ agonists pioglitazone, 202 rosiglitazone, 198–202 combination studies, 200–201 phase 2 studies, 198–199 phase 3 studies, 198–199 safety, 201–202 sulfonylurea, 200–201 troglitazone, 197–198 type 2 profile, 181–182, 196 didanosine, acquired immune deficiency syndrome treatment, 243

559

SUBJECT INDEX

diet cholesterol-lowering effects, 83, 89–90 diabetes management, 196 dihydrotestosterone, 143–174 clinical studies, 151–168 benign prostatic hyperplasia, 6, 151–161 action mechanisms, 151, 153–154, 160 inhibitor responsiveness predictors, 159–160 natural history assessment, 144, 151–153 phase III studies, 154–155 proscar efficacy and safety, 155–159 prostate gland growth, 151 serum prostate-specific antigen role, 159–161 female androgenetic alopecia, 169 female hirsutism, 169–170 male androgenetic alopecia, 162–168 action mechanisms, 148, 162, 164–165, 168 clinical aspects, 162–164 phase III studies, 165–168 serum prostate-specific antigen role, 168 treatment approaches, 162–164 prostate cancer, 161–162, 168 development, 148–151 chemistry, 148–149 pharmacodynamics, 150–151 pharmacokinetics, 150, 174 rationale, 148 identification and characterization, 144–148 discovery in humans, 145–146 historical perspectives, 144–145 isozymes, 146 physiology and pathophysiology, 146–148 natural inhibitors, 172–173 overview, 143–144, 173–174 DMP-323, structure-based design, 218, 221 DMP-450, structure-based design, 218, 222 DMP-850, structure-based design, 218, 222 DMP-851, structure-based design, 219, 222 droloxifene, properties, 297, 316 drug resistance, see specific drugs drug testing, see clinical trials; specific drugs DuP-697, properties, 121–122

E echinocandins antifugal properties, 430–433, 450, 457–466 LY303366, see LY303366 MK-0911, see MK-0911 efavirenz, acquired immune deficiency syndrome treatment, 243 EM-139, structure, 297 EM-652 antiestrogen activity, 318–357 AF-1 and AF-2 blocking mechanisms, 323–324, 329–331 binding characteristics, 318–323 bone loss prevention, 346–357 cholesterol inhibition, 349–357 description, 297, 318–319 DMBA-induced mammary tumor development inhibition, 331–335 human breast cancer inhibition, 336–346 MCF-7 cancer cell cycling comparison, 337–340 SCR-1 blocking mechanisms, 324–331 structure, 297 EM-800 antiestrogen activity binding characteristics, 318–323 cholesterol inhibition, 349–357 DMBA-induced mammary tumor development inhibition, 334–335 MCF-7 cancer cell cycling comparison, 337–340 properties, 297, 318–319 bone loss treatment, 347 structure, 297 enalapril characterization, 30, 44 clinical approvals, 30 congestive heart failure treatment, 5, 49 design considerations, 27–28 enalaprilat, 28–29, 37 hypertension treatment, 30, 44 properties, 29–30, 44 sulfhydryl group, 27–28 epristeride, properties, 171 eptifibatide, angina treatment, 4

560

SUBJECT INDEX

estrogen antiestrogens, see antiestrogens dehydroepiandrosterone conversion to estrogen, 296, 300–301 estrogen replacement therapy, 295, 299–302 intracrinology, 300–301 menopause, 298–301 estrogen-receptors activation mechanisms, 303–313 AP-1 response element, 307–309 β-receptors, 303–304 coactivators, 309–312 corepressors, 309–310, 312–313 estrogen-induced AF-2-receptors, 304–307 kinase-induced AF-1-receptors, 307 nuclear-receptors, 303 α-receptors, 303–304 antiestrogen activity, see antiestrogens etodolac, properties, 121–123

F female medicine, see women’s health needs fever, treatment, cyclooxygenase-2 inhibitors, 129–130 fibrates, cholesterol-lowering effects, 84, 91 finasteride androgenetic alopecia treatment, 144, 149, 163, 165–168 benign prostatic hypertrophy treatment, 6, 144, 153–158 female hirsutism treatment, 170 pharmacodynamics, 150–151 pharmacokinetics, 150, 174 FK463 antifugal properties, 465 structure, 435 flosulide, properties, 121–123 fluconazole, antifugal properties, 425, 440 flucytosine, antifugal properties, 426–427 fluvastatin, cholesterol-lowering effects, 83, 87–88 fosinopril clinical approval, 36 phosphorus-containing inhibitors, 34–35 properties, 35–36, 37 fungus infections, antifugal agents, 423–466 amino compound development, 439–457 action mechanisms, 443–457

Aspergillus models, 440–441, 452–453 Candida albicans model, 451–452 genetic insights, 443–457 glucan synthase gene models, 447–453 resistance mechanisms, 453–457 Saccharomyces cerevisiae model, 446–447 SAR development, 439–442 synthesis, 439–442 in vivo potency enhancement, 442–443, 453–457 antifungal resistance, 426–428, 453–457 cilofungin, 433–435 echinocandins, 430–433, 450, 457–466 FK463, 435, 465 fungal cell wall research cell wall screens, 434–437 early research, 430–437 properties, 428–430 LY303366, 462–465 pharmacology, 464–465 in vitro activity, 462–464 in vivo activity, 464 MK-0911, 455–462 pharmacology, 443, 460–461 therapeutic efficacy in humans, 462 in vitro activity, 457–459 in vivo activity, 459–460 mulundoncandin, 465 overview, 423–428, 466 papulacandins, 430–433 pneumocandins, 437–439 fermentation, 438–439 L-693,989 discovery, 435, 439, 442 pneumocystis carinii, 437–438 solubility issues, 438–439

G gastropathy clinical trials role in management, 3 nonsteroidal anti-inflammatory druginduced problems, 115–116, 127, 130–132 genomics, randomized controlled trials, 10–11 glucan synthase inhibitors, antifugal properties, 423–466 amino compound development, 439–457 action mechanisms, 443–457 Aspergillus models, 440–441, 452–453 Candida albicans model, 451–452

SUBJECT INDEX

genetic insights, 443–457 glucan synthase gene models, 447–453 resistance mechanisms, 453–457 Saccharomyces cerevisiae model, 446–447 SAR development, 439–442 synthesis, 439–442 in vivo potency enhancement, 442–443, 453–457 antifungal resistance, 426–428, 453–457 cilofungin, 433–435 echinocandins, 430–433, 450, 457–466 FK463, 435, 465 fungal cell wall cell wall screens, 434–437 early research, 430–437 properties, 428–430 LY303366, 462–465 pharmacology, 464–465 in vitro activity, 462–464 in vivo activity, 464 MK-0911, 455–462 pharmacology, 443, 460–461 therapeutic efficacy in humans, 462 in vitro activity, 457–459 in vivo activity, 459–460 mulundoncandin, 465 overview, 423–428, 466 papulacandins, 430–433 pneumocandins, 437–439 fermentation, 438–439 L-693,989 discovery, 435, 439, 442 Pneumocystis carinii, 437–438 solubility issues, 438–439 glycoprotein platelet receptor antagonists, angina treatment, 4 GW-2570, structure, 188

H hair loss, see androgenetic alopecia heparin, myocardial infarction treatment, 4 hepatotoxins, HMG co-A reductase inhibitors, 95 HER2, monoclonal antibody therapy target, 394–403 hirsutism, in females, 5α-reductase inhibitor studies, 169–170 HIV-1 protease inhibitors, 6, 213–245 clinical perspectives, 235–242 developmental milestones, 236–239 use issues, 239–242

561

development, 215–216 future research directions, 234–235, 244 identification, 223–227 mechanism-based characterization strategy, 227–234 overview, 213–214, 244–245 selection and validation, 214–215 structure-based design, 216–223 treament combinations, 242–244 HMG co-A reductase inhibitors, 77–107 cholesterol-lowering effects, 89–91 action mechanisms, 89 cholesterol controversy, 97–98 combination therapy, 89–91 bile acid sequestrants, 82, 90–91 diet, 83, 89–90 fibrate drugs, 84, 91 niacin, 83, 90–91, 93 statins, 90–91 clinical trials cancer, 103–105 noncardiovascular deaths, 102–103 outcome studies, 4, 98–100 coronary morbidity and mortality reduction, 100–102, 107 future research directions, 105–107 lipoprotein reaction, 84–88 chronopharmacology, 86 comparative effectiveness, 87–88 dose-response relationship, 86 outcome studies, 98–100 overview, 77–84 safety, 91–98, 102–105 tolerance, 91–98 central nervous system effects, 96–97 cholesterol controversy, 97–98 hepatotoxicity, 95 myopathy, 92–95 treatment intensity, 105–106 hyperplasia, see benign prostatic hyperplasia hypertension angiotensin II role, 16–17 normotension compared, 54–55 renin-angiotensin system link, 14–16, 54 treatment angiotensin-converting enzyme inhibitors, 15, 41–45, 54–55 captopril, 5, 26, 43–44 clinical trials role, 5

562

SUBJECT INDEX

hypertension (continued) enalapril, 30, 44 fosinopril, 36 lisinopril, 33, 48

I ibuprofen gastropathy incidence, 130–131 properties, 129–130 ICI-164,384, properties, 297, 313–314 ICI-182,780, properties, 297, 313–314 idoxifene, properties, 297, 316 immunotherapy, see monoclonal antibody therapy indinavir acquired immune deficiency syndrome treatment, 6, 237–239, 243 mechanism-based design, 231, 233 indomethacin, properties, 121–122 infliximab, rheumatoid arthritis treatment, 382–384 insulin peroxisome proliferator-activated receptor γ agonists, sensitization, 187–193 clinical experience, 197 direct cellular effects, 191–192 gene expression alterations, 188–189 target gene identification, 192–193 thiazolidinedione insulin sensitizers, 182–183, 197 in vivo efficacy, 187–188 in vivo physiology, 188–191 resistance, 181, 191, 195–197 intercellular adhesion molecule-1, monoclonal antibody therapy target, 386 interleukin-1, rheumatoid arthritis monoclonal antibody therapy, 385

K ketoconazole, antifugal properties, 425

L L-693,989 antifugal properties, 439, 442 structure, 435 L-705,589, antifugal properties, 440, 442–445

L-731,373, antifugal properties, 440, 442–445 L-733,560, antifugal properties, 440, 442–447, 450 L-764,406, structure, 188 L-796,449, structure, 188 lamivudine, acquired immune deficiency syndrome treatment, 243 lisinopril, 30–33 cardiovascular disease treatment, 33, 48 characterization, 32–33, 37 clinical approvals, 33 oral testing, 31–32 liver toxins, HMG co-A reductase inhibitors, 95 lopinavir, structure-based design, 218, 220–221 lovastatin cancer treatment, 103–105 cholesterol-lowering effects, 4, 82–85, 88, 99–102 hepatotoxicity, 95 long-term safety, 97 lupus, monoclonal antibody therapy, 386–387 LY121019, antifugal properties, 434 LY303366, antifugal properties, 462–465 pharmacology, 464–465 structure, 435 in vitro activity, 462–464 in vivo activity, 464 LY353381, antiestrogenic properties, 318

M meloxicam, properties, 121–123, 128 menopause, overview, 298–301 metformin, diabetes treatment, 200–201 MK-0386, properties, 171–172 MK-0663, properties, 121–122 MK-0911, antifugal properties, 455–462 pharmacology, 443, 460–461 therapeutic efficacy in humans, 462 in vitro activity, 457–459 in vivo activity, 459–460 MK-0991, structure, 435 moexipril, properties, 38 monoclonal antibody therapy, 369–403 antibody engineering, 372–373 antibody generation, 371 cancer treatment, 387–403 considerations, 387–390

563

SUBJECT INDEX

lymphocyte differentiation antigens, 390–394 CAMPATH-1, 394 CD19 targeting, 393 CD20 targeting, 390–393 CD22 targeting, 393 CD25 targeting, 393 idiotype, 390 radioimmunotherapy, 392–393 rituximab, 391–392 oncogene-product antigens, 394–403 bispecific antibodies, 401–402 CD64 targeting, 401 C225 radiotherapy, 403 epidermal growth factor race, 403 HER2 targeting, 394–403 immunoliposomes, 402 immunotoxins, 402 trastuzumab, 395–401 cardiomyopathy, 379 coronary artery therapy, 376–379 Crohn’s disease, 387 heart failure, 379 organ transplant therapy, 374–376 activation-associated T-cell antigens, 375–376 function-associated antigens, 374–375 T-cell differentiation, 374–375 overview, 369–371, 403 psoriatic arthritis, 387 rheumatoid arthritis, 381–386 interleukin-1 targeting, 385 T-cell targeting, 385–386 tumor necrosis factor-α blockade, 381–385 scleroderma, 387 septic shock, 379–381 antiendotoxin antibodies, 380 tumor necrosis factor-blocking agents, 379–380 systemic lupus erythematosus, 386–387 viral antigens, 381 mulundoncandin, antifugal properties, 465 myocardial infarction, treatment angiotensin-converting enzyme inhibitors, 5, 41, 49–50 clinical trials role, 4–5 myopathy HMG co-A reductase inhibitor tolerance, 92–95 monoclonal antibody therapy, 379

N naproxen, properties, 123, 129 nelfinavir acquired immune deficiency syndrome treatment, 239, 243 mechanism-based design, 229, 232 niacin, cholesterol-lowering effects, 83, 90–91, 93 nimesulide, properties, 121–123 nonsteroidal anti-inflammatory drugs, see also specific drugs efficacy, 129–130 historical perspectives, 116–117 medicinal chemistry, 124–127 myocardial infarction treatment, 4 overview, 115–116, 135–137 safety, 130–133 articular cartilage damage, 133 gastrointestinal problems, 115–116, 127, 130–132 renal problems, 132–133 selectivity, 120–124, 127–129 normotension, hypertension compared, 54–55 NS-398, properties, 121–122

O organ transplants, monoclonal antibody therapy, 374–376 activation-associated T-cell antigens, 375–376 function-associated antigens, 374–375 T-cell differentiation, 374–375 osteoarthritis, treatment cyclooxygenase-2 inhibitors, 123, 129 nonsteroidal anti-inflammatory drugs, 129, 133 osteoporosis incidence, 298–300 treatment alendronate, 5 dehydroepiandrosterone, 347 EM-652, 346–347 raloxifene, 5, 316, 346–347

P palinavir, mechanism-based design, 229 papulacandins, antifugal properties, 430–433

564

SUBJECT INDEX

perindopril, properties, 38 permixon, properties, 173 peroxisome proliferator-activated receptor γ agonists action mechanisms, 182–195 adverse consequences, 193–195 inflammation, 193–194 physiologic and pathophysiologic effects, 194–195 insulin sensitization, 187–193 direct cellular effects, 191–192 gene expression alterations, 188–189 target gene identification, 192–193 in vivo efficacy, 187–188 in vivo physiology, 188–191 ligand spectrum, 185–188 chemistry, 186–187 screening assays, 185–186 synthetic ligand structure, 187–188 structure and function, 183–185 activation, 185 isoforms, 183–184 tissue expression, 183–184 transcriptional regulation, 184–185 therapeutic indications, 193–195 atherosclerosis, 193–194 colon cancer, 194 thiazolidinedione insulin sensitizers, 182–183, 197 clinical experience, 195–202 β-cell failure, 196–197 glycemic control benefits, 196 insulin resistance treatment, 197 insulin sensitivity improvement, 197 pioglitazone, 188, 194, 202 rosiglitazone, 198–202 combination studies, 200–201 phase 2 studies, 198–199 phase 3 studies, 198–199 safety, 201–202 troglitazone, 197–198 type 2 antidiabetic treatment, 196 future research directions, 203 overview, 181–182, 203 pharmaceutical industry, see also specific drugs clinical trial support, 2, 9 knowledge-base advances, 40–41 new chemical entity development, 3–8 presenting protein strategy, 279–283

drug–target interaction enhancement, 280–281 presenter protein properties, 281–282 SH2 domain ligand affinity enhancement, 282–283 small molecule ligand affinity enhancement, 280–281 pioglitazone atherosclerosis treatment, 194 diabetes treatment, 202 structure, 188 pneumocandins, antifugal properties, 437–439 fermentation, 438–439 L-693,989 discovery, 435, 439, 442 Pneumocystis carinii, 437–438 solubility issues, 438–439 PNU-103017, HIV-1 protease inhibition, 225–226 PNU-140690, HIV-1 protease inhibition, 225–226 pravastatin cancer treatment, 103–105 central nervous system effects, 96–97 cholesterol-lowering effects, 4, 83, 88, 92–93, 99–102 long-term safety, 97 presenting protein strategy calcineurin inhibition, immunophilin/immunosuppressant complex inhibition, 275–279 complex structure, 275–276 FKBP-FK506-calcineurin ternary complex structure, 276–278 FKBP-rapamycin-FRB ternary complex structure, 278–279 description, 279–283 drug–target interaction enhancement, 280–281 presenter protein properties, 281–282 SH2 domain ligand affinity enhancement, 282–283 small molecule ligand affinity enhancement, 280–281 propanolol, myocardial infarction treatment, 5 proscar, benign prostatic hyperplasia treatment, 155–159 prostate cancer, clinical studies, 5αreductase inhibitor effects, 161–162, 168

565

SUBJECT INDEX

prostate-specific antigen benign prostatic hyperplasia diagnosis, 159–161 male androgenetic alopecia diagnosis, 168 prostatic hyperplasia, treatment, 6, 151–161 dihydrotestosterone role, 151, 153–154, 160 inhibitor responsiveness predictors, 159–160 natural history assessment, 144, 151–153 phase III studies, 154–155 proscar efficacy and safety, 155–159 prostate gland growth, 151 serum prostate-specific antigen role, 159–161 protease inhibitors, see HIV-1 protease inhibitors

Q quinapril, properties, 38

R raloxifene breast cancer treatment, 316–318 osteoporosis treatment, 5, 316, 346–347 structure, 297 ramipril congestive heart failure treatment, 5, 48 properties, 38 randomized controlled trials cholesterol hypothesis validation, 2–3 description, 1–2 genomics role, 10–11 natural history determination, 1–3, 8–9 patient safety, 9–10 pharmaceutical industry role, 2, 9 renal disease, treatment angiotensin-converting enzyme inhibitors normotension versus hypertension concepts, 54–55 renal insufficiency, 53–54 renin-angiotensin system, 45–48 clinical trials role, 5 rofecoxib, 132–133 renal function cyclooxygenase-2 inhibitor effects, 132–133

nonsteroidal anti-inflammatory drug effects, 132–133 renin-angiotensin system, 45–48 renin-angiotensin system angiotensin-converting enzyme inhibitors effects cardiac disease treatment, 46–49 renal disease treatment, 45–48 hypertension link, 14–16, 54 restenosis, treatment, angiotensinconverting enzyme inhibitors, 52–53 reteplase, myocardial infarction treatment, 4 rheumatoid arthritis, treatment cyclooxygenase-2 inhibitors, 129 infliximab, 382–384 monoclonal antibody therapy, 381–386 interleukin-1 targeting, 385 T-cell targeting, 385–386 tumor necrosis factor-α blockade, 381–385 ritonavir acquired immune deficiency syndrome treatment, 6, 237, 239, 243 structure-based design, 217, 220, 227 rituximab, cancer monoclonal antibody therapy, 391–392 rofecoxib arthritis treatment, 6, 123, 129 colon cancer treatment, 134 gastropathy incidence, 116, 123, 130–131 properties, 121–124, 129–130 renal function effects, 132–133 selectivity in humans, 127–129 rosiglitazone clinical experience in diabetes treatment, 198–202 combination studies, 200–201 phase 2 studies, 198–199 phase 3 studies, 198–199 safety, 201–202 peroxisome proliferator-activated receptor γ agonism, 187 structure, 188 S Saccharomyces cerevisiae, glucan synthase inhibitor antifugal properties amino compound development, 446–447 cell wall targeting, 428–430 FKS genes, 447–451

566

SUBJECT INDEX

saquinavir acquired immune deficiency syndrome treatment, 243 mechanism-based design, 227–228, 232–233 saralasin, properties, 37–38 SB-219994, structure, 188 SC-52151, mechanism-based design, 231, 234 septic shock, monoclonal antibody therapy, 379–381 antiendotoxin antibodies, 380 tumor necrosis factor-blocking agents, 379–380 serotherapy, see monoclonal antibody therapy simvastatin cancer treatment, 103–105 cholesterol-lowering effects, 4, 82–92, 99–102 hepatotoxicity, 95 long-term safety, 97 skin cancer, treatment, statins, 104 statins, 77–107 cholesterol-lowering effects, 4, 82–92 action mechanisms, 89 cholesterol controversy, 97–98 combination therapy, 89–91 bile acid sequestrants, 82, 90–91 diet, 83, 89–90 fibrate drugs, 84, 91 niacin, 83, 90–91, 93 clinical trials cancer, 103–105 noncardiovascular deaths, 102–103 outcome studies, 4, 98–100 coronary morbidity and mortality reduction, 100–102, 107 future research directions, 105–107 hepatotoxicity, 95 lipoprotein reaction, 84–88 chronopharmacology, 86 comparative effectiveness, 87–88 dose-response relationship, 86 outcome studies, 98–100 overview, 77–84 safety, 91–98, 102–105 tolerance, 91–98 central nervous system effects, 96–97 cholesterol controversy, 97–98

hepatotoxicity, 95 myopathy, 92–95 treatment intensity, 105–106 stavudine, acquired immune deficiency syndrome treatment, 243 streptokinase, myocardial infarction treatment, 4 sulfonylurea, diabetes treatment, 200–201 surrogate end point dilemma, 55–61 systemic lupus erythematosus, monoclonal antibody therapy, 386–387

T tamoxifen action mechanisms, 296–297, 314–316 breast cancer treatment, 6, 295–298, 302, 314 MCF-7 cancer cell cycling comparison, 337–340 T-cells, monoclonal antibody therapy activation-associated antigens, 375–376 function-associated antigens, 374–375 rheumatoid arthritis, 385–386 teprotide, clinical studies, 21–22, 27, 37 testosterone, see also dihydrotestosterone conversion by 5α-reductase, 145, 149 thiazolidinediones atherosclerosis treatment, 193–194 cancer treatment, 194 insulin sensitization, 182–183, 197 ligand chemistry, 186–187 physiologic and pathophysiologic effects, 194–195 timolol, myocardial infarction treatment, 5 tipranavir, HIV-1 protease inhibition, 225–226 tirofiban, angina treatment, 4 toremifene, properties, 297, 316 trandolapril atherosclerosis treatment, 48 properties, 38 transcription, regulation, peroxisome proliferator-activated receptor γ role, 184–185 trastuzumab, cancer monoclonal antibody therapy, 395–401 triphenylethylenes, antiestrogen activity, 297, 314–316

567

SUBJECT INDEX

troglitazone atherosclerosis treatment, 194 clinical experience in diabetes treatment, 197–198 structure, 188 tumor necrosis factor, monoclonal antibody therapy rheumatoid arthritis, 381–385 septic shock, 379–380

U U-96988, HIV-1 protease inhibition, 224 ulcers clinical trials role in management, 3 nonsteroidal anti-inflammatory druginduced problems, 115–116, 127, 130–132

V valdecoxib, properties, 121–122

W warfarin, HIV-1 protease inhibition, 224, 226 women’s health needs androgenetic alopecia, 169 antiestrogens, see antiestrogens

breast cancer treatment benzopyrans DMBA-induced mammary tumor development inhibition, 331–335 human breast cancer inhibition, 336–346 MCF-7 cancer cell cycling comparison, 337–340 clinical trials role, 6 estrogen replacement therapy, 295, 299–302 raloxifene, 316–318 statins, 103–105 tamoxifen, 6, 295–298, 302, 314 trastuzumab, 395–401 cardiovascular disease, see cardiovascular disease estrogen replacement therapy, 295, 299–302 hirsutism, 169–170 hypertension, see hypertension menopause, 298–301 osteoporosis, 5, 298–300, 316, 346–347 overview, 295

Z zidovudine, acquired immune deficiency syndrome treatment, 243

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