Combination Cancer Therapy: Modulators and Potentiators (Cancer Drug Discovery and Development)

COMBINATION CANCER THERAPY CANCER DRUG DISCOVERY AND DEVELOPMENT BEVERLY A. TEICHER, SERIES EDITOR Combination Cancer...

Author: Gary K. Schwartz

16 downloads 523 Views 2MB Size Report

This content was uploaded by our users and we assume good faith they have the permission to share this book. If you own the copyright to this book and it is wrongfully on our website, we offer a simple DMCA procedure to remove your content from our site. Start by pressing the button below!

Report copyright / DMCA form

DOWNLOAD PDF

COMBINATION CANCER THERAPY

CANCER DRUG DISCOVERY AND DEVELOPMENT BEVERLY A. TEICHER, SERIES EDITOR Combination Cancer Therapy: Modulators and Potentiators, edited by Gary K. Schwartz, 2005 Death Receptors in Cancer Therapy, edited by Wafik S. El-Deiry, 2005 Bone Metastasis: Experimental and Clinical Therapeutics, edited by Gurmit Singh and Shafaat A. Rabbani, 2005 The Oncogenomics Handbook, edited by William J. LaRochelle and Richard A. Shimkets, 2005 Camptothecins in Cancer Therapy, edited by Thomas G. Burke and Val R. Adams, 2005 Cancer Chemoprevention, Volume 2: Strategies for Cancer Chemoprevention, edited by Gary J. Kelloff, Ernest T. Hawk, and Caroline C. Sigman, 2005 Cancer Chemoprevention, Volume 1: Promising Cancer Chemopreventive Agents, edited by Gary J. Kelloff, Ernest T. Hawk, and Caroline C. Sigman, 2004 Proteasome Inhibitors in Cancer Therapy, edited by Julian Adams, 2004 Nucleic Acid Therapeutics in Cancer, edited by Alan M. Gewirtz, 2004 DNA Repair in Cancer Therapy, edited by Lawrence C. Panasci and Moulay A. Alaoui-Jamali, 2004 Hematopoietic Growth Factors in Oncology: Basic Science and Clinical Therapeutics, edited by George Morstyn, MaryAnn Foote, and Graham J. Lieschke, 2004 Handbook of Anticancer Pharmacokinetics and Pharmacodynamics, edited by William D. Figg and Howard L. McLeod, 2004

Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials, and Approval, Second Edition, edited by Beverly A. Teicher and Paul A. Andrews, 2004 Handbook of Cancer Vaccines, edited by Michael A. Morse, Timothy M. Clay, and Kim H. Lyerly, 2004 Drug Delivery Systems in Cancer Therapy, edited by Dennis M. Brown, 2003 Oncogene-Directed Therapies, edited by Janusz Rak, 2003 Cell Cycle Inhibitors in Cancer Therapy: Current Strategies, edited by Antonio Giordano and Kenneth J. Soprano, 2003 Chemoradiation in Cancer Therapy, edited by Hak Choy, 2003 Fluoropyrimidines in Cancer Therapy, edited by Youcef M. Rustum, 2003 Targets for Cancer Chemotherapy: Transcription Factors and Other Nuclear Proteins, edited by Nicholas B. La Thangue and Lan R. Bandara, 2002 Tumor Targeting in Cancer Therapy, edited by Michel Pagé, 2002 Hormone Therapy in Breast and Prostate Cancer, edited by V. Craig Jordan and Barrington J. A. Furr, 2002 Tumor Models in Cancer Research, edited by Beverly A. Teicher, 2002 Tumor Suppressor Genes in Human Cancer, edited by David E. Fisher, 2001 Matrix Metalloproteinase Inhibitors in Cancer Therapy, edited by Neil J. Clendeninn and Krzysztof Appelt, 2001

COMBINATION CANCER THERAPY MODULATORS AND POTENTIATORS Edited by

GARY K. SCHWARTZ, MD Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY

© 2005 Humana Press Inc. 999 Riverview Drive, Suite 208 Totowa, New Jersey 07512 www.humanapress.com All rights reserved. No part of this book may be reproduced, stored in a retrieval system, or transmitted in any form or by any means, electronic, mechanical, photocopying, microfilming, recording, or otherwise without written permission from the Publisher. The content and opinions expressed in this book are the sole work of the authors and editors, who have warranted due diligence in the creation and issuance of their work. The publisher, editors, and authors are not responsible for errors or omissions or for any consequence arising from the information or opinions presented in this book and make no warranty, express or implied, with respect to its contents. Due diligence has been taken by the publishers, editors, and authors of this book to assure the accuracy of the information published and to describe generally accepted practices. The contributors herein have carefully checked to ensure that the drug selections and dosages set forth in this text are accurate and in accord with the standards accepted at the time of publication. Notwithstanding, as new research, changes in government regulations, and knowledge from clinical experience relating to drug therapy and drug reactions constantly occurs, the reader is advised to check the product information provided by the manufacturer of each drug for any change in dosages or for additional warnings and contraindications. This is of utmost importance when the recommended drug herein is a new or infrequently used drug. It is the responsibility of the treating physician to determine dosages and treatment strategies for individual patients. Further it is the responsibility of the health care provider to ascertain the Food and Drug Administration status of each drug or device used in their clinical practice. The publisher, editors, and authors are not responsible for errors or omissions or for any consequences from the application of the information presented in this book and make no warranty, express or implied, with respect to the contents in this publication.

This publication is printed on acid-free paper. h ANSI Z39.48-1984 (American National Standards Institute) Permanence of Paper for Printed Library Materials. Production Editor: Tracy Catanese Cover design by Patricia F. Cleary Cover illustration: VEGF-induced corneal neovascularization in a rat eye. Figure 4 from Chapter 10, “Use of Animal Models to Evaluate Signal Transduction Inhibtiors As Modulators of Cytotoxic Therapy,” by Beverly A. Teicher. For additional copies, pricing for bulk purchases, and/or information about other Humana titles, contact Humana at the above address or at any of the following numbers: Tel.: 973-256-1699; Fax: 973-256-8341; E-mail: [email protected] or visit our Website at www.humanapress.com Photocopy Authorization Policy: Authorization to photocopy items for internal or personal use, or the internal or personal use of specific clients, is granted by Humana Press Inc., provided that the base fee of US $25.00 per copy is paid directly to the Copyright Clearance Center at 222 Rosewood Drive, Danvers, MA 01923. For those organizations that have been granted a photocopy license from the CCC, a separate system of payment has been arranged and is acceptable to Humana Press Inc. The fee code for users of the Transactional Reporting Service is: [0-58829-200-2/05 $25.00]. Printed in the United States of America. 10 9 8 7 6 5 4 3 2 1 e-ISBN: 1-59259-864-1 Library of Congress Cataloging-in-Publication Data Combination cancer therapy : modulators and potentiators / edited by Gary K. Schwartz. p. ; cm. -- (Cancer drug discovery and development) Includes bibliographical references and index. ISBN 1-58829-200-2 (alk. paper) 1. Cancer--Chemotherapy. 2. Apoptosis. 3. Cancer--Treatment. [DNLM: 1. Neoplasms--drug therapy. 2. Drug Therapy, Combination. QZ 267 C731 2005] I. Schwartz, Gary K. II. Series. RC271.C5C615 2005 616.99'4061--dc22 2004010543

PREFACE The concept of combining chemotherapeutic agents to increase cytotoxic efficacy has evolved greatly over the past several years. In the past, the rationale for combination chemotherapy centered on attacking different biochemical targets, overcoming drug resistance in heterogenous tumors, and increasing the dose-density of combination chemotherapy to take advantage of tumor growth kinetics. The overall goal was to improve clinical efficacy with acceptable clinical toxicity. We are now moving to a new generation of drug therapies. An understanding of the molecular basis of tumor cell growth and differentiation has resulted in the identification of new targets for cancer therapy. These targets range from proteins that regulate the cell cycle to cell-surface receptors that mediate signal transduction events. However, despite promising preclinical activity as single agents, early clinical trials with these drugs have proven disappointing, with rare clinical responses. This may be explained by the fact that these agents are not classic cytotoxic drugs. Instead, the interruption of intracellular signals is capable of inducing growth arrest with minimal cell death. Recent studies indicate that these new agents are potent potentiators of chemotherapy-induced apoptosis. It is now apparent that the future clinical development of these molecularly targeted therapies will depend on the modulation of molecular events that will enhance the efficacy of our classic cytotoxic drugs. Therefore, as these drugs become part of our clinical programs, it will be essential to understand how to combine them with traditional chemotherapy. For example, it is now apparent that the sequence of administration of cyclin-dependent kinase inhibitors relative to chemotherapy can either enhance or antagonize the chemotherapeutic effect. These sequence-dependent effects can be explained by cell cycle perturbations, or by pharmacodynamic interactions between the agents in combination. An understanding of these drug interactions will be critical for the successful introduction of these new agents into traditional clinical use. Thus, it does not appear that we will be abandoning traditional cytotoxic agents for the new molecularly based approach to oncology. Rather, current studies indicate that chemotherapy and cytotoxic agents will continue to serve as a foundation upon which the next generation of new small molecules will be added as modulators and potentiators. In Combination Chemotherapy: Modulators and Potentiators, we focus on novel drug combinations with new agents that hold the most promise for the future of medical oncology. They run the gamut of targeted therapies against cell surface receptors (EGF-R and HER2), the cell cycle (the CDKs), signal transduction events (PKC and NF-PB), apoptosis (bcl-2), as well v

vi

Preface

as novel targeted therapies in ovarian cancer, hematologic diseases, and breast cancer. The emphasis is on new translational approaches that are being moved from the laboratory bench top to the patient’s bedside for the future treatments in cancer therapy. Gary K. Schwartz, MD

CONTENTS Preface ............................................................................................................... v Contributors ...................................................................................................... ix 1 Targeting of the EGFR As a Modulator of Cancer Chemotherapy ..................................................................... 1 Jose Baselga 2 Cyclin-Dependent Kinase Inhibitors in Combination Chemotherapy .......................................................... 27 Manish A. Shah and Gary K. Schwartz 3 Development of Protein Kinase C and Cyclin-Dependent Kinase Inhibitors As Potentiators of Cytotoxic Drug Action in Leukemia ........................................................................................ 61 Steven Grant 4 Carboxyamidotriazole, an Inhibitor of Nonvoltage-Operated Calcium Entry: Single-Agent and Combination Therapy for Ovarian Carcinoma ....................................................................... 89 Chad M. Michener and Elise C. Kohn 5 Targeted F-Particle Therapy: A Rational Approach to Drug Development in Hematologic Diseases and Solid Tumors ............. 107 John M. Burke, David A. Scheinberg, and Joseph G. Jurcic 6 Pharmacological Modulation of Fluoropyrimidines: Building on the Lessons of the Past................................................. 133 Owen A. O’Connor 7 Development of Inhibitors of HER2 With Taxanes: New Directions in Breast Cancer Therapy ..................................... 175 Shanu Modi, Monica N. Fornier, and Andrew D. Seidman 8 Targeting NF-PB to Increase the Activity of Cisplatin in Solid Tumors ................................................................................ 197 Don S. Dizon, Carol Aghajanian, X. Jun Yan, and David R. Spriggs 9 Combinations of Chemotherapy and G3139, an Antisense Bcl-2 Oligonucleotide ................................................ 209 Luba Benimetskaya, Sridhar Mani, and C. A. Stein

vii

viii

Contents

10 Use of Animal Models to Evaluate Signal Transduction Inhibitors As Modulators of Cytotoxic Therapy ............................. 231 Beverly A. Teicher Index .............................................................................................................. 277

CONTRIBUTORS CAROL AGHAJANIAN, MD • Developmental Chemotherapy Service, Memorial Sloan-Kettering Cancer Center, New York, NY JOSE BASELGA, MD • Vall d'Hebron University Hospital, Universidad Autonoma, Barcelona, Spain LUBA BENIMETSKAYA, PhD • Department of Medical Oncology/Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, NY JOHN M. BURKE, MD • Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY DON S. DIZON, MD • Director of Medical Oncology, Program in Women’s Oncology, Women & Infants’ Hospital, Brown Medical School, Providence, RI MONICA N. FORNIER, MD • Breast Cancer Service, Memorial Sloan-Kettering Cancer Center, New York, NY STEVEN GRANT, MD • Department of Medicine, Medical College of Virginia, Virginia Commonwealth University, Richmond, VA JOSEPH G. JURCIC, MD • Hematology Service, Memorial Sloan-Kettering Cancer Center, New York, NY ELISE C. KOHN, MD • Molecular Signaling Section, Laboratory of Pathology, National Cancer Institute, Bethesda, MD SRIDHAR MANI, MD • Department of Medical Oncology/Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, NY CHAD M. MICHENER, MD • Molecular Signaling Section, Laboratory of Pathology, National Cancer Institute, Bethesda, MD SHANU MODI, MD • Breast Cancer Service, Memorial Sloan-Kettering Cancer Center, New York, NY OWEN A. O’CONNOR, MD, PhD • Lymphoma and Developmental Chemotherapy Services, Division of Hematologic Oncology, Memorial Sloan-Kettering Cancer Center, New York, NY DAVID A. SCHEINBERG, MD, PhD • Experimental Therapeutics Center, Leukemia Service, Memorial Sloan-Kettering Cancer Center, New York, NY ix

x

Contributors

GARY K. SCHWARTZ, MD • Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY ANDREW D. SEIDMAN, MD • Breast Cancer Service, Memorial Sloan-Kettering Cancer Center, New York, NY MANISH A. SHAH, MD • Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY DAVID R. SPRIGGS, MD • Division of Solid Tumor Oncology, Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY C. A. STEIN, MD, PhD • Department of Medical Oncology/Medicine, Montefiore Medical Center, Albert Einstein College of Medicine, Bronx, New York, NY BEVERLY A. TEICHER, PhD • Vice President and Director of Oncology Portfolio, Genzyme Corporation, Framingham, MA X. JUN YAN, MD • Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY

Chapter 1 / EGFR to Modulate Chemotherapy

1

1

Targeting of the EGFR As a Modulator of Cancer Chemotherapy Jose Baselga, MD CONTENTS SUMMARY THE EGF RECEPTOR AS A TARGET FOR CANCER THERAPY ANTI-EGF RECEPTOR STRATEGIES AND MECHANISMS OF ACTION CLINICAL DEVELOPMENT OF ANTI-EGF RECEPTOR MONOCLONAL ANTIBODIES LOW-MW EGF RECEPTOR TYROSINE KINASE INHIBITORS CHALLENGES IN THE DEVELOPMENT OF ANTI-EGF RECEPTOR COMPOUNDS DIRECTIONS IN THE COMBINED TREATMENT WITH ANTI-EGF AGENTS AND CHEMOTHERAPY IN THE TREATMENT OF CANCER

SUMMARY The epidermal growth factor (EGF) receptor (EGFR) is a tyrosine kinase receptor of the ErbB family that is abnormally activated in many epithelial tumors. In human tumors, receptor overexpression correlates with a more aggressive clinical course. These observations suggest that the EGFR is a promising target for cancer therapy, and monoclonal antibodies directed at the ligand-binding extracellular domain and low molecular weight (MW) inhibitors of the receptor’s tyrosine kinase inhibitors (TKI) are currently in advanced stages of clinical development. These agents prevent ligand-induced receptor activation and downstream signaling, which results in cell-cycle arrest, promotion of apoptosis, and inhibition of angiogenesis. In preclinical models, these agents markedly enhance the antitumor effects of chemotherapy and radiation therapy. In patients, antiFrom: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

1

2

Baselga

EGFR agents can be given safely at doses that fully inhibit receptor signaling, and single-agent activity has been observed against a variety of tumor types, including colon carcinoma, non-small-cell lung cancer (NSCLC), head and neck cancer, ovarian carcinoma, and renal-cell carcinoma. However, their antitumor activity is significant but modest, and to improve their efficacy, ongoing research efforts are being directed at the selection of patients with EGFR-dependent tumors, identification of the differences among the various classes of agents, and new clinical development strategies. One such strategy, derived from the preclinical models, is to combine these agents with chemotherapy. Clinical trials of antiEGFR agents in combination with chemotherapy have been conducted or are underway in a variety of tumor types and in different clinical settings. In NSCLC, a series of well-supported multinational phase III clinical trials have shown that the combined therapy with chemotherapy and anti-EGFR TKI is not superior to chemotherapy alone. On the other hand, in advanced colorectal cancer, the combined treatment with anti-EGFR monoclonal antibodies and conventional chemotherapy was found to be statistically superior in terms of disease-free survival when compared with chemotherapy alone. In addition, in smaller trials, the addition of anti-EGFR monoclonal antibodies to chemotherapy does result in a higher antitumor response rate than with chemotherapy alone. Taken together, antiEGFR agents are active antitumor agents, and the optimal way to combine these agents with conventional chemotherapy is still to determined and likely to be agent and tumor-type dependent. Intensive clinical research on how best to integrate these agents into treatment is warranted.

THE EGF RECEPTOR AS A TARGET FOR CANCER THERAPY The epidermal growth factor (EGF) receptor (EGFR) was the first identified of a family of receptors known as the type I receptor tyrosine kinases, or ErbB receptors. This receptor family is comprised of four related receptors: the EGFR itself (ErbB1/EGFR/HER1), ErbB2 (HER2/neu), ErbB3 (HER3), and ErbB4 (HER4) (1–3). These receptors trigger downstream signaling pathways that are not linear but consist of a rich, multilayered network, which allows for horizontal interactions and permits multiple combinatorial responses which may explain the specificity of cellular outcomes to receptor activation. Deregulation of these tightly regulated ErbB receptor signaling pathways leads frequently to malignant transformation. In order to simplify our understanding of EGFR signaling, it may be useful to dissect the process into sequential levels starting at the cell surface, subsequently moving into intracellular-signaling pathways that lead to gene transcription, and ending in a variety of cellular responses (1). The cell surface is where the initial ligand–receptor and receptor–receptor interactions occur. ErbB receptors are composed of an extracellular ligand-binding domain, a transmembrane segment, and an intracellular protein kinase domain

Chapter 1 / EGFR to Modulate Chemotherapy

3

with a regulatory carboxyl-terminal segment. ErbB receptors become activated by several mechanisms. Under physiological conditions, a variety of EGFR family ligands drive the formation of homo- or heterodimeric complexes among the four ErbB receptors, which provides for signal amplification and diversification (Fig. 1) (1). In tumor cells, these receptors can be activated by additional mechanisms. First, receptor overexpression in the tumor may lead to ligand-independent receptor dimerization. In some tumors, such as glioblastoma, mutant forms of the EGFR that arise from gene rearrangements result in ligand-independent constitutive receptor activation and impaired receptor downregulation (4). Heterologous ligand-dependent mechanisms are also at play, as demonstrated by the finding that stimulation of G protein-coupled receptors results in EGFR activation via metalloproteinase-mediated cleavage of precursor membrane-bound EGF ligands (5). Recently, a ligand-independent mechanism of EGFR activation via the urokinase plasminogen receptor has been identified (6). These findings suggest that tumor cells may have additional EGFR activation mechanisms beyond receptor overexpression, mutations, and autocrine ligand production. At the signal-processing level, activation of the intrinsic receptor protein tyrosine kinase and tyrosine autophosphorylation occurs. These events result in the recruitment and phosphorylation of several intracellular substrates, as well as binding of docking and adaptor molecules to specific phosphotyrosine sites on receptor molecules (Fig. 2) (7). A major downstream signaling route of the ErbB family is via the Ras-Raf-MAP-kinase pathway (8). Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPKs, ERK1, and ERK2 (9). ERK1/2 regulate transcription of molecules that are linked to cell proliferation, survival, and transformation in laboratory studies (9). Another important target in EGFR signaling is phosphatidylinositol 3-kinase (PI3K) and the downstream protein-serine/threonine kinase Akt (10–12). Akt transduces signals that trigger a cascade of responses from cell growth and proliferation to survival and motility (12). Another route for signaling is via the stress-activated protein kinase pathway, involving protein kinase C and Jak/Stat. The activation of these pathways translates in the nucleus into distinct transcriptional programs that mediate a variety of cellular responses, including cell division, survival (or death), motility, invasion, adhesion, and cellular repair (1). The EGFR was proposed almost 20 yr ago as a target for cancer therapy for a variety of reasons. First, as already mentioned, the EGFR is frequently overexpressed in human tumors. Examples include cancers of the breast, lung, glioblastoma, head and neck cancer, bladder carcinoma, colorectal cancer, ovarian carcinoma, and prostate cancer (13). The level of increased expression can reach an order of magnitude or greater. Gene amplification is not a commonly reported finding in tumors, with the exception of the glioblastomas. Furthermore, in some glioblastomas a mutant variant of the receptor, denominated EGFR vIII, has a deletion in the extracellular domain leading to constitutive activation of its tyrosine kinase (14–16).

4

Baselga

Fig. 1. Epidermal growth factor receptor signaling. The process of receptor signaling may be intracell into sequential levels starting at the cell surface, where ligand–receptor and receptor–receptor interactions occur, into key intracellular-signaling pathways that lead to gene transcription and cell-cycle progression. The end result is a variety of cellular responses that promote the malignant phenotype.

Second, increased EGFR expression correlates with a poorer clinical outcome in a number of malignancies, including bladder, breast, lung, and head and neck cancers (13,17). Third, increased receptor content is often associated with increased production of ligands, such as transforming growth factor (TGF)-_ by the same tumor cells (13,17,18). This establishes conditions conducive to receptor activation by an autocrine stimulatory pathway. And finally, in early studies by one of us (J. M.), a series of monoclonal antibodies (MAbs) directed at the EGFR were shown to inhibit the growth of cancer cells bearing high levels of EGFRs, both in culture and in nude mouse xenografts (19–22).

ANTI-EGF RECEPTOR STRATEGIES AND MECHANISMS OF ACTION There are several potential strategies for targeting the EGFR, including MAbs that interfere with receptor signaling and MAbs serving as carriers of radionuclides, toxins, or prodrugs (23); low-molecular-weight (MW) tyrosine kinase inhibitors that interfere with receptor signaling; antisense oligonucleotides or ribozymes, which block receptor translation (24,25); or prevention of receptor

Chapter 1 / EGFR to Modulate Chemotherapy

5

Fig. 2. Inhibition of signaling pathways by anti-epidermal growth factor (EGF) receptor therapies. In unperturbed conditions, major signaling routes of the EGF receptor are the ones constituted by the Ras-Raf-MAP-kinase and the phosphatidylinositol 3-kinase (PI3K), and the downstream protein-serine/threonine kinase Akt pathway. Activation of Ras initiates a multistep phosphorylation cascade that leads to the activation of MAPkinases. Akt transduces signals that fall into two main classes: regulation of apoptosis and regulation of cell growth. (B,C): Signal transduction via these pathways is efficiently blocked by anti-EGF receptor therapies as shown here in cultures of A431 cells treated with the EGF receptor tyrosine kinase inhbitor ZD1839. (Adapted from ref. 34.)

trafficking to the cell surface with intracellular single-chain Fv fragments of antibodies (26). Of these approaches, MAbs and the low-MW tyrosine kinase inhibitors are the ones in the most advanced stages of clinical development and will be reviewed in detail. The antibodies in clinical trials bind to the easily accessible extracellular domain of the receptor and compete with the ligand binding to the receptor. For example, the murine MAb 225 and its chimeric human:murine derivative cetuximab (cetuximab, Erbitux™) bind to the EGFR with high affinity (Kd = 0.39 nM for cetuximab), compete with ligand binding, and block activation of receptor tyrosine kinase by EGF or TGF-_ (19,20,27). In addition, MAb 225/ cetuximab induces antibody-mediated receptor dimerization resulting in receptor downregulation, and this effect may be important for its growth-inhibitory capacity (28). The low-MW inhibitors, on the other hand, compete with ATP for binding to the tyrosine kinase portion of the receptor, and thereby abrogate the

6

Baselga

receptor’s catalytic activity. Some of these small molecules can induce formation of inactive EGFR homodimers and EGFR/HER2 (ErbB1/ErbB2) heterodimers (29,30), which impair EGFR-mediated transactivation of the potent ErbB2 tyrosine kinase. In addition, because of the >80% homology in the kinase domain between the EGFR (ErbB1) and HER2 (ErbB2) (3), some ATP-competitive low-MW inhibitory molecules can block the catalytic activity of both receptors (reviewed in 31). These small molecules are also able to block the catalytic activity of EGFR mutants lacking the extracellular domain (32) and should be able to prevent ligand-independent activation of EGFR kinase activity as well. At the level of downstream receptor-dependent signaling pathways, EGFR antibodies and low-MW ATP-competitive inhibitors of the EGFR kinase have similar effects. Both strategies result in an efficient blockade of the main EGFR signal transduction pathways, including the MAPK and PI3K/Akt pathways (33–38) and the Jak/Stat pathway (39) (Fig. 3). As a result of their effects on the receptor and downstream signaling, antiEGFR MAbs and the low-MW tyrosine kinase inhibitors interfere with a number of key cellular functions regulated by the receptor that satisfactorily explain their antitumor effects. These are summarized below, with antibody studies described first in most cases because they were reported earlier: 1. Cell-cycle arrest. Initial experiments with MAb 225 demonstrated that the antibody induces G1-phase arrest due to elevated levels of the CDK2 inhibitor p27KIP1, which results in hypophospholyration of Rb protein (40,41). Similarly, low-MW tyrosine kinase inhibitors of the EGFR induce an accumulation of p27KIP1 and of hypophosphorylated Rb protein that leads to a G1 arrest (42,44). 2. Potentiation of apoptosis. In some cases G1 arrest is followed by apoptosis (45). In DiFi colon carcinoma cells, this can be attributed to induction of Bax and activation of caspase-8 (45–48). Activation of other proapoptotic molecules has also been reported. 3. Inhibition of angiogenesis. Blockade of EGFR activation by cetuximab and by low-MW tyrosine kinase inhibitors results in a significant decrease in tumor-cell production of angiogenic growth factors such as `FGF, VEGF, and IL-8 (45– 48). The decrease in angiogenic growth factors in turn correlates with a significant decrease in microvessel density and an increase in apoptotic endothelial cells in human tumor xenografts (45). 4. Inhibition of tumor-cell invasion and metastasis. Cetuximab inhibits lung metastasis in mice with established human tumor xenografts (47). Cetuximab and similar MAbs directed against the EGFR have also been shown to inhibit the expression and activity of several matrix metalloproteinases (MMPs) that play a key role in tumor-cell adhesion, including the gelatinase MMP-9. Several studies have correlated this antibody-mediated decrease in MMP production with both a significant reduction in in vitro tumor-cell invasion and the inhibition of tumor growth and metastasis in nude mice (49–51). The inhibitory effects

Chapter 1 / EGFR to Modulate Chemotherapy

7

Fig. 3. Restoration of sensitivity to irinotecan by the addition of cetuximab in a colorectal carcinoma model. Growth inhibition of CPT-11 refractory colorectal tumor xenografts in nude mice. Mice with established HT-29 tumors were treated with two cycles of CPT11 therapy (100 mg/kg) on d 0 and 7. Mice with tumors that did not respond to CPT-11 therapy (>2 × initial tumor volume at d 12; shown as dotted vertical line) were selected, randomized, and then treated with cetuximab at 1 mg/dose/q3d (•), continued CPT-11 at 100 mg/kg/wk (white box), or combination therapy (black box). Bars, ±SE (56).

on invasion, metastasis, and angiogenesis cells may explain why cetuximab treatment is often more effective in vivo than in vitro. 5. Augmentation of the antitumor effects of chemotherapy and radiation therapy. Based on an initial observation that an anti-EGFR antibody had the capacity to enhance the antitumor activity of cisplatin in a human tumor xenograft model (22), extensive studies of human tumor-cell xenografts were conducted with MAb 225 and cetuximab. The experiments demonstrated that these MAbs markedly augment the antitumor effects of different classes of chemotherapeutic agents including cisplatin, doxorubicin, topotecan, and paclitaxel (52–55). In addition to the enhanced antitumor effects when both class of agents are given together, studies in preclinical models have shown that anti-EGFR monoclonal antibodies can reverse chemotherapy resistance (56). In a refractory tumor model, combined treatment with cetuximab and CPT-11 significantly inhibited the growth of CPT-11 refractory DLD-1 and HT-29 colon tumors, whereas either agent alone did not control tumor growth. These findings are highly consistent with the clinical data that have emerged from the colorectal BOND trial (see Subheading 3.).

8

Baselga

Studies have also demonstrated that the low-MW EGFR tyrosine kinase inhibitors enhance the antitumor activity of conventional chemotherapeutic agents, both in cell culture and in human tumor xenografts (57,58). Similar findings are observed when these agents are given in combination with radiation therapy (59,60). There is, however, evidence that the mechanisms of action and the antitumor effects of MAbs and the low-MW tyrosine kinase inhibitors are not completely overlapping. Anti-EGFR MAbs (28), but not the low-MW tyrosine kinase inhibitors (35), have the capacity to form receptor-containing complexes that result in receptor internalization, an important mechanism for attenuating receptor signaling. In addition, cetuximab can elicit antibody-dependent cellular cytotoxicity (ADCC) (61), an antitumor mechanism that also could be important for the action of the anti-erbB2 MAb, trastuzumab (62). In contrast, the inhibition of more than one ErbB receptor type is unique to the low-MW tyrosine kinase inhibitors. Therefore, it is not surprising that in studies with cultured cancer cells maximally inhibited by low-MW EGFR tyrosine kinase inhibitors, the addition of anti-EGFR MAbs can result in further antitumor activity (63). This finding sets the stage for combining anti-EGFR MAbs and low-MW tyrosine kinase inhibitors in the clinic.

CLINICAL DEVELOPMENT OF ANTI-EGF RECEPTOR MONOCLONAL ANTIBODIES Among available anti-EGFR MAbs (Table 1), the one furthest ahead in clinical development is the chimeric human:murine MAb cetuximab (cetuximab, Erbitux™). Cetuximab is a potent inhibitor of the growth of cultured cancer cells that have an active autocrine EGFR loop, and it is capable of inducing complete regressions of well-established human tumor xenografts overexpressing the EGFR (64). A series of phase I/II studies of cetuximab given alone or in combination either with chemotherapy or radiation have now been completed. In these early studies, cetuximab was found to be safe, and the most prominent side effects included an acneiform skin rash and anaphylactoid or anaphylactic reactions that occurred in 2% of cases (data from ImClone Systems, Inc.). The allergic reactions occurred after the first infusion and responded well to standard therapy (65). Nonneutralizing human antibodies against chimeric antibodies (HACAs) were detected in 4% of patients and were not related to allergic or anaphylactic reactions, and the HACA responses had no effect upon the pharmacokinetics of repeated weekly infusions of cetuximab (66). The optimal biological dose, as determined by saturation of antibody clearance, was found to be in the range of 200 to 400 mg/m2 per week (67). These doses have been confirmed to block EGFR activation and downstream signaling in biopsy specimens from patients (68).

Chapter 1 / EGFR to Modulate Chemotherapy

9

Table 1 Anti-EGFR MAbs: Clinical Activity

Cetuximab (C225)

EMD72000 EBX-EGF

n

Response

218

22.9%

120 10.8% 11% 22

17%

Colorectal (CPT-11 refractory)

+ CPT-11

Single agent

111

Head and neck (CDDP refractory)

+ CDDP

Colorectal (phase 1) Colorectal Renal Cell

Single agent

75 19

11% 16%

Single agent Single agent

23 31

13% 6%

23%

Table 2 IMC-C225 Phase II Trials in Refractory CRC

No. patients evaluable Response rate Stable disease Median duration

C225

C225 + Irinotecan

57 11% (3–19%) 37% 182 d

121 17% (11–24%) 31% 164 d

In phase II studies, cetuximab was shown to be active in patients with CPT11-treated colorectal cancer and with cisplatin-resistant head and neck tumors. Following an initial clinical observation that the addition of cetuximab to CPT11 induced responses in CPT-11-refractory patients with advanced colorectal carcinoma (69), a phase II study was performed with patients with advanced colorectal carcinoma and progression on CPT-11 treatment. In this study, 120 patients were continued on the same dose and schedule of CPT-11, and cetuximab was added on a full-dose weekly schedule (Table 2). The combination was found to be safe and the response rate was 22.5%, with a median duration of response of 186 d (70). A retrospective analysis of this study has revealed an interesting correlation between the occurrence of skin rash and a greater response rate (71). Recently, single agent cetuximab in patients with CPT-11-refractory advanced colorectal carcinoma has shown an 11% response rate in a small phase II study (59). These studies by Saltz raised some important questions: first, were the

10

Baselga

patients that responded to cetuximab and CPT-11 in the colorectal carcinoma study truly CPT-11 refractory? The design of the phase II colorectal trial of adding cetuximab to CPT-11 by Saltz and colleagues was well suited to answer this question, since a minimal number of cycles of CPT-11 prior to adding cetuximab to CPT-11 was not required. Second, even if we assume that the patients were chemotherapy refractory, the observed antitumor activity with cetuximab could be the result of several possibilities: cetuximab may reverse chemotherapy resistance—an indication of synergy in vivo, an indication of single agent activity by cetuximab, or both: single activity on one hand and a capacity to reverse resistance to chemotherapy on the other. As mentioned above, a follow-up study demonstrated that cetuximab has single-agent activity in colorectal carcinomas, so the question has been partially answered (72). The second possibility has been recently addressed by a European study in which patients refractory to CPT-11 were randomized to receive cetuximab as a single agent or cetuximab plus CPT-11 at the same dose and schedule that they had progressed on. This study, named BOND (bowel oncology with cetuximab antibody), evaluated the antitumor activity of cetuximab treatment alone (111 patients) or in combination with CPT-11 (218 patients) in advanced colorectal cancer patients with EGFR-positive disease that had progressed on CPT-11 (73). In this truly CPT-11-refractory population, the antitumor activity was greater in the patients that were given the same dose and schedule of CPT-11 in combination with cetuximab (response rate 22.9%) that in patients treated with cetuximab alone (response rate 10.8%) (p = 0.0074). Similarly, a significantly better disease control (partial responses plus disease stabilization) and a prolonged median time to progression were observed in the combination as compared to the cetuximab combination arm ( 55.5% vs 32.4%, p = 0.0001; and 4.1 vs 1.5 mo, p < 0.0001). Survival was not increased in the combination arm (8.6 vs 6.9 mo), although this is likely to be related to the fact that upon progression on the cetuximab alone arm, crossover to combined treatment was allowed. The enhanced antitumor activity of the combination did not result in an increase in CPT11 specific toxicity. This study also confirmed the lack of correlation of levels between the levels of EGFR expression and antitumor activity of cetuximab, and a greater response rate in those patients that developed a skin rash (73). In patients with advanced head and neck tumors, a phase II study analyzed the addition of cetuximab to the treatment of patients who had received two cycles of cisplatin-based therapy and had either stable or progressing disease. In the progressing-on-chemotherapy subgroup, 5 responses were seen out of 22 treated patients, for a response rate of 23% (74). In a larger study involving 75 evaluable patients with refractory head and neck cancer who had documented progression after having received at least two cycles of platinum-based therapy, an 11% response rate was observed when cetuximab was added to the platinum regimen

Chapter 1 / EGFR to Modulate Chemotherapy

11

(75). In the latter study, the potential source of bias of treating with cetuximab with patients not truly refractory to chemotherapy was controlled for, because patients were required to have documented progression after having received at least two cycles of platinum-based therapy, prior to cetuximab being added to the platinum regimen (75). Additional studies with cetuximab in combination with chemotherapy have also been conducted. A small phase III study in head and neck tumors comparing cisplatin and placebo to cisplatin and cetuximab showed more than doubling of the response rate, but only a modest and nonsignificant improvement in time to disease progression in the cetuximab arm (76). A randomized phase II study in EGFR-positive, advanced NSCLC patients (LUCAS, the Lung Cancer Cetuximab Combination Study), first-line therapy with cisplatin-vinorelbine was compared to the same combination plus cetuximab (77). Interestingly, a higher response rate was observed in the combination arm than in the chemotherapy alone arm ( response rate 50% vs 29%) (77). These results are of interest, taking into consideration the absence of benefit of combining an anti-EGFR TKI to two drugcontaining chemotherapy regimens in the same patient population. Additional responses to chemotherapeutic agents given in combination with cetuximab were observed in phase II studies of gemcitabine in patients with advanced pancreatic carcinoma (78) and docetaxel in advanced NSCLC (79). Cetuximab can also be administered safely in patients with head and neck cancer, when given in combination with radiation therapy, with 13 complete responses and 2 partial responses in 16 patients (80). A phase III study of radiation ± cetuximab in patients with advanced head and neck tumors has recently completed accrual. Other anti-EGFR MAbs that have a similar mechanism of action to cetuximab are currently under clinical investigation. ABX-EGF is a fully human IgG2 antiEGFR MAb that binds with high affinity (Kd = 50 pM), inhibits ligand-dependent receptor activation, and effectively inhibits the growth of human tumor xenografts (81). In a phase II study of ABX-EGF in advanced renal-cell carcinoma, 31 patients who had failed or were unable to receive IL-2/IFN-_ completed one 8-wk cycle of ABX-EGF and were evaluable for response. Objective responses were seen in one patient each at 1 and 1.5 mg/kg/wk dose levels. Fifty-eight percent of patients showed minor response/stable disease, and 36% progressed (82). EMD 72000 is a humanized anti-EGFR monoclonal antibody that also prevents ligand-induced receptor activation and is currently in phase I studies (83). This antibody has shown single-agent activity in a variety of tumor types and has a prolonged half-life that may allow for a less frequent administration schedule than the other antibodies, which are given on a weekly basis. In an ongoing trial, preliminary efficacy and pharmacodynamic data suggest that a more convenient every 2–3 wk administration schedule may actually be fea-

12

Baselga

sible with EMD 72000 (84). As with cetuximab, studies of EMD 72000 in combination with chemotherapy are underway in NSCLC. A pilot study has reported that in patients with NSCLC, the combination of paclitaxel and EMD 72000 is feasible and active (85). Antibody h-R3 is another anti-EGFR monoclonal antibody that has entered clinical trials. The safety profile of all of these antibodies has been good and, not surprisingly, acneiform skin rashes are the most frequent side effect.

LOW-MW EGF RECEPTOR TYROSINE KINASE INHIBITORS There are a large number of low-MW inhibitors of EGFR tyrosine kinase that are under clinical development (Table 3). In an attempt to classify these antireceptor agents, we have grouped them by their degree of receptor specificity (restricted to the EGFR, or also inhibiting other ErbB kinases) and by their reversibility or irreversibility of action.

Class 1. Reversible EGFR-Specific Tyrosine Kinase Inhibitors These compounds are the furthest ahead in clinical development and can be exemplified by ZD1839 and ERLOTINIB. ZD1839 (gefinitib, Iressa®), inhibits the EGFR kinase in vitro with an IC50 of 0.02 μM and requires a dose almost 200fold higher to inhibit HER2 (3.7 μM) (86). Preclinical studies with ZD1839 have shown antitumor activity in a variety of cultured tumor cell lines and in human tumor xenografts, both as a single agent and in combination with chemotherapy and radiation therapy (48,57,58,60,86,87). An intriguing finding has been that cultured breast cancer cells that express high levels of HER2, even in the presence of a low number of EGFRs, are exquisitely sensitive to ZD1839 at concentrations that do not suppress HER2 tyrosine kinase activity (35–38). Phase I studies have demonstrated that daily administration of ZD1839 is safe, with dose-dependent pharmacokinetics, although with a high degree of interpatient variability (88–90). The most common side effects were an acneiform skin rash, generally mild and reversible on cessation of treatment, and diarrhea. In these early studies, the effects of ZD1839 on EGFR activation and receptor-dependent events in the skin, an EGFR-dependent tissue, were analyzed (68). ZD1839 significantly suppressed EGFR phosphorylation, inhibited MAPK activation, reduced keratinocyte proliferation, and increased p27KIP1 levels and apoptosis. Marked reduction in EGFR phosphorylation was observed at doses well below doses producing unacceptable gastrointestinal toxicity, a finding that strongly supports the use of an optimal biological dose instead of the maximally tolerated dose for these types of agents. Clinical responses were observed in patients with NSCLC (88–90).

Chapter 1 / EGFR to Modulate Chemotherapy

13

Table 3 Cetuximab-BOND Study Design Irinotecan + cetuximab n = 218 Patients with colorectal cancer progressed on or within 3 mo of irinotecan-based chemotherapy

Radomized Cetuximab n = 111

PD

Irinotecan + cetuximab

Phase II studies with two dose levels of ZD1839 (250 and 500 mg) have know been completed in patients with NSCLC that had progressed after first- or second-line chemotherapy for advanced disease. The first study was conducted in 210 patients previously treated with one or two chemotherapy regimens, and showed an 18.7% response rate and marked improvement in disease-related symptoms (91) (Table 4). Interestingly, the 250 mg/d dose was as active as the 500 mg/d dose and had a lower frequency of adverse events. In the second study of 216 patients who had failed at least two prior chemotherapy regimens, tumor response rates of 11.8% and 8.8% were observed for the 250 and 500 mg/d groups, respectively (92). The results of these trials have led to the regulatory approval of ZD1839 in Japan. Pilot trials of ZD1839 with carboplatin/paclitaxel or gemcitabine/cisplatin demonstrated these combinations to be well tolerated, with antitumor activity in patients with NSCLC (93,94). The preclinical experiments mentioned above and the feasibility of combining chemotherapy with ZD1839 as demonstrated in the pilot studies led to the design of two large phase III studies of chemotherapy ± ZD1839 in patients with advanced chemotherapy-naïve NSCLC. These studies, known as INTACT 1 and 2 (for Iressa NSCLC Trial Assessing Combination Treatment) were randomized, double-blind, placebo-controlled trials of chemotherapy ± ZD1839 (95,96). In the first study, the chemotherapy regimen was a combination of cisplatin and gemcitabine at the usual dose and schedule (six cycles of gemcitabine 1250 mg/m2 on d 1 and 8, plus cisplatin 80 mg/m2 on d 1) (96). Patients were randomized to chemotherapy + placebo, chemotherapy + 250 mg/d ZD1839, or chemotherapy + 500 mg/d ZD1839. A total of 1093 patients were entered. There were no differences in overall survival (median 11.1, 9.9, and 9.9 mo for placebo, 250-mg, and 500-mg arms respectively), progressionfree survival, and time to worsening of symptoms across the three arms. In the second trial, 1037 patients were entered into a similarly designed trial although with a chemotherapy consisting of carboplatin ( AUC 6) and paclitaxel (225 mg/ m2) every 3 wk for six cycles (96). There were again no differences in overall

14

Baselga Table 4 Cetuximab-BOND Response Rate/TTP IRC–ITT cohort Combination (n = 218)

[95% CI]

Monotherapy (n = 111)

[95% CI]

PR 22.9% [17.5–29.1] 10.8% [5.7–18.1] Deisease control* 55.5% [48.6–62.2] 32.4% [23.9–42.0] Median TTP 4.1 mo 1.5 mo

p-value 0.0074 0.0001 <0.0001

*CR + PR + SD

survival (median 9.9, 9.8, and 8.7 mo for placebo, 250-mg, and 500-mg arms respectively), progression-free survival, and time to worsening of symptoms across the three arms. The results in these two well-controlled trials have clearly demonstrated that in NSCLC the addition of ZD1839 to conventional chemotherapy does not result in enhanced clinical benefit over chemotherapy alone. Single-agent phase II studies with ZD1839 have also been conducted or are still ongoing in other tumor types, including head and neck carcinomas, colon, prostate, gastric, and breast cancer. In tumors of the head and neck, a phase II study has shown an 11% response rate (97). A study in patients with previously treated colorectal carcinoma with ZD1839 at a (high) dose of 750 mg has recently been reported. Although some evidence of antitumor and biological activity were observed, there were no documented responses (98). In a study in hormonerefractory prostate cancer, patients were randomized to receive either ZD1839 at 250 mg or 500 mg/d (99). A total of 40 patients were treated: no objective or PSA responses were seen, and *9 patients had a best response of treatment failure, meeting the protocol criteria for stopping the study. Erlotinib (OSI-774, Tarceva®, formerly known as CP-358, 774) is an orally available quinazoline that is a selective inhibitor of the EGFR. A phase I study with increasing daily doses of erlotinib demonstrated that the MTD was 150 mg/ d. At this recommended dose level, erlotinib resulted in a steady-state serum concentration higher than the concentration required to achieve full receptor inhibition in preclinical models. Similar to ZD1839, erlotinib inhibited EGFRdependent processes in skin and tumor biopsies (100). Clinical responses were also seen in phase II studies conducted in patients with NSCLC, head and neck tumors, and ovarian carcinoma (101–103) (Table 3). In NSCLC, 57 patients that had histologically documented stage IIIB/IV EGFR-positive disease that had failed prior systemic therapy were treated with erlotinib at a dose of 150 mg/d (101). There were two complete remissions and five partial remissions for an overall response rate of 12%. In addition, the median survival in that study was 9.3 mo and the 1-yr survival was an impressive 40% (101).

Chapter 1 / EGFR to Modulate Chemotherapy

15

As with gefitinib, a comprehensive phase III program in first-line NSCLC of erlotinib in combination with chemotherapy was initiated. Two multicenter, randomized, controlled phase III studies evaluated erlotinib at 150 mg/d in combination with standard chemotherapy in patients with stage IIIB/IV metastatic NSCLC. In the TRIBUTE study, 1050 patients were randomized to receive erlotinib plus carboplatin and paclitaxel or chemotherapy alone. In the TALENT study, 1200 patients were randomized to receive either erlotinib in combination with gemcitabine and cisplatin or chemotherapy plus a placebo. In a recent announcement by the pharmaceutical company sponsoring the trial (www.gene.com), the results of both studies were reported as failing to meet their primary endpoint (survival) in addition to other secondary endpoints. These results are highly similar to the results of the gefitinib studies in combination with chemotherapy in NSCLC. In addition to the TALENT and TRIBUTE studies, there is an ongoing 700patient phase III study in refractory NSCLC being conducted by the National Cancer Institute in Canada, in which patients are being randomized to receive erlotinib vs placebo, with survival as its primary endpoint. In ovarian carcinoma, 34 patients were treated and 2 patients had a confirmed response (overall response rate of 6%), in addition to 2 patients with unconfirmed responses and 14 patients with stable disease for >2 mo (103). In head and neck, a multicenter trial of 124 patients with locally recurrent and/or metastatic disease that had been previously treated were given erlotinib at the recommended dose of 150 mg/d: there were 6 patients with confirmed responses, for an overall response rate of 5% (102). In a phase II ongoing study in colorectal carcinoma, no clinical responses have been reported to date (104). Currently, phase II studies with erlotinib are underway in other tumor types, including breast cancer, and phase IB studies are exploring the feasibility of combining erlotinib with a variety of conventional chemotherapeutic agents.

Class 2. Irreversible EGFR-Specific Tyrosine Kinase Inhibitors This class is represented by EKB-569, an EGFR tyrosine kinase inhibitor that binds irreversibly to the EGFR and has an IC50 of 38.5 nM in vitro (105). To demonstrate that EKB-569 bound covalently to the EGFR, 14C-labeled EKB-569 was synthesized and incubated with cellular membranes from cell lines expressing the receptor; the reaction was terminated under reducing conditions, and continued binding of labeled EKB-569 to the EGFR was observed. EKB-569 exerts far less inhibition of the tyrosine kinase activities of other members of the EGFR family, displaying an IC50 30 times higher for HER2 than for the EGFR. In the A431 human tumor xenograft model, a single dose of EKB-569 resulted in a 50% inhibition of receptor phosphorylation at 24 h despite a serum half-life of 2 h, a finding consistent with its reported irreversibility (105). In an initial phase I study, EKB-569 has been reported to be safe both on an intermittent and a continuous dose schedule (106). The observed side effects were skin rashes and diarrhea, very similar to those observed with other compounds of this type.

16

Baselga

Class 3. Reversible PAN-HER (Human EGF Receptor Family) Tyrosine Kinase Inhibitors In those situations in which coexpression of the EGFR (ErbB1) and the HER2 (ErbB2) occurs (13), an inhibitor that simultaneously targets both receptors may have therapeutic advantages. GW2016, currently under clinical development, inhibits the kinase activity of the two receptors, with an IC50 of 10 nM for the EGFR and of 9 nM for HER2. However, GW2016 does not inhibit HER4 well, with an IC50 >30-fold higher (107,108). A relevant question is whether a dual inhibitor will be of greater efficacy than a receptor-specific tyrosine kinase inhibitor, taking into consideration the observation that EGFR-specific tyrosine kinase inhibitors can prevent activation of HER2 in vivo. If these agents, on the other hand, target better HER2 than the more selective EGFR inhibitors, they could also have an improved activity profile in tumors such as breast cancer, which are HER2 dependent. However, this improved activity could also be at the cost of additional toxicities.

Class 4. Irreversible EGF Receptor Family Tyrosine Kinase Inhibitors CI-1033 is a 4-anilinoquinazoline that irreversibly inhibits in vitro the three catalytically active members of the EGFR family: EGFR, HER2, and HER4. Irreversibility is achieved by virtue of the compound’s ability to covalently modify a specific cysteine residue in the ATP binding site of these receptors (cys773) (109). CI-1033 is currently under phase I evaluation (110). Reported adverse events include an acneiform rash, diarrhea, thrombocytopenia, and one episode of a reversible hypersensitivity reaction. One clinical response has been reported in a patient with advanced squamous cell carcinoma (110). At the present time it is not known whether these different classes of compounds—and the different compounds within the same class—will have a different activity and/or toxicity profile. As an example, it will be of interest to see whether the pan-HER inhibitors will have a greater efficacy in HER2-driven tumors such as breast cancer when compared to selective EGFR inhibitors. Another discussion point is whether irreversibility of action will be advantageous. Because these agents are given orally on a daily basis, a point can be made that reversible inhibitors could also result in permanent receptor inhibition. However, the effects of irreversible and reversible inhibitors on receptor degradation and synthesis are unknown.

CHALLENGES IN THE DEVELOPMENT OF ANTI-EGF RECEPTOR COMPOUNDS The finding that anti-EGFR agents have antitumor activity and a low toxicity has validated the EGFR as a target for cancer therapy. On the other hand, there is clearly

Chapter 1 / EGFR to Modulate Chemotherapy

17

a need to optimize their utilization, since their single-agent activity is modest and two large phase III combination studies with conventional chemotherapy in NSCLC have been negative. As with other targeted agents in their early stages of development, continued research in some key areas will hopefully result in an enhanced efficacy. Challenges include how to improve patient selection, identification of the differences among the various classes of agents, a reassessment of the predictive value of the currently used preclinical model, and new study designs.

Patient Selection The activity of these agents has been mostly observed in an unselected patient population. The majority of the studies have either not preselected their patient population or have just required a positive EGFR expression in the tumor. This latter approach could be questioned, since there is no standardized method to determine EGFR expression and, most importantly, the level of EGFR expression required in the tumor in order to obtain clinical benefit from these therapies is not known at the present time. Although it may be tempting to establish a parallelism between anti-EGFR agents and the anti-HER2 MAb trastuzumab (Herceptin®), which has activity only on cells displaying amplification of the HER2 target, the biology of the EGFR is quite different from that of HER2. The EGFR has a series of well-known ligands, and ligand binding to the receptor triggers homo- and heterodimer formation; in contrast, HER2 is a ligand-less receptor, and receptor overexpression may be required to activate downstream signaling. In addition, the data with cetuximab in colorectal carcinoma showed that the response rates were comparable in patients expressing 1+, 2+, or 3+ levels of the EGFR (70). The same holds true in patients with head and neck carcinomas, with similar response rates to cetuximab in patients with tumors expressing different levels of EGFR (75). No data are available in patients treated with low-MW inhibitors of the EGFR, but in cell lines there is no linear correlation between EGFR expression and response to tyrosine kinase inhibitors (46), as opposed to a linear relationship between receptor number and growth inhibition in breast-cancer cell lines treated with trastuzumab (111). We believe that factors other than receptor number determine whether a particular tumor is dependent upon the EGFR pathway for driving its proliferation and function. Therefore, it will be critical in the new series of clinical trials to analyze not only the level of EGFR expression in the tumor, but also the level of expression of its ligands, such as TGF-_ or EGF, which are required for the maintenance of an active EGFR autocrine loop. In addition, the expression levels of the other members of the same receptor family, the level of tyrosine phosphorylation on EGFRs and on downstream molecules such as MAPK, PI3K-Akt, p27, Stat3, Ki-67, and others should be measured. In cell culture, inhibition of phosphorylation of the EGFR and MAPK are necessary but not sufficient for cell-growth inhibition. On the other hand, cell lines such as MDA-468 that have

18

Baselga

a mutant PTEN and a high level of basally phosphorylated Akt, are more resistant to tyrosine kinase inhibitors (35). In preliminary data from a phase II study of ZD1839 in breast cancer, only patients who had low levels of phosphorylated Akt had a decrease in the proliferation marker Ki-67 (J. Baselga, personal communication). In a similar fashion, in a phase I study of EMD 72000 in patients with advanced colorectal carcinoma, inhibition of EGFR phosphorylation was observed in all tumors, irrespective of response. In one responding tumor for which biopsies pre- and posttherapy are available, the level of basally phosphorylated Akt was low and disappeared completely with treatment with EMD 72000. On the contrary, progressing tumors had higher basal phosphorylated Akt and it did not decrease with treatment (71). Therefore, it will be important to test in an orderly fashion whether these assays can identify markers that predict sensitivity to therapy with EGFR inhibitors. Furthermore, taking into consideration the vast complexity of the EGFR signaling network, instead of limiting the search to a limited number of markers, it will also be necessary to analyze much larger gene and protein expression levels at baseline and on therapy. Such an approach will imply the performance of repeated tumor biopsies in patients participating in these trials. While this approach has been shown to be feasible (84,98), new technologies including gene expression profiling in archived paraffin-embedded tumors may allow for easier prospective and retrospective correlations with clinical benefit (84).

Differences Among the Various Classes of Agents Emerging clinical data suggest that these agents, although they target the same receptor, may have different activity profiles. Interestingly, these differences in activity seem to be tumor-dependent. In advanced colorectal cancer, consistent single-agent activity has been observed with the MAbs cetuximab and EMD 72000 (72,83,84), whereas the tyrosine kinase inhibitors ZD1839 and erlotinib have been shown to be inactive to date (98,104). Likewise, antitumor activity has been reported in renal-cell carcinoma with the MAb ABX-EGF (82) but not with cetuximab and ZD1839. As a third example, in head and neck tumors, activity has been reported with the MAb cetuximab (74,75) and with the tyrosine kinase inhibitors erlotinib and ZD1839 (97,102). These findings imply that different agents (or classes of agents) may have to be tested separately in individual tumor types. This differential activity profile could also be an indication that these agents do not have completely overlapping mechanisms of action, and it provides a rationale for studying in the clinic the combined treatment with different anti-EGFR compounds such as tyrosine kinase inhibitors and MAbs. In support for this approach, preclinical studies in a panel of cell lines have shown that once maximal growth inhibition is achieved with one type of agent (MAbs or tyrosine kinase inhibitors), the addition of the other agent results in a remarkable additional antitumor effect (63).

Chapter 1 / EGFR to Modulate Chemotherapy

19

DIRECTIONS IN THE COMBINED TREATMENT WITH ANTI-EGFR AGENTS AND CHEMOTHERAPY IN THE TREATMENT OF CANCER Combinations of chemotherapy and anti-EGFR agents in a variety of preclinical models have consistently shown a synergistic interaction both with monoclonal antibodies and with low-MW tyrosine kinase inhibitors. However, the results in the clinic have not been uniform, with negative outcomes in some large studies. This point is well exemplified by the recently negative outcome of the two large phase III studies of chemotherapy ± ZD1839 in patients with advanced chemotherapy-naïve non-small-cell lung carcinoma (95,96), despite the existence of supporting preclinical data (57,58). On the other hand, a true reversal of CPT-11 resistance has been observed with cetuximab in advanced colorectal cancer in the BOND trial (73), in results that mimic what was observed in preclinical models. Similarly, a beneficial effect of the combined treatment with cetuximab and chemotherapy when compared with chemotherapy alone has been observed in head and neck cancer and in NSCLC (76,77). The results of these trials, taken together, suggest that there will not be a unifying approach on how to combine anti-EGFR agents and conventional chemotherapy agents. In the case of the negative lung cancer studies with gefitinib and erlotinib, several potential explanations have been proposed. There is the possibility that these results are just being seen with one class of agents and in one tumor type, NSCLC, where three drugs given concomitantly have never been shown to be superior to a two-drug regimen. However, in the case of triplet combinations of cytotoxic agents, it is generally necessary to reduce the dose of at least one of the three drugs in order to maintain tolerability, which could hamper the activity of the combination. This was not the case in the gefitinib trials, where full doses of chemotherapy and gefitinib were administered. In addition, the results of the NSCLC trial with cetuximab and chemotherapy suggest otherwise. There are other potential explanations for the lack of activity of the gefitinib plus cytotoxic combination chemotherapy. In these studies, patients were not selected on the basis of presence of EGFR or other yet unknown determinants of response: it is possible that if only a small number of patients are sensitive to gefitinib, the diluting effect may make small differences undetectable. The magnitude of the preclinical effects of the combination could have not been large enough to warrant a clinical effect. It is possible that in order to see an effect in the clinic, in the preclinical models a true proof of synergy or, as in the preclinical studies with cetuximab and cisplatin (112) or trastuzumab and paclitaxel (113), a complete and sustained eradication of well established xenografts may be required. Another potential explanation is that the cancer cell lines that are being used in cell culture and xenograft models may be too distant from, and therefore not predictive of, the behavior of human tumors. The impli-

20

Baselga

cation is that new models resembling more closely the clinical reality, such as tumor explants, would be highly desirable. However, with results from these models not yet available, we may have to consider different clinical development approaches based preferentially on single-agent studies in an enriched patient population of EGFR-sensitive tumors. In addition, for the combination studies, a new clinical trial methodology may be required. In light of the phase III studies with gefitinib and erlotinib, the type of pilot efficacy evidence required for a given combination may have to be redefined before proceeding to large phase III randomized trials. Likewise, other approaches of combining chemotherapy and antireceptor agents would need to be explored, such as sequential administration, in a fashion similar to how hormonal therapy and chemotherapy have been integrated in the treatment of breast cancer. In the case of gefitinib and erlotinib, given their clinical activity as single agents in second and third line in advanced NSCLC, a direction to be explored is the sequential administration of chemotherapy first, followed by the administration of the anti-EGFR TKIs as a maintenance therapy. In support of this approach is the result of one subset analysis that was performed in the INTACT2 trial (96). In this study, there was a trend towards improved survival in patients who had received chemotherapy *90 d, suggesting a possible effect of gefitinib monotherapy as maintenance therapy. The question of sequential administration of chemotherapy and gefitinib will be addressed in a phase III study that will compare gefitinib 250 mg/d vs placebo following chemoradiation and consolidation docetaxel in patients with inoperable stage IIIA/B NSCLC. In conclusion, the combined effect of anti-EGFR agents and chemotherapeutic agents will have to be carefully explored based on improved preclinical models, specific agents being studied, tumor type, and stage of disease. In addition, special attention may be required to the dose and scheduling of the tested combinations. At the end, it is likely that each individual combination will have to be tested in the clinic. If this is the case, it would seem more appropriate to conduct exploratory randomized phase II studies or to build in early stopping rules for large phase III trials prior to committing to full-scale studies.

REFERENCES 1. Yarden Y, Sliwkowski M. Untangling the ErbB signalling network. Nat Rev Mol Cell Biol 2001;2:127–137. 2. Klapper L, Waterman H, Sela M, et al. Tumor-inhibitory antibodies to HER-2/ErbB-2 may act by recruiting c-Cbl and enhancing ubiquitination of HER-2. Cancer Res 2000;60:3384–3388. 3. Olayioye M, Neve R, Lane H, et al. The ErbB signaling network: receptor heterodimerization in development and cancer. EMBO J 2000;19:3159–3167. 4. Kuan CT, Wikstrand CJ, Bigner DD. EGF mutant receptor vIII as a molecular target in cancer therapy. Endocr Relat Cancer 2001;8:83–96. 5. Prenzel N, Zwick E, Daub H, et al. EGF receptor transactivation by G-protein-coupled receptors requires metalloproteinase cleavage of proHB-EGF. Nature 1999;402:884–888. 6. Liu D, Ghiso JA, Estrada Y, et al. EGFR is a transducer of the urokinase receptor initiated signal that is required for in vivo growth of a human carcinoma. Cancer Cell 2002;1:445–457.

Chapter 1 / EGFR to Modulate Chemotherapy

21

7. Schlessinger J. Cell signaling by receptor tyrosine kinases. Cell 2000;103:211–225. 8. Alroy I, Yarden Y. The ErbB signaling network in embryogenesis and oncogenesis: signal diversification through combinatorial ligand-receptor interactions. FEBS Lett 1997;410:83–86. 9. Lewis TS, Shapiro PS, Ahn NG. Signal transduction through MAP kinase cascades. Adv Cancer Res 1998;74:49–139. 10. Chan TO, Rittenhouse SE, Tsichlis PN. AKT/PKB and other D3 phosphoinositide-regulated kinases: kinase activation by phosphoinositide-dependent phosphorylation. Annu Rev Biochem 1999;68:965–1014. 11. Blume-Jensen P, Hunter T. Oncogenic kinase signalling. Nature 2001;411:355–365. 12. Vivanco I, Sawyers CL. The phosphatidylinositol 3-kinase-Akt pathway in human cancer. Nat Rev Cancer 2002;2:489–501. 13. Salomon D, Brandt R, Ciardiello F, et al. Epidermal growth factor-related peptides and their receptors in human malignancies. Crit Rev Oncol Hematol 1995;19:183–232. 14. Tang CK. Epidermal growth factor receptor vIII enhances tumorigenicity in human breast cancer. 2000;60:3081–3087. 15. Moscatello DK. Frequent expression of a mutant epidermal growth factor receptor in multiple human tumors. Cancer Res 1995;55:5536–5539. 16. Nishikawa R. A mutant epidermal growth factor receptor common in human glioma confers enhanced tumorigenicity. Proc Natl Acad Sci USA 1994;91:7727–7731. 17. Grandis JR, Melhem MF, Gooding WE, et al. Levels of TGF-_ and EGFR protein in head and neck squamous cell carcinoma and patient survival. J Cancer Natl Inst 1998;90:824–832. 18. Rusch V, Baselga J, Cordon-Cardo C, et al. Differential expression of the epidermal growth factor receptor and its ligands in primary non-small cell lung cancers and adjacent benign lung. Cancer Res 1993;53:2379–2385. 19. Kawamoto T, Sato JD, Le A, et al. Growth stimulation of A431 cells by EGF: identification of high affinity receptors for epidermal growth factor by an anti-receptor monoclonal antibody. Proc Natl Acad Sci USA 1983;80:1337–1341. 20. Sato JD, Kawamoto T, Le AD, et al. Biological effect in vitro of monoclonal antibodies to human EGF receptors. Mol Biol Med 1983;1:511–529. 21. Rodeck U, Herlyn M, Herlyn D, et al. Tumor growth modulation by a monoclonal antibody to the epidermal growth factor receptor: immunologically mediated and effector cell-independent effects. Cancer Res 1987;47:3692–3696. 22. Aboud-Pirak E, Hurwitz, E., Pirak, M.E., Bellot, F., Schlessinger, J. and Sela, M.. Efficacy of antibodies to epidermal growth factor receptor against KB carcinoma in vitro and in nude mice. J Natl Can Inst 1988;80:1605–1611. 23. Azemar M, Schmidt M, Arlt F, et al. Recombinant antibody toxins specific for ErbB2 and EGF receptor inhibit the in vitro growth of human head and neck cancer cells and cause rapide regression in vivo. Int J Cancer 2000;86:269–275. 24. Ciardiello F, Caputo R, Troiani T, et al. Antisense oligonucleotides targeting the epidermal growth factor receptor inhibit proliferation, induce apoptosis, and cooperate with cytotoxic drugs in human cancer cell lines. Int J Cancer 2001;93:172–178. 25. Yamazaki H, Kijima H, Ohmishi Y, et al. Inhibition of tumor growth by ribozyme-mediated suppression of aberrant epidermal growth factor receptor gene expresssion. J Natl Cancer Inst 1998;90:558–559. 26. Jannot CB, Beerli RR, Mason S, et al. Intracellular expression of a single-chain antibody directed to the EGFR leads to growth inhibition of tumor cells. Oncogene 1994;13:275–282. 27. Gill GN, Kawamoto T, Cochet C, et al. Monoclonal anti-epidermal growth factor receptor antibodies which are inhibitors of epidermal growth factor binding and antagonists of epidermal growth factor-stimulated tyrosine protein kinase activity. J Biol Chem 1984;259:7755–7760. 28. Fan Z, Lu Y, Wu X, et al. Antibody-induced epidermal growth factor dimerization mediates inhibition of autocrine proliferation of A431 squamous carcinoma cells. J Biol Chem 1994;269:27,595–27,602.

22

Baselga

29. Arteaga CL, Ramsey TT, Shawver LK, et al. Unliganded epidermal growth factor receptor dimerization induced by direct interaction of quinazolines with the ATP binding site. J Biol Chem 1997;272:23,247–23,254. 30. Lichtner RB, Menrad A, Sommer A, et al. Signaling-inactive epidermal growth factor receptor/ligand complexes in intact carcinoma cells by quinazoline tyrosine kinase inhibitors. Cancer Res 2001;61:5790–5795. 31. Fry DW. Site-directed irreversible inhibitors of the erbB family of receptor tyrosine kinases as novel chemotherapeutic agents for cancer. Anticancer Drug Des 2000;15:3–16. 32. Lal A, Glazer CA, Martinson HM, et al. Mutant epidermal growth factor receptor up-regulates molecular effectors of tumor invasion. Cancer Res 2002;62:3335–3339. 33. Busse D, Doughty RS, Arteaga CL. HER-2/neu (erbB-2) and the cell cycle. Semin Oncol 2000;27:3–8; discussion 92–100. 34. Albanell J, Codony-Servat J, Rojo F, et al. Activated extracellular signal-regulated kinases: association with epidermal growth factor receptor/transforming growth factor alpha expression in head and neck squamous carcinoma and inhibition by anti-epidermal growth factor receptor treatments. Cancer Res 2001;61:6500–6510. 35. Anido J, Matar P, Albanell J, et al. ZD1839, a specific epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, induces the formation of inactive EGFR/HER2 and EGFR/ HER3 heterodimers and prevents heregulin signaling in HER2-overexpressing breast cancer cells. Clin Cancer Res 2003;9:1274–1283. 36. Moasser MM, Basso A, Averbuch SD, Rosen N. The tyrosine kinase inhibitor ZD1839 (“Iressa”) inhibits HER2-driven signaling and suppresses the growth of HER2overexpressing tumor cells. Cancer Res 2001;61:7184–7188. 37. Normanno N, Campiglio M, De LA, et al. Cooperative inhibitory effect of ZD1839 (Iressa) in combination with trastuzumab (Herceptin) on human breast cancer cell growth. Ann Oncol 2002;13:65–72. 38. Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson JF, Arteaga CL. Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/ neu (erbB2)-overexpressing breast cancer cells in vitro and in vivo. Cancer Res 2001;61:8887–8895. 39. Bromberg J, Wrzeszczynska M, Devgan G, et al. Stat3 as an oncogene. Cell 1999;98:295–303. 40. Fan Z, Shang BY, Lu Y, et al. Reciprocal changes in p27(Kip1) and p21(Cip1) in growth inhibition mediated by blockade or overstimulation of epidermal growth factor receptors. Clin Cancer Res 1997;3:1943–1948. 41. Peng D, Fan Z, Lu Y, et al. Anti-epidermal growth factor receptor monoclonal antibody 225 up-regulates p27KIP1 and induces G1 arrest in prostatic cancer cell line DU145. Cancer Res 1996;56:3666–3669. 42. Moyer JD, Barbacci ES, Iwata KT, et al. Induction of apoptosis and cell cycle arrest by CP358,774, an inhibitor of epidermal growth factor receptor tysosine kinase. Cancer Res 1997;57:4838–4848. 43. Busse D, Doughty R, Ramsey T, et al. Reversible G(1) arrest induced by inhibition of the epidermal growth factor receptor tyrosine kinase requires up-regulation of p27(KIP1) independent of MAPK activity. J Biol Chem 2000;275:6987–6995. 44. Budillon A, Di Gennaro E, Barbarino M, Bruzzese F. ZD1839, an epidermal growth factor receptor tyrosine kinase inhibitor, upregulates p27Kip1 inducing G1 arrest and enhancing the antitumor effect of interferon a. Proc Amer Assoc Cancer Res 2000;41:773. 45. Bruns CJ, Solorzano CC, Harbison MT, et al. Blockade of the epidermal growth factor receptor signaling by a novel tyrosine kinase inhibitor leads to apoptosis of endothelial cells and therapy of human pancreatic carcinoma. Cancer Res 2000;60:2926–2935. 46. Petit AM, Rak J, Hung MC, et al. Neutralizing antibodies against epidermal growth factor and ErbB-2/neu receptor tyrosine kinases down-regulate vascular endothelial growth factor production by tumor cells in vitro and in vivo: angiogenic implications for signal transduction therapy of solid tumors. Amer. J Pathol 1997;151:1523–1530.

Chapter 1 / EGFR to Modulate Chemotherapy

23

47. Perrotte P, Matsumoto T, Inoue K, et al. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopically in nude mice. Clin Cancer Res 1999;5:257–264. 48. Ciardiello F, Caputo R, Bianco R, et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (‘Iressa’), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clin Cancer Res 2001;7:1459–1465. 49. O-charoenrat P, Modjtahedi H, Rhys-Evans P, et al. Epidermal growth factor-like ligands differentially up-regulate matrix metalloproteinase 9 in head and neck squamous carcinoma cells. Cancer Res 2000;60:1121–1128. 50. O-charoenrat P, Rhys-Evans P, Court W, et al. Differential modulation of proliferation, matrix metalloproteinase expression and invasion of human head and neck squamous carcinoma cells by c-erbB ligands. Clin Exp Metastasis 1999;17:631–639. 51. Matsumoto T, Perrotte P, Bar-Eli M, et al. Blockade of EGF-R signaling with anti-EGFR monoclonal antibody (Mab) C225 inhibits matrix metalloproteinase-9 (MMP-9) expression and invasion of human transitional cell carcinoma (TCC) in vitro and in vivo. Proc Am Assoc Cancer Res 1998;39:83. 52. Baselga J, Norton L, Masui H, et al. Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies. J Natl Cancer Inst 1993;85:1327–1333. 53. Baselga J, Mendelsohn J. The epidermal growth factor receptor as a target for therapy in breast carcinoma. Breast Cancer Res Treat 1994;29:127–138. 54. Fan Z, Masui H, Altas I, Mendelsohn J. Blockade of epidermal growth factor receptor function by bivalent and monovalent fragments of 225 anti-epidermal growth factor receptor monoclonal antibodies. Cancer Res 1993;53:4322–4328. 55. Ciardiello F, Bianco R, Damiano V, et al. Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225. Clin Caner Res 1999;5:909–916. 56. Prewett MC, Hooper AT, Bassi R, et al. Enhanced antitumor activity of anti-epidermal growth factor receptor monoclonal antibody IMC-C225 in combination with irinotecan (CPT-11) against human colorectal tumor xenografts. Clin Cancer Res 2002;8:994–1003. 57. Ciardiello F, Caputo R, Bianco R, et al. Antitumor effect and potentiation of cytotoxic drugs activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptorselective tyrosine kinase inhibitor. Clin Cancer Res 2000;6:2053–2063. 58. Sirotnak FM, Zakowsky MF, Miller VA, et al. Efficacy of cytotoxic agents against human tumor xenographs is markedly enhanced by coadministration of ZD1839 (‘Iressa’) an inhibitor of tyrosine kinase. Clin Cancer Res 2000;6:4885–4892. 59. Milas L, Mason K, Hunter N, et al. In vivo enhancement of tumor radioresponse by C225 antiepidermal growth factor receptor antibody. Clin Cancer Res 2000;6:701–708. 60. Williams K, Telfer B, Stratford I, et al. Combination of ZD1839 (‘Iressa’), an EGFR tyrosine kinase inhibitor, and radiotherapy increases antitumour efficacy in a human colon cancer xenograft model. Proc Am Assoc Cancer Res 2001;42:Abstract 3840. 61. Naramura M, Gillies SD, Mendelsohn J, et al. Therapeutic potential of chimeric and murine anti-(epidermal growth factor receptor) antibodies in a metastasis model for human melanoma. Cancer Immunol Immunother 1993;37:343–349. 62. Clynes RA, Towers TL, Presta LG, et al. Inhibitory Fc receptors modulate in vivo cytoxicity against tumor targets. Nat Med 2000;6:443–446. 63. Bos M, Mendelsohn J, Kim YM, et al. PD153035, a tyrosine kinase inhibitor, prevents epidermal growth factor receptor activation and inhibits growth of cancer cells in a receptor number-dependent manner. Clin Cancer Res 1997;3:2099–2106. 64. Goldstein NI, Prewett M, Zuklys K, et al. Biological efficacy of a chimeric antibody to the epidermal growth factor receptor in a human tumor xenograft model. Clin Cancer Res 1995;1:1311–1318.

24

Baselga

65. Cohen R, Falcey JW, Paulter VJ, Fetzer KM, Waksal HW. Safety profile of the monoclonal antibody (MOAB) IMC-C225, an anti-epidermal growth factor receptor (EGFR) used in the treatment of EGFR-positive tumors. Proc Am Soc Clin Oncol 2000;A1862. 66. Khazaeli MB, LoBuglio AF, Falcey JW, et al. Low immunogenicity of a chimeric monoclonal antibody (MOAB), IMC-C225, used to treat epidermal growth factor receptor-positive tumors, Am Soc Clin Oncol 2000;A808. 67. Baselga J. New therapeutic agents targeting the epidermal growth factor receptor. J Cin Oncol 2000;18:54S–59S. 68. Albanell J, Rojo F, Averbuch S, et al. Pharmacodynamic studies of the epidermal growth factor receptor inhibitor ZD1839 in skin from cancer patients: histophathologic and molecular consequences of receptor inhibitor. J Clin Oncol 2002;20:110–124. 69. Rubin M, Shin D, Pasmantier M, et al. Monoclonal antibody (MoAb) IMC-C225, an antiepidermal growth factor receptor (EGFR), for patients with EGFR-positive tumors refractory to or in relapse from previous therapeutic regimens (meeting abstract). Proc Am Soc Clin Oncol 2000;19:1860. 70. Saltz L, Rubin M, Hochster H, et al. Cetuximab (IMC-C225) plus Irinotecan (CPT-11) is active in CPT-11-refractory colorectal cancer (CRC) that expresses epidermal growth factor receptor (EGFR). Proc Am Soc Clin Oncol 2001;20:7A. 71. Saltz L RM, Rubin MS, Hochster HS, et al. Acne-like rash predicts response in patients treated with Cetuximab (IMC-225) plus Irinotecan (CPT-11) in CPT-11 refractory colorectal cancer that expresses EGFR. AACR-NCI-EORTC, 2001. 72. Saltz L, Meropol NJ, Loehrer PJ, et al. Single agent IMC-C225 (Erbitux™) has activity in CPT-11-refractory colorectal cancer (CRC) that expresses the epidermal growth factor receptor (EGFR). Proc Am Soc Clin Oncol 2002;21:A504. 73. Cunningham D, Humblet Y, Siena S, et al. Cetuximab (Erbitux) in combination with irinotecan or as single agent in patients with EGFR-expressing, irinotecan-refractory metastatic colorectal cancer. Proc Am Soc Clin Oncol 2003;22:A1012. 74. Hong WK, Arquette M, Nabell L, et al. Efficacy and safety of the anti-epidermal growth factor antibody IMC-C225 in combination with cisplatin in patients with recurrent squamous cell carcinoma of the head and neck refractory to cisplatin containing chemotherapy. Proc Am Soc Clin Oncol 2001;20:A895. 75. Baselga J, Trigo JM, Bourthis J, et al. Cetuximab (C225) plus cisplatin/carboplatin is active in patients (pts) with recurrent/metastatic squamous cell carcinoma of the head and neck (SCCHN) progressing on a same dose and schedule platinum based agent. Proc Am Soc Clin Oncol 2002;21:A900. 76. Burtness BA, Li Y, Flood W, et al. Phase III trial comparing cisplatin (C) + placebo (P) to C + anti-epidermal growth factor antibody (EGF-R) C225 in patients (pts) with metastatic/ recurrent head & neck cancer (HNC). Proc Am Soc Clin Oncol 2002;21:A901. 77. Gatzemeier U, Rosell R, Ramlau R, et al. Cetuximab (C225) in combination with cisplatin/ vinorelbine alone in the first-line treatment of patients (pts) with epidermal growth factor (EGFR) positive advanced non-small-cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2003;22:A2582. 78. Abbruzzese JL, Rosenberg A, Xiong Q, et al. Phase II study of anti-epidermal growth factor receptor (EGFr) antibody Cetuximab (IMC-C225) in combination with gemcitabine in patients with advanced pancreatic cancer. Proc Am Soc Clin Oncol 2001;20:A518. 79. Kim ES, Mauer AM, Fossella FV, et al. A phase II study of Erbitux (IMC-C225), an epidermal growth factor receptor (EGFR) blocking antibody, in combination with docetaxel in chemotherapy refractory/resistant patients with advanced non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2002;21:A1168. 80. Robert F, Ezekiel MP, Spencer SA, et al. Phase I study of anti-epidermal growth factor receptor antibody cetuximab in combination with radiation therapy in patients with advanced head and neck cancer. J Clin Oncol 2001;19:3234–3243.

Chapter 1 / EGFR to Modulate Chemotherapy

25

81. Yang X-D, Jia X-C, Corvalan JRF, et al. Eradication of established tumors by a fully human monoclonal antibody to the epidermal growth factor receptor without concomitant chemotherapy. Cancer Res 1999;59:1236–1243. 82. Schwartz G, Dutcher JP, Vogelzang NJ, et al. Phase 2 clinical trial evaluating the safety and effectiveness of ABX-EGF in renal cell cancer (RCC). Proc Am Soc Clin Oncol 2002;21:A91. 83. Tewes M, Schleucher N, Dirsch O, et al. Results of a phase I trial of the humanized anti epidermal growth factor receptor (EGFR) monoclonal antibody EMD 72000 in patients with EGFR expressing solid tumors. Proc Am Soc Clin Oncol 2002;21:A378. 84. Tabernero J, Rojo F, JimÈnez E, et al. A phase I pharmacokinetic (PK) and serial tumor and skin pharmacodynamic (PD) study of weekly, every 2 weeks or every 3 weeks 1-hour (h) infusion EMD72000, an humanized monoclonal anti-epidermal growth factor receptor (EGFR) antibody, in patients (p) with advanced tumors known to overexpress the EGFR. Eur J Cancer 2002;38, Suppl 7:69 (abstract 216). 85. Kollmannsberger C, Schittenhelm M, Honecker F, et al. Epidermal growth factor receptor (EGFR) antibody EMD 72000 in combination with paclitaxel (P) in patients (pts) with EGFR-positive advanced non-small cell lung cancer (NSCLC): A phase-I study. Proc Am Soc Clin Oncol 2003;22:627 (abstr 2520). 86. Woodburn JR, Barker AJ, Gibson KH, et al. ZD1839, an epidermal growth factor tyrosine kinase inhibitor selected for clinical development (meeting abstract). Proc Am Assoc Cancer Res 1997;38:A4521. 87. Woodburn J, Kendrew J, Fennell M, et al. ZD1839 (‘Iressa’) a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI): inhibition of c-fos mRNA, an intermediate marker of EGFR activation, correlates with tumor growth inhibition., Proc Am Assoc Cancer Res 2000:A2552. 88. Ferry D, Hammond L, Ranson M, et al. Intermittent oral ZD1839 (Iressa), a novel epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), shows evidence of good tolerability and activity: final results from a Phase I study. Proc Am Soc Clin Oncol 2000;19:3a (abs 5E). 89. Nakagawa K, Yamamoto N, Kudoh S, et al. A phase I intermittent dose-escalation trial of ZD1839 (Iressa) in Japanese patients with solid malignant tumours. Proc Am Soc Clin Oncol 2000;19:183a. 90. Baselga J, Herbst R, LoRusso P, et al. Continuous administration of ZD1839 (Iressa), a novel oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), in patients with five selected tumor types: evidence of activity and good tolerability, Proc Am Soc Clin Oncol 2000:A686. 91. Fukuoka M, Yano S, G Giaccone G, et al. Final results from a phase II trial of ZD1839 (‘Iressa’) for patients with advanced non-small-cell lung cancer (IDEAL 1). Proc Am Soc Clin Oncol 2002;21:A1188. 92. Kris MG, Natale RB, Herbst RS, et al. A phase II trial of ZD1839 (‘Iressa’) in advanced nonsmall cell lung cancer (NSCLC) patients who had failed platinum- and docetaxel-based regimens (IDEAL 2). Proc Am Soc Clin Oncol 2002;21:A1166. 93. Miller V, Johnson D, Heelan R, et al. A pilot trial demonstrates the safety of ZD1839 (‘Iressa’), an oral epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), in combination with carboplatin (C) and paclitaxel (P) in previously untreated advanced nonsmall cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2001;20:326a (Abstract 1301). 94. Giaccone G, Gonzales-Larriba J, Smit E, et al. ZD1839 (‘Iressa’), an orally-active, selective, epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI), is well tolerated in combination with gemcitabine and cisplatin, in patients with advanced solid tumours: preliminary tolerability, efficacy and pharmacokinetic results. Eur J Cancer 2001;Suppl:Abstract 102. 95. Giaccone G, Johnson D, Manegold C, et al. A phase III trial of ZD1839 (‘Iressa’) in combination with gemcitabine and cisplatin in chemotherapy-naive patients with advanced nonsmall-cell lung cancer (INTACT 1). Ann Oncol Suppl 2002;5:2 (abstract 40).

26

Baselga

96. Johnson D, Herbst R, Giaccone G, et al. ZD1839 (‘Iressa’) in combination with paclitaxel & carboplatin in chemotherapy-naive patients with advanced non-small-cell lung cancer (NSCLC): Results from a phase III clinical trial (INTACT 2). Ann Oncol 2002;Suppl 5:127 (abstract 468). 97. Cohen EE, Rosen F, Dekker A, et al. Phase II study of ZD1839 (Iressa) in recurrent or metastatic squamous cell carcinoma of the head and neck (SCCHN). Proc Am Soc Clin Oncol 2002;21:abstract 899. 98. Seymour L, Goss G, Stewart D, et al. A translational research study of ZD1839 at a dose of 750 mg in patients with pretreated advanced or metastatic colorectal cancer: NCIC CTG IND.122. Ann Oncol Suppl 2002;5:73 (abstract 264). 99. Moore M, Winquist E, Pollak M, et al. A randomised phase II study of two doses of ZD1839 in patients (pts) with hormone refractory prostate cancer (HRPC): A NCI Canada Clinical Trials Group Study. Ann Oncol 2002;Suppl 5:90 (abstract 326). 100. Hidalgo M, Siu LL, Nemunaitis J, et al. Phase I and pharmacologic study of OSI-774, an epidermal growth factor receptor tyrosine kinase inhibitor, in patients with advanced solid malignancies. J Clin Oncol 2001;19(13):3267–3279. 101. Perez-Soler R, Chachoua A, Huberman M, et al. A Phase II trial of the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor OSI-774, following platinum-based chemotherapy, in patients (pts) with advanced, EGFR-expressing, non-small cell lung cancer (NSCLC). Proc Am Soc Clin Oncol 2001;20:1235A. 102. Senzer NN, Soulieres D, Siu L, et al. Phase 2 evaluation of OSI-774, a potent oral antagonist of the EGFR-TK in patients with advanced squamous cell carcinoma of the head and neck. Proc Am Soc Clin Oncol 2001;20:6A. 103. Finkler N, Gordon A, Crozier M, et al. Phase 2 evaluation of OSI-774, a potent oral antagonist of the EGFR-TK in patients with advanced ovarian carcinoma. Proc Am Soc Clin Oncol 2001;20:A831. 104. Townsley C, Major P, Siu LL, et al. Phase II study of OSI-774 in patients with metastatic colorectal cancer. Eur J Cancer 2002;38, Suppl 7:57 (abstract 179). 105. Torrance CJ, Jackson PE, Montgomery E, et al. Combinatorial chemoprevention of intestinal neoplasia. Nature Med 2000;6:1024–1028. 106. Hidalgo M, Erlichman C, Rowinsky EK, et al. Phase 1 trial of EKB-569, an irreversible inhibitor of the epidermal growth factor receptor (EGFR), in patients with advanced solid tumors. Proc Am Soc Clin Oncol 2002;21:A65. 107. Keith BR, Allen PP, Alligood KJ, et al. Anti-tumor activity of GW2016 in the ErbB-2 positive human breast cancer xenograft, BT-474. Proc Am Assoc Cancer Res 2001;42:A4308. 108. Rusnak DW, Lackey K, Aflleck K, et al. The effects of the novel, reversible Epidermal Growth Factor Receptor/ErbB-2 tyrosine kinase inhibitor , GW2016, on the growth of human normal and tumor-derived cell lines in vitro and in vivo. Mol Cancer Ther 2001;1:85–94. 109. Fry DW, Bridges AJ, Denny WA, et al. Specific, irreversible inactivation of the epidermal growth factor receptor and erbB2, by a new class of tyrosine kinase inhibitor. Proc Natl Acad Sci USA 1998;95:12,022–12,027. 110. Zinner RG, Nemunaitis JJ, Donato NJ, et al. A phase I clinical and biomarker study of the novel pan-erbB tyrosine kinase inhibitor, CI-1033, in patients with solid tumors, Proceedings AACR-NCI-EORTC Molecular Targets and Cancer Therapeutics. Miami, FL, 2001;566. 111. Lewis GD, Figari I, Fendly B, et al. Differential responses of human tumor cell lines to antip185HER2 monoclonal antibodies. Cancer Immunol Immunother 1993;37:255–263. 112. Fan Z, Baselga J, Masui H, et al. Antitumor effect of anti-EGF receptor monclonal antibodies plus cis-Diaminnedichloroplatinum (cis-DDP) on well established A431 cell xenografts. Cancer Res 1993;53:4637–4642. 113. Baselga J, Norton L, Albanell J, Kim YM, Mendelsohn J. Recombinant humanized antiHER2 antibody (Herceptin) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/neu overexpressing human breast cancer xenografts. Cancer Res 1998;58:2825–2831. Erratum in: Cancer Res 1999;59:2020.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

2

27

Cyclin-Dependent Kinase Inhibitors in Combination Chemotherapy Manish A. Shah, MD and Gary K. Schwartz, MD CONTENTS SUMMARY INTRODUCTION THE CELL CYCLE AND ITS REGULATION THE CELL CYCLE AS A TARGET FOR CANCER THERAPEUTICS CDK INHIBITORS IN COMBINATION THERAPY CYTOTOXIC CHEMOTHERAPY COMBINATIONS WITH CDK INHIBITORS

SUMMARY Cyclin-dependent kinase inhibitors (CDKIs) represent a new class of anticancer therapeutics. Perturbations in the cell cycle are commonly described in carcinogenesis. This novel class of anticancer therapeutics exploits these perturbations to achieve tumor-specific cytotoxicity. In the last several years, our understanding of cell-cycle regulation has improved with the emerging concepts of cell-cyclemediated drug resistance and cell-cycle modulation to improve cytotoxic drug efficacy. It is becoming increasingly apparent that CDKIs may improve cytotoxic drug efficacy by functioning as cell-cycle modulators. In this chapter, we review the field of CDKIs as novel anticancer therapeutics, with a focus on their efficacy in drug combinations.

From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

27

28

Shah and Schwartz

INTRODUCTION With advancements in our understanding of the basic mechanisms of oncogenesis, cell-cycle regulation, and the induction of apoptosis, we have also gained a better understanding of the effects of chemotherapy on normal and cancerous cells. With this knowledge, it is becoming increasingly apparent that the cell cycle plays a critical role in the development of resistance to chemotherapy. This observation has led to the development of a new class of anticancer therapeutics, namely drugs that target the motors of the cell cycle, the cyclin-dependent kinases (CDKs). The development of CDK inhibitors (CDKIs) has undergone a gradual evolution. This class of drugs was first applied to the treatment of malignancy as single agents, which are now more commonly used in combination with traditional cytotoxic drugs. Along with this shift in development has come an improved understanding of the role that the cell cycle plays in drug resistance. In this review, we describe the cell cycle and the rationale for cell-cycle modulation in drug development, as well as the emerging concept of cell-cycle-mediated drug resistance. Specifically, we describe the cell cycle and the various mechanisms by which novel CDKIs impact this cycle. We briefly describe the preclinical and clinical development of these agents, with a particular emphasis on the concept of cell-cycle-mediated drug resistance and how novel CDKIs are used to overcome this resistance to improve antitumor efficacy. We also highlight the importance of sequence of administration of combination chemotherapy as a mechanism to overcome cell-cycle-mediated resistance.

THE CELL CYCLE AND ITS REGULATION The cell cycle is a critical regulator of the processes of cell proliferation and growth as well as of cell division following DNA damage. It governs the transition from quiescence (G0) to cell proliferation, and, through its checkpoints, ensures the fidelity of the genetic transcript. It is the mechanism by which cells reproduce, and is typically divided into four phases. The periods associated with DNA synthesis (S phase) and mitosis (M phase) are separated by gaps of varying length called G1 and G2. Progression of a cell through the cell cycle is promoted by a number of CDKs which, when complexed with specific regulatory proteins called cyclins, drive the cell forward through the cell cycle. There exist corresponding cell-cycle inhibitory proteins (CDKIs) that serve as negative regulators of the cell cycle and stop the cell from proceeding to the next phase of the cell cycle (see Fig. 1). The INK4 (for inhibitor of cdk4) class of CDKIs, notably p16lnk4a, p15lnk4b, p18lnk4c, and p191nk4d, bind and inhibit cyclin D associated kinases (cdk2, 4, and 6). The KIP (kinase inhibitor protein) group of CDKIs, p21waf1, p27kip1, and p57kip2, negatively regulates cyclin E/CDK2 and cyclin A/ CDK2 complexes (1).

Fig. 1. The cell cycle is divided into four distincct phases (G1, S, G2, and M). G0 represents exit from the cell cycle in which the cell performs its routine functions, including the important function of cell grown. The progression of a cell through the cell cycle is promoted by cyclin-dependent kinases (CDKs), which are positively and negatively regulated by cyclins and CDK inhibitors, respectively. The Restriction Point governs the transition point beyond which a cells progression through the cell cycle is independent of external stimuli.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy 29

30

Shah and Schwartz

The pattern of cyclin expression varies with a cell’s progression through the cell cycle, and this specific cyclin expression pattern defines the relative position of the cell within the cell cycle (2,3). At least nine structurally related cdks (CDK1–CDK9) have been identified, though not all have clearly defined cellcycle regulatory roles. A considerable number of cyclins have been identified to date (cyclin A–cyclin T). CDK/cyclin complexes themselves become activated by phosphorylation at specific sites on the CDK by cdk7/cyclin H, also referred to as CDK-activating kinase (CAK) (4). Cyclin D isoforms (cyclin D1–D3) interact with CDK2, -4, and -6 and drive a cell’s progression through G1. The association of cyclin E with CDK2 is active at the G1/S transition and directs entry into S phase. S-phase progression is directed by the cyclin A/CDK2 complex, and the complex of cyclin A with CDK1 (also known as cdc2) is important in G2. CDK1/cyclin B is necessary for mitosis to occur. Normal cells need to decide when to divide (i.e., enter the cell cycle) and when to stay in G0. Although often termed a quiescent phase, G0 is in fact quite an active phase, in which cellular functions and cellular growth occur. It is necessarily tightly regulated because the alternative, i.e., uncontrolled cell division without cell growth, would lead to smaller cells with each division. The entry into the cell cycle (G1) is historically governed by the restriction point—a transition point beyond which cell progression through the cell cycle is independent of external stimuli such as exposure to nutrients or mitogen activation (5). This point of determination is thought to divide the early and late G1 phase of the cell cycle. Mitogenic signaling of a variety of growth signals is mediated by the RAS/ RAF/MAPK pathway, whose endpoint is the stimulation of D-type cyclin production. The retinoblastoma tumor suppressor gene product (Rb) governs the G1/S transistion (see Fig. 2). In its active state, Rb is hypophosphorylated and forms an inhibitory complex with a group of transcription factors known as E2FDP (E2F-1, -2, and -3), thus controlling the G1/S transition. The activity of Rb is modulated by the sequential phosphorylation by CDK4/6-cyclin D and CDK2/ cyclin E, reviewed by Malumbres (6). When Rb is partially phosphorylated by CDK4/6–cyclin D kinases, Rb remains bound to E2F-DP, but this transcription factor is still able to transcribe some genes such as cyclin E. Cyclin E then binds to CDK2, and this active complex then completely hyperphosphorylates Rb, thus releasing the E2F-DP complex and fully activating the E2F transcription factors, resulting in transcriptional activation of numerous S-phase proteins, such as thymidylate synthase (TS) and dihydrofolate reductase (DHFR). In addition to Rb, CDK2 phophorylates other substrates involved in DNA replication (7). Early in S phase, cyclins D and E are targeted by ubiquitination for proteosome degradation (8). The production of cyclin A and its complexing with CDK2 enables S-phase progression, with the production of other enzymes and proteins involved in DNA synthesis, including histones and proliferating cell nuclear antigen (PCNA) (9). During late S and throughout G2, cells prepare for mitosis

Fig. 2. Retinoblastoma gene product (Rb) governs entry into S phase. In its active state, Rb is hypophosphoorylated and forms a complex with a group of transcription factors known as E2F (E2F-1, E2F-2, and E2F-3). Cyclin-dependent kinase (CDK)4 and CDK6 partially phosphorylate Rb, resulting in partial activation of this transcription factor. Cyclin E is then transcribed and binds to inactivation and complete release of the E2F transcription factors. This results in the transcription of a wide range of targets involved in chemotherapy sensitivity including ribonucleotide reductase (RR), thymidylate synthase (TS), thymidine knase (TK), dihydrofolate reductase (DHFR), c-jun, c-myc, and c-fos.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy 31

32

Shah and Schwartz

by increasing levels of cyclins A and B. As the level of cyclin B rises, it forms a complex with cdc2 (CDK 1) in the cytoplasm, where it remains until mitosis, at which point it shuttles into the nucleus. Recently, an S-phase checkpoint has been described, also termed the replication checkpoint (10,11). This checkpoint monitors progression through S phase and slows the rate of ongoing DNA synthesis. The S-phase checkpoint is thought to involve activations of ATM and ATR kinases with subsequent activation of Chk1 and Chk2 (10–13). These pathways ultimately control the ability of a cell to enter mitosis, which is dependent on the completion of S phase. Entry into mitosis is determined by the activity of the cyclin B/cdc2 complex, which is tightly regulated by its phosphorylation status, both by an activating phosphorylation at Thr161 by CAK and inhibitory phosphorylations at Thr14 and Thr15. At the completion of S phase, wee1 kinase is degraded by proteolysis in a cdc34-dependent fashion, and the phosphatase, cdc25c, is activated by a regulatory phosphorylation, which leads to cyclin B/ cdc2 activation. This complex then rapidly is relocated into the nucleus, and mitosis begins (9,14). Upon DNA damage, however, ATM and ATR are activated, leading to a Chk1 and Chk2 phosphorylation, and an inhibitory phosphorylation of Cdc25c, which prevents the activation of cyclin B/cdc2 and halts further S-phase progression and the entry into mitosis (10–13). Progression through mitosis is dependent on the anaphase-promoting complex (APC)/cyclosome and the degradation of cyclin B (14). During mitosis, the assembly of a bipolar spindle is vital to the preservation of genetic fidelity between daughter cells, and is monitored by a checkpoint that senses microtubule defects (15) or aberrant kinetochore attachment (16,17). Survivin has been implicated in the regulation of the mitotic spindle and in the preservation of cell viability, due in large part to its expression during cell division in a cell-cycledependent manner and localization to the mitotic apparatus (18,19). Cyclin B1/ cdc2 activity during mitosis plays a critical role in survivin expression and function in cell viability (20).

THE CELL CYCLE AS A TARGET FOR CANCER THERAPEUTICS The rationale for targeting the cell cycle, and in particular the CDKs, in anticancer therapy lies in the frequency of their perturbations in human malignancy and the observation that cell-cycle arrest by CDK inhibition would induce apoptosis. Most tumor suppressor genes and oncogenes are components of signal transduction pathways that control several cellular functions, including cellcycle entry and exit (21,22). In contrast to normal cells, tumor cells are unable to stop at predetermined points of the cell cycle because of loss of checkpoint integrity. Targeting CDKs would recapitulate cell-cycle checkpoints that would necessarily limit a tumor cell’s ability to cycle, and this may then facilitate the induction of apoptosis (23).

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

33

Fig. 3. Flavopiridol is a pan-cyclin-dependent kinase (CDK) inhibitor of CDK2, CDK4, and CDK6 at nanopolar concentrations resulting in cell cycle arrest at both the G1/S transition and the G2/M transition.

This rationale led to the development of CDK inhibitors as novel antitumor agents. These compounds can inhibit CDKs by direct effects that target the catalytic CDK subunit, or by indirect means that target regulatory pathways that govern CDK activity (24). Several small-molecule CDKIs are currently in development, including flavopiridol, UCN-01, and bryostatin. Flavopiridol is a novel antineoplastic agent that originally was noted for its ability to inhibit the activity of a number of protein kinases. Flavopiridol is now best classified as a CDKI because of its considerable affinity for CDKs and its ability to induce cell-cycle arrest in a number of cell lines (25–28). It has been shown to bind to and directly inhibit CDK1(cyclin B1–cdc2 kinase), CDK2, CDK4, and CDK6 (see Fig. 3). Flavopiridol administration has been associated with the selective induction of apoptotic cell death, particularly in hematopoietic cell lines (29–31). This induction of apoptosis may be mediated by an early activation of the MAPK protein kinase family of proteins (MEK, p38, and JNK), leading to activation of caspases (32). Cell-cycle arrest and the induction of apoptosis have been also demonstrated in squamous head and neck cell lines and other preclinical models (33,34). The apoptotic effect appears to be independent of p53 and is associated with the depletion of cyclin D1 (35). Xenograft studies have confirmed these in vitro results (36,37), leading to single-agent clinical evaluation. The initial schedule of administration, based on the preclinical data, was a 72-h continuous infusion schedule administered every 2 wk (38,39). Gastrointestinal toxicity (i.e.,

34

Shah and Schwartz

Fig. 4. UCN-01 has cell cycle effects at both the G1/S and G2/M transition. At G1/S, UCN01 hypophosphorylates Rb and causes the destruction of free E2F-1 by ubiquitini-zation and targeting for porteosome degradation. The net effect is an arrest in G1 and a significant reduction in early S-phase proteins, including ribonucleotide reductase and TS. At the G2/M transition, through inhibition of Chk1, UCN-01 increases the activity of Cyclin B-CDC2 resulting in a G2 checkpoint abrogation and a pre-mature entry into M.

diarrhea) was dose-limiting, with the recommended phase II doses 40–50 mg/ m2/d × 3 d repeated every 2 wk. Mean plasma concentrations of flavopiridol were between 250 and 500 nM, and preliminary clinical activity was seen in patients with renal, prostate, and gastric carcinoma (38,39). Despite the preclinical data, phase II clinical evaluation of flavopiridol as a single agent has demonstrated no significant clinical activity in non-small-cell lung cancer, gastric cancer, colorectal cancer, melanoma, renal-cell cancer, or lymphoma (40–45). This limited activity may be related to the fact that, in solid tumor preclinical models, the induction of apoptosis occurred at 300 nM concentrations or higher (33). Protein binding may limit the free drug concentration, resulting in inadequate drug concentrations for single-agent activity. UCN-01 (7-hydroxystaurosporine) is a staurosporine analog isolated from the culture broth of Streptomyces species (46,47), and is a selective inhibitor of protein kinase C (47). UCN-01 is associated with a G1/S cell-cycle arrest (48– 50), associated with the induction of p21CIP/Waf1, dephosphorylation of CDK2 (51), and with the resultant dephosphorylation of the retinoblastoma gene product (pRb) (50,51). Hypophosphorylated pRb remains tightly bound to E2F-1,

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

35

thereby preventing cell-cycle progression into S phase (52). Additionally, UCN01 causes the degradation of E2F-1 by targeting the transcription factor for proteosome degradation by ubiquitinization (see Fig. 4) (53). UCN-01 also abrogates the G2 checkpoint. It inhibits the Chk1 kinase, which is involved in the regulation of Cdc25C and 14-3-3 proteins in response to DNA damage (54,55). The 14-3-3 protein binds to Cdc25C and prevents this phosphatase from entering the nucleus, where it dephosphorylates Cdc2. This dephosphorylation activates the cyclin B/Cdc2 kinase and allows cells to progress towards mitosis (see Fig. 4). The phosphorylation of Cdc25C on serine 216 by Chk1 prevents 14-3-3 protein binding, thereby allowing Cdc25C to enter the nucleus and dephosphorylate Cdc2. UCN-01 inhibits this phosphorylation of Cdc25C by Chk1 kinase (54,55). UCN-01 also inhibits Chk2 kinase, which also regulates Cdc25C phosphorylation (56). The first clinical trial of UCN-01 as a single agent was recently completed by the NCI (57). The initial schedule of a 72-h continuous infusion repeated every 2 wks was abandoned when it became apparent that UCN-01 is strongly bound, in a species-specific, high-affinity manner, to human _-1-acid glycoprotein (57– 59). The administration of UCN-01 every 2 wk was associated with drug accumulation, leading to an alternative schedule of every 4 wk, with the second and subsequent doses one-half of the cycle 1 dose. With this schedule, the recommended phase II dose of UCN-01 is 42.5 mg/m2/d for 72 h, followed by 42.5 mg/ m2/d for 36 h for cycle 2 and subsequent cycles (57). Free salivary drug concentrations were approx 100 nM, which has been shown to modulate cell-cycle processes. This was supported by demonstration of G2 checkpoint abrogation in an ex vivo assay, suggesting that UCN-01 did in fact reach its target. Major toxicities included hyperglycemia, nausea, and vomiting. There was preliminary evidence of activity with one partial response observed in a patient with melanoma, and 19 patients demonstrating stable disease with a median duration of 5 mo (57). Additional phase I studies are underway or have been completed but not reported, evaluating shorter infusion schedules (60,61). Dose-limiting toxicity in these studies has also included hypotension. The bryostatins are macrocyclic lactones with a unique polyacetate backbone. Bryostatin-1 was isolated from the marine invertebrate Bugula neritina and first characterized after showing high activity against the murine P388 lymphocytic leukemia. Currently there are 20 known natural bryostatins, distinguished by their substituents at C7 and C20 (62). The main binding sites in bryostatins include the C1, C19, and C26 oxygen atoms (63). Bryostatin-1 has a unique mechanism of action in that, like phorbol esters, it can be a potent activator of PKC (64). Short-term exposure of tumor cells to bryostatin-1 induces PKC activation and autophosphorylation, followed by translocation of the enzyme to the cellular or nuclear membrane. However, despite sharing the same binding sites, phorbol esters have tumor-promoting properties,

36

Shah and Schwartz

Fig. 5. The cell cycle effects of bryostatin-1 primarity are in cyclin-dependent kinase (CDK)2 inhibition, by the transient induction of p21 and by the decrease in cyclin B levels. The net result is a G2 cell-cycle arrest.

while bryostatin-1 has antineoplastic properties that, in fact, can inhibit phorbol ester-mediated tumor promotion (65). Prolonged exposure of tumor cells to bryostatin-1 induces PKC inhibition by causing depletion from the cell (66), likely through ubiquitin-mediated proteasomal degradation (67). Bryostatin-1 affects the activity of certain cell-cycle regulatory proteins, although the resulting effects on apoptosis are inconsistent. Bryostatin-1 produces transient induction of p21 and subsequent dephosphorylation and inactivation of cdk2. The degree of cdk2 dephosphorylation correlates with the inhibition of tumor cell growth (68). However, bryostatin-1 also interferes with the up-regulation of p21 produced by phorbol esters (69). In U937 cells, pretreatment with deoxycytidine blocked bryostatin-mediates p21 induction, and enhances apoptosis (70). Bryostatin also decreases cyclin B expression in tumor xenografts, resulting in the prevention of paclitaxel-mediated cdc2 kinase activation (71). The net effect is an arrest of cells in G2 (see Fig. 5). Bryostatin is lipid soluble and must be delivered either in an ethanol or polyethylene glycol formulation. These vehicles are associated with acute, treatmentrelated side effects, including dyspnea, flushing, and hypotension (72), as well as localized thrombophlebitis and cellulitis (73). Phase I studies of bryostatin have explored various infusion rates and dosing schedules (73,74), with doselimiting toxicity being myalgia. The MTD remains 25 μg/m2 when bryostatin-1 is infused over 24 h (75). The MTD of bryostatin, given as a 72-h continuous

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

37

infusion every 2 wk, was 120 μg/m2. In contrast to shorter infusion durations, hematologic toxicity was uncommon (76). Limited single-agent activity was noted with bryostatin in patients with melanoma, ovarian cancer, and non-Hodgkin’s lymphoma in a phase I trial (75). However, single-agent phase II trials have not demonstrated significant activity, including trials in melanoma (77–79), renal-cell carcinoma (80), colorectal cancer (81), non-Hodgkin’s lymphoma (82), and soft-tissue sarcoma (83).

CDK INHIBITORS IN COMBINATION THERAPY Cell-Cycle Mediated Drug Resistance: An Emerging Concept The concept of tumor resistance to chemotherapy is based in part on the work of Luria and Delbruck, who found that bacteria spontaneously developed mutations that made them resistant to bacteriophage (84,85). When applying this concept to cancer, Goldie and Coldman proposed that the probability that a given tumor will contain resistant clones at the time of diagnosis would be a function of the mutation rate of that cancer and the size of the tumor at diagnosis (86,87). Even with low mutation rates of 1 in 106 mitoses, it would be virtually certain that drug-resistant mutants would populate the cells of a clinically detectable 1-cm tumor deposit (109 tumor cells) (85–87). It has been suggested that the antitumor activity of many chemotherapeutic agents (e.g., cisplatin and etoposide) is a consequence of their induction of apoptosis (88–93). Apoptosis is an active, energy-dependent process in which the cell participates in its own destruction. Morphologic features of apoptosis include cell shrinkage, nuclear chromatin condensation, and the formation of pedunculated protuberances on the cell surface (apoptotic bodies) (94–100), a process which results in the degradation of genomic DNA by endonucleases (101,102). The molecular cascade of apoptosis is characterized by the early release of mitochondrial cytochrome c, activation of apoptotic protease activating factor (Apaf-1), activation of caspase 9, and subsequent cleavage of downstream, or effector caspases in a self-amplifying cascade. Effector caspases finally degrade a number of cellular proteins, such as poly-adenosine 5'-diphosphateribosyl polymerase (PARP), laminin, and `-actin (103–107). The biochemical cascade of apoptosis is subject to regulation at several levels. Members of the Bcl2 family of proteins may be either antiapoptotic in nature (Bcl-2, Bcl-XL, Bcl-W, Mcl-1, A1/Bfi-1), or proapoptotic, acting to enhance apoptosis (BAD, Bax, Bak, and others). Bcl-2 or Bcl-XL bind and inhibit Apaf-1 and consequently prevent the activation of caspases. In the presence of excess Bax, however, Bcl-2 is displaced from Apaf-1, allowing caspase cleavage and activation. Bax further promotes apoptosis by mediating the release of cytochrome c from mitochondria. Caspases themselves are subject to regulation. A number of inhibitors of apoptosis protein (IAP), or caspase inhibitors, have been identified (97).

38

Shah and Schwartz

The failure of many chemotherapeutic agents reflects an inability of these drugs to induce apoptosis (97,108–113). Indeed, neoplastic cells have acquired a number of cellular adaptations and mutations, which act as survival factors, thus preventing the induction of apoptosis. For example, tumor cells with a mutation in the p53 gene have shown resistance to undergoing apoptosis in the presence of chemotherapy (94–96,100,114,115). Other examples of tumor resistance to the induction of apoptosis is the overexpression of Bcl-2 (92), Bcl-xL (116,117), and the induction of NF-gB activity (118,119). The cell cycle and apoptosis are intimately related, as evidenced by the central role of p53, both in cell-cycle arrest and in the induction of apoptosis. p53 induction by DNA damage causes arrest in both the G1 and G2 phases of the cell cycle (120,121). G1 arrest is due primarily to the induction of p21, whereas the arrest in G2 is due to the induction of both p21 and 14-3-3m, a protein that normally sequesters cyclinB/cdc2 complexes in the nucleus (122–125). p53 is also critical in the induction of apoptosis by activating the transcription of multiple apoptosis-associated genes (126,127). Another example of this intimate relationship is demonstrated in human colon cancer cell lines that differ only in their p21 checkpoint status. Cells with wild-type p21, when irradiated with gamma radiation, undergo a cell-cycle growth arrest followed by clonogenic survival, whereas cells lacking p21, when irradiated with gamma radiation, do not undergo a cell-cycle growth arrest and proceed to apoptosis (128). Cells that undergo a growth arrest may be protected from apoptosis and may therefore be ultimately resistant to the cytotoxic agent. Cell-cycle-mediated drug resistance is best described as a relative insensitivity to a chemotherapeutic agent due to the position of the cell in the cell cycle, or more precisely, due to the activation of cell-cycle checkpoints that interrupt cell-cycle progression to allow time for repair. This is a recently recognized mechanism of resistance to cytotoxic chemotherapy. In combination chemotherapy, specifically, one chemotherapeutic agent can impact the cell cycle such that the next chemotherapeutic agent given immediately in sequence becomes less effective (129). Cell-cycle-mediated drug resistance that limits the efficacy of many standard cytotoxic drugs can be overcome by the appropriately scheduled administration of novel CDKIs. Below, we will provide preclinical evidence of cell-cycle-mediated drug resistance and how it is overcome by CDKIs, and the initial clinical evaluation of CDKIs in combination chemotherapy. To understand the role a cell-cycle modulator may play in combination therapy, one must first understand the cell-cycle effects of the cytotoxic agent. The cellular response to DNA damage is the activation of cell-cycle checkpoints that serve as natural surveillance mechanisms for DNA integrity. Different cytotoxic agents are more effective in certain points during the cell cycle, and may elicit different cell-cycle checkpoint responses. Below we describe the cell-cycle effects of several standard cytotoxic chemotherapeutic classes, and then provide

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

39

the preclinical and clinical data that support the use of CDK inhibitors to modulate their cytotoxicity in an effort to overcome cell-cycle-mediated drug resistance.

CYTOTOXIC CHEMOTHERAPY COMBINATIONS WITH CDK INHIBITORS Combinations of CDK Inhibitors With Taxanes TAXANE CELL-CYCLE EFFECTS The taxanes act by stabilizing microtubules, thereby causing a G2/M arrest followed by apoptosis. Unlike other known mitotic spindle inhibitors (vinca alkaloids, colchicine, and podophyllotoxin), which inhibit tubulin polymerization, taxanes markedly enhance microtubule assembly and disrupt the transition through mitosis. The two primary taxanes in clinical use today are paclitaxel (Paclitaxel®) and docetaxel (Taxotere®); however, significantly more preclinical and clinical information has been presented with paclitaxel. Paclitaxel is an anticancer agent with a broad spectrum of activity and is currently being used in patients with ovarian, breast, lung, head and neck, bladder, and esophageal cancers (130). Paclitaxel promotes microtubule assembly and stabilizes tubulin polymer formation (131), thereby interrupting the dynamic cellular reorganization necessary for mitosis (132) and resulting in a G2-M arrest (133). Paclitaxel is also associated with downregulation of CDK4 (134) with concomitant G1/S arrest. The primary effect of paclitaxel is to interfere with the assembly of the mitotic spindle, resulting in the failure of chromosomes to segregate (135). As a microtubule promoter, paclitaxel shifts the equilibrium in favor of the microtubule and thus decreases the concentration of tubulin necessary for subsequent assembly (136). Mitosis is initiated by the activation of the cyclin B1–CDK1 complex (also called cyclin B1–cdc2 kinase), and as mitosis progresses, cyclin B is destroyed by ubiquitin-mediated proteolysis. Cyclin B and Cdc2 kinase activity are closely related to paclitaxel function. Expression of cyclin B and the activation of CDK1 occur coincidentally with paclitaxel-induced apoptosis (137,138), and destruction of cyclin B1 can be inhibited by paclitaxel (139). Furthermore, a dominant-negative mutant of p34cdc2 blocks paclitaxel-induced apoptosis (140). Although cytotoxicity is maximal at G2-M and minimal at G1-S (141), paclitaxel may induce apoptosis by other mechanisms as well. In particular, paclitaxel exposure is also associated with hyperphosphorylation of bcl-2 and phosphorylation of c-Raf-1 (3,142,143), steps necessary for apoptosis. However, understanding of the exact mechanism of programmed cell death induced by paclitaxel has been confounded by seemingly contradictory laboratory observations (144). These observations occur in vitro due to cell-type specificity (145), as well as effects related to concentration and duration of exposure (144,146). Nanomolar concentrations appear to be sufficient to polymerize

40

Shah and Schwartz

tubulin, and micromolar concentrations have demonstrated tubulin-independent effects, and may, in fact, be clinically irrelevant. COMBINATIONS WITH FLAVOPIRIDOL AND BRYOSTATIN: LABORATORY EVALUATION The combination of each of these agents (flavopiridol or bryostatin-1) with paclitaxel demonstrates the concept of cell-cycle-mediated drug resistance. Flavopiridol was examined in combination with paclitaxel in various sequences in the MKN-74 human gastric cancer cell line as well as the human breast cancer cell line MCF-7, which are both heterozygous for p53 (147). Cell-cycle-mediated resistance was demonstrated when flavopiridol exposure was followed by paclitaxel. Flavopiridol’s multiple cell-cycle effects (including the inhibition of CDK4, CDK6, and CDK2 at G1, and the inhibition of cyclin B1–cdc2 kinase activity at G2 [147]), creates a cell-cycle arrest. This prevents cells from entering M phase, the phase during which paclitaxel is most active, and leads to a significant reduction in paclitaxel sensitivity in culture (147). Similarly, in a mouse mammary tumor xenograft system, treatment with bryostatin-1 followed by paclitaxel demonstrated bryostatin-1-mediated suppression of cyclin B1, and an associated decrease in cyclin B1–cdc2 kinase activity. This resulted in a significant reduction in paclitaxel sensitivity. In the mouse xenograft system, bryostatin1 followed by paclitaxel was associated with a significant decrease in tumor doubling time as compared to paclitaxel alone (9.3 ± 1.9 d vs 22.7 ± 2.5 d, p < 0.001) (71,148). Therefore, when either flavopiridol or bryostatin-1 is given first, as a consequence of cell-cycle-mediated drug resistance, paclitaxel sensitivity is markedly reduced. In the case of flavopiridol, cells are arrested in the cell cycle and are insensitive to paclitaxel, which asserts its activity as cells enter M phase. In the case of bryostatin-1, cyclin B1–cdc2 kinase activity is reduced, resulting in cells arresting in G2, as the cyclin B1–cdc2 kinase is associated with the activity of the spindle-assembly checkpoint (139), and is required to initiate entry into M phase (2). Koutcher et al. demonstrated the G2 cell-cycle arrest in vitro: treatment of human MKN-74 gastric cancer cells with bryostatin-1 followed by paclitaxel resulted in a decrease in cells entering M phase (23% vs 56% with paclitaxel alone), and a concomitant increase in cells in G2 (69% vs 21% with paclitaxel alone) (71). As fewer cells enter M phase, the net effect is a significant decrease in paclitaxel sensitivity. Cell-cycle-mediated drug resistance may be overcome by appropriate sequencing of the drug combination. The reverse sequence of paclitaxel followed by flavopiridol is associated with an increased induction of apoptosis (147,149), as evidenced by caspase-3 activation and poly-(ADP-ribose)-polymerase degradation (147). This sequence is associated with an accelerated exit of cells from mitosis, an event that may be critical for the sequence-dependent enhancement of paclitaxel-induced apoptosis by flavopiridol. In the case of paclitaxel followed by bryostatin-1, there is decreased tumor metabolism and blood flow (71),

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

41

which may impact on tumor growth. The increased sensitivity to paclitaxel when followed by bryostatin-1 may be explained in part by Bcl-2:Bax, the heterodimer pair that is closely associated with mitochondrial dysfunction and the initiation of apoptosis. Loss of the Bcl-2 phosphorylation loop domain (150) and ectopic expression of Bcl-xL (151) can protect human leukemia cells (U937) from paclitaxel-mediated apoptosis. Administration of bryostatin-1 following paclitaxel can overcome paclitaxel resistance in U937 cells ectopically expressing Bcl-xL (152), and is associated with an increase in the pro-apoptotic factor, Bax, with resultant increased sequence-dependent apoptosis (153). TAXANE COMBINATIONS WITH FLAVOPIRIDOL AND BRYOSTATIN: CLINICAL EVALUATION The sequential combination of paclitaxel followed by flavopiridol has been evaluated in a phase I study (154). The clinical results are remarkable for major responses in patients with chemotherapy-refractory malignancies (i.e., prostate and esophagus), including patients who have received prior paclitaxel therapy. In particular, five of seven patients with esophageal cancer responded to the combination treatment, three of whom received prior paclitaxel therapy (154). In this clinical trial, there was no effect of flavopiridol on paclitaxel pharmacokinetics. In a trial combining paclitaxel and bryostatin, patients were treated with a weekly dose of paclitaxel 80 mg/m2 followed 24 h later with increasing doses of bryostatin-1. Two partial responses were demonstrated, with 9/27 patients demonstrating stable disease, including a patient with metastatic pancreatic carcinoma whose disease remained radiographically stable for 15 mo. Again, we found no pharmacokinetic effects on paclitaxel by bryostatin-1 (71,148). Both studies are now in phase II evaluations in patients with esophageal cancer— phase II combination of paclitaxel followed by bryostatin-1 for upfront treatment and by flavopiridol for paclitaxel refractory patients with esophageal cancer.

Combination of CDK Inhibitors With Camptothecins CAMPTOTHECIN CELL-CYCLE EFFECTS Camptothecins induce their primary cytotoxicity during the period of DNA synthesis. These agents form a class of chemotherapeutic drugs derived from the Chinese tree Camptotheca acuminata (155). They are alkaloids that are potent inhibitors of the nuclear enzyme topoisomerase I (156), an enzyme that functions primarily in the S phase of the cell cycle. In fact, cells in S phase are 100 to 1000 times more sensitive to camptothecin than cells in G1 or G2 (157). Topoisomerase I induces transient single-stranded breaks of DNA, relieving torsional strain and permitting DNA unwinding ahead of the replication fork during S phase. Camptothecins stabilize the “cleavable complex” between topoisomerase I and DNA. When these cleavable complexes collide with the moving DNA replication fork, double-stranded DNA breaks occur, leading to cell death (158–160).

42

Shah and Schwartz

This apoptotic cell death is mediated by caspase activation, and inhibition of this caspase activation shifts the cells from apoptosis to transient G1 arrest followed by cell necrosis (161). Camptothecin treatment is associated with a G2 cell-cycle arrest (162) at low concentrations, and an S-phase arrest at higher concentrations (163). This arrest is p53 independent and is regulated via the ATM/ATR kinases, which are activated by DNA damage during S phase, with subsequent phosphorylation of Chk1 (11–13). Apoptosis from short-bolus exposure to camptothecin is not associated with changes in bcl-2, bax, p53, or p21 acutely; however, prolonged exposure (greater than 72 h) is associated with increased expression of bax (164). Finally, camptothecin treatment is associated with the transient and unscheduled stimulation of cyclin B1–cdc2 kinase activity prior to apoptosis in HL60 cells (165). The clinically important members of this class of chemotherapeutic agents include irinotecan and topotecan. Irinotecan has been approved for clinical use in the US for colorectal cancer (166,167), and both irinotecan and topotecan have been approved in Japan for small-cell lung cancer, non-small-cell lung cancer, uterine cancer, ovarian cancer, stomach cancer, colorectal cancer, breast cancer, skin cancer, and non-Hodgkin’s lymphoma (168). IRINOTECAN AND FLAVOPIRIDOL Cell-cycle-mediated drug resistance has been demonstrated in the human colon cancer cell line HCT-116 (with an intact p53-p21 axis), both with irinotecan alone and with the combination of flavopiridol and irinotecan. The p21 gene is transcriptionally activated by p53 and is responsible for the p53-dependent checkpoint that results in a G1 and G2 arrest following DNA damage (169,170). Treatment of human colon cancer HCT-116 cells with SN-38 alone is associated with an induction of p21, and a concomitant G2 arrest, thereby rendering these cells resistant to SN-38 (and therefore to irinotecan), which is 1000 times more effective in S phase (157). This cell-cycle-mediated drug resistance was demonstrated in culture by QFM analysis, where SN-38 exposure to HCT-116 cells resulted in only 1 ± 1% cell death (170). As a CDK inhibitor, flavopiridol itself induces a G1 and G2 cell-cycle arrest: therefore, when flavopiridol precedes irinotecan, cell-cycle-mediated drug resistance should again be demonstrated. When HCT-116 cells were exposed to the drug sequence of flavopiridol followed by SN-38, QFM analysis demonstrated only 15% ± 2% cell death. However, this cell-cycle-mediated resistance was again overcome by appropriate drug sequencing: SN-38 A flavopiridol resulted in significantly increased HCT-116 cell death at 44 ± 2% (p < 0.001) (170). This sequence (SN-38 A flavopiridol) demonstrated significant induction of apoptosis, as evidenced by PARP cleavage, caspase-3 activation, and DNA laddering. The augmentation of the irinotecan’s antitumor effect was also demonstrated in HCT-116 xenografts (170). HCT-116 tumor implants were treated with

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

43

irinotecan, flavopiridol, and irinotecan followed by flavopiridol separated by varying intervals from 2 to 24 h. In these experiments, the greatest tumor regression (the percent decrease in tumor volume) and cures were observed if the interval between irinotecan and flavopiridol was at least 7 to 16 h. Two weeks after the end of treatment (d 30), there was a 40 ± 25% regression of the tumor in mice treated with irinotecan alone; whereas in mice treated with CPT-11 followed after 7 or 16 h with flavopiridol, the tumor regression was 86 ± 9% or 82 ± 5%, respectively. The difference between the areas under the volume-time curve of CPT-11 alone and CPT-11 followed by flavopiridol after 7 h or 16 h were statistically significant (p = 0.0002 and p = 0.0005, respectively). The cure rates for irinotecan followed after 7 h or 16 h by flavopiridol was 30% (3 of 10) or 29% (2 of 7), respectively. This was in contrast to CPT-11 alone or CPT-11 followed by flavopiridol at 4 h, where no cures were found (170). CLINICAL EVALUATION OF IRINOTECAN AND FLAVOPIRIDOL The combination of irinotecan followed by flavopiridol is being examined in a phase I clinical trial with a 7-h interval prior to the administration of flavopiridol (171). This trial represents the first evaluation of flavopiridol as a 1-h infusion given weekly. Irinotecan was initially fixed at a dose of 100 mg/m2, and flavopiridol was escalated in sequential cohorts from 10 mg/m2 to 70 mg/m2. Myelosuppression resulting in dose delay occurred in two patients, and one patient developed significant diarrhea, requiring dose delay and intravenous hydration at a flavopiridol dose of 70 mg/m2. At flavopiridol doses below 70 mg/ m2, 45 out of 48 wk of therapy were administered without delay. Pharmacokinetic interactions between irinotecan and flavopiridol were examined by giving flavopiridol 24 h prior to irinotecan during wk 1 of cycle 2. Pharmacokinetic studies demonstrated weekly flavopiridol Cmax ranged from 2.04 ± 0.60 micromolar (flavopiridol dose level 50 mg/m2) to 2.89 ± 0.62 micromolar (flavopiridol dose level 70 mg/m2), exceeding the nanomolar concentrations necessary to enhance irinotecan-induced apoptosis in vitro. Pooled cycle 1 flavopiridol AUC (2.29 μM/h) was significantly lower than pooled cycle 2 flavopiridol AUC (3.10 μM /h) with p = 0.0078, suggesting an increase in flavopiridol metabolism when preceded by irinotecan. Preliminary antitumor efficacy was observed. Several patients with stable disease and one minor response patient with colorectal cancer who progressed on prior irinotecan therapy were noted. Additionally, investigators observed two patients with adrenocortical cancer who both had an extended duration of stable disease, with minor responses in lung and liver metastases (average time on study over 10 mo) (171). IRINOTECAN AND UCN-01 The G2/S arrest induced by irinotecan effectively results in cell-cycle-mediated drug resistance (170,172). This resistance is overcome by UCN-01, result-

44

Shah and Schwartz

ing in improved antitumor efficacy with the drug combination (173–176). Although this improvement in irinotecan cytotoxicity is apparent in both wild-type and mutant p53 models (56,174), p53 mutant tumor models suggest a greater effect on irinotecan-induced cytotoxicity by UCN-01 (174,176). In particular, although p53 exerts effects on both the G1 and G2 checkpoints, it is required for arrest in G1 and appears to strengthen the G2 arrest. Thus, irinotecan therapy leads to a G1 and G2 arrest in p53 wild-type cells and primarily a S/G2 arrest in p53 mutant models (177). This distinction may explain the relative specificity of irinotecan and UCN-01 for tumor cells, particularly those with p53 mutations, over p53 wild-type cells (174). In this study, investigators examined the cytotoxicity of the drug combination on normal endothelial cells. They found that, while normal endothelial cells showed a loss of S-phase-arrested cells with UCN-01 treatment, the cells accumulated in G1 and were resistant to cell death by irinotecan and UCN-01. However, with p53 mutant cells, increased cytotoxicity of the combination was observed (174). Tumor cells deficient in complementary G2/M checkpoint pathways that are parallel to that affected by UCN-01 will be selectively more sensitive to the combination. This concept is demonstrated with isogenic HCT-116 cells that differ only in p53 or p21 status (176). UCN-01 is now being investigated with irinotecan in a phase I clinical trial.

Combination With CDK Inhibitors With Fluorouracil FLUOROURACIL CELL-CYCLE EFFECTS Fluorouracil is an antimetabolite with broad activity in epithelial tumors arising in the breast, head and neck, gastrointestinal tract, and ovary, with singleagent response rates ranging from 10 to 30% (178). Upon cell entry, fluorouracil is converted to floxuridine (FUdR) by thymidine phosphorylase, which is then again phosphorylated by thymidine kinase to its active form, 5-fluoro-2'deoxyuridine monophosphate (FdUMP) (178). In the presence of a reduced folate cofactor, this active metabolite forms a stable complex with thymidylate synthase (TS), thereby limiting its ability to continually synthesize thymidine 5'monophosphase (179), with resultant inhibition of DNA synthesis. Increased TS expression is associated with fluorouracil resistance in multiple fluororuacilresistant cell lines (180). Fluorouracil also is extensively incorporated into both nuclear and cytoplasmic RNA species, interfering with normal RNA processing (reviewed by Allegra) (178). It is purely an S-phase active chemotherapeutic agent, with no activity when cells are in G0 or G1. Twenty-four-hour exposure to fluorouracil is associated with an accumulation of cells in S phase, as well as a transient induction of p53 and p21 (181). The accumulation of cells in early S phase is associated with expression of cyclin A, and an increase in cyclin A–cdk2 kinase activity (182).

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

45

FLUOROURACIL AND UCN-01 Appropriate sequencing of drug combinations is important to overcome the cell-cycle interactions that create cell-cycle-mediated drug resistance. UCN-01 increases chemosensitivity by suppressing the expression of critical events regulated in the cell cycle by the inhibition of E2F-1 and the consequent significant reduction in both TS and dihydrofolate reductase (DHFR) gene expression, which normally increase substantially during G1/S phase boundary of cell cycle (183,184). Decreased expression of TS and DHFR as a result of UCN-01’s cellcycle effects is relevant to its interaction with fluorouracil (53), with resultant increased antimetabolite chemosensitivity. Both concepts of cell-cycle-mediated drug resistance and augmentation of apoptosis by suppression of TS induction were demonstrated in vitro (53). In SKGT5 cells, a human gastric adenocarcinoma cell line with a mutated p53 gene, cell-cycle-mediated drug resistance was demonstrated when fluorouracil followed UCN-01. This sequence demonstrated only 17 ± 1% apoptotic cells by QFM analysis. Maximal apoptosis was demonstrated with the reverse sequence of fluorouracil A UCN-01, with 46% ± 1% apoptotic cells by QFM analysis (53). Exposure to UCN-01 resulted in a dose-dependent decrease in TS protein expression, as well as a dose-dependent decrease in TS mRNA, with associated reductions in E2F-1 protein levels, and consequent increased sensitivity to fluorouracil as evidenced by increased apoptosis (53). CLINICAL EVALUATION OF FLUOROURACIL AND UCN-01 UCN-01 and fluorouracil has been evaluated in a phase I clinical trial (185). Escalating doses of weekly 24-h infusional fluorouracil (250 A 2600 mg/m2) were given in combination with a fixed monthly dose of UCN-01 (45 mg/m2/d). Based on the single-agent maximum tolerated dose, UCN infusion began on day 2 of each 28-d cycle over 72 h for cycle 1 and over 36 h for subsequent cycles. We found that full weekly doses of fluorouracil may be given (2600 mg/m2/d) in combination with UCN-01. Toxicity consisted primarily of UCN-01-related toxicity, including hyperglycemia, and the syndrome of nausea, vomiting, and headache temporally associated with the UCN-01 infusion. A linear correlation between serum _-1 acid glycoprotin (AGP) and total UCN-01 concentrations was noted (185). This combination is currently being evaluated in a phase II clinical trial for pancreas cancer refractory to first-line gemcitabine-based therapy.

Cisplatin Combinations CELL-CYCLE EFFECTS Cisplatin belongs to the alkylating agent group of chemotherapy drugs. It binds to DNA base pairs, creating adducts, crosslinks, and strand breaks that

46

Shah and Schwartz

inhibit DNA replication. As such, cisplatin is not cell-cycle specific, though cells appear to be maximally sensitive to cisplatin in G1, just prior to the onset of DNA synthesis, and minimally sensitive in peak DNA synthesis, with entry into S phase resulting in a twofold decrease in sensitivity (140). Cells that are blocked at the G1/S-phase boundary during cisplatin treatment remain maximally sensitive after release (140). Detection of damaged DNA leads to the activation of cyclin-dependent kinase inhibitors such as p21 and wee1/mik1, which subsequently arrest cells in either G1 or G2. Cisplatin exposure increases the duration of S phase and blocks cells in G2 in a dose-dependent manner (186). This arrest is accompanied by the accumulation of inactive, phosphorylated p34cdc2 protein. Following a protracted delay, the p34cdc2 protein is dephosphorylated and an aberrant mitosis occurs. In fact, a number of agents that abrogate the G2 cellcycle checkpoint and induce premature mitosis have demonstrated enhancement of cisplatin-induced cytotoxicity (187–189). Resistance to cisplatin has been associated with increased glutathione levels, increased metallothioneins, decreased drug uptake, increased DNA repair, and the tolerance of the formation of platinum-DNA adducts (190). Though the precise mechanism by which platinum-DNA damage results in cell death remains unknown, unrepairable DNA damage often results in activation of the apoptotic pathway (191,192). P53 plays a significant role in DNA repair, proliferative arrest, and apoptosis (193), and has led to a correlation between p53 and cisplatin sensitivity (194–196). Apoptosis has been associated with an unscheduled activation of cdc2 kinase in cisplatin-resistant cells, and with the p53/p21Waf1 pathway in cisplatin-sensitive cells (197). CISPLATIN AND UCN-01 Detection of platinum-DNA adducts leads to the activation of cyclin-dependent kinase inhibitors such as p21 and wee1/mik1, which subsequently arrest cells in G2. Cisplatin exposure increases the duration of S phase and blocks cells in G2 in a dose-dependent manner (186). This arrest is accompanied by the accumulation of inactive, phosphorylated Cdc2 protein. Agents that abrogate the G2 cell-cycle checkpoint, like UCN-01, induce a premature mitosis and enhance cisplatin-induced cytotoxicity (187–189,198,199). This augmentation of apoptosis by UCN-01 occurs even in p53 mutant tumor-cell models (200,201). UCN-01 inhibits the Chk1 kinase that is involved in the regulation of Cdc25C and 14-3-3 proteins in response to DNA damage (54,55). The 14-3-3 protein binds to Cdc25C and prevents this phosphatase from entering the nucleus, where it dephosphorylates Cdc2. This dephosphorylation activates the cyclin B/Cdc2 kinase and allows cells to progress towards mitosis. The phosphorylation of Cdc25C on serine 216 by Chk1 prevents 14-3-3 protein binding, thereby allowing Cdc25C to enter the nucleus and dephosphorylate Cdc2. UCN-01 inhibits this phosphorylation of Cdc25C by Chk1 kinase (54).

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

47

CISPLATIN AND FLAVOPIRIDOL The addition of flavopiridol to cisplatin similarly improves antitumor efficacy. As shown in Fig. 6, MKN-74 gastric cancer cells were treated with 100 nM flavopiridol, 1 μM cisplatin, and combinations of the two drugs for 24 h. No drug (ND24) is the negative control. Neither flavopiridol nor cisplatin alone induced apoptosis, as evidenced by lack of PARP cleavage. However, the scheduledependent addition of flavopiridol to cisplatin does demonstrate the induction of apoptosis with cleavage of PARP (see Fig. 6). Importantly, when flavopiridol precedes cisplatin, PARP cleavage is not demonstrated. Bible et al. examined this drug combination using colony-formation assays, and also demonstrated synergy when cisplatin precedes flavopiridol (149). These preclinical data form the basis of an ongoing phase I clinical evaluation of cisplatin followed by 24h flavopiridol infusion on an every-3-wk schedule. Dose escalation continues.

Combinations of CDK Inhibitors With Gemcitabine CELL-CYCLE EFFECTS Gemcitabine (difluorodeoxycytidine; dFdC) is an agent with promising antitumor activity in a variety of solid tumors, with activity in pancreas, bladder, and lung cancer. Gemcitabine is an S-phase-active drug. It is a nucleoside analog of cytidine and is phosphorylated inside cells, leading to the diphosphate (dFdCDP) and triphosphate (dfdCTP) derivatives. The diphosphate derivative inhibits DNA synthesis indirectly through inhibition of ribonucleotide reductase (201). This inhibits de novosynthesis of DNA, thereby self-potentiating gemcitabine activity by decreasing intracellular concentrations of normal deoxynucleotide triphosphates. The triphosphate derivative of gemcitabine is incorporated into DNA by replication synthesis in the cytidine sites of the growing DNA strand (202– 204). Once incorporated into the DNA strand, an additional natural nucleoside is added, masking dFd-CTP and preventing DNA repair by 3'-5'-exonuclease activity. DNA polymerases are unable to proceed, leading to a process termed “masked DNA chain termination” (203,204). As might be anticipated given gemcitabine’s mechanism of action, cell-cycle analysis of gemcitabine’s effects reveals a G1/S-phase arrest or delay (205,206). However, unlike an immediate induction of apoptosis seen in CEM leukemic cells (206), the induction of apoptosis seen in an epithelial ovarian cell line was delayed by 24–48 h (205). The authors suggest that following release from a gemcitabine-induced G1/S-phase arrest, recycling cells experience subsequent blocks at G2/M and G1 again. There is a competition between recovery and apoptotic pathways that determine whether the majority of cells survive or die. The molecular determinants of this decision are, as yet, unidentified. However, p53 may play a critical role in this determination of cell-cycle arrest vs programmed cell death. This is supported by the observation that loss of p53 func-

48

Shah and Schwartz

tion leads to loss of cell-cycle control and alterations in the apoptotic cascade, conferring resistance to gemcitabine (207,208). The observation that gemcitabine-treated cells are most sensitive to subsequent induction of programmed cell death upon resuming an initial G1/S-phase arrest also has important implications with regard to combination therapies. This is demonstrated in vivo, where potentiation with other cytotoxic drugs is seen with a 24-h interval and coincides with the presence of recycled cells (205). This observation may additionally have important implications when considering combinations with CDKIs. GEMCITABINE AND FLAVOPIRIDOL Flavopiridol was found to potentiate gemcitabine-induced apoptosis in a sequence-dependent manner in several epithelial gastrointestinal cell lines (209). Cells were treated with gemcitabine, flavopiridol, and their combination in varying sequences and schedules. In pancreas, human gastric, and colon cancer cell lines, maximal antitumor effect was observed with the combination of gemcitabine followed by flavopiridol. This was demonstrated by the induction of apoptosis as assessed by QFM analysis, PARP cleavage, and cytochrome c release, and by colony-formation assays (209). The reverse sequence of flavopiridol followed by gemcitabine demonstrated no additional antitumor efficacy over that of flavopiridol or gemcitabine alone. This sequence dependence is due to a G1/S arrest induced by flavopiridol, as demonstrated by a significant reduction of incorporation of [3H]Thd into DNA (209). The synergy was associated with a reduction in RNA and protein levels of the M2 subunit of the early S-phase protein, ribonucleotide reductase. Ribonucleotide reductase consists of two subunits, M1 and M2. The M2 subunit is primarily involved with ribonucleotide reductase activity during S phase, and has been correlated with DNA synthesis and the invasive potential of cancer cells (210). Increased ribonucleotide reductase activity expands the deoxynucleotide triphosphate pool, thereby competitively inhibiting the incorporation of dFdCTP into the DNA. This expanded pool of deoxynucleotide triphosphates also downregulates the activity of deoxycytidine kinase, reducing the phosphorylation of gemcitabine and further reducing gemcitabine’s efficacy (211). Jung et al. demonstrated that the addition of flavopiridol is associated with a reduction in the M2 subunit of ribonucleotide reductase, thereby potentiating gemcitabine’s antitumor activity (209). The S-phase delay induced by gemcitabine may also play an important role in flavopiridol’s ability to potentiate gemcitabine’s activity (205,212). Flowcytometry studies demonstrate that the cells at risk for programmed cell death are those in the S-phase population, following a G1/S arrest. In a specific, sequenceand schedule-dependent manner, the subsequent delayed administration of flavopiridol is observed to induce maximal apoptosis (212). Based on these

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

49

Fig. 6. Flavopiridol augments cisplatin cytotoxicity. Western blot for PARP (polyadenosine 5'-diphosphate-ribosyl polymerase). MKN-74 gastric cancer cells are treated with 1 μM cisplatin for 24 h (Cis24), 100 nM flavopiridol for 24 h (F24), and their various combinations. Apoptosis is identified by cleavage of the PARP band to a lower 90 kD band. As shown above, cisplatin and flavopiridol do not induce apoptosis alone. However their combination (wen cisplatin is givien together wiht flavopiridol (Cis + F)24, or whien flavopiridol follows cisplatin (Cis24 A F24) is associated with a significant induction of apoptosis. The reverse sequence of flavopiridol floowed by cisplatin does not demonstrate PARP cleavage and the induction of apoptosis.

preclinical results, the NCI is now sponsoring a phase I clinical trial combining gemcitabine with flavopiridol in a sequence-dependent manner. GEMCITABINE AND UCN-01 Cell-cycle arrest plays a critical role in resistance to nucleoside analogs such as cytarabine, fludarabine, and gemcitabine. For example, when primary leukemic cells in the marrow are exposed to cytarabine, a substantial portion of the cells undergo a cell-cycle arrest, with resultant termination of DNA synthesis, and accumulation in S phase. Decreasing DNA synthesis results in less cytotoxicity of the nucleoside analog, and resultant protection from cell death (213,214). In vitro, UCN-01 has demonstrated an enhancement of the antitumor efficacy of these analogs, perhaps through G2 cell-cycle checkpoint abrogation (198). However, a recent study that confirms the UCN-01-mediated enhancement of apoptosis by gemcitabine addresses this question of overcoming the cell-cycle arrest with UCN-01 more closely. Shi et al. elegantly demonstrate that gemcitabine increases the percentage of cells arrested in S phase with a significant reduction in DNA synthesis. Subsequent exposure to UCN-01 demonstrated no reactivation of DNA synthesis, but rather an increase in nucleosomal DNA fragmentation, activation of caspase-3, and induction of apoptosis (214). Therefore, it appears that the augmentation of apoptosis by UCN-01 may not require cells to proceed through the S-phase arrest induced by nucleoside analogs such as gemcitabine.

50

Shah and Schwartz

CONCLUSIONS Cell-cycle perturbations are commonly observed in human malignancies. Exploiting this finding is the rationale for the development of CDK inhibitors as antitumor agents. Single-agent evaluation of several CDKIs has demonstrated limited clinical activity. The combination of CDKIs with standard cytotoxic agents is an emerging, alternative approach to anticancer therapy that also exploits the cell-cycle perturbations of malignancy. Preclinical studies demonstrate the concept of cell-cycle-mediated drug resistance, and suggest that the combination of standard cytotoxic agents with CDKIs will require thoughtful sequencing and scheduling. With this in mind, there are presently several clinical investigations underway examining the combination of a standard cytotoxic with a novel CDKI, with particular attention to sequence and scheduling. Although phase II evaluation of these combination studies will provide initial evidence of antitumor activity, definitive phase III studies will be needed to establish this class of agents in the care of patients with cancer.

REFERENCES 1. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13(12):1501–1512. 2. Grana X, Reddy EP. Cell cycle control in mammalian cells: role of cyclins, cyclin dependent kinases (CDKs), growth suppressor genes and cyclin-dependent kinase inhibitors (CKIs). Oncogene 1995;11:211–219. 3. Johnson DG, Walker CL. Cyclins and cell cycle checkpoints. Ann Rev of Pharmacol & Toxicol 1999;39:295–312. 4. Kaldis P, Russo AA, Chou HS, et al. Human and yeast cdk-activating kinases (CAKs) display distinct substrate specificities. Mol Biol Cell 1998;9(9):2545–2560. 5. Pardee AB. A restriction point control for normal animal cell proliferation. Proc Natl Acad Sci USA 1974;71:1286–1290. 6. Malumbres M, Barbacid M. To cycle or not to cycle: A critical decision in cancer. Nat Cancer Revs 2001;1(3):222–231. 7. Sherr CJ. The Pezcoller Lecture: cancer cell cycles revisited. Cancer Res 2000;60:3689–3695. 8. Elledge SJ, Harper JW. The role of protein stability in the cell cycle and cancer. Biochim Biophys Acta 1998;1377:M61–70. 9. Ford HL, Pardee AB. The S-phase: beginning, middle, and end: a perspective. J Cell Biochem 1998;30/31(Suppl):1–7. 10. Zhou BS, Elledge SJ. The DNA damage response: putting checkpoints in perspective. Nature 2000;408:433–439. 11. Xu B, Kim S-T, Kastan MB. Involvement of BRCA1 in S-phase and G2-phase checkpoints after ionizing radiation. Mol Cell Biol 2001;21:3445–3450. 12. Falck J, Mailand N, Syljuasen RG, et al. The ATM-Chk2-Cdc25A checkpoint pathway gaurds against radioresistant DNA synthesis. Nature 2001;410:842–847. 13. Zhou X, Wang X, Hu B, et al. An ATM-independent S phase checkpoint response involves Chk1 pathway. Cancer Res 2002;62:1598–1603. 14. Ford HL, Pardee, AB. Cancer and the cell cycle. J Cell Biochem 1999;32/33 Suppl:166–172. 15. Anderson SSL. Spindle assembly and the art of regulating microtubule dynamics by MAPs and stathmin/Op18. Trends Cell Biol 2000;10:261–267.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

51

16. Rudner AD, and Murray AW. The spindle assembly checkpoint. Curr Opin Cell Biol 1996;8:773–80. 17. Nicklas RB. How cells get the right chromosome. Science 1997;275:632–637. 18. Reed JC, Bischoff JR. Ringing chromosomes through cell division—and survivin’ the experience. Cell 2000;102:545–548. 19. Altieri DC. The molecular basis and potential role of survivin in cancer diagnosis and therapy. Trends Mol Med 2001;7:542–547. 20. O’Connor DS, Wall NR, Porter ACG, et al. A p34cdc2 survival checkpoint in cancer. Cancer Cell 2002;2:43–54. 21. Hartwell LH, Kastan MB. Cell cycle control and cancer. Science 1994;266:1821–1828. 22. Harper J, Elledge SJ. Cdk inhibitors in develpment and cancer. Curr Opin Genet Dev 1996;6:56–64. 23. Chen Y-N, Sharma SK, Ramsey TM, et al. Selective killing of transformed cells by cyclin/ cyclin-dependent kinase 2 antagonists. Proc Natl Acad Sci USA 1999;96:4325–4329. 24. Senderowicz AM. Cyclin-dependent kinases as targets for cancer therapy. In: (Giaccone G, Schilsky R, Sondel G, eds), Cancer Chemotherapy and Biological Response Modifiers, Annual 20. Elsevier Science, Amsterdam: 2002;169–188. 25. Losiewicz MD, Carlson BA, Kaur G, et al. Potent inhibition of cdc2 kinase activity by the flavonoid L86-8275. Biochem Biophys Res Commun 1994;201:589–595. 26. Carlson BA, Dubay MM, Sausville EA, et al. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase CDC2 and CDK4 in human breast carcinoma cells. Cancer Res 1996;56:2973–2978. 27. Carlson BA, Pearlstein RA, Naik RG, et al. Inhibition of CDK2, CDK4 and CDK7 by flavopiridol and structural anologs. Proc Am Assoc Cancer Res 1996;36. 28. Dumant JA. HMR Report No. B/98/0189/R, B/98/0188/R, 8/98/0187/R. HMR Oncology 1996. 29. Konig A, Schwartz GK, Mohammad RM, et al. The novel cyclin-dependent kinase inhibitor flavopiridol downregulates Bcl-2 and induces growth arrest and apoptosis in chronic B-cell leukemia cell lines. Blood 1997;90:4307–4312. 30. Byrd JC, Shinn, C, Waselenko JK, et al. Flavopiridol induces apoptosis in chronic lymphocytic leukemia cells via activation of caspase-3 without evidence of bcl-2 modulation or dependence of functional p53. Blood 1998;92:3804–3816. 31. Arguello F, Alexander M, Sterry JA, et al. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity in vivo against human leukemia and lymphoma xenografts. Blood 1998;91:2482–2490. 32. Cartee L, Wang Z, Decker RH, et al. The cyclin-dependent kinase inhibitor (CDKI) Flavopiridol disrupts phorbol 12-myristate 13-acetate-induced differentiation and CDKI expression while enhancing apoptosis in human myeloid leukemia cells. Cancer Res 2001;61(6):2583–2591. 33. Bible KC, Kaufmann SH. Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Res 1996;56(21):4856–4861. 34. Schrump DS, Mathews W, Chen GA, et al. Flavopiridol mediates cell cycle arrest and apoptosis in esophageal cancer cell lines. Clin Cancer Res 1998;4:2885–2890. 35. Carlson B, Lahusen T, Singh S, et al. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 1999;59(18):4634–4641. 36. Drees M, Dengler WA, Roth T, et al. Flavopiridol (L86-8275): selective antitumor activity in vitro and activity in vivo for prostate carcinoma cells. Clin Cancer Res 1997;3(2):271–279. 37. Patel V, Senderowicz AM, Pinto, D, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, suppresses the growth of head and neck squamous cell carcinomas by inducing apoptosis. J Clin Invest 1998;102(9):1674–1681.

52

Shah and Schwartz

38. Thomas JP, Tutsch KD, Cleary JF, et al. Phase I clinical and pharmacokinetic trial of cyclindependent kinase inhibitor flavopiridol. Cancer Chemother Pharmacol 2002;50:465–472. 39. Senderowicz AM, Headlee D, Stinson SF, et al. Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. J Clin Oncol 1998;16(9):2986–2999. 40. Stadler WM, Vogelzang NJ, Amato R, et al. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in metastatic renal cancer: a University of Chicago Phase II Consortium study. J Clin Oncol 2000;18(2):371–375. 41. Shapiro GI, Patterson A, Lynch C, et al. A phase II trial of flavopiridol in patients with stage IV non-small cell lung cancer. Proc Amer Soc Clin Oncol 1999: Abstract 2013. 42. Schwartz GK, Ilson D, Saltz L, Howard OM, Neuberg DS, et al. Phase II study of the cyclindependent kinase inhibitor flavopiridol administered to patients with advanced gastric carcinoma. J Clin Oncol 2001;19(7):1985–1992. 43. Lin TS, Howard OM, Neuberg DS, et al. Seventy-two hour continuous infusion flavopiridol in relapsed and refractory mantle cell lymphoma. Leuk Lymphoma 2002;43(4):793–797. 44. Burdette-Radoux S, Tozer RG, Lohmann R, et al. NCIC CTG phase II study of flavopiridol in patients with previously untreated metastatic malignant melanoma. Proc Amer Soc Clin Oncol 2002;Abstract 1382. 45. Bennett P, Mani S, O’Reilly S, et al. Phase II trial of flavopiridol in metastatic colorectal cancer: preliminary results. Proc Amer Soc of Clin Oncol 1999;Abstract 1065. 46. Takahashi I, Saitoh Y, Yoshida M. UCN-01 and UCN-02, new selective inhibitors of protein kinase C. Purification, physico-chemical properties, structural determination and biological activities. J Antibiot 1989;42:571–576. 47. Tamoaki T, Nakano H. Potent and specific inhibitors of PKC of microbial origin. Biotech 1990;8:732–735. 48. Seynaeve CM, Stetter-Stevenson M, Sebers S, et al. Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 1993;53(9):2081–2086. 49. Akinaga S, Nomura K, Gomik, et al. Effect of UCN-01, a selective inhibitor of protein kinase C, on the cell-cycle distribution of human epidermoind carcinoma, A431 cells. Cancer Chemother Pharmacol 1994;33(4):273–280. 50. Kawakami K, Futami H, Takahara J, et al. UCN-01, 7-hydroxylstaurosporine, inhibits kinase activity of cyclin-dependent kinases and reduces the phosphorylation of the retinoblastoma susceptibility gene product in A549 human lung cancer cell line. Biochem Biophys Res Commun 1996;219(3):778–783. 51. Akiyama T, Yoshida T, Tsujita T, et al. G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as the induction of CDK inhibitor p21/CIP/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 1997;57(8):1495–1501. 52. Fan G, Steer CJ. The retinoblastoma gene product is a negative modulator of the apoptotic pathway. Adv Enzyme Regul 1996;36:283–303. 53. Hsueh CT, Kelsen D, Schwartz GK. UCN-01 suppresses thymidylate synthase gene expression and enhances 5-fluorouracil-induced apoptosis in a sequence-dependent manner. Clin Cancer Res 1998;4(9):2201–2206. 54. Graves PR, Yu L, Schwarz JK, et al. The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 2000;275(8):5600–5605. 55. Busby EC, Leistritz DF, Abraham RT, et al. The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 2000;60(8):2108–2112. 56. Yu Q, Rose, JH, Zhang H, et al. UCN-01 inhibits p53 up-regulation and abrogates gammaradiation-induced G2-M Checkpoint independently of p53 by targeting both of the checkpoint kinases, Chk2 and Chk1. Cancer Res 2002;62:5743–5748.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

53

57. Sausville EA, Lush RD, Headlee D, et al. Phase I trial of 72-hour continuous infusion of UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001;19(8):2319–2333. 58. Fuse E, Tanii H, Kurata N, et al. Unpredicted clinical pharmacology of UCN-01 caused by specific binding to human alpha1-acid glycoprotein. Cancer Res 1998;58:3248–3253. 59. Sausville EA, Lush RD, Headlee D, et al. Clinical pharmacology of UCN-01: initial observations and comparison to preclinical models. Cancer Chemother Pharmacol 1998;42(Suppl):S54–S59. 60. Tamura T, Sasaki Y, Minami H, et al. Phase I study of UCN-01 by 3 hour infusion. Proc Amer Soc Clin Oncol 1999;18:159a. 61. Dees EC, O'Reilly S, Figg WD, et al. A phase I and pharmacologic study of UCN-01, a protein kinase C inhibitor. Proc Amer Soc Clin Oncol 2000;19:205a. 62. Mutter R, Wills M. Chemistry and clinical biology of the bryostatins. Bioorg Med Chem 2000;8:1841–1860. 63. Wender PA, Cribbs CM, Koehler KF, et al. Modeling of the bryostatins to the phorbol ester pharmacophore on protein kinase C. Proc Natl Acad Sci USA 1988;85:7197–7201. 64. Kraft AS, Smith JB, Berkow RL. Bryostatin, an activator of the calcium phospholipid-dependent protein kinase, blocks phorbol ester-induced differentiation of human promyelocytic leukemia cells HL-60. Proc Natl Acad Sci USA 1986;83:1334–1338. 65. Hennings H, Blumberg PM, Pettit GR, et al. Bryostatin-1, an activator of protein kinase C inhibits tumor promotion by phorbol esters in Sencar mouse skin. Carcinogenesis 1987;9:1343–1346. 66. Isakov N, Galron D, Mustelin T, et al. Inhibition of phorbol ester-induced T cell proliferation by bryostatin is associated with rapid degradation of protein kinase C. J Immunol 1993;150(4):1195–1204. 67. Lee HW, Smith L, Pettit GR, et al. Ubiquitination of protein kinase C-alpha and degradation by the proteasome. J Biol Chem 1996;271:20,973–20,976. 68. Asiedu C, Biggs J, Lilly M, et al. Inhibition of leukemic cell growth by the protein kinase C activator bryostatin-1 correlates with the dephosphorylation of cyclin-dependent kinase 2. Cancer Res 1995;55:3716–3720. 69. Vrana JA, Saunders AM, Chellappan SP, et al. Divergent effects of bryostatin 1 and phorbol myristate acetate on cell cycle arrest and maturation in human myelomonocytic leukemia cells (U937). Differentiation 1998;63:33–42. 70. Vrana, JA, Kramer LB, Saunders AM, et al. Inhibition of protein kinase C activator-mediated induction of p21cip1 and p27kip1 by deoxycytidine analogs in human leukemia cells: relationship to apoptosis and differentiation. Biochem Pharmacol 1999;58:121–131. 71. Koutcher JA, Motwani M, Dyke JP, et al. The in vitro effect of bryostatin-1 on paclitaxelinduced tumor growth, mitotic entry, and blood flow. Clin Cancer Res 2000;6:1498–1507. 72. Hickman PF, Kemp GJ, Thompson CH, et al. Bryostatin 1, a novel antineoplastic agent and protein kinase C activator, induces human myalgia and muscle metabolic defects: a 31P magnetic resonance spectroscopic study. Br J Cancer 1995;72:998–1003. 73. Prendiville J, Crowther D, Thatcher N, et al. A phase I study of intravenous bryostatin 1 in patients with advanced cancer. Br J Cancer 1993;68:418–424. 74. Philip, PA, Rea D, Thavasu P, et al. Phase I study of bryostatin 1: assessment of interleukin 6 and tumor necrosis factor alpha induction in vivo. The Cancer Research Campaign Phase I Committee. J Natl Cancer Inst 1993;85:1812–1818. 75. Jayson GC, Crowther D, Prendiville J, et al. A phase I trial of bryostatin 1 in patients with advanced malignancy using a 24-hour intravenous infusion. Br J Cancer 1995;72:461–468. 76. Varterasian ML, Mohammad RM, Eilender DS, et al. Phase I study of bryostatin 1 in patients with relapsed non-Hodgkin’s lymphoma and chronic lymphocytic leukemia. J Clin Oncol 1998;16:56–62.

54

Shah and Schwartz

77. Propper DJ, Macaulay V, O'Bryne KJ, et al. A phase II study of bryostatin 1 in metastatic malignant melanoma. Br J Cancer 1998;78:1337–1341. 78. Gonzalez, R, Ebbinghaus S, Henthorn TK, et al. Treatment of patients with metastatic melanoma with bryostatin-1—a phase II study. Melanoma Res 1999;9:599–606. 79. Bedikian AY, Plager C, Stewart JR, et al. Phase II evaluation of bryostatin-1 in metastatic melanoma. Melanoma Res 2001;11:183–188. 80. Pagliaro L, Daliani D, Amato R, et al. A phase II trial of bryostatin-1 for patients with metastatic renal cell carcinoma. Cancer 2000;89:615–618. 81. Zonder JA, Shields AF, Zalupski M, et al. A phase II trial of bryostatin 1 in the treatment of metastatic colorectal cancer. Clin Cancer Res 2001;7:38–42. 82. Blackhall FH, Ranson M, Radford JA, et al. A phase II trial of bryostatin 1 in patients with non-Hodgkin’s lymphoma. Br J Cancer 2001;84:249–254. 83. Brockstein B, Samuels B, Humerickhouse R, et al. Phase II studies of bryostatin-1 in patients with advanced sarcoma and advanced head and neck cancer. Invest New Drugs 2001;19:249–254. 84. Luria SE, Delbruck M. Mutations of bacteria from virus sensitivity to virus resistance. Genetics 1943;28:491. 85. DeVita VT. Principles of cancer management: chemotherapy. In: (DeVita VT, Rosenberg SA, Hellman S, eds), Cancer: Principles and Practice of Oncology, Lippincott-Raven, Philadelphia: 1997;333–347. 86. Goldie JH, Coldman AJ. A model for tumor response to chemotherapy: an integration of the stem cell and somatic mutation hypotheses. Cancer Invest 1985;3(6):553–564. 87. Goldie JH, Coldman AJ. A mathematic model for relating the drug sensitivity of tumors to their spontaneous mutation rate. Cancer Treat Rep 1979;63(11–12):1727–1733. 88. Ritke MK, Rusnak JM, Lazo JS, et al. Differential induction of etoposide-mediated apoptosis in human leukemia HL-60 and K562 cells. Mol Pharmacol 1994;46:605–611. 89. Evans DL, Dive C. Effects of cisplatin on the induction of apoptosis in proliferating hepatoma cells and nonproliferating immature thymocytes. Cancer Res 1993;53:2133–2139. 90. Elledge RM, Gray R, Mansour E, et al. Accumulation of p53 protein as a possible predictor of response to adjuvant combination chemotherapy with cyclophosphamide, methotrexate, fluorouracil, and prednisone for breast cancer. J Natl Cancer Inst 1995;87(16):1254–1256. 91. Rusch V, Klimstra D, Venkatraman E, et al. Aberent p53 expression predicts clinical resistance to cisplatin-based chemotherapy in locally advanced non-small cell lung cancer. Cancer Res 1995;55:5038–5042. 92. Eliopoulos AG, Kerr DJ, Herod J, et al. The control of apoptosis and drug resistance in ovarian cancer: influence of p53 and Bcl-2. Oncogene 1995;11:1217–1228. 93. Righetti SC, Della Torre G, Pilotti S, et al. A comparative study of p53 gene mutations, protein accumulation and response to cisplatin-based chemotherapy in advanced ovarian carcinoma. Cancer Res 1996;56(4):689–693. 94. Kerr JFR, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wide ranging implications in tissue kinetics. Br J Cancer 1972;26:239–257. 95. Wyllie AH, Kerr JFR, Currie AR. Cell death: the significance of apoptosis. Int Rev Cytol 1990;68:251–306. 96. Dive C, Evans CA, Whetton AD. Induction of apoptosis-new targets of cancer chemotherapy. Semin Cancer Biol 1992;3:417–427. 97. Reed JC. Dysregulation of apoptosis in cancer. J Clin Oncol 1999;117(9):2941–2953. 98. Vaux DL, Haecker G, Strasser A. An evolutionary perspective on apoptosis. Cell 1994;76:777–779. 99. Stewart BW. Mechanisms of apoptosis: integration of genetic, biochemical, and cellular indicators. J Natl Cancer Inst 1994;86:1286–1296. 100. Hockenbery D. Defining apoptosis. Am J Pathol 1995;146:16–19.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

55

101. Martin SJ, Green DR. Protease activation during apoptosis. Death by a thousand cuts? Cell 1995;82:349–352. 102. Wyllie AH. Glucocorticoid-induced thymocyte apoptosis is associated with endogenous endonuclease activation. Nature 1980. 284:555–556. 103. Lazebnik YA, Takahashi A, Moir RD, et al. Studies of the lamin proteinase reveal multiple parallel biochemical pathways during apoptotic execution. Proc Natl Acad Sci USA 1995;92:9042–9046. 104. Kaufmann SH, Desnoyers S, Ottaviano Y, et al. Specific proteolytic cleavage of poly (ADPribose) polymerase: an early marker of chemotherapy-induced apoptosis. Cancer Res 1993;53:3976–3985. 105. Lazebnik YA, Kaufman SH, Desnoyers S, et al. Cleavage of poly (ADP-ribose) polymerase by a proteinase with properties like ICE. Nature 1994;371:346–347. 106. Nicholson DW, Ali A, Thornberry NA, et al. Identification and inhibition of the ICE/CED3 protease necessary for mammalian apoptosis. Nature 1995;376:37–43. 107. Neamati N, Fernandez A, Wright S, et al. Degradation of lamin B1 precedes oligonucleosomal DNA fragmentation in apoptotic thymocytes and isolated thymocyte nuclei. J Immunol 1994;154:3788–3795. 108. Seller WR, Fisher DE. Apoptosis and cancer drug targeting. J Clin Invest 1999;104(12):1655–1661. 109. Fisher, DE. Apoptosis in cancer therapy: crossing the threshold. Cell 1994;78:539–542. 110. Kerr JF, Winterford CM, Harmon BV. Apoptosis. Its significance in cancer and cancer therapy. Cancer 1994;73:2013–2026. 111. Bellamy CO, Malcolmson RD, Harrison DJ, et al. Cell death in health and disease: the biology and regulation of apoptosis. Semin Cancer Biol 1995;6:3–16. 112. McDonnell TJ, Meyn RE, Robertson LE. Implications of apoptotic cell death regulation in cancer therapy. Semin Cancer Biol 1995;6:53–60. 113. Eastman A. Survival factors, intracellular signal transduction, and the activation of endonucleases in apoptosis. Semin Cancer Biol 1995;6:45–52. 114. Lowe SW, Ruley HE, Jact T, et al. p53-dependent apoptosis modulates the cytotoxicity of anticancer agents. Cell 1993;74:957–967. 115. Nabeya Y, Loganzo F. Maslak P, et al. The mutational status of p53 protein in gastric and esophageal adenocarcinoma cell lines predicts sensitivity to chemotherapeutic agents. Int J Cancer 1994;64:1–10. 116. Ibardo AM, Huang Y, Fang G, et al. Bcl-xl overexpression inhibits taxol-induced yama protease activity and apoptosis. Cell Growth and Differ 1996;7:1087–1094. 117. Datta R, Manome Y, Taneja N, et al. Overexpression of Bcl-xl by cytotoxic drug exposure confers resistance to ionizing radiation-induced internucleosomal DNA fragmentation. Cell Growth and Differ 1995;6:363–370. 118. Gajewski TF, Thompson CB. Apoptosis meets signal transduction: elimination of a BAD influence. Cell 1996;87(4):589–592. 119. Jarvis WD, Grant S. Protein kinase C targeting in antineoplastic treatment strategies. Invest New Drugs 1999;17(3):227–240. 120. Levine AJ. p53, the cellular gatekeeper for growth and division. Cell 1997;88:323–331. 121. El-Diery WS, Harper JW, O'Connor PM, et al. Waf1/Cip1 is induced in p53-mediated G1 arrest and apoptosis. Cancer Res 1994;54:1169–1174. 122. Brugaralas J, Chandrasekaran C, Gordon JI, et al. Radiation-induced cell arrest compromised by p21 deficiency. Nature 1995;377:552–557. 123. Chan TA, Hwang PM, Hermeking J, et al. Cooperative effects of genes controling the G2/ M checkpoint. Genes Dev 2000;14:1584–1588. 124. Bunz F, Dutriaux A, Lengauer C, et al. Requirement for p53 and p21 to sustain G2 arrest after DNA damage. Science 1998;282:1497–1501.

56

Shah and Schwartz

125. Bunz F, Hwang PM, Torrance C, et al. Disruption of p53 in human cancer cells alters the responses to therapeutic agents. J Clin Invest 1999;104(3):263–269. 126. Yu J, Zhang L, Hwang PM, et al. Identification and classification of p53-regulated genes. Proc Natl Acad Sci USA 1999;96:14,517–14,522. 127. Clarke AR, Gledhill S, Hooper ML, et al. p53 dependence of early apoptotic and proliferative responses within the mouse intestinal epithelium following gamma-irradiation. Oncogene 1994;9:1767–1773. 128. Waldman T, Yongyang Y, Diollehay L, et al. Cell cycle arrest versus cell death in cancer therapy. Nat Med 1997;3:1034–1036. 129. Shah MA, Schwartz GK. Cell Cycle mediated drug resistance: An emerging concept in cancer therapy. Clin Cancer Res 2001;7(8):2168–2181. 130. Rowinsky EK. The development and clinical utility of the taxane class of antimicrotubule chemotherapy agents. Annu Rev Med 1997;48:353–374. 131. Schiff PB, Fant J, and Horwitz SB. Promotion of microtubule assembly in vitro by taxol. Nature 1979;277(5698):665–667. 132. Schiff PB, Horwitz SB. Taxol assembles tubulin in the absence of exogenous guanosine 5'triphosphate or microtubule-associated proteins. Biochemistry 1981;20(11):3247–3252. 133. Schiff PB, Horwitz SB. Taxol stabilizes microtubules in mouse fibroblast cells. Proc Natl Acad Sci USA 1980;77(3):1561–1565. 134. Yoo YD, Park JK, Choi JY, et al. CDK4 down-regulation induced by paclitaxel is associated with G1 arrest in gastric cancer cells. Clin Cancer Res 1998;4(12):3063–3068. 135. Long BH, Fairchild CR. Paclitaxel inhibits progression of mitotic cells to G1 phase by interference with spindle formation without affecting other microtubule functions during anaphase and telephase. Cancer Res 1994;54(16):4355–4361. 136. Brown T, Havlin K, Weiss G, et al. A phase I trial of taxol given by a 6-hour intravenous infusion. J Clin Oncol 1991;9(7):1261–1267. 137. Donaldson KL, Goolsby GL, Kiener PA, Wahl AF. Activation of p34cdc2 coincident with taxol-induced apoptosis. Cell Growth Differ 1994;5(10):1041–1050. 138. Ling YH, Yank Y, Cansoli U, et al. Accumulation of cyclin B1, activation of cyclin B1dependent kinase and induction of programmed cell death in human epidermoid carcinoma KB cells treated with Taxol. Int J Cancer 1998;75:925–932. 139. Clute P, Pines, J. Temporal and spatial control of cyclin B1 destruction in metaphase. Nat Cell Biol 1999;1:82–86. 140. Yu D, Jing T, Liu B, et al. Overexpression of ErbB2 blocks taxol-induced apoptosis by upregulation of p21Cip1 which inhibits p34Cdc2 kinase. Mol Cell 1998;2:581–591. 141. Donaldson KL, Goolsby GL, Wahl AF. Cytotoxicity of the anticancer agents cisplatin and taxol during cell proliferation and the cell cycle. Int J Cancer 1994;57(6):847–855. 142. Blagosklonny MV, Giannakakou P, El-Deiry WS, et al. Raf-1/bcl-2 phosphorylation: a step from microtubule damage to cell death. Cancer Res 1997;57(1):130–135. 143. Chen CY, Faller DV Phosphorylation of Bcl-2 protein and association with p21Ras in Rasinduced apoptosis. J Biol Chem 1996;271(5):2376–2379. 144. Blagosklonny MV, Fojo T. Molecular effects of paclitaxel: myths and reality (a critical review). Int J Cancer 1999;83(2):151–156. 145. Trielli MO, Andreassen PR, Lacroix FB, et al. Differential Taxol-dependent arrest of transformed and nontransformed cells in the G1 phase of the cell cycle, and specific related mortality of transformed cells. J Cell Biol 1996;135:689–700. 146. Wang T-H, Wang H-S, Soong Y-K. Paclitaxel-Induced cell death. Cancer 2000;88:2619–2628. 147. Motwani M, Delohery TM, Schwartz GK. Sequential dependent enhancement of caspase activation and apoptosis by flavopiridol on paclitaxel-treated human gastric and breast cancer cells. Clin Cancer Res 1999;5(7):1876–1883.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

57

148. Kaubisch A, Kelsen DP, Saltz L, et al. A phase I trial of weekly sequential bryostatin (Bryo) and paclitaxel in patients with advanced solid tumors. Proc Amer Soc Clin Oncol 1999;18: Abstract 639. 149. Bible KC, Kaufmann SH. Cytotoxic synergy between flavopiridol (NSC 649890, L86-8275) and various antineoplastic agents: the importance of sequence of administration. Cancer Res 1997;57:3375–3380. 150. Wang S, Wang Z, Boise L, et al. Loss of the bcl-2 phosphorylation loop domain increases resistance of human leukemia cells (U937) to paclitaxel-mediated mitochondrial dysfunction and apoptosis. Biochem Biophys Res Commun 1999;259(1):67–72. 151. Schmitt E, Cimoli G, Steyaert A, et al. Bcl-xL modulates apoptosis induced by anticancer drugs and delays DEVDase and DNA fragmentation-promoting activities. Exp Cell Res 1998;240(1):107–121. 152. Wang S, Wang Z, Boise LH, et al. Bryostatin-1 enhances paclitaxel induced mitochondrial dysfunction and apoptosis in human leukemia cells (U937) ectopically expressing Bcl-xl. Leukemia 1999;13(10):1564–1573. 153. Wang S, Guo C, Castillo A, et al. Effect of bryostatin-1 on taxol-induced apoptosis and cytotoxicity in human leukemia cells (U937). Biochem Pharmacol 1998;56(5):635–644. 154. Schwartz GK, O'Reilly E, Ilson D, et al. Phase I and pharmacokinetic study of paclitaxel followed by bryostatin-1 in patients with advanced solid tumors. J Clin Oncol 2001;20:257–2170. 155. Wall ME, Wani MC, Cook CE, et al. Plant anti-tumor agents 1. The isolation and structure of camptothecin, a novel alkaloidal leukemia and tumor inhibitor from Camptotheca acuminata. J Am Chem Soc 1966;88:3888–3890. 156. Slichenmeyer WJ, Rowinsky EK, Donehower RC, et al. The current status of camptothecin analogues as antitumor agents. JNCI 1993;85:271–291. 157. Li LH, Fraser TJ, Olin EJ, et al. Action of camptothecin on mammalian cells in culture. Cancer Res 1972;32(12):2643–2650. 158. Creemers CJ, Lund B, Verwij J. Topoisomerase I inhibitors: topotecan and irinotecan. Cancer Treat Rev 1994;20:73–96. 159. Hsiang Y-H, Hertzberg R, Hecht S, et al. Camptothecin induces protein-linked DNA breaks via mammalian Topoisomerase I. J Biol Chem 1985;260:14,873–14,878. 160. Hsiang Y-H, Lihou MG, Liu F. Arrest of replication forks by drug-stabilized topoisomerase I-DNA cleavable complexes as a mechanism of cell killing by Camptotheicin. Cancer Res 1989;49:5077–5082. 161. Sane AT, Bertrand R. Caspase inhibition in camptothecin-treated U-937 cells is coupled with a shift from apoptosis to transient G1 arrest followed by necrotic cell death. Cancer Res 1999;59(15):3565–3569. 162. Tsao YP, D’Apra P, Liu LF. The involvement of active DNA synthesis in camptothecininduced G2 arrest: altered regulation of p34cdc2/cyclin B. Cancer Res 1992;52(7):1823–1829. 163. Kohn EA, Ruth ND, Brown MK, et al. Abrogation of the S phase DNA damage checkpoint results in S phase progression of premature mitosis depending on the concentration of 7hydroxystaurosporine and the kinetics of Cdc25C activation. J Biol Chem 2002;277(29):26,553–26,564. 164. Weller M, Winter S, Schmidt C, et al. Topisomerase-I inhibitors for human malignant glioma: differential modulation of p53, p21, bax, and bcl-2 expression and of CD95-mediated apoptosis by camptothecin and beta-lapachone. Int J Cancer 1997;73(5):707–714. 165. Shimizu T, O’Connor PM, Kohn KW, et al. Unscheduled activation of cyclin B1/cdc2 kinase in human promyelocytic leukemia cell line HL60 cells ungroing apoptosis induced by DNA damage. Cancer Res 1995;55:228–231. 166. Saltz LB, Konowitz J, Kemeny NE, et al. Phase I clinical and pharmacokinetic study of irinotecan, fluorouracil, and leucovorin in patients with advanced solid tumors. J Clin Oncol 1996;14(11):2959–2967.

58

Shah and Schwartz

167. Saltz LB, Cox JV, Blanke C, et al. Irinotecan plus fluorouracil and leucovorin for metastatic colorectal carcinoma. Irinotecan Study Group. New Engl J Med 2000;343(13):905–912. 168. Saijo N. Clinical trials of irinotecan hydrochloride (CPT, campto injection, topotecan injection) in Japan. Ann NY Acad Sci 1996;803:292–305. 169. Waldman T, Kinzler KW, Vogelstein B. P21 is necessary for the p53 mediated G1 arrest in human cancer cells. Cancer Res 1995;55:5187–5195. 170. Motwani M, Jung C, Sirotnak FM, et al. Augmentation of apoptosis and tumor regressions by flavopiridol in the presence of CPT-11 in HCT116 colon cancer monolayers and xenografts. Clin Cancer Res 2001;7:4209–4219. 171. Shah MA, Kortmansky J, Gonen M, et al. A phase I/pharmacologic study of weekly sequential irinotecan (CPT) and flavopiridol. Proc Am Soc Clin Oncol 2002: Abstract 373. Available at: http://www.asco.org. Accessed August 11, 2003. 172. Goldwasser F, Shimuzu T, Jackman J, et al. Correlations between S and G2 arrest and the cytotoxicity of camptothecin in human colon carcinoma cells. Cancer Res 1996;56(19):4430–4437. 173. Shao RG, Cao CX, Shimizu T, et al. Abrogation of an S-phase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function. Cancer Res 1997;57(18):4029–4035. 174. Jones CB, Clements MK, Wasi S, et al. Enhancement of camptothecin-induced cytotoxicity with UCN-01 in breast cancer cells: abrogation of S/G2 arrest. Cancer Chemother Pharmacol 2000;45:252–258. 175. Nieves-Neira W, Pommier Y. Apoptotic response to camptothecin and 7-hydroxystaurosporine (UCN-01) in the 8 human breast cancer cell lines of the NCI anticancer drugs screen: multifactorial relationships with topoisomerase I, protein kinase C, Bcl-2, p53, MDM-2, and caspace pathways. Int J Cancer 1999;82:396–404. 176. Tse A, Schwartz GK. Abrogation of the G2/M checkpoint by 7-hydroxystaurosporine (UCN01) on SN-38 treated cells: a strategy for targeting tumors with defective cell cycle checkpoints. Am Assoc Cancer Res 2002. 177. Bozko P, Larsen AK, Raymond E, et al. Influence of G2 arrest on the cytotoxicity of DNA topoisomerase inhibitors toward human carcinoma cells with different p53 status. Acta Biochimica Polonica 2002;49(1):109–119. 178. Allegra CJ, Grem JL. Antimetabolites. In: (DeVita VT, Hellman S, and Rosenberg SA, eds), Cancer: Principles and Practice of Oncology. Lippincott-Raven: Philadelphia: 1997; 432–452. 179. Santi DV, McHenry CS, Raines RT, et al. Kinetics and thermodynamics of the interaction of 5-fluoro-2'-deoxyuridylate with thymidylate synthase. Biochemistry 1987;26(26):8606–8613. 180. van Triest B, Pinedo HM, van Hensbergen, et al. Thymidylate synthase level as the main predictive parameter for sensitivity to 5-fluorouracil, but not for folate-based thymidylate synthase inhibitors, in 13 nonselected colon cancer cell lines. Clin Cancer Res 1999;5(3):643–654. 181. Grem JL, Nguyen D, Monahan DP, et al. Sequence-dependent antagonism between fluorouracil and paclitaxel in human breast cancer cells. Biochem Pharmacol 1999;58(3):477–486. 182. Takeda H, Haisa M, Naomoto Y, et al. Effect of 5-fluorouracil on cell cycle regulatory proteins in human colon cancer cell line. Jpn J Cancer Res 1999;90(6):677–684. 183. Farnham PJ, and Schimke RT. Transcriptional regulation of mouse dihydrofolate reductase in the cell cycle. J Biol Chem 1985;260(12):7675–7680. 184. Ayusawa D, Shimizu K, Koyama H, et al. Cell-cycle-directed regulation of thymidylate synthase messenger RNA in human diploid fibroblasts stimulated to proliferate. J Mol Biol 1986;190(4):559–567. 185. Kortmansky J, Shah MA, Kemeny N, et al. A phase I/Pharmacologic study of UCN-01 in combination with 5-fluorouracil in patients with advanced solid tumors. In NCI-EORTC Conference, 2001, Miami, FL.

Chapter 2 / CDK Inhibitors in Combination Chemotherapy

59

186. Sorenson CM, Eastman A. Mechanism of cis-diamminedichloroplatinum(II)-induced cytotoxicity: role of G2 arrest and DNA double strand breaks. Cancer Res 1988;48:4484–4488. 187. Schlegel R, Belinsky GS, Harris MO. Premature mitosis induced in mammalian cells by the protein kinase inhibitors 2-aminopurine and 6-dimethylaminopurine. Cell Growth Differ 1990;1(4):171–178. 188. Demarcq C, Bunch RT, Creswell D, et al. The role of cell cycle progression in cisplatininduced apoptosis in Chinese hamster ovary cells. Cell Growth Differ 1994;5(9):983–993. 189. Shi Y, Frankel A, Radvanyi LG, et al. Rapamycin enhances apoptosis and increases sensitivity to cisplatin in vitro. Cancer Res 1995;55(9):1982–1988. 190. O’Dwyer PJ, Johnson SW, Hamilton TC. Cisplatin and its analogues. In: (DeVita VT, Hellman S, and Rosenberg SA, eds), Cancer: Principles and Practice of Oncology, LippincottRaven, Philadelphia: 1997; 467–483. 191. Barry MA, Behnke CA, Eastman A. Activation of programmed cell death (apoptosis) by cisplatin, other anticancer drugs, toxins and hyperthermia. Biochem Pharmacol 1990;40(10):2353–2362. 192. Judson PL, Watson JM, Gehrig PA, et al. Cisplatin inhibits paclitaxel-induced apoptosis in cisplatin-resistant ovarian cancer cell lines: possible explanation for failure of combination therapy. Cancer Res 1999;59(10):2425–2432. 193. Kaufmann WK, Paules RS. DNA damage and cell cycle checkpoints. FASEB 1996;10:238–247. 194. Hawkins DS, Demers GW, Galloway DA. Inactivation of p53 enhances sensitivity to multiple chemotherapeutic agents. Cancer Res 1996;56:892–898. 195. Brown R, Clugson C, Burns P, et al. Increased accumulation of p53 protein in cisplatinresistant cell lines. Int J Cancer 1993;55:678–684. 196. Perego P, Giarola M, Righetti SC, et al. Association between cisplatin resistance and mutation of p53 gene and reduced bax expression in ovarian carcinoma cell systems. Cancer Res 1996;56:556–562. 197. Zaffaroni N, Slivertrini R, Orlandi L, et al. Induction of apoptosis by taxol and cisplatin and effect on cell cycle-related proteins in cisplatin-sensitive and -resistant human ovarian cancer cells. Br J Cancer 1998;77(9):1378–1385. 198. Monks A, Horris ED, Vaigro-Wolff A, et al. UCN-01 enhances the in vitro toxicity of clinical agents in human tumor cell lines. Invest New Drugs 2000;18(2):95–107. 199. Bunch RT, Eastman A. 7-Hydroxystaurosporine (UCN-01) causes redistribution of proliferating cell nuclear antigen and abrogates cisplatin-induced S-phase arrest in Chinese hamster ovary cells. Cell Growth Differ 1997;8(7):779–788. 200. Husain A, Yan XY, Rosales N, et al. UCN-01 enhances CDDP cytotoxicity in ovarian cancer cells independent of p53 gene status and abrogation of G2 block. Clin Cancer Res 1997;4:2089–2097. 201. Sugiyama K, Shimizu M, Akiyama T, et al. UCN-01 selectively enhances mitomycin C cytotoxicity in p53 defective cells which is mediated through S and/or G(2) checkpoint abrogation. Int J Cancer 2000;85(5):703–709. 202. Huang P, Plunkett W. Induction of apoptosis by gemcitabine. Semin Oncol 1995;22:19–25. 203. Plunkett W, et al. Gemcitabine: metabolism, mechanisms of action and self-potentiation. Semin Oncol 1995;22:3–10. 204. Huang P, Chubb S, Hertel LW, et al. Action of 2'2'-difluorodeoxycytidine on DNA synthesis. Cancer Res 1991;51:6110–6117. 205. Cappella P, Tomasoni D, Faretta M, et al. Cell cycle effects of gemcitabine. Int J Cancer 2001;93:401–408. 206. Huang P, Plunkett W. Fludarabine- and gemcitabine-induced apoptosis: incorporation of analogs into DNA is a critical event. Cancer Chemother Pharmacol 1995;36:181–188.

60

Shah and Schwartz

207. Chen M, Hough AM, Lawrence TS, et al. The role of p53 in gemcitabine-mediated cytotoxicity and radiosensitization. Cancer Chemother Pharmacol 2000;45:369–374. 208. Galmarini CM, Clarke ML, Falette N, et al. Expression of non-functional p53 affects the sensitivity of cancer cells to gemcitabine. Int J Cancer 2002;97:439–445. 209. Jung CP, Motwani MV, Schwartz GK. Flavopiridol increases sensitization to gemcitabine in human gastrointestinal cancer cell lines and correlates with down-regulation of ribonucleotide reductase M2 subunit. Clin Cancer Res 2001;7:2527–2536. 210. Zhou BS, Tsai P, Ker R, et al. Overexpression of transfected human ribonucleotide reductase M2 subunit in human cancer cells enhances their invasive potential. Clin Exp Metastasis 1998;16:43–49. 211. Goan YG, Zhou BS, Hu E, et al. Overexpression of ribonucleotide reductase as a mechanism of resistance to 2,2-difluorodeoxycytidine in human KB cancer cell line. Cancer Res 1999;59:4204–4207. 212. Matranga CB, Shapiro GI. Selective sensitization of transformed cells to flavopiridol-induced apoptosis following recruitment to S-phase. Cancer Res 2002;62:1707–1717. 213. Banker DE, Groudine M, Willman CL, et al. Cell cycle perturbations in acute myeloid leukemia samples following in vitro exposures to therapeutic agents. Leuk Res 1998;22:221–239. 214. Shi Z, Azuma A, Sampath D, et al. S-phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine. Cancer Res 2001;61:1065–1072.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

3

61

Development of Protein Kinase C and Cyclin-Dependent Kinase Inhibitors As Potentiators of Cytotoxic Drug Action in Leukemia Steven Grant, MD CONTENTS INTRODUCTION PKC INHIBITORS AS POTENTIATORS OF CYTOTOXIC DRUG ACTION IN LEUKEMIA INTERACTIONS BETWEEN CYCLIN-DEPENDENT KINASE INHIBITORS AND CYTOTOXIC AGENTS IN LEUKEMIA INTERACTIONS BETWEEN PKC/CELL-CYCLE INHIBITORS AND DIFFERENTIATION-INDUCING AGENTS IN LEUKEMIA

INTRODUCTION Targeted Antileukemic Strategies and Apoptosis During the last decade, the approach to cancer chemotherapy has been revolutionized by two major advances: an accelerated understanding of the cell-death process (apoptosis) (1) and progress in the development of molecularly targeted agents directed against specific oncogenes and enzymes responsible for neoplastic transformation, e.g., STI571 in the case of Bcr/Abl+ malignancies (2). Such efforts have served to focus attention on two general classes of agents: inhibitors of (a) cell-cycle regulation and (b) cytoprotective signal transduction pathways. The rationale for developing cell-cycle inhibitors is based on abundant evidence From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

61

62

Grant

that neoplastic cells are defective in cell-cycle regulation, i.e., loss of the G1 checkpoint (3). In addition, it is now recognized that a variety of neoplastic cells, particularly those of hematopoietic origin, exhibit increased activity of certain pro-survival signaling pathways, i.e., the Ras/Raf/MEK/MAP kinase cascade (4). Thus, interference with specific cell-cycle progression and/or signaling pathways represents a logical alternative (or adjunct) to the use of cytotoxic agents, which kill neoplastic cells through more general mechanisms. For a variety of reasons, hematologic malignancies, particularly the leukemias, provide a major target for the development of such agents. Foremost among these is the observation that hematopoietic cells, including those of leukemic origin, exhibit an innate capacity to undergo apoptosis (5). Apoptosis is a genetically regulated process of cell suicide in which cells engage a program that culminates in the activation of a family of proteases referred to as caspases (6). Several general reviews of the apoptotic process have recently appeared (7,8). Briefly, apoptosis is triggered by a wide variety of noxious stimuli, which engage the caspase cascade by either of two pathways. The first is the mitochondrial (intrinsic) pathway, in which proapoptotic proteins are released (e.g., cytochrome c, Smac/DIABLO, AIF) and mitochondrial membrane potential is lost (9). Cytochrome c release results in activation of a multiprotein complex referred to as the apoptosome, consisting of cytochrome c, apoptosis-initiating factor-1 (apaf1), dATP, and the initiator caspase procaspase-9 (10). The formation of the apoptosome leads to cleavage/activation of procaspase-9, and in so doing, activates effector caspases such as procaspase-3, which are directly responsible for degradation of diverse cellular constituents, including DNA, nuclear lamin, and actin, among many others (11). Alternatively, apoptosis may proceed through the extrinsic, receptor-related cascade in which engagement of various ligands (e.g., Fas/Apo) to members of the TNF family of receptors results in the formation of a multiprotein complex referred to as the DISC, which cleaves/activates procaspase-8 (12). Activated caspase-8, by cleaving effector caspases directly (e.g., caspases-3 and -6) can thereby initiate the apoptotic cascade directly without requiring mitochondrial injury. However, crosstalk between the intrinsic and extrinsic pathways exists, and activation of the extrinsic pathway can amplify the cell-death process triggered by various stimuli that primarily cause mitochondrial injury (e.g., cytotoxic drugs) (13).

Rationale for Targeting Protein Kinase C As a Chemopotentiating Strategy Protein kinase C (PKC) is a serine/threonine kinase consisting of a family of at least 12 isoforms that is intimately involved in multiple cell functions, including proliferation, differentiation, survival, and stimulus-response coupling, among others (14). PKC consists of a family of least 12 kinases, which have been divided into three major groups, which differ primarily with respect to the co-

Chapter 3 / PKC and CDK Inhibitors in Leukemia

63

Fig. 1. Representation of the structure of the conventional protein kinase C (PKC) isoforms. cPKC isoforms consist of a C-terminal regulatory domain and an N-terminal catalytic domain. They are comprised of five variable regions and four conserved regions. The C1 component of the regulatory domain binds to lipid activators (i.e., DAG), whereas the C2 component is involved in Ca+2 binding. The C3 and C4 components of the catalytic domain are responsible for ATP and substrate binding, respectively. The majority of pharmacologic PKC inhibitors interfere with the PKC ATP-binding site. The novel and atypical PKC isoforms contain ATP- and substrate-binding sites, as well as a C2-like domain, but lack a Ca+2-binding region.

factors required for activation. The conventional isoforms (_, `I, `II, and a) are Ca+2-dependent, and are activated by phosphatidylserine (PS) as well as diacylglycerol (DAG). The novel PKC isoforms (6, ¡, d, and e) are also activated by PS and DAG, but are Ca+2 independent. Finally, the atypical isoforms (c and f/h) are activated by PS but are Ca+2 and DAG independent (15). All PKC isoforms consist of an N-terminal regulatory domain and a C-terminal catalytic domain (Fig. 1). The conventional isoforms contain four conserved (C1–4) and five variable (V1–5) regions (16–20). The C1 region is involved in binding to DAG, whereas Ca+2 binding occurs at C2. The C3 and C4 regions are involved in binding to ATP and substrates, respectively. Both the novel and atypical isoforms contain a C2-like region that does not bind to Ca+2. Most pharmacologic PKC inhibitors inhibit the ATP-binding site. Once activated, PKC isoforms are ultimately targeted to the nucleus, where they engage a wide variety of transcription factors and genes involved in diverse cellular processes (16–20). A schematic diagram displaying the general structure of cPKC isoforms is provided in Fig. 1. From the standpoint of cell survival, a particularly important target of PKC is the cytoprotective Raf/MEK/MAP kinase module, most notably in hematologic malignancies such as acute myeloid leukemia (AML) (21). For example,

64

Grant

activation of the Ras/Raf/MEK/MAP kinase pathway is commonly observed in acute myeloid leukemia cells (22). Moreover, the survival of neoplastic cells has been linked to the relative outputs of cytoprotective MAP kinase and the stressrelated JNK pathaways (23). Consequently, the notion that increased activation of cytoprotective pathways downstream of PKC contributes to transformationassociated events in leukemia seems plausible. More direct evidence for a cytoprotective role for PKC signaling in leukemic cell survival stems from evidence that agents that activate PKC (e.g., phorbol 12-myristate 13-acetate; PMA) protect hematopoietic cells from growth factor deprivation-induced apoptosis (24) and leukemic drugs from certain forms of cytotoxic drug-induced cell death (25). Moreover, pharmacologic PKC inhibitors are among the most potent inducers of apoptosis in malignant cells, including those of hematopoietic origin (26,27). In addition to antagonizing leukemic cell survival, agents that interrupt PKC and downstream cytoprotective pathways represent logical candidates as potentiators of cytotoxic drug action. The underlying thesis is that such agents may lower the threshold for mitochondrial damage and apoptosis induced by antileukemic drugs. In fact, there is abundant preclinical evidence that this is the case, and clinical trials testing this concept are currently underway (see subsequent discussion).

Rationale for Targeting CDKs As a Chemotherapeutic Strategy The cell cycle in normal and neoplastic cells is regulated by a complex network of proteins, which include cyclins, cyclin-dependent kinases, and a variety of cyclin-dependent kinase inhibitors (CDKIs; e.g., p21CIP1, p27KIP1, p16INK4A, and so on); these govern progression of cells from one phase of the cell cycle to the next (28,29). Briefly, cyclins, whose abundance fluctuates throughout the cell cycle, interact with cyclin-dependent kinases to perform a number of functions, including regulation of cellular transit through G2M, as well as phosphorylation of the retinoblastoma protein (pRb), which plays a critical role of progression of cells into and through S phase (30). Generally, progression of cells from G0G1 into and through S phase is governed by cyclins D, A, and E in association with CDKs 2 and 4/6 (31), whereas G2M progression is under the control of cyclin B in association with CDK1 (32). The CDKIs, which can be induced by a variety of factors, including DNA damage or differentiation stimuli, provide another level of control by interacting stoichiometrically with CDKs to block cell-cycle progression (33). CDKIs such as p21CIP1 can also exert functions independent of their cell-cycle regulatory activities, including inhibition of apoptosis (34). A simplified summary of cell-cycle regulation is provided in Fig. 2. The observation that cell-cycle dysregulation (i.e., loss of the G1 checkpoint) represents a virtually universal feature of neoplastic cells (35) has served as the impetus for the development of pharmacologic agents that target the cell cycle.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

65

Fig. 2. Schematic diagram of the cell cycle and its regulation. Quiescent cells in G0 are stimulated to enter the cell cycle by diverse factors (e.g., growth factors, mitogens) and do so by passing through the restriction point, beyond which they are irreversibly committed to cell-cycle traverse. Entry of cells into and through G1 involves the actions of cyclins 4 and 6 in conjunction with the cyclin D family, as well as the cyclin-dependent kinase (CDK)2/cyclin E complex. Progression through S phase also involves cyclin A in conjunction with CDK2. Traverse of cells through G2 and M requires activation of p34cdc2 (CDK1) in concert with cyclins A and B. CDK1 and CDK2 activity is inhibited by members of the high-molecular-weight endogenous CDK inhibitor (CDKI) family, consisting of p21CIP1, p27KIP1, and p57KIP2. The low-molecular-weight p16INK4A family (p16INK4A, p15INK4B, p18INK4C, p19INK4D) is primarily involved in opposing activation of CDK4/6/cyclin D complexes. The activity of CDKs are also regulated by inhibitory phosphorylations on threonine 15 and tyrosine 16 residues, and are activated (on threonine 160/161 residues) by the cyclin-activating kinase (CAK; CDK7). The pharmacologic CDK inhibitor flavopiridol (FP) interferes with the ATP-binding site, and in so doing, blocks the activity of all of the CDKs, but particularly CDK1 and CDK2. FP may also disrupt the cell cycle by down-regulating cyclin D and by interfering with cell-cycle progression indirectly by inhibiting CAK.

Several of these, including flavopiridol and UCN-01, have now entered clinical trials in humans (35–36). However, in view of the close relationship that exists between the cell cycle and apoptotic machinery (38), it is not surprising, at least in retrospect, that cell-cycle inhibitors are also potent triggers of cell death, particularly in hematopoietic cells (39). While the mechanism by which this phenomenon occurs is not known with certainty, activation of both the extrinsic (40) and mitochondrial apoptotic pathways (41) has been invoked. More recently, there has been intense interest in efforts to combine CDK inhibitors with conventional cytotoxic drugs in the treatment of various malignancies, particularly acute leukemia. The rationale underlying these efforts is

66

Grant

that (a) CDK inhibitors are effective by themselves in triggering apoptosis in leukemic cells (42); and (b) the possibility exists that administration of cell-cycle inhibitors in conjunction with conventional cytotoxic agents may result in additional perturbations in, or disruptions of, cell-cycle regulation, culminating in apoptosis. The possible benefits of such a strategy, particularly in leukemia, must be weighed against the theoretical risk that CDK inhibitors may reduce the Sphase fraction of leukemic cells, thereby rendering them less susceptible to various S-phase-specific antileukemic agents (e.g., 1-`-D-arabinofuranosylcytosine—ara-C) (43). In this context, drug-scheduling and -sequence considerations may be particularly important in determining whether such combination regimens exert synergistic or potentially antagonistic antileukemic effects. In summary, PKC and CDK inhibitors offer the potential for enhancing the lethal effects of conventional cytotoxic agents in leukemia through several mechanisms. First, these agents may trigger apoptosis on their own, possibly by inducing mitochondrial injury and/or by interfering with downstream cytoprotective pathways. Second, they may modify cell-cycle and signaling pathways, the net effect of which may be to lower the threshold for cytotoxic drug-induced lethality. Lastly, they may, when combined with conventional agents, trigger additional perturbations in cell-cycle or signal-transduction pathways that lead to apoptosis. A summary of preclinical and early clinical studies designed to test the feasibility of these strategies follows.

PKC INHIBITORS AS POTENTIATORS OF CYTOTOXIC DRUG ACTION IN LEUKEMIA Bryostatin 1 (NSC339555) Bryostatin 1 is macrocyclic lactone derived from the marine bryozoan Bugula neritina (44). It was discovered through the NCI’s natural product development program, and was initially found to exhibit impressive preclinical activity in vitro and in vivo against a variety of hematopoietic and nonhematopoietic tumor types (45–50). Bryostatin 1, like the tumor promoter PMA, is an activator of PKC, and acute exposure of cells to this agent results in the early translocation of PKC from the cytosol to the plasma membrane (51). However, chronic exposure of cells to bryostatin 1 leads to extensive PKC downregulation (52), a phenomenon that stems from proteasomal degradation of the protein (53). In contrast to the phorbol esters, bryostatin 1 is non-tumor-promoting; in fact, bryostatin 1 can block certain PMA-related actions that it does not itself induce, including tumor promotion and differentiation induction (54,55). The basis for the disparate activity spectra of bryostatin 1 and phorbol esters is not known with certainty, but may reflect differential activation (56) or nuclear translocation (57)

Chapter 3 / PKC and CDK Inhibitors in Leukemia

67

of PKC isoforms. In any case, although bryostatin 1 cannot be viewed as a typical PKC inhibitor, its capacity to downregulate the enzyme on chronic exposure allows it to mimic the actions of pharmacologic agents that antagonize PKC activity. When administered alone, bryostatin 1 has been shown to exert antiproliferative effects toward human leukemia cells (58) while, intriguingly, stimulating the in vitro growth of their normal counterparts (59). Bryostatin 1 also variably induces maturation in primary human leukemic myeloblasts (60), an effect that may be enhanced by co-administration of hematopoietic growth factors (e.g., granulocyte-macrophage colony-stimulating factor [GM-CSF]) (61). The latter phenomenon may explain the earlier observation that combined treatment with GM-CSF and bryostatin 1 reduced the in vitro self-renewal capacity of primary human leukemia blast cells (62). Bryostatin 1 has also been found to induce maturation in several human lymphoma cell lines (63–65). Bryostatin 1mediated leukemic cell differentiation has recently been shown to be dependent upon activation of the MEK/MAP kinase pathway (66). INTERACTIONS WITH ARA-C Although bryostatin 1 may be viewed as a maturation-inducing agent in leukemia, it is considerably weaker in this regard than tumor-promoting phorbol esters such as PMA (67) and, as noted previously, can block PMA-related differentiation (55). The relative ineffectiveness of bryostatin 1 as a differentiation inducer may reflect multiple factors, including its extensive capacity to downregulate various PKC isoforms, which could disrupt the maturation process, or its inability to induce the cyclin-dependent kinase p21CIP1, which in leukemic cells is required for the normal differentiation process to occur (68). Although several studies have demonstrated synergistic interactions between bryostatin 1 and conventional cytotoxic agents in human solid tumor and lymphoma cell lines (69–72), studies in human leukemia cells have been restricted to a relatively small number of agents. Of these, interactions between bryostatin 1 and the nucleoside analog ara-C are perhaps the best characterized. The biologic activity of ara-C, arguably the most effective agent available for the treatment of acute leukemia (73), are closely intertwined with its effects on lipid signaling pathways. For example, ara-C is converted intracellularly to its lethal derivative ara-CTP, which inhibits DNA polymerase _ and is incorporated into elongating DNA strands, leading to chain termination and interference with chain elongation (74). However, exposure of cells to ara-C also leads to generation of diacylglycerol, whose primary target is PKC, as well as induction of the proapoptotic lipid second messenger ceramide (75). Thus, it is tempting to speculate that the response of leukemic cells to ara-C may depend upon the relative effects this agent on pro- and antiapoptotic signaling events, which may in turn be modulated by agents such as bryostatin 1.

68

Grant

Early studies demonstrated that bryostatin 1 potentiated ara-C-induced DNA damage and apoptosis in human leukemia cells (i.e., HL-60) (76), an effect that was temporally correlated with PKC downregulation (77). In primary human leukemic blasts, combined exposure to ara-C and bryostatin 1 resulted in a significant reduction in leukemic self-renewal capacity (78), a determinant that has been correlated with clinical outcomes in patients with acute leukemia receiving chemotherapy (79). Interestingly, the sequence dependence of the interaction between bryostatin 1 and ara-C may reflect the extent to which bryostatin 1 induces leukemic cell differentiation. For example, in HL-60 cells, which are relatively insensitive to bryostatin 1-mediated maturation, the sequence bryostatin 1 followed by ara-C optimizes apoptosis (77). In contrast, in U937 cells, which are partially responsive to bryostatin 1-related differentiation, the sequence ara-C followed by bryostatin 1 was associated with maximal induction of cell death (80). This phenomenon is consistent with reports that in human leukemia cells, sequential exposure to a DNA-damaging agent followed by a maturation stimulus is a potent initiator of apoptosis (81). In addition to promoting ara-C-induced cell death, treatment with bryostatin 1 also attenuates, at least in part, the cytoprotective effects of increased expression of Bcl-2 or Bcl-xL. This capacity has been temporally related to the ability of bryostatin 1 to induce Bcl-2 phosphorylation (82). It should be noted, however, that in lymphoid leukemia cells (REH), bryostatin 1-mediated Bcl-2 phosphorylation was associated with enhanced drug resistance (83). However, the finding that bryostatin 1 enhances ara-C-mediated lethality in human leukemic cells ectopically expressing a Bcl-2 protein lacking the N-terminal phosphorylation loop region (84) argues against a role for bryostatin 1-mediated Bcl-2 phosphorylation in synergistic interactions between these agents. Instead, bryostatin 1 appears to act by blocking the ability of antiapoptotic proteins such as Bcl-xL to prevent release of cytochrome c into the cytosol (85). Most recently, however, the capacity of bryostatin 1 to potentiate ara-C lethality in human leukemia cells (U937 and HL60) has been attributed to the PKC-dependent release of tumor necrosis factor (TNF)-_ and activation of the extrinsic apoptotic cascade (86). Such findings suggest that downregulation of PKC by bryostatin 1 does not play a critical role in the proapoptotic actions of bryostatin 1, at least in human leukemic cells exposed to ara-C. BRYOSTATIN 1/ARA-C: CLINICAL EXPERIENCE To test the possibility that in vivo administration of bryostatin 1 could induce downregulation of PKC, a phase I trial was launched in patients with refractory nonhematologic malignancies in which bryostatin 1 was administered according to various schedules (e.g., 1-h infusion, 24-h continuous infusion, and a split course infusion on days 1 and 4) on a q3 weekly basis (87). The bryostatin 1 MTD was identified as 50 μg/m2, with myalgias and myelosuppression representing

Chapter 3 / PKC and CDK Inhibitors in Leukemia

69

the major dose-limiting toxicities. Correlative laboratory studies revealed a reduction in total PKC activity in peripheral blood mononuclear cells in a subset of patients (87). Based upon these findings, a phase I trial was initiated in patients with refractory acute leukemia, in which escalating doses of bryostatin 1 were administered as a 24-h infusion before and after two courses of high-dose ara-C (1.5 g/m2 q 12 h on d 1, 2 and 9, 10) (88). The results of this trial identified a bryostatin 1 MTD of 50 μg/m2 when administered in this manner. Significantly, 5 out of a total of 28 highly resistant acute leukemia patients achieved objective responses with this regimen, raising the possibility that synergistic antileukemic interactions between bryostatin 1 and ara-C observed in vitro might occur in vivo. Although downregulation of total PKC activity was observed in the blasts of a subset of patients, no correlation was observed between modulation of PKC activity and ex vivo response of blasts to ara-C-mediated apoptosis. A multiinstitutional phase II trial designed to assess the activity of this regimen more rigorously is ongoing. INTERACTIONS WITH FLUDARABINE Fludarabine (2-fluoroadenine 9-`-D-arabinofuranoside) is a purine analog that has shown significant activity in lymphoid malignancies, particularly CLL (89). Several studies have demonstrated that fludarabine can interact synergistically with bryostatin 1 in a dose- and sequence-dependent manner. Vrana et al. reported that in human myelomonocytic leukemia cells (U937), sequential administration of fludarabine followed by bryostatin 1 resulted in a marked increase in cell death and loss of clonogenic survival (90). In contrast, bryostatin 1 failed to modify fludarabine pharmacodynamics—i.e., incorporation into DNA and formation of the lethal metabolite F-ara-ATP (90). However, using an in vivo CLL xenograft model, Mohammad et al. showed that sequential administration of bryostatin 1 followed, rather than preceded, by fludarabine was associated with optimal cell killing (91). Whether these disparate findings reflect cell-type-specific differences or differences between in vitro and in vivo models remains to be determined. Clinical Studies Involving Bryostatin 1 and Fludarabine. Based on the studies described above, a phase I clinical trial has been initiated in which bryostatin 1 has been combined with fludarabine in patients with progressive chronic lymphocytic leukemia (CLL) and indolent non-Hodgkin’s lymphoma (NHL). Because of the divergent results of the preclinical studies, two sequences of drug administration have been evaluated. In one arm of the study, patients receive escalating doses of bryostatin 1 as 24-h continuous infusion prior to a standard 5-d course of fludarabine (e.g., 25 mg/m2/d × 5 d) (92). In the other arm of the study, patients receive 5 d of fludarabine followed by bryostatin 1 as a 24h continuous infusion. Although the study is ongoing, the MTD for bryostatin 1 for both arms of the study appears to be 50 μg/m2. Clinical results of this phase

70

Grant

I study appear to be promising, with responses in 33% of patients, including six who have previously received fludarabine. As in the case of the bryostatin 1/araC trial, no clear relationship has yet emerged between clinical responses and effects of bryostatin 1 on PKC activity in circulating CLL cells. Currently, there has been no clear therapeutic superiority for one sequence over the other. Following completion of the phase I trial, phase II trials are planned, using one or possibly both sequences, examining the activity of bryostatin 1 and fludarabine, at their MTD, in patients with refractory lymphoid malignancies. INTERACTIONS WITH CHLORODEOXYADENOSINE Mohammed et al. reported enhanced antitumor activity of the combination of bryostatin 1 and the purine analog chlorodeoxyadenosine in a murine xenograft model system (93). As in the case of in vivo studies combining bryostatin 1 and fludarabine, the sequence bryostatin 1 followed by chlorodeoxyadenosine appeared to be optimal (93). Based upon these results, a phase I trial of bryostatin 1 and chlorodeoxyadenosine has been initiated in patients with lymphoid malignancies. Although this trial is ongoing, initial results appear to be promising, with responses noted in some patients who have failed prior aggressive chemotherapeutic regimens (94). INTERACTIONS WITH VP-16 Yalowich and co-workers reported that bryostatin 1 enhanced the activity of the topoisomerase II inhibitor VP-16 in human leukemia cells, possibly by phosphorylating and hence activating topoisomerase II activity (95). To date, clinical trials combining bryostatin 1 with VP-16 in leukemia or other malignancies have not yet been initiated. INTERACTIONS WITH VINCRISTINE Mohammad et al. described potentiation of vincristine toxicity by bryostatin 1 in a large-cell hymphoma xenograft model system (96). Remick et al. have conducted a phase I trial of bryostatin 1 and vincristine in patients with refractory hematologic malignancies, including multiple myeloma, and preliminary results appear to be promising (97).

Phorbol 12-Myristate 13-Acetate (PMA) PMA is a tumor-promoting phorbol ester that has long been known to induce maturation in human leukemia cells (98). PMA targets PKC, triggering its translocation from the cytosol to the membrane as well as to the nucleus (99). Thus, PMA mimics the action of the endogenous lipid second messenger diacylglycerol. However, although acute exposure of cells to PMA induces PKC activation, prolonged, chronic exposure leads, as in the case of bryostatin 1, to PKC downregulation (100). In this way, PMA may function as a PKC antagonist, i.e., by blocking the activity of PKC as well as its downstream targets. It has been

Chapter 3 / PKC and CDK Inhibitors in Leukemia

71

proposed that downregulation of PKC by PMA may promote apoptosis in malignant cells by disrupting Bcl-2 phosphorylation and, in so doing, interfere with its cytoprotective activities (101). The effects of PMA on cytotoxic drug action in leukemia cells appear to vary with the schedule of administration. For example, as previously noted, induction of differentiation in human leukemia cells by PMA protects them from apoptosis induced by certain chemotherapeutic drugs, i.e., VP-16 (25). On the other hand, Vrana et al. reported that pretreatment of human leukemia cells with deoxycytidine analogs—i.e., ara-C, gemcitabine—blocked PMA-associated induction of p21CIP1, and resulted in a pronounced increase in apoptosis (102). It was proposed that inhibition of DNA polymerase by these analogs, through a yet to be identified mechanism, opposes induction of this CDKI, and in so doing, blocks the differentiation response to PMA. Such results are consistent with evidence that interference with p21CIP1 induction in leukemia cells converts a maturation into a cell-death response. Several clinical trials involving the administration of PMA to humans have now been conducted (103–105). In some of these, PMA has been given to patients with hematological malignancies in conjunction with cytotoxic agents (103,104). Initial results suggest that under these circumstances, PMA may ameliorate the myelosuppressive effects of such drugs (103,104). No information is yet available concerning the net effect of PMA on the therapeutic index of these agents, or on the impact of scheduling considerations on drug interactions. However, the completion of phase I trials of PMA should allow successor trials combining PMA with standard cytotoxic agents to be initiated, at which point answers to these questions may become available.

UCN-01 UCN-01 (7-hydroxystaurosporine) is a staurosporine derivative that was originally developed as a selective inhibitor of PKC (106). In contrast to staurosporine, UCN-01 is active in vivo (107). However, recent studies suggest that despite its PKC inhibitory activities, a number of the biologic actions of UCN-01 occur through other mechanisms. For example, the ability of UCN-01 to induce apoptosis in human leukemia cells has been shown to be proceed through a PKCindependent process (108). In this regard, UCN-01 also functions as a CDK inhibitor, inducing cell-cycle arrest in G1 (109), as well as an abrogator of the G2M checkpoint (110). In this regard, UCN-01 has been shown to act as a potent inhibitor of Chk1, the enzyme responsible for phosphorylation of the cdc25 phosphatase, a process that promotes proteasomal degradation of this protein (111). Accumulation of cdc25 leads in turn to dephosphorylation of CDKs (e.g., p34cdc2) on inhibitory sites, and as a consequence, cell-cycle progression (112). As such, CDKs are intimately involved in cell-cycle arrest in cells subjected to DNA damage; the net effect of UCN-01 is that of a checkpoint abrogator. In fact,

72

Grant

synergistic interactions between UCN-01 and various DNA-damaging agents (e.g., ionizing radiation, mitomycin C, cisplatin, camptothecin) have been reported (113–116). Analogously, synergistic antileukemic interactions between UCN-01 and nucleoside analogs have been observed in multiple leukemia cell lines. INTERACTIONS BETWEEN UCN-01 AND ARA-C Several studies have demonstrated that UCN-01 can potentiate the lethality of nucleoside analogs such as ara-C in human leukemia cells. For example, Wang et al. and Tang et al. reported that UCN-01 interacted in a highly synergistic manner with ara-C to induce mitochondrial damage and apoptosis in human leukemia cells such as HL-60 and U937 (84,96). Furthermore, similar interactions were observed in leukemia cells overexpressing the antiapoptotic proteins Bcl-2 or Bcl-xL (84,86). These events were associated with enhanced mitochondrial damage, as well as perturbations in Bcl-2 phosphorylation status. Thus, UCN-01 appears to be able to antagonize the ability of antiapoptotic proteins to block ara-C-induced mitochondrial injury through a yet to be determined mechanism. Similarly, Shi et al. reported that UCN-01 increased the lethal effect of nucleoside analogs such as ara-C and gemcitabine through a process that involved abrogation of the G1 checkpoint (117). As the ara-C concentrations employed in the latter study were considerably lower than those used in the preceding reports, it remains to be determined whether perturbations in checkpoint control underlie synergistic antileukemic interactions between ara-C and UCN-01 under all circumstances. Clinical phase I trials involving the combination of UCN-01 and ara-C have recently been initiated, but it is too early to assess the antileukemic activity of this regimen. INTERACTIONS BETWEEN UCN-01 AND FLUDARABINE In a recent study, UCN-01 was shown to interact synergistically with fludarabine in human myeloid leukemia cells to induce mitochondrial injury and apoptosis (118). Analogous to results obtained in cells exposed to ara-C, UCN-01 failed to potentiate fludarabine pharmacodynamics (i.e., ara-C DNA incorporation). The observed potentiation of fludabine-related apoptosis was accompanied by a marked reduction in leukemic cell clonogeneic potential. More recently, potentiation of fludarabine-mediated lethality in human leukemia cells has been shown to be associated with loss of checkpoint control (119). Based in part upon these studies, phase I trials of UCN-01 in combination with fludarabine in patients with lymphoid malignancies have been initiated at several centers. It is too early to assess the efficacy of this or similar approaches, although in an individual case report, administration of UCN-01 with standard cytotoxic agents appeared to exert significant activity in a heavily pretreated patient with NHL (120).

Chapter 3 / PKC and CDK Inhibitors in Leukemia

73

Safingol Safingol (threo-dihydrosphingosine) is a PKC inhibitor that acts at the PKC regulatory site (121). In preclinical studies, safingol has been shown to reverse, at least in part, the multi-drug-resistance (MDR) phenotype and, in so doing, increase the lethality of P-glycoprotein (Pgp) substrates such as the anthracycline antibiotics (122). There have been very few studies investigating the effect of safingol on drug-induced lethality in leukemia cells, and these have generally involved nonPgp substrates, e.g., nucloside analogs. For example, in human promyelocytic leukemia cells (HL-60), safingol mimicked the ability of the PKC downregulator bryostatin 1 and the PKC inhibitor UCN-01 to promote ara-C-induced apoptosis (123). Moreover, the effects of each of these agents were related to interference with the downstream cytoprotective MEK/MAP kinase pathway. A phase I trial of safingol in combination with doxorubicin has been carried out in patients with solid tumor malignancies (124). The regimen was tolerable, although whether it is able to circumvent Pgp-related drug resistance remains to be determined. To date, no trials involving safingol in patients with leukemia have been carried out, in part due to limitations in drug availability.

PKC412 The staurosporine derivative PKC412 is an inhibitor of PKC as well as a variety of other kinses, including VEGF, c-kit, and KDR (125). It also acts as a Pgp inhibitor, and is capable of reversing the MDR phenotype in several neoplastic cell lines (126). More recently, the actions of PKC412 have been related to inhibition of Akt (127) and FLT3 (128). The latter function may be particularly relevant to leukemia, as 35% of AML patients exhibit mutations of the FLT3 receptor. PKC has also displayed radiosensitizing actions (129). In preclinical studies, PKC412 has shown activity against primary CLL cells and has also increased the sensitivity of these cells to conventional cytotoxic agents, including chlorambucil and chlordeoxyadenosine (130). Phase I trials of PKC412 have been carried out in patients with solid tumor malignancies, and have identified an MTD of 150 mg/d by the oral route (131). PKC412 has also shown some activity when administered at daily doses of 150– 225 mg/d in patients with refractory/progressive CLL and NHL (132). To date, studies combining PKC412 with conventional cytotoxic agents have not been carried out in patients with acute leukemia, but in view of the potential for activity of FLT3 inhibitors in a subset of AML patients, such studies will likely be initiated in the near future.

PKC Antisense Oligonucleotides A number of groups have investigated the role of individual PKC isoforms in leukemic cell survival using either pharmacologic inhibitors (e.g., RO 31-8220) or antisense oligonucleotides directed against specific isoforms (133–135). The

74

Grant

major finding to date is that interference with PKC or one or more of its isoforms tends to block leukemic cell maturation (134,135), supporting the notion that PKC plays a critical role in the maturation process in these cells. However, relatively few studies have addressed the issue of whether antisense strategies directed against PKC could sensitize leukemic cells to drug-induced apoptosis. One notable exception is the study by Whitman et al., which demonstrated that antisense oligonucleotides directed against PKC `II-sensitized human promyelocytic leukemia cells (HL-60) to ara-C-induced lethality (136). Analogously, evidence that PKC-iota protects human leukemia cells from paclitaxel-mediated lethality has appeared (137). In view of continuing efforts to develop PKC antisense oligonucleotides for the clinic (138), the concept that such agents could be used to enhance the antileukemic activity of established antileukemic agents represents a distinct possibility.

INTERACTIONS BETWEEN CYCLIN-DEPENDENT KINASE INHIBITORS AND CYTOTOXIC AGENTS IN LEUKEMIA Flavopiridol GENERAL CONSIDERATIONS Flavopiridol (L86-8275; NSC 649890) is a semisynthetic flavone derived from a rohitukine alkaloid (139). It is the first pharmacologic CDK inhibitor to enter clinical trials in humans (140). Flavopiridol binds to the ATP pocket of CDKs and, in so doing, blocks their activity (141). Flavopiridol broadly inhibits CDKs, particularly CDKs 1, 2, and 4/6 (142,143). It also inhibits, to varying degrees, the activities of PKC, PKA, and PDGF. Evidence suggesting that flavopiridol exhibits anti-angiogenic activity has also appeared (144). Interestingly, recent reports indicate that flavopiridol, by virtue of its ability to inhibit the CDK9TEFb complex, can act as a transcriptional repressor (145). In addition to inducing cell-cycle arrest (e.g., in G1) (146), flavopiridol is also a potent inducer of apoptosis in neoplastic cells. In this regard, flavopiridol appears to be a particularly potent inducer of cell death in malignant hematopoietic cells, e.g., leukemia and lymphoma (147,148). In lung-cancer cells, flavopiridol-mediated lethality has been linked to induction of the extrinsic, receptor-related pathway (149). However, in human leukemia cells, flavopiridolinduced cell death primarily stems from activation of the extrinsic, mitochondrial-related pathway, i.e., through cytosolic release of cytochrome c (150). The mechanism by which flavopiridol triggers the cell-death process is not known with certainty, but may be related to perturbations in expression of cell-cycleassociated proteins (e.g., downregulation of cyclin D1) (151) or diminished expression of certain antiapoptotic proteins (e.g., Bcl-2, XIAP, Mcl-1) (152,153). More recently, flavopiridol has been shown to block induction of the cyclindependent kinase inhibitor (CDKI) p21CIP1 in human leukemia cells exposed to

Chapter 3 / PKC and CDK Inhibitors in Leukemia

75

certain differentiation-inducing agents (e.g., phorbol myrsistate ester and the histone deacetylase inhibitors sodium butyrate and SAHA), a phenomenon associated with a marked increase in apoptosis (154–155). This may reflect loss of the direct antiapoptotic actions of p21CIP1 (156). INTERACTIONS BETWEEN FLAVOPIRIDOL AND CYTOTOXIC AGENTS IN LEUKEMIA In nonhematopoietic tumor cells, flavopiridol has been shown to potentiate the lethality of a variety of conventional cytotoxic agents. For example, flavopiridol has been reported to enhance the lethal effects of paclitaxel in gastric cancer cells through a mechanism involving release of cells from mitotic arrest and caspase activation (157). In lung-cancer cells, synergistic interactions were observed between flavopiridol and various cytotoxic agents, including cispatin and gemcitabine (158). In these and other studies also involving lung-cancer cells, such interactions were found to be highly sequence specific, in that maximal induction of apoptosis occurred in cells in which flavopiridol was administered after the cytotoxic drug (159). The superiority of this particular sequence may reflect the accumulation of cells in S phase, where they are more susceptible to flavopiridol-mediated lethality. Surprisingly, there has been a relative paucity of studies in which flavopiridol has been combined with conventional cytotoxic agents in leukemia. Preliminary results indicate that flavopiridol may promote ara-C lethality in such cells when it is administered according to a schedule in which the cells become synchronized (160). Whether factors other than cell-cycle synchronization are involved in the enhanced lethality of such regimens remains to be determined. CLINICAL TRIALS INVOLVING FLAVOPIRIDOL AND ARA-C In an initial phase I trial, flavopiridol was administered to refractoy/relapsed AML at doses of 50–60 mg/m2 daily × 2 prior to ara-C and mitoxantrone (160). The concept underlying this timed sequential therapy (TST) approach is that once leukemic cells are released from cell-cycle blockade secondary to flavopiridol administration, they will be more susceptible to the lethal actions of cytotoxic drugs such as ara-C. In fact, flavopiridol appeared to induce apoptosis by itself in leukemic blasts in vivo; moreover, responses were noted in three of seven evaluable patients. Clearly, further evaluation of the clinical strategy of combining flavopiridol with established cytotoxic agents in leukemia will be needed before its success can be assessed. However, given abundant preclinical evidence of the activity of flavopiridol against leukemic cells (147), this strategy warrants further investigation.

Purine Analog CDK Inhibitors The CDK inhibitors butyrolactone, olomoucine, and roscovitine are purine analogs that inhibit several CDKs, particularly CDK1 and CDK2 (161). Roscovitine also shows inhibitory activity toward CDK5 (162). Each of these

76

Grant

agents exhibits inhibitory effects toward leukemia cells (163–165). In general, low concentrations of CDK inhibitors induce cell-cycle arrest, whereas higher concentrations induce apoptosis (163–165). To date, interactions between purine analog CDK inhibitors and antileukemic agents have not been extensively investigated. The tri-substituted purine CGP74514A is a relatively selective inhibitor of CDK1 (166). At low concentrations (e.g., 1 μM), it induces G1 arrest in human leukemia cells (U937). However, like other purine analogs, at higher concentrations (e.g., 5 μM), it potently induces apoptosis (167). Antileukemic interactions between CGP74514A and conventional cytotoxic agents have not yet been investigated. CYC202 (R-roscovitine) is a recently described CDK inhibitor that principally targets CDK2/cyclin E (168). It displayed activity against a variety of human tumor cell lines, including the Lovo colorectal carcinoma cell line with an IC50 of approx 10 μM. Interestingly, CYC202 lethality was not restricted to cells in S phase, but occurred in cells throughout the cell cycle. Currently, no information exists concerning interactions between CYC202 and cytotoxic agents in leukemia, but based upon results obtained with flavopiridol, such studies would clearly be of interest.

INTERACTIONS BETWEEN PKC/CELL-CYCLE INHIBITORS AND DIFFERENTIATION-INDUCING AGENTS IN LEUKEMIA Although the preceding discussion has focused on combinations of PKC modulators or CDK inhibitors with conventional cytotoxic agents in leukemia, attention has recently focused on the concept of combining these agents with each other in leukemia therapy. The underlying concept is that simultaneous administration of agents that perturb PKC and cell-cycle regulatory pathways may lead to events that culminate in mitochondrial damage and apoptosis. Such a concept appears to be particularly relevant in the case of differentiation-inducing compounds. For example, there is considerable evidence that disruption of leukemic cell maturation (e.g., by dysregulation of individual PKC isoforms or the CDKI p21CIP1) may cause cells to engage an alternative, apoptotic program when exposed to a differentiation-inducing agent (169,170). A brief summary of attempts to develop antileukemic strategies based upon this concept follows.

Cell-Cycle Inhibitors and Differentiation-Inducing Agents One of the hallmarks of the differentiation process of leukemic cells is arrest of cells in G1 (171). Moreover, certain CDK inhibitors (e.g., flavopiridol) have been shown to induce maturation in some neoplastic cell types (172). Therefore, it seemed reasonable to postulate that combining CDK inhibitors with maturation-inducing agents would result in enhanced leukemic cell differentiation. To

Chapter 3 / PKC and CDK Inhibitors in Leukemia

77

test this possibility, Cartee et al. exposed human leukemia cells (U937) to the PKC activator PMA in conjunction with the CDK inhibitor flavopiridol. Contrary to expectations, flavopiridol did not promote PMA-induced maturation; instead, it resulted in multiple perturbations in cell-cycle regulatory and signaling events that culminated in extensive mitochondrial damage and apoptosis (173). Chief among these was flavopiridol-mediated interference with PMAmediated p21CIP1 induction, a phenomenon that may reflect flavopiridol-related inhibition of the CDK9/cyclin T complex and resulting transcriptional repression (145). Increased apoptosis in flavopiridol/PMA-treated cells involved, in addition to induction of mitochondrial injury, activation of the extrinsic pathway, a consequence of enhanced induction of TNF-_ (174). More recently, these findings have been extended to include bryostatin 1. Specifically, co-treatment of human myeloid leukemia cells with flavopiridol and bryostatin 1 did not result in enhanced maturation, but instead resulted in a marked potentiation of apoptosis (175). As in the case of PMA, synergistic interactions between flavopiridol and bryostatin 1 involved the PKC-dependent release of TNF-_, and resulting activation of the extrinsic apoptotic pathway. Parallel results were obtained when the PKC and Chk1 inhibitor UCN-01 was combined with PMA in human myelomonocytic leukemia cells (U937) (176). Interestingly, UCN-01, like flavopiridol, attenuated induction of p21CIP1 by PMA. Such findings, along with recent observations that other pharmacologic CDK inhibitors (i.e., butyrolactone) block p21CIP1 induction (177), raise the possibility that disruption of the cell-cycle arrest machinery by such agents may interfere with the “normal” differentiation program of leukemic cells, and thereby trigger mitochondrial injury and apoptosis.

SUMMARY AND FUTURE DIRECTIONS The development of molecular target therapies, i.e., those involving the Bcr/ Abl kinase inhibitor STI571, have helped to stimulate initiatives involving PKC and CDK inhibitors, particularly in leukemia. Protein kinase C represents an important component of a cytoprotective signal transduction pathway, and there is abundant evidence that interruption of this pathway can lower the threshold for drug-induced apoptosis in leukemia cells. Clinical trials involving the combination of the PKC modulators bryostatin 1 and UCN-01 in conjunction with conventional cytotoxic agents are currently underway. Analogously, there is accumulating evidence that flavopiridol, the prototypical CDK inhibitor, can substantially increase drug-induced apoptosis in diverse tumor cell types, including leukemias. However, it is becoming apparent that bryostatin 1 and UCN01 exert multiple actions in addition to their effects on PKC, and that flavopiridol is not simply a CDK inhibitor. These considerations raise questions with regard to the mechanism(s) by which these novel agents promote the antileukemic

78

Grant

activity of standard cytotoxic drugs. For example, questions remaining to be addressed include: (1) Are the actions of PKC/CDK inhibitors responsible for induction of apoptosis when administered alone the same as those implicated in potentiation of antileukemic drug activity? (2) What are the drug sequences, schedules, and doses that will yield optimal clinical results? (3) Can antileukemic synergism observed in the in vitro setting be recapitulated in vivo? (4) What is the theoretical basis for therapeutic selectivity for such approaches? Another promising antileukemic strategy may involve the rational combination of PKC and CDK inhibitors with other novel signaling/cell-cycle modulatory agents, i.e., histone deacetylase inhibitors. An additional example of this approach has been the combination of UCN-01 with inhibitors of the cytoprotective Raf/MEK/ ERK (extracellular signal-regulated kinase) pathway, which has shown substantial in vitro activity against leukemia and other malignant hematopoietic cell lines (178,179). With the arrival of newer and potentially more effective PKC and CDK inhibitors, as well as the development of novel inhibitors of other cytoprotective signal transduction pathways, the number of such combination approaches appears virtually limitless. The challenge will be to identify the most promising of these for clinical development, not only in leukemia, but also in other hematologic as well as nonhematologic malignancies.

REFERENCES 1. Yang E, Korsmeyer SJ. Molecular thanatopsis: a discourse on the BCL2 family and cell death. Blood 1996;88:386–401. 2. Shuai K, Halpern J, ten Hoeve J, Rao X, Sawyers CL. Constitutive activation of STAT5 by the BCR-ABL oncogene in chronic myelogenous leukemia. Oncogene 1996;13:247–254. 3. McDonald ER 3rd, El-Deiry WS. Checkpoint genes in cancer. Ann Med 2001;33:113–122. 4. Lee JT Jr, McCubrey JA. The Raf/MEK/ERK signal transduction cascade as a target for chemotherapeutic intervention in leukemia. Leukemia 2002;16:486–507. 5. Sachs L, Lotem J. Control of programmed cell death in normal and leukemic cells: new implications for therapy. Blood 1993;82:15–21. 6. Alnemri ES. Mammalian cell death proteases: a family of highly conserved aspartate specific cysteine proteases. J Cell Biochem 1997; 64:33–42. 7. Cory S, Adams JM. The Bcl2 family: regulators of the cellular life-or-death switch. Nat Rev Cancer 2002;2:647–656. 8. Igney FH, Krammer PH. Death and anti-death: tumour resistance to apoptosis. Nat Rev Cancer 2002;2:277–288. 9. Zamzami N, Kroemer G. The mitochondrion in apoptosis: how Pandora’s box opens. Nat Rev Mol Cell Biol 200;2:67–71. 10. Acehan D, Jiang X, Morgan DG, Heuser JE, Wang X, Akey CW. Three-dimensional structure of the apoptosome: implications for assembly, procaspase-9 binding, and activation. Mol Cell 2002; 9:423–432. 11. Chang HY, Yang X. Proteases for cell suicide: functions and regulation of caspases. Microbiol Mol Biol Rev 2000;64:821–846. 12. Peter ME, Krammer PH. Mechanisms of CD95 (APO-1/Fas)-mediated apoptosis. Curr Opin Immunol 1998;10:545–551.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

79

13. Sun XM, MacFarlane M, Zhuang J, Wolf BB, Green DR, Cohen GM. Distinct caspase cascades are initiated in receptor-mediated and chemical-induced apoptosis. J Biol Chem 1999;274:5053–5060. 14. Nishizuka Y. Protein kinase C and lipid signaling for sustained cellular responses. FASEB J 1995;9:484–496. 15. Mellor H, Parker PJ. The extended protein kinase C superfamily. Bochem J 1998;232:281–292. 16. Ventura C, Maioli M. Protein kinase C control of gene expression. Crit Rev Eukaryot Gene Expr 2001;11:243–267. 17. Nishizuka Y. Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992; 258:607–614. 18. Nishizuka Y. Studies and perspectives of protein kinase C. Science 1986;233:305–312. 19. Jaken S. Protein kinase C isozymes and substrates. Curr Opin Cell Biol 1996;8:168–173. 20. Mellor H, Parker PJ. The extended protein kinase C superfamily. Biochem J 1998;332:281–292. 21. Cai H, Smola U, Wixler V, et al. Role of diacylglycerol-regulated protein kinase C isotypes in growth factor activation of the Raf-1 protein kinase. Mol Cell Biol 1997;17:732–741. 22. Morgan MA, Dolp O, Reuter CW. Cell-cycle-dependent activation of mitogen-activated protein kinase kinase (MEK-1/2) in myeloid leukemia cell lines and induction of growth inhibition and apoptosis by inhibitors of RAS signaling. Blood 2001;97:1823–1834. 23. Xia Z, Dickens M, Raingeaud J, Davis RJ, Greenberg ME. Opposing effects of ERK and JNK-p38 MAP kinases on apoptosis. Science 1995;270:1326–1331. 24. Lotem J, Cragoe EJ Jr, Sachs L. Rescue from programmed cell death in leukemic and normal myeloid cells. Blood 1991;78:953–960. 25. Solary E, Bertrand R, Kohn KW, Pommier Y. Differential induction of apoptosis in undifferentiated and differentiated HL-60 cells by DNA topoisomerase I and II inhibitors. Blood 1993;81:1359–1368. 26. Jarvis WD, Turner AJ, Povirk LF, Traylor RS, Grant S. Induction of apoptotic DNA fragmentation and cell death in HL-60 human promyelocytic leukemia cells by pharmacological inhibitors of protein kinase C. Cancer Res 1994;54:1707–1714. 27. Bertrand R, Solary E, O’Connor P, Kohn KW, Pommier Y. Induction of a common pathway of apoptosis by staurosporine. Exp Cell Res 1994;21:314–321. 28. Sherr CJ. The Pezcoller lecture: cancer cell cycles revisited. Cancer Res 2000;60:3689–3695. 29. Nurse P. A long twentieth century of the cell cycle and beyond. Cell 2000;100:71–78. 30. Weinberg RA. The retinoblastoma protein and cell cycle control. Cell 995;81:323–330. 31. Bartek J, Lukas J. Pathways governing G1/S transition and their response to DNA damage. FEBS Lett 2001;490:117–122. 32. Smits VA, Medema RH. Checking out the G(2)/M transition. Biochim Biophys Acta 2001;1519:1–12. 33. Sherr CJ, Roberts JM. CDK inhibitors: positive and negative regulators of G1-phase progression. Genes Dev 1999;13:1501–112. 34. Wang J, Walsh K. Resistance to apoptosis conferred by Cdk inhibitors during myocyte differentiation. Science 1996;273:359–361. 35. McDonald ER 3rd, El-Deiry WS. Checkpoint genes in cancer. Ann Med 2001;33:113–122. 36. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001;19:2319–2333. 37. Thomas JP, Tutsch KD, Cleary JF, et al. Phase I clinical and pharmacokinetic trial of the cyclindependent kinase inhibitor flavopiridol. Cancer Chemother Pharmacol 2002;50:465–472. 38. Nahle Z, Polakoff J, Davuluri RV, et al. Direct coupling of the cell cycle and cell death machinery by E2F. Nat Cell Biol 2002;4:859–864. 39. Arguello F, Alexander M, Sterry JA, et al. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity In vivo against human leukemia and lymphoma xenografts. Blood 1998;91:2482–2490.

80

Grant

40. Achenbach TV, Muller R, Slater EP. Bcl-2 independence of flavopiridol-induced apoptosis. Mitochondrial depolarization in the absence of cytochrome c release. J Biol Chem 2000;275:32,089–32,097. 41. Decker RH, Dai Y, Grant S. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in human leukemia cells (U937) through the mitochondrial rather than the receptor-mediated pathway. Cell Death Differ 2001;8:715–724. 42. Parker BW, Kaur G, Nieves-Neira W, et al. Early induction of apoptosis in hematopoietic cell lines after exposure to flavopiridol. Blood 1998;91:458–465. 43. Crowther PJ, Cooper IA, Woodcock DM. Biology of cell killing by 1-beta-D-arabinofuranosylcytosine and its relevance to molecular mechanisms of cytotoxicity. Cancer Res 1985;45:4291–4300. 44. Smith JB, Smith L, Pettit GR. Bryostatins: potent, new mitogens that mimic phorbol ester tumor promoters. Biochem Biophys Res Commun 1985;132:939–945. 45. Jones RJ, Sharkis SJ, Miller CB, Rowinsky EK, Burke PJ, May WS. Bryostatin 1, a unique biologic response modifier: anti-leukemic activity in vitro. Blood 1990;75:1319–1323. 46. Lilly M, Tompkins C, Brown C, Pettit G, Kraft A. Differentiation and growth modulation of chronic myelogenous leukemia cells by bryostatin. Cancer Res 1990;50:5520–5525. 47. Kennedy MJ, Prestigiacomo LJ, Tyler G, May WS, Davidson NE. Differential effects of bryostatin 1 and phorbol ester on human breast cancer cell lines. Cancer Res 1992;52:1278–1283. 48. Szallasi Z, Du L, Levine R, et al. The bryostatins inhibit growth of B16/F10 melanoma cells in vitro through a protein kinase C-independent mechanism: dissociation of activities using 26-epi-bryostatin 1. Cancer Res 1996;56:2105–2111. 49. Hornung RL, Pearson JW, Beckwith M, Longo DL. Preclinical evaluation of bryostatin as an anticancer agent against several murine tumor cell lines: in vitro versus in vivo activity. Cancer Res 1992;52:101–107. 50. Kraft AS, William F, Pettit GR, Lilly MB. Varied differentiation responses of human leukemias to bryostatin 1. Cancer Res 1989;49:1287–1293. 51. Kiss Z, Deli E, Girard PR, Pettit GR, Kuo JF. Comparative effects of polymyxin B, phorbol ester and bryostatin on protein phosphorylation, protein kinase C translocation, phospholipid metabolism and differentiation of HL60 cells. Biochem Biophys Res Commun 1987;146:208–215. 52. Isakov N, Galron D, Mustelin T, Pettit GR, Altman A. Inhibition of phorbol ester-induced T cell proliferation by bryostatin is associated with rapid degradation of protein kinase C. J Immunol 1993;150:1195–1204. 53. Lee HW, Smith L, Pettit GR, Smith JB. Bryostatin 1 and phorbol ester down-modulate protein kinase C-alpha and -epsilon via the ubiquitin/proteasome pathway in human fibroblasts. Mol Pharmacol 1997;51:439–447. 54. Kraft AS, Smith JB, Berkow RL. Bryostatin, an activator of the calcium phospholipid-dependent protein kinase, blocks phorbol ester-induced differentiation of human promyelocytic leukemia cells HL-60. Proc Natl Acad Sci USA 1986;83:1334–1338. 55. Vrana JA, Saunders AM, Chellappan SP, Grant S. Divergent effects of bryostatin 1 and phorbol myristate acetate on cell cycle arrest and maturation in human myelomonocytic leukemia cells (U937). Differentiation 1998;63:33–42. 56. Szallasi Z, Smith CB, Pettit GR, Blumberg PM. Differential regulation of protein kinase C isozymes by bryostatin 1 and phorbol 12-myristate 13-acetate in NIH 3T3 fibroblasts. J Biol Chem 1994;269:2118–2124. 57. Hocevar BA, Fields AP. Selective translocation of beta II-protein kinase C to the nucleus of human promyelocytic (HL60) leukemia cells. J Biol Chem 1991;266:28–33. 58. Asiedu C, Biggs J, Lilly M, Kraft AS. Inhibition of leukemic cell growth by the protein kinase C activator bryostatin 1 correlates with the dephosphorylation of cyclin-dependent kinase 2. Cancer Res 1995;55:3716–3720.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

81

59. May WS, Sharkis SJ, Esa AH, et al. Antineoplastic bryostatins are multipotential stimulators of human hematopoietic progenitor cells. Proc Natl Acad Sci USA 1987;84:8483–8487. 60. Steube KG, Drexler HG. Differentiation and growth modulation of myeloid leukemia cells by the protein kinase C activating agent bryostatin-1. Leuk Lymphoma 1993;9:141–148. 61. Matsui WH, Gladstone DE, Vala MS, et al. The role of growth factors in the activity of pharmacological differentiation agents. Cell Growth Differ 2002;13:275–283. 62. Grant S, Pettit GR, Howe C, McCrady C. Effect of the protein kinase C activating agent bryostatin 1 on the clonogenic response of leukemic blast progenitors to recombinant granulocyte-macrophage colony-stimulating factor. Leukemia 1991;5:392–398. 63. Mohammad RM, al-Katib A, Pettit GR, Sensenbrenner LL. Differential effects of bryostatin 1 on human non-Hodgkin’s B-lymphoma cell lines. Leuk Res 1993;17:1–8. 64. al-Katib A, Mohammad RM, Mohamed AN, Pettit GR, Sensenbrenner LL. Conversion of high grade lymphoma tumor cell line to intermediate grade with TPA and bryostatin 1 as determined by polypeptide analysis on 2D gel electrophoresis. Hematol Oncol 1990;8:81–89. 65. Arbuck SG, Sorensen JM, Christian MC, Ho P, Pluda JM, Cheson BD. New drugs in nonHodgkin’s lymphoma. Ann Oncol 1997;1:119–128. 66. Wall NR, Mohammad RM, Al-Katib AM. Mitogen-activated protein kinase is required for bryostatin 1-induced differentiation of the human acute lymphoblastic leukemia cell line Reh. Cell Growth Differ 2001;12:641–647. 67. Ng SB, Guy GR. Two protein kinase C activators, bryostatin-1 and phorbol-12-myristate13-acetate, have different effects on haemopoietic cell proliferation and differentiation. Cell Signal 1992;4:405–416. 68. Wang Z, Su ZZ, Fisher PB, Wang S, VanTuyle G, Grant S. Evidence of a functional role for the cyclin-dependent kinase inhibitor p21(WAF1/CIP1/MDA6) in the reciprocal regulation of PKC activator-induced apoptosis and differentation in human myelomonocytic leukemia cells. Exp Cell Res 1998;244:105–116. 69. Al-Katib AM, Smith MR, Kamanda WS, et al. Bryostatin 1 down-regulates mdr1 and potentiates vincristine cytotoxicity in diffuse large cell lymphoma xenografts. Clin Cancer Res 1998;4:1305–1314. 70. Basu A, Lazo JS. Sensitization of human cervical carcinoma cells to cis-diamminedichloroplatinum(II) by bryostatin 1. Cancer Res 1992;52:3119–3124. 71. Basu A, Akkaraju GR. Regulation of caspase activation and cis-diamminedichloroplatinum(II)-induced cell death by protein kinase C. Biochemistry 1999;38:4245–4251. 72. Mohammad RM, Wall NR, Dutcher JA, Al-Katib AM. The addition of bryostatin 1 to cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy improves response in a CHOP-resistant human diffuse large cell lymphoma xenograft model. Clin Cancer Res 2000;6:4950–4956. 73. Grant S. Ara-C: cellular and molecular pharmacology. Adv Cancer Res 1998;72:197–233. 74. Beardsley Mikita T, Beardsley GP. Functional consequences of the arabinosylcytosine structural lesion in DNA. Biochemistry 1988;27:4698–4705. 75. Strum JC, Small GW, Pauig SB, Daniel LW. 1-beta-D-Arabinofuranosylcytosine stimulates ceramide and diglyceride formation in HL-60 cells. J Biol Chem 1994;269:15493–15497. 76. Grant S, Jarvis WD, Swerdlow PS, et al. Potentiation of the activity of 1-beta- Darabinofuranosylcytosine by the protein kinase C activator bryostatin 1 in HL-60 cells: association with enhanced fragmentation of mature DNA. Cancer Res 1992;52:6270–6278. 77. Jarvis WD, Povirk LF, Turner AJ, et al. Effects of bryostatin 1 and other pharmacological activators of protein kinase C on 1-[beta-D-arabinofuranosyl]cytosine-induced apoptosis in HL-60 human promyelocytic leukemia cells. Biochem Pharmacol 1994;47:839–852. 78. Grant S, Traylor R, Bhalla K, McCrady C, Pettit GR. Effect of a combined exposure to cytosine arabinoside, bryostatin 1, and recombinant granulocyte-macrophage colony-stimulating factor on the clonogenic growth in vitro of normal and leukemic human hematopoietic progenitor cells. Leukemia 1992;6:432–439.

82

Grant

79. Curtis JE, Messner HA, Hasselback R, Elhakim TM, McCulloch EA. Contributions of hostand disease-related attributes to the outcome of patients with acute myelogenous leukemia. J Clin Oncol 1984; 2:253–259. 80. Grant S, Turner AJ, Freemerman AJ, Wang Z, Kramer L, Jarvis WD. Modulation of protein kinase C activity and calcium-sensitive isoform expression in human myeloid leukemia cells by bryostatin 1: relationship to differentiation and ara-C-induced apoptosis. Exp Cell Res 1996;228:65–75. 81. Bhatia U, Traganos F, Darzynkiewicz Z. Induction of cell differentiation potentiates apoptosis triggered by prior exposure to DNA-damaging drugs. Cell Growth Differ 1995;6:937–944. 82. Wang S, Vrana JA, Bartimole TM, et al. Agents that down-regulate or inhibit protein kinase C circumvent resistance to 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human leukemia cells that overexpress Bcl-2. Mol Pharmacol 1997;52:1000–1009. 83. Ruvolo PP, Deng X, Carr BK, May WS. A functional role for mitochondrial protein kinase Calpha in Bcl2 phosphorylation and suppression of apoptosis. J Biol Chem 1998;273:25,436–25,442. 84. Tang L, Boise LH, Dent P, Grant S. Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells. Cancer Res 2001;61:5106–5115. 85. Wang Z, Wang S, Dai Y, Grant S. Bryostatin 1 increases 1-beta-D-arabinofuranosylcytosineinduced cytochrome c release and apoptosis in human leukemia cells ectopically expressing Bcl-x(L). J Pharmacol Exp Ther 2002;301:568–577. 86. Wang S, Wang Z, Grant S. Bryostatin 1 and UCN-01 Potentiate 1-beta-D-arabinofuranosylcytosine-induced apoptosis in human myeloid leukemia cells through disparate mechanisms. Mol Pharmacol 2003;63:232–242. 87. Grant S, Roberts J, Poplin E, et al. Phase Ib trial of bryostatin 1 in patients with refractory malignancies. Clin Cancer Res 1998;4:611–618. 88. Cragg LH, Andreeff M, Feldman E, et al. Phase I trial and correlative laboratory studies of bryostatin 1 (NSC 339555) and high-dose 1-`-D-arabinofuranosylcytosine in patients with refractory acute leukemia. Clin Cancer Res 2002;8:2123–2133. 89. Adkins JC, Peters DH, Markham A. Fludarabine. An update of its pharmacology and use in the treatment of haematological malignancies. Drugs 1997;53:1005–1037. 90. Vrana JA, Wang Z, Rao AS, et al. Induction of apoptosis and differentiation by fludarabine in human leukemia cells (U937): interactions with the macrocyclic lactone bryostatin 1. Leukemia 1999;13:1046–1055. 91. Mohammad RM, Limvarapuss C, Hamdy N, et al. Treatment of a de novo fludarabine resistant-CLL xenograft model with bryostatin 1 followed by fludarabine. Int J Oncol 1999;14:945–950. 92. Roberts J, Smith M, Feldman E, Grant S. Phase I trial of bryostatin 1 (NSC 399555) and fludarabine in patients with progressive CLL and indolent non-Hodgkin(s lymphoma. Clinical Lymphoma 2002;3:184–188. 93. Mohammad RM, Katato K, Almatchy VP, et al. Sequential treatment of human chronic lymphocytic leukemia with bryostatin 1 followed by 2-chlorodeoxyadenosine: preclinical studies. Clin Cancer Res 1998;4:445–453. 94. Ahmad I, Al-Katib AM, Beck FW, Mohammad RM. Sequential treatment of a resistant chronic lymphocytic leukemia patient with bryostatin 1 followed by 2-chlorodeoxyadenosine: case report. Clin Cancer Res 2000;6:1328–1332. 95. Ritke MK, Murray NR, Allan WP, Fields AP, Yalowich JC. Hypophosphorylation of topoisomerase II in etoposide (VP-16)-resistant human leukemia K562 cells associated with reduced levels of beta II protein kinase C. Mol Pharmacol 1995;48:798–805.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

83

96. Mohammad RM, Diwakaran H, Maki A, et al. Bryostatin 1 induces apoptosis and augments inhibitory effects of vincristine in human diffuse large cell lymphoma. Leuk Res 1995;19:667–673. 97. Dowlati A, Payne J, Robertson K, et al. Phase I trial of combination bryostatin 1 and vincristine in B-cell malignancies: final report. AACR-NCI-EORTC International Conference Abst 800, 2001. 98. Tanaka Y, Miyake R, Kikkawa U, Nishizuka Y. Rapid assay of binding of tumor-promoting phorbol esters to protein kinase C1. J Biochem (Tokyo) 1986;99:257–261. 99. Forsbeck K, Nilsson K, Hansson A, Skoglund G, Ingelman-Sundberg M. Phorbol esterinduced alteration of differentiation and proliferation in human hematopoietic tumor cell lines: relationship to the presence and subcellular distribution of protein kinase C. Cancer Res 1985;45:6194–6199. 100. Rodriguez-Pena A, Rozengurt E. Disappearance of Ca2+-sensitive, phospholipid-dependent protein kinase activity in phorbol ester-treated 3T3 cells. Biochem Biophys Res Commun 1984;120:1053–1059. 101. Chen CY, Faller DV. Direction of p21ras-generated signals towards cell growth or apoptosis is determined by protein kinase C and Bcl-2. Oncogene 1995;11:1487–1498. 102. Vrana JA, Kramer LB, Saunders AM, et al. Inhibition of protein kinase C activator-mediated induction of p21CIP1 and p27KIP1 by deoxycytidine analogs in human leukemia cells: relationship to apoptosis and differentiation. Biochem Pharmacol 1999;58:121–131. 103. Han ZT, Zhu XX, Yang RY, et al. Effect of intravenous infusions of 12-O-tetradecanoylphorbol-13-acetate (TPA) in patients with myelocytic leukemia: preliminary studies on therapeutic efficacy and toxicity. Proc Natl Acad Sci USA 1998;95:5357–5361. 104. Han ZT, Tong YK, He LM, et al. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced increase in depressed white blood cell counts in patients treated with cytotoxic cancer chemotherapeutic drugs. Proc Natl Acad Sci USA 1998;95:5362–5365. 105. Strair RK, Schaar D, Goodell L, et al. Administration of a phorbol ester to patients with hematological malignancies: preliminary results from a phase I clinical trial of 12-Otetradecanoylphorbol-13-acetate. Clin Cancer Res 2002;8:2512–2518. 106. Akahashi I, Kobayashi E, Asano K, Yoshida M, Nakano H. UCN-01, a selective inhibitor of protein kinase C from Streptomyces. J Antibiot (Tokyo) 1987;40:1782–1784. 107. Akinaga S, Gomi K, Morimoto M, Tamaoki T, Okabe M. Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human tumor models. Cancer Res 1991;51:4888–4892. 108. Wang Q, Worland PJ, Clark JL, Carlson BA, Sausville EA. Apoptosis in 7-hydroxystaurosporine-treated T lymphoblasts correlates with activation of cyclin-dependent kinases 1 and 2. Cell Growth Differ 1995;6:927–936. 109. Seynaeve CM, Stetler-Stevenson M, Sebers S, Kaur G, Sausville EA, Worland P. Cell cycle arrest and growth inhibition by the protein kinase antagonist UCN-01 in human breast carcinoma cells. Cancer Res 1993;53:2081–2086. 110. Wang Q, Fan S, Eastman A, Worland PJ, Sausville EA, O’Connor P.UCN-01: a potent abrogator of G2 checkpoint function in cancer cells with disrupted p53. J Natl Cancer Inst 1996;88:956–965. 111. Graves PR, Yu L, Schwarz JK, et al. The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 2000;275:5600–5605. 112. Walworth NC. DNA damage: Chk1 and Cdc25, more than meets the eye. Curr Opin Genet Dev 2001;11:78–82. 113. Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN. The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 2000;60:2108–2112.

84

Grant

114. Sugiyama K, Shimizu M, Akiyama T, et al. UCN-01 selectively enhances mitomycin C cytotoxicity in p53 defective cells which is mediated through S and/or G(2) checkpoint abrogation. Int J Cancer 2000;85:703–709. 115. Bunch RT, Eastman A. Enhancement of cisplatin-induced cytotoxicity by 7-hydroxystaurosporine (UCN-01), a new G2-checkpoint inhibitor. Clin Cancer Res 1996;2:791–797. 116. Shao RG, Cao CX, Shimizu T, O’Connor PM, Kohn KW, Pommier Y. Abrogation of an Sphase checkpoint and potentiation of camptothecin cytotoxicity by 7-hydroxystaurosporine (UCN-01) in human cancer cell lines, possibly influenced by p53 function. Cancer Res 1997;57:4029–4035. 117. Shi Z, Azuma A, Sampath D, Li YX, Huang P, Plunkett W. S-phase arrest by nucleoside analogues and abrogation of survival without cell cycle progression by 7-hydroxystaurosporine. Cancer Res 2001;61:1065–1072. 118. Harvey S, Decker R, Dai Y, et al. Interactions between 2-fluoroadenine 9-beta- Darabinofuranoside and the kinase inhibitor UCN-01 in human leukemia and lymphoma cells. Clin Cancer Res 2001;7:320–330. 119. Sampath D, Shi Z, Plunkett W. Inhibition of cyclin-dependent kinase 2 by the Chk1-Cdc25A pathway during the S-phase checkpoint activated by fludarabine: dysregulation by 7hydroxystaurosporine. Mol Pharmacol 2002;62:680–688. 120. Wilson WH, Sorbara L, Figg WD, et al. Modulation of clinical drug resistance in a B cell lymphoma patient by the protein kinase inhibitor 7-hydroxystaurosporine: presentation of a novel therapeutic paradigm. Clin Cancer Res 2000;6:415–421. 121. Schwartz GK, Jiang J, Kelsen D, Albino AP. Protein kinase C: a novel target for inhibiting gastric cancer cell invasion. J Natl Cancer Inst 1993;85:402–407. 122. Schwartz GK, Haimovitz-Friedman A, et al. Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells. J Natl Cancer Inst 1995;87:1394–1399. 123. Jarvis WD, Fornari FA Jr, Tombes RM, et al. Evidence for involvement of mitogen-activated protein kinase, rather than stress-activated protein kinase, in potentiation of 1-beta-Darabinofuranosylcytosine-induced apoptosis by interruption of protein kinase C signaling. Mol Pharmacol 1998;54:844–856. 124. Schwartz GK, Ward D, Saltz L, et al. A pilot clinical/pharmacological study of the protein kinase C-specific inhibitor safingol alone and in combination with doxorubicin. Clin Cancer Res 1997;3:537–543. 125. Fabbro D, Ruetz S, Bodis S, et al. PKC412—a protein kinase inhibitor with a broad therapeutic potential. Anticancer Drug Des 2000;15:17–28. 126. Swannie HC, Kaye SB. Protein kinase C inhibitors. Curr Oncol Rep 2002;4:37–46. 127. Tenzer A, Zingg D, Rocha S, et al. The phosphatidylinositide 3'-kinase/Akt survival pathway is a target for the anticancer and radiosensitizing agent PKC412, an inhibitor of protein kinase C. Cancer Res 2001;61:8203–8210. 128. Weisberg E, Boulton C, Kelly LM, et al. Inhibition of mutant FLT3 receptors in leukemia cells by the small molecule tyrosine kinase inhibitor PKC412. Cancer Cell 2002;1:433–443. 129. Zaugg K, Rocha S, Resch H, et al. Differential p53-dependent mechanism of radiosensitization in vitro and in vivo by the protein kinase C-specific inhibitor PKC412. Cancer Res 2001;61:732–738. 130. Ganeshaguru K, Wickremasinghe RG, Jones DT, et al. Actions of the selective protein kinase C inhibitor PKC412 on B-chronic lymphocytic leukemia cells in vitro. Haematologica 2002;87:167–176. 131. Propper DJ, McDonald AC, Man A, et al. Phase I and pharmacokinetic study of PKC412, an inhibitor of protein kinase C. J Clin Oncol 2001;19:1485–1492.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

85

132. Virchis A, Ganeshaguru K, Hart S, et al. A novel treatment approach for low grade lymphoproliferative disorders using PKC412 (CGP41251), an inhibitor of protein kinase C. Hematol J 2002;3:131–136. 133. Dieter P, Schwende H. Protein kinase C-alpha and -beta play antagonistic roles in the differentiation process of THP-1 cells. Cell Signal 2000;12:297–302. 134. Mallia CM, Aguirre V, McGary E, et al. Protein kinase calpha is an effector of hexamethylene bisacetamide-induced differentiation of Friend erythroleukemia cells. Exp Cell Res 1999;246:348–354. 135. Pessino A, Passalacqua M, Sparatore B, Patrone M, Melloni E, Pontremoli S. Antisense oligodeoxynucleotide inhibition of delta protein kinase C expression accelerates induced differentiation of murine erythroleukaemia cells. Biochem J 1995;312:549–554. 136. Whitman SP, Civoli F, Daniel LW. Protein kinase CbetaII activation by 1-beta-Darabinofuranosylcytosine is antagonistic to stimulation of apoptosis and Bcl-2alpha downregulation. J Biol Chem 1997;272:23481–23484. 137. Murray NR, Baumgardner GP, Burns DJ, Fields AP. Protein kinase C isotypes in human erythroleukemia (K562) cell proliferation and differentiation. Evidence that beta II protein kinase C is required for proliferation. J Biol Chem 1993;268:15,847–15,853. 138. Mani S, Rudin CM, Kunkel K, et al. Phase I clinical and pharmacokinetic study of protein kinase C-alpha antisense oligonucleotide ISIS 3521 administered in combination with 5-fluorouracil and leucovorin in patients with advanced cancer. Clin Cancer Res 2002;8:1042–1048. 139. Zhai S, Senderowicz AM, Sausville EA, Figg WD. Flavopiridol, a novel cyclin-dependent kinase inhibitor, in clinical development. Ann Pharmacother 2002;36:905–911. 140. Senderowicz AM, Headlee D, Stinson SF, et al. Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. J Clin Oncol 1998;16:2986–2999. 141. De Azevedo WF Jr, Mueller-Dieckmann HJ, Schulze-Gahmen U, Worland PJ, Sausville E, Kim SH. Structural basis for specificity and potency of a flavonoid inhibitor of human CDK2, a cell cycle kinase. Proc Natl Acad Sci USA 1996;93:2735–2740. 142. Gray N, Detivaud L, Doerig C, Meijer L. ATP-site directed inhibitors of cyclin-dependent kinases. Curr Med Chem 1999;6:859–875. 143. Senderowicz AM. Cyclin-dependent kinase modulators: a novel class of cell cycle regulators for cancer therapy. Cancer Chemother Biol Response Modif 2001;19:165–188. 144. Rapella A, Negrioli A, Melillo G, Pastorino S, Varesio L, Bosco MC. Flavopiridol inhibits vascular endothelial growth factor production induced by hypoxia or picolinic acid in human neuroblastoma. Int J Cancer 2002;99:658–664. 145. Chao SH, Price DH. Flavopiridol inactivates P-TEFb and blocks most RNA polymerase II transcription in vivo. J Biol Chem 2001;276:31,793–31,799. 146. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK) 2 and CDK4 in human breast carcinoma cells. Cancer Res 1996;56:2973–2978. 147. Arguello F, Alexander M, Sterry JA, et al. Flavopiridol induces apoptosis of normal lymphoid cells, causes immunosuppression, and has potent antitumor activity In vivo against human leukemia and lymphoma xenografts. Blood 1998;91:2482–2490. 148. Rapoport AP, Simons-Evelyn M, Chen T, et al. Flavopiridol induces apoptosis and caspase3 activation of a newly characterized Burkitt’s lymphoma cell line containing mutant p53 genes. Blood Cells Mol Dis 2001;27:610–24. 149. Achenbach TV, Muller R, Slater EP. Bcl-2 independence of flavopiridol-induced apoptosis. Mitochondrial depolarization in the absence of cytochrome c release. J Biol Chem 2000;275:32,089–32,097.

86

Grant

150. Decker RH, Dai Y, Grant S. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in human leukemia cells (U937) through the mitochondrial rather than the receptor-mediated pathway. Cell Death Differ 2001;8:715–724. 151. Carlson B, Lahusen T, Singh S, et al. Down-regulation of cyclin D1 by transcriptional repression in MCF-7 human breast carcinoma cells induced by flavopiridol. Cancer Res 1999;59:4634–4641. 152. Kitada S, Zapata JM, Andreeff M, Reed JC. Protein kinase inhibitors flavopiridol and 7hydroxy-staurosporine down-regulate antiapoptosis proteins in B-cell chronic lymphocytic leukemia. Blood 2000;96:393–397. 153. Gojo I, Zhang B, Fenton RG. The cyclin-dependent kinase inhibitor flavopiridol induces apoptosis in multiple myeloma cells through transcriptional repression and down-regulation of Mcl-1. Clin Cancer Res 2002;8:3527–3538. 154. Rosato RR, Almenara JA, Cartee L, Betts V, Chellappan SP, Grant S. The cyclin-dependent kinase inhibitor flavopiridol disrupts sodium butyrate-induced p21WAF1/CIP1 expression and maturation while reciprocally potentiating apoptosis in human leukemia cells. Mol Cancer Ther 2002;1:253–266. 155. Almenara J, Rosato R, Grant S. Synergistic induction of mitochondrial damage and apoptosis in human leukemia cells by flavopiridol and the histone deacetylase inhibitor suberoylanilide hydroxamic acid (SAHA). Leukemia 2002;16:1331–1343. 156. Asada M, Yamada T, Ichijo H, et al. Apoptosis inhibitory activity of cytoplasmic p21(Cip1/ WAF1) in monocytic differentiation. EMBO J 1999;18:1223–1234. 157. Motwani M, Delohery TM, Schwartz GK. Sequential dependent enhancement of caspase activation and apoptosis by flavopiridol on paclitaxel-treated human gastric and breast cancer cells. Clin Cancer Res 1999;5:1876–1883. 158. Matranga CB, Shapiro GI. Selective sensitization of transformed cells to flavopiridol-induced apoptosis following recruitment to S-phase. Cancer Res 2002;62:1707–1717. 159. Bible KC, Kaufmann SH. Cytotoxic synergy between flavopiridol (NSC 649890, L86-8275) and various antineoplastic agents: the importance of sequence of administration. Cancer Res 1997;57:3375–3380. 160. Karp JE, Yang WD, Tidwell ML, et al. Timed-sequential therapy (TST) of acute leukemia with flavopiridol (FP): in vitro model for a phase I clinical trial. Blood 2001;98:2482. 161. Lee AD, Ren S, Lien EJ. Purine analogs as CDK enzyme inhibitory agents: a survey and QSAR analysis (Review). Prog Drug Res 2001;56:155–93. 162. Filgueira de Azevedo W Jr, Gaspar RT, Canduri F, Camera JC Jr, Freitas da Silveira NJ. Molecular model of cyclin-dependent kinase 5 complexed with roscovitine. Biochem Biophys Res Commun 2002;297:1154–1158. 163. Somerville L, Cory JG. Apoptosis induced by inhibitors of nucleotide synthesis in deoxyadenosine-resistant leukemia L1210 cells that lack p53 expression. Anticancer Res 2000;20:4171–4178. 164. Meijer L, Borgne A, Mulner O, et al. Biochemical and cellular effects of roscovitine, a potent and selective inhibitor of the cyclin-dependent kinases cdc2, cdk2 and cdk5. Eur J Biochem 1997;243:527–536. 165. Vermeulen K, Strnad M, Krystof V, et al. Antiproliferative effect of plant cytokinin analogues with an inhibitory activity on cyclin-dependent kinases. Leukemia 2002;16:299–305. 166. Chang YT, Gray NS, Rosania GR, et al. Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors. Chem Biol 1999;6:361–375. 167. Dai Y, Dent P, Grant S. Induction of apoptosis in human leukemia cells by the CDK1 inhibitor CGP74514A. Cell Cycle 2002;1:143–152. 168. McClue SJ, Blake D, Clarke R, et al. In vitro and in vivo antitumor properties of the cyclin dependent kinase inhibitor CYC202 (R-roscovitine). Int J Cancer 2002;102:463–468.

Chapter 3 / PKC and CDK Inhibitors in Leukemia

87

169. de Vente J, Kiley S, Garris T, et al. Phorbol ester treatment of U937 cells with altered protein kinase C content and distribution induces cell death rather than differentiation. Cell Growth Differ 1995;6:371–382. 170. Wang Z, Su ZZ, Fisher PB, Wang S, VanTuyle G, Grant S. Evidence of a functional role for the cyclin-dependent kinase inhibitor p21(WAF1/CIP1/MDA6) in the reciprocal regulation of PKC activator-induced apoptosis and differentiation in human myelomonocytic leukemia cells. Exp Cell Res 1998;244:105–116. 171. Freytag SO. Enforced expression of the c-myc oncogene inhibits cell differentiation by precluding entry into a distinct predifferentiation state in G0/G1. Mol Cell Biol 1988; 8:1614– 1624. 172. Lee HR, Chang TH, Tebalt MJ 3rd, Senderowicz AM, Szabo E. Induction of differentiation accompanies inhibition of Cdk2 in a non–small cell lung cancer cell line. Int J Oncol 1999; 15:161–166. 173. Cartee L, Wang Z, Decker RH, et al. Synergistic induction of apoptosis in human myeloid leukemia cells by phorbol 12-myristate 13-acetate and flavopiridol proceeds via activation of both the intrinsic and tumor necrosis factor-mediated extrinsic cell death pathways. Mol Pharmacol 2002;61:1313–1321. 174. Cartee L, Smith R, Dai Y, et al. Synergistic induction of apoptosis in human myeloid leukemia cells by phorbol 12-myristate 13-acetate and flavopiridol proceeds via activation of both the intrinsic and tumor necrosis factor-mediated extrinsic cell death pathways. Mol Pharmacol 2002;61:1313–1321. 175. Cartee L, Maggio SC, Smith R, Sankala HM, Dent P, Grant S. Protein kinase C-dependent activation of the TNF receptor-mediated extrinsic cell death pathway is critical for the synergistic induction of apoptosis by bryostatin 1 and flavopiridol in human myeloid leukemia cells (U937 and HL-60). Mol Cancer Ther 2003;2(1):83–93. 176. Rahmani M, Grant S. UCN-01 (7-hydroxystauorsporine) blocks PMA-induced maturation and reciprocally promotes apoptosis in human myelomonocytic leukemia cells (U937). Cell Cycle 2002;1:273–281. 177. Sax JK, Dash BC, Hong R, Dicker DT, El-Deiry WS. The cyclin-dependent kinase inhibitor butyrolactone is a potent inhibitor of p21 (WAF1/CIP1 expression). Cell Cycle 2002;1:90–96. 178. Dai Y, Yu C, Singh V, et al. Pharmacological inhibitors of the mitogen-activated protein kinase (MAPK) kinase/MAPK cascade interact synergistically with UCN-01 to induce mitochondrial dysfunction and apoptosis in human leukemia cells. Cancer Res 2001; 61:5106–5115. 179. Dai Y, Landowski TH, Rosen ST, Dent P, Grant S. Combined treatment with the checkpoint abrogator UCN-01 and MEK1/2 inhibitors potently induces apoptosis in drug-sensitive and resistant myeloma cells through an IL-6-independent mechanism. Blood 2002;100:3333–3343.

Chapter 4 / CAI in Cancer Therapy

4

89

Carboxyamidotriazole, an Inhibitor of Nonvoltage-Operated Calcium Entry Single-Agent and Combination Therapy for Ovarian Carcinoma

Chad M. Michener, MD and Elise C. Kohn, MD CONTENTS INTRODUCTION THE PLEITROPIC ROLE OF CALCIUM IN CANCER CAI-INDUCED ALTERATIONS IN CELLULAR FUNCTION ON ANGIOGENESIS AND CAI CLINICAL APPLICATION OF CAI IN HUMAN CANCERS

INTRODUCTION Conventional cytotoxic chemotherapy has reached a critical point in cancer therapy. While marked improvements in treatment outcome and remission frequency have been made, improvement in cure rate for advanced-stage cancers remains unchanged. This sobering statistic has led investigators to search for novel targets and intervention combinations for the prevention and treatment of cancer. The advent of combination carboplatin/paclitaxel for the adjuvant treatment of ovarian cancer has led to improved response rates and prolonged survival (1). However, over 60% of patients relapse, and the 5-yr survival rate is still below 50%. Biochemical and biological mechanisms through which to intervene in the progression and dissemination of ovarian cancer are now being developed and studied clinically. An active approach for ovarian cancer and other solid tumors From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

89

90

Michener and Kohn

has been to limit the angiogenic and invasive events that lead to persistent and metastatic disease through targeted intervention. This molecular therapeutics direction has focused attention back to the etiology and biology of the tumors and their host environments, and has moved drug development away from the generally toxic approaches of chemotherapy, where injury is indiscriminate against the nuclear synthetic function of any cell unfortunate enough to be replicating. Molecular targets may occur at any point of intervention that is selectively functional in the cell of interest under the circumstances of tumor behavior. This may include the activation of proliferative and invasive behavior of vascular endothelium, activation of stromal fibroblasts during matrix remodeling, and/or the tumor cell functions themselves. Molecular therapeutics may re-regulate these altered events, resulting in a reduction in activity, or by interfering in a pathway may result in cellular apoptosis. The former approach may be cytostatic or invasostatic, and the latter could result in measurable reduction in tumor bulk. Our focus on invasostasis led to the identification of carboxyamidotriazole (CAI) as one agent that shows antiproliferative, antiinvasive, and antiangiogenic properties in vitro and in vivo at physiologically attainable concentrations reached with minimal toxicity. Further, early data suggest that some chemotherapeutic agents combine additively or synergistically with molecular targeted agents such as CAI.

THE PLEITROPIC ROLE OF CALCIUM IN CANCER The Local Tumor Microenvironment Many of the cell’s signaling mechanisms important for normal physiological processes have been shown to be perturbed in cancer cells (2). Alterations of these normal signaling mechanisms lead to changes in the way cells interact with one another as well as their surrounding microenvironment (3). It is this crosstalk between tumor cells and the surrounding stroma that causes dysregulation of processes important for the checks and balances that maintain normal tissue architecture and prevent aberrant growth of a single population of cells. Progression of cancer from a locally contained to a disseminated disease is based on the ability of tumor cells to acquire a migratory and invasive phenotype. Alteration of the normal tissue’s signaling milieu begins with release of a variety of regulatory molecules, either from the tumor cells themselves or from inflammatory and endothelial cells that have been recruited to the tumor–host interface (3–7). Communication between the tumor cells and these activated cell types is responsible for the release of numerous growth factors that are able to stimulate a variety of downstream signaling events. Calcium has been shown to be an important secondary mediator of many of these events within the cell, either through direct stimulation of calcium influx or internal release, regulation of

Chapter 4 / CAI in Cancer Therapy

91

phosphorylation or other posttranslational modifications of downstream proteins, or as a ligand itself.

G Proteins and Calcium Channels Regulate Cytoplasmic Calcium Concentration The first steps in tumor-cell dissemination are adhesion to and proteolysis of the basement membrane in order to establish a conduit for tumor-cell access to the lymph-vascular channels (8–11). It is within this context that carboxyamidotriazole (CAI) was discovered as a potential mediator of tumor-cell adhesion and migration from an array of compounds tested. It has been shown that one mechanism of tumor-cell adhesion and spreading is regulation through a guanine (G) nucleotide-binding, protein-dependent, receptor-stimulated mechanism following stimulation with autotaxin in more than 20 cell lines screened (10,12–16). G protein receptors are known to control several downstream signaling events, including generation of cyclic nucleotides, release of arachadonic acid metabolites, an alteration in calcium ion homeostasis (17–19), and secondary crosstalk to growth-factor receptors (20,21). Several mechanisms exist by which G protein activation causes these alterations in cell function. First, activation of the G protein-coupled receptor can activate phospholipase C-`, resulting in hydrolysis of phosphatidylinositol bisphosphate (PIP2) into inositol trisphosphate (IP3) and diacylglycerol (DAG). IP3 binds to its endoplasmic reticulum receptor and causes release of free calcium ion from intracellular stores. This process also creates a signal to the cell to refill the ER stores, which causes a secondary opening of depletion-operated calcium channels (also known as store-operated or capacitative calcium channels) at the cell surface and an influx of calcium (22). Classic and novel subclasses of protein kinase C (PKC) are activated by DAG; the former also require calcium for activation. PKC activity can secondarily regulate intracellular calcium balance, and is important in the metastatic process (23–25). Finally, activation of the G protein receptor complex can directly alter the state of calcium channels causing an influx of calcium into the cell in the form of receptoroperated calcium channels. Increases in cytoplasmic calcium through these mechanisms allow amplification of message caused by G protein stimulation.

Azoles and the Regulation of Intracellular Calcium The use of imidazole anti-mycotics in patients with various cancers provided additional insight into the importance of calcium in carcinogenesis. CAI was the first agent to link this class of compounds with calcium influx regulation of proliferation, metastasis, and cancer progression (26). Further support for imidazole compound regulation of intracellular calcium was provided by AlonsoTorre et al. (27). They showed that induction of intracellular calcium release and

92

Michener and Kohn

calcium uptake following treatment of human leukemic cells with dimethyl sulfoxide could be blocked by econazole and itraconazole. Other reports have shown that imidazole compounds are able to reduce calcium-stimulated calcium and potassium influx in erythrocytes, neutrophils, platelets, and T-lymphocytes (28– 34). The mechanism of inhibition of tumor-cell proliferation and calcium regulation was initially felt to be owing to inhibition of cytochrome P-450. However, a report from Walter Reed Medical Center showed that azole-induced inhibition of intracellular calcium changes could not be mimicked by inhibitors of the P450 enzyme system that were structurally dissimilar to the azole compounds (29). Najid and Ratinaud first identified direct effects of imidazoles on human cancer cells by showing that econazole and ketoconazole were able to reduce proliferation, DNA synthesis, and interrupt the cell cycle in MCF-7 breast-cancer cells in vitro (35). Similar inhibition of proliferation and cytokine secretion were seen in leukemic blast cells and T-lymphocytes treated with various azole compounds in vitro (36). Following the lead of the CAI story, clotrimazole was shown to regulate calcium homeostasis and cause a reduction in metastatic activity (37). An MMRU melanoma nude mouse xenograft was used to show an inhibition of mitogeninduced intracellular calcium increases following treatment with clotrimazole. A >70% reduction in pulmonary metastases was identified following treatment of these animals with subcutaneous clotrimazole. Zhang et al. subsequently showed that econazole was able to reduce proliferation of two breast-cancer cell lines in vitro and caused preferential cell death of the breast-cancer cell lines over that of human bone-marrow progenitor cells (38). Breast-cancer cell kill was enhanced by stimulation of cells with epidermal growth factor. The potential ability to isolate pure populations of blood-cell precursors in patients with circulating tumor cells provides us with a novel clinical application for molecules that function as inhibitors of calcium flux in human cancer cells. Suggestions of the importance of these compounds and this pathway also come from clinical observations. The use of imidazole compounds such as econazole, miconazole, and ketoconazole for antifungal prophylaxis and/or therapy in neutropenic patients receiving systemic chemotherapy caused an increase in toxicities normally associated with certain cytotoxic compounds. For example, children undergoing concomitant treatment with vincristine for leukemia or lymphoma and itraconazole for antifungal prophylaxis were shown to have more significant neurotoxicity than expected (39). Based on these small case reports it has been recommended that these two drugs not be used concomitantly in this setting. However, researchers have also found beneficial links between imidazole compounds and suppression of tumor-promoting hormones (40), inhibition of G protein/adenylate cyclase-directed release of anterior pituitary hormones (41), and potential reversal of daunorubicin resistance in murine leukemia cells (42).

Chapter 4 / CAI in Cancer Therapy

93

CAI-INDUCED ALTERATIONS IN CELLULAR FUNCTION CAI Inhibits Proliferation, Adhesion, and Migration Through Inhibition of Calcium Mobilization The link of CAI to calcium first occurred while confirming antiproliferative and antimetastatic properties of CAI in several cell lines in vitro and OVCAR3 cells in vivo (26). In this set of studies, CAI was shown to reversibly inhibit adhesion to plastic culture plates and migration to crude autotaxin preparation through collagen IV-coated membranes. Early mechanistic studies showed that CAI treatment reduced the hydrolysis of PIP2 to IP3 in response to crude autotaxin preparation in the melanoma cells. It was hypothesized that these effects were, at least partially, regulated through a G protein-mediated receptor-activated mechanism owing to the involvement of phospholipase C-` (Fig. 1). The G protein-coupled m5 muscarinic receptor was used by Felder et al. to characterize the effects of CAI on calcium balance. Here it was shown that CAI specifically inhibited mobilization of calcium through calcium influx, and that this blockade or reduction in calcium influx was responsible for inhibition of release of arachadonic acid (43). Importantly, the background cell for these studies, the Chinese hamster ovary line (CHO) that had been transfected with the m5 receptor, is devoid of voltage-operated calcium channels, providing us with the first evidence that CAI affects calcium homeostasis through a nonvoltageoperated calcium entry (NVOCE) mechanism (43,44). We later showed that this receptor also stimulated activation of phospholipase C-a, generally activated by receptor tyrosine kinase-mediated phosphorylation and, in this model, requiring calcium influx (45). This was an early suggestion of crosstalk between these two receptor types. This effect was blocked by addition of CAI to the culture medium and could be duplicated by removal of calcium from the culture medium or pretreatment with the calcium chelator BAPTA. These experiments provided evidence that CAI functions via inhibition of calcium influx in a nonvoltagedependent mechanism and not by preventing mobilization of intracellular calcium stores. Structurally, CAI contains a triazole ring and a benzophenone tail (Fig. 2). Structure analysis revealed that both of these components are required for calcium signal inhibition (46). However, there is some flexibility allowed in the substitution of the side chains on the triazole ring with retention of intracellular inhibitory activity.

CAI Alters the Invasive Phenotype of Human Cancer Cells We have shown that treatment of tumor cells with CAI limits the ability of tumor cells to proliferate and migrate, two key components of the metastatic cascade. However, the ability of tumor cells to gain access to vascular channels required for dissemination of disease is dependent on the cells’ ability to degrade

Fig. 1. Effect of CAI on intracellular signaling pathways, cytokine secretion, and calcium homeostasis.

94 Michener and Kohn

Chapter 4 / CAI in Cancer Therapy

95

Fig. 2. Structure of carboxyamidotriazole. The triazole ring (upper left) and benzophenone tail are both required for signal transduction inhibition.

surrounding extracellular matrix (ECM). Matrix metalloproteinases (MMPs) have long been known to have the capacity to degrade various ECM components. CAI was shown to inhibit production of both latent and active forms of MMP2 (gelatinase A) in several cell lines in vitro (47). The importance of calcium in reduction of MMP-2 by CAI was underscored by duplication of these findings following treatment of cells with an unrelated inhibitor of calcium influx. Inhibition of tumor cell growth, attachment, invasion, and migration by CAI in a broad array of cancer cell types in vitro and in vivo demonstrates the potential clinical utility of this compound. However, the ability of disseminated tumor cells to survive in a foreign setting is also dependent on their ability to interact with their microenvironment, to set up and maintain a network of vascular channels necessary for tumor cell growth (3).

ON ANGIOGENESIS AND CAI CAI Alters Function of Endothelial Cells The ability of tumor cells to induce formation of new blood vessels is dependent on an entire cascade of events, including duplication, proliferation, migration, and tube-forming capacity of endothelial cells. The importance of calcium homeostasis in these events was demonstrated initially both in vitro and in vivo following treatment of human umbilical vein endothelial cells (HUVEC) with CAI (48). A reduction in HUVEC proliferation under serum and growth factorstimulated conditions was observed in culture. Further, adhesion to different

96

Michener and Kohn

basement membrane component proteins and migration to those proteins was inhibited in a dose-dependent fashion following treatment with various concentrations of CAI. Moreover, the ability of endothelial cells to attach and spread on type IV collagen, a known calcium-dependent process, was inhibited by CAI (49,50). These alterations were caused by inhibition of activation and phosphorylation of focal adhesion kinase and calcium influx-dependent blockade of RhoA activation of actin stress fiber production. In addition, a reduction in gelatinase activity was identified in HUVEC treated with basic fibroblast growth factor (48). CAI exposure also caused a reduction in HUVEC vascular tube formation in the Matrigel model, caused a decrease in neovessel outgrowth in rat aortic ring assays, and significantly limited in vivo de novo developmental angiogenesis in the chick chorioallantoic membrane assay (48,51). In oral administration to melanoma-bearing syngenic mice, CAI treatment reduced the size of hepatic metastases significantly and inhibited vascularization selectively in the areas of tumor neovascularization without affecting non-tumor-bearing regions of the liver (52). These effects on in vivo angiogenesis stimulated further laboratory evaluation of CAI effects on proangiogenic factors.

CAI Inhibits Proangiogenic Cytokine Production Initiation of the proangiogenic cascade of primary or metastatic tumors can be caused by several different factors in the surrounding microenvironment (3). Proangiogenic switches cause downstream upregulation of angiogenic cytokines such as interlukin (IL)-8 and vascular endothelial growth factor (VEGF) (53– 56). The stimulatory effect of VEGF on neovascularization has been shown in various models (57–59). Recent data show that CAI treatment in human melanoma xenografts caused a marked reduction in tumor size as well as marked reduction in concentration of circulating VEGF and IL-8 (59a). In vitro studies demonstrated that CAI treatment reduces gene expression and protein quantity of HIF-1_ and VEGF in the melanoma cells. HUVEC exposed to CAI did not migrate to VEGF, as did controls. This suggests that CAI exposures regulated the local microenvironment by reducing the capacity of the tumor cells to secrete VEGF, and also of the endothelial cells to respond to it. Further in vivo confirmation of this effect was seen when in vivo neovessel growth into Matrigel plugs impregnated with VEGF was significantly reduced by oral treatment with 100 mg/kg/d of CAI. This supports the hypothesis that calcium is important for the production and secretion of VEGF in vivo and confirms the importance of calcium flux on the regulation of VEGF production. Taken together, these data show that CAI plays an important role in the mediation of tumor vessel formation both in vitro and in vivo, and may be at least partially regulated through VEGF production and secretion.

Chapter 4 / CAI in Cancer Therapy

97

CLINICAL APPLICATION OF CAI IN HUMAN CANCERS Preclinical Safety and Efficacy In vivo studies with CAI demonstrated a reduction in tumor take, size, and metastases of melanoma and ovarian cancer in mice treated with CAI compared with vehicle controls (60). Pretreatment of rat embryo fibroblast and human colon cancer cell lines with 20 μM CAI resulted in more than a 90% reduction in pulmonary metastases following tail-vein injection of cell lines. Study of tumor explants revealed necrosis and fewer vessels within the tumors, consistent with previous data. Treatment of mice with 100–200 mg/kg of oral CAI yielded CAI plasma concentrations of 1–10 μg/mL (2–20 μM), a range shown to be biologically relevant in vitro. Furthermore, microscopic examination of major organs showed that these levels were achieved with no physiologic consequences. Detailed preclinical IND-directed toxicity studies demonstrated vomiting and neurologic toxicity in dogs undergoing twice-daily dosing.

Dosage and Distribution Initial CAI phase I studies evaluated three oral formulations and two dosing schemas. Comparison of liquid and liquid gelatin capsule preparations identified greater bioavailability, shorter time to peak concentration, and almost twofold higher peak concentrations following a single oral dose of the gelcap preparation (61). CAI was shown to be 99.6% protein bound with a half-life of 11 h and a volume of distribution of 252 L. More variability between peak and trough concentrations was noted for patients receiving 330–400 mg/kg every other day compared to patients receiving daily doses at 100–300 mg/kg. However, all trough levels with both dosing regimens remained within the biologically active target range. Metabolism of CAI occurs mainly via hepatic pathways and is partly achieved through the cytochrome P-450 route (62). A history of hepatic disease and concomitant administration of drugs affecting the cytochrome P-450 pathway may necessitate dose adjustments in these patients. In addition, absorption of CAI is affected by concomitant intake of food. Plasma concentrations were found to be up to 2.6-fold higher in patients taking CAI with or in close proximity to meals (51,63). We therefore recommend that CAI be administered in the fasting state first thing in the morning or at a reduced dose in the evening after several hours of fasting after dinner. Dose-finding studies using the liquid and gelcap preparations identified doselimiting gastrointestinal and neurologic toxicities at 150 mg/m2/d. Gastrointestinal side effects were more common in patients taking the gelcap form, even after changing to bedtime administration. Complaints of adverse taste and viscosity of the liquid preparation led to development of a micronized powder capsule, which has a slower rate of absorption and lower bioavailability than the

98

Michener and Kohn

former liquid suspension (64). Peak concentrations were again in the targeted range, but lower milligram for milligram than for the liquid preparation. A phase I dose-finding study was completed using micronized CAI, with disease stability rates comparable to those seen with the liquid gelcap formulation and a better toxicity and side-effect profile (64). Recent studies have focused on treatment with the micronized preparation due to improved patient tolerability and similar response rates in the treatment of patients with refractory solid tumors.

Side Effects and Toxicity Profile Early trials with CAI in liquid or gelatin capsule formulations had dose-limiting toxicity of nausea and vomiting. Fatigue toxicity was much less frequent and less significant than gastrointestinal side effects (Table 1). Hematological toxicity was rare and usually mild. Peripheral neuropathy was expected, based on preclinical toxicity monitoring studies, but was rare with the micronized formulation. Central neurologic symptoms such as gait disturbances and seizures seen in preclinical dog studies were not observed during phase I trials at doses under 300 mg/m2/d (64). However, one patient receiving a micronized dose of 350 mg/m2/d developed cerebellar ataxia after 1 wk of therapy. These symptoms abated following discontinuation of the drug for several days, and she was able to tolerate a dose reduction to 300 mg/m2/d for several additional months of therapy. In a phase I trial at the University of Wisconsin, two patients developed reversible vision loss during treatment with micronized CAI at 300 mg/m2/d; one patient was rechallenged and had recurrence of the vision loss. No correlation of toxicities and plasma levels of CAI could be identified, even though CNS side effects were noted only at higher dosing levels (64). The use of oral CAI appears to be well tolerated, with mostly minor toxicities and only rare grade 3 or 4 side effects.

Tumor Response With Single-Agent CAI The NCI phase I dose-finding trial included 49 patients with a wide range of solid tumors who underwent treatment with either liquid or gelcap formulation (65). Whereas no patient had a partial or complete response to this therapy, 49% had disease stabilization and improvement in performance status for periods of 1–7 mo. Seven patients discontinued therapy solely due to unacceptable side effects, and only one patient each developed grade 3/4 peripheral neuropathy or myelosuppression. Disease stabilization was seen in 2 of 10 ovarian cancer patients, as well as in patients with renal carcinoma, colon cancer, pancreatobiliary cancer, non-small-cell lung cancer, and melanoma. A follow-up study using micronized CAI demonstrated a 47% disease stabilization rate, with less significant toxicity than observed for the liquid preparations with no patient withdrawals (64). An additional phase I study by Berlin et al. achieved a disease stabilization rate of approx 20% for 4–43 cycles of therapy using various doses

Chapter 4 / CAI in Cancer Therapy

99

Table 1 Common Toxicities of Carboxyamidotriazole in Clinical Trials Toxicity

Grade

Kohn (64)

Hussain (66)

Berlin (63)

Kohn (73)

Nausea

1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3 1 2 3

48 10 0 10 5 0 29 5 0 29 5 0 33 0 0 5 10 5 NR NR 0 0 10 0

NR 12 0 NR 6 3 NR 9 0 NR 6 6 NR NR NR NR NR NR NR 3 0 NR 3 6

39 21 4 32 11 4* 14 0 0 NR NR NR 39 11 4 NE NE NE 0 4 0 18 14 0

31 2 0 12 6 0 13 4 0 16 13 1 21 2 1 13 0 0 19 9 1 14 2 0

Vomiting

Anorexia

Fatigue

Peripheral neuropathy CNS toxicity

Anemia

Neutropenia

Percentage of patients with each toxicity is listed. All dose levels are combined for studies where there was a dose escalation (62,72). NR = not recorded. NE = Not evaluable due to multiple central nervous system toxicities listed. * = grade 4 toxicity.

of micronized CAI between 150 and 300 mg/m2/d (63). Recommendations from this dose-finding study were for use of a standard CAI dose of 250 mg at bedtime to limit untoward side effects while maintaining serum levels in the 3- to 10-μM range. Experience using CAI in the treatment of ovarian cancer patients has been limited to the patients accrued in the phase I studies and a recent phase II study at the National Cancer Institute. In the latter study, 39 women with recurrent ovarian cancer have been treated to date with daily oral CAI at the fasting morning starting dose of 250 mg/m2 (66). Thirty percent of patients attained disease stabilization for at least 6 mo and there was one partial response and two minor responses. This compares favorably with therapy for recurrent disease with other

100

Michener and Kohn

second-line chemotherapy agents, but with a much reduced toxicity frequency (67–72). This study is ongoing and has been linked to evaluation of potential surrogate molecular markers of treatment such as serum MMP-2, VEGF, and IL8 concentrations. The activity of CAI in phase II studies for ovarian cancer supports pursuit of this novel molecularly targeted compound for future studies.

CAI and Conventional Cytotoxic Agents As Combination Therapy The use of small-molecule signal transduction inhibitors in conjunction with cytotoxic chemotherapy agents is an attractive paradigm for the application of these new, molecularly targeted therapeutics. Current second-line therapy for the treatment of recurrent ovarian cancer has limited efficacy, with overall response rates ranging from 10 to 38% and progression-free intervals of 3 to 6 mo (67–72). Choosing two different classes of drugs with different mechanisms of action is a logical approach to optimizing the use of these agents in order to achieve a synergistic effect in vivo. CAI, a cytostatic signal transduction inhibitor, was combined with chemotherapy to maximize the differential target mix. The combination of CAI and carboplatin was the first combination to be tested in vitro in ovarian cancer cell lines, since platinum agents remain the cornerstone of chemotherapy for ovarian cancer patients. This combination demonstrated an unexpected antagonistic effect in vitro, with a loss of efficacy of carboplatin both in sensitive and resistant ovarian cancer cell lines (73). Paclitaxel has been used for primary therapy of ovarian cancer patients since the mid-1990s and is also used for treatment of recurrent disease (74–77). Although it inhibits mitosis through promotion and stabilization of polymerized microtubules, paclitaxel has also been shown to possess anti-angiogenic activity in vitro at concentrations below the common therapeutic schedules for the treatment of ovarian cancer. CAI and paclitaxel were investigated in colony-forming assays in platinum-sensitive and -resistant ovarian cancer cell lines in vitro after the determination of the inhibitory concentration for 10% of cells for paclitaxel (73). CAI markedly improved the growth-inhibitory activity when mixed with the IC10 dose of paclitaxel. Although not statistically different, the schedule of CAI prior to paclitaxel was preferred. This schedule was modeled in nude mice and was found not to have untoward toxicity, with no evidence of additive or synergistic end-organ toxicity. In unpublished work, CAI did not augment the myelosuppressive effects of paclitaxel in colony-forming assays in vitro. Exposure of a paclitaxel-resistant cell line to this combination of drugs did not reverse the paclitaxel resistance and resulted in a paradoxical increase in mean inhibitory concentration for CAI. An initial cohort of patients was treated with the combination of daily oral CAI with a 3-h infusion of paclitaxel (d 8) (73). The dose escalation study demonstrated modest activity in patients with a variety of recurrent solid tumors. Three partial responses, 2 minor responses, and 17 patients with disease stabilization

Chapter 4 / CAI in Cancer Therapy

101

were seen in this group (n = 39), with no clear dose–response relationship. There was a concern at the initiation of the study that peripheral neuropathy may be a dose-limiting toxicity for this combination, since both drugs are associated with peripheral neuropathy when used individually. However, neurotoxicity was neither additive nor common in this study. The adverse event seen most commonly was chemotherapy-associated neutropenia, at paclitaxel doses of 200 mg/m2 or greater, and particularly when combined with the higher doses of micronized CAI. Interestingly, pharmacokinetic studies demonstrated that paclitaxel administration caused a dose-dependent increase in CAI plasma concentration. No dose-limiting toxicity was observed, and accrual to this schedule was discontinued when the maximal safe administered doses of either of the two agents alone or in combination was reached: CAI 250 mg/m2/d fasting in the morning with paclitaxel 250 mg/ m2 over 3 h every 3 wk. Granulocyte colony-stimulating factor (G-CSF) was used in most of the high-dose patients owing to the neutropenia caused by paclitaxel. A cohort is currently accruing to test daily administration of oral CAI with paclitaxel begun at 1 wk and continued every 3 wk. No adverse dose-limiting events have been observed, and the use of G-CSF has been limited at CAI doses of 250 mg daily at bedtime with 250 mg/m2 paclitaxel over 3 h every 3 wk. Numerous partial responses in ovarian cancer have been seen, along with a minor response in cervical cancer and stable disease in renal-cell carcinoma. Although early in clinical trials, these data suggest a possible increase in therapeutic efficacy when CAI is combined with conventional cytotoxic drugs. Further investigation of additional combination regimens may elucidate even more effective combinations for treatment of recurrent ovarian cancer and lead us to the best approach to using CAI, a signal transduction modulator.

CONCLUSIONS CAI is a novel compound for the treatment of ovarian cancer and other solid tumors. Preclinical analysis of CAI activity in vitro confirmed the antiproliferative, antimigratory, and antiinvasive activity of this signal-transduction agent. Alteration of calcium-related signal-transduction pathways following treatment with CAI appears to play an important role in carcinogenesis. Available data suggest that these changes in cellular function translate to a clinical difference in disease progression. Data from phase II studies suggest that CAI limits disease progression and may cause regression of disease in some patients with recurrent ovarian cancer. Treatment with oral CAI is convenient and well tolerated in patients who are often already heavily treated with cytotoxic chemotherapy. The favorable toxicity profile of CAI lends itself to the potential for prolonged therapy with little consequence. Continued investigation of CAI as a biological mediator may uncover novel and improved therapeutic regimens giving patients and physicians new options for the treatment of ovarian cancer.

102

Michener and Kohn

REFERENCES 1. McGuire WP, Hoskins WJ, Brady MF, et al. Cyclophosphamide and cisplatin compared with paclitaxel and cisplatin in patients with stage III and stage IV ovarian cancer. N Engl J Med 1996;334:1–6. 2. Hanahan D, Weinberg RA. The hallmarks of cancer. Cell 2000;100:57–70. 3. Liotta LA, Kohn EC. The microenvironment of the tumour-host interface. Nature 2001;411:375–379. 4. Park CC, Bissell MJ, Barcellos-Hoff MH. The influence of the microenvironment on the malignant phenotype. Mol Med Today 2000;6:324–329. 5. Roberts AB, Lamb LC, Newton DL, Sporn MB, De Larco JE, Todaro GJ. Transforming growth factors: isolation of polypeptides from virally and chemically transformed cells by acid/ethanol extraction. Proc Natl Acad Sci USA 1980;77:3494–3498. 6. Silletti S, Raz A. Regulation of autocrine motility factor receptor expression in tumor cell locomotion and metastasis. Curr Top Microbiol Immunol 1996;213(Pt 2):137–169. 7. Elenbaas B, Weinberg RA. Heterotypic signaling between epithelial tumor cells and fibroblasts in carcinoma formation. Exp Cell Res 2001;264:169–184. 8. Kohn EC, Liotta LA, Schiffmann E. Autocrine motility factor stimulates a three-fold increase in inositol trisphosphate in human melanoma cells. Biochem Biophys Res Commun 1990;166:757–764. 9. Liotta LA. Tumor invasion and metastases—role of the extracellular matrix: Rhoads Memorial Award lecture. Cancer Res 1986;46:1–7. 10. Liotta LA, Mandler R, Murano G, et al. Tumor cell autocrine motility factor. Proc Natl Acad Sci USA 1986;83:3302–3306. 11. Aznavoorian S, Stracke ML, Krutzsch H, Schiffmann E, Liotta LA. Signal transduction for chemotaxis and haptotaxis by matrix molecules in tumor cells. J Cell Biol 1990;110:1427–1438. 12. Stracke ML, Krutzsch HC, Unsworth EJ, et al. Identification, purification, and partial sequence analysis of autotaxin, a novel motility-stimulating protein. J Biol Chem 1992;267:2524–2529. 13. Stracke M, Liotta LA, Schiffmann E. The role of autotaxin and other motility stimulating factors in the regulation of tumor cell motility. Symp Soc Exp Biol 1993;47:197–214. 14. Nam SW, Clair T, Kim YS, et al. Autotaxin (NPP-2), a metastasis-enhancing motogen, is an angiogenic factor. Cancer Res 2001;61:6938–6944. 15. Nam SW, Clair T, Campo CK, Lee HY, Liotta LA, Stracke ML. Autotaxin (ATX), a potent tumor motogen, augments invasive and metastatic potential of ras-transformed cells. Oncogene 2000;19:241–247. 16. Clair T, Lee HY, Liotta LA, Stracke ML. Autotaxin is an exoenzyme possessing 5'nucleotide phosphodiesterase/ATP pyrophosphatase and ATPase activities. J Biol Chem 1997;272:996–1001. 17. Burch RM, Luini A, Axelrod J. Phospholipase A2 and phospholipase C are activated by distinct GTP-binding proteins in response to alpha 1-adrenergic stimulation in FRTL5 thyroid cells. Proc Natl Acad Sci USA 1986;83:7201–7205. 18. Berridge MJ, Heslop JP, Irvine RF, Brown KD. Inositol trisphosphate formation and calcium mobilization in Swiss 3T3 cells in response to platelet-derived growth factor. Biochem J 1984;222:195–201. 19. Berridge MJ. Calcium signalling and cell proliferation. Bioessays 1995;17:491–500. 20. Hernandez M, Barrero MJ, Crespo MS, Nieto ML. Lysophosphatidic acid inhibits Ca2+ signaling in response to epidermal growth factor receptor stimulation in human astrocytoma cells by a mechanism involving phospholipase C(gamma) and a G(alpha) protein. J Neurochem 2000;75:1575–1582.

Chapter 4 / CAI in Cancer Therapy

103

21. Wu J, Cunnick JM. Trans-regulation of epidermal growth factor receptor by lysophosphatidic acid and G protein-coupled receptors. Biochim Biophys Acta 2002;1582:100–106. 22. Putney JW, Jr. A model for receptor-regulated calcium entry. Cell Calcium 1986;7:1–12. 23. Schwartz GK, Redwood SM, Ohnuma T, Holland JF, Droller MJ, Liu BC. Inhibition of invasion of invasive human bladder carcinoma cells by protein kinase C inhibitor staurosporine. J Natl Cancer Inst 1990;82:1753–1756. 24. Schwartz GK, Jiang J, Kelsen D, Albino AP. Protein kinase C: a novel target for inhibiting gastric cancer cell invasion. J Natl Cancer Inst 1993;85:402–407. 25. Schwartz GK, Haimovitz-Friedman A, Dhupar SK, et al. Potentiation of apoptosis by treatment with the protein kinase C-specific inhibitor safingol in mitomycin C-treated gastric cancer cells. J Natl Cancer Inst 1995;87:1394–1399. 26. Kohn EC, Liotta LA. L651582: a novel antiproliferative and antimetastasis agent. J Natl Cancer Inst 1990;82:54–60. 27. Alonso-Torre SR, Alvarez J, Montero M, Sanchez A, Garcia-Sancho J. Control of Ca2+ entry into HL60 and U937 human leukaemia cells by the filling state of the intracellular Ca2+ stores. Biochem J 1993;289 ( Pt 3):761–766. 28. Villalobos C, Fonteriz R, Lopez MG, Garcia AG, Garcia-Sancho J. Inhibition of voltage-gated Ca2+ entry into GH3 and chromaffin cells by imidazole antimycotics and other cytochrome P450 blockers. FASEB J 1992;6:2742–2747. 29. Hornstein EH, Vassilopoulos D, Thomas DE, Friedman FK, Tsokos GC. Modulation of human T-lymphocyte plasma membrane Ca2+ permeability by imidazole antimycotics. Immunopharmacol Immunotoxicol 1996;18:237–245. 30. Coupry I, Armsby CC, Alper SL, Brugnara C, Parini A. Clotrimazole and efaroxan inhibit red cell Gardos channel independently of imidazoline I1 and I2 binding sites. Eur J Pharmacol 1996;295:109–112. 31. Brugnara C, Armsby CC, Sakamoto M, Rifai N, Alper SL, Platt O. Oral administration of clotrimazole and blockade of human erythrocyte Ca(++)-activated K+ channel: the imidazole ring is not required for inhibitory activity. J Pharmacol Exp Ther 1995;273:266–272. 32. Brugnara C, De Franceschi L, Armsby CC, et al. A new therapeutic approach for sickle cell disease. Blockade of the red cell Ca(2+)-activated K+ channel by clotrimazole. Ann N Y Acad Sci 1995;763:262–271. 33. Montero M, Garcia-Sancho J, Alvarez J. Comparative effects of cytochrome P-450 inhibitors on Ca2+ and Mn2+ entry induced by agonists or by emptying the Ca2+ stores of human neutrophils. Biochim Biophys Acta 1993;1177:127–133. 34. Sargeant P, Clarkson WD, Sage SO, Heemskerk JW. Calcium influx evoked by Ca2+ store depletion in human platelets is more susceptible to cytochrome P-450 inhibitors than receptormediated calcium entry. Cell Calcium 1992;13:553–564. 35. Najid A, Ratinaud MH. Comparative studies of steroidogenesis inhibitors (econazole, ketoconazole) on human breast cancer MCF-7 cell proliferation by growth experiments, thymidine incorporation and flow cytometric DNA analysis. Tumori 1991;77:385–390. 36. Bruserud O. Effects of azoles on human acute myelogenous leukemia blasts and T lymphocytes derived from acute leukemia patients with chemotherapy-induced cytopenia. Int Immunopharmacol 2001;1:2183–2195. 37. Benzaquen LR, Brugnara C, Byers HR, Gatton-Celli S, Halperin JA. Clotrimazole inhibits cell proliferation in vitro and in vivo. Nat Med 1995;1:534–540. 38. Zhang Y, Crump M, Berge SA. Purging of contaminating breast cancer cells from hematopoietic progenitor cell preparations using activation enhanced cell death. Breast Cancer Res Treat 2002;72:265–278. 39. Kamaluddin M, McNally P, Breatnach F, et al. Potentiation of vincristine toxicity by itraconazole in children with lymphoid malignancies. Acta Paediatr 2001;90:1204–1207.

104

Michener and Kohn

40. Ayub M, Levell MJ. Inhibition of testicular 17 alpha-hydroxylase and 17,20-lyase but not 3 beta-hydroxysteroid dehydrogenase-isomerase or 17 beta-hydroxysteroid oxidoreductase by ketoconazole and other imidazole drugs. J Steroid Biochem 1987;28:521–531. 41. Stalla GK, Stalla J, von Werder K, et al. Nitroimidazole derivatives inhibit anterior pituitary cell function apparently by a direct effect on the catalytic subunit of the adenylate cyclase holoenzyme. Endocrinology 1989;125:699–706. 42. Gupta S, Kim J, Gollapudi S. Reversal of daunorubicin resistance in P388/ADR cells by itraconazole. J Clin Invest 1991;87:1467–1469. 43. Felder CC, Ma AL, Liotta LA, Kohn EC. The antiproliferative and antimetastatic compound L651582 inhibits muscarinic acetylcholine receptor-stimulated calcium influx and arachidonic acid release. J Pharmacol Exp Ther 1991;257:967–971. 44. Felder CC, MacArthur L, Ma AL, Gusovsky F, Kohn EC. Tumor-suppressor function of muscarinic acetylcholine receptors is associated with activation of receptor-operated calcium influx. Proc Natl Acad Sci USA 1993;90:1706–1710. 45. Gusovsky F, Lueders JE, Kohn EC, Felder CC. Muscarinic receptor-mediated tyrosine phosphorylation of phospholipase C-gamma. An alternative mechanism for cholinergic-induced phosphoinositide breakdown. J Biol Chem 1993;268:7768–7772. 46. Kohn EC, Felder CC, Jacobs W, et al. Structure-function analysis of signal and growth inhibition by carboxyamido-triazole, CAI. Cancer Res 1994;54:935–942. 47. Kohn EC, Jacobs W, Kim YS, Alessandro R, Stetler-Stevenson WG, Liotta LA. Calcium influx modulates expression of matrix metalloproteinase-2 (72- kDa type IV collagenase, gelatinase A). J Biol Chem 1994;269:21,505–21,511. 48. Kohn EC, Alessandro R, Spoonster J, Wersto RP, Liotta LA. Angiogenesis: role of calciummediated signal transduction. Proc Natl Acad Sci USA 1995;92:1307–1311. 49. Alessandro R, Masiero L, Lapidos K, Spoonster J, Kohn EC. Endothelial cell spreading on type IV collagen and spreading-induced FAK phosphorylation is regulated by Ca2+ influx. Biochem Biophys Res Commun 1998;248:635–640. 50. Masiero L, Lapidos KA, Ambudkar I, Kohn EC. Regulation of the RhoA pathway in human endothelial cell spreading on type IV collagen: role of calcium influx. J Cell Sci 1999;112:3205–3213. 51. Bauer KS, Figg WD, Hamilton JM, et al. A pharmacokinetically guided Phase II study of carboxyamido-triazole in androgen-independent prostate cancer. Clin Cancer Res 1999;5:2324–2329. 52. Luzzi KJ, Varghese HJ, MacDonald IC, et al. Inhibition of Angiogenesis in Liver Metastases by Carboxyamidotriazole (CAI): Computer Assisted Analysis. Angiogenesis 1999;2:373–379. 53. Folkman J, Watson K, Ingber D, Hanahan D. Induction of angiogenesis during the transition from hyperplasia to neoplasia. Nature 1989;339:58–61. 54. Folkman J, Hanahan D. Switch to the angiogenic phenotype during tumorigenesis. Princess Takamatsu Symp 1991;22:339–347. 55. Hanahan D. Signaling vascular morphogenesis and maintenance. Science 1997;277:48–50. 56. Benjamin LE, Keshet E. Conditional switching of vascular endothelial growth factor (VEGF) expression in tumors: induction of endothelial cell shedding and regression of hemangioblastoma-like vessels by VEGF withdrawal. Proc Natl Acad Sci USA 1997;94:8761–8766. 57. Neufeld G, Cohen T, Gengrinovitch S, Poltorak Z. Vascular endothelial growth factor (VEGF) and its receptors. Faseb J 1999;13:9–22. 58. Wellner M, Maasch C, Kupprion C, Lindschau C, Luft FC, Haller H. The proliferative effect of vascular endothelial growth factor requires protein kinase C-alpha and protein kinase Czeta. Arterioscler Thromb Vasc Biol 1999;19:178–185. 59. Mukhopadhyay D, Nagy JA, Manseau EJ, Dvorak HF. Vascular permeability factor/vascular endothelial growth factor-mediated signaling in mouse mesentery vascular endothelium. Cancer Res 1998;58:1278–1284.

Chapter 4 / CAI in Cancer Therapy

105

60. Kohn EC, Sandeen MA, Liotta LA. In vivo efficacy of a novel inhibitor of selected signal transduction pathways including calcium, arachidonate, and inositol phosphates. Cancer Res 1992;52:3208–3212. 61. Figg WD, Cole KA, Reed E, et al. Pharmacokinetics of orally administered carboxyamidotriazole, an inhibitor of calcium-mediated signal transduction. Clin Cancer Res 1995;1:797–803. 62. Soltis MJ, Yeh HJ, Cole KA, Whittaker N, Wersto RP, Kohn EC. Identification and characterization of human metabolites of CAI [5- amino-1-1(4'-chlorobenzoyl-3,5-dichlorobenzyl)1,2,3-triazole- 4- carboxamide). Drug Metab Dispos 1996;24:799–806. 63. Berlin J, Tutsch KD, Arzoomanian RZ, et al. Phase I and pharmacokinetic study of a micronized formulation of carboxyamidotriazole, a calcium signal transduction inhibitor: toxicity, bioavailability and the effect of food. Clin Cancer Res 2002;8:86–94. 64. Kohn EC, Figg WD, Sarosy GA, et al. Phase I trial of micronized formulation carboxyamidotriazole in patients with refractory solid tumors: pharmacokinetics, clinical outcome, and comparison of formulations. J Clin Oncol 1997;15:1985–1993. 65. Kohn EC, Reed E, Sarosy G, et al. Clinical investigation of a cytostatic calcium influx inhibitor in patients with refractory cancers. Cancer Res 1996;56:569–573. 66. Hussain M, Sarosy G, Kotz H, et al. A phase II trial of carboxyamidotriazole (CAI) In Patients with refractory eithelial ovarian cancer. Proceedings of the Association for Cancer Research, 93rd Annual Meeting, San Francisco, CA, 2002. 67. ten Bokkel Huinink W, Gore M, Carmichael J, et al. Topotecan versus paclitaxel for the treatment of recurrent epithelial ovarian cancer. J Clin Oncol 1997;15:2183–2193. 68. Rose PG, Blessing JA, Mayer AR, Homesley HD. Prolonged oral etoposide as second-line therapy for platinum-resistant and platinum-sensitive ovarian carcinoma: a Gynecologic Oncology Group study. J Clin Oncol 1998;16:405–410. 69. Rose PG, Fusco N, Fluellen L, Rodriguez M. Tolerance of twenty-four hour paclitaxel and carboplatin as first-line therapy in ovarian, peritoneal and fallopian tube carcinoma. Int J Gynecol Cancer 1999;9:448–451. 70. Lund B, Hansen OP, Theilade K, Hansen M, Neijt JP. Phase II study of gemcitabine (2',2'difluorodeoxycytidine) in previously treated ovarian cancer patients. J Natl Cancer Inst 1994;86:1530–1533. 71. Markman M, Hakes T, Reichman B, et al. Ifosfamide and mesna in previously treated advanced epithelial ovarian cancer: activity in platinum-resistant disease. J Clin Oncol 1992;10:243–248. 72. Muggia FM, Hainsworth JD, Jeffers S, et al. Phase II study of liposomal doxorubicin in refractory ovarian cancer: antitumor activity and toxicity modification by liposomal encapsulation. J Clin Oncol 1997;15:987–993. 73. Kohn EC, Reed E, Sarosy GA, et al. A phase I trial of carboxyamido-triazole and paclitaxel for relapsed solid tumors: potential efficacy of the combination and demonstration of pharmacokinetic interaction. Clin Cancer Res 2001;7:1600–1609. 74. Thigpen JT, Blessing JA, Ball H, Hummel SJ, Barrett RJ. Phase II trial of paclitaxel in patients with progressive ovarian carcinoma after platinum-based chemotherapy: a Gynecologic Oncology Group study. J Clin Oncol 1994;12:1748–1753. 75. Abu-Rustum NR, Aghajanian C, Barakat RR, Fennelly D, Shapiro F, Spriggs D. Salvage weekly paclitaxel in recurrent ovarian cancer. Semin Oncol 1997;24:S62–S67. 76. Uziely B, Groshen S, Jeffers S, et al. Paclitaxel (Taxol) in heavily pretreated ovarian cancer: antitumor activity and complications. Ann Oncol 1994;5:827–833. 77. Kohn EC, Sarosy G, Bicher A, et al. Dose-intense taxol: high response rate in patients with platinum-resistant recurrent ovarian cancer. J Natl Cancer Inst 1994;86:18–24.

Chapter 5 / Targeted F-Particle Therapy

5

107

Targeted F-Particle Therapy A Rational Approach to Drug Development in Hematological Diseases and Solid Tumors

John M. Burke, MD David A. Scheinberg, MD, PhD and Joseph G. Jurcic, MD CONTENTS INTRODUCTION RATIONALE FOR TARGETED F-PARTICLE THERAPY MECHANISMS OF RADIATION-INDUCED CELL DEATH SELECTED F-PARTICLE-EMITTING RADIOISOTOPES RADIOLABELING DOSIMETRY PRECLINICAL AND CLINICAL STUDIES POTENTIAL TOXICITIES

INTRODUCTION The role of monoclonal antibodies (MAbs) in the treatment of malignancies has increased dramatically over the past decade. Three unlabeled MAbs— rituximab (Rituxan; IDEC Pharmaceuticals, San Diego, CA, and Genentech, Inc., South San Francisco, CA), trastuzumab (Herceptin; Genentech, Inc., South San Francisco, CA), and alemtuzumab (Campath-1H; Burroughs Wellcome, United Kingdom)—have been approved by the US Food and Drug Administration (FDA) for the treatment of lymphoma, breast cancer, and chronic lymphocytic leukemia, respectively. These and other unlabeled MAbs kill tumor cells

From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

107

108

Burke, Scheinberg, and Jurcic

by mediating complement-dependent cytotoxicity and antibody-dependent cellular cytotoxicity (1) and by directly causing apoptosis (2,3). Despite these clinical successes, many native antibodies have only weak antitumor effects. To overcome this limitation, MAbs have been conjugated to chemotherapeutic agents and to toxins, such as Pseudomonas exotoxin, ricin, gelonin, and diphtheria toxin. For example, gemtuzumab ozogamicin (Mylotarg; Wyeth Laboratories, Philadelphia, PA) consists of an anti-CD33 antibody conjugated to the antitumor antibiotic calicheamicin; it has activity against CD33-positive acute myeloid leukemia and was approved for clinical use by the FDA in 2000 (4). Another strategy to increase the antitumor activity of MAbs is radioimmunotherapy, in which antibodies act as carrier molecules to deliver radioisotopes to target cells, where the emitted F- or G-particles damage DNA, resulting in cell death. To date, the only radioimmunoconjugate approved by the FDA for clinical use is yttrium-90 (90Y) ibritumomab tiuxetan (Zevalin; IDEC Pharmaceuticals, San Diego, CA) for the treatment of patients with relapsed or refractory follicular or transformed non-Hodgkin’s lymphoma (5). Most radioisotopes used in clinical trials have been G-particle emitters. Gparticles are negatively charged particles equivalent to electrons that are emitted from the nucleus of radioisotopes. They have a relatively long range in tissue (several millimeters) and low energies (approx 300–2000 keV). Because of these physical properties, approx 10,000 G-particles traversing a cell nucleus are required to kill a target cell. Examples of G-emitters used in clinical radioimmunotherapy trials include iodine-131 (131I), 90Y, and rhenium-188 (188Re). Despite the predominant use of G-emitters in radioimmunotherapy trials, investigators have recognized the potential advantages of using F-particle emitters for years. F-particles are positively charged helium nuclei with a shorter range (50–80 (μm) and higher energy (5000–8000 keV) than G-particles. Examples of F-particle emitters under investigation include astatine-211 (211At), bismuth-212 (212Bi), bismuth-213 (213Bi), and actinium-225 (225Ac). In this chapter, we will review the characteristics of various isotopes for the targeted Fparticle therapy of cancer, issues in radiolabeling and dosimetry, and recent preclinical and clinical studies. The reader is also referred to other published reviews on this subject (6–8).

RATIONALE FOR TARGETED F-PARTICLE THERAPY The different physical properties of F- and G-particles confer theoretical advantages and disadvantages to each type of particle in various clinical situations. G-emitters should be more effective than F-emitters in the treatment of large solid tumor masses. In large solid tumor masses, the vasculature may be unevenly distributed, and target antigen expression within the tumor may vary. As a result, the distribution of antibody binding is not necessarily uniform, and

Chapter 5 / Targeted F-Particle Therapy

109

many malignant cells may escape binding by the antibody. Because G-particles have a long range, they should damage malignant cells not directly bound by antibody molecules, resulting in a “crossfire effect.” In contrast, because Fparticles have a range of only a few cell diameters, they are likely to miss malignant cells not directly bound by the antibody, thus failing to eradicate the tumor. Whereas G-emitters should be more effective in the treatment of larger solid tumors, F-emitters should be more effective in the treatment of micrometastatic disease and circulating tumor cells like leukemia. In both clinical settings, Gemissions may result in significant damage to normal “bystander” cells (9). In a microdosimetric model using single-cell conditions, one cell-surface decay of the F-emitter 211At would result in the same degree of cell killing as about 1000 cell-surface decays of the G-emitter 90Y (10). Thus, because of the short range and high energy of F particles, F-particle immunotherapy should provide more efficient and specific killing of tumor cells, particularly in the setting of leukemia, microscopic disease, or tumors that spread as thin sheets within compartments. Based on these considerations, F-particle therapy has been investigated in a variety of settings, including leukemias, lymphomas, gliomas after surgical resection, neoplastic meningitis, and peritoneal carcinomatosis.

MECHANISMS OF RADIATION-INDUCED CELL DEATH Although the mechanisms by which radiation induces cell death are not completely understood, several processes have been implicated (11,12). Radiation induces single- and double-stranded DNA breaks (13), causes the cleavage of sphingomyelin in cell membranes leading to the formation of ceramide and the subsequent induction of apoptosis (14), and results in the induction of p53 by ATM-dependent phosphorylation, resulting in delays in the G1 phase of the cell cycle (15,16). Death of cells exposed to F-particles occurs only when the particles traverse the nucleus; low doses of F-particle radiation directed at the nucleus are lethal, whereas high doses directed at the cytoplasm have no effect on cell proliferation (17). Linear energy transfer (LET) and relative biological effectiveness (RBE) are essential radiobiological concepts. LET refers to the number of ionizations caused by that radiation per unit of distance traveled. F-particles cause a large number of ionizations in a relatively short distance, and thus have a high LET. G-particles and L rays, on the other hand, cause a lower number of ionizations over a longer distance, and therefore have a lower LET. For example, the F-particle emitted by astatine-211 (211At) has a mean range in tissue of 70 μm and a LET of 97 keV/ μm, whereas the G-particle emitted by 90Y has a mean range of 3960 μm and a LET of 0.2 keV/μm (7). The RBE for a type of radiation (e.g., F-particles) refers to the dose of a reference radiation (usually X-rays) that produces the same biological effect as

110

Burke, Scheinberg, and Jurcic

the type of radiation in question. Depending on their emission characteristics, the RBE of F-particles for cell sterilization ranges from 3 to 7. The RBE of a type of radiation is a function of the LET of that radiation; RBE is highest at an LET of about 100 keV/μm (18). F-particle radiation has a high RBE because the LET of F-particles is often close to 100 keV/μm. The dependency of RBE on LET can be explained by several differences in the type and extent of cellular damage caused by low- and high-LET radiations. First, high-LET radiation generally causes more irreparable clustered and doublestranded DNA breaks than low-LET radiation (19,20). The maximum rate of double-stranded DNA breaks occurs at LETs of 100–200 keV/μm because at these LETs, the distance between ionizations caused by the radiation approximates the diameter of double-stranded DNA (2 nm) (18). At LETs below 100 keV/μm or above 200 keV/μm, the rates of double-stranded DNA breaks are lower. Second, high-LET radiation causes more severe chromosomal damage, including shattered chromosomes at mitosis and complex chromosomal rearrangements, than low-LET radiation (11). The highest frequency of chromosome breaks and complex chromosomal rearrangements occurs with LETs of approx 100–150 keV/μm (11,19). Third, high-LET F-irradiation causes more pronounced G2-phase delays than low-LET L-irradiation (21,22). The mechanisms behind these differences in cell-cycle effects have not been fully elucidated, but may be related to differences in gene expression induced by the lowand high-LET radiations (23). Many studies have demonstrated the high potency of F-irradiation. In one of the first, Munro demonstrated that only a few F-particles traversing a nucleus can kill Chinese hamster fibroblasts (17). Bird et al. investigated the cytotoxicity of helium-3 (3He) ions in Chinese hamster V79 cells in different phases of the cell cycle—either at the G1/S transition (induced by hydroxyurea) or in late S phase (24). About four nuclear traversals of 3He were required to kill cells at the G1/ S transition, whereas five to eight were required in late S phase. Other studies using a variety of cell lines irradiated in vitro with F particles (usually from a 238Pu or 239Pu source) also showed that a mean of about two to six F-particle nuclear traversals are required to kill cells (20,25–30). One “outlying” study found that a larger number (10–20) of F-particle traversals were required to kill a cell (31). In this study, mouse embryo fibroblasts from the C3H 10T1/2 cell line were irradiated with 5.6-MeV F-particles (LET 85 keV/μm). One possible explanation for the relatively large number of F-particles required to kill these cells is that the cells, when plated in Petri dishes, became flattened, causing the nuclear surface area to became much larger (mean 313 μm2) than that of the cell lines used in the other studies.

Chapter 5 / Targeted F-Particle Therapy

111

SELECTED F-PARTICLE-EMITTING RADIOISOTOPES Because more than 100 radioisotopes emit F-particles, and most of them decay too quickly to be of therapeutic use, we will confine our discussion of specific F-emitters to those that have therapeutic potential and have been investigated in animal models or humans.

Actinium-225 209Bi, 225Ac

In its decay to stable (half-life, 10.0 d) generates francium-221 (221Fr), 217At, 213Bi, and lead-209 (209Pb) and emits four F particles (Fig. 1). 225Ac can be produced by the natural decay of uranium-233 (233U) (8) or by reactor- or accelerator-based methods (32). In the first method, 233U decays to thorium-229 (229Th), which then decays with a 7340-yr half-life to 225Ra. 225Ra (half-life, 14.8 d) emits an F-particle as it decays to 225Ac. Following the collection of 229Th, 225Ra and 225Ac are then separated using a series of ion-exchange columns (33). The second method of production of 225Ac, developed by the Institute for Transuranium Elements in Karlsruhe, Germany, involves the neutron irradiation of 226Ra by successive n,G capture decay reactions via 227Ac, 228Th to 229Th (32). Other reactor-based methods are under development. 225Ac has both advantages and disadvantages compared with other F-emitters. Because it emits four F-particles with each decay and has a relatively long halflife, 225Ac is more potent than other isotopes that emit just a single F-particle and have shorter half-lives (34). However, after the decay of 225Ac, the daughter isotopes (221Fr, 217At, 213Bi, and 209Pb) are released from the chelator and can therefore result in nonspecific cytotoxicity. One strategy to overcome this obstacle is the use of internalizing antibodies so that the daughters remain predominantly within target cells.

Bismuth-213 213Bi

(half-life, 45.6 min) is produced from the decay of 225Ac (Fig. 1). 213Bi decays by a branched pathway to 209Pb and then to stable 209Bi, emitting an Fparticle and two G-particles. Additionally, a 440-keV photon emission allows detailed biodistribution, pharmacokinetic, and dosimetry studies to be performed. A clinically approved 213Bi generator consists of 225Ac dispersed onto a cation exchange resin. The 213Bi is eluted from the generator, and antibody molecules appended with the C-functionalized trans-cyclohexyldiethylenetriamine pentaacetic acid moiety, CHX-A-DTPA, readily chelate the 213Bi radionuclide (35–37). Clinical studies in acute myeloid leukemia, non-Hodgkin’s lymphoma, and melanoma are underway with this isotope.

112

Burke, Scheinberg, and Jurcic

Fig. 1. The 229Th decay scheme.

Astatine-211 211At

The halogen (half-life, 7.2 h) decays through a branched pathway (Fig. 2). Each branch results in the production of an F-particle and in decay to stable 207Pb. The F-particle produced by the decay of 211At has a mean energy of 6.8 MeV, a mean LET of 97–99 keV/μm, and a range in tissue of 55–80 μm. 211At

Chapter 5 / Targeted F-Particle Therapy

113

Fig. 2. The 211At decay scheme. EC = electron capture.

is produced by the bombardment of bismuth with F-particles in a cyclotron via the 209Bi(F, 2n)211At nuclear reaction (38). After production, 211At can be isolated from the cyclotron target using a dry distillation procedure (39). It has been labeled to various types of carrier molecules, including antibodies and antibody fragments (40), tellurium colloid (41,42), the naphthoquinone derivative 2-methyl-1,4-naphthoquinol disphosphate (astato-MNDP) (43), methylene blue (44, 45), 2'-deoxyuridine (29), benzylguanidine (28,46), a derivative of the vitamin biotin (47), and bisphosphonates (48). There are both advantages and disadvantages to the therapeutic use of 211Atlabeled molecules. The 7.2-h half-life enables 211At-labeled constructs to be used even when the targeting molecule does not gain immediate access to tumor cells. Another advantage is that the polonium-211 (211Po) daughter emits K X-rays that permit photon counting of samples and external imaging for biodistribution studies. A disadvantage is that only a few institutions have cyclotrons capable of producing 211At. Furthermore, after internalization into cells, 211At is retained less well than other F-emitting radiometals like 212Bi, 213Bi, and 212Pb (49).

Bismuth-212 212Bi

has a half-life of 60.6 min and emits an F-particle with a mean energy of 7.8 MeV. 212Bi is produced from the decay of 228Th (Fig. 3). A generator that uses 224Ra as the parent radionuclide facilitates the production of 212Bi for labeling to antibodies (50). 212Bi decays by a branched pathway to stable 208Pb (Fig. 3). The thallium-208 (208Tl) produced by the decay of 212Bi emits a 26-MeV Lray along with other medium to high-energy L-particles that require heavy shielding to minimize radiation exposure to personnel and have limited the clinical use of 212Bi. 212Bi-labeled antibodies have been used in cell lines and animal models but not in humans (51–57).

114

Burke, Scheinberg, and Jurcic

Fig. 3. The 228Th decay scheme.

Lead-212 Like 212Bi, 212Pb is produced from the decay of 228Th and from a 224Ra generator system (Fig. 3) (50). 212Pb has a half-life of 10.6 h and emits a G-particle in its decay to 212Bi. 212Pb acts as an F-particle emitter only by serving as a source of 212Bi. Thus, when administered clinically, 212Pb would serve as an in situ 212Bi generator. 212Pb has been conjugated to antibodies using various chelators (58–60).

Chapter 5 / Targeted F-Particle Therapy

115

Radium-223 223Ra

(half-life, 11.4 d) can be obtained from uranium mill tailings in large quantities. A generator system has been developed using a 227Ac parent (halflife, 21.8 yr). Like 225Ac, 223Ra emits four F-particles over its decay scheme. A clinical phase I study using cationic 223Ra in the treatment of skeletal metastases in patients with prostate and breast cancer demonstrated pain relief and reduction in tumor marker levels (61).

RADIOLABELING Several techniques can be used to label F-emitting radioisotopes to carrier molecules. For a labeling technique to be useful, it must produce the immunoconjugate in high yield in a time compatible with the half-life of the radioisotope. Furthermore, it must not alter the specificity and affinity of the carrier molecule for the target antigen. Finally, the radioimmunoconjugate must be stable in vivo. Finding a suitable approach for labeling radioisotopes to carrier molecules has often been a limiting factor in the development of radiolabeled molecules for therapeutic use. Many F-emitters—including 225Ac, 212Bi, and 213Bi—require bifunctional chelators that bind both radioisotopes and carrier proteins. Figure 4 depicts the molecular structures of a number of chelators that have been developed. Many of these chelators are derived from diethylene triamine penta-acetic acid (DTPA), including the isobutylcarboxylcarbonic anhydride derivative (54,55), the cyclic dianhydride derivative (CA-DTPA) (51,56), 2-(p-isothiocyanatobenzyl)-DTPA (SCN-Bz-DTPA) (62), 2-(p-isothiocyanatobenzyl)-5(6)-methyl-DTPA (MxDTPA) (62), the cyclohexyl derivative (Cy-DTPA) (63), and the cyclohexylbenzyl derivative (CHX-A-DTPA) (52,64). 1,4,7,10-tetraaza-cyclododecane-1,4,7,10tetraacetic acid (DOTA) (62,63) and its derivatives (34) have also been used. The isobutylcar-boxylcarbonic anhydride and the cyclic dianhydride of DTPA are not useful in chelating bismuth radioisotopes to antibodies because the radioimmunoconjugates produced have poor stability in vivo (58,62). Use of SCN-Bz-DTPA, Mx-DTPA, and DOTA results in improved stability of bismuth-labeled antibodies (62). However, the formation of bismuth-DOTA complexes is slow (63). The most effective bismuth chelator to date has been CHX-A-DTPA, which can link bismuth radioisotopes to a variety of antibodies. When CHX-A-DTPA is used to label bismuth to the anti-CD33 antibody HuM195, the labeling procedure takes a mean of 23 min and results in a radiolabeling reaction efficiency of 81% and in production of a final product of 99% purity (64). The resulting immunoconjugates are stable (52,65) and have been used effectively in human clinical trials (36,37). Although the development of a chelator that binds 225Ac has been slow (66), bifunctional derivatives of DOTA were recently found to be effective (34). 225Ac-DOTA-containing radioimmunoconjugates are stable both in vitro and in mice.

116

Burke, Scheinberg, and Jurcic

Fig. 4. Chemical structures of selected chelators derived from diethylene triamine pentaacetic acid (DTPA) and 1,4,7,10-tetraazacyclododecane-1,4,7,10-tetraacetic acid (DOTA).

Chapter 5 / Targeted F-Particle Therapy 211At

117

has been labeled to antibodies and to other types of carrier molecules.

211At is usually labeled to antibodies by incorporation of an aryl carbon–astatine

bond into the antibody, not by use of a chelator (38). Various methods can be used to create the aryl carbon–astatine bond, most of which involve an astatodemetallation reaction using a tin, silicon, or mercury precursor (38,39). When 211At has been labeled to other carrier molecules besides antibodies, different labeling methods have been used; a complete discussion of these methods is beyond the scope of this review.

DOSIMETRY The dosimetry of F-emitting radionuclides is distinguished from that of Gemitters by a number of characteristics. Few F-particle emitters decay to stable or short-lived daughter products. Those that do have half-lives that are considerably shorter than the commonly used G-emitters. Additionally, the shorter range and higher LET of F-particles compared to G-particles result in an RBE for cell sterilization of 3 to 7. Radioimmunotherapy with short-lived F-particle emitters such as 213Bi results in markedly different pharmacology than with longer-lived G-emitters. With longer-lived isotopes, pharmacokinetics are determined predominantly by the biological clearance of the antibody. The distribution of the antibody within the first several minutes to hours after administration yields residence times that are negligible in proportion to the overall residence times achieved in target and normal organs. In contrast, for 213Bi, with its 46-min half-life, 20% of the total F emissions occur within the first 15 min after injection, and after 3 h, only 6% of the total emissions remain. Conventional medical internal radiation dosimetry (MIRD), based on imaging-derived pharmacokinetics, can be performed, assuming that all F and electron emissions arising from the decay of the parent are locally deposited. The absorbed dose is given by the cumulated activity concentration, [Ã], multiplied by the energy emitted per decay as electrons, )e, and the F particles, )F. To determine a radiation dose equivalent (in Sieverts), the F particle contribution to the dose should be adjusted for the RBE: D = [Ã] × ()e + RBE × )F) (67). All of the F-particle emitters considered for radioimmunotherapy yield radioactive daughters. The fate and biodistribution of these daughters following decay of the parent must be considered in dosimetry estimates. If the daughters remain in the vicinity of the parent, then the parameters )e and )F should include the energy associated with their emissions, weighted by the yield of each daughter. If the half-life of the daughters is long in relation to the rate of diffusion of the daughter, information regarding the redistribution must be incorporated into the dosimetric estimates.

118

Burke, Scheinberg, and Jurcic

Given the high energy of F-particles delivered over a short range, however, conventional methodologies that estimate mean absorbed dose over a specific organ volume may not always yield biologically meaningful information. Because of the physical properties of F emissions, targeted cells may receive high absorbed radiation doses, while adjacent cells may receive no radiation at all. Therefore, microdosimetric or stochastic analyses that account for the spatial distribution of various cell types and the distribution of F decays within the organ will be necessary to estimate the absorbed dose to tumor cells and normal tissues more accurately. Because the geometric relationship between the radionuclide and the target cell is not uniform, F-particle hits cannot be assumed to be a Poisson distribution. Several distributions have been modeled, and microdosimetric spectra, expressed as specific energy probability densities, have been calculated. Based on this work, methods have been developed to perform basic microdosimetric assessments that account for the probability of the number of hits and the mean specific energy from a single hit (68).

PRECLINICAL AND CLINICAL STUDIES Mouse Models In one of the earliest studies of F-particle therapy in a mouse model of cancer, mice were inoculated intraperitoneally with EL-4 murine lymphoma cells, which express Thy-1.2, resulting in malignant ascites (51). One day later the mice were treated with intraperitoneal injections of either anti-Thy-1.2 conjugated to 212Bi using the cyclic dianhydride of DTPA or relevant controls. Survival in the treated mice was prolonged compared with controls. However, within 2 h after injection, up to 30% of the injected dose of 212Bi was present in the renal collecting system and bladder. Because free 206Bi is excreted in the urine (69), the presence of 212Bi in the kidneys and bladder suggested dissociation of the 212Bi from the antibody. In another study, athymic nude mice were inoculated intraperitoneally with LS174T colon cancer cells, which secrete the mucin antigen TAG-72 (57). Seven to 13 d later, the mice were treated with intraperitoneal injections of either 212Bilabeled anti-TAG-72 or controls. The radiolabeled antibody decreased tumor burdens and may have prolonged survival. However, as in the previous study, much of the injected dose of 212Bi was taken up by the kidney, indicating instability of the radioimmunoconjugate. To overcome the problem of unstable radioimmunoconjugates, a different chelating agent—CHX-A-DTPA—was used to conjugate 212Bi to the anti-gp70 antibody 103A (52). BALB/c mice were inoculated with the Rauscher leukemia virus, resulting in erythroleukemia. In this model, infected cells express the viral envelope glycoprotein gp70. Treatment of the mice with 212Bi-103A at 8 to 13 d after inocu-

Chapter 5 / Targeted F-Particle Therapy

119

lation resulted in decreased splenic tumor growth and prolonged median survival. In contrast to the previous studies, the radioimmunoconjugate was stable. Hartmann et al. used CHX-A-DTPA to conjugate 212Bi to anti-Tac, which targets CD25 (70). In an experiment designed to simulate the treatment of smallvolume disease, athymic nude mice were inoculated subcutaneously with murine plasmacytoma cells engineered to express human CD25. Three days later, prior to the development of overt tumor masses, the mice were treated intravenously with either 212Bi-anti-Tac or controls. Treatment with 212Bi-anti-Tac prolonged the time to tumor occurrence and completely prevented the development of tumors in 30% of mice. However, 212Bi-anti-CD25 did not eradicate established tumors in mice. These results support the hypothesis that F-particle radio-immunotherapy may be more effective in the treatment of small-volume disease than in the treatment of bulky tumors. Similar results were found in a mouse model of ovarian cancer (60). Female athymic nude mice were inoculated subcutaneously with SK-OV-3 ovarian cancer cells. Three days later the mice were treated with the anti-HER2/neu monoclonal antibody AE1 conjugated to 212Pb. The treatment resulted in improved tumor-free survival compared with controls. However, when the treatment was delayed until larger-volume tumors had developed, no beneficial effects occurred. Only a few studies have directly compared radioimmunotherapy with F-emitters to radioimmunotherapy with G-emitters in animal models. In the first, mice with intraperitoneal growth of murine ovarian cancer were treated with 211Attellurium colloid or with radiocolloids of dysprosium-165 (165Dy), phosphorus32 (32P), or 90Y (42). Although all the treatments prolonged survival compared with controls, 211At-tellurium colloid was the most effective and was often curative. Behr et al. investigated the toxicity and antitumor efficacy of the CO17-1A monovalent Fab' fragment labeled with either 213Bi or 90Y in a human colon cancer xenograft model in nude mice (71). CO17-1A is an antibody directed against a glycoprotein found on normal and malignant gastrointestinal cells. At equitoxic doses, 213Bi-labeled Fab' prevented tumor growth and prolonged survival compared with 90Y-labeled Fab'. The maximum tolerated absorbed doses to blood were similar for the two conjugates. Finally, Andersson et al. compared 211At-labeled MOv18 with the G-emitting 131I-MOv18 (72). MOv18 targets a membrane folate-binding glycoprotein on human ovarian carcinomas. A previous study had demonstrated that 211At-MOv18 has antitumor activity when injected intraperitoneally into nude mice with microscopic cancer (73). In the comparison study, 211At-MOv18 prevented the growth of microscopic disease more effectively than 131I-MOv18 did (72). Thus, in the few studies that have directly compared equivalent doses of F-emitters and G-emitters in animal mod-

120

Burke, Scheinberg, and Jurcic

els, F-emitters have been more effective in preventing tumor growth and prolonging survival.

Spheroids Further evidence that F-particle radioimmunotherapy may be most useful in the treatment of small-volume disease comes from studies of spheroids. Spheroids are clusters of malignant cells that serve as models for micrometastatic disease. In one study using relatively small spheroids (200 μm in diameter) composed of CD44-expressing cells, 213Bi-labeled anti-CD44 killed a large proportion of the cells (74). In another study, 213Bi labeled to the anti-prostatespecific membrane antigen (PSMA) antibody J591 reduced the volume of spheroids containing cells of the prostate cancer line LNCaP-LN3 (75). The amount of reduction in spheroid volume was inversely proportional to initial spheroid size; 130-μm spheroids responded better than 180-μm spheroids. In another study demonstrating the importance of spheroid size, 212Bi bound to the monoclonal antibody NRLU-10 (which targets a carcinoma antigen) and its Fab fragment killed single human colon adenocarcinoma cells but did not shrink large spheroids (450–1000 μm in diameter) (76). 213

Bi-Labeled Anti-CD33 for Myeloid Leukemia

Preclinical biodistribution studies using bismuth-labeled anti-CD33 monoclonal antibody HuM195, which targets myeloid leukemia cells, were conducted in BALB/c mice. These studies showed no loss or uptake of isotope to any normal tissues, including the kidneys, which are know to have an avidity for free bismuth (65). Up to 10 mCi/kg of 213Bi-HuM195 could be injected intravenously into BALB/c mice without significant toxicity. In vitro studies of 213Bi-labeled HuM195 and the murine anti-prostate membrane-specific antigen (PSMA) antibody J591 demonstrated dose-dependent and specific-activitydependent killing of cells expressing the relevant antigenic targets (77). In nude mice bearing prostate carcinoma xenografts, treatment with 213Bi-J591 decreased prostate-specific antigen (PSA) levels and prolonged survival compared with controls (77). Based on these data, human clinical trials have been performed at Memorial Sloan-Kettering Cancer Center (MSKCC) in patients with myeloid leukemias, using 213Bi conjugated to HuM195 with CHX-A-DTPA. In a phase I doseescalation trial, 18 patients with relapsed or refractory acute myeloid leukemia (AML) or chronic myelomonocytic leukemia (CMMoL) were treated with 0.28– 1.0 mCi/kg of 213Bi-HuM195 in three to seven fractions over 2–4 d (36). Within 10 min of administration, the 213Bi was taken up in the bone marrow, liver, and spleen, where it remained throughout its half-life. No significant uptake was seen in any other organ. The absorbed dose ratios between marrow, liver, and spleen and the whole body were 1000 times greater with 213Bi-HuM195 than

Chapter 5 / Targeted F-Particle Therapy

121

with G-emitting HuM195 constructs used in similar patients in previous trials (67). Toxicities included myelosuppression in all patients and transient minor liver function abnormalities in 6 patients. Fourteen of 15 evaluable patients (93%) had reductions in circulating blasts after therapy, and 14 of the 18 patients (78%) had decreases in the percentage of bone-marrow blasts. However, no complete remissions occurred. This trial demonstrated that 213Bi-HuM195 has antileukemic activity, but the lack of complete remissions suggested that it remained difficult to target one to two atoms of 213Bi to all leukemia cells in patients with advanced disease. To test the hypothesis that 213Bi-HuM195 should be more effective in the treatment of cytoreduced disease, we are conducting a phase I/II trial in which patients with advanced AML are treated with cytarabine (200 mg/m2/d for 5 d) followed by 213Bi-HuM195 (0.5–1.25 mCi/kg) (37). To date, 12 patients (median age, 63 yr) have been treated. Nine of 10 evaluable patients had reductions in the percentage of bone-marrow blasts. Two patients, both treated at the 1.0 mCi/kg dose level, achieved complete remissions. A third patient achieved a partial remission (4–9% bone-marrow blasts and normalization of peripheral blood counts) that lasted 4 mo. Myelosuppression was common; however, most patients had severe neutropenia and thrombocytopenia prior to therapy. Although these results are preliminary, sequential administration of cytarabine and 213BiHuM195 appears safe and can produce complete remissions in patients with advanced AML. 213

Bi-Labeled Antibodies As Conditioning for Nonmyeloablative Allogeneic Marrow Transplantation Although potentially curative for a number of malignancies, allogeneic marrow transplantation using myeloablative preparative regimens causes significant toxicity. To decrease the toxicity of the procedure, nonmyeloablative preparative regimens have been developed. Many of these nonmyeloablative preparative regimens use low doses of total-body irradiation (TBI), with or without additional chemotherapeutic agents (78,79). Recently, 213Bi-labeled anti-CD45 was investigated as a preparative regimen in dogs (80). Seven dogs were treated with escalating doses of 213Bi-anti-CD45 (0.1 to 5.9 mCi/kg) without marrow transplantation. At higher doses, significant declines in peripheral blood counts occurred; 213Bi doses of 3.7 to 5.9 mCi/kg had similar effects on neutrophil counts similar to those of 200 to 300 cGy of TBI. Subsequently, three dogs were treated with 213Bi-anti-CD45 (3.6 to 8.8 mCi/kg) followed by infusion of marrow from DLA-identical littermates and by immunosuppression with mycophenolate mofetil and cyclosporine. In all dogs, engraftment occurred promptly, and stable mixed hematopoietic chimerism occurred as early as 2–3 wk after transplantation. Subsequent studies revealed that doses of >2 mCi/kg of 213Bi-anti-CD45 were necessary to allow engraftment. Toxicities included transient cytopenias

122

Burke, Scheinberg, and Jurcic

and liver function abnormalities. One dog developed intractable ascites; at autopsy, histologic examination showed marked periportal fibrosis, irregular sinusoidal fibrosis, and Kupffer cell aggregates. In a subsequent study by the same group of investigators, four dogs underwent treatment with 213Bi labeled to an antibody directed at the T-cell receptor (TCR) FG followed by marrow transplantation (81). Because T-cells that express TCRFG are thought to be involved in marrow graft rejection (82), interfering with the function of these T-cells could result in sufficient immunosuppression to allow engraftment of allogeneic marrow. The four dogs received doses of 213Bi-anti-TCRFG ranging from 3.7 to 5.6 mCi/kg, followed by marrow transplantation from DLA-identical littermates and by immunosuppression. In all dogs, engraftment occurred rapidly, and stable mixed hematopoietic chimerism occurred as early as 1 wk after transplantation. As with 213Bi-anti-CD45, toxicities included transient cytopenias and liver-function abnormalities. One dog developed sustained liver-function test abnormalities; at autopsy, hepatic pericellular and periportal fibrosis was found. These studies show that targeted Fparticle therapy alone can provide adequate immunosuppression to permit nonmyeloablative marrow transplantation. 225

Ac Atomic Nanogenerators

225Ac

has been stably conjugated to a variety of antibodies using derivates of DOTA. In vitro studies of 225Ac-J591, 225Ac-HuM195, and 225Ac-B4 (anti-CD19) demonstrated dose-dependent and specific-activity-dependent killing of tumor cells at doses 1000 times less than 213Bi-containing constructs (34). In nude mice bearing prostate cancer xenografts, single nanocurie-level doses of 225Ac-J591 decreased PSA levels and cured a substantial fraction of animals (34). Similarly, in mice bearing lymphoma xenografts, treatment with 225Ac-B4 also improved survival compared with controls (34). The increased potency of these 225Ac constructs compared with 213Bi analogs can be explained by the longer (10-d) half-life of 225Ac and by the ability of 225Ac conjugates to act as atomic nanogenerators, emitting four F particles within an individual tumor cell as it decays. A phase I trial of 225Ac-HuM195 for advanced myeloid leukemias is planned. 211

At-Labeled Anti-Tenascin for Gliomas

A group of investigators at Duke University has extensively studied 211At81C6, a chimeric antibody that targets tenascin, and other 211At-labeled constructs (30,83–85). Tenascin is an extracellular matrix glycoprotein that is overexpressed on gliomas relative to normal brain tissue (86). 211At-labeled 81C6 and 211Atlabeled Mel-14, a chimeric antichondroitin sulfate antibody, are cytotoxic to glioma and melanoma cells, respectively (30). Reduction in cell survival to 37% requires a mean of 1–2 hits to the cell nucleus. 211At-labeled 81C6 was investigated in rats that had been injected intrathecally with human rhabdomyosarcoma cells to cause

Chapter 5 / Targeted F-Particle Therapy

123

neoplastic meningitis (83). Treatment with 211At-labeled 81C6 4–8 d after inoculation prolonged survival compared with controls. Based on these data, a phase I dose-escalation trial of 211At-81C6 was initiated in patients with malignant gliomas (85). Surgical resection of the brain tumors was followed by instillation of up to 10 mCi of 211At-81C6 into the tumor cavity. At the time of a preliminary report, 12 patients (11 with malignant glioma and one with anaplastic oligodendroglioma) had been treated (85). Pharmacokinetic studies and gamma camera images showed that 99% of the 211At decays occurred within the tumor cavity, indicating high in vivo stability and only low levels of leakage into the circulation. Early results suggest that adjuvant therapy with 211At81C6 prolonged survival in these patients, compared with historical controls. No dose-limiting toxicities were observed, and dose escalation continues. 211

At-Labeled Methylene Blue for Melanoma

A group in the United Kingdom has investigated the use of methylene blue labeled with 211At in the treatment of melanoma (87–89). Methylene blue is a chemical phenothiazine derivative (3,7-dimethylamino-phenazathionium chloride) that binds to melanin and is taken up by melanoma cells in vitro and in vivo (87). In one study, female nude mice were injected with HX34 human melanoma cells, followed by treatment with 211At-methylene blue either 1 or 7 d later (87). The treatment decreased the number and size of melanoma metastases. In a second mouse study, 211At-methylene blue inhibited the growth of cutaneous melanomas and spontaneous lymph-node metastases (90). Based on these and other data, human clinical trials were initiated in 1997 (88). 211

At- and 213Bi-Labeled Rituximab for Lymphoma

Aurlien et al. studied the effects of 211At labeled to the chimeric anti-CD20 antibody rituximab on CD20-expressing RAEL and K422 lymphoma cells in vitro (91, 92). 211At-rituximab bound more strongly to lymphoma cells than to control bone-marrow cells and was cytotoxic to both lymphoma cell lines. Biodistribution studies in BALB/c mice showed targeting of blood, lung, liver, heart, and other organs. The biodistribution of 211At-rituximab was similar to that of 125I-rituximab. The cytotoxicity of 211At-labeled rituximab was not compared with that of 125I-labeled rituximab or unlabeled rituximab. A clinical trial of 211At-rituximab in patients with relapsed B-cell lymphomas is planned. A phase I clinical trial of 213Bi-labeled rituximab in patients with refractory B-lineage non-Hodgkin’s lymphoma is underway (93). In preclinical studies, 213Bi-rituximab was cytotoxic to lymphoma cells. The construct was stable in vivo and caused no toxic side effects other than myelosuppression. In the clinical trial, nine patients with refractory lymphomas were treated with escalating doses of 213Bi-rituximab (10 to 44 mCi) (93). Grade 1 leukopenia occurred in two patients. One patient achieved a minimal response, and another had stable disease. Enrollment to the study continues.

124

Burke, Scheinberg, and Jurcic

Pretargeting Studies In an effort to reduce radiation doses to normal organs and improve tumor-tonormal organ dose ratios, pretargeted methods of radioimmunotherapy have been developed. Most of these techniques take advantage of the rapid, highaffinity, and specific binding between streptavidin and the small molecule biotin (94). First, a monoclonal antibody or engineered targeting molecule, conjugated to streptavidin, is administered. Second, a clearing agent, consisting of biotinylated galactosyl human serum albumin, is given after the antibodystreptavidin construct has bound to tumor targets. The biotin component of the clearing agent binds to the streptavidin component of the circulating antibodystreptavidin conjugate. The galactose moiety of the clearing agent then binds to galactose receptors on hepatocytes, and the complex is cleared from the circulation. Finally, radiolabeled biotin is administered. Because excess antibodystreptavidin conjugated has been cleared from the circulation, the radiolabeled biotin can bind specifically to “pretargeted” streptavidin at the tumor. Unbound radiolabeled biotin is rapidly excreted in the urine. Zhang et al. have utilized the pretargeting approach in a murine model of adult T-cell leukemia (ATL) (95). Humanized anti-CD25 was conjugated to streptavidin and injected into nonobese diabetic/severe combined immunodeficient mice bearing the ATL cell line MET-1, which expresses CD25. After administration of the clearing agent, 213Bi-labeled or 90Y-labeled DOTA-biotin was given. Tumor-bearing mice treated with 213Bi had reductions in the concentrations of surrogate tumor markers human G2-microglobulin and soluble CD25 as well as improved survival compared with controls. Treatment with 213Bi was more effective than treatment with 90Y. Furthermore, use of the pretargeted approach was more effective than use of 213Bi linked to an intact monoclonal antibody. Despite these exciting and encouraging results, a single course of therapy with 213Bi did not completely eliminate the leukemia from the mice.

POTENTIAL TOXICITIES In deciding whether to bring an F-emitting radioimmunoconjugate from an animal model into human clinical trials, both efficacy and potential toxicities need to be considered. For example, in one study, 213Bi-labeled antithrombomo-dulin targeted pulmonary blood vessels and destroyed pulmonary micrometastases in a mouse model (96). However, the treatment was complicated by pulmonary fibrosis. Attempts to avoid pulmonary fibrosis by the use of several techniques to block tumor necrosis factor (TNF)-F—antibodies to TNF-F, the dimeric fusion protein etanercept, and mice genetically deficient for TNF-F production—were unsuccessful (97). Therefore, until the problem of pulmonary fibrosis can be circumvented, the use of 213Bi-labeled antithrombomodulin would seem unfit for clinical trials.

Chapter 5 / Targeted F-Particle Therapy

125

Several studies have investigated the toxicity of 211At—either free or bound to carrier molecules. Like iodine (another halogen), 211At is selectively concentrated in the thyroid gland. Intravenous injections of potentially lethal doses of sodium [211At]astatide in mice resulted in ablation of thyroid follicles as well as reduction of lymphocytes in the blood, spleen, and lymph nodes, marrow toxicity, abnormalities of the testes and ovaries, necrosis of the submandibular glands, and necrosis in the crypts of the stomach (98). Lower doses of [211At]astatide produced fibrosis of the thyroid, mild cytopenias, and severe reduction in reproductive cells in the testis (99). In a study designed to determine the lethal dose of [211At]astatide in two mouse models, tail-vein injections caused dose-related toxicity to the bone marrow, heart, testes, spleen, and stomach (100). [211At]astatide may also cause cancer in rats (101). When high doses of 211At were labeled to the antibody 81C6 and administered to mice, perivascular fibrosis of the intraventricular septum of the heart, myelosuppression, splenic white pulp atrophy, and spermatic maturational delay occurred (102). When labeled to methylene blue and administered to mice bearing melanoma xenografts, 211At caused minor abnormalities in the thyroid gland, regional lymph nodes, and lungs (103,104). Other F-emitters have different toxicity profiles. Hepatic toxicity has been observed in several studies of 213Bi-labeled antibodies (36,80,81). Mice treated with lethal doses of 225Ac-HuM195 developed gastrointestinal sloughing and marrow hypoplasia (David Scheinberg, unpublished data). Cynomolgus monkeys given repeated injections of 225Ac-HuM195 over a 9-month period developed renal failure, anemia, and hepatic injury (David Scheinberg, unpublished data). These toxicities are likely related to the effects of F-emitting daughters produced by the decay of 225Ac. The use of chelators to scavenge these free daughter isotopes from the circulation is a promising approach to ameliorate potential toxicity.

SUMMARY The role of monoclonal antibodies in the treatment of cancer is increasing. Most radioimmunotherapy trials have been performed with G-emitting isotopes. In contrast to G-emitters, the shorter range and higher LET of F-particles allow for more efficient and selective killing of individual tumor cells. Although some experimental models indicate that F-particle immunotherapy may eradicate large tumor burdens, the physical properties of F-irradiation and the clinical trials to date suggest that radioimmunotherapy with F-emitters may be best suited for the treatment of small-volume disease. While results of early studies appear promising, there are several obstacles to the widespread use of F-particle immunotherapy. To address these difficulties, new sources and methods of production of F-emitters and improved chelation chemistry must be developed. Additional

126

Burke, Scheinberg, and Jurcic

preclinical and clinical investigations are necessary to define optimal radioisotopes, dosing regimens, and therapeutic strategies.

ACKNOWLEDGMENTS The authors would like to thank Dr. Christophe Antczak for his expertise in chelation chemistry and assistance in preparing this manuscript.

REFERENCES 1. Reff ME, Carner K, Chambers KS, et al. Depletion of B cells in vivo by a chimeric mouse human monoclonal antibody to CD20. Blood 1994;83:435–445. 2. Shan D, Ledbetter JA, Press OW. Apoptosis of malignant human B cells by ligation of CD20 with monoclonal antibodies. Blood 1998;91:1644–1652. 3. Maloney DG, Smith B, Rose A. Rituximab: mechanism of action and resistance. Semin Oncol 2002;29:2–9. 4. Bross PF, Beitz J, Chen G, et al. Approval summary: gemtuzumab ozogamicin in relapsed acute myeloid leukemia. Clin Cancer Res 2001;7:1490–1496. 5. Witzig TE, Gordon LI, Cabanillas F, et al. Randomized controlled trial of yttrium-90-labeled ibritumomab tiuxetan radioimmunotherapy versus rituximab immunotherapy for patients with relapsed or refractory low-grade, follicular, or transformed B-cell non-Hodgkin’s lymphoma. J Clin Oncol 2002;20:2453–2463. 6. Vaidyanathan G, Zalutsky MR. Targeted therapy using alpha emitters. Phys Med Biol 1996;41:1915–1931. 7. Zalutsky MR, Bigner DD. Radioimmunotherapy with alpha-particle emitting radioimmunoconjugates. Acta Oncol 1996;35:373–379. 8. McDevitt MR, Sgouros G, Finn RD, et al. Radioimmunotherapy with alpha-emitting nuclides. Eur J Nucl Med 1998;25:1341–1351. 9. Humm JL. Dosimetric aspects of radiolabeled antibodies for tumor therapy. J Nucl Med 1986;27:1490–1497. 10. Humm JL. A microdosimetric model of astatine-211 labeled antibodies for radioimmunotherapy. Int J Radiat Oncol Biol Phys 1987;13:1767–1773. 11. Blakely EA, Kronenberg A. Heavy-ion radiobiology: new approaches to delineate mechanisms underlying enhanced biological effectiveness. Radiat Res 1998;150:S126–S145. 12. Jonathan EC, Bernhard EJ, McKenna WG. How does radiation kill cells? Curr Opin Chem Biol 1999;3:77–83. 13. Nunez MI, Villalobos M, Olea N, et al. Radiation-induced DNA double-strand break rejoining in human tumour cells. Br J Cancer 1995;71:311–316. 14. Kolesnick RN, Haimovitz-Friedman A, Fuks Z. The sphingomyelin signal transduction pathway mediates apoptosis for tumor necrosis factor, Fas, and ionizing radiation. Biochem Cell Biol 1994;72:471–474. 15. Pandita TK, Lieberman HB, Lim DS, et al. Ionizing radiation activates the ATM kinase throughout the cell cycle. Oncogene 2000;19:1386–1391. 16. Kastan MB, Lim DS. The many substrates and functions of ATM. Nat Rev Mol Cell Biol 2000;1:179–186. 17. Munro TR. The relative radiosensitivity of the nucleus and cytoplasm of Chinese hamster fibroblasts. Radiat Res 1970;42:451–470. 18. Hall EJ. Radiobiology for the Radiologist. Fifth ed. Lippincott Williams & Wilkins, Philadelphia:2000.

Chapter 5 / Targeted F-Particle Therapy

127

19. Kampf G. Induction of DNA double-strand breaks by ionizing radiation of different quality and their relevance for cell inactivation. Radiobiol Radiother (Berl) 1988;29:631–658. 20. Jenner TJ, deLara CM, O’Neill P, Stevens DL. Induction and rejoining of DNA doublestrand breaks in V79-4 mammalian cells following gamma- and alpha-irradiation. Int J Radiat Biol 1993;64:265–273. 21. Gadbois DM, Crissman HA, Nastasi A, et al. Alterations in the progression of cells through the cell cycle after exposure to alpha particles or gamma rays. Radiat Res 1996;146:414–424. 22. Lucke-Huhle C, Blakely EA, Chang PY, Tobias CA. Drastic G2 arrest in mammalian cells after irradiation with heavy-ion beams. Radiat Res 1979;79:97–112. 23. Woloschak GE, Chang-Liu CM. Differential modulation of specific gene expression following high- and low-LET radiations. Radiat Res 1990;124:183–187. 24. Bird RP, Rohrig N, Colvett RD, et al. Inactivation of synchronized Chinese Hamster V79 cells with charged-particle track segments. Radiat Res 1980;82:277–289. 25. Todd P, Wood JC, Walker JT, Weiss SJ. Lethal, potentially lethal, and nonlethal damage induction by heavy ions in cultured human cells. Radiat Res Suppl 1985;8:S5–S12. 26. Roberts CJ, Goodhead DT. The effect of 238Pu alpha-particles on the mouse fibroblast cell line C3H 10T1/2: characterization of source and RBE for cell survival. Int J Radiat Biol Relat Stud Phys Chem Med 1987;52:871–882. 27. Raju MR, Eisen Y, Carpenter S, Inkret WC. Radiobiology of alpha particles. III. Cell inactivation by alpha-particle traversals of the cell nucleus. Radiat Res 1991;128:204–209. 28. Strickland DK, Vaidyanathan G, Zalutsky MR. Cytotoxicity of alpha-particle-emitting m[211At]astatobenzylguanidine on human neuroblastoma cells. Cancer Res 1994;54:5414–5419. 29. Vaidyanathan G, Larsen RH, Zalutsky MR. 5-[211At]astato-2'-deoxyuridine, an alpha particle-emitting endoradiotherapeutic agent undergoing DNA incorporation. Cancer Res 1996;56:1204–1209. 30. Larsen RH, Akabani G, Welsh P, Zalutsky MR. The cytotoxicity and microdosimetry of astatine-211-labeled chimeric monoclonal antibodies in human glioma and melanoma cells in vitro. Radiat Res 1998;149:155–162. 31. Lloyd EL, Gemmell MA, Henning CB, et al. Cell survival following multiple-track alpha particle irradiation. Int J Radiat Biol Relat Stud Phys Chem Med 1979;35:23–31. 32. Methods of production of 225Ac and 213Bi for alpha immunotherapy. ITU Annual Report 1995 (EUR 16368)—Basic Actinide Research:55–56. 33. Geerlings MW, Kaspersen FM, Apostolidis C, van der Hout R. The feasibility of 225Ac as a source of alpha-particles in radioimmunotherapy. Nucl Med Commun 1993;14:121–125. 34. McDevitt MR, Ma D, Lai LT, et al. Tumor therapy with targeted atomic nanogenerators. Science 2001;294:1537–1540. 35. McDevitt MR, Finn RD, Sgouros G, et al. An 225Ac/213Bi generator system for therapeutic clinical applications: construction and operation. Appl Radiat Isot 1999;50:895–904. 36. Jurcic JG, Larson SM, Sgouros G, et al. Targeted alpha particle immunotherapy for myeloid leukemia. Blood 2002;100:1233–1239. 37. Burke JM, Jurcic JG, Divgi CR, et al. Sequential cytarabine and alpha-particle immunotherapy with bismuth-213-labeled anti-CD33 monoclonal antibody HuM195 in acute myeloid leukemia (AML). Blood 2002;100:339a (abstract #1314). 38. Zalutsky MR, Vaidyanathan G. Astatine-211-labeled radiotherapeutics: an emerging approach to targeted alpha-particle radiotherapy. Curr Pharm Des 2000;6:1433–1455. 39. Zalutsky MR, Narula AS. Astatination of proteins using an N-succinimidyl tri-n-butylstannyl benzoate intermediate. Int J Rad Appl Instrum [A] 1988;39:227–232. 40. Zalutsky MR, Garg PK, Friedman HS, Bigner DD. Labeling monoclonal antibodies and F(ab')2 fragments with the alpha-particle-emitting nuclide astatine-211: preservation of immunoreactivity and in vivo localizing capacity. Proc Natl Acad Sci USA 1989;86:7149–7153.

128

Burke, Scheinberg, and Jurcic

41. Bloomer WD, McLaughlin WH, Neirinckx RD, et al. Astatine-211—tellurium radiocolloid cures experimental malignant ascites. Science 1981;212:340–1. 42. Bloomer WD, McLaughlin WH, Lambrecht RM, et al. 211At radiocolloid therapy: further observations and comparison with radiocolloids of 32P, 165Dy, and 90Y. Int J Radiat Oncol Biol Phys 1984;10:341–348. 43. Vaidyanathan G, Zalutsky MR. 1-(m-[ 211At]astatobenzyl)guanidine: synthesis via astato demetalation and preliminary in vitro and in vivo evaluation. Bioconjug Chem 1992;3:499–503. 44. Brown I, Carpenter RN, Link E, Mitchell JS. Potential diagnostic and therapeutic agents for malignant melanoma: synthesis of heavy radiohalogenated derivatives of methylene blue by electrophilic and nucleophilic methods. J Radioanal Nucl Chem, Letters 1986;107:337–351. 45. Link EM, Brown I, Carpenter RN, Mitchell JS. Uptake and therapeutic effectiveness of 125Iand 211At-methylene blue for pigmented melanoma in an animal model system. Cancer Res 1989;49:4332–4337. 46. Vaidyanathan G, Strickland DK, Zalutsky MR. Meta-[211At]astatobenzylguanidine: further evaluation of a potential therapeutic agent. Int J Cancer 1994;57:908–913. 47. Foulon CF, Schoultz BW, Zalutsky MR. Preparation and biological evaluation of an astatine-211 labeled biotin conjugate: biotinyl-3-[ 211 At]astatoanilide. Nucl Med Biol 1997;24:135–143. 48. Murud KM, Larsen RH, Hoff P, Zalutsky MR. Synthesis, purification, and in vitro stability of 211At- and 125I-labeled amidobisphosphonates. Nucl Med Biol 1999;26:397–403. 49. Yao Z, Garmestani K, Wong KJ, et al. Comparative cellular catabolism and retention of astatine-, bismuth-, and lead-radiolabeled internalizing monoclonal antibody. J Nucl Med 2001;42:1538–1544. 50. Atcher RW, Friedman AM, Hines JJ. An improved generator for the production of 212Pb and 212Bi from 224Ra. Int J Rad Appl Instrum [A] 1988;39:283–286. 51. Macklis RM, Kinsey BM, Kassis AI, et al. Radioimmunotherapy with alpha-particle-emitting immunoconjugates. Science 1988;240:1024–1026. 52. Huneke RB, Pippin CG, Squire RA, et al. Effective alpha-particle-mediated radioimmunotherapy of murine leukemia. Cancer Res 1992;52:5818–5820. 53. Junghans RP, Dobbs D, Brechbiel MW, et al. Pharmacokinetics and bioactivity of 1,4,7,10tetra-azacylododecane off’,N’’,N’’’-tetraacetic acid (DOTA)-bismuth-conjugated anti-Tac antibody for alpha-emitter (212Bi) therapy. Cancer Res 1993;53:5683–5689. 54. Kozak RW, Atcher RW, Gansow OA, et al. Bismuth-212-labeled anti-Tac monoclonal antibody: alpha-particle-emitting radionuclides as modalities for radioimmunotherapy. Proc Natl Acad Sci U S A 1986;83:474–478. 55. Kurtzman SH, Russo A, Mitchell JB, et al. 212Bismuth linked to an antipancreatic carcinoma antibody: model for alpha-particle-emitter radioimmunotherapy. J Natl Cancer Inst 1988;80:449–452. 56. Macklis RM, Lin JY, Beresford B, et al. Cellular kinetics, dosimetry, and radiobiology of alpha-particle radioimmunotherapy: induction of apoptosis. Radiat Res 1992;130:220–226. 57. Simonson RB, Ultee ME, Hauler JA, Alvarez VL. Radioimmunotherapy of peritoneal human colon cancer xenografts with site-specifically modified 212Bi-labeled antibody. Cancer Res 1990;50:985s–988s. 58. Gansow OA, Brechbiel MW, Pippin CG, et al. Lead and bismuth complexes of functionalized DTPA ligands and of the polyazacycloalkane-N-acetic acid DOTA. Utility for radioimmunoimaging and radioimmunotherapy. Antibody Immunoconj Radiopharm 1991;4:413–425. 59. Kumar K, Magerstadt M, Gansow OA. Lead(II) and bismuth(III) complexes of the polyazacycloalkane-N-acetic acids nota, dota, and teta. J Chem Soc Chem Commun 1989:145–146.

Chapter 5 / Targeted F-Particle Therapy

129

60. Horak E, Hartmann F, Garmestani K, et al. Radioimmunotherapy targeting of HER2/ neu oncoprotein on ovarian tumor using lead-212-DOTA-AE1. J Nucl Med 1997;38:1944–1950. 61. Larsen RH. Biomedical application of alpha-particle-emitting radium-223. 225th American Chemical Society National Meeting 2003:40 (Abstract 111). 62. Ruegg CL, Anderson-Berg WT, Brechbiel MW, et al. Improved in vivo stability and tumor targeting of bismuth-labeled antibody. Cancer Res 1990;50:4221–4226. 63. Brechbiel MW, Pippin CG, McMurry TJ, et al. An effective chelating agent for labelling of monoclonal antibody with 212Bi for alpha-particle mediated radioimmunotherapy. J Chem Soc Chem Commun 1991:1169–1170. 64. McDevitt MR, Finn RD, Ma D, et al. Preparation of alpha-emitting 213Bi-labeled antibody constructs for clinical use. J Nucl Med 1999;40:1722–1727. 65. Nikula TK, McDevitt MR, Finn RD, et al. Alpha-emitting bismuth cyclohexylbenzyl DTPA constructs of recombinant humanized anti-CD33 antibodies: pharmacokinetics, bioactivity, toxicity and chemistry. J Nucl Med 1999;40:166–176. 66. Kaspersen FM, Bos E, Doornmalen AV, et al. Cytotoxicity of 213Bi- and 225Ac-immunoconjugates. Nucl Med Commun 1995;16:468–476. 67. Sgouros G, Ballangrud AM, Jurcic JG, et al. Pharmacokinetics and dosimetry of an alphaparticle emitter labeled antibody: 213Bi-HuM195 (anti-CD33) in patients with leukemia. J Nucl Med 1999;40:1935–1946. 68. Humm JL, Roeske JC, Fisher DR, Chen GT. Microdosimetric concepts in radioimmunotherapy. Med Phys 1993;20:535–541. 69. Russ GA, Bigler RE, Tilbury RS, et al. Metabolic studies with radiobismuth. I. Retention and distribution of 206Bi in the normal rat. Radiat Res 1975;63:443–454. 70. Hartmann F, Horak EM, Garmestani K, et al. Radioimmunotherapy of nude mice bearing a human interleukin 2 receptor alpha-expressing lymphoma utilizing the alpha-emitting radionuclide- conjugated monoclonal antibody 212Bi-anti-Tac. Cancer Res 1994;54:4362–4370. 71. Behr TM, Behe M, Stabin MG, et al. High-linear energy transfer (LET) alpha versus lowLET beta emitters in radioimmunotherapy of solid tumors: therapeutic efficacy and doselimiting toxicity of 213Bi- versus 90Y-labeled CO17-1A Fab' fragments in a human colonic cancer model. Cancer Res 1999;59:2635–2643. 72. Andersson H, Palm S, Lindegren S, et al. Comparison of the therapeutic efficacy of 211Atand 131I-labelled monoclonal antibody MOv18 in nude mice with intraperitoneal growth of human ovarian cancer. Anticancer Res 2001;21:409–412. 73. Andersson H, Lindegren S, Back T, et al. Radioimmunotherapy of nude mice with intraperitoneally growing ovarian cancer xenograft utilizing 211At-labelled monoclonal antibody MOv18. Anticancer Res 2000;20:459–462. 74. Kennel SJ, Stabin M, Roeske JC, et al. Radiotoxicity of bismuth-213 bound to membranes of monolayer and spheroid cultures of tumor cells. Radiat Res 1999;151:244–256. 75. Ballangrud AM, Yang WH, Charlton DE, et al. Response of LNCaP spheroids after treatment with an alpha-particle emitter (213Bi)-labeled anti-prostate-specific membrane antigen antibody (J591). Cancer Res 2001;61:2008–2014. 76. Langmuir VK, Atcher RW, Hines JJ, Brechbiel MW. Iodine-125-NRLU-10 kinetic studies and bismuth-212-NRLU-10 toxicity in LS174T multicell spheroids. J Nucl Med 1990;31:1527–1533. 77. McDevitt MR, Barendswaard E, Ma D, et al. An alpha-particle emitting antibody ([213Bi]J591) for radioimmunotherapy of prostate cancer. Cancer Res 2000;60:6095–6100. 78. McSweeney PA, Niederwieser D, Shizuru JA, et al. Hematopoietic cell transplantation in older patients with hematologic malignancies: replacing high-dose cytotoxic therapy with graft-versus-tumor effects. Blood 2001;97:3390–3400.

130

Burke, Scheinberg, and Jurcic

79. Niederwieser D, Maris M, Shizuru JA, et al. Low-dose total body irradiation (TBI) and fludarabine followed by hematopoietic cell transplantation (HCT) from HLA-matched or mismatched unrelated donors and postgrafting immunosuppression with cyclosporine and mycophenolate mofetil (MMF) can induce durable complete chimerism and sustained remissions in patients with hematological diseases. Blood 2003;101:1620–1629. 80. Sandmaier BM, Bethge WA, Wilbur DS, et al. Bismuth 213-labeled anti-CD45 radioimmunoconjugate to condition dogs for nonmyeloablative allogeneic marrow grafts. Blood 2002;100:318–326. 81. Bethge WA, Wilbur DS, Storb R, et al. Selective T-cell ablation with bismuth-213 labeled anti-TCRFG as nonmyeloablative conditioning for allogeneic canine marrow transplantation. Blood 2003;101(12):5068–5075. 82. Kikly K, Dennert G. Evidence for a role for T cell receptors (TCR) in the effector phase of acute bone marrow graft rejection. TCR V beta 5 transgenic mice lack effector cells able to cause graft rejection. J Immunol 1992;149:3489–3494. 83. Zalutsky MR, McLendon RE, Garg PK, et al. Radioimmunotherapy of neoplastic meningitis in rats using an alpha- particle-emitting immunoconjugate. Cancer Res 1994;54:4719–4725. 84. Zalutsky MR, Zhao XG, Alston KL, Bigner D. High-level production of alpha-particleemitting (211)At and preparation of (211)At-labeled antibodies for clinical use. J Nucl Med 2001;42:1508–1515. 85. Zalutsky MR, Cokgor I, Akabani G, et al. Phase I trial of alpha-particle-emitting astatine211 labeled chimeric anti-tenascin antibody in recurrent malignant glioma patients. Proc Am Assoc Cancer Res 2000;41:544 (abstract #3465). 86. Bourdon MA, Wikstrand CJ, Furthmayr H, et al. Human glioma-mesenchymal extracellular matrix antigen defined by monoclonal antibody. Cancer Res 1983;43:2796–2805. 87. Link EM, Carpenter RN. 211At-methylene blue for targeted radiotherapy of human melanoma xenografts: treatment of micrometastases. Cancer Res 1990;50:2963–2967. 88. Link EM, Blower PJ, Costa DC, et al. 211At-methylene blue for treatment of disseminated melanoma. Melanoma Res 1997;7(Suppl 1):s44 (abstract). 89. Link EM. Targeting melanoma with 211At/131I-methylene blue: preclinical and clinical experience. Hybridoma 1999;18:77–82. 90. Link EM, Carpenter RN. 211At-methylene blue for targeted radiotherapy of human melanoma xenografts: treatment of cutaneous tumors and lymph node metastases. Cancer Res 1992;52:4385–4390. 91. Aurlien E, Kvinnsland Y, Larsen RH, Bruland OS. Radiation doses to non-Hodgkin’s lymphoma cells and normal bone marrow exposed in vitro. Comparison of an alpha-emitting radioimmunoconjugate and external gamma-irradiation. Int J Radiat Biol 2002;78:133–142. 92. Aurlien E, Larsen RH, Kvalheim G, Bruland OS. Demonstration of highly specific toxicity of the alpha-emitting radioimmunoconjugate(211)At-rituximab against non-Hodgkin’s lymphoma cells. Br J Cancer 2000;83:1375–1379. 93. Heeger S, Moldenhauer G, Egerer G, et al. Alpha radioimmunotherapy of B-lineage nonHodgkin’s lymphoma using 213Bi-labeled anti-CD19- and anti-CD20-CHX-A”-DTPA conjugates. 225th American Chemical Society National Meeting 2003:29 (Abstract 78). 94. Axworthy DB, Reno JM, Hylarides MD, et al. Cure of human carcinoma xenografts by a single dose of pretargeted yttrium-90 with negligible toxicity. Proc Natl Acad Sci USA 2000;97:1802–1807. 95. Zhang M, Yao Z, Garmestani K, et al. Pretargeting radioimmunotherapy of a murine model of adult T-cell leukemia with the alpha-emitting radionuclide, bismuth 213. Blood 2002;100:208–216. 96. Kennel SJ, Boll R, Stabin M, et al. Radioimmunotherapy of micrometastases in lung with vascular targeted 213Bi. Br J Cancer 1999;80:175–184.

Chapter 5 / Targeted F-Particle Therapy

131

97. Davis IA, Kennel SJ. Radioimmunotherapy using vascular targeted 213Bi: the role of tumor necrosis factor alpha in the development of pulmonary fibrosis. Clin Cancer Res 1999;5:3160s–3164s. 98. Cobb LM, Harrison A, Butler SA. Toxicity of astatine-211 in the mouse. Hum Toxicol 1988;7:529–534. 99. Cobb LM, Butler SA, Harrison A. The effect of the alpha-particle emitter astatine-211 in the mouse at the minimum toxic dose. Hum Exp Toxicol 1990;9:289–293. 100. McLendon RE, Archer GE, Garg PK, et al. Radiotoxicity of systematically administered [211At]astatide in B6C3F1 and BALB/c (nu/nu) mice: a long-term survival study with histologic analysis. Int J Radiat Oncol Biol Phys 1996;35:69–80. 101. Durbin PW, Asling CW, Johnston ME, et al. The induction of tumors in the rat by astatine211. Radiat Res 1958;9:378–397. 102. McLendon RE, Archer GE, Larsen RH, et al. Radiotoxicity of systemically administered 211At-labeled human/mouse chimeric monoclonal antibody: a long-term survival study with histologic analysis. Int J Radiat Oncol Biol Phys 1999;45:491–499. 103. Link EM, Michalowski AS, Rosch F. 211At-methylene blue in targeted radiotherapy of disseminated melanoma. Pigment Cell Res 1994;7:358–362. 104. Link EM, Michalowski AS, Rosch F. 211At-methylene blue for targeted radiotherapy of disseminated melanoma: microscopic analysis of tumour versus normal tissue damage. Eur J Cancer 1996;32A:1986–1994.

Chapter 6 / Fluoropyrimidine Modulation

6

133

Pharmacological Modulation of Fluoropyrimidines Building on the Lessons of the Past

Owen A. O’Connor, MD, PhD CONTENTS INTRODUCTION BIOCHEMICAL PHARMACOLOGY MODULATING THE EFFECTS OF FLUORINATED PYRIMIDINES RIBONUCLEOTIDE REDUCTASE INHIBITORS SO, WHERE DO WE GO FROM HERE?

INTRODUCTION Few drugs in the present pharmacopia of antineoplastic pharmacology have exemplified the importance of the bench-to-bed paradigm that has become the cornerstone of present-day developmental chemotherapy. From its rational synthesis by Heidelberger in 1957 (1) to quantification of the molecular determinants of fluorinated pyrimidine (fluoropyrimidine) activity, our understanding of fluoropyrimidine pharmacology has helped mold our concepts of everything from target-oriented drug development to molecular phenotyping, and drug resistance to drug synergy. It is from our thorough understanding of the biochemical pharmacology of 5-fluorouracil (5-FU) that we have learned how to exploit this biochemistry to improve the cytotoxicity of 5-FU. Regrettably, although this augmented understanding has improved overall response rates and time to disease progression parameters in select clinical trials, it infrequently results in improved survival—the end-point that probably matters most. Pharmacological modulation of any drug revolves around understanding those critical relationships between biochemical targets in the cell, and how we can exploit From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

133

134

O’Connor

their underlying biology in a way that renders any combination of agents complementary. Over the past 45 yr, tomes have been written on virtually every aspect of this paradigm with regard to fluoropyrimidines. I will not reiterate the details of this experience. Rather, in this chapter, I will briefly review the historical experience in order to provide a context for the formulation of new concepts and approaches. Unquestionably, however, future innovations in the use of the fluoropyrimidines, or any other class of antineoplastic agents for that matter, will be markedly augmented by understanding the essential elements of the drug’s biochemical pharmacology.

BIOCHEMICAL PHARMACOLOGY Fluorinated Pyrimidine Biochemistry The class of drugs comprised under the larger rubric of fluoropyrimidines is a diverse group of compounds that have the capacity to obstruct DNA synthesis by inhibiting nucleotide synthesis, or by posing as fraudulent substrates of their natural counterparts (Fig. 1). The observation that ultimately led to the development these compounds came from the early observation by Rutman (2) that rat hepatomas utilized uracil-2-C14 more avidly than did normal tissues. In an effort to rationally develop a compound that could interfere with the cellular demands of uracil in tumor cells, Heidelberger (1) and his colleagues synthesized a series of uracil analogs, eventually identifying 5-FU as one compound with significant antitumor activity. The fluorine (F) substitution for the hydrogen atom at the 5-position of uracil creates a molecule that is only modestly different from uracil, given the similar van der Waals radii of fluorine and hydrogen (Fig. 1). The major molecular difference between the two compounds lies in the nature of the C–F bond, which is considerably stronger than the normal C–H bond. As a result of the greater bond strength between the carbon and fluorine, the fluorine atom cannot be eliminated like the hydrogen atom on the native molecule, and thus the subsequent methylation of the 5 position cannot occur. In the presence of the polyglutamated derivatives of the methyl-donor cofactor 5,10-methylene tetrahydrofolate (CH2-THF), the fluoropyrimidine forms a tight, although reversible, ternary complex with thymidylate synthase (TS), which functionally inhibits the enzymatic activity of TS. The thymidylate synthase enzyme consists of two identical subunits (36 kDa each), each of which possesses a nucleotidebinding site (usually reserved for uracil nucleotides), and two separate folatebinding sites, one for the CH2-THF and one for the oxidized dihydrofolate (DHF). The thermodynamics of the methylation reaction are dependent upon the ordered interaction of nucleotide and folate, with the nucleotide binding first. This ordered binding effects a conformational change in TS that results in a greater binding affinity for the polyglutamylated CH2-THF. When the fluoropyrimidine

Chapter 6 / Fluoropyrimidine Modulation

135

Fig. 1. Chemical structures of the pyrimidines uracil, thymine, 5-FU, and FdUMP.

5-fluorodexyuridine monophosphate (FdUMP) is substituted for uracil nucleotides in the ternary complex, the methylene group of the folate forms a noncovalent linkage with the C-5 position of FdUMP to create a poorly dissociable complex. In comparison, substitution at the 5-position with the larger halogen atoms bromine or iodine generates an analog that behaves more like thymidine rather than uracil, given the similarities in van der Waals radii between bromine or iodine and the methyl moiety. Phosphorylation of 5-iododeoxyuridine (idoxuridine), for example, results in a fraudulent nucleotide that is incorporated into DNA in place of thymidine nucleotides. Whereas other naturally occurring folates can promote the binding of FdUMP to TS, these complexes are typically less stable and are more readily dissociable (Fig. 2). The metabolic activation of 5-FU inside the cell leads to the generation of a panoply of different metabolites, many of which can have distinctly different effects on the cell. These pathways are summarized in Fig. 2. The mandatory initial steps in this activation include ribosylation and phosphorylation. Typically, 5-FU can be metabolized to fluorouridine (FUrd) or fluorodeoxyuridine (FdUR) by uridine or thymidine phosphorylase, respectively. These reactions generate substrates for the intracellular kinases (uridine and thymidine kinase), which catalyze the conversion of the nucleoside to the nucleotide. As can be seen from Fig. 2, it is the triphosphate derivatives FUTP or FdUTP that can be theoretically utilized by either RNA or DNA polymerase, leading to the fraudulent

136

O’Connor

Fig. 2. Metabolic pathway of 5-FU anabolism and catabolism.

incorporation of the fluoropyrimidine. In addition, 5-fluorouridine diphosphate (the ribose form of the nucleotide) can be converted to the 5-fluorodeoxyuridine diphosphate (the deoxyribose-form of the nucleotide) through ribonucleotide reductase, a metabolic step that can be inhibited with hydroxyurea. The alternative and likely dominant pathway of 5-FU activation involves the conversion of 5-FU to FUMP by orotate phophoribosyltransferase (OPRT) using the cofactor 5-phosphoribosyl-1-pyrophoasphate (PRPP). This pathway generates an intermediate that could go on to be incorporated into RNA, DNA, or converted to FdUMP, the eventual TS inhibitor. These steps can also be bypassed to a certain degree with another well known and commonly employed fluoropyrimidine, 5-fluorodeoxy-uridine (floxuridine; FdUR; or FUDR). FUDR has been found to be more active than 5-FU when given as a continuous infusion, because its activation requires only a single step by thymidine kinase to produce FdUMP(3,4). FUDR can be catabolized by thymidine phosphorylase to 5-FU, and is itself not a substrate for the catabolic enzyme dihydropyrimidine dehydrogenase (DPD). As can be seen in Fig. 2, 5-FU is a prodrug. Its cytotoxic effects are absolutely dependent on its metabolic activation and conversion to anyone of a number of different metabolites, as outlined above. The essential reaction sequence that appears to account for the predominant fraction of the drug’s cytotoxicity, however, appears to proceed through the conversion of 5-FU to FUDP, to FdUDP, and eventually to FdUMP, which results in the inhibition of de novo thymidylate synthesis.

Chapter 6 / Fluoropyrimidine Modulation

137

Cellular Consequences of Fluorinated Pyrimidine Exposure The mechanism of action of 5-FU is thought to involve at least three distinct cellular effects: (1) inhibition of thymidylate synthase (TS), leading to insufficient quantities of the nucleotide required for DNA biosynthesis, and eventually apoptosis due to the thymineless state; (2) fraudulent incorporation of fluorouridine triphosphate (FUTP) into RNA; and (3) fraudulent incorporation of FdUTP and dUTP into DNA. Although it is difficult at best to decipher the precise causeand-effect mechanism responsible for the ultimate cytotoxic effect of 5-FU inside the cell, it is clear that different schedules of drug administration and different co-administered agents can influence particular pathways of cytotoxicity. Sobrero et al. (5) have shown that pulsed 5-FU produces a higher transient plasma level of 5-FU and appeared to increase the degree of fluorouridine triphosphate incorporation into RNA, which itself could directly inhibit the de novo synthesis of thymidylate at the transcriptional level. In contrast, a low-dose continuous infusion schedule for 5 to 7 d produced significantly less RNA incorporation and more prolonged TS inhibition. Interestingly, it has been shown in intestinal epithelial cells, which undergo apoptosis with sometimes only minor physiological perturbations, that 5-FU induced apoptosis is p53 dependent and is mediated by incorporation of fraudulent FUTP into RNA6. Clinically, patients found to be resistant to one schedule of administration have responded to the alternative schedule, supporting this dual mechanism of activity and lack of cross resistance (5). Nonetheless, the inhibition of TS, and the RNA incorporation effects, are thought to be the major effects resulting in cell death. Efficient DNA synthesis is absolutely dependent upon the sufficient supply of deoxyribonucleotides, including dTTP, dCTP, dATP, and dGTP. The enzymatic step catalyzed by TS is considered the rate-limiting step in the synthesis of thymidine nucleotide precursors. Depletion of these nucleotides in cells that are actively dividing can lead to mis-incorporation of other nucleotides (uracil, fluorinated pyrimidines), leading to apoptosis. Central to the maintenance of these reactions is the recycling and regeneration of reduced folate cofactors (Fig. 3). Following methylation of dUMP, the cofactor CH2-THF is oxidized to dihydrofolate, and is no longer in a form that allows participation as a cofactor for TS. Dihydrofolate is subsequently reduced by dihydrofolate reductase (DHFR) to tetrahydrofolate (THF), and then converted to the 5,10-methylene tetrahydrofolate cofactor by the enzyme serine transhydroxymethylase. These enzymes and cofactors constitute the core of an important set of enzymatic reactions inside the cell known as the thymidylate cycle (Fig. 4).

MODULATING THE EFFECTS OF FLUORINATED PYRIMIDINES There is little doubt that our present understanding of the effects that other agents have on modulating fluorinated pyrimidines has occurred because there

138

O’Connor

Fig. 3. The role of N5, N10-methylenetetrahydrofolate in thymidylate biosynthesis.

are so few drugs that have proven to be better than 5-FU alone in the treatment of advanced colorectal cancer (CRC). Save recent evidence regarding the benefits in time to progression and overall survival from the addition of irinotecan with 5-FU and leucovorin (LV) (7–9) (Table 1), there has been remarkably little to no impact from other newer single agents or combination chemotherapy regimens on clinical outcome in patients with advanced or recurrent CRC (4). Unquestionably, many thousands of lives have been saved by adjuvant treatment with combination regimens including 5-FU with or without leucovorin. The addition of newer agents offers new opportunities to enhance the effectiveness of adjuvant therapy and improve the survival of patients with advanced metastatic CRC and other malignancies (4).

Reduced Folic Acid Derivatives Both preclinical and clinical data have clearly established that the reduced folate leucovorin (5-formyltetrahydrofoale; citrovorum factor; CHO-FH4) is effective in the biomodulation of fluorouracil. To date, an extensive literature has emerged that documents the clinical outcome of various schedules of 5-FU with

Chapter 6 / Fluoropyrimidine Modulation

139

Fig. 4. The thymidylate cycle.

or without leucovorin. Although many of these results are well summarized in great detail elsewhere (10,11), it is clear that the addition of LV to 5-FU produces response rates that are greater than those noted in patients receiving singleagent bolus 5-FU, and likely does not add to the results of infusional schedules of 5-FU (12–18). The basis for this effect revolves around the formation of the ternary complex as discussed above. Ternary complex formation is dependent upon the intracellular concentration of reduced 5,10-methylene tetrahydrofolate, with greater intracellular folate pools favoring ternary complex formation and more durable TS inhibition (19–24). These observations are validated to some extent by studies that have determined the degree of TS inhibition in malignant tissue derived from patients receiving treatment. In one such study, Swain (25) showed that in tissue from patients with metastatic breast cancer, the median TS inhibition in tumor tissue following treatment with 5-FU alone was 30%, compared to 71% in tumors from patients receiving 5-FU and highdose LV. In a slightly different study, Peters (26) showed that the addition of LV reduced the residual TS activity in tumor biopsy specimens from patients with colorectal cancer from 74% to 49%, and that the number of free FdUMPbinding sites on the TS enzyme decreased from 49 to 24%, suggesting more effective TS inhibition in the tumor. Despite the favorable evidence to date that leucovorin can modulate the effects of bolus 5-FU, there is considerable controversy regarding the best schedule of leucovorin to use with the fluoropyrimidines. Many questions remain, including:

140

O’Connor Table 1 Targets That, When Modulated, Augment Fluoropyrimidine Cytotoxicity in Preclinical and Clinical Models

Modulated target

Drug(s)

Mechanism of action

Thymidylate synthase (TS) (specifically the antifol binding sites on the TS enzyme) PRPP Accumulation

Leucovorin

Increased binding affinity of fluoropyrimidine, stabilizes formation of ternary complex

Methotrexate Trimatrexate Raltitrexed

Increased PRPP synthesis leads to increased levels of the cofactor required for orate phosphoribosyl-transferase conversion of 5-FU to 5-FUR Impairs the synthesis of de novo thymidylate synthesis, reduces dUMP levels Inhibits the catabolic degradation of 5-FU leading to more protracted levels of 5-FU

Ribonucleotide reductase

Dihydropyrimidine dehydrogenase (DPD)

Thymidine kinase

Hydroxyureas Gemcitabine Triapine Uracil CHDP Eniluracil CNDP AZT Acyclovir

Thymidine phosphorylase (TP) Taxotere Radiation Interferons Paclitaxel Mitomycin C Nucleoside transporters Dipyridamole Draflazine NBMPR

Impairs thymidine salvage pathways by inhibiting the phosphorylation of transported nucleoside Increased level of TP broadens the therapeutic window of the oral fluoropyrimidine carbamate ester capecitabine Impairs salvage of extracellular thymidine

PRPP, 5-phosphoribosyl-1-pyrophoasphate; CHDP, 5-chloro-2,4-dihydroxypyridine; CNDP, 3-cyano-2,6-dihydroxypyridine; AZT, 3'-Azido-3'-deoxythymidine; NBMPR, nitrobenzylthioinosine.

1. Are there schedule- or sequence-dependent factors to consider in the administration (i.e., which drug first?)? 2. Does the pharmacokinetic profile (i.e., dose, frequency of administration, and so on) of either drug influence the modulation? and 3. Does preferential absorption of the L-stereoisomer in the oral formulation of LV improve results given the possibility for competition between stereoisomers?

Chapter 6 / Fluoropyrimidine Modulation

141

Leucovorin (a racemic mixture of L [active] and D [inactive] stereoisomers) is metabolized to 5,10-methylene tetrahydrofolate, expanding that specific cofactor pool. What appears evident from preclinical studies is that 1 μM of LV is probably insufficient to expand the intracellular folate pools of CH2-THF, and that target concentrations of at least a log higher may be more efficacious. As one might expect, the concentration of LV required to expand the intracellular pools of CH2-THF decreases as the duration of LV exposure increases. Because LV is metabolized to CH2-THF, some investigators have used this as a rationale to support the use of LV first. Others, recognizing the ordered binding of nucleotide to TS first, have invoked schedules where the fluorouracil is given either first or simultaneously with the LV. Increasingly, there is an emerging sense that the results may be very dependent upon the cell lines and model studied, and may be influenced to varying levels by a host of variable molecular determinants. To date, the published results of at least 14 randomized trials comparing single-agent 5-FU to modulated 5-FU have shown a response rate of 25.3% among 1262 patients treated with 5-FU/LV compared to 13.8% among 995 patients receiving single-agent 5-FU (10,12,13,16,27–38). The median time to progression and overall survival for the modulated cohort was 6 mo and 12.4 mo, compared to 4.2 mo and 11 mo, respectively. An early meta-analysis reported by Piedbois (12) demonstrated an improvement in time to progression but not in overall survival. Except for one study reported by Poon (16), subsequent studies exploring different schedules (monthly vs weekly) revealed no statistically significant differences in response rate, or other outcome measures, though the dose-limiting toxicities were slightly different. On the weekly schedules, diarrhea was more common, while on the monthly schedules, patients developed both diarrhea and mucositis. Poon, using the monthly schedule, (16) did report a statistically significant survival advantage in the LV modulated 5-FU arm. Pharmacologically, we know that both the duration of exposure and concentration of 5-FU are key determinants of 5-FU cytotoxicity. Continuous infusion of 5-FU allows for a relatively high area under the concentration-time curve (AUC), and theoretically affects more cells as they eventually progress through the S phase of the cell cycle. Several studies have demonstrated the benefit of infusional 5-FU over intravenous bolus strategies (39–41). One such study, conducted by the Eastern Cooperative Oncology Group (ECOG), found a response rate of 28.3% for infusional 5-FU and 16.3% for bolus 5-FU (40), while a randomized study conducted by the Mid-Atlantic Oncology Program, comparing 300 mg/m2/d as a protracted infusion, produced a fourfold higher response rate compared with bolus 5-FU (29.9% vs 6.9%). Regrettably, none of these individual studies have ever reported a statistically significant survival advantage for the infusional schedules. A meta-analysis of six randomized trials reporting on 1219 patients with advanced colorectal cancer did find both a statistically

142

O’Connor

significantly response rate for infusional therapy over bolus therapy (22% vs 14%), with a statistically significant survival advantage in favor of the infusional arm (12.1 vs 11.3 mo, p = 0.04). In one interesting trial design developed by de Gramont and colleagues (15), they incorporated elements of both a bolus and infusional 5-FU regimen. The regimen (now known as the de Gramont regimen) administered 400 mg/m2 of 5FU as a bolus with 200 mg/m2 of LV over 2 h followed by 600 mg/m2 of 5-FU over 22 h on d 1 and 2 every other week. When the regimen was compared to the Mayo Clinic regimen, a higher response rate and longer time to progression in favor of the de Gramont regimen was found. Incorporation of leucovorin into the these infusional schedules appears to produce variable results with regard to response rates (some studies show improvement, others show no difference), and none has yet demonstrated any survival advantage to the arms containing LV (32,42). Whereas a variety of infusional 5-FU schedules have been studied with essentially equivalent results, the toxicity profile among the schedules is slightly different, with each carrying its own distinct advantages and disadvantages. The major drawback to the infusional schedules, however, relates to catheter-related complications. Approximately 20% of patients will require removal of the catheter for thrombosis, sepsis, malposition, or breakage, and an additional 10 to 15% of patents will develop an infection requiring antibiotics (10). The success with these infusional therapies, and the fact that oral formulations appear to pharmacokinetically mimic the infusional schedules without the need for catheters, has driven the development of oral fluorinated pyrimidines (UFT, a combination of uracil and tegafur; capecitabine). One conceivable approach that might lead to an improved benefit from fluoropyrimidine-based strategies is to define subpopulations of patients that might be more likely to benefit based upon the molecular phenotype of their disease. Such strategies could well normalize different populations of patients based upon the expression of key determinants of fluoropyrimidine sensitivity or resistance. The major molecular determinants of 5-FU activity include levels of TS, thymidine phosphorylase (TP), and dihydropyrimidine dehydrogenase (DPD) expression, with increases in all three enzymes portending greater resistance to 5-FU, and low-level expression portending greater sensitivity. In one such study, Salonga (43) correlated the response to 5-FU/LV to the level of TS, TP, and DPD expression. From the initial population of 33 patients, all 11 patients who responded to 5-FU demonstrated low expression levels of all three genes, and all 11 patients demonstrated significantly longer survival intervals than patients with high levels of TS, TP, and DPD gene expression. There was no correlation among the levels of expression of TS, TP, or DPD. These sorts of strategies will continue to play an important and emerging role in the tailored treatment of patients with colorectal and other solid-tumor malignancies. Based on our relatively comprehensive knowledge of 5-FU biochemical pharmacol-

Chapter 6 / Fluoropyrimidine Modulation

143

Fig. 5. Chemical structures of methotrexate and the nonclassical antifolate trimetrexate.

ogy, it is conceivable that the future treatment of patients with any specific drug regimen will be based upon their pattern of intratumoral gene expression As discussed, there have been many clinical studies seeking to identify the best schedule of administration of 5-FU with or without LV. Overall, administration of 5-FU by either a protracted intravenous infusion or as a weekly 24-h infusion appears to be equivalent to LV-modulated intravenous bolus 5-FU. While LV adds to the response rates of bolus 5-FU, its benefit in continuous infusion schedules remains unresolved, though the evidence does not suggest any dramatic overall benefit. The future of the field may be heading towards the use of molecular phenotyping as one way to risk stratify and tailor individual treatment programs, coupled with the integration of novel agents that affect tumor growth by alternative mechanisms of action.

Antifolates The biochemical modulation of 5-fluorouracil with antifolates is based on a number of theoretical considerations. The first revolves around the consideration that inhibition of DHFR by drugs like methotrexate (MTX) (Fig. 5), will result in accumulation of dihydrofolate polyglutamates, which are themselves sub-

144

O’Connor

strates for TS, capable of forming a ternary complex with FdUMP and TS. The second consideration revolves around the increase in intracellular levels of 5phophosribosyl-1-pyrophosphate (PRPP), presumably owing to the inhibition of purine biosynthesis. Dihydrofolate polyglutamates inhibit purine biosynthesis through the depletion of tetrahydrofolates, while the increase in PRPP increases the conversion of 5-FU to FUMP (Fig. 2). Collectively, the available data suggest that antifolates like MTX modulate 5-FU cytotoxicity by at least two separate mechanisms—the first involving the binding of FdUMP to TS, forming a ternary complex with dihydrofolate polyglutamates, and the second through the increased conversion of 5-FU to fluoropyrimidine nucleotides with a commensurate increase in fraudulent nucleotide incorporation. Many studies have clearly demonstrated that the combination of methotrexate and 5-FU are schedule dependent (44). Synergistic antitumor activity is appreciated only when the cells are exposed to methotrexate followed by 5-FU, with antagonistic effects being seen when the reverse schedule is used. Several pharmacodynamic studies have concluded that a 12- to 24-h interval between the administration of methotrexate and 5-FU leads to the most significant expansion of PRPP pools compared to other schedules (44–46). Several phase III studies have compared methotrexate administered at various intervals prior to 5-FU. In one study, the investigators directly compared a 1-h and a 24-h interval between 5-FU and MTX, and demonstrated superiority of the 24-h interval, successfully corroborating the pharmacodynamic experience (47–49). Interestingly, a metaanalysis of eight randomized studies comparing 5-FU vs MTX-modulated 5-FU revealed a significantly higher response rate (19% vs 10%), and overall survival advantage (10.7 vs 9.1 mo) in favor of the MTX-modulated 5-FU arm. Unfortunately, many of the study arms contained patients who simultaneously received LV, making it impossible to definitively conclude a benefit in favor of MTX, without the confounding influence of LV (50). Recently, two large randomized studies reported on the benefit of adding trimatrexate (Fig. 5) to the combination of 5-FU and LV (51,52). Trimatrexate is a nonclassical antifolate (2,4-diaminoquinazoline) that enters the cell by passive diffusion, does not require the reduced folate carrier (RFC-1), and hence does not compete with LV for cellular uptake. In addition, because the molecule does not possess a terminal glutamate, it is not a substrate for FPGS and therefore does not undergo polyglutamylation. These key features of the drug render it an attractive antifol alternative in cases where prior MTX exposure could have led to downregulation of RFC-1 and/or FPGS as part of an acquired mechanism of resistance. Based on promising preclinical and phase II studies, which suggested a benefit to the combination, and the hypothesis that trimetrexate (TMTX) was more effective at increasing PRPP levels, TMTX was added to 5FU/LV based on some of the biomodulatory principles discussed above with other antifolates (53–55).

Chapter 6 / Fluoropyrimidine Modulation

145

In one of these studies, Punt (52) treated 365 patients from 22 European centers (roughly 10% untreated) on an intention to treat analysis. Patients were randomized to either: (1) LV 200 mg/m2 as a 1-h intravenous infusion followed directly by 5-FU at a dose of 600 mg/m2 as an intravenous bolus (the control arm), or, (2) TMTX at a dose of 110 mg/m2 as a 1-hour infusion followed 22 to 24 h later by LV at a dose of 200 mg/m2 as a 1-h intravenous (iv) infusion, followed directly by 5-FU at a dose of 500 mg/m2 as an iv bolus, with 15 mg of oral LV every 6 h for seven doses, starting 6 h after the 5-FU administration (experimental arm). Their data revealed that there was a statistically significant increase in the median progression-free survival (PFS) in patients treated with TMTX compared to the 5-FU/LV alone arm. The PFS in the TMTX arm was 5.4 mo, compared to 4.1 mo in the 5-FU/LV alone arm, with a trend towards a benefit in overall survival (13.4 mo in the TMTX arm vs 10.5 mo in the 5-FU/LV alone arm; p = 0.08). Tumor response, quality of life, and toxicity were comparable between the two arms. In a slightly different study, Blanke (51) conducted a double-blind placebocontrolled randomized phase III study of 5-FU/LV plus or minus TMTX, in patients with previously untreated advanced colorectal cancer. Eligible patients (383 in total) were randomized to receive placebo or TMTX at a dose of 110 mg/ m2 intravenously, followed 24 h later by intravenous LV 200 mg/m2 with 5-FU 500 mg/m2 plus oral LV rescue. The authors noted that 41% of patients in the TMTX arm developed grade 3 or 4 diarrhea, while only 28% in the placebocontrolled arm developed similar toxicity. In addition, there was no statistically significant difference in progression-free survival between the arms (5.4 mo in the TMTX arm vs 4.4 mo in the placebo arm). Overall survival was also not statistically different (15.8 mo vs 16.8 mo in the TMTX and placebo-controlled arms, respectively). So, what is the present state of understanding regarding the merits of TMXT biomodulation, and why are, once again, the clinical results so disheartening despite what seemed like exciting preclinical evidence? Several important differences between these studies have been elucidated (56), including: (1) the dose of 5-FU used in the control arms was different in the two studies; (2) additional LV was given in the placebo-control US study; (3) grade 3 and 4 diarrhea was less common in the European study, which has been attributed to more aggressive use of loperamide; (4) the criteria for declaring time to progression in the two studies differed. Central to understanding these data, however, is the recognition that both of these studies were designed to detect a statistically significant difference in median time to progression of 80%, which requires a sample size of at least 300 patients. In addition, neither study was adequately powered to detect survival differences, an endpoint that has been emphasized more often by the US FDA. Despite the convincing preclinical evidence, it is possible that the addition

146

O’Connor

of other agents, or the integration of molecular determinants of 5-FU sensitivity, could be exploited to improve the synergy of these agents in the clinical arena. Another antifolate that has been studied and compared with 5-FU/LV is raltitrexed (Tomudex). Raltitrexed is slightly different from other classical antifolates, in that it is primarily a TS inhibitor. The cytotoxic activity is dependent upon active uptake into cells through the RFC-1, after which it is rapidly and extensively polyglutamylated. Interestingly, Longo (57) has shown synergistic cell kill in HCT-8 cells (colon cancer cell line) after the cells were treated for 24 h with raltitrexed followed by a 4-h exposure to 5-FU. A marginal increase in cytotoxicity was appreciated when the cells were exposed to 5-FU for 5 consecutive days. The reverse sequence, however (either 4 h or 5 d), resulted in less than additive cell kill. The synergy was not attributed to increased inhibition of TS, based on measurements of TS enzyme activity, but rather on an increase in the intracellular pools of PRPP. Levels of PRPP were found to be elevated for 24 h after exposure to raltitrexed, suggesting that raltitrexed and/or its polyglutamates directly or indirectly inhibited purine biosynthesis. The elevated levels of PRPP were associated with increased formation of 5-FU nucleotides and enhanced RNA incorporation. A number of phase I studies have established the safety of the combination with bolus 5-FU and raltitrexed (58–60). In general, the doselimiting toxicities included febrile neutropenia, thrombocytopenia, and diarrhea, with a response rate ranging from 9 to 46%, with several complete remissions. Several large randomized phase II studies have directly compared the activity of raltitrexed monotherapy to 5-FU/LV, all essentially revealing equivalent response rates (14.3 to 19.3 vs 15.2 to 18.1%) (61–63). This paradigm of exploiting other novel antifolates offers one reasonable approach to further advancing our opportunities to develop other biomodulatory strategies. Presently, compounds like pemetrexed (Alimta; Eli Lilly and Co., Indianapolis) and 10-propargyl-10-deazaaminopterin (PDX; Allos Therapeutics, Westminster, CO) represent two such examples of new-generation antifols with unique properties that could distinguish themselves from the standard compounds discussed above. Premetrexed (PMTX), for example, is a novel antifolate with potent activity against TS, and other essential folate-requiring enzymes including DHFR and glycinamide ribonucleotide formyltransferase (GARFT) (64). In one xenograft study, for example, premetrexed followed by 5-FU was shown to be more synergistic than 5-FU, MTX, or PMTX alone, and at least four to five times more effective at producing tumor growth delay than any combination with MTX and 5-FU (65). Clearly, these new-generation antifols and the potential to couple these agents with now newer active drugs like oxaliplatin and irinotecan, offers ample fodder for future improvements in biomodulating fluoropyrimidines. However, if coupled, as already mentioned, with some of the emerging concepts in defining molecular cohorts of patients based upon their molecular phenotypes,

Chapter 6 / Fluoropyrimidine Modulation

147

it is conceivable that our response rates with these combinations will improve, with potentially improved toxicity profiles and improvements in overall survival.

RIBONUCLEOTIDE REDUCTASE INHIBITORS The concept that ribonucleotide reductase inhibition could be synergistic with fluoropyrimidines has been around for decades. The strategy has now become an integral component of many combination chemotherapy programs for malignancies of the head and neck, stomach, and esophagus, for example. Ribonucleotide reductase is a dimeric enzyme consisting of a nucleotide-binding subunit (M1) and a metal-binding subunit (M2). The enzyme’s activity has been shown to be rate-limiting for DNA synthesis and, consequently, would be predicted to be rate-limiting for cellular replication. The rationale for inhibiting ribonucleotide reductase revolves around the idea that inhibition of the enzyme could lead to a decreased generation of dUMP. This decrease in dUMP not only markedly limits de novo thymidylate synthesis, but also eliminates any potential competition between FdUMP and dUMP binding to TS. These mechanisms provide the basis for potential synergy with fluorinated pyrimidines (5-FU would require use of ribonucleotide reductase as part of the anabolic pathway through the PRPP and orate phosphoribosyl transferase reaction). Hydroxyurea, the only clinically approved ribonucleotide inhibitor, acts by inhibiting the metal-binding M2 subunit of the enzyme. Using the L1210 murine leukemia model, Moran (22) showed that treatment with hydroxyurea at an intraperitoneal dose of 100 mg/kg daily and fluorodeoxyuridine at an intraperitoneal dose of 75 mg/d resulted in markedly improved survival compared to the best therapeutic dose of either drug alone. Following some preclinical studies, which supported the biochemical modulation of 5-FU by hydroxyurea (66), several phase II clinical trials had established the safety and potential benefit of the combination (67–71). In one study (72), patients with advanced colorectal cancer were randomized to receive LV (500 mg/m2 over a 2-h infusion) and 5-FU (600 mg/m2 given as a 1-h bolus after LV, every week for 6 wk), followed by a 2-wk rest period with or without hydroxyurea (35 mg/m2/d orally every 8 h). While there was no significant difference in the median time to progression and median survival, the authors concluded that the “double” modulation of 5-FU with LV and hydroxyurea did not appear better than the classic combination of 5-FU and LV alone. It is not entirely clear from these studies that the optimal schedule was employed, given that some of the preclinical data demonstrating biomodulation between fluorinated pyrimidine and hydroxyurea suggest a strong sequence dependency. Recently, in an effort to exploit essentially the same rationale, gemcitabine, a nucleoside analog of deoxycytidine (2',2'-difluorodeoxycytidine) was studied in combination with 5-FU. Once inside the cell, gemcitabine is rapidly phosphorylated by deoxycytidine kinase, the rate-limiting enzyme for the formation of

148

O’Connor

the active metabolites gemcitabine diphosphate and gemcitabine triphosphate. Gemcitabine diphosphate is a known and potent inhibitor of the nucleotide M1 binding site on ribonucleotide reductase. Recently, a phase I study demonstrated the safety and tolerability of the combination administered together using the fixed rate infusion of gemcitabine, which is likely more effective that other administration strategies (73,74). To date, whereas little to no data exist on the efficacy of this strategy, the combined use of ribonucleotide reductase inhibitors with fluorinated pyrimidines continues to be widely studied. New-generation inhibitors of this enzyme have begun to make their way into the clinic. One such novel compound, 3-aminopyridine-2-carboxaldehyde thiosemicarbazone (3-AP, Triapine; Vion, New Haven, CT), belongs to a novel class of agents selectively binding to the M2 subunit of ribonucleotide reductase, and are 65 to 5000 times more potent than hydroxyurea (75,76). Preclinical and subsequent clinical strategies that can formulate the schedule requirements of these agents certainly hold some promise in the development of new modulation strategies for the fluorinated pyrimidines.

Inhibitors of Dihydropyrimidine Dehydrogenase (DPD) Dihydropyrimidine dehydrogenase (DPD) is the first and rate-limiting enzyme in the three-step catabolic pathway for dissimilating thymine and uracil. DPD is responsible for converting more than 85% of the clinically administered dose of 5-FU to its inactive metabolites. Significant interpatient differences in the pharmacokinetic profiles of patients receiving bolus or continuous infusion 5-FU have been largely attributed to the variability in DPD activity, which also follows a circadian rhythm. The variability in DPD activity, in fact, has been shown to be considerable between individual patients (t1/2 range from 4 to 25 min), and is thought to account for most of the variability seen in patients receiving 5-FU. Less than 5% of the population has DPD activity that is significantly below the normal distribution of the enzyme’s activity, and that could theoretically place select patient populations at high risk for toxicity. This important population genetics feature to DPD makes this a true pharmacogenetic syndrome. Perhaps even more interesting is the observation that DPD levels in patients follow a circadian pattern when plotted on a cosine wave (77–79). This observation has laid the foundation for studying the administration of 5-FU in a chronologically modified manner, so that peak levels of 5-FU administration coincide with the nadir of DPD enzyme activity, producing a greater overall exposure to 5-FU. This understanding of the biology of DPD has established several important facts regarding the role of DPD in the biochemical pharmacology of fluorinated pyrimidines (80): (1) the circadian variation of DPD has implications in timemodified 5-FU therapy; (2) the differences in 5-FU pharmacokinetic are related to DPD; (3) variability in 5-FU bioavailability is related to DPD; (4) the defi-

Chapter 6 / Fluoropyrimidine Modulation

149

ciency or reduced activity of DPD in select patient populations makes this a true pharmacogenetic syndrome; and (5) the importance of DPD in 5-FU catabolism, as noted above, makes it a rational target for drug development. As can be seen from Fig. 2, inhibition of the primary catabolic enzyme of 5FU would push more 5-FU toward the anabolic reactions, leading to increased intracellular pools, which would theoretically simulate the continuous or protracted infusion schedules of 5-FU. To date, a variety of strategies for inhibiting DPD have been explored, including drugs like UFT, eniluracil, S-1, and BOFA2. While a detailed discussion of the biochemical pharmacology and the preclinical and clinical data of each of these agents is beyond the scope of this chapter, we will briefly discuss some of the pertinent features of this modulatory strategy. UFT is a 1:4 molar combination of uracil and tegafur. Tegafur is an orally active prodrug of 5-FU which undergoes hydroxylation and conversion to 5-FU, which was first synthesized in 1967, and although it has good oral bioavailability, it led to low 5-FU plasma levels. In the presence of uracil (called UFT), the combination leads to more protracted levels of 5-FU by competing with 5-FU at DPD. Several phase I and phase II studies have shown the combination of UFT plus LV to be well tolerated and active (81–87). In one recent study (88), UFT was administered at a dose of 300 mg/m2/d in three divided doses with oral leucovorin at a dose of 150 mg/d over 28 d, repeating the cycle every 5 wk until progression of disease or unacceptable toxicity. One hundred thirty-six patients were evaluable for response and 141 patients for toxicity. The authors noted a 19.9% overall response rate, with a median time to progression of 5.6 mo and an overall survival of 11.6 mo. The toxicity profile was considered low, with only 17% of patients experiencing grade 3 or 4 diarrhea. At least six large randomized phase III studies to date have not confirmed an improvement in median survival, and found oral tegafur to have unacceptable toxicity, including a high incidence of CNS toxicity. Thus, while the drug remains to be prescribed abroad for the treatment of colorectal and breast cancer, its development in the United States has been discontinued. Another example of a DPD-based strategy includes S-1, an orally active triple drug combination, consisting of the prodrug tegafur, the DPD inhibitor 5-chloro2,4-dihydroxypyridine (CHDP), and potassium oxonic acid. The oxonic acid is an inhibitor of phosphoribosyl transferase and is thought to protect against the diarrhea related to release of the 5-FU into the gastrointestinal tract, while the 5chloro-2,4-dihydroxypyridine is an inactivator of DPD. Phase I studies have established a safe dose of 40 mg/m2 twice daily on d 1 through 28 every 5 wk in patients exposed to little or no prior therapy, and 35 mg/m2 for more heavily pretreated patients. In the phase I, diarrhea was the dose-limiting toxicity (89). While data regarding its true benefit compared to the “gold standards” of care have yet to be published, phase II studies have shown a response rate of approx

150

O’Connor

35% in patients with metastatic colorectal cancer as upfront therapy (90). Ethynyuracil (eniluracil) is a relatively new inhibitor of DPD. The drug is a pyrimidine analog that has features similar to both uracil and 5-FU, that is itself not a prodrug of 5-FU. Tumor samples taken from patients receiving ethynluracil revealed near 100% inhibition of DPD activity in the tissue (91,92). Phase I studies combining the DPD inhibitor and oral fluorouracil have established doses of 50 mg/m2/d and 15 mg/m2/d, respectively, with myelosuppression, diarrhea, and mucositis being the primary dose-limiting toxicities (93). A phase II study conducted in the ECOG treated 53 previously untreated and 46 previously treated patients (one regimen only) with eniluracil at a dose of 10 mg/m2 and 5-FU 1 mg/ m2 twice daily for 28 d, with cycles repeated after a 7-d rest until progression of disease or prohibitive toxicity (94). The response rate among the untreated patients was 13.2%, with a mean duration of response of 6.3 mo, and an overall survival of 11.1 mo. Of the previously treated patients, only 2.2% achieved a response, with a duration of remission of approx 3 mo and an overall survival of only 9 mo. Although the regimen was noted to be quite tolerable with no significant untoward toxicity, it is apparent that the regimen in this small phase II study is significantly inferior in patients who have received prior therapy, though the results in the untreated population of patients is in line with that reported for other 5-FU-containing regimens. BOF-A2 is yet another example of a combination of fluoropyrimidine and DPD inhibitor that includes 1-ethoxymethyl-5-fluorouracil and the known DPD inhibitor 3-cyano-2,6-dihydroxypyridine (CNDP). Although preclinical studies have shown the drug to be active, few clinical trails are ongoing with the regimen, due to concerns regarding severe fluoropyrimidine toxicity. Obviously, inhibitors of DPD offer a unique opportunity to inhibit the catabolic pathways leading to the detoxification of 5-FU. Many of these strategies involve oral agents, which appear to be well tolerated, though few studies have shown superiority to more traditional fluorouracil regimens. The development and integration of other DPD-modulating strategies with other oral fluoropyrimidine-based regimens is a rational approach that may offer patients a more convenient and potentially less toxic alternative to intravenous approaches, though to date there is no clear therapeutic advantage in favor of the strategy, and in select cases it may even be associated with greater toxicity.

Interferons The interferons are a family of proteins with antiproliferative, antiviral, and immunomodulatory activity. Although these agents are felt to be minimally active against colorectal cancer as single agents, combinations of fluorinated pyrimidines and interferon (IFN)-F, -G, and -L have been extensively studied (95,96). The precise mechanisms by which IFN modulates 5-FU has been related to one of several mechanisms. The first revolves around the induction of thymi-

Chapter 6 / Fluoropyrimidine Modulation

151

dine phosphorylase. IFN has been shown to increase the metabolism of 5-FU to its active metabolite 5-fluorodeoxyuridine monophosphate (FdUMP) in both colon carcinoma and leukemia cell lines (97,98). This increase is associated with an eightfold induction of TP, which facilitates the conversion of 5-FU to 5fluorodeoxy-uridine (Fig. 2). Furthermore, TP levels have been shown to correlate with the sensitivity of colon cancer cell lines to 5-FU, and treatment of patients with IFN-F or IFN-G results in a rapid induction of TP in peripheral blood mononuclear cells within 1 to 2 h (99). In addition to TP, interferons can have a variety of other effects that can influence 5-FU activity. Treatment of human colon cancer cell lines with 5-FU and IFN-F has been shown to produce greater depletion of thymidine nucleotide pools than that seen with 5-FU alone, an effect that is also correlated with an increase in the number of double-strand DNA breaks. Interestingly, the DNA strand breaks were increased in the presence of 5-FU and LV. Another important effect of IFN on the modulation of 5FU pertains to the levels of TS. Typically, increased levels of TS expression, which occur following 5-FU exposure, are associated with resistance to fluoropyrimidines. For example, treatment of the colon cancer cell line H630 with 5-FU alone leads to increased TS activity, which is ameliorated by the coadministration of 5-FU and IFN-L (100,101). Both the 5-FU-mediated TS induction and its subsequent inhibition by IFN-L appear to be regulated at the post-transcriptional level, as no acute changes in TS mRNA have been observed after 5-FU treatment. As we have noted with many of the other fluoropyrimidine modulation strategies, there are very important schedule-dependence considerations with IFN and 5-FU as well. To date, all of the published studies have demonstrated equivalence in overall survival between 5-FU/LV alone vs 5-FU/LV plus IFN (102– 104). Some studies, however, have demonstrated improvement in response rate and progression-free survival in favor of the IFN-containing regimen (105). One consistent finding among the published studies is the increased toxicity seen in the IFN-containing arms. As pointed out by Makower and Wadler (95,96), however, the disappointing results from the randomized studies need to take into account the fact that these randomized studies all used slightly different schedules of IFN, and typically employed only IFN-F. One study gave IFN several hours after the 5-FU, while other studies failed to actually specify the IFN schedule. Preclinical studies have established that both 5-FU and IFN should be given simultaneously for synergy to occur (95,96). Since 5-FU can undergo its metabolic conversions within approx 2 h of administration, it is possible that schedule differences in randomized studies contributed to the generally unimpressive results noted to date (95) . Recently, an interesting phase I study exploring the potentiation of 5-FU and LV cytotoxicity by IFN-L in patients with colorectal cancer has been reported (106). Patients were treated with intravenous 5-FU at a dose of 370 mg/m2 and

152

O’Connor

LV 200 mg/m2 daily for 5 consecutive days with escalating doses of subcutaneous IFN-L (10–100 μg/m2) on d 1, 3, and 5 every 28 d. Besides exploiting the mechanisms discussed above, the authors studied the effects of 5-FU modulation on the Fas signaling pathways in colon cancer cells. The cell-surface receptor Fas (APO-1; CD95) is a member of the tumor necrosis factor receptor family of death receptors, which induce apoptosis in sensitive cells on binding to their specific death ligands. Fas has been shown to be highly expressed in normal human colonic epithelial cells, and its expression has been shown to become progressively less prominent during the progression of normal epithelium to adenocarcinoma in approx 50% of the cases (107,108). Several published reports have established, both in vitro and in vivo, that activation of Fas is one apparently new mechanism that may be modulated in a manner that is complimentary to the effect of 5-FU. The thymineless death induced by inhibition of TS from 5-FU/ LV occurs downstream of DNA damage, signaling through the Fas death receptor. Their preclinical experience successfully demonstrated that IFN-L induced the upregulation of Fas expression and sensitized human colon cancer cell lines to 5-FU/LV in a Fas-dependent manner, and that this effect was independent of the p53 status (109). They also demonstrated that IFN-L does not sensitize cells to 5-FU/LV when the mechanism is RNA directed, and that the Fas dependency of IFN-L may be quite specific to the cytotoxic agent used to damage DNA, since other agents, like doxorubicin and etoposide, do not appear to exert their cytotoxic mechanism in a Fas-dependent fashion. Their pharmacokinetic data revealed that at doses of 100 mg/m2 of IFN-F, the plasma concentrations of IFN-L persisted above 5 units/mL for up to 6.5 h, and above 1 unit/mL for >28.5 h. The authors reported that the pharmacokinetic parameters of IFN-L correlated with a two- to threefold upregulation of Fas expression at 24 h in CD15+ cells in the peripheral blood. The dose-limiting toxicity was stomatitis, which occurred most frequently at 100 μg/m2 IFN-L. The other grade 3 or greater toxicities noted in patients who received at least three doses of IFN included fatigue (1/19 patients), neutropenia (3/10), hand-foot syndrome (1/19), and one case of a small-bowel obstruction related to the patient’s underlying disease. Minor response or stable disease were observed in 2 of 9 patients, and in 4 of 12 patients at dose levels less than 50 mg/m2 and >75 mg/ m2, respectively. In three chemotherapy-naïve patients receiving 100 mg/m2 (either once or followed by IFN-L at 75 mg/m2), two partial responses and one complete response were seen. The complexity of deciphering and advancing our understanding of this biology will obviously be dependent upon a thorough understanding of the underlying signaling pathways. In addition to the schedule-dependence issues raised above, it is clear that other factors regarding the modulating effects of interferons will need to take into account the different cellular receptors that different types of interferons are capable of binding. Obviously, different types of interferons

Chapter 6 / Fluoropyrimidine Modulation

153

elicit different types of effects on their target cells depending upon the specific post-receptor signaling pathways. For example, IFN-2F has not been demonstrated to up-regulate Fas expression in human colon cancer cell lines. Critical in advancing this particular modulatory strategy will be understanding the concentration effect relationships as they pertain to the influence of various IFNs on not only Fas signaling, but also the determinants of 5-FU cytotoxicity, including levels of thymidine phosphorylase and intracellular thymidine pools.

Nucleoside Transport and Nucleotide Salvage Inhibitors One potential mechanism through which cells become resistant to conventional anticancer drugs, especially antimetabolites, involves the upregulation of proteins inside the cell that can lead to the increased salvage of exogenous nucleosides. Such strategies have the benefit of supplying the cell with sometimes large amounts of nucleotides, thereby circumventing the de novo synthetic pathways. Salvage pathways, by definition, are metabolic pathways used by cells for the synthesis of nucleotides from preformed nucleosides or nitrogenous bases. Obviously, these pathways tend to be less energetically taxing to the cell than the corresponding de novo synthetic pathways, and offer sometimes challenging barriers to effective chemotherapy development. Using a stepwise selection process in the presence of FUDR, Sobrero and colleagues (110) generated HCT-8-resistant cell lines that were over 700-fold resistant to FUDR compared to the parent strain. The subsequent molecular studies demonstrated that the rapid transport of the FUDR into the cell was essentially absent in the drug-resistant cells, but intact in the sensitive cells. They further demonstrated that treatment of the HCT-8 cell lines with dipyridamole and nitrobenzylthioinosine (NBMPR) rendered the sensitive cells resistant to the FUDR, mimicking the pattern of resistance seen in the resistant cell lines generated originally. Obviously, elucidating and understanding these mechanisms of acquired drug resistance may afford new opportunities for the biomodulation of the activity of 5-FU. A detailed discussion on the biology and implications of these strategies can be found elsewhere (111–113). One logical mechanism of resistance to fluorinated pyrimidines is the ability to both transport into the cell, and then metabolically salvage extracellular thymidine. At least seven functionally distinct nucleoside transporters have been described in human cells (114–119). The functional uptake processes can be broadly divided into two categories based on the primary mechanism of transport: equilibrative nucleoside transporters and concentrative transporters. An equilibrative nucleoside transporter is a membrane protein that accelerates the influx of hydrophilic nucleoside molecules across biological membranes. These transporters are also known as facilitative diffusion systems because they mediate a much more rapid influx of molecules than simple diffusion would allow across the cell membrane. These equilibrate transporters are subdivided into

154

O’Connor

equilibrative sensitive (es) and equilibrative insensitive (ei) transporters, based on their sensitivity to NBMPR. The equilibrative sensitive transporters are potently inhibited by NBMPR (Ki 0.1 to 10 nM), while the equilibrative insensitive transporters are minimally affected by NBMPR (116,120). In addition to NBMPR, these transporters, though usually es moreso than ei, are also inhibited by the coronary vasodilators dipyridamole, dilazep, and draflazine. These transporters typically are widely distributed among most, if not all, cell types in the body. Concentrative nucleoside transporters are membrane proteins that catalyze the co-transport (also know as symport) of nucleosides and an ion, typically sodium or a hydrogen atom, across the cell membrane. Because these ionic gradients provide the energy to drive the transport of nucleosides against the concentration gradient, the transporters are commonly referred to as active transporters, and are usually insensitive to NBMPR. These transporters constitute the first of two processes that can play a major role in the salvaging nucleosides, potentially protecting cells from the growth-inhibitory properties of anticancer drugs in general and the antimetabolites in particular. Aside from functioning as salvage pathways for the cell, nucleoside transporters play an important role in transporting many antineoplastic drugs, including purine analogs like cladribine (2-chloro-2'-deoxyadnoosine) and fludarabine (9G-D-arabinosyl-2-fluoroadenine), and the pyrimidine analogs cytarabine (1-[Gb-Darabinofuranosyl] cytosine) and gemcitabine (2'2'-difluoro-2'-deoxycytidine). Mediated transport of these molecules is a key determinant for the in vitro and in vivo effectiveness of these molecules (112). The correctly administered sequence of inhibitors of thymidine salvage and fluorinated pyrimidines offers one possible venue for modulating the cytotoxicity of 5-FU. As discussed above, dipyridamole is a well known and potent inhibitor of the facilitated nucleoside transporter. To date, at least two clinical trials in which dipyridamole and 5-FU were given by concurrent continuous intravenous infusion failed to successfully modulate the cytotoxicity of 5-FU, and few clinical responses were noted. These negative results are attributed to the high-affinity binding of dipyridamole to the serum protein acidic glycoprotein (AGP). In healthy volunteers, AGP is present at levels between 0.3 and 1 mg/mL, but in patients with cancer, the levels are 1.5- to 3.8-fold greater. Although the maximum steady-state total plasma concentration of DP achievable by intravenous and oral routes of administration is approx 12 to 16 μM, the maximal free dipyridamole levels are only 27 and 38 nM (121–123). Based on in vitro studies, these concentrations are insufficient to inhibit the equilibrative nucleoside transporter. One way to circumvent the pharmacological liabilities associated with dipyridamole is to develop more selective agents that do not bind to AGP and have the ability to inhibit the transporter. New dipyridamole derivatives with this property (for example NU3076, NU3084, NU3108, and NU3121) have been developed by the Cancer Research Unit at the University of Newcastle upon

Chapter 6 / Fluoropyrimidine Modulation

155

Tyne (124). These analogs have been shown to inhibit thymidine uptake in L1210 leukemia cells, and they do not bind AGP. Smith and colleagues (121) have shown that these analogs successfully inhibit thymidine and hypoxanthine rescue in A549 and COR L23 human lung cancer cell lines despite the presence of 2.5 mg/mL of AGP, whereas the activity of dipyridamole is completely abolished. To date, we await the results of clinical studies that will further explore this strategic rationale to modulate 5-FU. These compounds, in addition to other promising molecules that have demonstrated abilities to impair nucleoside transport, including BIBW22BS, and the fungus-derived transport inhibitor C3368A, may offer new venues for modulating 5-FU activity in humans (125,126). The second essential step, at least in the case of thymidine salvage pathways, involves the phosphorylation of thymidine (the nucleoside) to thymidylate (the nucleotide) through the enzyme thymidine kinase (TK). Effective nucleotide salvage involves not only transport of extracellular thymidine, but also phosphorylation of the nucleoside to the nucleotide level. Thymidine kinase (TK) is the principal intracellular enzyme responsible for phosphorylating thymidine to dTMP (thymidylate), and its regulation is felt to be dependent on the cellular pools of dTTP. Regrettably, there are very few drugs with the ability to selectively inhibit human TK effectively. Drugs like acyclovir and ganciclovir, and their derivatives, are potent and almost exclusive inhibitors of viral TK. One drug capable of affecting mammalian TK is azidothymidine (3'-Azido-3'-deoxythymidine), or AZT. AZT, originally developed as an antineoplastic agent, was found to have some, but certainly not dramatic, single-agent activity against various cancers, and has since found valuable utility in the treatment of AIDS. The cytotoxicity of AZT is attributed to its incorporation as AZT-triphosphate (AZTTP) into DNA, leading to chain termination. This effect is markedly augmented in thymine-depleted states, and in the presence of drugs that inhibit TS and DHFR. Several preclinical studies have demonstrated synergy with 5-FU in vitro and in vivo (127–129). Whereas AZT did not affect FdUMP-mediated toxicity or RNA incorporation, it presumably competed with dTTP at the DNA polymerase, leading to more AZT-TP incorporation. Another mechanism of action of AZT involves the ability of the AZT diphosphate to inhibit TK. The inhibition of TK markedly impairs the back half of the thymidine salvage pathways, decreasing dTTP and leading to increased AZT-TP incorporation. Other studies have also shown that AZT can competitively inhibit the transport of thymidine (Km = 0.23 mM) into human erythrocytes with a Ki of 1.0 mM (130). The principal human metabolite of AZT, the 5'-glucuronide (GAZT), is a weak inhibitor of the nucleoside transporter, while the minor metabolite of AZT (3'amino-3'-deoxythymidine [AMT]) competitively inhibits the transporter with a Ki of 0.1 mM. To date, no clinical trials have demonstrated successful modulation of 5-FU activity with AZT, though one study has demonstrated a response rate of 44% (15 objective responses among 34 treated patients). Although this

156

O’Connor

Fig. 6. Metabolic conversion of capecitabine by compartment.

rationale is certainly clear, it is likely that future studies in this general area will need to occur in the setting of newer oral-based fluoropyrimidine regimens.

Thymidine Phosphorylase Unquestionably, one of the newest and most exciting fluorinated pyrimidines to come into recent development has been the novel prodrug capecitabine (Roche, Nutley, NJ). Capecitabine (N-[1-(5-deoxy-G-D-ribofuranosyl)-5-fluoro-1,2dihydro-2-oxo-4-pyrimidinyl]-n-pentyl carbamate), a prodrug of the prodrug 5FU, is a fluoropyrimidine carbamate. The drug is a product of many years of rationale drug design, which focused on the synthesis of a compound that would have minimal gastrointestinal toxicity and enhanced tumor selectivity based upon a unique enzymatic activation pathway found predominantly in malignant tissues. Capecitabine undergoes a series of specific metabolic reactions in the liver, and tumor-specific reactions, that leads to very high intracellular concentrations of 5FU (Fig. 6). This unique set of reactions has two major consequences: (1) it markedly reduces the systemic exposure to the toxic intermediates of 5-FU; and (2) it mimics the pharmacokinetic profiles seen with infusional 5-FU. Following rapid absorption, capecitabine passes unchanged into the liver, where it is converted by the enzyme carboxyesterase to 5'-deoxy-5-fluorocytidine (5'-DFCR), which is a

Chapter 6 / Fluoropyrimidine Modulation

157

Fig. 7. Metabolic activation of capecitabine to 5-FU.

substrate for cytidine deaminase. 5'-DFCR undergoes oxidative deamination by cytidine deaminase in the liver to produce 5'-deoxy-5-fluorouridine (5'DFUR, doxifluridine, Furtuton®) (131–134). Figure 7 presents the metabolic pathway for capecitabine. 5'-DFUR, which was the original lead compound for development before capecitabine, has been shown to be equally or more effective that 5-FU in preclinical models, with an improved safety profile in various murine tumor models. The drug has been used extensively for the treatment of breast and colorectal cancer in Japan. 5'-DFUR is a substrate for cytidine deaminase (CD), the major enzyme responsible for the inactivation of cytosine arabinoside to uracil arabinoside, which is highly expressed in liver, kidney, and malignant tissues. CD is minimally expressed in immature hematopoietic cells compared to mature granulocytes, which accounts for the myelotoxicity of Ara-C (135–137). The final step involves cleavage of the sugar moiety form the nitrogenous base to generate 5-FU, a reaction catalyzed exclusively by thymidine phosphorylase. Thymidine phosphorylase (dThdPase) is a member of the pyrimidine nucleoside phosphorylase family, is essential for DNA synthesis, and is often a ratelimiting step in cellular proliferation (138). The enzyme has been shown to be identical to platelet-derived endothelial cell growth factor (PDEGF), which has been shown to possess potent angiogenic activity in vivo (139–141). One of the enzymatic products of dThdPase from thymidylate is 2-deoxy-D-ribose, which has chemotactic activity in vitro and angiogenic activity in vivo. What makes this

158

O’Connor

Fig. 8. Relative expression of thymidine phosphorylase activity in human tissues.

target so critical is the fact that dThdPase expression is 3 to 10 times higher in tumor tissue compared to the corresponding normal tissues, including malignancies derived from the gastrointestinal tract (Fig. 8) (142–147), pancreas, breast, and urinary bladder. Several reports have also demonstrated that a higher level of dThdPase expression correlates with a poorer prognosis and more aggressive invasive potential (138,148). Besides the intrinsically elevated levels of dThdPase that exist in tumor tissues, recent data have begun to demonstrate that various other factors can contribute to a further induction in dThdPase, thereby offering a potential synergy with capecitabine. These factors include a variety of chemotherapeutic drugs (including paclitaxel and docetaxel, vinca alkaloid, cyclophosphamide, gemcitabine, and mitomycin C), radiation, cytokines (including tumor necrosis factor [TNF]-F, IFN-L, IFN-F, interleukin [IL]-1, IL-12), growth factors (including GFGF), hypoxia, and acidic pH (149–154) (Fig. 9). Among the chemotherapeutic agents, the taxanes have emerged as promising agents that have marked synergy with capecitabine in human cancer xenograft models. Using a WiDR human colon cancer xenograft model, Sawada and colleagues (149) have demonstrated that paclitaxel, docetaxel, and mitomycin C markedly increased levels of human dThdPase in tumors, while simultaneously increasing the levels of TNF-F, which itself is a potent inducer of dThdPase. The efficacy of fluorouracil in combination with paclitaxel and docetaxel was found to be synergistic in xenograft models, while the toxicity (i.e., weight loss) was found not to be increased. More recently, it has been shown that the antitumor activity of fluorouracil and docetaxel is schedule dependent, at least in murine xenograft

Chapter 6 / Fluoropyrimidine Modulation

159

Fig. 9. Induction of thymidine phosphorylase by conventional cytotoxic drugs.

models of a human breast-cancer cell line (MAXF401). These data suggested that administering the docetaxel during the middle part of the capecitabine regimen was synergistic, whereas the other possible combinations of drug administration were less active. Interestingly, in the human colon-cancer xenograft model with WiDR, early administration of the docetaxel was most synergistic with the taxane. The increase in dThdPase was far greater in the WiDR line, suggesting that the earlier administration led to an induction of an enzyme that was necessary for prodrug activation, while in the MAXF401 line, there was little to no increase in dThdPase, suggesting another potential mechanism for the synergistic interaction. Clinically, the efficacy of capecitabine has been demonstrated in a number of large clinical trials. In one large multicenter phase II study reported by Blum (155), 162 patients with refractory metastatic breast cancer who were previously treated with paclitaxel and an anthracycline received oral capecitabine at a dose of 2510 mg/m2/d divided into two doses for 2 out of every 3 wk. Of the 135 patients evaluable for response, the overall response rate was 20% (with 2.2% complete responses, and 17.8% partial remissions), whereas 40% of patients had stable disease. The median duration of response was 241 d. In the pivotal trial, a randomized phase II study in 605 patients with advanced or metastatic colorectal cancer, the overall response rate was 24.8% compared to a response rate of 11.6% for 5FU/LV (p = 0.0001) (156). There was no difference between the groups with regard to overall survival (12.5 vs 13.3 mo respectively), median duration of response (9.1 vs 9.5 mo), or median time to disease progression (4.3 vs 4.7 mo). Another randomized phase III trial of 602 patients comparing 5-FU plus LV to

160

O’Connor

capecitabine in patients with advanced or metastatic colorectal cancer, reported an overall response rate of 19% with capecitabine compared to 15% in patients receiving 5-FU/LV (157). Again, no significant differences were noted between the two groups with regard to overall survival, median duration of response, median time to disease progression, and time to treatment failure (157). Chemotherapynaïve patients received either capecitabine 1250 mg/m2 orally twice a day for 14 d followed by a 7-d rest period or rapid intravenous injection of LV at a dose of 20 mg/m2 followed by a bolus of 425 mg/m2 of 5-FU on d 1 through 5 every 4 wk. A markedly lower incidence of stomatitis, grade 3 and 4 neutropenia, and alopecia (p < 0.0001), and a significantly higher incidence of hand-foot syndrome (p < 0.00001) and uncomplicated grade 3 and 4 hyperbilirubinemia were observed in those patients receiving capecitabine compared to 5-FU/LV. These studies have established that oral capecitabine has at least equivalent efficacy compared to 5FU/LV with a clinically meaningful safety advantage (157). Building further on the merits of the oral agent, Twelves and colleagues (158) have reported that capecitabine was shown to substantially reduce medical resource use and to improve response rate and tolerability compared to the Mayo Clinic regimen of 5-FU/ LV in patients with metastatic colorectal cancer. Validating some of the preclinical experiences with dThdPase-inducing drugs and the consequential synergistic interactions with capecitabine, a large randomized phase III clinical trial in anthracycline pre-treated patients with breast cancer has shown a statistically significant improvement in overall response rate, overall survival, and time to disease progression with the combination of capecitabine and docetaxel (255 patients) compared to single-agent docetaxel (256 patients) (159). The combination therapy consisted of capecitabine at a dose of 1250 mg/m2 twice daily for 14 d followed by a 1-wk rest and docetaxel at a dose of 75 mg/m2 intravenous every 3 wk. In the monotherapy arm, docetaxel was administered at a dose of 100 mg/m2 every 3 wk. On comparison of the combination arm and the monotherapy arm, respectively, the overall response rates were 32% vs 22%, the median overall survival was 14.5 vs 11.5 mo, and the median time to disease progression was 186 d vs 128 d, all in favor of the combination therapy. These results, and continued emerging data from a variety of ongoing studies, continue to confirm the single-agent activity of capecitabine. Undoubtedly, future studies will explore the integration of other conventional agents with capecitabine, many hopefully exploiting the underlying biology with regard to thymidine phosphorylase induction. Based on the data to date, the modulation strategies revolving around TPase appear to have clinical benefit.

SO, WHERE DO WE GO FROM HERE? Since the introduction of 5-FU into the clinic over 40 yr ago, a considerable amount of time has been dedicated to trying to optimize and improve on the use

Chapter 6 / Fluoropyrimidine Modulation

161

of fluorouracil. While many studies have established a sound scientific rationale for a panoply of strategies in the laboratory, few phase II or III studies have confirmed the findings in the clinic. While in select cases improvements in response rate, time to progression, or duration of response may be better in the “modulated arm,” gains in overall survival remain elusive. Perhaps the most aggravating conundrum in the story of fluoropyrimidine modulation is, “Why do the in vitro and murine in vivo models continue to so poorly predict what happens in our patients?” Is it possible that we have reached our maximum potential with fluorinated pyrimidines? Is it possible that the best we can do is infusional 5-FU, or bolus 5-FU perhaps modulated by LV? Clearly, our understanding of the underlying biology and biochemistry has provided a basis for developing new testable theories, and there is no foreseeable end to the number of new hypotheses we can generate. The most important question is, which of these hypotheses deserves our attention? Part of the past emphasis on developing biomodulation strategies with 5-FU emerges from the fact that there are very few drugs with sufficient single-agent activity in diseases like colorectal cancer. Recently, at least in colorectal cancer, a host of new drugs with activity as single agents and in combination with 5-FU have come to the forefront. Drugs like irinotecan, oxaliplatin, antiangiogenic factors (the anti-vascular endothelial growth factor antibody bevacizumab, Genentech, CA), and monoclonal antibodies that target the epidermal growth factor receptor (C225, Imclone, NY), represent new developments that will undoubtedly play a role in the future management of our patients. Recently, a survival advantage was demonstrated in a randomized phase II study in patients receiving bevacizumab with a 5-FU (21.5 vs 13.8 mo) compared to patients not receiving the monoclonal antibody (160). These data, among the first in several decades to demonstrate a survival advantage through the addition of a second agent to a fluorpyrimidine, provide an enormous motivation to continue to pursue the study of alternative targets beyond the traditional fluoropyrimidine targets, in the pursuit of more novel regimens that have the potential of improving patient survival. Certainly, finding newer and better agents that can modulate TS would be one potential venue to pursue. Recently, Wu and Dolnick (161) reported on the development of a novel screening assay to identify novel modulators of TS. Taking advantage of the fact that TS can serve as a repressor of its own synthesis by binding to its own mRNA through TS-specific biding elements (TBEs), the authors developed a luciferase reporter plasmid containing two TS-binding elements, which can be used as a tool for the identification and initial profiling of compounds that modulate TS activity, levels, or ability to bind mRNA. The model was validated using available inhibitors of the enzyme. These sorts of novel assays that allow high throughput screening of large combinatorial librar-

162

O’Connor

ies, in search of drugs that can affect a specific target, offer new opportunities to identify perhaps more promising TS-specific agents. There is no denying the fact that oral fluorinated pyrimidines, most likely in the form of capecitabine in the United States, are here to stay, and their role in a host of solid tumors will continue to be defined over the years to come. While a number of economic issues and concerns have been raised and aptly discussed by others (7), it is clear that these economic issues will become even more complicated as the oral therapies become integrated components of combination regimens that will necessarily include parenterally administered agents. Presently, we await the results of more clinical studies that have begun to move the use of these oral agents and combination regimens into front-line treatment strategies. It appears that these oral agents are as well or better tolerated than any parenteral fluoropyrimidine-based therapy, and are no worse, though possibly no better, than their intravenous counterparts. Pharmacologically, these fluorinated pyrimidines mimic infusional 5-FU strategies, and appear to exploit the underlying ThdPase biology indicative of many malignancies. Perhaps one of the most significant areas of future development in improving fluorouracil modulation is to better define discrete molecular phenotypes of patients, especially as they pertain to the determinants of fluoropyrimidine cytotoxicity. As we have discussed, there is a strong link between understanding the mechanisms of acquired drug resistance in cancer cells and identifying potential targets that may be amenable to some modulatory strategy. There are many molecular determinants that have been correlated with fluoropyrimidine resistance (Table 2), including elevated levels of thymidylate synthase, reduced expression of nucleoside transporters, increased expression of the catabolic enzyme DPD, and changes in any of the anabolic enzymes that lead to 5-FU activation. In addition to the significance of TS mRNA expression, some sets of data have begun to demonstrate that the TS genotype might also provide supplemental information in interpreting TS protein expression and predicting response to TS-targeted therapy (162,163). The expression of TS is controlled in part by a multiple number of tandem repeats of a 28-basepair sequence in the 5’-promoter enhancer region (TSER) of the gene. Alleles containing two, three, four, five, and nine copies of the repeated sequence have been described (TSER*2, TSER*3, TSER*4, TSER*5, TSER*9), with TSER*2 and TSER*3 being the most commonly described (164–167). Multiple studies have shown that increasing the number of repeats leads to an increase in TS mRNA levels and protein expression. One such study reported a 3.6-fold increase in TS mRNA levels in individuals homozygous for TSER*3 compared to TSER*2 homozygotes, and a 1.7-fold increase between patients heterozygous for TSER*3 and patients heterozygous for TSER*2/TSER*3. Subsequent translational studies have demonstrated that patients with168 lower numbers of TSER repeats had higher response rates to traditional 5-FU, while a decrease in median survival was

Chapter 6 / Fluoropyrimidine Modulation

163

Table 2 Correlation Between Molecular Determinants of Fluoropyrimidine Sensitivity and Effect Molecular targets TS

a

DPD2

TSa

Observation

Ref.

Intratumoral colorectal cancer

TS and TPa

DPD, TS, TP

Tissue

a

Statistically significant association 169 between TS and survival and response. TS/G-actin ratios ranged from 0.1 to 18.2 (10–3), median 3.5). Median ratio in responding patients 1.7 vs 5.6 in nonresponding patients. Median survival in patients with TS ratios <3.5 was 13.6 mo vs 8.2 mo for ratios >3.5 Intratumoral Low expression of TS and TP predicted 170 colorectal very high response rate (11 of 14) to cancer 5-FU/LV and longer survival, where 0 of 24 patients with high TS and TP expression responded. –3 Intratumoral DPD:G-actin ratios >2.5 × 10 43 colorectal (14 of 33 samples) responded to 5-FU, tissue prewhile ratios <2.5 × 10–3 had a 5-FU from response rate of 50%. Patients with 33 biopsies the lowest levels of TS, TP, and DPD had longer survival. Peripheral Circadian variation of 5-FU plasma 79 blood concentrations—peak levels at 11 mononuclear AM, trough levels at 11 PM, with cells and nearly a fivefold difference in 5-FU PK 5-FU concentrations. DPD activity in 7 patients peak at 1 AM, and trough at 1 PM. An inverse relationship between 5-FU levels and DPD activity. Malignant 77% of sensitive (low TS) patients had 171 tissue from response, whereas only 9% of resistant hepatic patients had a response—survival metastasis advantage for patients with low in patients TS levels. pre-HAI. Many types of cancer included. (continued on next page )

164

O’Connor Table 2 (continued)

Molecular targets

Tissue

TKb, TSd, DPDa

Human cancer Moderately strong correlation between 10 cell lines 5-FU and FUDR GI , weak 50% from the moderate correlation between TS National mRNA and protein. Neither TS Cancer nor TK expression correlated Institute with 5-FU/FUDR GI , low DPD 50% Database expression favored increased 5-FU based on sensitivity. Cell lines with high growth doubling times and wild-type inhibition p53 more sensitive. Malignant TS expression was an independent 172 colorectal prognostic factor for disease-free tissue from and overall survival, and suggested 862 patients that patients with the highest levels of with Dukes TS were more likely to benefit from B or C adjuvant 5-FU-based therapy. disease. Malignant No correlations between survival, 173 metastatic response, or levels of TP, DPD, colorectal RFC-1, or FPGS. Statistically tissue presignificant relationship with low raltitrexed. TS and responders (21.7 vs 5.7 mo)

TS

c

TSc, TPa, DPDa, FPGSa, RFC-1a

a

Observation

Ref.

b

Levels determined by reverse-transcription polymerase-chain reaction ; enzyme activity; immunohistochemistry; dWestern Blot; TS, thymidine synthase; TP, thymidine phosphorylase; LV, leucovorin; DPD, dihydropyrimidine dehydrogenase; HAI, hepatic artery infusion; RFC, reduced folate carrier.

c

correlated with increasing TSER repeats (166). While the allure of risk-stratifying patients on the basis of these pharmacologically based phenotypes at a molecular level looms large, there is substantial work to be done. While the weight of published literature supports the prognostic value of TS in various tumor types, the use of these molecular phenotypes to dictate treatment selection will require definitive randomized clinical trials in the future. Integrating our understanding of TS polymorphisms and genotype into the treatment algorithm in the future can provide a potentially valuable tool for identifying those patients at risk for excess toxicity, and perhaps those most likely to benefit from some biologically tailored therapy. At present, until these observations can be validated accordingly, their application in the clinic may be premature. Undoubtedly, there is the approach of reiterating the themes of the past, which are almost certainly in the queue. If methotrexate effectively modulates 5-FU,

Chapter 6 / Fluoropyrimidine Modulation

165

how will newer-generation antifolates like premetrexed, PDX, and others compare? If dipyridamole modulates 5-FU by inhibiting thymidine salvage, how will new derivatives of drugs with improved pharmacologic parameters affect the modulation of 5-FU? Although these questions are almost certain to be addressed in future clinical studies, it is likely that they will need to be addressed in a slightly different context. Integrating these potential scenarios into the most recent developments in oral-based therapies and molecular phenotyping sooner rather that later will most assuredly afford new opportunities for chemotherapy development. The addition of newer drugs that effect tumor cell death by alternative mechanisms of action (antiangiogenesis, anti-EGFR monoclonal antibodies, and so on) provide an important venue that will lead to the development of more novel and effective chemotherapy regimens. Regardless of the agents tested, there is an essential need to pay close attention to the biology at hand and to design trials that respect that understanding.

ACKNOWLEDGMENTS The author would like to thank Alexandra Sedehi for all her assistance in preparing the manuscript. OAO is a Scholar in Research from the Leukemia and Lymphoma Society.

REFERENCES 1. Heidelberger C, Chaudhuri NK, Danneberg P, et al. Fluorinated pyrimidines, a new class of tumour-inhibitory compounds. Nature 1957;179:663–666. 2. Rutman RJ, Cantarow A, Paschkis KE. Studies in 2-acetylaminofluorene carcinogenesis. III. The utilization of uracil-2-C14 by preneoplastic rat liver and rat hepatoma. Cancer Res 1954;14:119–23. 3. Schmoll HJ, Buchele T, Grothey A, Dempke W. Where do we stand with 5-flourouracil? Semin Oncl 1999;26(6):589–605. 4. Bertino JR. Chemotherapy of colorectal cancer: history and new themes. Semin Oncol 1997;24:S18-3–S18-7. 5. Sobrero AF, Aschele C, Guglielmi AP, et al. Synergism and lack of cross-resistance between short-term and continuous exposure to fluorouracil in human colon adenocarcinoma cells. J Natl Cancer Inst 1993;85:1937–1944. 6. Pritchard DM, Watson AJ, Potten CS, et al. Inhibition by uridine but not thymidine of p53dependent intestinal apoptosis initiated by 5-fluorouracil: evidence for the involvement of RNA perturbation. Proc Natl Acad Sci USA 1997;94:1795–1799. 7. Saltz LB, Cox JV, Blanke C, et al. Irinotecan plus fluorouracil and leucovorin for metastatic colorectal cancer. Irinotecan Study Group. N Engl J Med 2000;343:905–914. 8. Saltz LB, Douillard JY, Pirotta N, et al. Irinotecan plus fluorouracil/leucovorin for metastatic colorectal cancer: a new survival standard. Oncologist 2001;6:81–91. 9. Moehler M, Hoffmann T, Zanke C, et al. Safety and efficacy of outpatient treatment with CPT-11 plus bolus folinic acid/5-fluorouracil as first-line chemotherapy for metastatic colorectal cancer. Anticancer Drugs 2003;14:79–85. 10. Grem JL. Biochemical modulation of 5-FU in systemic treatment of advanced colorectal cancer. Oncology (Huntingt) 2001;15:13–19.

166

O’Connor

11. Grem JL. 5-Fluoropyrimidines. In: Chabner BA, Longo DA (eds). Cancer Chemotherapy and Biotherapy. Principles and Practice, 2nd Ed. Lippincott-Raven, Philadelphia: 1996;149. 12. Piedbois P BM, Rustum Y. Modulation of fluorouracil by leucovorin in patients with advanced colorectal cancer: evidence in terms of response rate. Advanced Colorectal Cancer Meta-Analysis Project. J Clin Oncol 1992;10:896–903. 13. Doroshow JH, Multhauf P, Leong L, et al. Prospective randomized comparison of fluorouracil versus fluorouracil and high-dose continuous infusion leucovorin calcium for the treatment of advanced measurable colorectal cancer in patients previously unexposed to chemotherapy. J Clin Oncol 1990;8:491–501. 14. De Gramont A, Krulik M, Cady J, et al. High-dose folinic acid and 5-fluorouracil bolus and continuous infusion in advanced colorectal cancer. Eur J Cancer Clin Oncol 1988;24:1499–1503. 15. de Gramont A, Bosset JF, Milan C, et al. Randomized trial comparing monthly low-dose leucovorin and fluorouracil bolus with bimonthly high-dose leucovorin and fluorouracil bolus plus continuous infusion for advanced colorectal cancer: a French intergroup study. J Clin Oncol 1997;15:808–815. 16. Poon MA, O’Connell MJ, Moertel CG, et al. Biochemical modulation of fluorouracil: evidence of significant improvement of survival and quality of life in patients with advanced colorectal carcinoma. J Clin Oncol 1989;7:1407–1418. 17. Poon MA, O’Connell MJ, Wieand HS, et al. Biochemical modulation of fluorouracil with leucovorin: confirmatory evidence of improved therapeutic efficacy in advanced colorectal cancer. J Clin Oncol 1991;9:1967–1972. 18. Wolmark N, Rockette H, Fisher B, et al. The benefit of leucovorin-modulated fluorouracil as postoperative adjuvant therapy for primary colon cancer: results from National Surgical Adjuvant Breast and Bowel Project protocol C-03. J Clin Oncol 1993;11:1879–1887. 19. Evans RM, Laskin JD, Hakala MT. Effect of excess folates and deoxyinosine on the activity and site of action of 5-fluorouracil. Cancer Res 1981;41:3288–3295. 20. Cao S, Frank C, Rustum YM. Role of fluoropyrimidine Schedule and (6R,S)leucovorin dose in a preclinical animal model of colorectal carcinoma. J Natl Cancer Inst 1996;88:430–436. 21. Keyomarsi K, Moran RG. Folinic acid augmentation of the effects of fluoropyrimidines on murine and human leukemic cells. Cancer Res 1986;46:5229–5235. 22. Moran RG, Danenberg PV, Heidelberger C. Therapeutic response of leukemic mice treated with fluorinated pyrimidines and inhibitors of deoxyuridylate synthesis. Biochem Pharmacol 1982;31:2929–2935. 23. Houghton JA, Williams LG, de Graaf SS, et al. Relationship between dose rate of [6RS]Leucovorin administration, plasma concentrations of reduced folates, and pools of 5,10-methylenetetrahydrofolates and tetrahydrofolates in human colon adenocarcinoma xenografts. Cancer Res 1990;50:3493–3502. 24. Zhang ZG, Harstrick A, Rustum YM. Modulation of fluoropyrimidines: role of dose and schedule of leucovorin administration. Semin Oncol 1992;19:10–15. 25. Swain SM, Lippman ME, Egan EF, et al. Fluorouracil and high-dose leucovorin in previously treated patients with metastatic breast cancer. J Clin Oncol 1989;7:890–899. 26. Peters GJ, van der Wilt CL, van Groeningen CJ, et al. Thymidylate synthase inhibition after administration of fluorouracil with or without leucovorin in colon cancer patients: implications for treatment with fluorouracil. J Clin Oncol 1994;12:2035–2042. 27. Petrelli N, Herrera L, Rustum Y, et al. A prospective randomized trial of 5-fluorouracil versus 5-fluorouracil and high-dose leucovorin versus 5-fluorouracil and methotrexate in previously untreated patients with advanced colorectal carcinoma. J Clin Oncol 1987;5:1559–1565. 28. Petrelli N, Douglass HO, Herrera L, et al. The modulation of fluorouracil with leucovorin in metastatic colorectal carcinoma. A prospective randomize phase III trial. J Clin Oncol 1991;7:1419–1426.

Chapter 6 / Fluoropyrimidine Modulation

167

29. Martoni A, Cricca A, Guaraldi M, et al. Randomized clinical trial with a weekly regimen of 5-FU vs 5-FU + intermediate-dose folinic acid in the treatment of advanced colo-rectal cancer. Ann Oncol 1992;3:87–88. 30. Nobile MT, Canobbio L, Sobrero A, et al. A randomized trial of 5-fluorouracil alone versus 5-flourouracil and high dose leucovorin in untreated advanced colorectal cancer patients. Adv Exp Med Biol 1998;244:213–218. 31. Laufman LR, Bukowski RM, Collier MA, et al. A randomized, double-blind trial of fluorouracil plus placebo versus fluorouracil plus oral leucovorin in patients with metastatic colorectal cancer. J Clin Oncol 1993;11:1888–1893. 32. Leichman CG, Fleming TR, Muggia FM, et al. Phase II study of fluorouracil and its modulation in advanced colorectal cancer: a Southwest Oncology Group study. J Clin Oncol 1995;13:1303–1311. 33. Erlichman C, Fine S, Wong A, et al. A randomized trial of fluorouracil and folinic acid in patients with metastatic colorectal carcinoma. J Clin Oncol 1988;6:469–475. 34. Valone FH, Friedman MA, Wittlinger PS, et al. Treatment of patients with advanced colorectal carcinomas with fluorouracil alone, high-dose leucovorin plus fluorouracil, or sequential methotrexate, fluorouracil, and leucovorin: a randomized trial of the Northern California Oncology Group. J Clin Oncol 1989;7:1427–1436. 35. Labianca R, Pancera G, Aitini E, et al. Folinic acid + 5-fluorouracil (5-FU) versus equidose 5-FU in advanced colorectal cancer. Phase III study of ’GISCAD’ (Italian Group for the Study of Digestive Tract Cancer). Ann Oncol 1991;2:673–679. 36. Di Costanzo F, Bartolucci R, Calabresi F, et al. Fluorouracil-alone versus high-dose folinic acid and fluorouracil in advanced colorectal cancer: a randomized trial of the Italian Oncology Group for Clinical Research (GOIRC). Ann Oncol 1992;3(5):371–376. 37. Petrioli R, Lorenzi M, Aquino A, et al. Treatment of advanced colorectal cancer with highdose intensity folinic acid and 5-fluorouracil plus supportive care. Eur J Cancer 1995;31A:2105–2108. 38. Borner MM, Castiglione M, Bacchi M, et al. The impact of adding low-dose leucovorin to monthly 5-fluorouracil in advanced colorectal carcinoma: results of a phase III trial. Swiss Group for Clinical Cancer Research (SAKK). Ann Oncol 1998;9:535–541. 39. Lokich JJ, Ahlgren JD, Gullo JJ, et al. A prospective randomized comparison of continuous infusion fluorouracil with a conventional bolus schedule in metastatic colorectal carcinoma: a Mid-Atlantic Oncology Program Study. J Clin Oncol 1989;7:425–432. 40. Hansen RM, Ryan L, Anderson T, et al. Phase III study of bolus versus infusion fluorouracil with or without cisplatin in advanced colorectal cancer. J Natl Cancer Inst 1996;88:668–674. 41. Rougier P, Paillot B, LaPlanche A, et al. 5-Fluorouracil (5-FU) continuous intravenous infusion compared with bolus administration. Final results of a randomised trial in metastatic colorectal cancer. Eur J Cancer 1997;33:1789–1793. 42. Kohne CH, Schoffski P, Wilke H, et al. Effective biomodulation by leucovorin of high-dose infusion fluorouracil given as a weekly 24-hour infusion: results of a randomized trial in patients with advanced colorectal cancer. J Clin Oncol 1998;16:418–426. 43. Salonga D, Danenberg KD, Johnson M, et al. Colorectal tumors responding to 5-fluorouracil have low gene expression levels of dihydropyrimidine dehydrogenase, thymidylate synthase, and thymidine phosphorylase. Clin Cancer Res 2000;6:1322–1327. 44. Bertino JR. Biomodulation of 5-fluorouracil with antifolates. Semin Oncol 1997;24:S1852–S18-56. 45. Benz C, Cadman E. Modulation of 5-fluorouracil metabolism and cytotoxicity by antimetabolite pretreatment in human colorectal adenocarcinoma HCT-8. Cancer Res 1981;41:994–999. 46. Mini E, Mazzei T, Coronnello M, et al. Effects of 5-methyltetrahydrofolate on the activity of fluoropyrimidines against human leukemia (CCRF-CEM) cells. Biochem Pharmacol 1987;36(18):2905–2911.

168

O’Connor

47. Marsh JC, Bertino JR, Katz KH, et al. The influence of drug interval on the effect of methotrexate and fluorouracil in the treatment of advanced colorectal cancer. J Clin Oncol 1991;9:371–380. 48. Kemeny NE, Ahmed T, Michaelson RA, et al. Activity of sequential low-dose methotrexate and fluorouracil in advanced colorectal carcinoma: attempt at correlation with tissue and blood levels of phosphoribosylpyrophosphate. J Clin Oncol 1984;2:311–315. 49. Mackintosh J, Coates A, Swanson C, et al. Chemotherapy of advanced colorectal cancer. A randomized trial of sequential methotrexate and 5-fluorouracil. Am J Clin Oncol 1987;10:210–212. 50. Piedbois P BM, Blijham G. Meta-analysis of randomized trials testing the biochemical modulation of fluorouracil by methotrexate in metastatic colorectal cancer. Advanced Colorectal Cancer Meta-Analysis Project. J Clin Oncol 1994;12:960–969. 51. Blanke CD, Shultz J, Cox J, et al. A double-blind placebo-controlled randomized phase III trial of 5-fluorouracil and leucovorin, plus or minus trimetrexate, in previously untreated patients with advanced colorectal cancer. Ann Oncol 2002;13:87–91. 52. Punt CJ, Keizer HJ, Douma J, et al. Trimetrexate as biochemical modulator of 5-fluorouracil/ leucovorin in advanced colorectal cancer: final results of a randomised European study. Ann Oncol 2002;13:81–86. 53. Sobrero A, Romanini A, Russello O, et al. Sequence-dependent enhancement of HCT-8 cell kill by trimetrexate and fluoropyrimidines: implications for the mechanism of this interaction. Eur J Cancer Clin Oncol 1989;25:977–982. 54. Romanini A, Li WW, Colofiore JR, et al. Leucovorin enhances cytotoxicity of trimetrexate/ fluorouracil, but not methotrexate/fluorouracil, in CCRF-CEM cells. J Natl Cancer Inst 1992;84:1033–1038. 55. Elliott WL, Howard CT, Dykes DJ, et al. Sequence and schedule-dependent synergy of trimetrexate in combination with 5-fluorouracil in vitro and in mice. Cancer Res 1989;49:5586–5590. 56. Bertino JR. Modulation of 5-fluorouracil/leucovorin by trimetrexate—did it work? Ann Oncol 2002;13:10–11. 57. Longo GS, Izzo J, Chang YM, et al. Pretreatment of colon carcinoma cells with Tomudex enhances 5-fluorouracil cytotoxicity. Clin Cancer Res 1998;4:469–473. 58. Schwartz GK, Harstrick A, Gonzalez Baron M. Raltitrexed (Tomudex) in combination with 5-fluorouracil for the treatment of patients with advanced colorectal cancer: preliminary results from phase I clinical trials. Eur J Cancer 1999;35(Suppl 1):S9–13. 59. Schwartz GK, Harstrick A, Gonzalez Baron M. Raltitrexed (Tomudex) in combination with 5-fluorouracil for the treatment of patients with advanced colorectal cancer: preliminary results from phase I clinical trials. Eur J Cancer 1999;35(Suppl 1):S9–S13. 60. Caponigro F, Avallone A, McLeod H, et al. Phase I and pharmacokinetic study of tomudex combined with 5-fluorouracil plus levofolinic acid in advanced head and neck cancer and colorectal cancer. Clin Cancer Res 1999;5:3948–3955. 61. Cocconi G, Cunningham D, Van Cutsem E, et al. Open, randomized, multicenter trial of raltitrexed versus fluorouracil plus high-dose leucovorin in patients with advanced colorectal cancer. Tomudex Colorectal Cancer Study Group. J Clin Oncol 1998;16:2943–2952. 62. Cocconi G, Cunningham D, Van Cutsem E, et al. Open, randomized, multicenter trial of raltitrexed versus flourouracil plus high-dose leucovorin in patients with advanced colorectal cancer. Tomudex Colorectal Cancer Study Group. J Clin Oncol 1998;16(9):2943–2952. 63. Cunningham D, Zalcberg JR, Rath U, et al. Final results of a randomised trial comparing ’Tomudex’ (raltitrexed) with 5-fluorouracil plus leucovorin in advanced colorectal cancer. “Tomudex” Colorectal Cancer Study Group. Ann Oncol 1996;7:961–965.

Chapter 6 / Fluoropyrimidine Modulation

169

64. Shih C, Chen VJ, Gossett LS, et al. LY231514, a pyrrolo[2,3-D]pyrimidine-based antifolate that inhibits multiple folate-requiring enzymes. Cancer Res 1997;57:1116–1123. 65. Teicher BA, Chen V, Shih C, et al. Treatment regimens including the multitargeted antifolate LY231514 in human tumor xenografts. Clin Cancer Res 2000;6:1016–1023. 66. Matei D, Hagag N, Madajewicz S, et al. Preclinical and clinical evaluation of 5-fluorouracil biochemical modulation with folinic acid and hydroxyurea for patients with colorectal carcinoma. Cancer 1995;76:1330–1335. 67. Engstrom PF, MacIntyre JM, Mittelman A, et al. Chemotherapy of advanced colorectal carcinoma: fluorouracil alone vs. two drug combinations using fluorouracil, hydroxyurea, semustine, dacarbazine, razoxane, and mitomycin. A phase III trial by the Eastern Cooperative Oncology Group (EST: 1278). Am J Clin Oncol 1984;7:313–318. 68. Engstrom PF, MacIntyre JM, Schutt AJ, et al. Chemotherapy of large bowel carcinoma—— fluorouracil (FU) + hydroxyurea (HU) vs. methyl-CCNU, oncovin, fluorouracil, and streptozotocin (MOF-Strep). An Eastern Cooperative Oncology Group study. Am J Clin Oncol 1985;8:358–361. 69. Di Costanzo F, el-Taani H, Parriani D, et al. Hydroxyurea may increase the activity of fluorouracil plus folinic acid in advanced gastrointestinal cancer: phase II study. Cancer Invest 1996;14:234–238. 70. Di Costanzo F, Tagliaventi M, Carlini P, et al. High-dose folinic acid and fluorouracil with or without ifosfamide is an inactive combination in advanced pancreatic cancer. A randomized phase II study of the Italian Oncology Group for Clinical Research (G.O.I.R.C.). Am J Clin Oncol 1996;19:307–310. 71. de Gramont A, Louvet C, Bennamoun M, et al. Dual modulation of 5-fluorouracil with folinic acid and hydroxyurea in metastatic colorectal cancer. J Infus Chemother 1996;6:97–101. 72. Di Costanzo F, Gasperoni S, Malacarne P, et al. High-dose folinic acid and 5-fluorouracil alone or combined with hydroxyurea in advanced colorectal cancer: a randomized trial of the Italian Oncology Group for Clinical Research. Am J Clin Oncol 1998;21:369–375. 73. Madajewicz S, Hentschel P, Burns P, et al. Phase I chemotherapy study of biochemical modulation of folinic acid and fluorouracil by gemcitabine in patients with solid tumor malignancies. J Clin Oncol 2000;18:3553–3557. 74. Plunkett W, Huang P, Gandhi V. Preclinical characteristics of gemcitabine. Anticancer Drugs 6 Suppl 1995;6:7–13. 75. Liu MC, Lin TS, Sartorelli AC. Synthesis and antitumor activity of amino derivatives of pyridine-2-carboxaldehyde thiosemicarbazone. J Med Chem 1992;35:3672–3677. 76. Liu MC, Lin TS, Penketh P, et al. Synthesis and antitumor activity of 4- and 5-substituted derivatives of isoquinoline-1-carboxaldehyde thiosemicarbazone. J Med Chem 1995;38:4234–4243. 77. Harris BE, Song RL, He YJ, et al. Circadian rhythm of rat liver dihydropyrimidine dehydrogenase. Possible relevance to fluoropyrimidine chemotherapy. Biochem Pharmacol 1988;37:4759–4762. 78. Harris BE, Song RL, Soong SJ, et al. Circadian variation of 5-fluorouracil catabolism in isolated perfused rat liver. Cancer Res 1989;49:6610–6614. 79. Harris BE, Song R, Soong SJ, et al. Relationship between dihydropyrimidine dehydrogenase activity and plasma 5-fluorouracil levels with evidence for circadian variation of enzyme activity and plasma drug levels in cancer patients receiving 5-fluorouracil by protracted continuous infusion. Cancer Res 1990;50:197–201. 80. Diasio RB. Clinical implications of dihydropyrimidine dehydrogenase on 5-FU pharmacology. Oncology (Huntingt) 2001;15:21-26; discussion 27.

170

O’Connor

81. Pazdur R, Lassere Y, Diaz-Canton E, et al. Phase I trials of uracil-tegafur (UFT) using 5 and 28 day administration schedules: demonstration of schedule-dependent toxicities. Anticancer Drugs 1996;7:728–733. 82. Muggia FM, Wu X, Spicer D, et al. Phase I and pharmacokinetic study of oral UFT, a combination of the 5-fluorouracil prodrug tegafur and uracil. Clin Cancer Res 1996;2:1461–467. 83. Pazdur R, Lassere Y, Diaz-Canton E, et al. Phase I trial of uracil-tegafur (UFT) plus oral leucovorin: 14-day schedule. Invest New Drugs 1997;15:123–128. 84. Meropol NJ, Rustum YM, Petrelli NJ, et al. A phase I and pharmacokinetic study of oral uracil, ftorafur, and leucovorin in patients with advanced cancer. Cancer Chemother Pharmacol 1996;37:581–586. 85. Saltz LB, Leichman CG, Young CW, et al. A fixed-ratio combination of uracil and Ftorafur (UFT) with low dose leucovorin. An active oral regimen for advanced colorectal cancer. Cancer 1995;75:782–785. 86. Sanchiz F, Milla A. Tegafur-uracil (UFT) plus folinic acid in advanced rectal cancer. Jpn J Clin Oncol 1994;24:322–326. 87. Pazdur R, Lassere Y, Rhodes V, et al. Phase II trial of uracil and tegafur plus oral leucovorin: an effective oral regimen in the treatment of metastatic colorectal carcinoma. J Clin Oncol 1994;12:2296–2300. 88. Aranda E, Anton-Torres A, Sastre J, et al. Tegafur and uracil plus leucovorin in advanced colorectal cancer: a phase II trial. Clin Colorectal Cancer 2001;1:43–46. 89. van Groeningen CJ, Peters GJ, Schornagel JH, et al. Phase I clinical and pharmacokinetic study of oral S-1 in patients with advanced solid tumors. J Clin Oncol 2000;18:2772–2779. 90. Ohtsu A, Baba H, Sakata Y, et al. Phase II study of S-1, a novel oral fluorophyrimidine derivative, in patients with metastatic colorectal carcinoma. S-1 Cooperative Colorectal Carcinoma Study Group. Br J Cancer 2000;83:141–145. 91. Ahmed FY, Johnston SJ, Cassidy J, et al. Eniluracil treatment completely inactivates dihydropyrimidine dehydrogenase in colorectal tumors. J Clin Oncol 1999;17:2439–2445. 92. Spector T, Harrington JA, Porter DJ. 5-Ethynyluracil (776C85): inactivation of dihydropyrimidine dehydrogenase in vivo. Biochem Pharmacol 1993;46:2243–2248. 93. Schilsky RL, Hohneker J, Ratain MJ, et al. Phase I clinical and pharmacologic study of eniluracil plus fluorouracil in patients with advanced cancer. J Clin Oncol 1998;16:1450–1457. 94. Benson AB, 3rd. Oral eniluracil/5-FU for advanced colon and breast carcinomas. Oncology (Huntingt) 2001;15:57–63; discussion 64. 95. Makower D, Wadler S. Interferons as biomodulators of fluoropyrimidines in the treatment of colorectal cancer. Semin Oncol 1999;26:663–671. 96. Wadler S, Horowitz R, Mao X, et al: Effect of interferon on 5-fluorouracil-induced perturbations in pools of deoxynucleotide triphosphates and DNA strand breaks. Cancer Chemother Pharmacol 1996;38:529-535. 97. Schwartz EL, Hoffman M, O’Connor CJ, et al. Stimulation of 5-fluorouracil metabolic activation by interferon-alpha in human colon carcinoma cells. Biochem Biophys Res Commun 1992;182:1232–1239. 98. Schwartz EL, Baptiste N, Wadler S, et al. Thymidine phosphorylase mediates the sensitivity of human colon carcinoma cells to 5-fluorouracil. J Biol Chem 1995;270:19073–19077. 99. Makower D, Wadler S, Haynes H, et al. Interferon induces thymidine phosphorylase/plateletderived endothelial cell growth factor expression in vivo. Clin Cancer Res 1997;3:923–929. 100. Chu E, Zinn S, Boarman D, et al. Interaction of gamma interferon and 5-fluorouracil in the H630 human colon carcinoma cell line. Cancer Res 1990;50:5834–5840. 101. Chu E, Koeller DM, Johnston PG, et al. Regulation of thymidylate synthase in human colon cancer cells treated with 5-fluorouracil and interferon-gamma. Mol Pharmacol 1993;43:527–533. 102. Hill M, Norman A, Cunningham D, et al. Royal Marsden phase III trial of fluorouracil with or without interferon alfa-2b in advanced colorectal cancer. J Clin Oncol 1995;13:1297–1302.

Chapter 6 / Fluoropyrimidine Modulation

171

103. Greco FA, Figlin R, York M, et al. Phase III randomized study to compare interferon alfa2a in combination with fluorouracil versus fluorouracil alone in patients with advanced colorectal cancer. J Clin Oncol 1996;14:2674–2681. 104. Corfu. A Study Group: Phase III randomized study of two fluorouracil combinations with either interferon alfa-2a or leucovorin for advanced colorectal cancer. J Clin Oncol 1995;13:921–928. 105. Dufour P, Husseini F, Dreyfus B, et al. 5-Fluorouracil versus 5-fluorouracil plus alphainterferon as treatment of metastatic colorectal carcinoma. A randomized study. Ann Oncol 1996;7:575–579. 106. Schwartzberg LS, Petak I, Stewart C, et al. Modulation of the Fas signaling pathway by IFNgamma in therapy of colon cancer: phase I trial and correlative studies of IFN-gamma, 5fluorouracil, and leucovorin. Clin Cancer Res 2002;8:2488–2498. 107. Moller P, Koretz K, Leithauser F, et al. Expression of APO-1 (CD95), a member of the NGF/ TNF receptor superfamily, in normal and neoplastic colon epithelium. Int J Cancer 1994;57:371–377. 108. Leithauser F, Dhein J, Mechtersheimer G, et al. Constitutive and induced expression of APO1, a new member of the nerve growth factor/tumor necrosis factor receptor superfamily, in normal and neoplastic cells. Lab Invest 1993;69:415–429. 109. Tillman DM, Petak I, Houghton JA. A Fas-dependent component in 5-fluorouracil/leucovorin-induced cytotoxicity in colon carcinoma cells. Clin Cancer Res 1999;5:425–430. 110. Sobrero AF, Moir RD, Bertino JR, et al. Defective facilitated diffusion of nucleosides, a primary mechanism of resistance to 5-fluoro-2'-deoxyuridine in the HCT-8 human carcinoma line. Cancer Res 1985;45:3155–3160. 111. Mackey JR, Mani RS, Selner M, et al. Functional nucleoside transporters are required for gemcitabine influx and manifestation of toxicity in cancer cell lines. Cancer Res 1998;58:4349–4357. 112. Mackey JR BS, Young JD, Cass CE. Nucleoside transport and its significance for anticancer drug resistance. Drug Resistance Updates 1998;1:310–324. 113. Baldwin SA, Mackey JR, Cass CE, et al. Nucleoside transporters: molecular biology and implications for therapeutic development. Mol Med Today 1999;5:216–224. 114. Griffiths M, Yao SY, Abidi F, et al. Molecular cloning and characterization of a nitrobenzylthioinosine-insensitive (ei) equilibrative nucleoside transporter from human placenta. Biochem J 1997;328(Pt 3):739–743. 115. Ritzel MW, Yao SY, Huang MY, et al. Molecular cloning and functional expression of cDNAs encoding a human Na+-nucleoside cotransporter (hCNT1). Am J Physiol 1997;272:C707–714. 116. Crawford CR, Patel DH, Naeve C, et al. Cloning of the human equilibrative, nitrobenzylmercaptopurine riboside (NBMPR)-insensitive nucleoside transporter ei by functional expression in a transport-deficient cell line. J Biol Chem 1998;273:5288–5293. 117. Griffiths M, Beaumont N, Yao SY, et al. Cloning of a human nucleoside transporter implicated in the cellular uptake of adenosine and chemotherapeutic drugs. Nat Med 1997;3:89–93. 118. Wang J, Su SF, Dresser MJ, et al. Na(+)-dependent purine nucleoside transporter from human kidney: cloning and functional characterization. Am J Physiol 1997;273:F1058–1065. 119. Ritzel MW, Yao SY, Ng AM, et al. Molecular cloning, functional expression and chromosomal localization of a cDNA encoding a human Na+/nucleoside cotransporter (hCNT2) selective for purine nucleosides and uridine. Mol Membr Biol 1998;15:203–211. 120. Griffith DA, Jarvis SM. Nucleoside and nucleobase transport systems of mammalian cells. Biochim Biophys Acta 1996;1286:153–181. 121. Smith PG, Thomas HD, Barlow HC, et al. In vitro and in vivo properties of novel nucleoside transport inhibitors with improved pharmacological properties that potentiate antifolate activity. Clin Cancer Res 2001;7:2105–2113.

172

O’Connor

122. Fischer PH, Willson JK, Risueno C, et al. Biochemical assessment of the effects of acivicin and dipyridamole given as a continuous 72-hour intravenous infusion. Cancer Res 1988;48:5591–5596. 123. Budd GT, Jayaraj A, Grabowski D, et al. Phase I trial of dipyridamole with 5-fluorouracil and folinic acid. Cancer Res 1990;50:7206–7211. 124. Curtin NJ, Bowman KJ, Turner RN, et al. Potentiation of the cytotoxicity of thymidylate synthase (TS) inhibitors by dipyridamole analogues with reduced alpha1-acid glycoprotein binding. Br J Cancer 1999;80:1738–1746. 125. Jansen WJ, Pinedo HM, van der Wilt CL, et al. The influence of BIBW22BS, a dipyridamole derivative, on the antiproliferative effects of 5-fluorouracil, methotrexate and gemcitabine in vitro and in human tumour xenografts. Eur J Cancer 1995;31A:2313–2319. 126. Su J, Zhen YC, Qi CQ, et al. Antibiotic C3368-A, a fungus-derived nucleoside transport inhibitor, potentiates the activity of antitumor drugs. Cancer Chemother Pharmacol 1995;36:149–154. 127. Weber G, Ichikawa S, Nagai M, et al. Azidothymidine inhibition of thymidine kinase and synergistic cytotoxicity with methotrexate and 5-fluorouracil in rat hepatoma and human colon cancer cells. Cancer Commun 1990;2:129–133. 128. Weber G, Nagai M, Prajda N, et al. AZT: a biochemical response modifier of methotrexate and 5-fluorouracil cytotoxicity in human ovarian and pancreatic carcinoma cells. Cancer Commun 1991;3:127–132. 129. Brunetti I, Falcone A, Calabresi P, et al. 5-Fluorouracil enhances azidothymidine cytotoxicity: in vitro, in vivo, and biochemical studies. Cancer Res 1990;50:4026–4031. 130. Zimmerman TP, Domin BA, Mahony WB. Inhibition of thymidine transport by 3'-azido-3'deoxythymidine and its metabolites. Oncol Res 1993;5:483–487. 131. Ishikawa T, Sekiguchi F, Fukase Y, et al. Positive correlation between the efficacy of capecitabine and doxifluridine and the ratio of thymidine phosphorylase to dihydropyrimidine dehydrogenase activities in tumors in human cancer xenografts. Cancer Res 1998;58:685–690. 132. Ishitsuka H. Capecitabine: preclinical pharmacology studies. Invest New Drugs 2000;18:343–354. 133. Miwa M, Ura M, Nishida M, et al. Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue. Eur J Cancer 1998;34:1274–1281. 134. Schuller J, Cassidy J, Dumont E, et al. Preferential activation of capecitabine in tumor following oral administration to colorectal cancer patients. Cancer Chemother Pharmacol 2000;45:291–297. 135. Camiener GW, Smith CG. Studies of the enzymatic deamination of cytosine arabinoside. I. Enzyme distribution and species specificity. Biochem Pharmacol 1965;14:1405–1416. 136. Giusti G, Mangoni C, De Petrocellis B, et al. Deoxycytidylate deaminase and deoxycytidine deaminase in normal and neoplastic human tissues. Enzymol Biol Clin (Basel) 1970;11:375–383. 137. Chabner BA, Johns DG, Coleman CN, et al. Purification and properties of cytidine deaminase from normal and leukemic granulocytes. J Clin Invest 1974;53:922–931. 138. Matsuura T, Kuratate I, Teramachi K, et al. Thymidine phosphorylase expression is associated with both increase of intratumoral microvessels and decrease of apoptosis in human colorectal carcinomas. Cancer Res 1999;59:5037–5040. 139. Sumizawa T, Furukawa T, Haraguchi M, et al. Thymidine phosphorylase activity associated with platelet-derived endothelial cell growth factor. J Biochem (Tokyo) 1993;114:9–14. 140. Haraguchi M, Miyadera K, Uemura K, et al. Angiogenic activity of enzymes. Nature 1994;368:198. 141. Ishikawa F, Miyazono K, Hellman U, et al. Identification of angiogenic activity and the cloning and expression of platelet-derived endothelial cell growth factor. Nature 1989;338:557–562.

Chapter 6 / Fluoropyrimidine Modulation

173

142. Takebayashi Y, Akiyama S, Akiba S, et al. Clinicopathologic and prognostic significance of an angiogenic factor, thymidine phosphorylase, in human colorectal carcinoma. J Natl Cancer Inst 1996;88:1110–1117. 143. Takebayashi Y, Miyadera K, Akiyama S, et al. Expression of thymidine phosphorylase in human gastric carcinoma. Jpn J Cancer Res 1996;87:288–295. 144. Maeda K, Chung YS, Ogawa Y, et al. Thymidine phosphorylase/platelet-derived endothelial cell growth factor expression associated with hepatic metastasis in gastric carcinoma. Br J Cancer 1996;73:884–888. 145. Takebayashi Y, Yamada K, Maruyama I, et al. The expression of thymidine phosphorylase and thrombomodulin in human colorectal carcinomas. Cancer Lett 1995;92:1–7. 146. Takebayashi Y, Yamada K, Miyadera K, et al. The activity and expression of thymidine phosphorylase in human solid tumours. Eur J Cancer 1996;32A:1227–1232. 147. Shomori K, Sakatani T, Goto A, et al. Thymidine phosphorylase expression in human colorectal mucosa, adenoma and carcinoma: role of p53 expression. Pathol Int 1999;49:491–499. 148. Kitazono M, Takebayashi Y, Ishitsuka K, et al. Prevention of hypoxia-induced apoptosis by the angiogenic factor thymidine phosphorylase. Biochem Biophys Res Commun 1998;253:797–803. 149. Sawada N, Ishikawa T, Fukase Y, et al. Induction of thymidine phosphorylase activity and enhancement of capecitabine efficacy by taxol/taxotere in human cancer xenografts. Clin Cancer Res 1998;4:1013–1019. 150. Eda H, Fujimoto K, Watanabe S, et al. Cytokines induce uridine phosphorylase in mouse colon 26 carcinoma cells and make the cells more susceptible to 5'-deoxy-5-fluorouridine. Jpn J Cancer Res 1993;84:341–347. 151. Eda H, Fujimoto K, Watanabe S, et al. Cytokines induce thymidine phosphorylase expression in tumor cells and make them more susceptible to 5'-deoxy-5-fluorouridine. Cancer Chemother Pharmacol 1993;32:333–338. 152. Tevaearai HT, Laurent PL, Suardet L, et al. Interactions of interferon-alpha 2a with 5'-deoxy5-fluorouridine in colorectal cancer cells in vitro. Eur J Cancer 1992;28:368–372. 153. Tanaka Y EM, Ouchi K, Mori K, Ishitsuka H. Augmentation of antitumor activity of 5'deoxy5-fluorouridine by IL-12 through the up-regulation of pyrimidine nucleoside phosphorylase in murine tumor models. Eur J Cancer 1997;33:176–177. 154. Ishitsuka H, Ishikawa t, Fukase Y, et al. Capecitabine and the dThdPase up-regulators IFN or Taxol showed synergistic activity in human cancer xenografts. Proc Am Soc Clin Oncol 1996;37:2766. 155. Blum JL, Jones SE, Buzdar AU, et al. Multicenter phase II study of capecitabine in paclitaxelrefractory metastatic breast cancer. J Clin Oncol 1999;17:485–493. 156. Hoff PM, Ansari R, Batist G, et al. Comparison of oral capecitabine versus intravenous fluorouracil plus leucovorin as first-line treatment in 605 patients with metastatic colorectal cancer: results of a randomized phase III study. J Clin Oncol 2001;19:2282–2292. 157. Van Cutsem E, Twelves C, Cassidy J, et al. Oral capecitabine compared with intravenous fluorouracil plus leucovorin in patients with metastatic colorectal cancer: results of a large phase III study. J Clin Oncol 2001;19:4097–4106. 158. Twelves C, Boyer M, Findlay M, et al. Capecitabine (Xeloda) improves medical resource use compared with 5-fluorouracil plus leucovorin in a phase III trial conducted in patients with advanced colorectal carcinoma. Eur J Cancer 2001;37:597–604. 159. O’Shaughnessy J, Miles D, Vukelja S, et al. Superior survival with capecitabine plus docetaxel combination therapy in anthracycline-pretreated patients with advanced breast cancer: phase III trial results. J Clin Oncol 2002;20:2812–2823. 160. Kabbinavar F, Hurwitz HI, Fehrenbacher L, et al. Phase II, randomized trial comparing bevacizumab plus fluorouracil (FU)/leucovorin (LV) with FU/LV alone in patients with metastatic colorectal cancer. J Clin Oncol 2003;21:60–65. 161. Wu Q, Dolnick BJ. Detection of thymidylate synthase modulators by a novel screening assay. Mol Pharmacol 2003;63:167–73.

174

O’Connor

162. Kawakami K, Omura K, Kanehira E, et al. Polymorphic tandem repeats in the thymidylate synthase gene is associated with its protein expression in human gastrointestinal cancers. Anticancer Res 1999;19:3249–3252. 163. Kawakami K, Salonga D, Park JM, et al. Different lengths of a polymorphic repeat sequence in the thymidylate synthase gene affect translational efficiency but not its gene expression. Clin Cancer Res 2001;7:4096–4101. 164. Kaneda S, Takeishi K, Ayusawa D, et al. Role in translation of a triple tandemly repeated sequence in the 5'-untranslated region of human thymidylate synthase mRNA. Nucleic Acids Res 1987;15:1259–1270. 165. Horie N, Aiba H, Oguro K, et al. Functional analysis and DNA polymorphism of the tandemly repeated sequences in the 5'-terminal regulatory region of the human gene for thymidylate synthase. Cell Struct Funct 1995;20:191–197. 166. Marsh S, McKay JA, Cassidy J, et al. Polymorphism in the thymidylate synthase promoter enhancer region in colorectal cancer. Int J Oncol 2001;19:383–386. 167. Luo HR, Lu XM, Yao YG, et al. Length polymorphism of thymidylate synthase regulatory region in Chinese populations and evolution of the novel alleles. Biochem Genet 2002;40:41–51. 168. Pullarkat ST, Stoehlmacher J, Ghaderi V, et al. Thymidylate synthase gene polymorphism determines response and toxicity of 5-FU chemotherapy. Pharmacogenomics J 2001;1:65–70. 169. Leichman CG, Lenz HJ, Leichman L, et al. Quantitation of intratumoral thymidylate synthase expression predicts for disseminated colorectal cancer response and resistance to protracted-infusion fluorouracil and weekly leucovorin. J Clin Oncol 1997;15:3223–3229. 170. Metzger R, Danenberg K, Leichman CG, et al. High basal level gene expression of thymidine phosphorylase (platelet-derived endothelial cell growth factor) in colorectal tumors is associated with nonresponse to 5-fluorouracil. Clin Cancer Res 1998;4:2371–2376. 171. Link KH, Kornmann M, Butzer U, et al. Thymidylate synthase quantitation and in vitro chemosensitivity testing predicts responses and survival of patients with isolated nonresectable liver tumors receiving hepatic arterial infusion chemotherapy. Cancer 2000;89:288–296. 172. Edler D, Glimelius B, Hallstrom M, et al. Thymidylate synthase expression in colorectal cancer: a prognostic and predictive marker of benefit from adjuvant fluorouracil-based chemotherapy. J Clin Oncol 2002;20:1721–1728. 173. Farrugia DC, Ford HE, Cunningham D, et al. Thymidylate synthase expression in advanced colorectal cancer predicts for response to raltitrexed. Clin Cancer Res 2003;9:792–801.

Chapter 7 / Inhibitors of HER2 With Taxanes

7

175

Development of Inhibitors of HER2 With Taxanes New Directions in Breast Cancer Therapy

Shanu Modi, MD, Monica N. Fornier, MD, Andrew D. Seidman, MD CONTENTS INTRODUCTION TARGETING HER2 HERCEPTIN IN COMBINATION WITH TAXANES INTEGRATION OF TAXANE/TRASTUZUMAB COMBINATIONS INTO ADJUVANT THERAPY NOVEL INHIBITORS OF HER2

INTRODUCTION The development of superior treatment strategies directed against metastatic breast cancer remains a major challenge. Because this condition is historically treatable but not curable, the primary goal of clinical management is often to create an optimal equilibrium between maximizing response, minimizing toxicity, improving survival, and optimizing symptom palliation. The search for new drugs that might provide these characteristics, together with an expanding understanding of the molecular basis of cancer, has prompted the development and investigation of “biologic” agents, which offer high tumor selectivity together with a favorable therapeutic ratio.

From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

175

176

Modi, Fornier, and Seidman

TARGETING HER2 The HER2/neu oncogene, also referred to as c-erb-2, neu, encodes a protein with a molecular weight of 185,000 daltons (p185). The gene product is a transmembrane tyrosine kinase receptor belonging to a family of epidermal growth factor receptors structurally related to the human epidermal growth factor receptor (EGFR) (1,2). The HER2 gene product is composed of a cytoplasmic domain with tyrosine kinase activity, a transmembrane domain, and an extracellular domain (ECD) that may be shed from the surface of breast cancer cells. The other members of this family are HER1 (also known as EGFR), HER3, and HER4. This receptor family is known as the type 1-receptor tyrosine kinases. The HER2 receptor has partial homology to the EGFR; however, to date, unlike for EGFR, HER3, and HER4, no ligand for HER2 has been identified (3). It is hypothesized that the main role of HER2 may be to dimerize with the other members of the HER family of receptors. The HER2 gene was first identified as a transforming oncogene in DNA from chemically induced neuroblastomas in the rat (4). HER2 is overexpressed or amplified in approx 20–25% of human breast cancers (5). Evidence exists to support a direct role for HER2 over-expression in the pathogenesis and poor clinical outcome of human tumors: HER2 has been shown to be overexpressed in an array of human carcinomas, including, but not limited to, breast, ovarian, gastric, colon, and non-small-cell lung cancer (6). In particular, amplification of the HER2 proto-oncogene, and overexpression of its protein product in patients with breast cancer, has been linked to a poor prognosis, with more aggressive clinical course and shortened survival (7). Strategies to antagonize the abnormal function of overexpressed HER2 were developed with creation of antibodies directed against the rat neu receptor and the HER2 receptor on human cell lines. The 4D5 murine monoclonal antibody, directed against the extracellular domain of HER2/neu gene product, is a potent inhibitor of growth in vitro and in xenograft models of HER2 overexpressing breast cancer (8). As murine antibodies are limited clinically because they are immunogenic, a recombinant humanized monoclonal antibody that binds with high affinity and specificity to the extracellular domain of the p185 HER2 protein (Herceptin, Genentech, Inc., South San Francisco, CA) was produced and showed antitumor activity as a single agent in phase I and II trials for patients with metastatic breast carcinoma overexpressing HER2. Selection of HER2positive patients for trastuzumab therapy is supported by preclinical studies demonstrating that the antibody has little or no effect on HER2-negative cells (9); in fact, preclinical observations suggest that HER2 receptor density in excess of 100,000 receptors per cell would be required for maximal trastuzumab benefit (10). The efficacy and safety of single-agent trastuzumab was first evaluated by Baselga et al. in a phase II clinical trial involving patients with

Chapter 7 / Inhibitors of HER2 With Taxanes

177

metastatic breast cancer whose tumors overexpressed HER2, most of them extensively pretreated (11). The treatment plan consisted of a loading dose of trastuzumab of 250 mg administered intravenously, which was followed by 10 weekly doses of 100 mg each. Upon completion of this treatment period, patients with no disease progression could receive a weekly maintenance dose of 100 mg, to be continued until disease progression. Pharmacokinetic studies were also performed: the goal was to achieve trastuzumab trough serum concentrations exceeding 10 μg/mL, a level associated with optimal inhibition of cell growth in preclinical models (12). Over 90% of the examined patients (41 patients) had trastuzumab trough levels above the targeted 10 μg/mL level, with a mean serum half-life of trastuzumab of 8.3 ± 5.0 d. Antibodies against trastuzumab were not detected in any patients. Treatment with the antibody was remarkably well tolerated, with the most common side effect being fever and chills occurring after the first administration of trastuzumab. An overall response rate of 11.6% was reported. The single-agent activity of trastuzumab was subsequently confirmed in larger trials in heavily pretreated and minimally pretreated patients with metastatic breast cancer (13,14).

HERCEPTIN IN COMBINATION WITH TAXANES Notably, in vitro and in vivo preclinical studies showed that administration of trastuzumab in combination with some chemotherapeutic agents, such as the taxanes, doxorubicin, platinum salts, etoposide, and vinblastine, significantly inhibited the growth of breast tumor-derived cell lines that overexpress the HER2 gene product (15,16). Treatment of athymic mice bearing BT-474 breast cancer xenografts overexpressing HER2 with paclitaxel and trastuzumab resulted in greater inhibition of growth than that observed with either agent alone, yielding a striking rate of tumor eradication, and providing evidence that trastuzumab can effectively enhance the tumoricidal effects of paclitaxel. Following a 5-wk treatment period, 59% of mice who had received trastuzumab and paclitaxel were tumor free, compared with 17.3% of mice treated with paclitaxel alone (p = 0.004). Moreover, the increased effects were observed in the absence of additional animal toxicity. These observations prompted the use of trastuzumab in conjunction with chemotherapy. Motivated by these findings, a multicenter, multinational, randomized phase III trial examined the activity of trastuzumab in combination with chemotherapy (17). This four-armed trial enrolled 469 patients with HER2-positive metastatic breast cancer to receive chemotherapy with or without trastuzumab. All patients eligible for trial entry had tumors that were scored as 2+ or 3+ for HER2 by immunohistochemistry. Women without prior exposure to anthracycline were randomized to receive doxorubicin (or epirubicin) plus cyclophosphamide with or without trastuzumab. Patients who had previously received adjuvant

178

Modi, Fornier, and Seidman

anthracycline were treated with “q3-weekly” paclitaxel as a single agent or paclitaxel plus trastuzumab. Trastuzumab was delivered weekly at the dose of 2 mg/kg, after a loading dose of 4 mg/kg. Despite a crossover design that allowed patients initially assigned to chemotherapy alone to receive subsequent trastuzumab when they progressed (approximately two-thirds of patients did so), greater clinical benefit was noted among patients who received trastuzumab plus chemotherapy, with improved ORR (50% vs 32%, p < 0.001), median time to disease progression (TTP) (7.4 vs 4.6 mo, p < 0.001) and median OS (25 vs 20 mo, p = 0.01), compared with those who received chemotherapy alone. The overall survival advantage of adding trastuzumab was seen both in the AC and in the paclitaxel group; however, the combination of trastuzumab and an anthracycline-containing regimen resulted in an increased incidence of subclinical and clinical cardiac toxicity. A retrospective analysis of patient response on the basis of HER2 status as determined by fluorescent in situ hybridization (FISH) was also performed (18). Patients whose disease showed evidence of HER2 gene amplification by FISH had a higher response rate to the combination regimen compared to those patients whose disease did not show gene amplification. (54% vs 38%, respectively), and identified those patients who derived a survival benefit from the addition of trastuzumab (relative risk of mortality = 0.71 vs 1.11, respectively). Moreover, patients with tumors lacking HER2 amplification (FISH negative), the ORR was similar regardless of treatment groups (38%, with comparable 95% confidence intervals [CIs]), thus suggesting that trastuzumab might not confer benefit in this group of patients. Of note, this group included many patients with “2+ overexpressing” breast cancer as assessed by IHC. In a collaborative trial between Memorial Sloan-Kettering Cancer Center and the M. D. Anderson Cancer Center, 95 patients with metastatic breast carcinoma, both overexpressing and nonoverexpressing HER2, were treated with weekly trastuzumab at 4 mg/kg initial dose followed by 2 mg/kg weekly intravenously plus weekly 1-h paclitaxel at 90mg/m2 (19). Therapy was generally well tolerated, with neuropathy being the most common nonhematologic toxicity; significant neuropathy occurred in 28% of patients, with grade 4 toxicity occurring in one patient. Three patients pretreated with anthracycline had serious cardiac complications. The intent-to-treat response rate for all 95 patients enrolled was 56.8%. The regimen produced statistically significantly higher response rates in patients with HER2-overexpressing metastatic breast cancer compared with HER2 nonoverexpressors, as determined by tissue analysis with a panel of four antibodies: Dako HercepTest, TAB 250, CB11, and pAb1. For example, response rates were 81% and 43% for HER2-positive and HER2-negative patients, respectively, as determined using TAB250 (p = 0.001). In patients with FISHpositive disease, the response rate was found to be 75% compared with 44% in FISH-negative patients (p = 0.004). Of note, in this study, IHC was as effective

Chapter 7 / Inhibitors of HER2 With Taxanes

179

Fig. 1. Modification of Cancer and Leukemia Group B (CALGB): dose-dense vs standard paclitaxel.

as FISH as a predictor of response to combination therapy with paclitaxel and trastuzumab. On the basis of these positive results, trastuzumab was incorporated into a phase III clinical study (CALGB 9840) to compare weekly 1-h paclitaxel and q3-weekly 3-h paclitaxel in patients with metastatic breast cancer (Fig. 1). Results for the 735 patients analyzed (which included 158 patients treated with q3-weekly paclitaxel on the CALGB 9342 trial) revealed that weekly paclitaxel was superior to standard q3-weekly paclitaxel with respect to response rate (40% vs 28%, p = 0.017) and time to progression (9 vs 5 mo, p = 0.0008). The overall survival was 24 mo vs 16 mo in favor of weekly paclitaxel, but this was not statistically significant (p = 0.17). Furthermore, there was no observed benefit with the addition of trastuzumab to paclitaxel for HER2-normal metastatic breast cancer. Although weekly paclitaxel caused more grade 3 sensory/motor neuropathy (8% vs 4%, p = 0.001), it was associated with less grade *3 neutropenia (8% vs 15%, p = 0.013) (19a). In addition to centralized HER2 assessment by FISH and IHC, serial evaluation of circulation serum levels of the cleaved extracellular domain (p105) of HER2 and quality of life assessments were also performed in parallel with this trial and will be reported at a later date. Furthermore, clinical trials are currently being conducted to evaluate alternative dose schedules for trastuzumab: data from a phase I-II trial of trastuzumab plus paclitaxel, both given tri-weekly, in women with HER2-positive metastatic breast cancer, were presented. The primary aim of the study was to characterize the pharmacokinetic profile of trastuzumab 6 mg/kg by intravenous administration every 3 wk, following an initial loading dose of 8 mg/kg. Preliminary pharmacologic data from this study indicated that the tri-weekly dosing schedule of

180

Modi, Fornier, and Seidman

trastuzumab is compatible with maintenance of biologically active levels of the drug: the measured trough levels were approx 40, 55, and 60 μg/mL, consistent with those observed following weekly administration using the standard dose schedule. There were no unexpected toxicities, and overall response rate was 53% (20). A multicenter study of q3-weekly trastuzumab monotherapy for metastatic breast cancer has already enrolled more than 90 patients. Docetaxel, the second taxane to enter clinical development, is one of the most active single agents currently available for the treatment of breast cancer (21–23). In addition, preclinical studies revealing a synergistic interaction between docetaxel and trastuzumab have provided a compelling rationale for testing this combination in the clinic (24). In a study from M. D. Anderson Cancer Center, 30 women with Her2-overexpressing metastatic breast cancer were treated with weekly docetaxel and trastuzumab as first- or second-line therapy (25). Both docetaxel (35 mg/m2) and trastuzumab (4 mg/kg load and then 2 mg/kg) were delivered in 4-wk cycles consisting of three weekly treatments followed by 1 wk of rest. The intent-to-treat overall response rate was 63% (95% CI, 44–80%) and the overall response rate in patients whose tumors demonstrated HER2 amplification by FISH was 67% (95% CI, 45–84%). Median time to progression on trial was 9 mo. Acute toxicity, including myelosuppression, was mild; however, fatigue, fluid retention, and excessive lacrimation were more frequent with cumulative drug delivery. Cardiotoxicity was not a significant factor, as reflected by the maintenance of a relatively consistent mean left ventricular ejection fraction through 1 yr of therapy on trial. There was only one reported case of clinical congestive heart failure, which was associated with a 12% reduction in left ventricular ejection fraction; however, this patient improved with medical treatment, and follow-up echocardiography revealed a return to normal left ventricular function. In another pilot phase II trial, patients with HER2-overexpressing metastatic breast cancer received weekly trastuzumab (4 mg/kg load followed by 2 mg/kg/ wk) plus tri-weekly docetaxel given at 75 mg/m2 as first- or second-line therapy (26). HER2 overexpression was determined using immunohistochemistry, for which both 2+ and 3+ overexpressors were allowed entry. Of the 21 patients enrolled, 16 are evaluable for efficacy, and preliminary results reveal an overall response rate of 44%, including six partial responses and one complete response. An additional 3 patients have documented minor responses and continue to receive treatment. When subdivided by HER2 status, patients with 3+ overexpression had a response rate of 55% vs 20% for 2+ overexpressors. Grade 3 myelosuppression was the primary adverse event noted, with 5 patients requiring granulocyte colonystimulating factor during treatment and 1 patient needing hospitalization for febrile neutropenia. A single patient was removed from study secondary to dermatitis. Notably, there was no significant decline in left ventricular ejection fraction and there were no cases of symptomatic congestive heart failure.

Chapter 7 / Inhibitors of HER2 With Taxanes

181

Table 1 Ongoing Phase II Trials of Docetaxel/Trastuzumab in HER2-Positive Metastatic Breast Cancer Investigator

Trial design

Esteva et al., 2002 (25)

D: 35 mg/m /wk (3 of 4 wk) T: 4 mg/kg load A 2 mg/kg/wk (3 of 4 wk) D: 75 mg/m2 every 3 wk T: 4 mg/kg load A 2 mg/kg/wk 2 D: 35 mg/m /wk (6 of 8 wk) T: 4 mg/kg load A 2 mg/kg/wk 2 D: 35 mg/m /wk (6 of 7 wk) T: 4 mg/kg load A 2 mg/kg/wk D: 33 mg/m2/wk T: 4 mg/kg A 2 mg/kg/wk

Burris, 2001 (26) Uber et al., 2001 (27) Meden et al., 201 (28) Sparano et al., 2000 (29)

2

Patients (N)

Response rate

30

63%

16

44%

19

63%

12

50%

6

83%

D, docetaxel; T, trastuzumab.

Most recently, results from a Europe-wide multicenter randomized phase II trial comparing docetaxel monotherapy vs docetaxel plus trastuzumab (26a) were presented at the San Antonio Breast Cancer Symposium in December 2003. One hundred eighty-eight patients with previously untreated metastatic breast cancer were enrolled; on HER2 testing, 95% had IHC 3-positive and/or FISH-positive disease. Docetaxel was administered at a dose of 100 mg/m2 every 3 wk × 6; conventional weekly trastuzumab was given until progression. Patients who had progression on docetaxel alone were allowed to cross over and receive trastuzumab. The results revealed that the addition of trastuzumab to docetaxel significantly improved response rate (61% vs 36%, p = 0.001) and time to progression (10.6 vs 6.1 mo, p = 0.0001). Overall survival also favored the combination arm (2.7 vs 18.3 mo, p = 0.0002), despite at least 44% of the docetaxel patients crossing over to receive trastuzumab. Moreover, these improvements in clinical outcomes were seen in the absence of significant additional grade 3/4 toxicities. There are yet additional trials underway, and several others that are being planned to determine the optimal administration schedule for docetaxel and trastuzumab in combination (Table 1). Thus far, available data suggest that this combination, similar to paclitaxel plus trastuzumab, is well tolerated, with a toxicity profile resembling that of the individual taxane alone, and appears to be an active therapeutic option in HER2-overexpressing metastatic breast cancer. There are yet additional trials underway, and several others that are being planned to determine the optimal administration schedule for docetaxel and

182

Modi, Fornier, and Seidman

trastuzumab in combination (Table 1). Furthermore, a Europe-wide multicenter phase III registration trial has been initiated comparing docetaxel monotherapy vs docetaxel plus trastuzumab; this study is designed to enroll 156 patients with HER2-overexpressing metastatic breast cancer. Pending these important results, the available data suggest that the combination of docetaxel and trastuzumab, similar to paclitaxel plus trastuzumab, is well tolerated, with a toxicity profile resembling that of the individual taxane alone, and appears to be an active therapeutic option in HER2-overexpressing metastatic breast cancer.

INTEGRATION OF TAXANE/TRASTUZUMAB COMBINATIONS INTO ADJUVANT THERAPY The survival advantage noted for the use of trastuzumab with chemotherapy in metastatic breast carcinoma has motivated the incorporation of this monoclonal antibody into adjuvant chemotherapy trials for patients with node-positive, HER2-overexpressing breast cancer. However, concern regarding potential cardiac toxicity with the concurrent delivery of anthracyclines and trastuzumab has presented a challenge to this process. This is particularly problematic as retrospective data indicates substantial activity for anthracyclines in the treatment of HER2-positive breast cancer (30–32). Thus several different strategies have been developed in efforts to minimize this possible cardiac complication. In one approach, investigators have separated the anthracycline and trastuzumab by using a sequential chemotherapy schedule, adopting the Cancer and Leukemia Group B (CALGB) 93-44 regimen of doxorubicin and cyclophosphamide (60 and 600 mg/m2 every 3 wk) for four cycles followed by paclitaxel (175 mg/ m2 every 3 wk) for four cycles as the control arm (NSABP-B-31). In this trial, patients are randomized to either the control arm or the control arm plus trastuzumab, given conventionally as a 4-mg/kg loading dose followed by 2 mg/ kg on a weekly basis, starting at the same time as the paclitaxel chemotherapy and continuing for a total of 52 wk (Fig. 2). The Intergroup N9831 trial has a similar design; however, in this case paclitaxel is delivered weekly at 80 mg/m2 for a total of 12 wk. Trastuzumab is administered on a weekly basis concurrently with the paclitaxel (19) and continued for another 40 wk afterwards. Additionally, this trial has a third arm in which patients receive standard AC followed by weekly paclitaxel; however, the weekly trastuzumab (for 52 wk) is started after the completion of the paclitaxel chemotherapy (Fig. 3). Similarly, two European adjuvant trials have also incorporated sequential delivery of trastuzumab into their trial design. The HER2 European adjuvant (HERA) study will recruit patients who have completed any recognized adjuvant chemotherapy regimen and randomize them to either observation, 1 yr, or 2 yr of trastuzumab, where the trastuzumab will be administered on a tri-weekly schedule (loading bolus of 8 mg/kg followed by doses of 6 mg/kg every 3 wk).

Fig. 2. NSABP B-31 adjuvant trial for HER2+ breast cancer.

Chapter 7 / Inhibitors of HER2 With Taxanes 183

184

Modi, Fornier, and Seidman

The second trial, to be conducted in Belgium and France, will randomize patients with node-positive breast cancer to either six cycles of FEC100 or six cycles of docetaxel plus epirubicin (both given at 75 mg/m2 given every 3 wk). Following this, patients who are HER2 positive will be randomized to either observation or tri-weekly trastuzumab for 1 yr. In another approach, investigators have combined trastuzumab with chemotherapy regimens using less cardiotoxic anthracyclines, such as liposomal doxorubicin and epirubicin. In a phase I/II trial, 37 patients with Her2-overexpressing advanced breast cancer were treated with weekly trastuzumab plus the liposomal doxorubicin TLC D-99 (60 mg/m2 every 3 wk) (33). Patients were allowed up to one prior trastuzumab regimen and less than two cytotoxic regimens for advanced breast cancer, and a prior cumulative doxorubicin exposure of <240 mg/ m2; left ventricular ejection fraction (LVEF) was monitored on study with serial MUGA scans starting at baseline and after every two cycles of therapy. One patient had a significant but asymptomatic decline in LVEF, and a second patient developed congestive heart failure. Both patients had prior therapy with doxorubicin and both recovered cardiac function off trial. Other toxicities were minimal, and the response rate in evaluable patients was 58% (19/33). Overall, this combination was active and well tolerated, and further studies are planned. Cardiac safety and efficacy were also assessed in a dose-finding phase I study of TLC D99 (doses ranging from 40 to 60 mg/m2 every 3 wk), paclitaxel (doses of 60–80 mg/m2 weekly), and weekly trastuzumab in patients with untreated HER2positive locally advanced or metastatic breast cancer (34). Fifteen patients have been treated thus far with a dose-limiting toxicity of severe dermatological reaction (one case); another three patients are planned for enrollment to confirm the recommended dose level. There was no clinical cardiac toxicity; however, there were two cases of reversible asymptomatic decline in LVEF. Of the 13 patients evaluable for response, there were 8 partial and 3 complete responses, for an overall response rate of 85%. A phase II study in 68 patients is under planning. Also in a recent pilot trial of neoadjuvant therapy for operable breast cancers, weekly trastuzumab plus epirubicin and docetaxel (35 and 30 mg/m2 given weekly for 6 of 7 wk for one cycle), revealed no evidence of acute cardiac toxicity in 9 patients who received two full preoperative cycles (35). These early but encouraging safety results have led to expanded testing of these less cardiotoxic anthracyclines in trastuzumab-chemotherapy combinations. Investigators at M.D. Anderson Cancer Center have reported initial results from a randomized neoadjuvant trial in patients with operable HER2-positive breast cancer where the primary endpoint evaluation was of pathological responses (35a). Patients were randomized to receive four cycles of paclitaxel followed by four cycles of 5-fluorouracil + epirubicin + cyclophosphamide (FEC), or the same chemotherapy with simultaneous weekly trastuzumab for 24 wk. Although the planned sample size was 164 patients, the study was stopped after 34 patients had completed

Fig. 3. Intergroup N9831 adjuvant trial for HER2+ breast cancer.

Chapter 7 / Inhibitors of HER2 With Taxanes 185

186

Modi, Fornier, and Seidman

therapy, owing to the superiority of the trastuzumab-containing arm. The pathological complete response rate was 67% vs 25% in favor of the trastuzumabtreated patients (p = 0.02), and no patients had clinical congestive heart failure or other clinically relevant cardiac toxicity. Presently the National Cancer Institute (NCI) is sponsoring a phase II study of liposomal doxorubicin and docetaxel with or without trastuzumab in women with untreated metastatic breast cancer (ECOG 3198). Finally, prompted by evidence of preclinical synergy (15,24,36,37) and a desire to avoid any possibility of cardiac toxicity in potentially curable patients in the adjuvant setting, the Breast Cancer International Research Group (BCIRG) has withdrawn anthracyclines altogether and proposed a novel combination regimen of trastuzumab, docetaxel, and a platinum salt. They have now completed testing of this triplet therapy in two parallel multicenter phase II trials (38,38a). In both trials, patients with HER2-positive metastatic breast cancer were treated with docetaxel at 75 mg/m2 every 3 wk and weekly trastuzumab. In the first trial, patients also received cisplatin at 75 mg/m2 every 3 wk; in the second they received carboplatin administered at an AUC of 6 every 3 wk. Both trials required testing of tumor samples for HER2 by immunohistochemistry (either 2+ or 3+) and FISH analysis for gene amplification. In the cisplatin trial, patients could not have received prior chemotherapy for metastatic disease; however, in the carboplatin trial, one prior regimen was permitted, including prior monotherapy with a taxane. The overall response rate among 62 evaluable patients on the cisplatin trial was 70% (95% confidence interval [CI], 66–89%), and 58% (95% CI, 44–70%) for the patients (n = 59) on the carboplatin trial. For FISHpositive patients, the response rates were 77% (CI, 59–90%) and 62.5% (CI, 46– 77%), respectively. With a median follow-up of 37.7 mo for the cisplatin study, the overall survival was 28.1 mo (CI, 18.2–not estimable); median survival was 27 mo for the FISH-positive and 31.5 mo for the FISH-negative subgroups. For the carboplatin trial, where the median follow-up was 34.3 mo, the overal survival has not yet been reached, with 54.8% of patients remaining alive. Treatment was well tolerated in both trials, with the most common grade 3/4 toxicities being febrile neutropenia, anemia, asthenia, and nausea. Cardiac toxicity was minimal, with NCI grade 3 cardiac dysfunction observed in one patient on the cisplatin trial and two patients on the carboplatin trial (Table 2). In an independent third study, Brufsky and colleagues have treated patients with HER2-positive metastatic breast cancer (2+ or 3+ by IHC) also with docetaxel (75 mg/m2 every 3 wk), trastuzumab (4 mg/kg load followed by 2 mg/ kg/wk), and carboplatin (AUC 5 every 3 wk) (39). Thus far, four of five evaluable patients have responded; accrual to this study is ongoing. The North Central Cancer Treatment Group has conducted two concurrent phase II trials assessing the efficacy and tolerability of two schedules of paclitaxel, carboplatin, and trastuzumab as first-line therapy for HER2-positive metastatic

Chapter 7 / Inhibitors of HER2 With Taxanes

187

breast cancer. The first schedule delivered paclitaxel at 200 mg/m2, carboplatin AUC of 6, and trastuzumab at 6 mg/kg every 3 wk for eight cycles (6 mo), followed by trastuzumab alone until progrssion; the second delivered weekly paclitaxel at 80 mg/m2, carboplatin AUC of two weekly for 3 of 4 wk, and trastuzumab at 2 mg/kg weekly for six cycles (6 mo), followed by trastuzumab alone until progression. Ninety-six eligible patients have been enrolled and of the 36 evaluable for efficacy, the interim results reveal a response rate of 78% vs 50% and a median progression-free survival of 13.4 mo vs 8.8 mo in favor of the weekly schedule. Similarly, the toxicity profile favored the weekly schedule, with fewer grade 3/4 toxicities: neutropenia (52% vs 88%), thrombocytopenia (2% vs 30%), anemia (5% vs 15%), red blood cell transfusions (5% vs 25%) febrile neutropenia (0% vs 18%), neurosensory (2% vs 20%), myalgias (0% vs 15%), and arthralgias (0% vs 13%) (39a). Currently, the US Oncology group is conducting a phase III study of trastuzumab and paclitaxel with and without carboplatin in patients with untreated HER-2/neu-positive advanced breast cancer. Prelimiary results from the 188 eligible patients reveals an overall response rate of 52% vs 36% (p = 0.04) and time to progression of 11.9 mo vs 6.8 no (p = 0.02) in favor of the carboplatin/paclitaxel/trastuzumab arm. In the HER-2/neu 3-positive subset of patients, the corresponding results were: 57% vs 37% (p = 0.03) and 14.6 mo vs 6.9 mo (p = 0.006). There was a trend in the overall survival in favor of the carboplatin-containing arm of 42.1 mo vs 33.3 mo (p = 0.27), and 42.1 mo vs 30.6 mo (p = 0.11) in the IHC 3-positive subset. Although there was an increased frequency of grade 3 and 4 neutropenia and thrombocytopenia for the triplet combination, there were no differences between the two arms in terms of neurologic toxicities, neutropenic fever, asthenia, or cardiovascular or pulmonary toxicities (39b). Further exploration with other trastuzumab-containing combinations and novel agents is ongoing. Beyond the metastatic setting, trastuzumab is also under investigation in several multicenter, randomized phase III trials for the treatment of HER2-overexpressing breast cancer in the adjuvant and neoadjuvant setting. Finally, a neoadjuvant study of taxane/platinum/trastuzumab has been reported in which patients with locally advanced or inflammatory breast cancer received both docetaxel and cisplatin at 70mg/m2 every 3 wk for four doses and conventional trastuzumab (4 mg/kg loading dose and then 2 mg/kg/wk) for a total of 12 wk of induction therapy (40). All 16 study patients had tumor HER2 expression of either 2+ or 3+ by IHC. Standard doxorubicin and cyclophosphamide (60/600 mg/m2) for four cycles was subsequently administered post-operatively. The clinical response rate was 100%, comprised of 9 complete and 7 partial responses. Eight patients underwent surgery, two of these having had a complete pathologic response (25%) and one patient with residual microscopic disease. Grade 3 toxicities were as follows: three patients with hyperglycemia, one patient with nausea and vomiting, and one case of reduced LVEF.

188

Modi, Fornier, and Seidman

Table 2 Docetaxel/Trastuzumab/Platinum Combinations in HER2-Positive Breast Cancer Investigator

Trial design

Slamon et al. (38a)

D: 75 mg/m q3 wk IV 62 T: 4 mg/kg load A 2 mg/kg/wk 2 P: 75 mg/m q3 wk 2 D: 75 mg/m q3 wk IV 59 T: 4 mg/kg load A 2 mg/kg/wk B: AUC 6 q3 wk 2 D: 75 mg/m q3 wk IV 5 T: 4 mg/kg load A 2 mg/kg/wk B: AUC 5 q3 wk 2 D: 70 mg/m q3 wk III 16 T: 4 mg/kg load A 2 mg/kg/wk (neoadjuvant) P: 70 mg/m2 q3 wk

Slamon et al. (38a)

Brufsky et al. (39)

Hurley et al. (40)

2

Stage

Pts (N)

RR 79%

58%

80%

100% (25% pCR)

D, docetaxel; T, trastuzumab; P, cisplatin; B, carboplatin; pCR, pathologic complete response; Pts, patients; RR, response rate.

Overall, the studies of docetaxel, trastuzumab, and a platinum salt in combination have demonstrated an acceptable safety and efficacy profile, which in turn has motivated the testing of this non-anthracycline triplet regimen in two large adjuvant multicenter randomized trials. In BCIRG Trial 007, patients with HER2overexpressing metastatic breast cancer are randomized to receive docetaxel and trastuzumab or docetaxel and trastuzumab plus a platinum salt (either carboplatin or cisplatin). This trial is open to patients with untreated metastatic breast cancer with HER2-amplified tumors by FISH analysis (Fig. 4). A total of 550 patients are planned for enrollment. In contrast, BCIRG Trial 006 is an adjuvant trial for patients with node-positive and high-risk node-negative breast cancers. Patients in this trial are randomized to one of three arms: the control arm receives doxorubicin/cyclophosphamide for four cycles followed by four cycles of docetaxel. The first experimental arm receives doxorubicin/cyclophosphamide for four cycles followed by four cycles of docetaxel and concurrent weekly trastuzumab followed by weekly trastuzumab alone for an additional 40 wk. The third arm receives docetaxel and a platinum salt (either cisplatin or carboplatin) both for six cycles with concurrent weekly trastuzumab followed by trastuzumab alone given at a dose of 6 mg/kg every 3 wk, for a total of 52 wk (1 yr) of trastuzumab. This trial is also open to patients with HER2-amplified tumors, with a target enrollment of 3150 participants (Fig. 5).

Fig. 4. BCIRG trial 007: metastatic breast cancer trial for untreated HER2-amplified disease.

Chapter 7 / Inhibitors of HER2 With Taxanes 189

190

Modi, Fornier, and Seidman

NOVEL INHIBITORS OF HER2 Given the importance of the HER2 signaling pathway in the pathogenesis and progression of breast cancer, investigators are exploring other unique strategies for inhibiting the activity of this receptor tyrosine kinase. 2C4, a fully humanized monoclonal antibody that targets a different epitope on the extracellular domain of the HER2 receptor, has demonstrated in vitro and in vivo growth inhibition in several breast and prostate tumor models, and is currently in phase I trials being conducted by Genentech (41,42). 17-allylamino-geldanamycin (17-AAG) is a modified ansamycin antibiotic that binds and inactivates heat shock protein 90 (Hsp90) and thereby removes its protective effect over HER2. Subsequently, HER2 undergoes ubiquitin-dependent degradation in the proteasome (43). Treatment of HER2-overexpressing breast cancer cells with 17-AAG induces growth arrest and apoptosis, and results in a downregulation of HER2 protein expression (43). Several groups have evaluated the effects of 17-AAG in xenograft models and have demonstrated that the findings in cell-culture models are reproducible in animals with acceptable toxicity (44). Moreover, in a non-small-cell lung cancer model, administration of 17-AAG enhanced the antiproliferative effects of paclitaxel and significantly prolonged survival of the xenograft-bearing mice (45). Phase I clinical trials of this agent are underway, and thus far, 17-AAG appears safe. Dose-limiting toxicities have been diarrhea, thrombocytopenia, and reversible hepatotoxicity (46– 48). Cardiotoxicity has not been observed. CI-1033 is a small-molecule tyrosine kinase inhibitor that targets and irreversibly inactivates all four members of the HER family of growth-factor receptors, including HER2 (49). In a phase I study, 53 patients with incurable solid tumors were treated with oral CI-1033 for 7 d every 3 wk at doses of 50–750 mg/d (49). Overall, the safety profile was acceptable, with the most common toxicities being mild to moderate acneiform rash, nausea/vomiting, diarrhea, and mucositis. The maximum tolerated dose (MTD) on this schedule was 750 mg/d for doselimiting toxicities of diarrhea and vomiting. There was one partial response and 18 cases of stable disease, including one patient with heavily pretreated breast cancer. In another phase I trial of 68 patients with advanced solid tumors, the MTD achievable was 220 mg/d when CI-1033 was administered on a schedule of 28 d on followed by 7 d off (50). A randomized phase II study for patients with metastatic breast cancer who have progressed following treatment with a maximum of two cytotoxic chemotherapy regimens is currently underway. CI-1033 will be administered orally daily for 21 consecutive d in a 28-d cycle. Similarly, the dual small molecule kinase inhibitor, GW572016, which potently inhibits both EGFR and HER2 tyrosine kinases inducing growth arrest in EGFR- and HER2-dpendent tumor cell lines, is undergoing clinical testing in patients with advanced-stage breast cancer (50a).

Fig. 5. BCIRG trial 006: adjuvant breast cancer trial for HER2-amplified, node-positive or high risk node-negative disease.

Chapter 7 / Inhibitors of HER2 With Taxanes 191

192

Modi, Fornier, and Seidman

Also in development are HER2-based vaccines. Although HER2 vaccines have been shown to provide benefit in animal models and to be immunogenic in humans, the optimal vaccine formulation is not yet known, and the therapeutic efficacy of these agents in humans has not yet been evaluated (51).

CONCLUSION The introduction of trastuzumab as the first clinically available biologic therapy for the treatment of solid tumors has heralded a new era of rationally designed and targeted drug development. The remarkable 20% reduction in the risk of death with the addition of trastuzumab to chemotherapy has set a precedent in the treatment of HER2-driven metastatic breast cancer, a disease where previously few therapies have had more than a palliative impact. Several clinical trials are now underway, examining the effects of adding trastuzumab to the adjuvant setting and exploring optimal combinations, doses, and delivery of this monoclonal antibody. Other promising novel agents are also under various stages of clinical development, including inhibitors of growth factor receptors and their ligands, signal transduction proteins, angiogenesis factors, cell-cycle regulators, and transcription factors. Assessment of their toxicity profiles, single-agent activity, and efficacy in combinations with cytotoxic therapies, in particular with the taxanes, will be the focus for this next generation of biological agents.

REFERENCES 1. Stern DF, Heffernan PA, Weinberg RA. p185, a product of the neu proto-oncogene, is a receptor-like protein associated with tyrosine kinase activity. Mol Cell Biol 1986;6(5):1729–1740. 2. Yamamoto T, Ikawa S, Akiyama T, et al. Similarity of protein encoded by the human c-erbB2 gene to epidermal growth factor receptor. Nature 1986;319:226–230. 3. Pinkas-Kramarski R, Shelly M, Guarino BC, et al. ErbB tyrosine kinases and the two neuregulin families constitute a ligand-receptor network. Mol Cell Biol 1998;18:6090–6101. 4. Padhy LC, Shih C, Cowing D, et al. Identification of a phosphoprotein specifically induced by the transforming DNA of rat neuroblastomas. Cell 1982;28:865–871. 5. Slamon DJ, Godolphin W, Jones LA, et al. Studies of the HER-2/neu proto-oncogene in human breast and ovarian cancer. Science 1989;244:707–712. 6. Press MF, Cordon-Cardo C, Slamon DJ: Expression of the HER2/neu proto-oncogene in normal human adult and fetal tissues. Oncogene 1990;5:953–962. 7. Slamon DJ, Clark GM, Wong SG, et al. Human breast cancer: correlation of relapse and survival with amplification of the HER-2/neu oncogene. Science 1987;235:177–181. 8. Hudziak RM, Lewis GD, Winget M, et al. p185HER2 monoclonal antibody has antiproliferative effects in vitro and sensitizes human breast tumor cells to tumor necrosis factor. Mol Cell Biol 1989;9:1165–1172. 9. Pietras RJ, Fendly BM, Chazin VR, et al. Antibody to HER-2/neu receptor blocks DNA repair after cisplatin in human breast and ovarian cancer cells. Oncogene 1994;9:1829–1838. 10. Mass R. The role of HER2 expression in predicting response to therapy in breast cancer. Sem Oncol 2000;27(6, Supp 11):46–52.

Chapter 7 / Inhibitors of HER2 With Taxanes

193

11. Baselga J, Tripathy D, Mendelsohn J, et al. Phase II study of weekly intravenous recombinant humanized anti-p185 HER2 monoclonal antibody in patients with HER2/neu overexpressing metastatic breast cancer. J Clin Oncol 1996;14:737–744. 12. Carter P, Presta L, Gorman CM, et al. Humanization of an anti-p185HER2 antibody for human cancer therapy. Proc Natl Acad Sci USA 1992;89:4285–4289. 13. Cobleigh MA, Vogel CL, Tripathy D, et al. Multinational study of the efficacy and safety of humanized anti-HER2 monoclonal antibody in women who have HER2-overexpressing metastatic breast cancer that has progressed after chemotherapy for metastatic disease. J Clin Oncol 1999;17:2639–2648. 14. Vogel CL, Cobleigh M, Tripathy D, et al. Superior outcomes with Herceptin (trastuzumab) in fluorescence in situ hybridization (FISH)-selected patients. Proc Am Soc Clin Oncol 2001;20, abstract 86. 15. Pegram M, Hsu S, Lewis G, et al. Inhibitory effects of combinations of HER2/neu antibody and chemotherapeutic agents used for treatment of human breast cancers. Oncogene 1999;18:2241–2251. 16. Baselga J, Norton L, Albanell J, et al. Recombinant humanized anti-HER2 antibody (Herceptin ™) enhances the antitumor activity of paclitaxel and doxorubicin against HER2/ neu over-expressing human breast cancer xenografts. Cancer Res 1998;58:2825–2831,. 17. Slamon, D J, Leyland-Jones, B, Shak, S, et al. Use of chemotherapy plus a monoclonal antibody against HER2 for metastatic breast cancer that over-expresses HER2. N Engl J Med 2001;344:783–792. 18. Mass RD, Press M, Anderson S, et al. Improved survival benefit from Herceptin (trastuzumab) in patients selected by fluorescence in situ hybridization (FISH). Proc Am Soc Clin Oncol 2001;20, abstract 85. 19. Seidman AD, Fornier MN, Esteva FJ, et al. Weekly trastuzumab and paclitaxel therapy for metastatic breast cancer with analysis of efficacy by HER2 immunophenotype and gene amplification. J Clin Oncol 2001;19:2587–2595. 19a. Seidman AD, Berry D, Cirrincione C, et al. CALGB 9840: phase III study of weekly paclitaxel via 1-hour infusion versus standard 3h infusion every third week in the treatment of metastatic breast cancer (MBC), with trastuzumab (T) for HER2 positive MBC and randomized for T in HER2 normal MBC. J Clin Oncol 2004;22(14S):6s (abst 512). 20. Gelmon K, Arnold A, Verma S, et al. Pharmacokinetics and safety of trastuzumab when administered every three weeks to women with metastatic breast cancer. Proc Am Soc Clin Oncol 2001;abs 271. 21. Nabholtz JM, Senn HJ, Bezwoda WR, et al. Prospective randomized trial of docetaxel versus mitomycin plus vinblastine in patients with metastatic breast cancer progressing despite previous anthracycline-containing chemotherapy. J Clin Oncol 1999;17(5):1413–1424. 22. Chan S, Friedrichs K, Noel D, et al. Prospective randomized trial of docetaxel versus doxorubicin in patients with metastatic breast cancer. J Clin Oncol 1999;17(8):2341–2354. 23. Sjostrom J, Blomqvist C, Mouridsen H, et al. Docetaxel compared with sequential methotrexate and 5-fluorouracil in patients with advanced breast cancer after anthracycline failure: a randomised phase III study with crossover on progression by the Scandinavian Breast Group. Eur J Cancer 1999;35(8):1194–1201. 24. Konecny G, Pegram MD, Beryt M, et al. Therapeutic advantage of chemotherapy drugs in combination with Herceptin against human breast cancer cells with HER2/neu overexpression. Breast Cancer Res Treat 1999;57:114 (abst 467). 25. Esteva FJ, Valero V, Booser D, et al. Phase II study of weekly docetaxel and trastuzumab for patients with HER-2-overexpressing metastatic breast cancer. J Clin Oncol 2002;20(7):1800–1808.

194

Modi, Fornier, and Seidman

26. Burris HA. Docetaxel plus trastuzumab in breast cancer. Sem Oncol 2001;28:38–44. 26a. Extra JM, Cognetti F, Chan S, et al. First-line trastuzumab plus docetaxel versus docetaxel alone in women with HER2-positive metastatic breast cancer: results from a randomized phase II trial (M77001). Breast Cancer Res Treat 2003;82(suppl 1):S47 (abst 217). 27. Uber KA, Nicholson BP, Thor AD, et al. A phase II trial of weekly docetaxel and Herceptin as first- or second-line treatment in HER2 over-expressing metastatic breast cancer. Proc Am Soc Clin Oncol 2001;20:50b (abst 1949). 28. Meden H, Beneke A, Hesse T, et al. Weekly intravenous recombinant humanized antiP185HER2 monoclonal antibody (herceptin) plus docetaxel in patients with metastatic breast cancer: a pilot study. Anticancer Res 2001;21(2B):1301–1305. 29. Sparano JA, Malik U, Manalo J, et al. Phase II trial of weekly docetaxel alone or in combination with trastuzumab in patients with metastatic breast cancer. Breast Cancer Res Treat 2000;64:80 (abst 317). 30. Muss HB, Thor AD, Berry DA, et al. c-erbB-2 expression and response to adjuvant therapy in women with node-positive early breast cancer. N Engl J Med 1994;330(18):1260–1266. 31. Thor AD, Berry DA, Budman DR, et al. erbB-2, p53, and efficacy of adjuvant therapy in lymph node-positive breast cancer. J Natl Cancer Inst 1998;90(18):1346–1360. 32. Paik S, Bryant J, Park C, et al. erbB-2 and response to doxorubicin in patients with axillary lymph node-positive, hormone receptor-negative breast cancer. J Natl Cancer Inst 1998;90(18):1361–1370. 33. Theodoulou M, Campos SM, Batist G, et al. TLC D99 and Herceptin is safe in advanced breast cancer: final cardiac safety and efficacy analysis. Proc Am Soc Clin Oncol 2002;21:55a (abst 216). 34. Trigo J, Climent MA, Gil M, et al. Cardiac safety and activity of a phase I study of 3-weekly myocet in combination with weekly herceptin and paclitaxel in HER2-positive locally advanced or metastatic breast cancer. Proc Am Soc Clin Oncol 2002;21:61a (abst 242). 35. Steger GG, Wenzel C, Locker GJ, et al. Pilot trial of trastuzumab + weekly epidoxorubicin/ docetaxel in the neoadjuvant treatment of primary breast cancer- preliminary results. Proc Am Soc Clin Oncol 2002;21:39b (abst 1966). 35a. Buzdar AU, Hunt K, Smith T, et al. Signficantly higher pathological complete remission rate following neoadjuvant therapy with trastuzumab, paclitaxel, and anthracycline-containing chemotherapy: initial results of a randomized trial in operable breast cancer with HER2 positive disease. J Clin Oncol 2004;22(14S):7s (abst 520). 36. Pegram MD, Finn RS, Arzoo K, et al. The effect of HER-2/neu overexpression on chemotherapeutic drug sensitivity in human breast and ovarian cancer cells. Oncogene 1997;15:537–547. 37. Pegram MD, Lipton A, Hayes DF, et al. Phase II study of receptor-enhanced chemosensitivity using recombinant humanized anti-p185HER2/neu monoclonal antibody plus cisplatin in patients with HER2/neu-overexpressing metastatic breast cancer refractory to chemotherapy treatment. J Clin Oncol 1998;16:2659–2671. 38. Nabholtz JA, Reese DM, Lindsay M, et al. Docetaxel in the treatment of breast cancer: an update on recent studies. Sem Oncol 2002;29(3):28–34. 38a. Slamon D, Yeon CH, Pienkowski T, et al. Survival analysis from two open-label nonrandomized phase II trials of trastuzumab combined with docetaxel and platinums (cisplatin or carboplatin) in women with HER2+ advanced breast cancer. J Clin Oncol 2004;22(14S):37s (abst 642). 39. Brufsky A, Lebish J, Shanahan C, et al. Phase II trials of carboplatin/docetaxel and carboplatin/docetaxel/trastuzumab as first line therapy for metastatic breast cancer. Breast Cancer Res Treat 2000;64:81 (abst 321). 39a. Perez EA, Rowland KM, Suman VJ, et al. N98-32-52: efficacy and tolerability of two schedules of paclitaxel, carboplatin, and trastuzumab in women with HER2 positive meta-

Chapter 7 / Inhibitors of HER2 With Taxanes

39b.

40. 41. 42. 43. 44. 45. 46. 47. 48. 49. 50. 50a.

51.

195

static breast cancer: a North Central Cancer Treatment Group randomized phase II trial. Breast Cancer Res Treat 2003;82(suppl 1):S47 (abst 216). Robert N, Leyland-Jones B, Asmar L, et al. Randomized phase III study of trastuzmab, paclitaxel, and carboplatin versus trastuzumab and paclitaxel in women with HER-2 overexpressing metastatic breast cancer: an update including survival. Proc Am Soc Clin Oncol 2004;23:20 (abst 573). Hurley J, Doliny P, Silva O, et al. Neoadjuvant herceptin/taxotere/cisplatin in the treatment of locally advanced and inflammatory breast cancer. Proc Am Soc Clin Oncol 2002;21:50a (abst 196). Baselga J: A new anti-ErbB2 strategy in the treatment of cancer: prevention of liganddependent ErbB2 receptor heterodimerization. 2002;Cancer Cell 2(2):93. Agus D, Akita R, Fox W, et al. Targeting ligand-activated ErbB2 signaling inhibits breast and prostate tumor growth. Cancer Cell 2002;2(2):127. Munster PN, Marchion DC, Basso AD, Rosen N. Degradation of HER2 by ansamycins induces growth arrest and apoptosis in cells with HER2 overexpression via a HER3, phosphatidylinositol 3'-kinase-AKT-dependent pathway. Cancer Res 2002;62:3132–3137. Basso AD, Solit DB, Munster PN, Rosen N. Ansamycin antibiotics inhibit Akt activation and cyclin D expression in breast cancer cells that overexpress HER2. Oncogene 2002;21:1159–1166. Nguyen DM, Lorang D, Chen A, et al. Enhancement of paclitaxel-mediated cytotoxicity in lung cancer cells by 17-allylamino geldanamycin: in vitro and in vivo analysis. Ann Thorac Surg 2001;72:371–379. Wilson RH, Takimoto CH, Agnew EB, et al. Phase I pharmacologic study of 17-(allylamino)17-demethoxygeldanamycin (AAG) in adult patients with advanced tumors. Proc Am Soc Clin Oncol 2001;20:82a. Banerji U, O’Donnell A, Scurr M, et al. Phase I trial of the heat shock protein 90 inhibitor 17-allylamino-17-demethoxygeldanamycin (17AAG). Pharmacokinetic profile and pharmacodynamic endpoints. Proc Am Soc Clin Oncol 2001;20:82a. Munster PN, Tong W, Schwartz L, et al. Phase I trial of 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) in patients with advanced solid malignancies. Proc Am Soc Clin Oncol 2001;20:83a. Allen LF, Lenehan PF, Eiseman IA, Elliot WL, Fry DW. Potential benefits of the irreversible pan-erbB inhibitor, CI-1033, in the treatment of breast cancer. Sem Oncol 2002;29(3 suppl 11):11–21. Rinehart JJ, Wilding G, Willson J, et al. A phase I clinical and pharmacokinetic study of oral CI-1033, a pan-erbB tyrosine kinase inhibitor, in patients with advanced solid tumors. Proc Am Soc Clin Oncol 2002;21:11a. Zhou H, Kim YS, Peletier A, et al. effectsof the EGFR/HER2 kinase inhibitor GW572016 on EGFR- and HER2-overexpressing breast cancer cell proliferation, radiosensitization, and resistance. Int J Radiat Oncol Biol Phys 2004;58(2):344–352. Foy TM, Fanger GR, Hand S, et al. Designing HER2 vaccines. Sem Oncol 2002;29(3):53–61.

Chapter 8 / NF-gB and Cisplatin Activity

8

197

Targeting NF-gB to Increase the Activity of Cisplatin in Solid Tumors Don S. Dizon, MD, Carol Aghajanian, MD, X. Jun Yan, MD, and David R. Spriggs, MD CONTENTS INTRODUCTION HISTORY MECHANISM OF ACTION RESISTANCE NF-gB IN PLATINUM RESISTANCE DEGRADATION OF IgB: THE UBIQUITIN–PROTEASOME PATHWAY MODIFYING PLATINUM RESISTANCE—PROTEASOME INHIBITION

INTRODUCTION For nearly 50 yr, cisplatin has been a mainstay of treatment of multiple cancers, including ovarian, lung, and testicular cancer. However, the development of resistance impacts the utility of the drug in many patients, particularly in the setting of recurrent disease. Although the exact mechanism of cisplatin resistance needs to be delineated, it is widely accepted that multiple factors may contribute to cisplatin resistance. We will discuss the history and clinical pharmacology of cisplatin and discuss the mechanisms of resistance, placing particular emphasis on the NF-gB pathway, which in multiple cancers has been shown to be associated with drug resistance. Finally, we will discuss novel approaches that may enable the circumventing of platinum resistance. From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

197

198

Dizon et al.

HISTORY The discovery of cis-diaminedichloroplatinum (II) (CDDP) dates back to 1845, when Peyrone first synthesized the compound later known as Peyrone’s chloride. However, it was not until 1983 that the chemical structure was elucidated by Alfred Werner. Its role as a chemotherapeutic agent was first discovered in the 1960s in a series of experiments unrelated to cancer therapy by Dr. Saul Rosenberg (1). As part of the preliminary investigations into the effect of magnetic fields on cellular growth, Rosenberg applied a platinum electrode to a culture dish containing Escherichia coli. With activation, he observed that bacterial growth appeared to decrease in the presence of platinum. He realized that the effect of cisplatin was inhibitory to cellular division. From this original observation came the discovery that a chemical reaction between the platinum electrode and ammonium chloride produced diamine-dichloroplatinum (DDP) and that activity was confined to the cis-isomer of the molecule. In 1968, Rosenberg and colleagues proved the antiproliferative effect of the drug in the murine sarcoma 180 model (2). This was eventually followed by the publication of the first phase I clinical trial by the National Cancer Institute (NCI) in 1970, followed by the introduction of cisplatin into widespread clinical practice in 1978 (3,4). Although numerous compounds have been synthesized, only carboplatin (CBDCA) has succeeded in gaining approval in the United States. Carboplatin differed from cisplatin in structure by the incorporation of a cyclobutane ring ligand in place of the two chloride groups of cisplatin. This resulted in a more stable molecule and was felt to be less reactive and likely less toxic. The initial phase I trial, conducted by Calvert and colleagues, revealed that carboplatin produced no neurotoxicity, ototoxicity, or nephrotoxicity in the first 60 patients treated, a vast improvement over the toxicity seen with cisplatin (5,6). Delayed myelosuppression, particularly thrombocytopenia, proved to be the dose-limiting toxicity. Clinical activity was seen in several tumor types, including ovarian cancer, prompting subsequent investigations. However, although safer than cisplatin, it did not solve the problem of cisplatin resistance.

MECHANISM OF ACTION Within the extracellular hyperchloremic compartment, cisplatin exists in a stable, nonreactive form. Once it crosses cellular membranes, a series of aquation reactions occurs, displacing each chloride ion. With the initial loss of the first chloride ion, it binds to DNA preferentially by linking N(7) sites at GC sequences or GG, respectively (7,8). Following the second aquation, the remaining chloride is lost and the Pt-DNA adduct is closed, creating a bifunctional lesion. It is important to note that the diaquated platinum species does not interact with DNA alone, but can also react with RNA and proteins.

Chapter 8 / NF-gB and Cisplatin Activity

199

90% of adducts formed are 1,2-intrastrand crosslinks between purine residues. The antitumor efficacy of each crosslink has not been established, but the intrastrand adduct, which causes local kinking and unwinding of the DNA, is felt to be an important mediator of the cytotoxicity. The interstrand adduct may also be important, as it has been shown to block both DNA and RNA polymerases (9). Furthermore, increased DNA repair activity of the interstrand adducts within ovarian cancer cell lines has also been linked to resistance (10). There is now substantial evidence that cisplatin kills cells by activating apoptosis. Overexpression of anti-apoptotic proteins bcl-xl and bcl-2 can prevent cisplatin-induced cell death (11,12). However, cell-cycle arrest at G2 has also been associated with cytotoxicity.

RESISTANCE Multiple, independent mechanisms have been implicated in both intrinsic and acquired cisplatin resistance. The multiplicity of these mechanisms is a mark of the kind of resistance seen in DNA damaging agent chemotherapeutics. Each mechanism described below has generally been characterized as important but insufficient to explain all of the observed resistant phenotype. Studies in cell lines with acquired platinum resistance have revealed that it is likely multifactorial. Decreased intracellular drug accumulation has been shown to develop rapidly in vivo (13). Gately has proposed a mechanism of cellular accumulation of cisplatin based on several lines of data suggesting both passive diffusion and the existence of a protein-mediated transport mechanism (14). He proposed that cisplatin entry occurs by both mechanisms and that the transport is mediated by a phosphorylation cascade, based on evidence that cisplatin uptake is modifiable with agents that inhibit the Na-K ATPase and the membrane surface. More recently, a copper transporter has been implicated (15,16). The regulation of expression for these transporters may be an potential target for intervention. Another mechanism involves increased levels of glutathiones (GSH) or glutathione-S-transferase activity. Cisplatin acts as an electrophile that is particularly reactive with sulfur-containing nucleophiles. Binding to these molecules may be an important method of detoxification. In several cisplatin-resistant cell lines, including the ovarian cancer cell line SKOV-3R, high levels of intracellular thiol have been documented (17). However, the degree to which intracellular platinum interacts with glutathione in vivo is still unclear. This mechanism has been targeted by clinical agents depleting glutathione (18). Other studies have utilized a prodrug cleaved by glutathione S-transferase pi (19,20). Another important mechanism is that of mismatch repair (MMR). Alteration of MMR may confer intrinsic resistance to cisplatin and carboplatin (21). Fink and colleagues showed that loss of the MMR protein MSH2 resulted in twofold

200

Dizon et al.

resistance to cisplatin that was sufficient to impair response to cisplatin in vivo (22). The rationale behind this is that MMR acts as a detector system for DNA damage. When it senses damage, it may generate a series of events ultimately leading to apoptosis. This association between loss of MMR and decreased cell apoptosis has been shown in ovarian cancer cell lines (23,24). It is also widely recognized that loss of MMR proteins is associated with resistance to a number of chemotherapeutic agents, including cisplatin. Cells may also be able to replicate DNA past the site of the platinum-DNA adduct in a process termed replicative bypass. This may prove to be another important mechanism of clinical resistance. The data for this come from preclinical studies in both murine and human cancer cell lines. Gibbons et al. studied the effect of two types of platinum adducts—ethylenediamminedichloroplatinum(II) (en-Pt) and diamminecyclohexylamine-platinum (DACH-Pt)—on both a parental and resistant murine leukemia cell line (L1210) with resistance to either cisplatin (L1210/DDP) or DACH-Pt compounds (L1210/DACH) (25). In the L1210/DDP cell line, the en-Pt was fourfold less effective than the DACH-Pt adduct in inhibition of chain elongation. In the L1210/DACH cell line, the reverse was true: the DACH-Pt was 2.7-fold less effective than the en-Pt adduct in inhibition, suggesting that replicative bypass was carrier specific. This work was subsequently confirmed in a parental and cisplatin-resistant ovarian cancer cell line (26).

NF-gB IN PLATINUM RESISTANCE Based on the diversity of possible resistance mechanisms, it is logical to consider how such diverse processes are induced during the acquisition of resistance. Shared transcriptional activation is one possible mechanism. NF-gB is a transcription factor that has been linked to apoptosis rescue, inflammation, cell adhesion, differentiation, and cell growth. Since its discovery in 1986 by Sen and Baltimore, it has been the subject of investigation as a means of controlling various cellular processes, including tumorigenicity (27). Within the cell, NF-gB is sequestered in the cytoplasm, where it is inactive and bound to IkB protein through binding to the RelA-p50 subunit of NF-gB. This interaction is a key regulatory step in the activation of NF-gB. All stimulatory signals of NF-gB, such as tumor necrosis factor (TNF)-_, growth factors, or cytokines, result in the activation of an IKK dimer that acts in a serine-specific fashion. That is, signal activation of IKK results in the phosphorylation of members of the IkB family at specific serine residues near the N-terminus, which subsequently target IKB species for degradation via the ubiquitin-proteasome pathway. When it dissociates from IkB, NF-gB is freed and is transported to the nucleus, where it binds to specific sequences and results in expression of target genes. Serial experiments have shown that the cellular response to NF-gB is not uniform; instead, there is variability on this response, depending on both the cell

Chapter 8 / NF-gB and Cisplatin Activity

201

type and the type of stimulus (28). Despite this, constitutive activation of NF-gB has been descried in a number of human cancers, including ovarian (29,30), breast (31,32), colon (33), and cervical cancer (34), to name but a few. NF-gB not only plays a role in these cellular processes, but also is felt to be a potential mediator of both chemosensitivity and drug resistance by blocking apoptosis induced by chemotherapy. In our studies, we have examined the relationship between NF-gB activation and the acquisition of cisplatin resistance. When we examined a variety of platinum-sensitive and -resistant cell lines, it was clear that in every case cisplatin resistance was associated with increases in NF-gB binding activity. In Fig. 1A, several different cell lines are examined for NF-gB activity and compared to the platinum-resistant phenotype. In each case, the CDDP-resistant line shows increased NF-gB-binding activity. In Fig. 1B, SK-OV-3 cells are exposed to cisplatin for various times, and an assay of IKK activity is shown for each time period. It is possible to use the IkB/IKK systems to establish an unequivocal linkage between platinum resistance and activation of the NF-gB system. When a vector expressing IKK_ is transfected into SK-Ov-3 cells, it is possible to select for coexpression of a selectable marker such as Neomycin resistance (Fig. 2A). Those cells with IKK_ expression are substantially more resistant to cisplatin cytotoxicity (Fig. 2B). Similarly, when a mutated IKK_ or IKKB is transfected into the SK-Ov-3 cells, viable clones can be selected (Fig. 3A and 3B, respectively). These inactive kinases act as dominant-negative genes, silencing the normal IKK dimer and leading to increased platinum sensitivity (Fig. 4).

DEGRADATION OF IKB: THE UBIQUITIN–PROTEASOME PATHWAY As previously discussed, the dissociation of IkB and NFgB is the key step in the activation of NFgB. This requires phosphorylation of NFgB, which subsequently targets it for ubiquination and degradation by the 26S proteasome. Transfectants with either wild-type or resistant IKK will cause predictable changes in IkB. As shown in Fig. 5, exposure to CDDP or transfection with a wild-type IKK results in a decrease of IkB_ and IkBB by Western blot. In contrast, the mutated IKK_ and IKKB vectors prevent the loss of the IkB family members following platinum treatment. In normal cells, the ubiquitin-proteasome pathway is responsible for the orderly breakdown of multiple proteins and thereby helps to define protein turnover rates. Proteasomes, large complexes of proteolytic enzymes, break down these intracellular proteins, recognizing them by their ubiquitin molecular tags. In addition to degrading unwanted proteins, the proteasome is involved in the regulation of cellular signals that govern the cell-division cycle, as well as cell growth and differentiation.

202

Dizon et al.

Fig. 1. (A) Gel-binding studies of the cell lines indicated. In each case, the wild-type cell is paired with the cisplatin-resistant clone. (B) In vitro kinase assay using cell extracts from SK-Ov-3 cells, treated with CDDP for times indicated. Phosphorylation on IkB was measured by densitometery to provide the bar graph below. Note that the resistant line is not treated with CDDP.

Selective inhibition of proteasome activity has numerous effects, including attenuating the activity of NF-gB as well as inhibiting the activity of bcl-2, a gene involved in cell survival. Both NF-gB and bcl-2 are used by cancer cells to help them survive treatment with standard chemotherapy agents. In tumor cells, proteasome inhibition produces overwhelming cellular stress by stabilizing cellcycle regulatory proteins and disrupting cellular proliferation, ultimately leading to apoptosis.

Chapter 8 / NF-gB and Cisplatin Activity

203

Fig. 2. (A) Transfected SK-Ov-3 cells using a selectable IKK_ expression vector with a neomycin resistance marker. (B) The effect of the IKK_ expression vector on CDDP killing, as measured by vital dye conversion.

Fig. 3. (A) Transfection with a kinase inactive mutated IKK_ expression vector selected by neomycin resistance. The Western blot illustrates the presence of the mutated kinase. (B) Transfection with a kinase-inactive mutated IKK` expression vector selected by neomycin resistance. The Western blot illustrates the presence of the mutated kinase.

204

Dizon et al.

Fig. 4. Effect of mutated IKK_ and mutated IKK` on platinum sensitivity in a 96-well vital dye cytoxicity assay.

MODIFYING PLATINUM RESISTANCE— PROTEASOME INHIBITION Given the importance of the RelA–IkB complex in inhibiting NF-gB from translocating into the nucleus, there has been a significant effort into blocking this dissociation. One of these has been to block the 26S proteasome, which degrades IkB. Of the agents in clinical trial, PS-341 (bortezomib, Velcade®, Millenium Pharmaceuticals) is the furthest in development. PS-341 is a dipeptidyl boronic acid inhibitor with high specificity for the proteasome. PS-341 acts through the ubiquitin-proteasome pathway to alter the expression of important proteins necessary for accelerated and uncontrolled cellular division. Because it targets the proteasome, it is not specifically targeting the NF-gB signaling pathway. However, this effect is felt to be central to the activity of this drug, particularly in melanoma. The phase I trials of this agent have been completed in both solid and hematologic malignancies. In the phase I trial in solid tumors, patients received escalating doses of PS-341 twice weekly for 2 wk followed by a 1-wk break. Forty-three patients were treated, and PK data demonstrated a dose-related inhibition of the 20S proteasome with increasing doses. Evidence of tumor activity was seen in this cohort, with one patient with non-small-cell lung carcinoma exhibiting a partial response (35). In the phase I trial for refractory hematological malignancies, patients were treated twice weekly for 4 wk followed by a 2-wk rest period. Twenty-seven patients were treated; PD data showed that 20S proteasome inhibition occurred in a time-dependent fashion and that this was dose-related. Complete responses

Chapter 8 / NF-gB and Cisplatin Activity

205

Fig. 5. Effect of cisplatin and the various expression vectors on IkB family member protein levels before and after CDDP.

were seen in patients with heavily pre-treated plasma cell dyscrasias. Other responses were seen in patients with mantle-cell and follicular lymphoma (36). Based on this preliminary evidence, the drug has been launched into phase II and III trials in multiple disease sites. Preliminary phase II results of PS-341 in patients with melanoma who had failed two prior regimens were presented at the 2002 annual meeting of the American Society of Clinical Oncology (37). Seventy-eight patients received PS-341 as a twice-weekly infusion for 2 wk followed by a week off. The authors reported that 77% had a reduction or stabilization in their M protein, which was used as a surrogate for their tumor burden. This included 20% who had 90% reduction in their M protein. Bortezomib is now approved for use in myeloma and under study in a variety of other settings.

CONCLUSION NF-gB is a key regulator in multiple processes from inflammation to tumorigenicity and chemotherapy resistance. We have only begun to explore methods of manipulating this pathway. PS-341 can serve as proof of principle that blocking the activation of NF-gB can lead to responses. Clinical trials of PS-341 and cisplatin are ongoing in ovarian cancer to evaluate the theory that blocking the proteasome will decrease cisplatin resistance.

206

Dizon et al.

REFERENCES 1. Rosenberg B, Van Camp L, Grimley EB, Thomson AJ. The inhibition of growth or cell division in Escherichia coli by different ionic species and platinum (IV) complexes. J Biol Chem 1967;242(6):1347–1352. 2. Rosenberg B, VanCamp L. The successful regression of large solid sarcoma 180 tumors by platinum compounds. Cancer Res 1970;30:1799–1802. 3. Talley RW, O’Bryan RM, Gutterman JU, Brownlee RW, McCredie KB. Clinical evaluation of toxic effects of cis-diamminedichloroplatinum (NSC119875)—Phase I clinical study. Cancer Chemother Rep 1973;57(4):465–471. 4. Lebwohl D, Canetta R. Clinical development of platinum complexes in cancer therapy: an historical perspective and an update. Eur J Cancer 1998;34(10):1522–1534. 5. Calvert A, Harland SJ, Newell DR, et al. Early clinical Studies of cis-diammine-1,1cyclobutane dicarboxylate platinum II. Cancer Chemother Pharmacol 1982;9(3):140–147. 6. Calvert AH, Harland SJ, Newell DR, Siddik ZH, Harrap KR. Phase I studies with carboplatin at the Royal Marsden Hospital. Cancer Treat Rev 1985;12 Suppl A:51–57. 7. Eastman A. Reevaluation of interaction of cis-dichloro(ethylenediamine)platinum(II) with DNA. Biochemistry 1986;25(13):3912–3915. 8. Lemaire MA, Schwartz A, Rahmouni AR, Leng M. Interstrand cross-links are preferentially formed at the d(GC) sites in the reaction between cis-diamminedichloroplatinum (II) and DNA. Proc Natl Acad Sci USA 1991;88(5):1982–1985. 9. Corda Y, Job C, Anin MF, Leng M, Job D. Spectrum of DNA—platinum adduct recognition by prokaryotic and eukaryotic DNA-dependent RNA polymerases. Biochemistry 1993;32(33):8582–8588. 10. Zhen W, Link CJ Jr, O’Connor PM, et al. Increased gene-specific repair of cisplatin interstrand cross-links in cisplatin-resistant human ovarian cancer cell lines. Mol Cell Biol 1992;12(9):3689–3698. 11. Minn AJ, Rudin CM, Boise LH, Thompson CB. Expression of bcl-xL can confer a multidrug resistance phenotype. Blood 1995;86(5):1903–1910. 12. Miyashita T, and Reed JC. Bcl-2 oncoprotein blocks chemotherapy-induced apoptosis in a human leukemia cell line. Blood 1993;81(1):151–157. 13. Andrews PA, Jones JA, Varki NM, Howell SB. Rapid emergence of acquired cisdiamminedichloroplatinum(II) resistance in an in vivo model of human ovarian carcinoma. Cancer Commun 1990;2(2):93–100. 14. Gately DP, and Howell SB. Cellular accumulation of the anticancer agent cisplatin: a review. Br J Cancer 1993;67(6):1171–1176. 15. Komatsu M, Sumizawa T, Mutoh M, et al. Copper-transporting P-type adenosine triphosphatase (ATP7B) is associated with cisplatin resistance. Cancer Res 2000;60(5):1312–1316. 16. Katano K, Kondo A, Safaei R, et al. Acquisition of resistance to cisplatin is accompanied by changes in the cellular pharmacology of copper. Cancer Res 2002;62(22):6559–6565. 17. Mistry P, Kelland LR, Abel G, Sidhar S, Harrap KR. The relationships between glutathione, glutathione-S-transferase and cytotoxicity of platinum drugs and melphalan in eight human ovarian carcinoma cell lines. Br J Cancer 1991;64(2):215–220. 18. Bailey HH, Mulcahy RT, Tutsch KD, et al. Phase I clinical trial of intravenous L-buthionine sulfoximine and melphalan: an attempt at modulation of glutathione. J Clin Oncol 1994;12(1):194–205. 19. Rosen LS, Laxa B, Boulos L, et al. Phase I study of TLK286 (glutathione S-transferase P11 activated glutathione analogue) in advanced refractory solid malignancies. Clin Cancer Res 2003;9(5):1628–1638. 20. Townsend DM, Shen H, Staros AL, Gate L, Tew KD. Efficacy of a glutathione S-transferase pi-activated prodrug in platinum-resistant ovarian cancer cells. Mol Cancer Ther 2002;1(12):1089–1095.

Chapter 8 / NF-gB and Cisplatin Activity

207

21. Fink D, Nebel S, Aebi S, et al. The role of DNA mismatch repair in platinum drug resistance. Cancer Res 1996;56(21):4881–4886. 22. Fink D, Zheng H, Nebel S, et al. In vitro and in vivo resistance to cisplatin in cells that have lost DNA mismatch repair. Cancer Res 1997;57(10):1841–1845. 23. Toney JH, Donahue BA, Kellett PJ, Bruhn SL, Essigmann JM, Lippard SJ. Isolation of cDNAs encoding a human protein that binds selectively to DNA modified by the anticancer drug cis-diamminedichloroplatinum(II). Proc Natl Acad Sci USA 1989;86(21):8328–8332. 24. Pil PM, and Lippard SJ. Specific binding of chromosomal protein HMG1 to DNA damaged by the anticancer drug cisplatin. Science 1992;256(5054):234–237. 25. Gibbons GR, Kaufmann WK, Chaney SG. Role of DNA replication in carrier-ligand-specific resistance to platinum compounds in L1210 cells. Carcinogenesis 1991;12(12):2253–2257. 26. Mamenta EL, Poma EE, Kaufmann WK, Delmastro DA, Grady HL, Chaney SG. Enhanced replicative bypass of platinum-DNA adducts in cisplatin-resistant human ovarian carcinoma cell lines. Cancer Res 1994;54(13):3500–3505. 27. Sen R, and Baltimore D. Inducibility of kappa immunoglobulin enhancer-binding protein Nfkappa B by a posttranslational mechanism. Cell 1986;47(6):921–928. 28. Bours V, Bonizzi G, Bentires-Alj M, et al. NF-kappaB activation in response to toxical and therapeutical agents: role in inflammation and cancer treatment. Toxicology 2000;153(13):27–38. 29. Huang S, Robinson JB, Deguzman A, Bucana CD, Fidler IJ. Blockade of nuclear factorkappaB signaling inhibits angiogenesis and tumorigenicity of human ovarian cancer cells by suppressing expression of vascular endothelial growth factor and interleukin 8. Cancer Res 2000;60(19):5334–5339. 30. Dejardin E, Deregowski V, Chapelier M, et al. Regulation of NF-kappaB activity by I kappaBrelated proteins in adenocarcinoma cells. Oncogene 1999;18(16):2567–2577. 31. Sovak MA, Bellas RE, Kim DW, et al. Aberrant nuclear factor-kappaB/Rel expression and the pathogenesis of breast cancer. J Clin Invest 1997;100(12):2952–2960. 32. Nakshatri H, Bhat-Nakshatri P, Martin DA, Goulet RJ Jr, Sledge GW Jr. Constitutive activation of NF-kappaB during progression of breast cancer to hormone-independent growth. Mol Cell Biol 1997;17(7):3629–3639. 33. Lind DS, Hochwald SN, Malaty J, et al. Nuclear factor-kappa B is upregulated in colorectal cancer. Surgery 2001;130(2):363–369. 34. Nair A, Venkatraman M, Maliekal TT, Nair B, Karunagaran D. NF-kappaB is constitutively activated in high-grade squamous intraepithelial lesions and squamous cell carcinomas of the human uterine cervix. Oncogene 2003;22(1):50–58. 35. Aghajanian C, Soignet S, Dizon DS, et al. A phase I trial of the novel proteasome inhibitor PS341 in advanced solid tumor malignancies. Clin Cancer Res 2002;8(8):2505–2511. 36. Orlowski RZ, Stinchcombe TE, Mitchell BS, et al. Phase I trial of the proteasome inhibitor PS-341 in patients with refractory hematologic malignancies. J Clin Oncol 2002;20(22):4420–4427. 37. Richardson PG, Barlogie B, Berenson J, et al. A phase 2 study of bortezomib in relapsed, refractory myeloma. N Engl J Med 2003;348(26):2609–2617.

Chapter 9 / Chemotherapy and G3139

9

209

Combinations of Chemotherapy and G3139, an Antisense Bcl-2 Oligonucleotide Luba Benimetskaya, PhD, Sridhar Mani, MD, and C. A. Stein, MD, PhD CONTENTS INTRODUCTION BCL-2—A KEY REGULATOR OF APOPTOSIS ANTISENSE STRATEGY: APPLICATION AND LIMITATIONS THE MECHANISM OF ACTION OF ANTISENSE OLIGONUCLEOTIDES THE PROBLEM OF NONSPECIFICITY PRECLINICAL STUDIES OF COMBINATION OF G3139 (GENASENSE, OBLIMERSEN) AND CHEMOTHERAPY COMBINATIONS OF G3139 PLUS CYTOTOXIC CHEMOTHERAPY ANTISENSE BCL-2 OLIGONUCLEOTIDE IN THE CLINIC G3139 (GENASENSE) UNIQUE ASPECTS OF G3139 METABOLISM/PHARMACOLOGY AND DRUG–DRUG INTERACTIONS

INTRODUCTION Apoptosis, or programmed cell death, is essential for maintaining homeostasis in multicellular organisms (1). It plays an important role in many physiological processes, especially in the immune and nervous systems, and in development (2,3). Deregulation of apoptosis can lead to autoimmune diseases, cancer, and the clinical manifestations of acquired immunodeficiency syndrome (AIDS) (2). The apoptotic process involves a sequence of events including cell shrinkage, increased cytoplasmic density, chromatin condensation, and the formation of From: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

209

210

Benimetskaya, Mani, and Stein

membrane-bound, smooth-surface apoptotic bodies (4,5). In addition, it is accompanied by the activation of endonucleases that cleave DNA at internucleosomal sites to produce multiple fragments of approx 180 basepairs (bp) (6). Subsequently, neighboring cells or macrophages phagocytose apoptotic cells without eliciting an inflammatory reaction. These morphological and biochemical processes are predominately mediated by death effectors, such as proteases, and ultimately lead to nuclear and cellular fragmentation. There are two principal pathways leading to apoptosis in mammalian cells: the extrinsic (cell-surface death receptor mediated) and intrinsic (mitochondrial mediated) (7). The mitochondrial pathway of apoptosis is activated from within the cell itself—for example, in response to stress initiated by damage-inducing agents such as cytotoxic drugs. Apoptosis mediated through this pathway involves the release of mitochondrial proteins such as cytochrome c, which is an essential cofactor required for the activation of the caspase cascade. The release of cytochrome c from the mitochondrion is regulated by a large and growing number of proteins, including the Bcl-2 family of proteins, which are upstream of caspase activation (8). Although the process of cellular apoptosis can be initiated by chemotherapeutic agents, its efficiency is relatively low. This resistance to apoptosis induction is commonly associated with the overexpression of antiapoptotic proteins such as Bcl-2. Strategies that aim to decrease the expression of such proteins in cancer cells could, at least in theory, enhance the effectiveness of the treatment of cancer patients. A strategy that is designed to decrease expression of bcl-2 protein involves the utilization of antisense oligonucleotides. These short, single-stranded DNA or modified RNA molecules bind to the target mRNA, and elicit the activity of Rnase H, a ubiquitous intracellular (and intranuclear) enzyme that cleaves the mRNA strand of the duplex formed by the mRNA and the antisense oligonucleotide. The degradation of the target mRNA results in the lack of new translation of protein, resulting, after catabolism of existing protein, in significant downregulation (up to 90–95%) of target expression. Whereas when employed clinically, antisense oligonucleotides may be useful as single agents, their major roles may in fact be to sensitize tumor cells to chemotherapeutic agents. Indeed, this is predominantly the way they are being used in ongoing clinical trials. In this case, it is possible that either less chemotherapy might be required to produce an equivalent therapeutic effect (resulting in less systemic toxicity), or that a greater therapeutic effect may be achieved with similar doses of cytotoxic agent.

BCL-2—A KEY REGULATOR OF APOPTOSIS The Bcl-2 protein is a member of a growing family that plays an important role in the regulation of apoptosis (9). This founding member of the family was originally identified at the chromosomal breakpoint of a t(14;18)-bear-

Chapter 9 / Chemotherapy and G3139

211

ing B-cell lymphoma (10). The overexpression of Bcl-2 protein was initially shown to prevent apoptosis induced by growth-factor deprivation in certain hematopoietic cells (11). The discovery that Bcl-2, unlike previously studied oncogenes, functions in preventing apoptosis, instead of promoting proliferation, established a new class of oncogenes, which may be divided into two groups: the antiapoptotic members (Bcl-2, Bcl-xL, Bcl-w, Mcl-1, A1, and Boo) and those that are proapoptotic (Bax, Bak, Bcl-xS, Bad, Bik, Bid, Hrk, Bim, and Blk) (9). Although the concept of apoptosis was introduced more than 30 yr ago (12), the precise mechanisms that control apoptosis are far from being fully understood, and in any case over 100 proteins have been identified as being in one way or another contributory. Antiapoptotic members promote survival and share conserved motifs that are known as Bcl-2 homology (BH) domains (BH1, BH2, BH3, and BH4), although some members lack an apparent BH4 domain. Proapoptotic members promote cell death and share sequence homology in the BH1, BH2, and BH3 domains only, though significant homology in the BH4 domain has been noted in some members (9,13). The three-dimensional structures of the Bcl-2 and Bcl-xL proteins, based on X-ray and nuclear magnetic resonance (NMR) studies, reveal a hydrophobic-binding pocket that mediates protein–protein interactions involving Bcl-2 family members (14,15). Indeed, proteins of the Bcl-2 family homo- and heterodimerize with each other (16,17), and the latter process, when occurring between pro- and antiapoptotic members, modulates proapoptotic activity, possibly by altering the mitochondrial permeability transition. In the Bcl-2 protein, the NH2-terminal BH4 domain appears to be required for heterodimerization with Bax, as well as for its antiapoptotic activity. In addition, deleting either the BH1 or BH2 domain also eliminates the ability of Bcl-2 to form heterodimers with Bax, and to suppress apoptosis (18). The BH3 domain of Bax is required for its ability to homodimerize and to heterodimerize with Bcl-2 and Bcl-xL, and to induce apoptosis (19,20). The ratio of Bcl family member homodimers to heterodimers has been proposed as a main regulator of the rate of apoptotic death or, alternately, of cell survival (16). A relatively high homodimer/heterodimer ratio leads to the release of cytochrome c from the mitochondria into the cytosol. Released cytochrome c forms a complex with dATP and with Apaf-1, a 130-kDa protein containing a homology region with caspase-9. This complex then combines with procaspase-9 to form a new complex known as the apoptosome. In the apoptosome, procaspase-9 is cleaved to capsase-9, which then cleaves procaspase-3 to caspase-3, which is one of the major effector downstream proteases in the apoptotic process. However, as mentioned earlier, this entire proapoptotic process can be aborted by cells that contain high levels of Bcl-2 protein, thus suggesting that elimination of Bcl-2 protein expression may in fact promote cellular apoptosis.

212

Benimetskaya, Mani, and Stein

ANTISENSE STRATEGY: APPLICATION AND LIMITATIONS The antisense strategy is designed to inhibit gene expression at the RNA level (21–33). The underlying aim is the achievement of specificity, produced via Watson–Crick binding of a defined nucleotide sequence to a complementary base sequence of a target messenger RNA (mRNA) transcript. The subsequent formation of the mRNA-DNA duplex can lead, probably via RNase H degradation of the mRNA strand, to the suppression of its expression of the target mRNA and hence translation into a corresponding protein. There are several classes of antisense molecule. One is a relatively short (up to 30 nucleotides, but much more commonly 18–20mer in length) synthetic single-stranded DNA molecule (“antisense” oligonucleotide). These molecules are currently being administered to cancer patients in phase I, II, and III clinical trials by intravenous infusion (34,35). Another type of antisense oligonucleotide is long-chain antisense RNA, which is typically intracellularly expressed following transient or stable transfection with antisense genes (36). A third type are ribozymes, which contain antisense arms for target binding, and a catalytic RNA core for cleavage of the target RNA at a specific consensus sequence (37). More recently, short (21–23 mer) double-stranded RNA molecules (siRNA) have shown interesting and significant activity in mammalian systems, although their mechanism of action is somewhat uncertain at present. The “antisense” approach to treating cancer was first suggested more than 35 yr ago by Grineva et al. (38), based on the observation that an RNA sequence can serve as an endogenous biological inhibitor of gene expression in prokaryotes (39). Some of the first antisense experiments were performed by Stevenson and Zamecnik in 1978 (40,41). They synthesized a series of phosphodiester oligonucleotides complementary to the terminal reiterated sequences of the Rous sarcoma virus mRNA, and found that reverse transcriptase activity was decreased in the supernatants of infected chicken-embryo fibroblast cells. However, phosphodiester oligonucleotides (i.e., normal DNA) can not be used as drugs because, among other reasons, they are easily digested by ubiquitous intra- and extracellular nucleases (42). To generate oligonucleotides more resistant to nuclease digestion, the phosphorothioate modification was developed, initially by Stec and colleagues. Here, a sulfur atom is substituted for a nonbridging oxygen atom at each phosphorus in the oligonucleotide chain (43), retaining the polyanionic character of the molecule, and its property of aqueous solubility. Phosphorothioate oligonucleotides are nuclease resistant (though by no means nuclease-proof) both in vitro (44) and in vivo (45), available commercially, relatively inexpensive, and are the class of oligonucleotide most commonly used in cell-culture and animal experiments, and most recently in human therapeutic clinical trials as well (46). In addition, there are also several other classes of oligonucleotides that are nuclease resistant, and which may also act as antisense agents. For example, in

Chapter 9 / Chemotherapy and G3139

213

the methylphosphonate modification, a methyl group replaces one of the nonbridging oxygen atoms at phosphorus (47). However, the net negative charge is lost, and with it, the property of aqueous solubility, which has led to serious formulation difficulties. For this and other reasons, methylphosphonate oligonucleotides are no longer commonly experimentally employed. An additional important class of antisense effector molecule consists of oligoribonucleotides modified by an alkyl group at the 2'-O position of the ribose moiety. The oligonucleotide backbone remains phosphorothioate, but the 2'-O-methyl modification cannot elicit RNase H activity (see next section). Other, more drastic chemical modifications of the oligonucleotide backbone, produce uncharged morpholino oligonucleotides (48); and peptide nucleic acids (PNAs), in which phosphate backbone has been removed entirely and replaced with polyamide structures to which the nitrogenous bases are linked (49). Both of these classes of oligonucleotide may well eventually find some interesting niche uses, and commercial efforts to develop them have been underway for some time.

THE MECHANISM OF ACTION OF ANTISENSE OLIGONUCLEOTIDES Detailed reviews of the mechanisms of target gene downregulation by antisense oligonucleotides are now available (34,35). The most commonly accepted antisense mechanism implicates RNase H-mediated hydrolysis of the target mRNA strand of the RNA–DNA duplex (50), but it is also possible that enzymes other than RNase H, such as RNase P (51), RNase III (52), and RNase L (53), may also participate under some circumstances in mRNA degradation. RNase-independent mechanisms of oligonucleotide activity include steric blockade of splicing (54,55), translational arrest (56), and the prevention of 5' capping of the mRNA, which affects nuclear transport of RNA (57,58). To all commercial intents and purposes, only phosphodiester and phosphorothioate oligonucleotides are substrates for RNase H activity (59,60), which appears to be required for the production of antisense effects at nanomolar oligonucleotide concentrations. When antisense effects are produced by RNase H-independent mechanisms by, for example, methylphosphonate oligodeoxynucleotides and 2'-O-methyloligoribonucleotides (60), micromolar oligonucleotide concentrations are invariably required. One of the major antisense oligonucleotide classes currently utilized in the laboratory is known as a “gap-mer.” This type of effector molecule contains a phosphorothioate backbone for nuclease resistance, and usually several (two to six) 2'-O-alkylated (methyl or methoxyethoxy [MOE]) oligoribonucleotides at the 3' and 5' molecular termini. These terminal modifications increase the melting temperature of the mRNA-oligonucleotide duplex, and diminish certain nonspecific aspects of RNase H activity. The central region of the molecule remains oligodeoxyribonucleotide, to provide a recognition site for the required

214

Benimetskaya, Mani, and Stein

RNase H cleavage. The use of gap-mers in tissue culture can produce extremely specific downregulaton of target expression in the low nanomolar range, but whether they will be therapeutically effective is highly uncertain at present.

THE PROBLEM OF NONSPECIFICITY Although in theory oligonucleotides have the potential for exquisite specificity, in reality they also produce nonspecific effects, which occur when the oligonucleotides interact with molecules other than their intended targets. Because phosphorothioate oligonucleotides are polyanions, they bind, in a length- and somewhat sequence-dependent manner, to heparin-binding proteins such as bFGF (61,62), PDGF, VEGF (61,62) and its receptors, EGF-R (63), CD4 (64), gp120 (65), Mac-1 (CD11/CD18) (66), fibronectin, laminin (67), and to many other proteins as well. The nonspecific properties of oligonucleotides can in some cases be a function of sequence: Oligonucleotides containing CpG motifs are well known to be highly immunostimulatory, even in SCID rodents (68,69), though probably less so in human beings. In addition, oligonucleotides with four contiguous guanosine residues can form G quartets and tetraplexes via Hoogsteen basepair formation (70–73). These highly negatively charged molecules can be extremely biologically active, but their activity may have little or nothing to do with the potential antisense properties of the single strand. Not only can the length of the phosphorothioate backbone produce increased nonspecificity, but increasing numbers of bases can, paradoxically, increase nonsequence specificity. This is because RNase H does not require 100% complementarity between the target mRNA and the incoming oligonucleotide for elicitation mRNA cleaving ability. Short, partial homology, consisting of perhaps a 5- to 10-bp contiguous region of hybridization, may well, under certain circumstances, be sufficient for RNase H-mediated cleavage of the hybrid (74). This partial hybridization can thus result in the degradation of nontargeted mRNAs throughout the transciptome bearing similar sense sequences (75,76). The problem may be suppressed, but not eliminated, by the use of gap-mers, in which the 3' and 5' molecular termini can no longer elicit RNase H activity, as mentioned above.

PRECLINICAL STUDIES OF COMBINATION OF G3139 (GENASENSE, OBLIMERSEN) AND CHEMOTHERAPY In some of the earliest experiments, phosphodiester and phosphorothioate antisense oligonucleotides targeted to the translation initiation site of the human Bcl-2 mRNA were successfully used to inhibit the proliferation of 697 human leukemia cells. Subsequently, Kitada et al. (77) found that an 18-mer phosphodiester oligonucleotide, complementary to the first six codons of the Bcl-2 mRNA, selectively downregulated Bcl-2 protein expression in cell-free rabbit

Chapter 9 / Chemotherapy and G3139

215

reticulocyte lysate. These oligonucleotides were not delivered with cationic lipids or other delivery vectors, and the concentrations employed were in the micromolar range, now considered to be unacceptably high. Furthermore, as mentioned above, phosphodiester oligonucleotides cannot be used as drugs. Scientists at Genta, Inc. (Berkeley Heights, NJ) developed an antisense 18mer phosphorothioate oligonucleotide (alternatively called G3139, Genasense, or Oblimersen) directed against the first six codons of the open reading frame of the human Bcl-2 mRNA (5'-TCTCCCAGCGTGCGCCAT-3'). This antisense oligonucleotide was evaluated in RS11846 (77), SU-DHL-4 (78), DoHH2 lymphoma (79), prostate cancer, and other tumor cell lines. Down-regulation of both Bcl-2 protein and mRNA expression could be convincingly demonstrated both in vitro and in vivo, leading to the employment of single-agent G3139 in phase I and II clinical trials against chemotherapy-refractory non-Hodgkin’s lymphoma. These trials were conducted by Cotter and colleagues in Great Britain, but were somewhat incomplete, as a maximum tolerated dose was apparently not defined. A great deal of study has been devoted to the treatment of prostate cancer and related cell lines (80–82). Treatment of LNCaP (80) and Shionogi (an androgendependent mouse mammary tumor that mimics aspects of prostate cancer cell physiology) tumor cells (81,82) in vitro with G3139 inhibited Bcl-2 mRNA and protein expression by approx 90%, in a dose-dependent and sequence-specific manner. Bcl-2 mRNA levels returned to pretreatment levels by 48 h after discontinuing the treatment (80). Because Bcl-2 protein is significantly membrane bound, its half-life can exceed 24 h. Therefore, it has been repeatedly observed that while downregulation of Bcl-2 mRNA by G3139 can reach 90% or more after 1 d, equal downregulation of protein expression will take approx 3 d. In the LNCaP prostate tumor model, two sets of in vivo experiments were performed. In the first set, athymic male mice bearing the LNCaP tumor were castrated and injected intraperitoneally with 12.5 mg/kg/d of G3139, beginning 1 d after castration in group 1 and 7 d after castration in group 2 (six mice in each group). The tumor volume in the mice treated with the mismatched control oligonucleotide increased 2.5-fold by 6 wk after castration, and fivefold by 12 wk after castration. In contrast, tumor volume in both G3139-treated groups gradually decreased during the first 2–3 wk of treatment after castration, with stabilization thereafter. By 6 wk after castration, the mean tumor volume was 53% of baseline in group 1 and 61% in group 2. By 12 wk after castration, tumor volume remained below precastrate baseline tumor volume in both groups (75% of baseline in group 1 and 95% in group 2). In the second set of experiments, all mice were treated with G3139 once daily intraperitoneally, beginning 1 d after castration, for the duration of the experiment. The tumor volume in the mice treated with mismatched control oligonucleotide increased 2.2-fold by 9 wk after castration, and by more than sevenfold by 14 wk after castration. In contrast, tumor

216

Benimetskaya, Mani, and Stein

volumes in the G3139-treated group decreased by 40% by 9 wk after castration and stabilized thereafter. In the Shionogi model, mice were castrated 2 to 3 wk after tumor implantation. Beginning 1 d after castration, 12.5 mg/kg G3139 was administered once a day by intraperitoneal injection. This treatment significantly delayed recurrence of androgen-independent tumors compared to mismatched control oligonucleotide treatment. During the observation period of 50 d postcastration, androgen-independent tumors recurred in five of seven mice after a median of 44 d in the G3139 treatment group, while androgen-independent tumors recurred in all of the mice after a median of 29 d in the mismatched control oligonucleotide treatment group. In addition, despite significant reductions of Bcl-2 expression in tumor tissues, G3139 had no effect on Bcl-2 expression in normal mouse organs, including spleen, thymus, and brain (81). Zangemeister-Wittke et al. recently designed a novel 2'-O-methoxy-ethoxymodified phosphorothioate antisense oligonucleotide designed to downregulate the expression of both Bcl-2 and Bcl-xL proteins, both of which are strongly antiapoptotic (83). The authors demonstrated that this “bi-specific” oligonucleotide (denoted 4625), which is completely homologous to the Bcl-2 mRNA, but which has three mismatches vs a highly homologous region of the Bcl-xL mRNA, simultaneously inhibited both Bcl-2 and Bcl-xL protein expression in SW2 lungcancer cells. Furthermore, this bi-specific activity induced cellular apoptosis, as demonstrated by a 10- to 100-fold increase in caspase-3-like protease activity, and by increased nuclear condensation and fragmentation as assessed by staining with Hoechst 33342. At a concentration of 600 nM, in complex with Lipofectin, 4625 reduced Bcl2 and Bcl-xL protein and mRNA levels in breast carcinoma cells in a dosedependent manner, and also induced death in cell lines of lung, breast, colorectal, prostate, and melanoma tumors. After in vivo administration, 4625 significantly inhibited the growth of breast and colorectal carcinoma xenografts in nude mice. This oligonucleotide was also evaluated in A375 melanoma cells, and in a series of primary melanoma cell lines of different clinical stages. In both, a decrease of Bcl-2 and Bcl-xL protein expression was demonstrated, which was attended by a reduction in cell growth, and an increase in cell death and apoptosis.

COMBINATIONS OF G3139 PLUS CYTOTOXIC CHEMOTHERAPY Downregulation of the Bcl-2 protein expression has been shown to reverse the chemoresistance of tumor cells, and to sensitize them to multiple anticancer drugs. The combination of Oblimersen with cytotoxic agents has been studied in both tissue culture and in a variety of solid tumor and hematopoietic xenografts in vivo.

Chapter 9 / Chemotherapy and G3139

217

The combination of G3139 with cyclophosphamide was evaluated in SCID mice bearing a human Do HH2 lymphoma xenograft (84). For both doses (15 and 35 mg/kg) of cyclophosphamide tested, increasing the dose of oligonucleotide from 2.5 to 5 mg/kg/injection resulted in longer median survivals and an overall increase in long-term survivors. The benefits are best illustrated at the lowest doses. For example, cyclophosphamide alone at 15 mg/kg resulted in no measurable increase in the life span of mice bearing the DoHH2 tumors. Alternatively, G3139 given alone at a dose of 2.5 mg/kg/injection increased median survival to 62 d, and one of six animals survived beyond 90 d. In combination, the median survival times were 72 and 84 d when treated with drug at 15 mg/kg and G3139 at 2.5 and 5 mg/ kg/injection, respectively. Both of these treatment groups exhibited long-term survival rates of 50% or more. An interesting result was achieved when a completely ineffective dose of drug (15 mg/kg; median survival 36 d and no long-term survivors) was combined with a modestly effective dose of oligonucleotide (2.5 mg/kg; 61-d median survival and 16% long-term survivors). This produced a 72d median survival and 50% long-term survival. This finding suggests that chemotherapy at very modest drug doses may be effective when combined with antisense oligonucleotides, without producing much in the way of toxicity to normal tissues. Evaluation of G3139 in combination with monoclonal antibody therapy was performed by Lacy et al. (85), who combined it with rituximab in the SCID-human chimeric model of posttransplant lymphoproliferative disease. A delayed treatment schedule was designed that permitted detection of an enhanced antitumor effect of the combined treatment. The mice (seven per group) were injected intraperitoneally with 20 million lymphoblastoid B-cells (LCLs) on d 0; on d 15 treatment was initiated with G3139 alone (10 mg/kg/d over 12 d in five intraperitoneal divided doses at 72-h intervals), rituximab alone (10 mg/kg intraperitoneally weekly × 4), or G3139 plus rituximab (same doses and schedule used for monotherapy). Untreated control animals, those treated with rituximab alone, and those treated with G3139 alone died due to progressive tumor at an average of 44, 59, and 71 d, respectively, after LCL injection. In contrast, when G3139 was combined with rituximab, five of seven mice remained tumor free at the time of sacrifice (150 d), and median survival of the combined treatment arm was 150 + d. Tauchi et al. (86) evaluated G3139 for its ability to inhibit BCR-ABL-mediated transformation in nude mice. Mice were transplanted with Gleevec-resistant BCR-ABL-transformed TF-1 cells. Untreated mice died within 10 wk (n = 5), while mice treated with G3139 (7 mg/kg/d intraperitoneally, 14 d, n = 5) survived for more than 6 mo and demonstrated reduced tumor volume (3/5 complete regressions). The authors also observed that myeloid leukemia cells harvested from nude mice treated with G3139 (7 mg/kg/d, intraperitoneally for 7 d) were more sensitive to subsequent treatment with the antileukemic agents Gleevec, daunorubicin, cytosine arabinoside, or etoposide, which demonstrated additive or synergistic effects on the induction of apoptosis.

218

Benimetskaya, Mani, and Stein

Gazitt et al. (87) evaluated pretreatment with G3139 (0.2–2.0 μM) on chemotherapy-induced apoptosis in myeloma cells. 6MM cells were pretreated with 10 μg/mL of G3139 for 3 d, followed by treatment with dexamethasone, paclitaxel, or adenovirally delivered p53 for 2 additional days. G3139 sensitized cells to the various cytotoxic agents whether they expressed low or high levels of Bcl-2. For example, pretreatment of the high-Bcl-2-expressing myeloma cell lines ARH-77 and U266 increased the fraction undergoing apoptosis from 15 to 20% with dexamethasone alone to 40 to 80% with the combination of dexamethasone plus G3139. Similar results were obtained with paclitaxel (from 10 to 24% to 56 to 85%) and adenovirally delivered p53 (from 6 to 12% to 40 to 50%). Additional data in vitro were obtained by Konopleva (88), who demonstrated that the combined treatment of HL60 myeloid leukemic cells with G3139 and cytosine arabinoside significantly reduced the amount of antisense oligonucleotide (from 12 to 4 μM) necessary to reach maximal cytotoxicity. However, this difference is relatively small and may be within experimental error. Much interesting and somewhat controversial data have been generated in SCID mice with established 518A2 human melanoma. Combined treatment with G3139 and dacarbazine, the most effective chemotherapeutic drug for human melanoma, resulted in complete and specific ablation of the tumor (89). The mice (six per group) were injected subcutaneously with 2.5 × 107 518A2 melanoma cells on d 0; on d 7 after cell inoculation, treatment was initiated with G3139 alone (5 mg/kg/d for 14 d). Dacarbazine was administered on d 12–16 at a dosage of 80 mg/kg/d. The animals were evaluated 21 d after initial cell inoculation. The mean tumor weights of untreated control mice, those treated with dacarbazine alone, and those treated with reverse control or two-base Bcl-2 antisense mismatched oligonucleotide plus drug were 0.44, 1.53, 0.39, and 0.41 g, respectively. In contrast, combination treatment of G3139 and dacarbazine resulted in complete ablation of the tumor in three of six animals (mean tumor weight of 0.02 g). No tumors could be detected in a repeat experiment after combined G3139/ dacarbazine treatment. Extensive studies of G3139 in prostate-cancer models have also been performed. In vitro treatment of Shionogi tumor cells with a mouse Bcl-2 antisense oligonucleotide in combination with docetaxel decreased the concentration of drug that reduced cell viability by 50% from 100 nM to 10 nM. Cellular apoptosis, as characterized by DNA laddering and cleavage of PARP protein (90,91), was also observed. Furthermore, combined administration of the Bcl-2 antisense oligonucleotide and polymeric micellar paclitaxel in male DD/S mice bearing Shionogi tumors delayed progression to androgen independence compared with either agent alone, and synergistically induced Shionogi tumor regression and growth inhibition. In these experiments, male mice were castrated 2 to 3 wk after tumor implantation, at which time tumors were 1–2 cm in diameter. Beginning 1 d postcastration, 12.5 mg/kg G3139 was injected intraperitoneally once a day

Chapter 9 / Chemotherapy and G3139

219

for 14 d. Beginning d 10 postcastration, 0.5 mg micellar paclitaxel was administered intravenously once daily for 5 d. By d 40 postcastration, androgen-independent tumors recurred in mice treated with G3139 alone or mismatched oligonucleotide plus paclitaxel after a median of 23 or 28 d, respectively. When mice were treated with a combination of G3139 plus drug, androgen-independent tumors recurred in three of six animals after a median of 37 d. The mean tumor volumes of the mice treated with G3139 plus paclitaxel were 91% lower than the tumor volumes of mice treated with G3139 alone, and 86% lower than the tumor volumes of mice treated with the mismatched control oligonucleotide plus drug. The authors also demonstrated an in vivo decrease in Bcl-2 mRNA and protein levels. Unfortunately, however, the number of animals in these experiments was too small to draw firm conclusions. However, a similar study in the LNCaP prostate tumor model demonstrated that treatment with G3139 markedly enhanced paclitaxel chemosensitivity in vitro, reducing cell viability after treatment with 1 nM paclitaxel from 76% to 42% (92). Adjuvant in vivo administration of combined G3139 and paclitaxel after castration of male athymic mice bearing LNCaP tumors resulted in a significant delay of the emergence of androgen-independent recurrent tumors compared with either agent alone. By 15 wk postcastration, tumor volumes in mice treated with either G3139 alone or with a mismatched, control oligonucleotide plus paclitaxel were more than threefold higher than in mice treated with combined therapy with G3139 and drug. In mice bearing androgen-dependent Shionogi tumors, downregulation of Bcl-2 expression by G3139 (500 nM) treatment enhanced by one log the IC50 of mitoxantrone-induced cell death. This occurred in a dose-dependent, sequencespecific manner when compared to mitoxantrone alone, or to a mismatched oligonucleotide plus drug (93). Treatment with G3139 of estrogen-receptor negative MDA435/LCC6 human breast cancer cells that were high Bcl-2 protein expressing, estrogen-receptor positive MCF-7, and low Bcl-2 expressing, produced more than an 80% reduction in Bcl-2 protein expression in a sequence-specific manner (94). Combined treatment with G3139 and doxorubicin, paclitaxel, or cisplatin resulted in additive cytotoxicity at low drug concentrations in both cell lines. However, under most conditions studied, the direct cytotoxic effect of G3139 was not synergistic with the cytotoxic agents. For example, when the degree of cell kill was normalized to the number of untreated controls, pretreatment of both cell lines with G3139 prior to 48-h exposure to doxorubicin (100 nM) led to significantly increased cytotoxicity of the combination. However, although this result demonstrates that combination of G3139 plus drug increases cytotoxicity vs the individual agents, this in itself does not necessarily demonstrate the existence of Bcl-2-dependent effects. Thus, when percentage cell kill with the combination was normalized to that of the cells treated with G3139 alone, no chemosensitization was observed.

220

Benimetskaya, Mani, and Stein

Synergistic tumor regression of human breast cancer MDA435/LCC6 xenografts in SCID mice was also demonstrated with combination therapies of G3139 and the P-glycoprotein inhibitor (PSC833) and liposomal doxorubicin (95). In another study, in the MDA435/LCC6 human xenograft solid-tumor model, the same authors demonstrated that synergistic effects of G3139 combined with chemotherapy were observed only under certain conditions (96). For example, free doxorubicin and G3139 were beneficial in small-volume, earlystage tumors, while G3139 and liposomal doxorubicin (encapsulated in small liposomes containing polyethylene glycol) was more effective in larger solid tumors (96). When administered in combination, G3139 (5 mg/kg intraperitoneally on d 17–21, 24–28, and 31–35) and liposomal doxorubicin (10 mg/kg intravenously on d 20, 27, and 34) resulted in d-35 tumor weights of 0.12 g, which were smaller than for equivalent doses of the individual treatments (0.21 g). By d 35 in the early-treatment model, combination G3139/doxorubicin-related tumor-growth inhibition was 48% relative to the control tumors, whereas inhibition was reduced by 22% in mice treated with free doxorubicin at 5 mg/kg alone. Lopes de Menezes et al. (97) also evaluated the pharmacokinetic (PK) properties of G3139 when combined with doxorubicin. When doxorubicin was co-administered intravenously with intraperitoneal G3139, the plasma doxorubicin PKs were relatively unaltered in the presence of G3139 in plasma. Changes in plasma G3139 PKs, but no substantial changes in the organ distribution of G3139, were observed in the presence of drug. For example, the G3139 Cmax was enhanced in the presence of doxorubicin (11.2 μg/mL compared to 6.9 μg/mL without drug). In the presence of drug, the volume of distribution increased to 7.7 mL (compared to 7.1 mL without drug). Although the presence of doxorubicin had a substantial effect on the t1/2 (0.8 h vs 0.2 h, G3139 with and without doxorubicin, respectively), the mean areas under the plasma concentration-time curve (AUC) were not significantly different (19.7 μg × h/mL in the presence of drug vs 17.4 μg × h/mL without drug). Vrignaud et al. (98) tested the effects of the combination of G3139 and docetaxel in a three-arm dose–response study in human NCI-H460 non-smallcell lung cancer xenografts. Swiss nude mice were treated with G3139 (subcutaneously twice a day from d 8 to 14 posttumor implantation), or docetaxel (intravenously on d 9, 12, and 15), or their combination. At the highest nontoxic dose tested (8 mg/kg per dose, total dose of 112 mg/kg), G3139 as a single agent was found active with a 1 log cell kill (log cell kill = tumor growth delay/3.32 × tumor doubling time). The highest nontoxic dose of docetaxel alone (20.8 mg/ kg per injection, total dose 60.9 mg/kg) was found to be active (2 logs cell kill). Significant antitumor activity was obtained at a nontoxic combination (5 mg/kg/ dose of G3139, 20.8 mg/kg/injection of docetaxel) with 3.1 logs of cell kill observed in four of seven mice. This combination was well tolerated, with the

Chapter 9 / Chemotherapy and G3139

221

only observed side effect being reversible edema that occurred at the site of the subcutaneous injection. A study performed by Zangemeister-Wittke and colleagues (99) analyzed the effects of combined treatment of the Bcl-2 antisense oligonucleotide 2009 (targeted to Bcl-2 mRNA codons 141–147) and etoposide, doxorubicin, and cisplatin on small-cell lung cancer cell lines expressing high (NCII-H69), intermediatehigh (SW2), and low (NCI-H82) levels of Bcl-2. In these experiments, cells were treated with serial dilutions of 2009 and drugs individually, and with fixed ratios of oligonucleotide and drugs simultaneously at doses at which viability was inhibited by 50% (IC50). For each level of cytotoxicity, a combination index (CI) was calculated. Synergy was indicated by a CI less than 1, additivity by a CI equal to 1, and antagonism by a CI greater than 1. In the NCII-H69 and SW2 cells, all combinations resulted in synergistic cytotoxicity. For example, when NCII-H69 cells were exposed to a 1:330 ratio of 2009 and etoposide, the CI values of the interaction were approx 0.2 at the IC10 and approx 0.9 at the IC90 of the combination. When SW2 cells were exposed to fixed ratios of 2009 and etoposide (1:330), doxorubicin (1:1), or cisplatin (1:83) at doses that corresponded to the IC50 values, the cytotoxic effects of the combinations were always greater than those observed with the single agents alone. For example, the CI of the treatment with 2009 and etoposide, doxorubicin, and cisplatin were approx 0.6, 0.6, and 0.83, respectively. However, in the NCI-H82 cells, most of combinations were slightly antagonistic (CI > 1). Synergistic cytotoxity was obtained only if 2009 was combined with cisplatin (1:43) at the IC10 and IC50 of the combination, with CI values of approx 0.72 and 0.95, respectively. In a human gastric cancer–SCID mouse xenotransplantation model, the combined treatment with G3139 (10 mg/kg/d) plus an intraperitoneal bolus of 9 mg/ kg cisplatin on d 7 led to a dramatic reduction in tumor volume compared to control treatment. After 14 d of continuous infusion of G3139 and single-bolus administration of cisplatin on d 7, mice showed more than 70% reduction in mean tumor volume (0.19 cm3) compared to mice treated with cisplatin alone (0.71 cm3) or treated with cisplatin plus mismatched control oligonucleotide (0.67 cm3). The combination with cisplatin markedly enhanced the antitumor effect of the drug (70% tumor size reduction compared to drug alone), and prolonged survival compared with drug alone or oligonucleotide alone by more than 50%, without adding significant drug-related toxicity (100,101). To evaluate the effects of variable Bcl-2 expression levels on chemosensitivity in the human hepatocellular QGYY-7703 carcinoma cell line, Bcl-2 expression was modulated via sense and antisense gene transfection approaches (102). These transfected cells were evaluated for chemosensitivity to taxol and doxorubicin. The authors demonstrated that cells with reduced Bcl-2 protein levels became more sensitive to the drugs than control cells with elevated Bcl-2 levels.

222

Benimetskaya, Mani, and Stein

Xu-Bao Shi et al. (103) also employed an antisense gene transfection approach, and demonstrated that downregulation of Bcl-2 protein could sensitize LNCaP prostate cancer cells to adriamycin, which exerts its cytotoxic action by damaging DNA through inhibition of topoisomerase II and the formation of free radicals.

ANTISENSE BCL-2 OLIGONUCLEOTIDE IN THE CLINIC The clinical development of antisense oligonucleotides (see Table 1) has moved forward considerably since the introduction of the first antisense drug (formivirsen) approved by the US Food and Drug Administration for the treatment of cytomegalovirus-induced retinitis in patients with AIDS. Despite the mode of administration of formivirsen by direct intravitreal injection, the proofof-principle of clinical benefit with antisense oligonucleotides shows that other well chosen targets for antisense inhibition may also be feasible. Oligonucleotides are relatively devoid of side effects. However, there are dose-dependent side effects that require careful vigilance of patients on therapy. These include febrility (flu-like illness), putatively from cytokine (e.g., interleukin [IL]-6) induction, asthenia, alterations in coagulation parameters (e.g., prolongation of thromboplastin time and activation of the complement cascade), secondary to oligonucleotide binding and the stabilization of the ternary coagulation complex, thrombocytopenia, hypotension, and elevations in transaminases, suggesting liver dysfunction.

G3139 (GENASENSE) The bcl-2 gene product provides a rational target for antisense strategies, because overexpression leads to cellular resistance to apoptosis in vitro. Bcl-2 is overexpressed in the majority of low-grade and approx 50% of high-grade nonHodgkin’s lymphomas (NHL) as well as a number of solid tumors (>90% in melanomas). A number of different molecular mechanisms are responsible for the upregulation of the Bcl-2 protein, the most common being a translocation between chromosomes 14 and 18 that brings the bcl-2 gene under the transcriptional control of the immunoglobulin heavy-chain promoter. There is evidence for an etiologic role of Bcl-2 overexpression in lymphoma. Transgenic mice with deregulated Bcl-2 expression initially develop lymphoid hyperplasia with extended B-cell survival. In some cases, this progresses to diffuse large B-cell lymphoma, often with the accumulation of additional genetic abnormalities, such as rearrangement of the c-myc gene. Three recent studies have examined the prognostic significance of Bcl-2 expression in diffuse large B-cell NHL. In multivariate analyses, Bcl-2 expression was found to be an independent poor prognostic factor in these patients. Antisense oligonucleotides targeting bcl-2 have been demonstrated to reduce bcl-2 mRNA and protein levels in vitro and to reverse chemoresistance of Bcl-

14

35 26

(104)

(106) (105)

Predominant tumor

0.6–6.5 mg/kd/d CIV 14d Stage IV Melanoma plus DTIC q21 d 0.6–6.9 mg/kg/d CIV 14 or 21d Stage IV Prostate 0.6 to 5.0 mg/kg/d CIV 14 d Stage IV prostate plus mitoxantrone 4–12 mg/m2 on d8 q 28 d

Dose/Schedule Bcl2 in melanoma tissue Apoptosis Bcl2 Protein Expression Bcl2 in PBLs ;PSA

Biologic endpoints

6.9 mg/kg/db NDc

NDa

MTD/toxicities

Dose-dependent transient liver function abnormalities; reversible aPTT changes; few episodes of cytokine induced symptoms. MTD, maximum tolerated dose. aND= not described but full doses of ASO delivered without dose-limiting effects bTransient liver function abnormalities, clinically inconsequential lymphopenia and fatigue. cND = not described, but doses up to 5.0 mg/kg/d tolerable with mitoxantrone; predominant side effects include fatigue and nausea. Liver function abnormalities and aPTT changes were infrequent.

N

Ref

Table 1

Chapter 9 / Chemotherapy and G3139 223

224

Benimetskaya, Mani, and Stein

2-expressing lymphoma cell lines. It has also been demonstrated that eradication of lymphoma can be achieved in a severe combined immune deficient mouse model of human NHL by a 14-d subcutaneous infusion of G3139. These observations provided the rationale for a phase I trial of antisense oligonucleotide G3139 in NHL patients. Twenty-one patients with Bcl-2-positive relapsed NHL received a 14-d subcutaneous infusion of G3139. Plasma pharmacokinetics were measured by anion exchange high-performance liquid chromatography. Response was assessed by computed tomography. Changes in Bcl-2 expression were measured by fluorescence-activated cell sorting of patients’ tumor samples. Eight cohorts of patients received doses between 4.6 and 195.8 mg/m2/d. No significant systemic toxicity was seen at doses up to 110.4 mg/m2/d. All patients displayed skin inflammation at the subcutaneous infusion site. Dose-limiting toxicities were thrombocytopenia, hypotension, fever, and asthenia. The maximum tolerated dose was 147.2 mg/m2/d. Plasma levels of G3139 equivalent to the efficacious plasma concentration in in vivo models were produced with doses above 36.8 mg/m2/d. Plasma levels associated with dose-limiting toxicity were greater than 4 μg/mL. By standard criteria, there was one complete response, two minor responses, nine cases of stable disease, and nine cases of progressive disease. Bcl-2 protein was reduced in 7 of 16 assessable patients. This reduction occurred in tumor cells derived from lymph nodes in two patients and from peripheral blood or bonemarrow mononuclear cell populations in the remaining five patients (104). The use of antisense techniques has led to some spectacular successes in the laboratory; however, many basic investigators have found it difficult to routinely modify gene expression in living cells with antisense molecules. Of interest, in the Waters article (104), clinical outcomes were not correlated with the dose of oligonucleotide delivered or with the degree of downregulation of the targeted protein. The latter is a critical measurement, because it is the one objective assessment of whether an antisense effect had been achieved. Data presented in the article reveal that expression of the targeted gene’s protein, Bcl-2, was diminished in 5 of 16 patients in whom this was measured. This is encouraging, but it must be pointed out that mean inhibition was only approx 24%, a result of uncertain biologic significance. Further, it cannot be stated with certainty that this small decline in Bcl-2 expression was specific, because the oligonucleotide’s effect on the expression of other proteins was not reported. Nevertheless, prompted by individual responses, other phase I studies have been published in diseases with high expression rates of Bcl-2, including melanomas (105), prostate cancer (106), and other advanced solid tumors (107) (Table 1). Based on the responses observed with these phase I studies, G3139 is in phase III trials for malignant melanoma, for which it has been awarded fast track status. Genasense received orphan drug status in August 2000. In September 2000, the company announced that pivotal phase III trials in multiple melanoma, chronic

Chapter 9 / Chemotherapy and G3139

225

lymphocytic leukemia (CLL), and acute myelocytic leukemia (AML) were underway by 2001. By January 2001, trials in AML and CLL had been initiated. A phase III trial in patients with advanced multiple myeloma at 65 centers in the United States, Canada, and Great Britain began in February 2001. The trial will examine whether the addition of Genasense can improve response rates, response duration, and quality of life compared with dexamethasone therapy alone.

UNIQUE ASPECTS OF G3139 METABOLISM/PHARMACOLOGY AND DRUG–DRUG INTERACTIONS The majority of Bcl-2 antisense oligonucleotide is plasma protein bound with little lipoprotein association and no significant movement between different lipoprotein and lipoprotein-deficient fractions in plasma. Plasma protein binding other than lipoprotein binding may be responsible for the difference in cellular uptake of free antisense as opposed to cationic lipoplexes (108). These data would impact design of trials with other drugs in combination that may be affected by protein binding. G3139 may be safely delivered with cytotoxic drugs like doxorubicin (or liposomal doxorubicin), as there are modest PK–PD interactions between the two agents. In fact, the combination is favorable in that elevated tumor DOX levels are achieved without compromising G3139 tumor uptake, or significantly altering plasma drug concentrations (96,97). Although antisense bcl-2 may sensitize cells to chemotherapy, the sensitization pathways are complex and likely would involve more than just bcl-2 as a target (109). This is demonstrated in another study in breast-cancer cell lines in which direct cytotoxic activity of G3139 antisense was not synergistic with several cytotoxic agents. These results suggest that while Bcl-2 clearly constitutes an attractive therapeutic target due to its role in regulating apoptosis in breast cancer cells, additional mechanisms are important in the control of apoptosis arising from exposure to anticancer agents in vitro (94).

REFERENCES 1. Vaux DL, Strasser A. The molecular biology of apoptosis. Proc Natl Acad Sci USA 1996;93: 2239–2244. 2. Krammer PH. CD95(APO-1/Fas)-mediated apoptosis: live and let die. Adv Immunol 1999;71:163–210. 3. Vaux DL, Korsmeyer SJ. Cell death in development. Cell 1999;96:245–254. 4. Raff MC. Social controls on cell survival and cell death. Nature 1992;356:397–400. 5. Cleary ML, Sklar J. Nucleotide sequence of a t(14;18) chromosomal breakpoint in follicular lymphoma and demonstration of a breakpoint-cluster region near a transcriptionally active locus on chromosome 18. Proc Natl Acad Sci USA 1985;82:7439–7443. 6. Bakhshi A, Jensen JP, Goldman P, et al. Cloning the chromosomal breakpoint of t(14;18) human lymphomas: clustering around JH on chromosome 14 and near a transcriptional unit on 18. Cell 1985;41:899–906. 7. Strasser A, O’Connor L, Dixit VM. Apoptosis signaling. Annu Rev Biochem 2000;69: 217–245.

226

Benimetskaya, Mani, and Stein

8. Gross A, McDonnell JM, Korsmeyer SJ. BCL-2 family members and the mitochondria in apoptosis. Genes Dev 1999;13:1899–1911. 9. Tsujimoto Y, and Shimizu S. Bcl-2 family: life-or-death switch. FEBS Lett 2000;466:6–10. 10. McDonnell TJ, Deane N, Platt FM, et al. bcl-2-immunoglobulin transgenic mice demonstrate extended B cell survival and follicular lymphoproliferation. Cell 1989;57:79–88. 11. Vaux DL, Cory S, and Adams JM. Bcl-2 gene promotes haemopoietic cell survival and cooperates with c- myc to immortalize pre-B cells. Nature 1988;335:440–502. 12. Kerr JF, Wyllie AH, Currie AR. Apoptosis: a basic biological phenomenon with wideranging implications in tissue kinetics. Br J Cancer 1972;26:239–257. 13. Kroemer G. The proto-oncogene Bcl-2 and its role in regulating apoptosis. Nat Med 1997;3:614–620. 14. de Jong D, Prins FA, Mason DY, et al. Subcellular localization of the bcl-2 protein in malignant and normal lymphoid cells. Cancer Res 1994;54:256–260. 15. Schlesinger PH, Gross A, Yin XM, et al. Comparison of the ion channel characteristics of proapoptotic BAX and antiapoptotic BCL-2. Proc Natl Acad Sci USA 1997;94:11357– 11362. 16. Oltvai ZN, Milliman CL, Korsmeyer SJ. Bcl-2 heterodimerizes in vivo with a conserved homolog, Bax, that accelerates programmed cell death. Cell 1993;74:609–619. 17. Sato T, Hanada M, Bodrug S, et al. Interactions among members of the Bcl-2 protein family analyzed with a yeast two-hybrid system. Proc Natl Acad Sci USA 1994;91:9238–9242. 18. Yin XM, Oltvai ZN, Korsmeyer SJ. BH1 and BH2 domains of Bcl-2 are required for inhibition of apoptosis and heterodimerization with Bax. Nature 1994;369:321–323. 19. Chittenden T, Flemington C, Houghton AB, et al. A conserved domain in Bak, distinct from BH1 and BH2, mediates cell death and protein binding functions. EMBO J 1995;14:5589–5596. 20. Zha H, Aime-Sempe C, Sato T, et al. Proapoptotic protein Bax heterodimerizes with Bcl-2 and homodimerizes with Bax via a novel domain (BH3) distinct from BH1 and BH2. J Biol Chem 1996;271:7440–7444. 21. Ficazzola MA, Taneja SS. Prospects for gene therapy in human prostate cancer. Mol Med Today 1998;4:494–504. 22. Tonkinson JL, and Stein CA. Antisense oligodeoxynucleotides as clinical therapeutic agents. Cancer Invest 1996;14:54–65. 23. Bower M, Waxman J. Gene therapy for prostate cancer. Semin Cancer Biol 1997;8:3–9. 24. Hrouda D, Dalgleish AG. Gene therapy for prostate cancer. Gene Ther 1996;3:845–852. 25. Harrington KJ, Spitzweg C, Bateman AR, et al. Gene therapy for prostate cancer: current status and future prospects. J Urol 2001;166:1220–1233. 26. Kuss B, Cotter F. Antisense—time to shoot the messenger. Ann Oncol 1999;10:495–503. 27. Galderisi U, Cascino A, and Giordano A. Antisense oligonucleotides as therapeutic agents. J Cell Physiol 1999;181:251–257. 28. Wraight CJ, White PJ. Antisense oligonucleotides in cutaneous therapy. Pharmacol Ther 2001;90:89–104. 29. Crooke ST. Potential roles of antisense technology in cancer chemotherapy. Oncogene 2000;19:6651–6659. 30. Khuri FR, Kurie JM. Antisense approaches enter the clinic. Clin Cancer Res 2000;6: 1607–1610. 31. Lebedeva I, Stein CA. Antisense oligonucleotides: promise and reality. Annu Rev Pharmacol Toxicol 2001;41:403–419. 32. Miyake H, Hara I, Kamidono S, et al. Novel therapeutic strategy for advanced prostate cancer using antisense oligodeoxynucleotides targeting anti-apoptotic genes upregulated after androgen withdrawal to delay androgen-independent progression and enhance chemosensitivity. Int J Urol 2001;8:337–349.

Chapter 9 / Chemotherapy and G3139

227

33. Morris MJ, Scher HI. Novel strategies and therapeutics for the treatment of prostate carcinoma. Cancer 2000;89:1329–1348. 34. Stein CA, Cheng YC. Antisense oligonucleotides as therapeutic agents—is the bullet really magical? Science 1993;261:1004–1012. 35. Wagner RW, Flanagan WM. Antisense technology and prospects for therapy of viral infections and cancer. Mol Med Today 1997;3:31–38. 36. Nellen W, Lichtenstein C. What makes an mRNA anti-sense-itive? Trends Biochem Sci 1993;18:419–423. 37. Marshall P, Thompson JB, Eckstein F. Inhibition of gene expression with ribozymes. Cell Mol Neurobiol 1994;14:5523–5538. 38. Belikova AM, Zarytova VF, Grineva NI. Synthesis of ribonucleosides and diribonucleo-side phosphates containing 2-chloroethylamine and nitrogen mustard residues. Tetrahedron Lett 1967;37:3557–3562. 39. Mizuno T, Chou MY, Inouye M. A unique mechanism regulating gene expression: translational inhibition by a complementary RNA transcript (micRNA). Proc Natl Acad Sci USA 1984;81:1966–1970. 40. Zamecnik PC, Stephenson ML. Inhibition of Rous sarcoma virus replication and cell transformation by a specific oligodeoxynucleotide. Proc Natl Acad Sci USA 1978;75:280–284. 41. Stephenson ML, Zamecnik PC. Inhibition of Rous sarcoma viral RNA translation by a specific oligodeoxyribonucleotide. Proc Natl Acad Sci USA 1978;75:285–288. 42. Eder PS, DeVine RJ, Dagle JM, et al. Substrate specificity and kinetics of degradation of antisense oligonucleotides by a 3' exonuclease in plasma. Antisense Res Dev 1991;1:141–151. 43. Stec W, Zon G, Egan V. Automated solid-phase synthesis, separation and stereochemistry of phosphorothioate analoges of oligodeoxyribonucleotides. J Amer Chem Soc 1984;106: 6077–6079. 44. Stein CA, Subasinghe C, Shinozuka K, et al. Physicochemical properties of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 1988;16:3209–3221. 45. Crooke ST. Therapeutic applications of oligonucleotides. Annu Rev Pharmacol Toxicol 1992;32:329–376. 46. Stein CA, Tonkinson JL, Yakubov L. Phosphorothioate oligodeoxynucleotides—anti-sense inhibitors of gene expression? Pharmacol Ther 1991;52:365–384. 47. Miller PS, Ts’o PO. A new approach to chemotherapy based on molecular biology and nucleic acid chemistry: Matagen (masking tape for gene expression). Anticancer Drug Des 1987;2:117–128. 48. Summerton J, Weller D. Morpholino antisense oligomers: design, preparation, and properties. Antisense Nucleic Acid Drug Dev 1997;7:187–195. 49. Nielsen PE, Egholm M, Berg RH, et al. Sequence-selective recognition of DNA by strand displacement with a thymine-substituted polyamide. Science 1991;254:1497–1500. 50. Dash P, Lotan I, Knapp M, et al. Selective elimination of mRNAs in vivo: complementary oligodeoxynucleotides promote RNA degradation by an RNase H-like activity. Proc Natl Acad Sci USA 1987;84:7896–7900. 51. Ma M, Benimetskaya L, Lebedeva I, et al. Intracellular mRNA cleavage induced through activation of RNase P by nuclease-resistant external guide sequences. Nat Biotechnol 2000;18:58–61. 52. Wu H, MacLeod AR, Lima WF, et al. Identification and partial purification of human double strand RNase activity. A novel terminating mechanism for oligoribonucleotide antisense drugs. J Biol Chem 1998;273:2532–2542. 53. Torrence PF, Xiao W, Li G, et al. Recruiting the 2-5A system for antisense therapeutics. Antisense Nucleic Acid Drug Dev 1997;7:203–206. 54. Dominski Z, Kole R. Restoration of correct splicing in thalassemic pre-mRNA by antisense oligonucleotides. Proc Natl Acad Sci USA 1993;90:8673–8677.

228

Benimetskaya, Mani, and Stein

55. McManaway ME, Neckers LM, Loke SL, et al. Tumour-specific inhibition of lymphoma growth by an antisense oligodeoxynucleotide. Lancet 1990;335:808–811. 56. Agrawal S, Goodchild J, Civeira MP, et al. Oligodeoxynucleoside phosphoramidates and phosphorothioates as inhibitors of human immunodeficiency virus. Proc Natl Acad Sci USA 1988;85:7079–7083. 57. Westermann P, Gross B, Hoinkis G. Inhibition of expression of SV40 virus large T-antigen by antisense oligodeoxyribonucleotides. Biomed Biochim Acta 1989;48:85–93. 58. Baker BF, Miraglia L, Hagedorn CH. Modulation of eucaryotic initiation factor-4E binding to 5'-capped oligoribonucleotides by modified anti-sense oligonucleotides. J Biol Chem 1992;267:11,495–11,499. 59. Itaya M, Kondo K. Molecular cloning of a ribonuclease H (RNase HI) gene from an extreme thermophile Thermus thermophilus HB8: a thermostable RNase H can functionally replace the Escherichia coli enzyme in vivo. Nucleic Acids Res 1991;19:4443–4449. 60. Crooke ST, Lemonidis KM, Neilson L, et al. Kinetic characteristics of Escherichia coli RNase H1: cleavage of various antisense oligonucleotide-RNA duplexes. Biochem J 1995;312:599–608. 61. Guvakova MA, Yakubov LA, Vlodavsky I, et al. Phosphorothioate oligodeoxynucleotides bind to basic fibroblast growth factor, inhibit its binding to cell surface receptors, and remove it from low affinity binding sites on extracellular matrix. J Biol Chem 1995;270:2620–2627. 62. Fennewald SM, Rando RF. Inhibition of high affinity basic fibroblast growth factor binding by oligonucleotides. J Biol Chem 1995;270:21,718–21,721. 63. Rockwell P, O’Connor WJ, King K, et al. Cell-surface perturbations of the epidermal growth factor and vascular endothelial growth factor receptors by phosphorothioate oligodeoxynucleotides. Proc Natl Acad Sci USA 1997;94:6523–6528. 64. Yakubov L, Khaled Z, Zhang LM, et al. Oligodeoxynucleotides interact with recombinant CD4 at multiple sites. J Biol Chem 1993;268:18,818–18,823. 65. Dorai T, Goluboff ET, Olsson CA, et al. Development of a hammerhead ribozyme against BCL-2. II. Ribozyme treatment sensitizes hormone-resistant prostate cancer cells to apoptotic agents. Anticancer Res 1997;17:3307–3312. 66. Benimetskaya L, Loike JD, Khaled Z, et al. Mac-1 (CD11b/CD18) is an oligodeoxynucleotide-binding protein. Nat Med 1997;3:414–420. 67. Khaled Z, Benimetskaya L, Zeltser R, et al. Multiple mechanisms may contribute to the cellular anti-adhesive effects of phosphorothioate oligodeoxynucleotides. Nucleic Acids Res 1996;24:737–745. 68. Krieg AM, Yi AK, Matson S, et al. CpG motifs in bacterial DNA trigger direct B-cell activation. Nature 1995;374:546–549. 69. Ballas ZK, Rasmussen WL, Krieg AM. Induction of NK activity in murine and human cells by CpG motifs in oligodeoxynucleotides and bacterial DNA. J Immunol 1996;157:1840–1845. 70. Wang Y, Patel DJ. Solution structure of a parallel-stranded G-quadruplex DNA. J Mol Biol 1993;234:1171–1183. 71. Sen D, Gilbert W. Novel DNA superstructures formed by telomere-like oligomers. Biochemistry 1992;31:65–70. 72. Wyatt JR, Stein CA. G-quartet inhibitory effects of phosphorothioate oligonucleotides. In: (Rabbani R, ed). Applications of antisense therapies to restenosis. Kluwer Academic, Norwell, MA: 1999. 73. Vilenchik M, Benimetsky L, Kolbanovsky A, et al. Evidence for higher-order structure formation by the c-myb 18-mer phosphorothioate antisense (codons 2–7) oligodeoxynucleotide: potential relationship to antisense c-myb inhibition. Antisense Nucleic Acid Drug Dev 2001;11:87–97. 74. Wagner RW, Matteucci MD, Grant D, et al. Potent and selective inhibition of gene expression by an antisense heptanucleotide. Nat Biotechnol 1996;14:840–844.

Chapter 9 / Chemotherapy and G3139

229

75. Stein CA. Is irrelevant cleavage the price of antisense efficacy? Pharmacol Ther 2000;85:231–236. 76. Benimetskaya L, Takle GB, Vilenchik M, et al. Cationic porphyrins: novel delivery vehicles for antisense oligodeoxynucleotides. Nucleic Acids Res 1998;26:5310–5317. 77. Kitada S, Takayama S, De Riel K, et al. Reversal of chemoresistance of lymphoma cells by antisense-mediated reduction of bcl-2 gene expression. Antisense Res Dev 1994;4:71–79. 78. Kitada S, Miyashita T, Tanaka S, et al. Investigations of antisense oligonucleotides targeted against bcl-2 RNAs. Antisense Res Dev 1993;3:157–169. 79. Cotter FE, Johnson P, Hall P, et al. Antisense oligonucleotides suppress B-cell lymphoma growth in a SCID-hu mouse model. Oncogene 1994;9:3049–3055. 80. Gleave M, Tolcher A, Miyake H, et al. Progression to androgen independence is delayed by adjuvant treatment with antisense Bcl-2 oligodeoxynucleotides after castration in the LNCaP prostate tumor model. Clin Cancer Res 1999;5:2891–2898. 81. Miyake H, Tolcher A, Gleave ME. Antisense Bcl-2 oligodeoxynucleotides inhibit progression to androgen-independence after castration in the Shionogi tumor model. Cancer Res 1999;59:4030–4034. 82. Miyake H, Tolcher A, Gleave ME. Chemosensitization and delayed androgen-independent recurrence of prostate cancer with the use of antisense Bcl-2 oligodeoxynucleotides. J. Natl. Cancer Inst. 1999;92:34–41. 83. Zangemeister-Wittke U, Leech SH, Olie RA, et al. A novel bispecific antisense oligonucleotide inhibiting both bcl-2 and bcl-xL expression efficiently induces apoptosis in tumor cells. Clin Cancer Res 2000;6:2547–2555. 84. Klasa RJ, Bally MB, Ng R, et al. Eradication of human non-Hodgkin’s lymphoma in SCID mice by BCL-2 antisense oligonucleotides combined with low-dose cyclophosphamide. Clin Cancer Res 2000;6:2492–2500. 85. Lacy J, Loomis R. Bcl-2 antisense (G3139, Genasense) enhances the in vitro and in vivo response of EBV-associated lymphoproliferative disease to rituximab. Proc Am Assoc Cancer Res 2002;43:575 [Abstract 2853]. 86. Tauchi T, Nakajima A, Sumi M, et al. G3139 (Bcl-2 antisense oligonucleotide) is active against Glleevec-resistant Bcr-Abl-positive cells. Proc Am Assoc Cancer Res 2002;43:949 [Abstract 4702]. 87. Gazitt Y, Liu QY, Vesole D. Bcl-2 antisense oligonucleotides (ASO) enhances apoptosis and cytotoxicity in drug-resistant myeloma cells. Blood 2001;98:641a [Abstract 2688]. 88. Konopleva M, Tari AM, Estrov Z, et al. Liposomal Bcl-2 antisense oligonucleotides enhance proliferation, sensitize acute myeloid leukemia to cytosine-arabinoside, and induce apoptosis independent of other antiapoptotic proteins. Blood 2000;95:3929–3938. 89. Jansen B, Schlagbauer-Wadl H, Brown BD, et al. bcl-2 antisense therapy chemosensitizes human melanoma in SCID mice. Nat Med 1998;4:232–234. 90. Gleave ME, Miayake H, Goldie J, et al. Targeting bcl-2 gene to delay androgen-independent progression and enhance chemosensitivity in prostate cancer using antisense bcl-2 oligodeoxynucleotides. Urology 1999;54:36–46. 91. Miayake H, Tolcher A, Gleave ME. Chemosensitization and delayed androgen-independent recurrence of prostate cancer with the use of antisense Bcl-2 oligodeoxynucleotides. J Natl Cancer Inst 2000;92:34–41. 92. Leung S, Miyake H, Zellweger T, et al. Synergistic chemosensitization and inhibition of progression to androgen independence by antisense Bcl-2 oligodeoxynucleotide and paclitaxel in the LNCaP prostate tumor model. Int J Cancer 2001;91:846–850. 93. Tolcher A, Miayake H, Gleave M. Downregulation of Bcl-2 expression by antisense-oligonucleotide (AS-ODN) treatment enhanced mitoxantrone cytotoxicity in the androgen-dependent Shionogi tumor model. Proc Am Assoc Cancer Res 1999;40:484 [Abstract 3198].

230

Benimetskaya, Mani, and Stein

94. Chi KC, Wallis AE, Lee CH, et al. Effects of Bcl-2 modulation with G3139 antisense oligonucleotide on human breast cancer cells are independent of inherent Bcl-2 protein expression. Breast Cancer Res Treat 2000;63:199–212. 95. Lopes de Menezes DE, Mayer LD. Combination of Bcl-2 antisense oligodeoxynucleotide (3139), p-glycoprotein inhibitor (PSC833) and liposomal doxorubicin can supress the growth of drug-resistant human breast cancer xenografts in SCID mice. Proc Am Assoc Cancer Res 2001;42:375 [Abstract 2018]. 96. Lopes de Menezes DE, Hudon N, McIntosh N, et al. Molecular and pharmacokinetic properties associated with the therapeutics of bcl-2 antisense oligonucleotide G3139 combined with free and liposomal doxorubicin. Clin Cancer Res 2000;6:2891–2902. 97. Lopes de Menezes DE, Mayer LD. Pharmacokinetics of Bcl-2 antisense oligonucleotide (3139) combined with doxorubicin in SCID mice bearing human breast cancer solid tumor xenografts. Cancer Chemother Pharmacol 2002;49:57–68. 98. Vrignaud P, Lejeune P, Klem RE, et al. Combination of G3139 (Genasense), a Bcl-2 antisense oligonucleotide, with docetaxel (Taxotere) is active in a murine xenograft model of human non-small cell lung cancer. Proc Am Assoc Cancer Res 2002;43:578 [Abstract 2865]. 99. Zangemeister-Wittke U, Schenker T, Luedke GH, et al. Synergistic cytotoxicity of bcl-2 antisense oligodeoxynucleotides and etoposide, doxorubicin and cisplatin on small-cell lung cancer cell lines. Br J Cancer 1998;78:1035–1042. 100. Wacheck V, Heere-Ress E, Halaschek-Wiener J, et al. Bcl-2 antisense oligonucleotides chemosensitize human gastric cancer in a SCID mouse xenotransplantation model. J Mol Med 2001;79:587–593. 101. Wacheck V, Heere-Ress E, Halaschek-Wiener J, et al. Bcl-2 antisense therapy prolongs survival of gastric cancer in a SCID mice. Proc Am Assoc Cancer Res 2001;43:848 [Abstract 4552]. 102. Luo D, Cheng SC, Xie H, et al. Chemosensitivity of human hepatocellular carcinoma cell line QGY-7703 is related to bcl-2 protein levels. Tumour Biol 1999;20:331–340. 103. Shi XB, Gumerlock PH, Muenzer JT, et al. BCL2 antisense transcripts decrease intracellular Bcl2 expression and sensitize LNCaP prostate cancer cells to apoptosis-inducing agents. Cancer Biother Radiopharm 2001;16:421–429. 104. Waters JS, Webb A, Cunningham D, et al. Phase I clinical and pharmacokinetic study of bcl2 antisense oligonucleotide therapy in patients with non-Hodgkin’s lymphoma. J Clin Oncol 2000;18:1812–1823. 105. Jansen B, Wacheck V, Heere-Ress E, et al. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet 2000;356:1728–1733. 106. Chi KN, Gleave ME, Klasa R, et al. A phase I dose-finding study of combined treatment with an antisense Bcl-2 oligonucleotide (Genasense) and mitoxantrone in patients with metastatic hormone-refractory prostate cancer. Clin Cancer Res 2001;7:3920–3927. 107. Morris MJ, Tong WP, Cordon-Cardo C, et al. Phase I Trial of BCL-2 Antisense Oligonucleotide (G3139) Administered by Continuous Intravenous Infusion in Patients with Advanced Cancer. Clin Cancer Res 2002;8:679–683. 108. Wasan EK, Waterhouse D, Sivak O, et al. Plasma protein binding, lipoprotein distribution and uptake of free and lipid-associated BCL-2 antisense oligodeoxynucleotides (G3139) in human melanoma cells. Int J Pharm 2002;241:57–64. 109. Benimetskaya L, Miller P, Benimetsky S, et al. Inhibition of potentially anti-apoptotic proteins by antisense protein kinase C-alpha (Isis 3521) and antisense bcl-2 (G3139) phosphorothioate oligodeoxynucleotides: relationship to the decreased viability of T24 bladder and PC3 prostate cancer cells. Mol Pharmacol 2001;60:1296–1307.

Chapter 10 / Signal Transduction Inhibitor Evaluation

10

231

Use of Animal Models to Evaluate Signal Transduction Inhibitors As Modulators of Cytotoxic Therapy Beverly A. Teicher, PhD CONTENTS INTRODUCTION RECEPTOR TYROSINE KINASES PROTEIN SERINE-THREONINE KINASE INHIBITORS INHIBITORS OF CELL-CYCLE SIGNAL TRANSDUCTION

INTRODUCTION Cancer cells survive and thrive because they are very similar to normal cells expressing growth and invasion “programs” that many cell types recognize and respond to in “programmed” patterns (1). As the understanding of cancer has increased, the complexity of the molecular events that comprise malignant disease has become evident (2). Interactions involved in intertwining signaling networks including membrane receptors, enzymes along with activators, deactivators and regulators, protein–protein interactions, protein–nucleic acid interactions, and small-molecule effectors in multiple cell types are all recognized targets for therapeutic attack. Agents are targeted to specific abnormalities in the sequence and expression of genes/proteins that operate in a stepwise, combinatorial manner to permit the malignant disease to progress (3). Cell growth, motility, differentiation, and death are regulated by signals received from the environment (4). Signals may come from interactions with other cells or components of the extracellular matrix, or from binding of soluble signaling molecules to specific receptors at the cell membrane, thereby initiating different signaling pathways inside of the cell. Cancer may be visualized as a permanent perturbaFrom: Cancer Drug Discovery and Development: Combination Cancer Therapy: Modulators and Potentiators Edited by: G. K. Schwartz © Humana Press Inc., Totowa, NJ

231

232

Teicher

tion of signaling pathways. Over the last 10 yr, numerous compounds have been rationally targeted toward components in signaling pathways shown to be abnormal in cancer (5). Some of these rationally targeted agents have progressed through clinical trials, and some are now approved clinical treatment agents. Genomics and proteomics technologies have been applied to many cancers to identify markers of the disease and to elucidate the intracellular and intercellular signaling pathways that support the malignant disease (6–14). Cancer cure requires eradication of all malignant cells. Cancer growth, however, requires proliferation of malignant cells and normal cells. The several anticancer treatment modalities currently available, including surgery, chemotherapy, radiation therapy, and immunotherapy, have been envisioned to target primarily the malignant cells. Research over the past 35 yr has reinforced the hypothesis put forth by Folkman that, without the proliferation of normal cells, especially endothelial cells, a tumor cannot grow beyond the size of a colony (15). The consequence of this finding is that both the normal cells and the malignant cells involved in tumor growth, as well as the chemical and mechanical signaling pathways that interconnect them, are valid targets for therapeutic intervention. The integration of therapeutics directed toward the vascular components, extracellular matrix components, and stromal and infiltrating cells, with classical cytotoxic anticancer therapies, may be regarded as a systems approach to cancer treatment (16). Although new noncytotoxic agents directed toward normal cells and extracellular enzymatic activities target processes critical to tumor growth, it is highly unlikely that treatment with these new agents alone will lead to tumor cure. The question arises of how to integrate these new therapeutic agents into existing cancer treatment regimens. Thus, by choosing multiple cellular and process targets for therapeutic attack, a systems approach to anticancer therapy regimen development may lead to the cure of systemic malignant disease (17).

RECEPTOR TYROSINE KINASES In complex multicellular organisms, intercellular signal transduction networks mediating growth, differentiation, migration, and death are regulated, in part, by polypeptide growth factors that activate cell-surface receptors acting in either an autocrine or paracrine manner (18). Receptor tyrosine kinases (RTKs) are key mediators of many normal cellular processes and are often deregulated in human cancers. Several signaling pathways controlled by tyrosine kinases have been selected as important targets for anticancer therapeutic intervention. The epidermal growth factor receptor (EGFR) autocrine pathway contributes to several processes important to the deregulated invasion growth that is the hallmark of malignant disease (19). The family of ligands for EGFR include epidermal growth factor, transforming growth factor (TGF)-_, amphiregulin,

Chapter 10 / Signal Transduction Inhibitor Evaluation

233

heparin-binding epidermal growth factor, and betacellulin. TGF-_ is a key modulator in cell proliferation in both normal and malignant cells. The receptor family includes EGFR (ErbB-1), HER-2/neu (ErbB-2), HER-3 (ErbB-3), and HER-4 (ErbB-4) (20). The TGF-_-EGFR autocrine pathway is activated in cancer cells by mechanisms ranging from overexpression of EGFR and increased concentration of ligand(s) to decreased phosphatase activity, decreased receptor turnover, or the presence of mutant receptors such as EGRFvIII, which lacks domains I and II of the extracellular domain and cannot bind ligand but has a constitutively activated kinase domain (21,22). EGFR is being targeted both by monoclonal antibodies (MAbs) to prevent ligand binding and small-molecule inhibitors of the tyrosine kinase enzymatic activity to inhibit auto-phosphorylation and downstream intracellular signaling. Mendelsohn first proposed the blockade of EGFR via a specific MAb as a cancer therapy in the 1980s (23,24). Mendelsohn’s group isolated a mouse MAb to EGFR designated MAb 225. The murine MAb 225 antibody was shown to have antitumor activity against human A431 epidermoid carcinoma and human MDA-MB-468 breast carcinoma grown as xenografts in nude mice, in combination with doxorubicin or cisplatin (25,26). For administration to patients, the MAb 225 antibody was humanized and designated C225 (IMC-C225). The humanized antibody C225 has been studied in combination with gemcitabine, topotecan, paclitaxel, and radiation therapy in several human tumor xenograft models (27–30). The combination of C225 with cytotoxic therapies has demonstrated greater antitumor activity than the cytotoxic therapy alone. In the fastgrowing GEO human colon carcinoma, C225 (10 mg/kg, intraperitoneally [ip], twice weekly for 5 wk) produced a tumor growth delay of 24 d; topotecan (2 mg/ kg, ip, twice weekly for 5 wk), a camptothecin analog, produced a tumor growth delay of 14 d; and the combination regimen produced a tumor growth delay of 86 d (Fig. 1) (27). At least part of the activity of C225 can be attributed to antiangiogenic activity (31,32). Bruns et al. (28) implanted L3.6pl human pancreatic carcinoma cells into the pancreas of nude mice and beginning on d 7 after tumor cell implantation began treatment with C225 (40 mg/kg, ip, twice weekly for 4 wk), gemcitabine (250 mg/kg, ip, twice weekly for 4 wk) or the combination. The animals were sacrificed on d 32 just at completion of the treatment regimens. Gemcitabine appeared to be most effective against the liver and lymphnode metastasis, and C225 appeared to be most effective against the primary disease. The combination regimen appeared to be more effective than either treatment alone. Combination treatment regimens including C225 with radiation therapy appeared to produce at least additive tumor growth delay in two head and neck squamous carcinoma xenograft models (30). C225 has undergone three consecutive phase I clinical trials, a phase Ib clinical trial, several single-agent and combination phase II trials, and is currently in phase III clinical trial (23,32). Among the several small-molecule ATP-binding-site competitive inhibitors of EGFR kinase activity currently in development, ZD1839 (Iressa) has pro-

234

Teicher

Fig. 1. Antitumor activity of topotecan and MAb C225 on established GEO human colon carcinoma xenografts. Mice were injected subcutaneously in the dorsal flank with 107 human GEO colon carcinoma cells. After 7 d (average tumor size, 0.2 cm3), mice were treated ip with topotecan alone (2 mg/kg/dose, twice weekly on d 1 and 2 of each wk for 2 wk) or with MAb C225 alone (0.25 mg/dose, twice weekly on d 3 and 6 of each wk for 5 wk) or with both drugs with the same sequential schedule. Each group consisted of 10 mice. The experiment was repeated three times. Data represent the average on a total of 30 mice for each group; bars, standard deviation. Student’s t test was used to compare tumor sizes among different treatment groups at d 29 after tumor-cell implantation. MAb C225 vs control (p < 0.001); topotecan vs control (p < 0.001); topotecan followed by MAb C225 vs control (p < 0.001); topotecan followed by MAb C225 vs MAb C225 (p < 0.001); topotecan followed by MAb C225 vs topotecan (p < 0.001).

gressed the furthest toward clinical approval. ZD1839 is [4-(3-chloro-4fluoroanilino)-7-methoxy-6-(3-morpholinopropoxy)quinazoline] (MW 447) and is an orally active antitumor agent in mice at daily doses of 12.5–200.0 mg/ kg (33). ZD1839 has been studied in combination with cisplatin, carboplatin, oxaliplatin, paclitaxel, docetaxel, doxorubicin, etoposide, ralitrexed, and radiation therapy in human tumor xenograft models (34–39). It has been shown that the contribution of ZD1839 to anticancer activity of combination treatment regimens is due, at least in part, to its activity as an antiangiogenic agent (37,40). When nude mice bearing the fast-growing human GEO colon carcinoma were treated with ZD1839 daily for 5 d per wk for 4 wk by intraperitoneal injection for doses of 50, 100, or 200 mg/kg, tumor growth delays of 4, 6, and 18 d, respec-

Chapter 10 / Signal Transduction Inhibitor Evaluation

235

tively, resulted (34). The 100 mg/kg dose of ZD1839 was selected for combination studies. Using the GEO colon xenograft tumor model, Ciardiello et al. (34) found that ZD1839 administered daily by intraperitoneal injection for 5 d per week for 4 wk produced 6 to 10 d for tumor growth delay, whereas standard regimens for paclitaxel (20 mg/kg), topotecan (2 mg/kg), and tomudex (12.5 mg/ kg) resulted in 9, 7, and 10 d of tumor growth delay. The combination treatment regimens of ZD1839 with each cytotoxic agent resulted in 33, 27, and 25 d of tumor growth delay, respectively. Sirotnak et al. (35) administered ZD1839 (150 mg/kg) orally daily for 5 d for 2 wk to nude mice bearing A431 human vulvar epidermoid carcinoma, A549, SK-LC-16, or LX-1 human non-small-cell lung carcinomas, or PC-3 or TSU-PR1 human prostate carcinomas as a single agent or along with cisplatin, carboplatin, paclitaxel, docetaxel, doxorubicin, edatexate, gemcitabine, or vinorelbine. ZD1839 was a positive addition to all of the treatment combinations except gemcitabine, where it did not alter the antitumor activity compared with gemcitabine alone, and vinorelbine, where the combination regimen was toxic. For example, in the LX-1 non-small-cell lung carcinoma xenograft, ZD1839 (150 mg/kg, po) produced a tumor growth delay of 8 d, paclitaxel (25 mg/kg, ip) produced a tumor growth delay of 16 d, and the combination treatment regimens resulted in a tumor growth delay of 26 d. Working in the human GEO colon carcinoma, Ciardiello et al. (37) found that ZD1839 (150 mg/kg, ip, daily for 5 d per week for 3 wk) was a more powerful antiangiogenic therapy than paclitaxel (20 mg/kg, ip, once per week for 3 wk) and that the combination treatment regimen was most effective. Given these results, it is unlikely that ZD1839 would be a highly effective single agent in the clinic, but it could be a useful component in combination treatment reigmens. Expanding on these studies, Tortora et al. (41) examined combinations of an antisense oligonucleotide targeting protein kinase A, a taxane, and ZD1839 in the fastgrowing human GEO colon carcinoma xenograft. The tumor growth delays were 8 d with the taxane IDN5109 (60 mg/kg, po), 20 d with ZD1839 (150 mg/kg, po), 23 d with the antisense AS-PKAI (10 mg/kg, po), and 61 d with the three-agent combination treatment regimen. Recently, Naruse et al. (42) found that a subline of human K562 leukemia made resistant to the phorbol ester TPA and designated K562/TPA was more sensitive to ZD1839 administered intravenously or subcutaneously to nude mice bearing subcutaneously implanted tumors than was the parental K562 line. The mechanism by which the K562/TPA line multidrug resistance occurs is unknown. The K562/TPA line does not express GP170 Pglycoportein or MDR-1 protein. ZD1839 has been evaluated in five phase I clinical trials including 254 patients, and it appears that response to ZD1839 does not correspond to EGFR expression (43). A pilot clinical study of 24 non-smallcell lung cancer patients showed that ZD1839 could be combined with caboplatin/ paclitaxel. A phase I study of 26 colorectal cancer patients showed that ZD1839 could be combined with 5-fluorouracil and leucovorin safely (44). Two large

236

Teicher

multicenter phase III clinical trials of ZD1839 (250 or 500 mg daily) in combination with carboplatin/paclitaxel or cisplatin/gemcitabine as first-line treatment in nonoperable stage III and stage IV non-small-cell lung cancer patients are underway (37,43,45). Other small-molecule inhibitors of EGFR that are progressing through development are OSI-774, PD183805/CI-1033, PKI-1033, PKI166, and GW2016 (46,47). The deregulated tyrosine kinase activity of the BCR-ABL fusion protein has been established as the causative molecular event in chronic myelogenous leukemia. The BCR-ABL has proven to be an ideal protein receptor kinase for pharmacological inhibition. SRI571 (Gleevec; Glivec; CGP57148B), a phenylamino-pyrimidine derivative, is a potent inhibitor of the Abl tyrosine kinase that is present on the malignant cells in 95% of patients with chronic myelogenous leukemia (CML). The compound selectively inhibits proliferation of v-Abl and Bcr-Abl expressing cells and has antitumor activity as a single agent in animal models at well-tolerated doses (48–57). Unlike many other tyrosine kinase inhibitors that are cytostatic, STI571 is cytotoxic toward chronic myeloid leukemia-derived cell lines, as demonstrated in colony-formation assays using the surviving fraction endpoint (58). In cell culture, STI571 adds to the cytotoxicity of other cytotoxic agents such as etoposide in cells that express the BCR-ABL mutation (58,59). In cell-culture studies modeling combinations that may be used for bone-marrow pretransplantation conditioning regimens, using the BV173 and EM-3 BCR-ABL-positive cell lines with an MTT growth inhibition endpoint, Topaly et al. (60) found that STI571 produced greater-than-additive growth inhibition in combination with radiation therapy and produced additive to less-than-additive growth inhibition with busulfan and treosulfan by the combination index method. Mice reconstituted with P210(BCR/ABL)-transduced bone-marrow cells succumb to a rapidly fatal leukemia (61). When these animals were treated with STI571, survival time was markedly increased. In contrast to the polyclonal leukemia in control mice, STI571-treated mice develop a CMLlike leukemia that is generally oligoclonal, suggesting that STI571 eliminated or severely suppressed certain leukemic clones. None of the STI571-treated mice were cured of the CML-like myeloproliferative disorder, and STI571-treated murine CML transplanted with high efficiency to fresh recipient animals. Progression of chronic myelogenous leukemia to acute leukemia (blast crisis) in humans has been associated with acquisition of secondary chromosomal translocations frequently resulting in the NUP98/HOXA9 fusion protein. Dash et al. (62) developed a murine model expressing BCR/ABL and NUP98/HOXA9 to cause blast crisis. The phenotype depends upon expression of both mutant proteins, and the tumor retains sensitivity to STI571. Despite the success of STI571, resistance can develop to this agent in the clinic (63,64). It has been recognized for some time that STI571 is not a specific inhibitor of BCR/ABL and is, indeed, a potent inhibitor of other tyrosine kinases, including

Chapter 10 / Signal Transduction Inhibitor Evaluation

237

the receptor tyrosine kinase KIT and the platelet-derived growth factor receptor (PDGFR). These other tyrosine kinases are involved in many malignant diseases. About 90% of malignant gastrointestinal stromal tumors (GISTs) have a mutation in c-kit leading to KIT receptor autophosphorylation and ligand-independent activation. Initial clinical studies have found that about 50% of GISTs respond to STI571 (65–72). PDGFR is expressed in several other human cancers and is also expressed by tumor endothelial cells, thus, enabling STI571 as an antiangiogenic agent for a wide variety of malignant diseases. Receptor tyrosine kinases have also been a focus for drug development in the area of antiangiogenic therapy (73–76). The therapeutically revolutionary concept of therapy directed toward the process of angiogenesis has been to focus the therapeutic attack of cancer away from the malignant cell and toward a “normal” cell, one of several types of stromal cell, the endothelial cell (1). The validation for this concept is the recognition that cancer is a disease process directed by the malignant cells but critically requiring the active involvement of a variety of normal cells to enable tumor growth, invasion, and metastasis (77–86). Numerous receptor tyrosine kinases have been identified that are directly or indirectly involved in angiogenesis, including VEGFR2 (Flk-1, KDR), VEGFR1 (Flt-1), PDGFR, bFGFRs, Tie-1, and Tie-2 (87). SU5416 has been under development as a selective inhibitor of VEGFR2 (Flk-1, KDR) kinase activity, and SU6668 is under development as a more broad-spectrum receptor kinase inhibitor blocking VEGFR2, bFGFRs, and PDGFR kinase activity. Early in vivo work with SU5416 suffered from the use of DMSO as a vehicle for the compound, administered by intraperitoneal injection to mice once daily beginning 1 d after tumorcell implantation (88). Using the DMSO vehicle, tumor growth delays of 0.5, 3, 6, 8, and 13 d were obtained in the human A375 melanoma xenograft with daily doses of SU5416 of 1, 3, 6, 12.5, and 25 mg/kg, respectively. The murine CT-26 colon carcinoma was used to assess the effect of SU5416 and SU6668 on the growth of liver metastases (89). For the study, 104 CT-26 cell were implanted beneath the capsule for the spleens of male Balb/c mice. Four days later, treatment with SU5416 or SU6668 began. SU5416 (12 mg/kg) was administered in 99% PEG-300/1% Tween-80, and SU668 (60 mg/kg) was administered in 30% PEG-300/phosphate-buffered saline (pH 8.2). The compounds were injected once daily until the end of the experiment on d 22 after tumor cell implantation. The mean number of liver nodules was decreased to about 9 with SU5416 treatment, and about 8 with SU6668 treatment, from about 19 in the control animals. SU5416 has a plasma half-life of 30 min in mice (90). The activity of SU5416, despite the short circulating half-life, led to cell-culture studies, which indicated that exposure to 5 μM SU5416 for 3 h inhibited the proliferation for HUVEC for 72 h. These studies also showed that SU5416 accumulated intracellularly. Geng et al. (91) found that SU5416 increased the sensitivity of the murine B16 melanoma and the murine GL261 glioma to radiation therapy. When the GL261

238

Teicher

glioma was grown subcutaneously in C57BL mice, administration of SU5416 (30 mg/kg, ip, twice weekly for 2 wk) produced a tumor-growth delay of 4.5 d. Fractionated radiation therapy (3 Gy × 8) resulted in 8.5 d of tumor-growth delay. The combination regimen involving SU5416 administration along with and after completion of the radiation resulted in 16 d of tumor-growth delay. SU5416 and SU6668 have been tested as single agents and in combination with fractionated radiation therapy in C3H mice bearing SCC VII squamous carcinomas (92–94). SU5416 (25 mg/kg, daily for 5 d) or SU6668 (75 mg/kg, daily for 5 d) were administered before or after radiation (2 Gy daily for 5 d). The tumor growth delay with SU5416 was 2 d, which increased to 6.5 d when combined with radiation therapy. The tumor-growth delay with SU6668 was 3.3 d, which increased to 11.9 d when combined with radiation therapy. Administration of the compounds before or after radiation delivery did not affect the tumor response. SU5416 and SU6668 are undergoing clinical trial (69–72,95). Like STI571, SU5416 and SU6668 have been found to inhibit c-kit (KIT), the stem-cell factor receptor tyrosine kinase (96–98). C-kit is essential for the development of normal hematopoietic cells and has been proposed to play a functional role in acute myeloid leukemia (AML). Among the six indoline-family tyrosine kinase inhibitors tested, including SU5416, SU6668, and SU6597, SU5416 was the most potent growth inhibitor of a series of human small-cell lung cancer cells expressing c-kit. Mesters et al. (98) reported a 4-mo response in a patient with acute myeloid leukemia after treatment with SU5416. SU5416 and similar agents may also be useful for the treatment of von Hippel–Lindau syndrome patients (99). While SU5416 and similar agents appear to be quite tolerable as single agents, SU5416 was difficult to administer in combination with cisplatin and gemcitabine owing to the incidence of thromboembolic events (100–103). Other small-molecule tyrosine kinase inhibitors showing promise in early clinical trial include PTK787/ZK222584 and ZD6474. PTK787/ZK222584 has shown activity in several solid-tumor models (104–108). When the RENCA murine renalcell carcinoma was grown in the subrenal capsule of Balb/c mice, the animals developed a primary tumor and metastases to the lung and to the abdominal lymph nodes. Daily oral treatment with PTK787/ZK222584 (50 mg/kg) resulted in a significant decrease of 61 to 67% in primary tumors after 14 and 21 d, respectively. The occurrence of lung metastases was reuced 98% and 78% on d 14 and 21, respectively; and lymph-node metastases appeared only on d 21 (Fig. 2) (105). A similar cohort of animals were treated with TNP-470, with treatment administered alternate days at 30 mg/kg, subcutaneously. While similar tumor response was seen, the compound was toxic, and therapy was discontinued early. The major alternative therapeutic methodology being developed to inhibit the vascular endothelial growth factor (VEGF) signaling pathway is anti-VEGF neutralizing MAbs (109,110). Bevacizumab, an anti-VEGF antibody, is showing promise in clinical trial (111).

Chapter 10 / Signal Transduction Inhibitor Evaluation

239

Fig. 2. (A) Effect of PTK787/ZK 222584 on tumor volume and number of metastases in murine renal cell carcinoma. PTK787/ZK 222584 was administered daily at 50 mg/kg orally. Therapy was initiated 1 d after inoculation of RENCA cells into the subcapsular space of the left kidney of syngeneic BALB/c mice. Animals were sacrificed after either 14 (n = 12) or 21 (n = 20) d, and primary tumor volume, number of lung metastases, and number of visible lymph nodes were assessed. (B) Effects of TNP-470 on tumor volume and number of metastases. BALB/c mice were sacrificed 14 (n = 10) or 21 (n = 10) d after inoculation of RENCA with TNP-470 (30 mg/kg administered sc every other day) was initiated 1 d after inoculation of RENCA cells. The control group received vehicle only. In the group that was sacrificed after 21 d, TNP-470 treatment had to be discontinued in all animals on d 13 because of strong side effects, such as weight loss > 20%, and ataxia. Values are means; bars, SE. Ps were calculated by comparing means of the treated group and means of the control group using the Mann Whitney t test. *, significant.

PROTEIN SERINE-THREONINE KINASE INHIBITORS Protein kinase C isoforms are centrally involved in signaling transduction pathways related to regulation of the cell cycle, apoptosis, angiogenesis, differentiation, invasiveness, senescence, and drug efflux (112–114). Protein kinase C is a gene family consisting of at least 12 isoforms (115–117). Based on differing substrate

240

Teicher

specificity, activator requirements, and subcellular compartmentalization, it is hypothesized that activation of individual protein kinase C isoforms preferentially elicits specific cellular responses (116,117). Treatment of adrenal cortex endothelial cells with VEGF resulted in protein kinase C activation and elevated endothelial nitric oxide synthase (eNOS) expression. Inhibition of protein kinase C with isoform-specific inhibitors abolished VEGF-induced eNOS upregulation (118). When protein kinase C pathways were activated in human glioblastoma U973 cells by phorbol 12-myristate 13-acetate (PMA), VEGF mRNA expression was upregulated via a posttranscriptional mRNA stabilization mechanism (119). PMA increased VEGF mRNA half-life from 0.8 to 3.6 h, which was blocked by protein kinase C inhibitors (staurosporine or calphostin C) but not by protein kinase A or cyclic nucleotide-dependent protein kinase inhibitors. Recent results provide evidence for the involvement of protein kinase C in the invasiveness of breast cancer cells through regulation of urokinase plasminogen activator (120,121). In cervical cancer cells in culture, protein kinase C inhibitors or high concentrations of a phorbol ester decreased telomerase activity in the cells (122). Several studies have associated specific isoforms of protein kinase C with metabolic pathways in prostate cancer cells (123–126). Protein kinase C ¡ may be involved in the induction of P-glycoprotein-mediated drug resistance in LNCaP human prostate carcinoma cells. Protein kinase C 6 may be involved in activating anticancer drug-induced apoptosis signaling by amplifying the ceramide-mediated death pathway. Protein kinase C c may be involved in the proliferation and responsiveness to apoptotic signals in murine TRAMP prostate cancer cells in culture. Activation of protein kinase C by exposure of LNCaP prostate carcinoma cells to a phorbol ester increased the secretion of prostatic acid phosphatase by the cells in culture. Protein kinase C has also been identified as an interesting therapeutic target for the treatment of malignant gliomas (127,128). Inhibitors of protein kinase C have been shown to decrease proliferation and invasion in preclinical glioma models. The most clear-cut, direct-acting, most frequently found angiogenic factor in cancer patients is VEGF (77,128). The signal transduction pathways of the KDR/ Flk-1 and Flt-1 receptors include tyrosine phosphorylation, activation of PLC, diacylglycerol generation, and PI-3 kinase, with downstream activation of protein kinase C and activation of the MAP kinase pathway (130–133) or, possibly, by translocation of protein kinase C into the cell nucleus (134,135). To assess the contribution of protein kinase C activation to VEGF signal transduction leading to neovascularization and enhanced vascular permeability, the effects of a protein kinase C` selective inhibitor that blocks the protein phosphorylation activity of conventional and novel protein kinase C isoforms via an interaction at the ATP binding site was studied (136–140). At concentrations predicted to selectively inhibit protein kinase C completely, the compound abrogated VEGF-stimulated growth of bovine aortic endothelial cells (136). Oral administration of the inhibitor decreased neovascularization in an ischemia-

Chapter 10 / Signal Transduction Inhibitor Evaluation

241

dependent model of in vivo retinal angiogenesis and blocked increases in retinal vascular permeability stimulated by the intravitreal instillation of VEGF (137– 139). Administration of LY333531 to animals bearing BNL-HCC hepatocellular carcinoma xenografts transfected with the VEGF gene under tetracycline control markedly decreased tumor growth subcutaneously and orthotopically, and decreased VEGF overexpression in the tumors (140). This protein kinase C`selective inhibitor has also demonstrated antitumor activity alone and in combination with standard cancer therapies in the murine Lewis lung carcinoma and in several human tumor xenografts (141). The National Cancer Institute 60-cell line identified the protein kinase C inhibitor UCN-01, 7-hydroxy-staurosporine, as of interest. UCN-01 has undergone phase I clinical trial (142–144). UCN-01 has been shown to inhibit the in vitro and in vivo growth of many types of tumor cells including breast, lung and colon cancer (145–147). However, the growth inhibitory properties of UCN-01 may correlate more closely with its ability to block the activity of cell-cycle progression by inhibition of chk1 rather than with inhibition of protein kinase C (148–153). Kruger et al. (154) showed that the protein kinase C inhibitor UCN-01, at concentrations lower than those necessary to inhibit cancer cell growth, inhibited proliferation of human endothelial cells in vitro, prevented microvessel outgrowth from explant cultures of rat aortic rings, and abrogated hypoxia-mediated transactivation of hypoxia-inducible factor (HIF-1)-responsive promoters. Based upon this background, a search was made for the optimal protein kinase C` inhibitor for application in oncology. The compound LY317615 is a potent and selective inhibitor of protein kinase C`. When compared in a panel of kinases with the well-known kinase inhibitor staurosporine, the compound LY317615 demonstrated marked selectivity for inhibition of the protein kinase C` isoforms vs other protein kinase C isoforms as well as protein kinase A, calcium calmodulin kinase, casein kinase src-tyrosine kinase, and rat brain protein kinase C (Table 1) (155). In monolayer culture, human umbilical vein endothelial cells (HUVEC) in basal medium were stimulated to proliferate by exposure to human VEGF (20 ng/ mL). When various concentrations of LY317615 were added to the cultures for 72 h, the proliferation of the VEGF-stimulated HUVEC was profoundly inhibited by 600 nM of the compound (Fig. 3). The IC50 for the assay was 150 nM of LY317615. In a similar experiment, when human SW2 small-cell lung carcinoma cells were exposed to various concentrations of LY317615 for 72 h, a potency differential in the effect of the compound on the malignant cells vs the HUVEC was apparent. The IC50 for LY317615 in the SW2 cells was 3.5 μM; thus there was a 23-fold selectivity in the concentration of LY317165 for the growth inhibition of HUVEC compared to human SW-2 small-cell lung carcinoma cells in culture (155). The cornea is normally an avascular tissue. Surgical implantation of a small filter disc impregnated with vascular endothelial growth factor (VEGF) into the

242

LY317615 Staurosporine

Compound

0.8 0.045

_

0.03 0.023

`I

0.03 0.019

`II 2 1 0.11 0.028

PKC isozyme a 6 0.3 0.018

¡ d

8 0.4 >1.5 0.005

c >100 0.1

PKA

10 0.004

Ca calmoulin

>100 14

Casein kinase

Table 1 Protein Kinase C Isozyme and Other Kinase IC5 Values (μM) for LY317615 and Staurosporine

>100 0.001

srcTK

1 0.19

rat-brain PKC

242 Teicher

Chapter 10 / Signal Transduction Inhibitor Evaluation

243

Fig. 3. Concentration-dependent growth inhibition of human umbilical vein endothelial cells and human SW2 small-cell lung carcinoma cells after 72 h exposure to various concentrations of LY317615 as determined by WST-1 assay. Points are the means of three determinations; bars are SEM.

cornea of a rat will result in robust neoangiogenesis that is quantifiable in 7 to 10 d. Administration of LY317615 orally twice per day on d 1 through 10 after surgical implant of VEGF-impregnated filters resulted in markedly decreased vascular growth in the cornea of Fisher 344 female rats. A dose of 10 mg/kg of LY317615 decreased vascular growth to about one-half that of the VEGF-stimulated controls, whereas a dose of 30 mg/kg of LY317615 decreased vascular growth to the level of the unstimulated surgical control (Fig. 4) (155). Basic fibroblast growth factor (bFGF) is another major angiogenic factor in tumors and is known to be a substrate for phosphorylation by protein kinase C. Surgical implantation of a small filter disc impregnated with bFGF into the cornea of Fisher 344 female rats resulted in robust neoangiogenesis that was quantifiable in 7 to 10 d. Administration of LY317615 (30 mg/kg) orally twice per day on d

Fig. 4. Photographic image of VEGF-induced corneal neovascularization in a rat eye taken at 10 d postimplantation of the stimulus after no treatment or after treatment of the animals with LY317615 (10 or 30 mg/kg) orally twice daily for 10 d. Images are representative of each treatment group.

244

244 Teicher

Chapter 10 / Signal Transduction Inhibitor Evaluation

245

1 through 10 after surgical implantation of bFGF resulted in decreased vascular growth to a level of 26% of that of the bFGF control. Nude mice bearing human SW2 small-cell lung carcinoma growing as a subcutaneous xenograft on the thigh of the animals were treated with LY317615 orally twice daily on d 14 through 30 after tumor cell implantation. On d 31, tumors were collected, preserved in 10% phosphate-buffered formalin, and 5mm-thick sections were immunohistochemically stained for expression of endothelial specific markers, either factor VIII or CD31. The number of intratumoral vessels in the samples was quantified by counting stained regions in 10 high-power microscope fields (×200). There was a LY317615 dose-dependent decrease in the number of countable intratumoral vessels in the human SW2 xenograft tumors. The number of intratumoral vessels stained by factor VIII was decreased to one-half that of the controls in animals treated with LY317615 (30 mg/kg), and the number of vessels stained by CD31 was decreased to one-quarter that of the controls in animals treated with LY317615 (30 mg/kg) (Fig. 5) (155). The plasma levels of VEGF in mice bearing the human SW2 SCLC, HCT116 colon, and Caki-1 renal-cell carcinomas treated or untreated with LY317615 were measured by the Luminex assay (156). For studies using the SW2 human SCLC, plasma samples were obtained every 3 d starting on d 7 after implantation and carried through treatment, as well as after the termination of treatment. Three individual mice were obtained from the control group and the treatment group at each time point. Plasma VEGF levels were undetectable in both treatment groups in SW2 tumor-bearing mice until d 17, when tumor volumes were near 600 mm3 and plasma VEGF levels reached 20 pg/mL (Fig. 6). Plasma VEGF levels were similar between the treated and untreated groups through d 20, when plasma VEGF levels reached 75 pg/mL. Plasma VEGF levels in the SW2 control group continued to increase throughout the study, reaching values of 400 pg/mL on d 40 after implantation. Beginning on d 23, 9 d after beginning therapy, plasma VEGF levels in the LY317615-treated SW2-bearing animals were similar through the duration of therapy. Upon termination of treatment, plasma VEGF levels slightly increased to 100 ng/mL, which were still significantly decreased compared to the untreated control group. Plasma VEGF in Caki-1-bearing mice was not detectable in the untreated animals until the tumor volume reached near 500 mm3 on d 27 (35 pg/mL), 5 d after beginning treatment. At this time, plasma VEGF levels in the LY317615 treatment group were already decreased compared to control values. The VEGF levels in the control Caki-1 group continued to increase through the study and peaked at 225 pg/mL on d 49 after tumor implantation. In the treatment group, the plasma levels remained suppressed compared to those of controls throughout the treatment period (d 21–39). The plasma VEGF levels, reaching a maximum of 37 pg/mL, remained suppressed out to d 53, which was 14 d after terminating treatment. Plasma VEGF levels in HCT116-bearing animals were not detectable until d 21 after tumor implantation

246

Teicher

(10 pg/mL), when tumor volumes averaged 900 mm3. The highest plasma VEGF levels in the untreated control group reached 50 pg/mL, and remained at this level for the duration of the study. However, treatment with LY317615 did not decrease plasma VEGF levels in HCT116-bearing animals (156). In patients, serum VEGF levels have also been found to change during therapy and to be related to treatment response (157–160). A sequential treatment regimen was used to examine the efficacy of LY317615 in the SW2 small-cell lung cancer xenograft. The compound LY317615 was effective in this tumor model. Administration of LY317615 alone on d 14 through 30 after tumor implantation over a dosage range from 3 to 30 mg/kg produced tumor-growth delays between 7.4 and 9.7 d (Fig. 7). The SW2 tumor is quite responsive to paclitaxel, and treatment with that drug alone produced a 25-d tumor-growth delay. Treatment with paclitaxel followed by LY317615 (30 mg/ kg) resulted in over 60 d of tumor-growth delay, a 2.5-fold increase in the duration of tumor response. The SW2 small-cell lung cancer was less responsive to carboplatin, which produced a tumor-growth delay of 4.5 d. Sequential treatment with carboplatin followed by LY317615 resulted in 13.1 d of tumor-growth delay (155). The antitumor activity of LY317615 alone and in combination with cytotoxic antitumor agents has been explored in several human tumor xenografts growing subcutaneously in nude mice (155,156,161–164). While in most of the tumor models, the tumor-growth delay produced by treatment with LY317615 as a single agent was not sufficient to predict single-agent activity in the clinic, in combination regimens LY317615 was a useful addition to the therapeutic regimen. Unexpectedly, administration of LY317615 to animals bearing human tumor xenografts that secrete measurable VEGF into circulating blood, markedly decreased the VEGF in the plasma of the animals (156). LY317615 is currently in phase I clinical trial (165). TNP-470, a synthetic derivative of fumagillin, an antibiotic that has little antibacterial or antifungal activity, but does have marked amebicidal activity (166), is a potent inhibitor of endothelial cell migration (167), endothelial cell proliferation (168), and capillary tube formation (169). TNP-470 also inhibits angiogenesis as demonstrated in chick CAM, the rabbit and rodent cornea (169). TNP-470 has been shown to inhibit the growth of primary and metastatic murine tumors, as well as human tumor xenografts (170–179). When administered to animals bearing the Lewis lung carcinoma, subcutaneously on alternate days beginning on d 4 after tumor implantation and continuing until day 18 after tumor implantation, TNP-470 was a moderately effective modulator of the cytotoxic therapies (Table 2). TNP-470 was most effective with melphalan, BCNU, and radiation, increasing the tumor-growth delay produced by these treatments 1.8to 2.4-fold. TNP-470, along with minocycline, administered intraperitoneally daily on d 4 through d 18, comprised a highly effective antiangiogenic agent

Chapter 10 / Signal Transduction Inhibitor Evaluation

247

Fig. 5. Countable intratumoral vessels in human SW2 small-cell lung carcinoma xenograft tumors after treatment of the tumor-bearing animals with LY317615 (3, 10, or 30 mg/kg) orally twice per day on d 14 through 30 after tumor implantation. Tumors were immunohistochemically stained for Factor VIII or CD31. Intratumoral vessels were counted manually. Data are the means of 10 determinations; bars are SEM.

combination. The increases in tumor-growth delay produced by the modulator combination TNP-470/minocycline, along with the cytotoxic therapies, ranged from twofold to fourfold. In the treatment group receiving TNP-470/minocycline and cyclophosphamide, approx 40% of the animals were long-term (>120 d) survivors. Each of the cytotoxic therapies (including radiation, which was delivered locally to the tumor-bearing limb) produced a reduction in the number of lung metastases found on d 20 (Table 3). Neither TNP-470/minocycline nor the combination of antiangiogenic agents altered the number of lung metastases or the percentage of large (vascularized) lung metastases on d 20 after tumor implantation. The modulators did not alter the number of lung metastases from

Fig. 6. Plasma VEGF levels in nude mice bearing human SW2 SCLC, Caki-1 renal cell carcinoma, or HCT116 colon carcinoma xenograft tumors, either untreated controls or treated with LY317615 orally twice daily, d 14–30 (21–39 for Caki-1-bearing mice). The data represent the average results for three trials, with each point being the average of nine individual tumors. Bars represent SEM. Asterisk indicates statistically significant differences (p < 0.05).

248 Teicher

248

Chapter 10 / Signal Transduction Inhibitor Evaluation

249

Fig. 7. Growth delay of the human SW2 small-cell lung carcinoma after treatment with LY317615 (3, 10, or 30 mg/kg) orally twice per day on d 14 through 30 alone or along with paclitaxel (24 mg/kg, intravenously) on d 7, 9, 11, 13, or carboplatin (50 mg/kg, intraperitoneally) on d 7. Points are the means of five animals; bars are SEM.

those obtained with the cytotoxic therapies, except in the case of cyclophosphamide, in which many animals treated with the drug and antiangiogenic agent combination had very few metastases on d 20, and most of those were very small (180–188). The efficacy of the modulator combination of TNP-470/minocycline against the primary Lewis lung tumor is compared with that of other potential antiangiogenic modulator combinations in Table 4. The most effective combination with cisplatin was 14(SO4)`-cyclodextrin/tetrahydrocortisone/minocycline; with the other cytotoxic therapies, the three antiangiogenic agent combinations, along with cyclophosphamide, were highly effective therapies, resulting in 40– 50% long-term survivors. None of the antiangiogenic agent combinations alone was effective against metastatic disease, although in each case the percentage of large metastases on d 20 was reduced (Table 5). There was a trend toward the combination of 14(SO4)`-cyclodextrin/tetrahydrocortisone/minocycline being

250

Teicher

Table 2 Growth Delay of Lewis Lung Tumor Produced by Various Anticancer Treatments Alone or in Combination With Potential Antiangiogenic Modulators Tumor growth delay, daysa Treatment group

Alone

— — CDDP (10 mg/kg) 4.5 ± 0.3 Cyclophosphamide 21.5 ± 1.7 (3 × 150 mg/kg) Melphalan (10 mg/kg) 2.7 ± 0.3 BCNU (3 × 15 mg/kg 3.6 ± 0.4 X-rays (5 × 3 Gy 4.4 ± 0.3 a

b

+Minocycline

+TNP-470

+TNP-470-MINO

1.2 ± 0.4 5.1 ± 0.3 32.4 ± 1.8

2.1 ± 0.4 6.0 ± 0.5 25.3 ± 2.2

1.8 ± 0.4 10.9 ± 0.8 44.8 ± 2.8c

4.3 ± 0.3 5.2 ± 0.4 7.8 ± 0.6

6.0 ± 0.5 6.3 ± 0.5 10.6 ± 1.1

8.5 ± 0.6 14.6 ± 1.0 15.3 ± 1.2 3

Tumor-growth delay is the difference in days for treated tumors to reach 500 mm , compared with untreated control tumors. Untreated control tumors reach 500 mm3 in about 14 d. Mean ± SE of 15 animals. b Minocycline (10 mg/kg) was administered ip daily on d 4–18. TNP-470 (32Pøg/kg) was administered subcutaneously on alternate days for eight injections, beginning on d 4. CDDP and melphalan were administered intraperitoneally (ip) on d 7. Cyclophosphamide and BCNU were administered ip on d 7, 9, and 11. X-rays were delivered daily on d 7–11 locally to the tumorbearing limb. c Five of 12 long-term survivors (>180 d).

the most effective antiangiogenic agents along with cytotoxic therapies against metastatic disease (180–182). Given the relatively modest impact of established combination chemotherapy regimens on survival in advanced non-small-cell lung cancer, the development of new treatments for this very common malignancy is imperative. Among the newer chemotherapeutic agents, the taxane paclitaxel has demonstrated significant activity against metastatic non-small-cell lung cancer as a single agent, with much improved median survival (189,190). Since platinum-based therapeutic combinations have been historically important in the treatment of non-small-cell lung cancer, several phase II studies were conducted combining administration of paclitaxel and carboplatin (191–195). These phase II studies produced promising results, showing that the combination of paclitaxel and carboplatin is an active and generally well tolerated regimen for non-small-cell lung cancer. This two-drug regimen produced response rates between 30 and 50%, and prolonged median survival >1 yr. Paclitaxel/carboplatin is not curative in advanced nonsmall-cell lung cancer, and complete responses are rare. Paclitaxel administered by intravenous injection on d 7 through 11 after tumor-cell implantation produced 4.6 d of tumor-growth delay, which was increased 1.4-fold to 6.4 d of tumor-growth delay when administered along with TNP-470 and minocycline (Table 6) (196). A single intraperitoneal injection of carboplatin on d 7 after

Chapter 10 / Signal Transduction Inhibitor Evaluation

251

Table 3 Number of Lung Metastases on Day 20 From Subcutaneous Lewis Lung Tumors, After Various Anticancer Therapies Alone or in Combination With Potential Antiangiogenic Modulators Mean number of lung metastases (% large) Treatment group — CDDP (10 mg/kg) Cyclophosphamide (3 × 150 mg/kg) Melphalan (10 mg/kg) BCNU (3 × 15 mg/kg) X-rays (5 × 3 Gy)

Alone

+Minocycline

+TNP-470

+TNP-470-MINO

20 (62) 13 (58)

20 (50) 11 (48)

21 (51) 14.5 (34)

18 (54) 14 (50)

12 (40) 13 (48) 16 (53) 15 (40)

6 (33) 11 (50) 15 (38) 13 (30)

6 (30) 15 (47) 15.5 (45) 10 (40)

2 (25) 15 (45) 13 (38) 12 (42)

tumor-cell implantation produced a tumor-growth delay of 4.2 d. When carboplatin was administered along with TNP-470 and minocycline, a tumorgrowth delay of 7.8 d resulted, a 1.9-fold increase compared with carboplatin alone. The combination of the cytotoxic anticancer drugs paclitaxel and carboplatin was well tolerated by the animals and produced a tumor-growth delay of 6.6 d. The complete regimen, including TNP-470 and minocycline along with paclitaxel and carboplatin, produced a tumor-growth delay of 10.5 d, a 1.6fold increase compared with the cytotoxic drug combination alone. Treatment with the antiangiogenic agent combination decreased the number of lung metastases on d 20 after Lewis lung tumor implantation to 68% of the number found in untreated control animals (Table 6) (196). Both of the cytotoxic chemotherapeutic agents also decreased the number of lung metastases on d 20. Paclitaxel administration decreased the number of lung metastases to 55% of the control number, which was not significantly altered by the addition of co-administration of TNP-470/minocycline. Treatment with carboplatin decreased the number of lung metastases to 63% of the number in the untreated control animals. Addition of TNP-470/minocycline administration to treatment with carboplatin did not significantly alter the number of lung metastases compared with carboplatin alone. The combination of the cytotoxic drugs reduced the number of lung metastases to 33% of the number in the control animals. With the addition of treatment with TNP-470/minocycline to the combination of cytotoxic anticancer drugs, the number of lung metastases was reduced to 20% of the number in the untreated control animals. The antiangiogenic combination of TNP-470 and minocycline administered for 2 wk did not alter the growth of the Lewis lung carcinoma, the EMT-6 mammary carcinoma, the 9L gliosarcoma, or the FsaII fibrosarcoma (181–188).

252

Teicher

Table 4 Growth Delay of Lewis Lung Tumor Produced by Various Anticancer Treatments Alone or in Combination With Potential Antiangiogenic Modulators Tumor growth delay, days b

Treatment group

Alone

— — CDDP (10 mg/kg) 4.5 ± 0.3 Cyclophosphamide 21.5 ± 1.7 a (3 × 150 mg/kg) (5/12) Melphalan (10 mg/kg) 2.7 ± 0.3 BCNU (3 × 15 mg/kg 3.6 ± 0.4 X-rays (5 × 3 Gy) 4.4 ± 0.3 a

b

THC-MINO

+ 14 (SO4) CDTHC-TNP-470

+ 14 (SO4) CD+ MINO/TNP-470

1.2 ± 0.4 26.2 ± 2.5 48.8 ± 3.3 a (6/12) 10.5 ± 0.9 9.8 ± 0.8 12.6 ± 1.2

1.5 ± 0.3 10.6 ± 0.7 49.2 ± 3.4 a (5/12) 12.2 ± 1.4 10.6 ± 1.1 10.3 ± 0.9

1.8 ± 0.4 10.9 ± 0.8 44.8 ± 2.8 8.5 ± 0.6 14.6 ± 1.0 15.3 ± 1.2

Lived a normal lifespan (approx 2 yr).

Table 5 Number of Lung Metastases on Day 20 From Subcutaneous Lewis Lung Tumors After Various Anticancer Therapies Alone or in Combination With Potential Antiangiogenic Modulators Mean number of lung metastases (% large) b

b

Treatment group

Alone

THC-MINO

+ 14 (SO4) CDTHC-TNP-470

— CDDP (10 mg/kg) Cyclophosphamide (3 × 150 mg/kg) Melphalan (10 mg/kg) BCNU (3 × 15 mg/kg) X-rays (5 × 3 Gy)

20 (62) 13 (58) 12 (40)

17 (46) 8 (42) 1 (0)

18 (50) 15 (40) 2 (50)

18 (54) 14 (50) 2 (25)

13 (48) 16 (53) 15 (40)

7 (50) 14 (45) 9 (43)

15 (40) 14 (43) 11 (36)

15 (45) 13 (58) 12 (42)

+ 14 (SO4) CD+MINO-TNP-470

However, when TNP-470 and minocycline were added to treatment with cytotoxic anticancer therapies, tumor response was markedly increased. When C3H mice bearing the FSaIIC fibrosarcoma were treated with TNP-470/minocycline for 5 d prior to intravenous injection of the fluorescent dye Hoechst 33342, there was a shift toward greater brightness of the entire tumor-cell population, so that the 10% brightest and the 20% dimmest cell subpopulations in the control tumor were fivefold dimmer than the same populations in the TNP-470-treated tumors (Fig. 8) (181,183). The TNP-470/minocycline treated tumors were more easily

Chapter 10 / Signal Transduction Inhibitor Evaluation

253

Table 6 Growth Delay of Lewis Lung Carcinamo, and Number of Lung Metastases on Day 20 After Treatment of the Animals With Paclitaxel and/or Carboplatin With or Without Antiangiogenic Agents Treatment group Controls TNP-470 (30 mg/kg) sc, alt. d 4–18 + minocycline (10 mg/kg) ip, d 4–18 Paclitaxel (36 mg/kg) iv d 7–11 TNP-mino-paclitaxel Carboplatin (50 mg/kg) ip, d 7 TNP-mino-carboplatin Paclitaxel/Carboplatin TNP/MINO/Paclitaxel/ Carboplatin

a

Tumor growth delay (d)

Number of lung metastases

— 1.0 ± 0.3

40 ± 7 27 ± 5

4.6 ± 0.3

22 ± 4

6.4 ±0.4 4.2 ± 0.3

b

20 ± 4 25 ± 4

c

21 ± 3 13 ± 2 8±1

7.8 ± 0.5 6.6 ± 0.4 b 10.5 ± 0.6

sc, subcutaneously; ip, intraperitoneally; iv, intravenously. a Tumor growth delay is the difference in days for treated tumors to reach 500 mm3, compared 3 with untreated control tumors. Untreated control tumors reach 500 mm in about 12.4 ± 0.3 d. Mean ± SE of 15 animals. b Significantly increased tumor growth delay, compared with the cytotoxic therapy alone, p < 0.01. c p < 0.005.

penetrated by the lipophilic dye. This was the first indication that TNP-470 and minocycline treatment might allow greater distribution of small molecules into tumors. To determine whether the TNP-470/minocycline affected cyclophosphamide tissue distribution, animals were injected intraperitoneally with [14C]cyclophosphamide on d 8; they were killed 6 h later and tissue levels of 14C were determined. There was an increased level of 14C in all of the tissues from TNP470/minocycline treated animals except blood, compared with levels in [14C]cyclophsophamide only treated animals. The largest increases were 2.6-fold in the tumor, 2.3-fold in the kidney, 3.2-fold in the heart, 5.6-fold in the gut, and 7.9fold in skeletal muscle (181,183). In a similar study, Lewis lung tumor-bearing mice pretreated with TNP-470/ minocycline, or untreated, were injected intraperitoneally with a single dose of cisplatin on d 8, then killed 6 h later, and tissue levels of platinum were determined. There were increased levels of platinum in all of the tissues taken from animals treated with TNP-470/minocycline (except blood) compared with ani-

254

Teicher

mals that were treated with cisplatin only. The largest increases were 5.2-fold in the tumor, 3.8-fold in the gut, 3.0-fold in the skin, and 2.5-fold in the skeletal muscle (181,183). Both cyclophosphamide and cisplatin are cytotoxic through the formation of crosslinks in cellular DNA. DNA alkaline elution from tumors treated in vivo showed that there was increasing DNA crosslinking with increasing dose of cyclophosphamide (Table 7). Treatment with cyclophosphamide alone resulted in a crosslinking factor of 4.7; treatment with the same dose of cyclophosphamide in animals pretreated with TNP-470/minocycline resulted in a crosslinking factor of 6.2, which extrapolates to an equivalency of about 650 mg/kg of cyclophosphamide. Increased DNA crosslinking also was detected with an increasing dose of cisplatin. Treatment with cisplatin alone resulted in a crosslinking factor of 2.0; treatment with the same dose of cisplatin in animals pretreated with TNP470/minocuycline resulted in a crosslink (181,183). [14C]paclitaxel was administered to Lewis lung carcinoma-bearing animals pretreated with TNP-470/minocycline, or not pretreated, on d 8 after tumor implantation, and tissues were collected over 24 h (196). At early time points (1 and 15 min) after intravenous administration of the [14C]paclitaxel, there was a fivefold higher concentration of the drug in the tumors of animals that had been pretreated with TNP-470/minocycline; however, by 24 h, there was a twofold greater concentration of [14C]paclitaxel in the tumors of the animals pretreated with TNP-470/minocycline compared with those that had not received the antiangiogenic therapy. The pattern of [14C]paclitaxel distribution into the other tissues was similar, with greater peak levels of [14C]paclitaxel in the tissues of animals pretreated with TNP-470/minocycline. In the liver, however, there was a prolonged increased level of [14C]paclitaxel of the pretreated animals. By far, the highest levels of [14C]paclitaxel were found in the lungs of the animals, in which the peak level in the pretreated animals reached 4800 μg/g tissue. Other tissues with relatively high paclitaxel concentrations were gut and heart. Concentrations of platinum from carboplatin were two- and threefold higher in the tumors of animals pretreated with TNP-470/minocycline at 15 and 30 min after drug administration. Between 6 and 24 h after carboplatin administration, platinum levels in the tumors of pretreated animals remained about twofold greater than in animals that did not receive the antiangiogenic therapy. Overall, the tissues of the animals pretreated with TNP-470/minocuyline had higher platinum levels, with the greatest differentials being in kidney, brain, muscle, and liver. The highest platinum levels overall were in kidney, gut, liver, and brain. To determine whether pretreatment with TNP-470/minocycline might also alter the tissue distribution of large molecules into tumor and tissues, [14C]albumin was administered to TNP-470/minocycline pretreated and nonpretreated animals. There was a two- to threefold higher concentration of [14C]albumin in the tumors of TNP-470/minocyline-treated animals over the first hour after protein injection

Chapter 10 / Signal Transduction Inhibitor Evaluation

255

Fig. 8. Fluorescence distribution in FSaIIC tumor cells after iv injection of tumor-bearing animals with Hoechst 33342 (2 mg/kg). The data shown are for an untreated control tumor and a tumor treated with TNP-470 (3 × 30 mg/kg, subcutaneously) and minocycline (5 × 10 mg/kg, intraperitoneally).

and a concentration differential with higher concentrations in those tumors. This effect persisted over the 24 h examined. A similar pattern pertained for the other tissues. The highest peak levels of [14C]albumin were in liver and lung (196). There has been much discussion regarding the best designs for early clinical trials for antiangiogenic agents and cytostatic agents (197–199). TNP-470 has undergone clinical trial both as a single agent and in combination with cytotoxic anticancer therapies, with results warranting further investigation (200–205).

INHIBITORS OF CELL-CYCLE SIGNAL TRANSDUCTION The most frequent alteration in human malignant disease thus far recognized is the overexpression, mutation, and/or disregulation of cyclin D (206–208). The cyclin D1 gene CCND1 is amplified in about 20% of breast cancers, and the protein cyclin D1 is overexpressed in about 50% of breast cancers (209–213). Overexpression of cyclin D1 has been reported in proliferative breast disease and in ductal carcinoma in situ, indicating that this change is important at the earliest stages of breast oncogenesis (211,213). Kamalati et al. (210) overexpressed cyclin D1 in normal human epithelial cells and found that the transfected cells had reduced growth-factor dependency, a shortened cell-cycle time, thus provid-

256

Teicher Table 7 a DNA Crosslinking Factors From Lewis Lung Tumors by Alkaline Elution

Treatment group TNP-470-minocycline Cyclophosphamide 150 mg/kg 300 mg/kg 500 mg/kg TNP-470-minocycline cyclophosphamide (300 mg/kg) CDDP 10 mg/kg 20 mg/kg 30 mg/kg TNP-470-minocycline-CDDP (20 mg/kg)

DNA crosslinking factorb 1.2 3.9 4.7 5.6 6.2 1.7 2.0 2.8 8.9

aFor DNA alkaline elution studies, Lewis Lung Carcinoma-bearing animals were treated with TNP-470 (30 mg/kg) subcutaneously on d 4, 6, and 8, with minocycline (10 mg/kg) intraperitoneally (ip) daily on d 4–8; and/or with cyclophosphamide (150, 300, or 500 mg/kg) ip or CDDP (10, 20, or 30 mg/kg) ip on d 8 after tumor cell implantation. [14C]-thymidine was administered ip on d 7 and 8. The animals were sacrificed on d 9. bA DNA crosslinking factor of 1.0 indicates no crosslinks.

ing the cells with a growth advantage. In 123 colorectal carcinoma specimens, those staining strongly for cyclin D1 corresponded to patients with a 5-yr survival rate of 53.3%, while those that were negative or weakly staining had 5-yr survival rates of 96.2 and 78.8% (214,215). Amplification of CCND1 was found in 25% of dysplastic head-and-neck lesions, 22% of head-and-neck carcinomas, and overexpression of cyclin D1 was found in 53% of head-and-neck carcinomas, indicating that in this disease, like breast cancer, alterations in cyclin D1 occur at the very earliest stages of tumorigenesis (216,217). In a study of 218 specimens of esophageal squamous-cell carcinoma, patients with cyclin D1positive tumors had significantly worse survival than patients with cyclin D1negative tumors (218). In eight human esophageal carcinoma cell lines, seven (87.5%) and six (75%) cell lines had homozygous deletions of the p16 and p15 genes, respectively (219). All of the p16-negative cell lines express high levels of cyclin D1 and cdk4. In a transgenic mouse in which the Epstein–Barr virus ED-L2 promoter was linked to human cyclin D1 cDNA, the transgene protein localizes to squamous epithelium in the tongue and esophagus, resulting in a dysplastic phenotype associated with increased cell proliferation and indicating that cyclin D1 overexpression may be a tumor-initiating event (220,221). In a series of 84 specimens of soft-tissue sarcomas, there was no amplification of the

Chapter 10 / Signal Transduction Inhibitor Evaluation

257

CCND1 gene, but there was overexpression of cyclin D1 in 29% of cases, and the overexpression of cyclin D1 was significantly associated with worse overall survival (222,223). Marchetti et al. (224) found that abnormalities of cyclin D1 and/or Rb at the gene and/or expression level were present in more than 90% of of a series of non-small-cell lung cancer specimens, indicating that cyclin D1 and/or Rb alterations represent an important step in lung tumorigenesis. In 49 out of 50 pancreatic carcinomas (98%), the Rb/p16 pathway was abrogated exclusively through inactivation of the p16 gene (225). Mantle-cell lymphoma is defined as a subentity of malignant lymphomas characterized by the chromosomal translocation t(11;14)(q13;q32) resulting in overexpression of cyclin D1 and, in addition, about 50% of these tumors have deletion of the p16 gene (226,227). In a series of 17 hepatoblastomas, 76% showed overexpression of cyclin D1 and 88% showed overexpression of cdk4 (222). There was a correlation between high-level cyclin D1 expression and tumor recurrence. Six distinct classes of small molecules from natural products have been idenitified as inhibitors of cdks: the purine-based compound olomoucine and analogs, butyrolactone, flavopiridol, staurosporine, UCN-01, suramin, and 9hydroxyellipticine (228–235). All of these molecules bind at the ATP-binding site of the enzyme and are competitive with ATP. Olomoucine is an inhibitor of Cdc2, cdk2, cdk5, and MAP kinase in micromolar concentrations and has much weaker effects toward cdk4 and cdk6 (234). Flavopiridol, a novel synthetic flavone, potently inhibits several cyclin-dependent kinases, including cdk1, cdk2, cdk4, and cdk7 (236–243). Exposure to flavopiridol can cause cells to arrest in both the G1 and G2 phases of the cell cycle, at concentrations similar to those required for cell-growth inhibition (236,243). Flavopiridol inhibits the cdks in a manner competitive with ATP and noncompetitive with the substrate. Flavopiridol also inhibits other protein kinases, such as protein kinase C, protein kinase A, and EGFR, but at concentrations of 10 μM/L or greater. Flavopiridol is an active antitumor agent in several human tumor xenograft models, including Colo-205 colon carcinoma and DU145 and LNCaP prostate carcinomas, MCF-7 and MB-468 breast carcinomas, and SW-2 and H82 small-cell lung carcinomas (206–208,236,240). Tumor growth delay was used to determine the sensitivity of human tumor xenografts to the cell-cycle agents flavopiridol and olomoucine. The human breast carinoma cell lines MCF-7 and MB-468 were grown as xenograft tumors in female nude mice (Fig. 10). Flavopiridol was administered orally to the mice over a dosage range once daily for 5 d per wk for 3 wk beginning on d 7 after tumor cell implantation. The MB-468 tumor was more responsive to treatment with flavopiridol than was the MCF-7 tumor. The tumor-growth delay at the dose of 10 mg/kg of flavopiridol in the MCF-7 tumor was 6.2 d, while the tumor-growth delay for the same treatment in the MB-468 tumor was 10.9 d. Olomoucine was administered to animals bearing the MCF-7 tumor or the MB-468 tumor by

258

Teicher

intravenous injection over a dosage range once daily for 5 d for 3 wk beginning on d 7 after tumor-cell implantation. The MB-468 tumor was also more responsive to olomoucine than was the MCF-7 tumor. The MCF-7 tumor showed no dose response to olomoucine with a tumor-growth delay of 7.6 to 8.6 d over the dosage range of 10 to 50 mg of the agent per kg. The MB-468 tumor, on the other hand, showed a marked dose response to olomoucine such that the tumor-growth delay was 4.8 d at a dose of 10 mg/kg olomoucine and 20.8 d at a dose of 50 mg/ kg olomoucine. When animals bearing the SW2 tumor or the H82 tumor were treated with flavopiridol over a dosage range orally once daily for 5 d per wk for 3 wk, the SW-2 tumor was markedly more responsive to treatment with flavopiridol than was the H82 tumor (Fig. 9) (240). The tumor-growth delay with flavopiridol over the dosage range tested in the H82 tumor was 4.8 to 5.1 d. The tumor-growth delay of the SW-2 tumor over the same dosage range of flavopiridol was 3.9 to 13.7 d. A similar pattern pertained when animals bearing the SW2 tumor or the H82 tumor were treated with intravenously administered olomoucine over a dosage range once daily for 5 d per week for 3 wk. The SW2 tumor was very responsive to olomoucine, with tumor-growth delays ranging from 11.3 to 16.7 d, while the H82 tumor was nonresponsive to olomoucine (240). Flavopiridol has completed several phase I clinical trial administered as a single agent or in combination with cytotoxic anticancer therapies, and phase II trials are planned (206–208,242,244,245). Other cell-cycle inhibitors currently in early clinical trial include Ro 31-7453, whose molecular target is unknown, the Raf-1 kinase inhbitor BAY 43-9006, and the MEK inhibitor CI-1040 (246–248). The ras family of GTPases form important regulatory switches coordinating response to extracellular stimulus with intracellular biological response pathways (249–251). The ras gene is mutated in about 30% of human tumors. The Kras mutations are frequent in non-small-cell lung cancers, colon cancers, and pancreatic carcinomas, and usually involve point mutations at codon 12, 13, or 61 forming the ras oncogene, which is constitutively activated. A critical posttranslational modification of the ras protein is the attachment of the isoprenoid lipid farnesol, catalyzed by the enzyme farnesyl transferase. Thus, a great effort has gone to the development of farnesyl transferase inhibitors. Other proteins acted on by farnesyl transferase will also be affected by these inhibitors and may lead to alternative modifications of the proteins, especially geranylgeranylation by the enzyme geranylgeranyl transferase-1 (252–255). Farnesyl transferase inhibitors, including farnesyl diphosphate analogs, peptidomimetics, and bisubstrate analogs, exhibit activity in cell culture and in tumor models (250,255– 258). It has become clear that response of cells to farnesyl transferase inhibitors does not correlate with ras mutational status. For example, SCH66336 inhibits the growth of cell lines bearing the wild-type ras genotype as well as cell lines with K-ras mutations (259–261). Farnesyl transferase inhibitors have demonstrated antitumor activity as single agents and in combination with standard

Chapter 10 / Signal Transduction Inhibitor Evaluation

259

anticancer therapies (262–264). Nielsen et al. (265) administered SCH66336 alone and in combination with SCH58500 (p53 adenovirus) to nude mice bearing intraperitoneally implanted human DU-145 prostate carcinoma, and found that SCH66336 decreased tumor burden 35%, SCH58500 decreased tumor burden 45%, and the combination decreased tumor burden 69% (Fig. 10). Treatment of animals bearing well-established, subcutaneously implanted DU-145 human prostate carcinoma xenografts with SCH66336 (40 mg/kg, po, 2 × daily) resulted in 4.5–5.5 d of tumor-growth delay and treatment with SCH58500 adenovirus produced a similar effect (Fig. 10) (265). The combination of SCH66336 and SCH58500 resulted in about 9.5 d of tumor-growth delay. Based on preclinical results, several farnesyl transferase inhibitors, including SCH66336, R115777, L-778,123, and BMS-214662, have entered clinical trial (266–273). SCH66336 and R115777 are currently undergoing phase II testing (274–276).

CONCLUSION The therapeutic agents described herein as signal-transduction inhibitors, although only a fraction of the molecules under study in the field, represent a wide variety of molecular structures with a wide variety of biological effects and targets. The diversity in this group of molecules gives strength to the potential of this approach in therapeutic applications. The biological and biochemical pathways involved in signal transduction are numerous and redundant. It is likely that blockade of more than one pathway related to signaling will be necessary to have impact on the natural progress of a malignant disease. There has been a tendency in recent years to emphasize endpoints in preclinical in vivo models that have a low predictability of clinical activity. The endpoint described as T/ C% at a selected day is one of the less predictive measures of tumor response (277). More reliable endpoints such as tumor-growth delay require waiting for the growth rate of the tumors in the treated groups to regain the unperturbed tumor growth rate (278–280). Thus, the emphasis on shortening timelines to the clinic has resulted in a reliance on short-term, less predictive endpoints. Examination of the preclinical tumor-response data for many signal-transduction inhibitors indicates that very few would be predicted to have substantial single-agent activity in patients. Another factor is the kinetics of tumor response. Especially for angiogenesis inhibitors, preclinical tumor models make it apparent that tumors respond slowly or late to these agents. In the patient, the growth thrust of the malignant cells may outpace the therapeutic effect of angiogenesis inhibitors on the vasculature. Overall, therefore, the best speculation is that signal-transduction inhibitors, whether targeted to malignant cells or to endothelial cells, will improve therapeutic response when used in combination with cytotoxic therapies. The incorporation of signal-transduction inhibitors into therapeutic regimens represents an

Fig. 9. Growth delay of the human MCF-7 breast carcinoma (䊉) and the MDA-MB-468 breast carcinoma (䊊) grown as xenografts, and of the human SW-2 small-cell lung carcinoma (䊉) and the human H82 small-cell lung carcinoma (䊊) grown as xenografts in nude mice after treatment with flavopiridol (3, 5, or 10 mg/kg) orally, d 7–11, 14–18, 21–25; or olomoucine (10, 25, or 50 mg/kg) intravenously, d 7–11, 14–18, 21–25. Points are the means of at least two independent experiments; bars are the SEM.

260

260 Teicher

261

Fig. 10. Response of the DU-145 human tumor xenograft model to SCH58500 (3.5–4.0 × 108 C.I.U.) by intraperitoneal injection on days 7, 9, 11, 14, 16, and 18, and SCH66336 (40 mg/kg) orally twice daily on d 7 through 18 (intraperitoneal study) or on d 0 through 16 (subcutaneous study) after tumor implant. A: Intraperitoneal tumors were harvested and weighed on day 29. Mean tumor weights ± SEM are shown (n = 10). B: Subcutaneous tumors were followed by tumor volume measurements. Mean tumor volumes ± SE are shown (n = 10).

Chapter 10 / Signal Transduction Inhibitor Evaluation 261

262

Teicher

important challenge. The successful treatment of cancer requires the eradication of all malignant cells, and therefore treatment with cytotoxic therapies. The compatibility of signal-transduction inhibitors with cytotoxic therapies is not obvious and will require clinical trial of combinations to elucidate therapeutic synergies and toxicities. The goal of the addition of any noncytotoxic pathway inhibitor to a therapeutic regimen is to take a good therapy and, without additional toxicity, push it to cure. It is likely that combinations of signal-transduction inhibitors will evoke greater tumor response to therapy than treatment with single agents of this class. The early phases of the clinical testing (phase I and II) of signal-transduction inhbitors are being performed with a high degree of science, including measurements of markers for the biological activity of these agents. The true strength of signal-transduction inhibitors will be in their use in combination with traditional cytotoxic therapies, in which they will add a new dimension to the anticancer armamentarium.

REFERENCES 1. Teicher BA. Malignant cells, directors of the malignant process: role of transforming growth factor beta. Cancer Metastasis Rev 2001;20:133–143. 2. Simpson RJ, Dorow DS. Cancer proteomics from signaling networks to tumor markers. Trends Biotechnol 2001;19:S40–S48. 3. Workman P. Scoring a bull’s-eye against cancer genome targets. Curr Opin Pharmacol 2001;1:342–352. 4. Heldin CH. Signal transduction: multiple pathways, multiple options for therapy. Stem Cells 2001;19:295–303. 5. Elsayed YA, Sausville EA. Selected novel anticancer treatments targeting cell signaling proteins. Oncologists 2001;6:517–537. 6. Hondermarck H, Vercoutter-Edouart AS, Revillion F, et al. Proteomics of breast cancer for marker discovery and signal pathway profiling. Proteomics 2001;1:1216–1232. 7. Arteaga CL, Khuri F, Krystal G, Sebti S. Overview of rationale and clinical trials with signal transduction inhibitors in lung cancer. Semin Oncol 2002;29(1 suppl 4):15–26. 8. Fodde R, Smits R, Clevers H. APC, signal transduction and genetic instability in colorectal cancer. Nature Rev Cancer 2001;1:55–67. 9. Graff JR. Emerging targets in the AKT pathway for treatment of androgen-independent prostatic adenocarcinoma. Exp Opin Therap Targets 2002;6:103–113. 10. Lango MN, Shin DM, Grandis JR. Targeting growth factor receptors: integration of novel therapeutics in the management of head and neck cancer. Curr Opin Oncol 2001;13:168–175. 11. Bode AM, Dong Z. Signal transduction pathways: targets for chemoprevention of skin cancer. Lancet Oncol 2000;1:181–188. 12. Heymach JV. Angiogenesis and antiangiogenic approaches to sarcomas. Curr Opin Oncology 2001;13:261–269. 13. Reddy SA. Signaling pathways in pancreatic cancer. Cancer Journal 2001;7:274–286. 14. Lieberman SM, Horig H, Kaufman HL. Innovative treatments for pancreatic cancer. Surg Clin N Amer 2001;81:715–739. 15. Folkman J. Tumor angiogenesis: therapeutic implications. New Engl J Med 1971;285:1182–1186. 16. Teicher BA. Systems approach to cancer therapy (antiangiogenics + standard cytotoxics’ mechanism(s) of interaction). Cancer Metastasis Rev 1996;15:247–272.

Chapter 10 / Signal Transduction Inhibitor Evaluation

263

17. Teicher BA, ed. Antiangiogenic Agents in Cancer Therapy. Humana, Totowa, NJ: 1999. 18. Zwick E, Bange J, Ullrich A. Receptor tyrosine signaling as a target for cancer intervention strategies. Endocrine-Related Cancer 2001;8:161–173. 19. Ciardiello F, Tortora G. A novel approach in the treatment of cancer: targeting the epidermal growth factor receptor. Clin Cancer Res 2001;7:2958–2970. 20. Sedlacek HH. Kinase inhibitors in cancer therapy. A look ahead. Drugs 2000;59:435–476. 21. Moscatello DK, Holgado-Madruga M, Emlet DR, Montgomery RB, Wong AJ. Constitutive activation of phosphatidylinositol 3-kinase by a naturally occurring mutant epidermal growth factor receptor. J Biol Chem 1998;273:200–206. 22. Voldborg BR, Damstrup L, Spang-Thomsen M, Poulsen HS. Epidermal growth factor receptor (EGFR) and EGFR mutations, function and possible role in clinical trials. Ann Oncol 1997;8:1197–1206. 23. Mendelsohn J. Blockade of receptors for growth factors: an anticancer therapy. Clin Cancer Res 2000;6:747–753. 24. Mendelsohn J. Epidermal growth factor receptor inhibition by a monoclonal antibody as anticancer therapy. Clin Cancer Res 1997;3:2703–2707. 25. Fan Z, Baselga J, Masui H, Mendelsohn J. Antitumor effect of anti-epidermal growth factor receptor monoclonal antibodies plus cis-diamminedichloroplatinum on well established A431 cell xenografts. Cancer Res 1992;53:4637–4642. 26. Baselga J, Norton L, Masui H, Pandiella A, Coplan K, Miller WH, Mendelsohn J. Antitumor effects of doxorubicin in combination with anti-epidermal growth factor receptor monoclonal antibodies. J Natl Cancer Inst 1993;85:1327–1333. 27. Ciardiello F, Bianco R, Damiano V, et al. Antitumor activity of sequential treatment with topotecan and anti-epidermal growth factor receptor monoclonal antibody C225. Clin Cancer Res 1999;5:909–916. 28. Bruns CJ, Harbison MT, Davis DW, et al. Epidermal growth factor receptor blockade with C225 plus gemcitabine results in regression of human pancreatic carcinoma growing orthotopically in nude mice by antiangiogenic mechanisms. Clin Cancer Res 2000;6:1936–1948. 29. Inoue K, Slaton JW, Perrotte P, et al. Paclitaxel enhances the effects of the anti-epidermal growth factor receptor monoclonal antibody ImClone C225 in mice with metastatic human bladder transitional cell carcinoma. Clin Cancer Res 2000;6:4874–4884. 30. Huang S-M, Harari P. Modulation of radiation response after epidermal growth factor receptor blockade in squamous cell carcinomas: inhibition of damage repair, cell cycle kinetics and tumor angiogenesis. Clin Cancer Res 2000;6:2166–2174. 31. Perrotte P, Matsumoto T, Inoue K, et al. Anti-epidermal growth factor receptor antibody C225 inhibits angiogenesis in human transitional cell carcinoma growing orthotopicallly in nude mice. Clin Cancer Res 1999;5:257–265. 32. Ciardiello F, Bianco R, Damiano V, et al. Antiangiogenic and antitumor activity of antiepidermal growth factor receptor C225 monoclonal antibody in combination with vascular endothelial growth factor antisense oligonucleotide in human GEO colon cancer cells. Clin Cancer Res 2000;6:3739–3747. 33. Woodburn JR, Kendrew J, Fennell M, Wakeling AE. ZD1839 (“Iressa”) a selective epidermal growth factor receptor tyrosine kinase inhibitor (EGFR-TKI): inhibition of c-fos mRNA, an intermediate marker of EGFR activation, correlates with tumor growth inhibition. Proc Am Assoc Cancer Res 2000;41:402. 34. Ciardiello F, Caputo R, Bianco R, et al. Antitumor effect and potentiation of cytotoxic drug activity in human cancer cells by ZD-1839 (Iressa), an epidermal growth factor receptorsensitive tyrosine kinase inhibitor. Clin Cancer Res 2000;6:2053–2063. 35. Sirotnak FM, Zakowsky MF, Miller VA, Scher HI, Kris MG. Efficacy of cytotoxic agents against human tumor xenografts is markedly enhanced by coadministration of ZD 1839 (Iressa) an inhibitor of EGFR tyrosine kinase. Clin Cancer Res 2000;6:4885–4892.

264

Teicher

36. Ohmori T, Ao Y, Nishio K, Saijo N, Arteaga CL, Kurki T. Low dose cisplatin can modulate the sensitivity of human non-small cell lung carcinoma cells to EGFR tyrosine kinase inhibitor (ZD1839; Iressa) in vivo. Proc Am Assoc Cancer Res 2000;41:482. 37. Ciardiello F, Caputo R, Bianco R, et al. Inhibition of growth factor production and angiogenesis in human cancer cells by ZD1839 (Iressa), a selective epidermal growth factor receptor tyrosine kinase inhibitor. Clin Cancer Res 2001;7:1459–1465. 38. Williams K, Telfer BA, Stratford IJ, Wedge SR. An evaluation of the EGFR tyrosine kinase inhibitor ZD1839 (Iressa) in combination with ionizing radiation. 11th NCI-EORTC-AACR Symposium on New Drugs in Cancer Therapy. Abs. LB3, Amsterdam, November 7–10, 2000. 39. Harari PM, Huang SM. Radiation response modification following molecular inhibition of epidermal growth factor receptor signaling. Semin Radiat Oncol 2001;11:281–289. 40. Hirata A, Ogawa S-I, Kometani T, et al. ZD1839 (Iressa) induces antiangiogenic effects through inhibition of epidermal growth factor receptor tyrosine kinase. Cancer Res 2002;62:2554–2560. 41. Tortora G, Caputo R, Damiano V, et al. Oral administration of a novel taxane, an antisense oligonucelotide targeting protein kinase A, and the epidermal growth factor receptor inhibitor Iressa causes cooperative antitumor and antiangiogenic activity. Clin Cancer Res 2001;7:4156–4163. 42. Naruse I, Ohmori T, Ao Y, et al. Antitumor activity of the selective epidermal growth factor receptor-tyrosine kinase inhibitor (EGFR-TKI) Iressa (ZD1839) in an EGFR-expressing multidrug-resistant cell line in vitro and in vivo. Int J Cancer 2002;98:310–315. 43. Drucker BJ, Schwartz L, Marion S, Motzer R. Phase II trial of ZD1839 (Iressa), an EGF receptor inhibitor, in patients with advanced reanl cell carcinoma. Proc Amer Soc Clin Oncol 2002; 38:abstract 720. 44. Cho CD, Fisher GA, Halsey JZ, Advani RH, Yuen AR, Sikic BI. A phase I study ZD1839 (Iressa) in combination with oxaliplatin, 5-fluororuacil (5-FU) and leucovorin (LV) in advanced solid malignancies. Proc Amer Soc Clin Oncol 2002; 38:abstract 38. 45. Albanell J, Rojo F, Baselga J. Pharmacodynamic studies with the epidermal growth factor receptor tyrosine kinase inhibitor ZD1839. Semin Oncol 2001;28(5 suppl 16):56–66. 46. Murren JR, Papadimitrakopoulou VA, Sizer KC, Vaidyanathan S, Ravera C, Abbruzzese JL. A phase I dose-escalating study to evaluate the biological activity and pharmacokinetics of PKI166, a novel tyrosine kinase inhibitor, in patients with advanced cancers. Proc Amer Soc Clin Oncol 2002; abstract 377. 47. Hoekstra R, Dumez H, van Oosterom AT, et al. A phase I and pharmacological study of PKI166, an epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, administered orally in a two weeks on, two weeks off scheme to patients with advanced cancer. Proc Amer Soc Clin Oncol 2002; abstract 340. 48. Traxler P, Bold G, Buchdunger E, et al. Tyrosine kinase inhibitors: from rational design to clinical trials Med Res Rev 2001;21:499–512. 49. Mauro MJ, Druker BJ. STI571: targeting BCR-ABL as therapy for CML. Oncologist 2001;6:233–238. 50. Mauro MJ, Druker BJ. STI571: a gene product-targeted therapy for leukemia. Curr Oncol Reports 2001;3:223–227. 51. Mauro MJ, O’Dwyer M, Heinrich MC, Druker BJ. STI571: a paradigm of new agents for cancer therapies. J Clin Oncol 2002;20:325–334. 52. Griffin J. The biology of signal transduction inhibition: basic science to novel therapies. Semin Oncol 2001;28 (5 suppl 17):3–8. 53. Thambi P, Sausville EA. STI571 (imatinib mesylate): the tale of a targeted therapy. AntiCancer Drugs 2002;13:111–114. 54. Olavarria E, Craddock C, Dazzi F, et al. Imatinib mesylate (STI571) in the treatment of relapse of chronic myeloid leukemia after allogenic stem cell transplantation. Blood 2002;99:3861–3862.

Chapter 10 / Signal Transduction Inhibitor Evaluation

265

55. Gorre ME, Sawyer CL. Molecular mechanisms of resistance to STI571 in chronic myeloid leukemia. Curr Opin Hematol 2002;9:303–307. 56. O’Dwyer ME, Mauro MJ, Druker BJ. Recent advances in the treatment of chronic myelogenous leukemia. Ann Rev Med 2002;53:369–381. 57. La Rosee P, O’Dwyer ME, Druker BJ. Insights from pre-clinical studies for new combination treatment regimens with the Bcr-Abl kinase inhibitor imatinib mesylate (Gleevec/ Glivec) in chronic myelogenous leukemia: a translational perspective. Leukemia 2002;16:1213–1219. 58. Liu WM, Stimson LA, Joel SP. The in vitro activity of the tyrosine kinase inhibitor STI571 in BCR-ABL positive chronic myeloid leukemia cells: Synergistic interactions with antileukemic agents. Br J Cancer 2002;86:1472–1478. 59. Marley SB, Davidson RJ, Goldman JM, Gordon MY. Effects of combinations of therapeutic agents on the proliferation of progenitor cells in chronic myeloid leukemia. Br J Hematol 2002;116:162–165. 60. Topaly J, Fruehauf S, Ho AD, Zeller WJ. Rationale for combination therapy of chronic myelogenous leukemia with imatinib and irradiation or alkylating agents: implications for pretransplant conditioning. Br J Cancer 2002;86:1487–1493. 61. Wolff NC, Ilaria RL. Establishment of a murine model for therapy-treated chronic myelogenous leukemia using the tyrosine kinase inhibitor STI571. Blood 2001;98:2808–2816. 62. Dash AB, Williams IR, Kutok JL, et al. A murine model of CML blast crisis induced by cooperation between BCR/ABL and NUP98/HOXA9. Proc Natl Acad USA 2002;99:7622–7627. 63. Krystal GW. Mechanisms of resistance to imatinib (STI571) and prospects for combination with conventional chemotherapeutic agents. Drug Resistance Updates 2001;4:16–21. 64. Weisberg E, Griffin JD. Mechanisms of resistance imatinib (STI571) in preclinical models and in leukemia patients. Drug Resistance Updates 2001;4:22–28. 65. Demetri GD. Targeting c-kit mutations in solid tumors: scientific rationale and novel therapeutic options. Semin Oncol 2001;28(5 suppl 17):19–26. 66. Joensuu H, Dimitrijevic S. Tyrosine kinase inhibitor imatinib (STI571) as an anticancer agent for solid tumors. Ann Med 2001;33:451–455. 67. Heinrich MC, Blanke CD, Druker BJ, Corlees CL. Inhibition of KIT tyrosine kinase activity: a novel molecular approach to the treatment of KIT-positive malignancies. J Clin Oncol 2002;20:1692–1703. 68. Joensuu H, Krause A, Demetri GD, et al. Gastrointestinal stromal tumor (GIST) patients who respond to imatinib (STI571, Gleevec) show marked decline of circulating levels of VRGF, KIT, and bFGF in serum, but not stem cell factor (SCF) levels. Proc Amer Soc Clin Oncol 2002; 38:abstract 552. 69. Kuenen B, Ruijter R, Hoekman K, Scigalla P, Giaccone G, Pinedo H. Dose finding study of SU6668 given thrice daily by oral route under fed conditions in patients with advanced malignancies. Proc Amer Soc Clin Oncol 2002; 38:437. 70. Britten CD, Rosen LS, Kabbinavar F, et al. Phase I trial of SU6668, a small molecule receptor tyrosine kinase inhibitor, given twice daily in patients with advanced cancers. Proc Amer Soc Clin Oncol 2002; 38:abstract 1922. 71. Brahmer JR, Kelsey S, Scigalla P, et al. A phase I study of SU6668 in patients with refractory solid tumors. Proc Amer Soc Clin Oncol 2002; 38:abstract 335. 72. Zahalsky AJ, Wong RJ, Lis E, et al. Phase II trial of SU5416 in patients with advanced incurable head and neck cancer. Proc Amer Soc Clin Oncol 2002; 38:abstract 902. 73. Mendel DB, Laird AD, Smolich BD, et al. Development of SU5416, a selective small molecule inhibitor of VEGF receptor tyrosine kinase activity, as an anti-angiogenic agent. Anti-Cancer Drug Design 2000;15:29–41. 74. Rosen LS. Angiogenesis inhibition in solid tumors. Cancer J 2001;7(suppl 3):S120–S128.

266

Teicher

75. Liekens S, De Clercq E, Neyts J. Angiogenesis: regulators and clinical applications. Biochem Pharmacol 2001;61:253–270. 76. Carter SK. Clinical strategy for the development of angiogenesis inhibitors. Oncologist 2000;5:51–54. 77. Teicher BA (ed). Antiangiogenic Agents in Cancer Therapy. Humana , Totowa, NJ: 1999. 78. Ellis LM, Liu W, Ahmad SA, et al. Overview of angiogenesis: biologic implications for antiangiogenic therapy. Semin Oncol 2001;28(5 suppl 16):94–104. 79. Jain RK. Normalizing tumor vasculature with anti-angiogenic therapy: a new paradigm for combination therapy. Nature Med 2001;7:987–989. 80. Miller KD, Sweeney CJ, Sledge GW. Redefining the target: chemotherapeutics as antiangiogenics. J Clin Oncol 2001;19:1195–1206. 81. Beecken W-DC, Fernandez A, Joussen AM, et al. Effect of antiangiogenic therapy on slowly growing, poorly vascularized tumors in mice. J Natl Cancer Inst 2001;93:382–387. 82. Kerbel RS, Viloria-Petit A, Klement G, Rak J. Accidental antiangiogenic drugs, antioncogene directed signal transduction inhibitors and conventional chemotherapeutic agents as examples. Eur J Cancer 2000;36:1248–1257. 83. Kerbel RS. Tumor angiogenesis: past, present and the near future. Carcinogenesis 2000;21:505–515. 84. Rosen L. Antiangiogenic strategies and agents in clinical trials. Oncologist 2000;5:20–27. 85. Gasparini G. The rationale and future potential of angiogenesis inhibitors in neoplasia. Drugs 1999;58:17–38. 86. Cherrington JM, Strawn LM,Shawver LK. New paradigms for the treatment of cancer: The role of anti-angiogenesis agents. Adv Cancer Res 2000;1–37. 87. Shepherd FA. Angiogenesis inhibitors in the treatment of lung cancer. Lung Cancer 2001;34(suppl 3):S81–S89. 88. Fong TA, Shawver LK, Sun L, et al. SU5416 is a potent and selective inhibitor of the vascular endothelial growth factor (Flk-1/KDR) that inhibitors tyrosine kinase catalysis, tumor vascularization and growth of multiple tumor types. Cancer Res 1999;59:99–106. 89. Shaheen RM, Davis DW, Liu W, et al. Antiangiogenic therapy targeting the tyrosine kinase receptor for vascular endothelial growth factor receptor inhibits the growth of colon cancer liver metastasis and induces tumor and endothelial cell apoptosis. Cancer Res 1999;59:5412–5416. 90. Mendel DB, Schreck RE, West DC, et al. The angiogenesis inhibitor SU5416 has longlasting effects on vascular endothelial growth factor receptor phosphorylation and function. Clin Cancer Res 2000;6:4848–4858. 91. Laird AD, Vajkoczy P, Shawver LK, et al. SU6668 is a potent antiangiogenic and antitumor agent that induces regression of established tumors. Cancer Res 2000;60:4152–4160. 92. Geng L, Donnelly E, McMahon G, et al. Inhibition of vascular endothelial growth factor receptor signaling leads to reversal of tumor resistance to radiotherapy. Cancer Res 2001;61:2413–2419. 93. Ning S, Laird D, Cherrington JM, Knox SJ. The antiangiogenic agents SU5416 and SU6668 increase the antitumor effects of fractionated irradiation. Radiat Res 2002;157:45–51. 94. Smolich BD, Yuen HA, West KA, Giles FJ, Albitar M, Cherrington JM. The antiangiogenic protein kinase inhibitors SU5416 and SU6668 inhibit the SCF receptor (c-kit) in a human myeloid leukemia cell line and in acute myeloid leukemia blasts. Blood 2001;97:1413–1421. 95. O’Farrell A-M, Yuen HA, Louie SG, et al. Analysis of mechanism of action and biomarkers for kinase inhibitor SU5416 in AML patients. Proc Amer Soc Clin Oncol 2002; 38:abstract 1077. 96. Krystal GW, Honsawek S, Kiewlich D, et al. Indoline tyrosine kinase inhibitors block kit activation and growth of small cell lung cancer cells. Cancer Res 2001;61:3660–3668. 97. Fiedler W, Staib P, Kuse R, et al. Role of angiogenesis inhibitors in acute myeloid leukemia Cancer J 2001;7(suppl 3):S129–S133.

Chapter 10 / Signal Transduction Inhibitor Evaluation

267

98. Mesters RM, Padro T, Bieker R, et al. Stable remission after administration of the receptor tyrosine kinase inhibitor SU5416 in a patient with refractory acute myeloid leukemia. Blood 2001;98:241–243. 99. Harris AL. Von Hippel-Landau syndrome: target for anti-vascular endothelial growth factor (VEGF) receptor therapy. Oncologist 2000;5(suppl 1):32–36. 100. Rosen LS. Clinical experience with angiogenesis signaling inhibitors: focus on vascular endothelial growth factor (VEGF) blockers. Cancer Control 2002;9(suppl 2):36–44. 101. Kuenen BC, Rosen L, Smit EF, et al. Dose-finding and pharmacokinetics study of cisplatin, gemcitabine and SU5416 in patients with solid tumors. J Clin Oncol 2002;20:1657–1667. 102. Hoekman K, Kuenen B, Levi M, et al. Activation of the coagulation cascade and endothelial cell perturbation during treatment with cisplatin, gemcitabine, and the angiogenesis inhibitor SU5416. Proc Amer Soc Clin Oncol 2002; 38:abstract 21. 103. Aklilu M, Kindler HL, Gajewski TF, Vogelzang NJ, Vokes EE, Stadler WM. Toxicities of the antiangiogenic agent SU5416 in phase II studies. Proc Amer Soc Clin Oncol 2002; 38:abstract 1921. 104. Wood JM, Bold G, Buchdunger E, et al. PTK787/ZK 222584, a novel and potent inhibitor of vascular endothelial growth factor receptor tyrosine kinases, impairs vascular endothelial growth factor-induced responses and tumor growth after oral administration. Cancer Res 2000;60:2178–2189. 105. Drevs J, Hofmann I, Hugenschmidt H, et al. Effects of PTK787/ZK 222584, a specific inhibitor of vascular endothelial growth factor receptor tyrosine kinases, on primary tumor, metastasis, vessel density and blood flow in a murine renal cell carcinoma model. Cancer Res 2000;60:4819–4824. 106. Yung WKA, Friedman H, Jackson E, et al. A phase I trial of PTK787/ZK222584 (PTK/ZK), a novel oral VEGFR TK inhibitor in recurrent glioblastoma. Proc Amer Soc Clin Oncol 2002; 38:abstract 315. 107. Drevs J, Schmidt-Gersbach CI, Mross K, et al. Surrogate markers for the assessment of biological activity of the VEGF-receptor inhibitor PTK787/ZK222584 (PTK/ZK) in two clinical phase I trials. Proc Amer Soc Clin Oncol 2002; 38:abstract 337. 108. Hurwitz H, Holden SN, Eckhardt SG, et al. Clinical evaluation of ZD6474, an orally active inhibitor of VEGF signaling, in patients with solid tumors. Proc Amer Soc Clin Oncol 2002; 38:abstract 325. 109. Schlaeppi JM, Wood JM. Targeting vascular endothelial growth factor (VEGF) for antitumor therapy, by anti-VEGF neutralizing monoclonal antibodies or by VEGF receptor tyrosine-kinase inhibitors. Cancer Met Rev 1999;18:473–481. 110. Borgstroem P, Gold DP, Hillan KJ, Ferrara N. Importance of VEGF for breast cancer angiogenesis in vivo: implications from intravital microscopy of combination treatments with an anti-VEGF neutralizing monoclonal antibody and doxirubicin. Anticancer Res 1999;19:4203–4214. 111. Yang JC, Haworth L, Steinberg SM, Rosenberg SA, Novotny W. A randomized double-blind placebo-controlled trial of bevacizumab (anti-VEGF antibody) demonstrating a prolongation in time to progression in patients with matastatic renal cancer. Proc Amer Soc Clin Oncol 2002; 38:abstract 15. 112. Goekjian PG, Jirousek MR. Protein kinase C inhibitors as novel anticancer drugs. Exp Opin Invest Drugs 2001;10:2117–2140. 113. O’Brian CA, Ward NE, Stewart JR, Chu F. Prospects for targeting protein kinase C isozymes in the therapy of drug-resistant cancer- an evolving story. Cancer Mets Rev 2001;20:95–100. 114. Swannie HC, Kaye SB. Protein kinase C inhibitors. Curr Oncol Rep 2002;4:37–46. 115. Way KJ, Chou E, King GL. Identification of PKC-isoform-specific biological actions using pharmacological approaches. TiPS 2000;21:181–187. 116. Nishizuka Y, Intracellular signaling by hydrolysis of phospholipids and activation of protein kinase C. Science 1992;8:607–614.

268

Teicher

117. Blumberg PM, Acs P, Bhattacharyya DK, Lorenzo PS. Inhibitors of protein kinase C and related receptors for the lipophilic second-messenger sn-1,2-diacylglycerol. In: (Gutkind JS, ed). Cell Cycle Control: The Molecular Basis of Cancer and Other Diseases. Humana, Totowa, NJ: 2000;349–366. 118. Shen B-Q, Lee DY, Zioncheck TF. Vascular endothelial growth factor governs endothelial nitric-oxide synthase expression via a KDR/flk-1 receptor and protein kinase C signaling pathway. J Biol Chem 1999;274:33,057–33,063. 119. Shih S-C, Mullen A, Abrams K, Mukhopadhyay M, Claffey KP. Role of protein kinase C isoforms in phorbol ester-induced vascular endothelial growth factor expression in human glioblastoma cells. J Biol Chem 1999;274:15,407–15,414. 120. Silva D, English D, Lyons D, Lloyd FP. Protein kinase C induces moltility of breast cancers by upregulating secretion of urokinase-type plsaminogen activator through activation of AP1 and NF-kappaB. Biochem Biophys Res Commun 2002;290:552–557. 121. Bhat-Nakshatri P, Sweeney CJ, Nakshatri H. Identification of signal transduction pathways involved in constitutive NF-kappaB activation in breast cancer cells. Oncogene 2002;21:2066–2078. 122. Kim YW, Hur SY, Kim TE, et al. Protein kinase C modulates telomerase activity in human cervical cancer cells. Exp Molec Med 2001;33:156–163. 123. Flescher E, Rotem R. Protein kinase C epsilon mediates the induction of P-glycoprotein in LNCaP prostate carcinoma cells. Cellular Signaling 2002;14:37–43. 124. Sumitomo M, Ohba M, Asakuma J, et al. Protein kinase C delta amplifies ceramide formation via mitochondrial signaling in prostate cancer cells. J Clin Invest 2002;109:827–836. 125. Ghosh PM, Bedolla R, Mikhailova M, Kreisberg JI. RhoA-dependent murine prostate cancer cell proliferation and apoptosis: role of protein kinase C zeta. Cancer Res 62:2630–2636, 2002. 126. Lin MF, Zhang XQ, Dean J, Lin FF. Protein kinase C pathway is involved in regulating the secretion of prostatic phosphatase in human prostate cancer cells. Cell Biol Int 2001;25:1139–1148. 127. Da Rocha AB, Mans DR, Regner A, Schwartsmann G. Targeting protein kinase C: a new therapeutic opportunity against high-grade malignant glioma? Oncologist 2002;7:17–33. 128. Andratschke N, Grosu AL, Molls M, Nieder C. Perspectives in the treatment of malignant gliomas in adults. Anticancer Res 2001;21:3541–3550. 129. Ellis LM, Takahashi Y, Liu W, Shaheen RM. Vascular endothelial growth factor in human colon cancer: biology and therapeutic implications. Oncologist 2000;5(suppl. 1):11–15. 130. Xia P, Aiello LP, Ishii H, et al. Characterization of vascular endothelial growth factor’s effect on the activation of protein kinase C, its isoforms, and endothelial cell growth. J Clinical Invest 1996;98:2018–2026. 131. Guo D, Jia Q, Song HY, Warren RS, Donner DB. Vascular endothelial cell growth factor promotes tyrosine phosphorylation of mediators of signal transduction that contain SH2 domains. Association with endothelial cell proliferation. J Biol Chem 1995;270:6729–6733. 132. Sawano A, Takahashi T, Yamaguchi S, Shibuya M. The phosphorylated 1169-tyrosine containing region of flt-1 kinase (VEGFR-1) is a major binding site for PLCgamma. Biochem Biophys Res Commun 1997;238:487–491. 133. McMahon G. VEGF receptor signaling in tumor angiogenesis. The Oncologist 2000;5(suppl1):3–11. 134. Buchner K. The role of protein kinase C in the regulation of cell growth and in signaling to the cell nucleus. J Cancer Res Clin Oncol 2000;126:1–11. 135. Martelli AM, Sang N, Borgatti P, Capitani S, Neri LM. Multiple biological responses activated by nuclear protein kinase C. J Cellular Biochem 1999;74:499–521. 136. Jirousek MR, Gillig JR, Gonzalez CM, et al. (S)-13-[(dimethylamino)methyl]-10,11,14,15tetrahydro-4,9:16, 21-dimetheno-1H, 13H-dibenzo[e,k]pyrrolo[3,4-h][1,4,13]oxadiazacyclohexadecene-1,3(2H)-dione (LY333531) and related analogues: isozyme selective inhibitors of protein kinase C beta. J Med Chem 1996;39:2664–2671. 137. Danis RP, Bingaman DP, Jirousek M, Yang Y. Inhibition of intraocular neovascularization caused by retinal ischemia in pigs by PKCbeta inhibition with LY333531, Investigative Ophthalmol Visual Sci 1998;39:171–179.

Chapter 10 / Signal Transduction Inhibitor Evaluation

269

138. Aiello LP, Bursell SE, Clermont A, et al. Vascular endothelial growth factor-induced retinal permeability is mediated by protein kinase C in vivo and suppressed by an orally effective beta-isoform-selective inhibitor. Diabetes 1997;46:1473–80. 139. Ishii H, Jirousek MR, Koya D, et al. Amelioration of vascular dysfunctions in diabetic rats by an oral PKC beta inhibitor [see comments]. Science 1996;272:728–731. 140. Yoshiji H, Kuriyama S, Ways DK, et al. Protein kinase C lies on the signaling pathway for vascular endothelial growth factor-mediated tumor development and angiogenesis. Cancer Res 1999;59:4413–4418. 141. Teicher BA, Alvarez E, Mendelsohn LG, Ara G, Menon K, Ways KD. Enzymatic rationale and preclinical support for a potent protein kinase Cb inhibitor in cancer therapy. Advs Enzyme Regul 1999;39:313–327. 142. Sausville EA, Arbuck SG, Messmann R, et al. Phase I trial of 72-hour continuous infusion UCN-01 in patients with refractory neoplasms. J Clin Oncol 2001;19:2319–2333. 143. Sausville EA, Elsayed Y, Monga M, Kim G. Signal transduction-directed cancer treatments. Ann Rev Pharmacol Toxicol 2003;43:199–231. 144. Grosios K. UCN-01 Kyowa Hakko Kogyo Co. Curr Opin Invest Drugs 2001;2:287–297. 145. Abe S, Kubota T, Otani Y, et al. UCN-01 (7-hydroxystaurosporine) inhibits in vivo growth of human cancer cells through selective perturbation of G1 phase checkpoint machinery. Jpn J Cancer Res 2001;92:537–545. 146. Akinaga S, Gomi K, Morimoto M, Tamaoki T, Okabe M. Antitumor activity of UCN-01, a selective inhibitor of protein kinase C, in murine and human models. Cancer Res 1991;51:4888–4892. 147. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst 2000;92:376–387. 148. Akiyama T, Yoshida T, Tsujita T, et al. G1 phase accumulation induced by UCN-01 is associated with dephosphorylation of Rb and CDK2 proteins as well as induction of CDK inhibitor p21/Cip1/WAF1/Sdi1 in p53-mutated human epidermoid carcinoma A431 cells. Cancer Res 1997;57:1495–1501. 149. Sugiyama K, Akiyama T, Shimizu M, et al. Decrease in susceptibility toward induction of apoptosis and alteration in G1 checkpoint function as determinants of resistance of human lung cancer cells against the antisignaling drug UCN-01 (7-hydroxystaurosporine). Cancer Res 1999;59:4406–4412. 150. Sarkaria JN, Busby EC, Tibbetts RS, et al. Inhibition of ATM and ATR kinase activities by the radiosensitizing agent, caffeine. Cancer Res 1999;59:4375–4382. 151. Busby EC, Leistritz DF, Abraham RT, Karnitz LM, Sarkaria JN. The radiosensitizing agent 7-hydroxystaurosporine (UCN-01) inhibits the DNA damage checkpoint kinase hChk1. Cancer Res 2000;60:2108–2112. 152. Graves PR, Yu L, Schwarz JK, et al. The Chk1 protein kinase and the Cdc25C regulatory pathways are targets of the anticancer agent UCN-01. J Biol Chem 2000;275:5600–5605. 153. Chen X, Lowe M, Keyomarsi K. UCN-01-mediated G1 arrest in normal but not tumor breast cells is Rb-dependent and p53-independent. Oncogene 1999;18:5691–5702. 154. Kruger EA, Blagosklonny MV, Dixon SC, Figg WD. UCN-01, a protein kinase C inhibitor, inhibits endothelial cell proliferation and angiogenic hypoxic response. Invasion Metastasis 1999;18:209–218. 155. Teicher BA, Alvarez E, Menon K, et al. Antiangiogenic effects of a protein kinase C betaselective small molecule. Cancer Chemo Pharmacol 2002;49:69–77. 156. Thornton DE, Keyes K, Mann L, et al. Determination of cancer growth factor biomarkers in plasma from mice bearing subcutaneous human tumor xenografts using Luminex Multiplex technology. Proc Amer Assoc Cancer Res 2002;43:897. 157. Dudek AZ, Malhaseth H. Time course of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), endostatin and thrombospondin-1 (TSP1) levels in non-

270

158.

159.

160.

161.

162.

163.

164.

165. 166. 167. 168. 169. 170. 171. 172. 173. 174.

175.

176.

Teicher small cell lung cancer (NSCLC) patients during chemotherapy and their correlation to patient survival. Proc Amer Soc Clin Oncol 2002; abstract 2969. Brattstrom D, Bergqvist M, Wagenius G, Hesselius P, Larsson A, Brodin O. Repeated measurements of serum bFGF and VEGF during therapy for non-small cell lung cancer. Proc Amer Soc Clin Oncol 2002; abstract 2992. Mani S, Rudin CM, Kunkel K, et al. Phase I clinical and pharmacokinetic study of protein kinase C-alpha antisense oligonucleotide ISIS 3521 administered in combination with 5-fluorouracil and leucovorin in patients with advanced cancer. Clin Cancer Res 2002;8:1042–1048. Go RS, Horstman AL. Circulating basic fibroblast growth factor (b-FGF) and vascular endothelial growth factor (VEGF) levels in cancer patients: implications for anti-angiogenic therapy. Proc Amer Soc Clin Oncol 2002; abstract 3031. Teicher BA, Menon K, Alvarez E, Galbreath E, Shih C, Faul MM. Antiangiogenic and antitumor effects of a protein kinase C beta inhibitor in human HT-27 colon carcinoma and human Caki-1 renal cell carcinoma xenografts. Anticancer Res 2001;21:3175–3184. Teicher BA, Menon K, Alvarrez E, Galbreath E, Shih C, Faul MM. Antiangiogenic and antitumor effects of a protein kinase C beta inhibitor in murine Lewis lung carcinoma and human Calu-6 non-small cell lung carcinoma xenografts. Cancer Chemother Pharmacol 2001;48:473–480. Teicher BA, Menon K, Alvarez E, Liu P, Shih C, Faul MM. Antiangiogenic and antitumor effects of a protein kinase C beta inhibitor in human hepatocellular and gastric cancer xenografts. In Vivo 2001;15:185–193. Teicher BA, Menon K, Alvarez E, Galbreath E, Shih C, Faul MM. Antiangiogenic and antitumor effects of a protein kinase C Beta inhibitor in human T98G glioblastoma multiforme xenografts. Clin Cancer Res 2001;7:634–640. Herbst RS, Thornton DE, Kies MS, Sinha V, Flanagan S, Cassidy CA, Carducci MA. Phase 1 study of LY317615, a protein kinase C` inhibitor. Amer Soc Clin Oncol 2002; abstract 326. Killough JH, Magill GB, Smith RC. Treatment of amebiasis with fumagillin. Science 1952;115:71–72. Katznelson H, Jamieson CA. Control of nosema disease of honeybees with fumagillin. Science 1952;115, 70–71. Brem H, Ingber D, Blood CH, Bradley D, Urioste S, Folkman J. Suppression of tumor metastasis by angiogenesis inhibition. Surg Forum 1991;42:439–441. Ingber D, Fujita T, Kishimoto S, et al. Synthetic analogues of fumagillin that inhibit angiogenesis and suppress tumor growth. Nature 1990;348:555–557. Kusaka M, Sudo K, Fujita T, et al. Potent antiangiogenic action of AGM-1470, comparison to the fumagillin parent. Biochem Biophys Res Commun 1991;174:1070–1076. Brem H, Folkman J. Analysis of experimental antiangiogenic therapy. J Ped Surg 1993;28:445–451. Beecken WD, Fernandez A, Panigrahy D, et al. Efficacy of antiangiogenic therapy with TNP470 in superficial and invasive bladder cancer models in mice. Urology 2000;56:521–526. Brem H, Gresser I, Grossfeld J, Folkman J. Combination of antiangiogenic agents to inhibit primary tumor growth and metastasis. J Ped Surg 1993;28:445–451. Kamei S, Okada H, Inoue Y, Yoshioka T, Ogawa Y, Toguchi H. Antitumor effects of angiogenesis inhibitor TNP-470 in rabbits bearing VX-2 carcinoma by arterial administration of microspheres and oil solution. J Pharmacol Exper Ther 1993;264:469–474. Yamaoka M, Yamamoto T, Masaki T, Ikeyama S, Sudo K, Fujita T. Inhibition of tumor growth and metastasis of rodent tumors by the angiogenesis inhibitor O-(chloroacetylcarbamoyl)fumagillin (TNP-470; AGM-1470). Cancer Res 1993;53:4262–4267. Konno H. Antitumor effect of the angiogenesis inhibtor TNP-470 on human digestive organ malignancy. Cancer Chemother Pharmacol 1999;43:S85–S89.

Chapter 10 / Signal Transduction Inhibitor Evaluation

271

177. Ogawa H, Sato Y, Kondo M, et al. Combined treatment with TNP-470 and 5-fluorouracil effectively inhibits growth of murine colon cancer cells in vitro and liver metastasis in vivo. Oncol Reps 2000;7:467–472. 178. Takatsuka S, Yamada N, Sawada T, et al. Contribution of angiogenesis to the progression of colon cancer: possible inhibitory effect of angiogenesis inhibitor TNP-470 on tumor growth and hepatic metastasis. Int J Oncol 2000;17:253–258. 179. Lund EL, Bastholm L, Kristjansen PE. Therapeutic synergy of TNP-470 and ionizing radiation: effects on tumor growth, vessel morphology and angiogenesis in human glioblastoma multiforme xenografts. Clin Cancer Res 2000;6:971–978. 180. Teicher BA, Holden SA, Ara G, et al. Potentiation of cytotoxic cancer therapies by TNP-470 alone and with other antiangiogenic agents. Int J Cancer 1994;57:920–925. 181. Teicher BA, Dupuis NP, Robinson M, Emi Y, Goff D. Antiangiogenic treatment (TNP-470/ minocycline) increases tissue levels of anticancer drugs in mice bearing Lewis lung carcinoma Oncol Res 1995;7:237–243. 182. Teicher BA, Alvarez E, Huang ZD, et al. b-Cyclodextrin tetradecasulfate/tetrahydrocortisol + minocycline as modulators of cancer therapies in vitro and in vivo against primary and metastatic Lewis lung carcinoma. Cancer Chemother Pharmacol 1993;33:229–238. 183. Teicher BA, Holden SA, Ara G, Northey D. Response of the FsaII fibrosarcoma to antiangiogenic modulators plus cytotoxic agents. AntiCancer Res 1993;13:2101–2106. 184. Alvaez Sotomayor E, Teicher BA, Schwartz GN, et al. Minocycline in combination with chemotherapy or radiation therapy in vitro and in vivo. Cancer Chemother Pharmacol 1992;30:377–384. 185. Teicher BA, Alvarez E, Huang ZD. Antiangiogenic agents potentiate cytotoxic cancer therapies against primary and metastatic disease. Cancer Res 1992;52:6702–6704. 186. Teicher BA, Dupuis N, Kusomoto T, et al. Antiangiogenic agents can increase tumor oxygenation and response to radiation therapy. Radiat Oncol Invest 1995;2:269–276. 187. Teicher BA, Holden SA, Dupuis NP, et al. Potentiation of cytotoxic therapies by TNP-470 and minocycline in mice bearing EMT-6 mammary carcinoma. Breast Cancer Res Treat 1995;36:227–236. 188. Teicher BA, Holden SA, Ara G, et al. Influence of an antiangiogenic treatment on 9L gliosarcoma: oxygenation and response to cytotoxic therapy. Int J Cancer 1995;61:732–737. 189. Chang A, Garrow G. Pilot study of vinorelbine (Navelbine) and paclitaxel (Taxol) in patients with refractory breast cancer and lung cancer. Semin Incol 1995;22(suppl5):66. 190. Murphy W, Fossella F, Winn R. Phase II study of taxol in patients with untreated advanced non–small cell lung cancer. J Natl Cancer Inst 8:384, 1993. 191. Johnson D, Paul D, Hande K. Paclitaxel plus carboplatin in advanced non-small cell lung cancer. J Clin Oncol 1996;14:2054. 192. Langer C, Leighton J, Comis R. Paclitaxel and carboplatin in combination in the treatment of advanced non-small cell lung cancer: a phase II toxicity, response and survival analysis. J Clin Oncol 1995;12:1860. 193. Langer C, Leighton J, Comis R. Taxol and carboplatin in combination in stage I., V. and IIIB non-small cell lung cancer (NSCLC): a phase II trial. Proc Am Soc Clin Oncol 1994;13:338. 194. Rowinsky EK, Sartorious SE, Bowling MK. Paclitaxel on a 3-hour schedule and carboplatin in non-small cell lung cancer, use of maximally tolertated and clinically relevant single-agent doses in combination is feasible. Proc Am Soc Clin Oncol 1995;14:1075. 195. Muggia FM, Vafai D, Natale R, et al. Paclitaxel 3-hour infusion given alone and combined with carboplatin: preliminary results of dose-escalation trials. Semin Oncol 1995;22(4 Suppl 9):63–66. 196. Herbst RS, Takeuchi H, Teicher BA. Paclitaxel/carboplatin administration along with antiangiogenic therapy in non-small cell lung and breast carcinoma models. Cancer Chemother Pharmacol 1998;41:497–504.

272

Teicher

197. Gasparini G. The rationale and future potential of angiogenesis inhibitors in neoplasia. Drugs 1999;58:17–38. 198. Carter SK, Clinical strategy for the development of angiogenesis inhibitors. Oncologist 2000;5(suppl 1):51–54. 199. Toppmeyer D. Phase I trial design and methodology. In: (Teicher BA, ed). Anticancer Drug Development: Preclinical Screening, Clinical Trial, and Approval. Humana, Totowa NJ: 1997:227–247. 200. Kudelka AP, Levy T, Verschraegen CF, et al. A phase I study of TNP-470 administered to patients with advanced squamous cell cancer of the cervix. Clin Cancer Res 1997;3:1501–1505. 201. Moore JD, Dezube BJ, Gill P, Zhou XJ, Acosta EP, Sommadossi JP. Phase I dose escalation pharmacokinetics of O-(chloroacetylcarbamoyl)fumagillol (TNP-470) and its metabolites in AIDS patients with Kaposi’s sarcoma. Cancer Chemother Pharmacol 2000;46:173–179. 202. Offodile R, Walton T, Lee M, Stiles A, Nguyen M. Regression of metastatic breast cancer in a patient treated with the antiangiogenic drug TNP-470. Tumori 1999;85:51–53. 203. Bhargava P, Marshall JL, Rizvi N, et al. A phase I and pharmacokinetic study of TNP-470 administered weekly to patients with advanced cancer. Clin Cancer Res 1999;5:1989–1995. 204. Dezube BJ, Von Roenn JH, Holden-Wiltse J, et al. Fumagillin analog in the treatment of Kaposi’s sarcoma: a phase I AIDS clinical trial group study. J Clin Oncol 1998;16:1444–1449. 205. Blumenschein GR, Fossella FV, Pisters KM, et al. A phase I study of TNP-470 continuous infusion alone or in combination with paclitaxel and carboplatin in adult patients with NSCLC and other solid tumors. Proc Amer Soc Clin Oncol 2002; 38:abstract 1254. 206. Senderowicz AM, Sausville EA. Preclinical and clinical development of cyclin-dependent kinase modulators. J Natl Cancer Inst 2000;92:376–387. 207. Senderowicz AM. Modulators of cyclin-dependent kinases. In: (Giordano A, Soprano KJ, eds). Cell Cycle Inhibitors in Cancer Therapy: Current Strategies. Humana, Totowa NJ: 2002:177–202. 208. Imoto M, Doki Y, Jiang W, Han EKH, Weinstein B. Effects of cyclin D1 overexpression on G1 progression-related events. Exp Cell Res 1997;236:173–180. 209. Barnes DM, Gillett CE. Cyclin D1 in breast cancer. Breast Cancer Res Treat 1998;52:1–15. 210. Kamalati T, Davies D, Titley J, Crompton MR. Functional consequences of cyclin D1 overexpression in human mammary luminal epithelial cells. Clin Exp Metastasis 1998;16:415–426. 211. Steeg PS, Zhou Q. Cyclins and breast cancer. Breast Cancer Res Treat 1998;52:17–28. 212. Landberg G, Roos G. The cell cycle in breast cancer. APMIS 1997;105:575–589. 213. Alle KM, Henshall SM, Field AS, Sutherland RL. Cyclin D1 protein is over expressed in hyperplasia and intraductal carcinoma of the breast. Clin Cancer Res 1998;4:847–854. 214. Maeda K, Ching YS, Kang SM, et al. Cyclin D1 overexpression and prognosis in colorectal adenocarcinoma. Oncology 1998;55:145–151. 215. Palmqvist R, Stenling R, Oberg A, Landberg G. Expression of cyclin D1 and retinoblastoma protein in colorectal cancer. Europ J Cancer 1998;34:1575–1581. 216. Kyomoto R, Kumazawa H, Toda Y, et al. Cyclin-D1-gene amplification is a more potent prognostic factor than its protein over-expression in human head-and-neck squamous-cell carcinoma. Int J Cancer (Pred Oncol) 1997;74:576–581. 217. Pignataro L, Pruneri G, Carboni N, et al. Clinical relevance of cyclin D1 protein overexpression in laryngeal squamous cell carcinoma. J Clin Oncol 1998;16:3069–3077. 218. Sarbia M, Stahl M, Fink U, et al. Prognostic significance of cyclin D1 in esophageal squamous cell carcinoma patients treated with surgery alone or combined therapy modalities. Int J Cancer (Pred Oncol) 1999;84:86–91. 219. Kitahara K, Yasui W, Yokozaki H, et al. Expression of cyclin D1, CDK4 and p27 kip1 is associated with the p16MTS1 gene status in human esophageal carcinoma cell lines. J Exp Therap Oncol 1996;1:7–12.

Chapter 10 / Signal Transduction Inhibitor Evaluation

273

220. Mueller A, Odze R, Jenkins TD, et al. A transgenic mouse model with cyclin D1 overexpression results in cell cycle, epidermal growth factor receptor and p53 abnormalities. Cancer Res 1997;57:5542–5549. 221. Nakagawa H, Wang TC, Zukerberg L, et al. The targeting of the cyclin D1 oncogene by an Epstein-Barr virus promoter in transgenic mice causes dysplasia in the tongue, esophagus and forestomach. Oncogene 1997;14:1185–1190. 222. Kim SH. Inhibition of cyclin-dependent kinases by purine analogues: crystal structure of human cdk2 complexed with roscovitine. Eur J Biochem 1997;243:518–526. 223. Yao J, Pollock RE, Lang A, et al. Infrequent mutation of the p16/MTS1 gene and overexpression of cyclin-dependent kinase 4 in human primary soft-tissue sarcoma. Clin Cancer Res 1998;4:1065–1070. 224. Marchetti A, Doglioni C, Barbareschi M, et al. Cyclin D1 and retinoblastoma susceptibility gene alterations in non-small cell lung cancer. Int J Cancer 1998;75:187–192. 225. Schutte M, Hruban RH, Geradts J, et al. Abrogation of the Rb/p16 tumor-supressive pathway in virtually all pancreatic carcinomas. Cancer Res 1997;57:3126–3130. 226. Dreyling MH, Bullinger L, Ott G, et al. Alterations of the cyclin D1/p16-pRB pathway in mantle cell lymphoma. Cancer Res 1997;57:4608–4614. 227. Taniguchi T, Fujita A, Takahashi S, et al. Cyclin D1 overexpression detected by a simple competitive reverse transcription-polymerase chain reaction assay for lymphoid malignancies. Jpn J Cancer Res 1998;89:159–166. 228. Coleman KG, Lyssikatos JP, Yang BV. Chapter 17. Chemical inhibitors of cyclin-dependent kinases. Ann Reps Med Chem 1997;32:171–179. 229. Collins K, Jacks T, Pavletich NP. The cell cycle and cancer. Proc Natl Acad Sci USA 1997;94:2776–2778. 230. De Azevedo WF, Leclerc S, Meijer L, Havlicek L, Strand M, Kim SH. Inhibition of cyclindependent kinases by purine analogues: Crystal structure of human cdk2 complexed with roscovitine. Eur J Biochem 1997;243:518–526. 231. Orr MS, Reinhold W, Yu L, Schreiber-Agus N, O’Connor PM. An important role for the retinoblastoma protein in staurosporine-induced G1 arrest in murine embryonic fibroblasts. J Biol Chem 1998;278:3803–3807. 232. Kakeya H, Onose R, Liu PCC, Onozawa C, Matsumura F, Osada H. Inhibition of cyclin D1 expression and phosphorylation of retinoblastoma protein by phosmidosine, a nucleotide antibiotic. Cancer Res 1998;58:704–710. 233. Harper JW. Cyclin dependent kinase inhibitors. Cancer Surveys 1997;29:91–107. 234. Garrett MD, Fattaey A. CDK inhibition and cancer therapy. Current Opin Genetics Develop 1999;9:104–11. 235. Mgbonyebi OP, Russo J, Russo IH. Roscovitine induces cell death and morphological change indicative of apoptosis in MDA-MB-231 breast cancer cells. Cancer Res 1999;59:1903– 1910. 236. Sedlacek HH, Czech J, Naik R, et al. Flavopiridol (L86 8275; NSC 649890), a new kinase inhibitor for tumor therapy. Int J Cancer 1996;65:1143–1168. 237. Carlson BA, Dubay MM, Sausville EA, Brizuela L, Worland PJ. Flavopiridol induces G1 arrest with inhibition of cyclin-dependent kinase (CDK)2 and CDK4 in human breast carcinoma cells. Cancer Res 1996;56:2973–2978. 238. Bible KC, Kaufman SH. Flavopiridol: a cytotoxic flavone that induces cell death in noncycling A549 human lung carcinoma cells. Cancer Res 1996;56:4856– 4861. 239. Schrump DS, Matthews W, Chen GA, Mixon A, Altorki NK. Flavopiridol mediates cell cycle arrest and apoptosis in esophageal cancer cells. Clin Cancer Res 1998;4:2885–2890. 240. Lu K, Shih C, Teicher BA. Expression of pRb, cyclin/cyclin dependent kinases and E2F1/ DP1 in human tumor lines in cell culture and in xenograft tissues and response to cell cycle agents. Cancer Chemother Pharmacol 2000;46:293–304.

274

Teicher

241. Brusselbach S, Nettelbeck DM, Sedlacek HH, Muller R. Cell cycle-independent induction of apoptosis by the anti-tumor drug flavopiridol in endothelial cells. Int J Cancer 1998;77:146–152. 242. Senderowicz AM, Headlee D, Stinson SF, et al. Phase I trial of continuous infusion flavopiridol, a novel cyclin-dependent kinase inhibitor, in patients with refractory neoplasms. J Clin Oncol 1998;16:2986–2999. 243. Jager W, Zembsch B, Wolschann P, et al. Metabolism of the anticancer drug flavopiridol, a new inhibitor of cyclin dependent kinases in rat liver. Life Sciences 1998;62:1861–1873. 244. Gries J-M, Kasimis B, Schwarzenberger P, et al. Phase I study of HMR1275 (flvopiridol) in non–small cell lung cancer (NSCLC) patients after 24hr IV administration in combination with paclitaxel and carboplatin. Proc Amer Soc Clin Oncol 2002; 38:abstract 372. 245. Murthy A, Karp J, Bauer K, Passaniti A. Correlative studies of acute leukemia patients treated with flavopiridol: endothelial cell (EC) proliferation as a marker of antiangiogenesis. Proc Amer Soc Clin Oncol 2002; 38:abstract 1089. 246. Papadimitrakopoulou V, Henderson E, Breimer L, et al. A phase I clinical and pharmacokinetic study of oral Ro 31-7453, a novel cell cycle inhibitor, administered in combination with paclitaxel. Proc Amer Soc Clin Oncol 2002; 38:abstract 1830. 247. Moore M, Hirte H, Oza A, et al. Phase I study of the Raf-1 kinase inhibitor BAY 43-9006 in patients with advanced refractory solid tumors. Proc Amer Soc Clin Oncol 2002; 38:abstract 1816. 248. LoRusso PM, Adjei AA, Meyer MB, et al. A phase I clinical and pahrmacokinetic evaluation of the oral MEK inhibitor, CI-1040, administered for 21 consecutive days, repeated every 4 weeks in patients with advanced cancer. Proc Amer Soc Clin Oncol 2002; 38:abstract 321. 249. Rak J, Yu JL, Klement G, Kerbel RS. Oncogenes and angiogenesis: signaling three-dimensional tumor growth. J Invest Dermatol 2000;5:24–33. 250. Prendergast GC, Rane N. Farnesyltransferase inhibitors: mechanism and applications. Exp Opin Invest Drugs 2001;10:2105–2116. 251. Malaney S, Daly RJ. The ras signaling pathway in mammary tumorigenesis and metastasis. J Mammary Gland Biol Neoplasia 2001;6:101–113. 252. Lebowtiz PF, Casey PJ, Prendergast GC, Thissen JA. Farnesyltransferase inhibitors alter the prenylation and growth-stimulating function of RhoB. J Biol Chem 1997;272:15,591–15,594. 253. Lebowtitz PF, Prendergast GC. Non-ras targets of franesyltransferase inhibitors: focus on Rho. Oncogene 1998;17:1439–1445. 254. Adjei AA, Davis JN, Bruzek LM, Erlichman C, Kaufmann SH. Synergy of the protein farnesyl transferase inhbitor SCH66336 and cisplatin in human cancer cell lines. Clin Cancer Res 2001;7:1438–1445. 255. Shi B, Yaremko B, Hajian G, et al. The farnesyl protein transferase inhibitor SCH66336 synergizes with taxanes in vitro and enhances their antitumor activity in vivo. Cancer Chemother Pharmacol 2000;46:387–393. 256. Rowinsky EK, Windle JJ, Von Hoff DD. Ras protein farnesyltransferase: a strategic target for anticancer therapeutic development. J Clin Oncol 1999;17:3631–3652. 257. Liu M, Bryant MS, Chen J, et al. Antitumor activity of SCH66336, an orally bioavailable tricyclic inhibitor of farnesyl protein transferase, in human tumor xenograft models and wapras trangenic mice. Cancer Res 1998;58:4947–4956. 258. Eskens FALM, Stoter G, Verweij J. Farnesyl transferase inhibitors. Current developments and future perspectives. Cancer Treat Rev 2000;26:319–332. 259. Petit T, Izbicka E, Lawrence RA, Bishop WR, Weitman S, Von Hoff DD. Activity of SCH66336, a tricyclic farnesyltransferase inhibitor, against human tumor colony-forming units. Ann Oncol 1999;10:449–453. 260. Kirschmeier P, Carr D, Gray K. SCH66336, an orally bioavailable tricyclic farnesyl transferase inhibitor blocks anchorage-independent growth of ras transformed fibroblasts and human tumor cell lines. Proc Amer Assoc Cancer Res 1998;39:318.

Chapter 10 / Signal Transduction Inhibitor Evaluation

275

261. Ashar HR, James L, Gray K, et al. The farnesyl transferase inhibitor SCH66336 induces a G2->M or G1 pause in sensitive human tumor cell lines. Exp Cell Res 2001;262:17–27. 262. Sun J, Blaskovich MA, Knowles D. Antitumor efficacy of a novel class of non-thiol-containing petidomimetic inhibitors of farnesyltransferase and geranylgeranyltransferase I: combination therapy with cytotoxic agents cisplatin, taxol and gemcitabine. Cancer Res 1999;59:4919–4926. 263. Prevost GP, Pradines A, Brezak M-C, et al. Inhibition of human tumor cell growth in vivo by an orally bioavailable inhibitor of human farnesyltransferase, BIM-46228. Int J Cancer 2001;91:718–722. 264. Cohen-Jonathan E, Evans SM, Koch CJ, et al. The farnesyltransferase inhibitor L774,832 reduces hypoxia in tumors expressing activated H-ras. Cancer Res 2001;61:2289–2293. 265. Nielsen LL, Shi B, Hajian G, et al. Combination therapy with the farnesyl protein transferase inhbitor SCH66336 and SCH58500 (p53 adenovirus) in preclinical cancer models. Cancer Res 1999;59:5896–5901. 266. Adjei AA, Erlichman C, Davis JN, et al. A phase I trial of the farnesyltransferase inhibitor SCH66336: evidence for biological and clinical activity. Cancer Res 2000;60:1871–1877. 267. Zujewski J, Horak ID, Bol CJ, et al. Phase I and phamracokinetic study of farnesyl protein tranferase inhibitor R115777 in advanced cancer. J Clin Oncol 2000;18:927–941. 268. Johnston SRD. Farnesyltransferase inhibitors: a novel targeted therapy for cancer. Lancet Oncol 2001;2:18–26. 269. Sebti SM, Hamilton AD. Farnesyltransferase and geranylgeranyltransferase I inhibitors in cancer therapy: important mechanistic and bench to bedside issues. Exp Opin Invest Drugs 2000;9:2767–2782. 270. Eskens FALM, Awada A, Cutler DL, et al. Phase I and phaemacokinetic study of the oral farnesyltransferase inhibitor SCH66336 given twice daily to patients with advanced solid tumors. J Clin Oncol 2001;19:1167–1175. 271. Lipton A, Ready N, Bukowski RM, et al. Phase I study of continuous oral lonafarib plus weekly paclitaxel for advanced cancer. Proc Amer Soc Clin Oncol 2002; 38:abstract 364. 272. Sprague E, Vokes EE, Garland LL, et al. Phase I study of continuous lonafarnib plus paclitaxel and carboplatin in refractory or advanced solid tumors. Proc Amer Soc Clin Oncol 2002; 38:abstract 1920. 273. Lobell RB, Liu D, Buser CA, et al. Preclinical and clinical pharmacodynamic assessment of L-778,123, a dual inhbitor of farnesyl:protein transferase and geranylgeranyl:protein transferase type-I. Mol Cancer Therap 2002;1:747–758. 274. Reuter CW, Morgan MA, Bergmann L. Targeting the ras signalling pathway: a rational mechanism-based treatment for hematologic malignancies? Blood 2000;96:1655–1669. 275. Kelland LR, Smith V, Valenti M, et al. Preclinical antitumor activity and pharmacodynamic studies with the farnesyl protein transferase inhibitor R11577 in human breast cancer. Clin Cancer Res 2001;7:3544–3550. 276. Karp JE, Kauffmann SH, Adjei AA, Lancet JE, Wright JJ, End DW. Current status of clinical trials of farnesyl transferase inhibitors. Curr Opin Oncol 2001;13:470–476. 277. Johnson JI, Decker S, Zaharevitz D, et al. Relationships between drug activity in NCI preclinical in vitro and in vivo models and early clinical trials. Brit J Cancer 2001;84:1424–1431. 278. Teicher BA. Tumor Models in Cancer Therapy. Humana, Totowa, NJ: 2002. 279. Corbett TH, Valeriote FA, LaRusso P, et al. In vivo methods for screening and Preclinical testing: use of rodent solid tumors for drug discovery. In: (Teicher BA, ed) Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials and Approval. Humana, Totowa, NJ: 1997:75–100. 280. Plowman J, Dykes DJ, Hollingshead M, Simpson-Herren L, Alley MC. Human tumor xenograft models in NCI drug development. In: Anticancer Drug Development Guide: Preclinical Screening, Clinical Trials and Approval (Teicher BA, ed). Humana, Totowa, NJ: 1997:101–126.

Index

277

Index

Azidothymidine (AZT), fluorinated pyrimidine cytotoxicity augmentation, 155, 156 AZT, see Azidothymidine

A ABX-EGF, epidermal growth factor receptor inhibition, 11 17-Allylamino-geldanamycin, HER2/neu inhibition, 190 F-particle therapy, see Radioimmunoconjugates Antisense oligonucleotides, Bcl-2 targeting, see G3139 cancer treatment rationale, 212 gene transfection, 221, 222 mechanism of action, 213, 214 modified oligonucleotides, 212, 213 nonspecific effects, 214 protein kinase C inhibition, 73, 74 Apoptosis, Bcl-2 regulation, 210, 211 cell features, 37, 209, 210 flavopiridol induction, 33 leukemia cell susceptibility, 62 pathways, 37, 62, 210 tumor resistance to chemotherapeutic agents, 38, 39 Ara-C, bryostatin-1 combination therapy in leukemia, 67–69 flavopiridol combination therapy in leukemia, 75 G3139 combination therapy, 218 UCN-01 combination therapy in leukemia, 72

B Bcl-2, antisense oligonucleotide targeting, see G3139 apoptosis regulation, 210, 211 dimerization, 211 structure, 211 BCR-ABL, STI571 inhibition, 236 Bone marrow transplantation, nonmyeloablative conditioning with F-particle therapy, 121, 122 Bortezomib, see PS-341 Breast cancer, trastuzumab combination therapies, 177–182, 184, 186–188 Bryostatin-1 clinical trials, 37 combination therapy in leukemia, ara-C, 67–69 chlorodeoxyadenosine, 70 fludarabine, 69, 70 vincristine, 70 VP-16, 70 dosing, 36, 37 formulations, 36 kinase specificity, 35, 66 mechanism of action, 35, 36, 66, 67 277

278 paclitaxel combination therapy, 40, 41 C CAI, see Carboxyamidotriazole Calcium, entry inhibitors, see Carboxyamidotriazole intracellular concentration regulation, 91 local tumor microenvironment, 90, 91 Capecitabine, efficacy studies, 159, 160 metabolic activation, 156, 157 Carboplatin, historical perspective, 198 TNP-470/minocycline combination therapy, 250, 251, 254 trastuzumab combination therapy in breast cancer, 186, 187 Carboxyamidotriazole (CAI), antitumor mechanisms, angiogenesis inhibition, cytokine production inhibition, 96 endothelial cell proliferation inhibition, 95, 96 matrix metalloproteinase downregulation, 93, 95 overview, 91, 92 proliferation, adhesion, and migration inhibition, 93 dosing and distribution, 97, 98 invasostatic action, 90 preclinical testing, 97 side effects and toxicity, 98, 99 tumor response, combination therapy with cytotoxic agents, 100, 101 monotherapy, 98, 100 CDKIs, see Cyclin-dependent kinase inhibitors

Index Cell cycle, cyclin-dependent kinase inhibitors, see Cyclindependent kinase inhibitors drug resistance mediation, 37–39 regulation overview, 28, 30, 32 Cetuximab, antitumor activity, 6, 7 CPT-11 combination therapy in colorectal cancer, 9, 10 dosing, 8 epidermal growth factor receptor binding affinity, 5 head and neck cancer trials, 10, 11 mechanism of action, 5 safety, 8 CHDP, see 5-Chloro-2,4dihydroxypyridine Chlorodeoxyadenosine, bryostatin-1 combination therapy in leukemia, 70 5-Chloro-2,4-dihydroxypyridine (CHDP), fluorinated pyrimidine cytotoxicity augmentation, 149, 150 CI-1033, epidermal growth factor receptor inhibition and antitumor activity, 16 HER2/neu inhibition, 190, 192 Cisplatin, cetuximab combination therapy in head and neck cancer, 10, 11 cyclin-dependent kinase inhibitor combination therapy, 46, 47 G3139 combination therapy, 219, 221 historical perspective, 198 mechanism of action, 45, 46, 198, 199 nuclear factor-PB role in resistance and modulation, 200–202, 204, 205

Index resistance mechanisms, 197, 199, 200 TNP-470/minocycline combination therapy, 253, 254 trastuzumab combination therapy in breast cancer, 186 CNDP, see 3-Cyano-2,6dihydroxypyridine Colorectal cancer, CPT-11/ cetuximab combination therapy, 9, 10 CPT-11, cetuximab combination therapy in colorectal cancer, 9, 10 3-Cyano-2,6-dihydroxypyridine (CNDP), fluorinated pyrimidine cytotoxicity augmentation, 150 Cyclin D1, overexpression in tumors, 255–257 Cyclin-dependent kinase inhibitors (CDKIs), see also specific inhibitors, cancer therapy rationale, 32–37 cell cycle regulation, 28, 30, 32, 64, 65 combination therapy, campothecins, 41–44 cisplatin, 45–47 fluorouracil, 44, 45 gemcitabine, 47–49 prospects for study, 50 taxanes, 39–41 overview of types, 257 therapeutic targeting rationale in leukemia, 64–66 Cyclophosphamide, G3139 combination therapy, 217 TNP-470/minocycline combination therapy, 253 D Dacarbazine, G3139 combination therapy, 218

279 Dihydropyrimidine dehydrogenase (DPD), circadian expression pattern, 148 fluorinated pyrimidine metabolism, 148, 149 inhibitors for fluorinated pyrimidine cytotoxicity augmentation, 148–150 Dipyramidole, fluorinated pyrimidine cytotoxicity augmentation, 154 Docetaxel, G3139 combination therapy, 218, 220, 221 mechanism of action, 39 trastuzumab combination therapy in breast cancer, 180–182, 186, 190 Doxorubicin, G3139 combination therapy, 220, 221 trastuzumab combination therapy in breast cancer, 184 DPD, see Dihydropyrimidine dehydrogenase E EGFR, see Epidermal growth factor receptor EKB-569, epidermal growth factor receptor inhibition and antitumor activity, 15 EMD 7200, epidermal growth factor receptor inhibition, 11, 12 Eniluracil, fluorinated pyrimidine cytotoxicity augmentation, 150 Epidermal growth factor receptor (EGFR), see also HER2/neu, classification, 2, 176, 233 ligands, 232, 233 low-molecular-weight tyrosine kinase inhibitors, antitumor activity, 8 irreversible inhibitors, 15, 16

280 mechanisms of action, 5, 6 reversible inhibitors, 12–16 monoclonal antibodies, see also Cetuximab, ABX-EGF, 11 EMD 7200, 11, 12 historical perspective, 233 h-R3, 12 types, 9 signal transduction, 2, 3 study design for inhibitors, combination chemotherapy trials, 19, 20 differential activity profile considerations, 18 patient selection, 17, 18 therapeutic targeting rationale, 1–4 tumor expression, 1, 3 Epirubicin, trastuzumab combination therapy in breast cancer, 18 ErbB, see Epidermal growth factor receptor Erbitux, see Cetuximab Erlotinib, epidermal growth factor receptor inhibition and antitumor activity, 14, 15 Etoposide, G3139 combination therapy, 221 F Flavopiridol, apoptosis induction, 33 cisplatin combination therapy, 47 combination therapy in leukemia, 74, 75 dosing, 34 gemcitabine combination therapy, 48, 49 irinotecan combination therapy, 42, 43 kinase specificity, 33, 74, 257

Index mechanism of action, 74, 75, 257 paclitaxel combination therapy, 40, 41 preclinical studies, 257, 258 Fludarabine, bryostatin-1 combination therapy in leukemia, 69, 70 UCN-01 combination therapy in leukemia, 72, 73 5-Fluorouracil, augmentation of fluorinated pyrimidine cytotoxicity, antifolates, 143–147 dihydropyrimidine dehydrogenase inhibitors, 148–150 interferons, 150–153 leucovorin, 138–141 nucleoside transport and nucleotide salvage inhibitors, 153–156 prospects, 160, 161, 163, 164 ribonucleotide reductase inhibitors, 147, 148 table of agents, 140 thymidine phosphorylase modulators, 156–160 continuous infusion versus bolus administration, 141, 142 cyclin-dependent kinase inhibitor combination therapy, 45 enzyme expression and antitumor activity effects, 142, 143, 162–164 mechanism of action, 44, 134, 135, 137 metabolic activation, 135, 136 G G3139, combination with other agents, clinical trials, 222–225 preclinical studies,

Index ara-C, 218 cisplatin, 219, 221 cyclophosphamide, 217 dacarbazine, 218 docetaxel, 218, 220, 221 doxorubicin, 220, 221 etoposide, 221 mitoxantrone, 219 overview, 214–216 paclitaxel, 218, 219 PSC833, 220 rituximab, 217 design and development, 215 metabolism and pharmacology, 225 Gemcitabine, cyclin-dependent kinase inhibitor combination therapy, 48, 49 fluorinated pyrimidine cytotoxicity augmentation, 147, 148 mechanism of action, 47, 48 Genasense, see G3139 Glioma, F-particle therapy with anti-tenascin radioimmunoconjugate, 122, 123 GW2016, epidermal growth factor receptor inhibition and antitumor activity, 16 H Head and neck cancer, cisplatin/ cetuximab combination therapy, 10, 11 HER2/neu, see also Epidermal growth factor receptor, antibody targeting, see Trastuzumab novel inhibitors, 17-allylamino-geldanamycin, 190 CI-1033, 190, 192 prospects, 192

281 vaccines, 192 overexpression in tumors, 176 structure, 176 Herceptin, see Trastuzumab h-R3, epidermal growth factor receptor inhibition, 12 7-Hydroxystaurosporine, see UCN-01 Hydroxyurea, fluorinated pyrimidine cytotoxicity augmentation, 147 I Interferons, fluorinated pyrimidine cytotoxicity augmentation, 150–153 Irinotecan, cyclin-dependent kinase inhibitor combination therapy, 42–44 mechanism of action, 41, 42 L Leucovorin, fluorinated pyrimidine cytotoxicity augmentation, 138–141 metabolism, 141 Leukemia, see also BCR-ABL, F-particle therapy with anti-CD33 radioimmunoconjugate, 120, 121 apoptosis induction in treatment, 61, 62 cyclin-dependent kinase inhibitor therapy, flavopiridol, 74, 75 protein kinase C inhibitor combination therapy, 76, 77 purine analog inhibitors, 75, 76 prospects for kinase inhibitor studies, 77, 78 protein kinase C,

282 inhibitor therapy, see Bryostatin1; Phorbol 12-myristate 13acetate; UCN-01 therapeutic targeting rationale, 62–64 Lung cancer, TNP-470/minocycline combination therapy, 246, 247, 249–255 LY317615, protein kinase C and cancer treatment, 241–243, 245, 246 M Matrix metalloproteinases, downregulation by carboxyamidotriazole, 93, 95 Melanoma, F-particle therapy with methylene blue conjugate, 123 Methotrexate (MTX), fluorinated pyrimidine cytotoxicity augmentation, 143, 144 Minocycline, TNP-470 combination therapy, 246, 247, 249–255 Mitoxantrone, G3139 combination therapy, 219 Monoclonal antibodies, F-particle therapy, see Radioimmunoconjugates epidermal growth factor receptor targeting, see Cetuximab; Epidermal growth factor receptor immunoconjugate strategies, 108 MTX, see Methotrexate N NF-PB, see Nuclear factor-PB Nuclear factor-PB (NF-PB), cisplatin resistance, proteasome inhibitors for prevention, 204, 205 role, 201 inhibitor protein,

Index activation, 200 degradation pathway, 201, 202 tumor expression, 200, 201 O Oblimersen, see G3139 Ovarian cancer, paclitaxel/ carboxyamidotriazole combination therapy, 100, 101 P Paclitaxel, carboxyamidotriazole combination therapy in ovarian cancer, 100, 101 cyclin-dependent kinase inhibitor combination therapy, 40, 41 G3139 combination therapy, 218, 219 mechanism of action, 39 TNP-470/minocycline combination therapy, 250, 251, 254 trastuzumab combination therapy in breast cancer, 178–180, 184 Phorbol 12-myristate 13-acetate (PMA), leukemia, cyclin-dependent kinase inhibitor combination therapy, 77 trials, 71 mechanism of action, 70, 71 PKC, see Protein kinase C PKC412, protein kinase C inhibition and leukemia trials, 73 PMA, see Phorbol 12-myristate 13acetate Premetrexed, fluorinated pyrimidine cytotoxicity augmentation, 146 Prostate cancer, G3139 combination with chemotherapy, 215, 216, 219

Index Protein kinase C (PKC), domains, 63 inhibitors, see also Bryostatin-1; Phorbol 12-myristate 13acetate; UCN-01 antisense oligonucleotides, 73, 74 LY317615, 241–243, 245, 246 PKC412, 73 safingol, 73 isoforms, 63, 239, 240 therapeutic targeting rationale, leukemia, 62–64 solid tumors, 240 PS-341, cisplatin resistance modification, 204, 205 PSC833, G3139 combination therapy, 220 R Radioimmunoconjugates, F-particle therapy, advantages, 108, 109 dosimetry, 117, 118 glioma studies, 122, 123 leukemia studies, 120, 121 lymphoma studies, 123 melanoma studies, 123 mouse model studies, 118–120 nonmyeloablative allogeneic bone marrow transplantation, 121, 122 pretargeting studies, 124 prospects, 125, 126 radioisotopes, actinium-225, 111, 122 astatine-211, 112, 113 bismuth-212, 113 bismuth-213, 111 lead-212, 114 radium-223, 115 spheroid studies, 120 toxicity, 124, 125

283 cell death induction mechanisms, 109, 110 emitter types, 108, 109 radiolabeling techniques, 115–117 Raltitrexed, fluorinated pyrimidine cytotoxicity augmentation, 146 Ras, farnesylation inhibitors in cancer treatment, 258, 259 Ribonucleotide reductase, inhibitors for fluorinated pyrimidine cytotoxicity augmentation, 147, 148 Rituximab, F-particle therapy with radioimmunoconjugates in lymphoma, 123 G3139 combination therapy, 217 S Safingol, protein kinase C inhibition and leukemia trials, 73 SCH66336, farnesylation inhibition in cancer treatment, 258, 259 Signal transduction, derangements in cancer, 231, 232 therapeutic targeting prospects, 259, 262 STI571 BCR-ABL inhibition, 236 kinase specificity, 236, 237 SU5416, receptor tyrosine kinase inhibition, 237, 238 SU6668, receptor tyrosine kinase inhibition, 237, 238 T Taxanes, see Docetaxel; Paclitaxel Thymidine phosphorylase, modulators for fluorinated pyrimidine cytotoxicity augmentation, 156–160 tumor and tissue expression, 157, 158

284 Thymidylate cycle, overview, 137, 139 Thymidylate synthase, expression regulation, 162, 164 fluorinated pyrimidine cytotoxicity augmentation, 161, 162 TMTX, see Trimetrexate TNP-470, angiogenesis inhibition, 246 minocycline combination therapy, 246, 247, 249–255 Tomudex, see Raltitrexed Trastuzumab, combination therapy, adjuvant therapy, 182, 184, 186–188, 190 breast cancer, 177–182, 184, 186–188 carboplatin, 186, 187 cisplatin, 186 docetaxel, 180–182, 186, 190 doxorubicin, 184 epirubicin, 184 paclitaxel, 178–180, 184 monotherapy trials, 176, 177 Triapine, fluorinated pyrimidine cytotoxicity augmentation, 148 Trimetrexate (TMTX), fluorinated pyrimidine cytotoxicity augmentation, 144–146 Tyrosine kinase inhibitors, see Epidermal growth factor receptor U UCN-01, cisplatin combination therapy, 46 combination therapy in leukemia,

Index ara-C, 72 fludarabine, 72 dosing, 35 fluorouracil combination therapy, 45 gemcitabine combination therapy, 49 irinotecan combination therapy, 43, 44 kinase specificity, 34, 71 mechanism of action, 34, 35, 71, 72 preclinical studies, 241 UFT, see Uracil:tegafur Uracil:tegafur (UFT), fluorinated pyrimidine cytotoxicity augmentation, 149 V Vascular endothelial growth factor (VEGF), carboxyamidotriazole downregulation, 96 receptor targeting in cancer therapy, 237 signal transduction in angiogenesis, 240, 243, 245 VEGF, see Vascular endothelial growth factor, Vincristine, bryostatin-1 combination therapy in leukemia, 70 VP-16, bryostatin-1 combination therapy in leukemia, 70 Z ZD1839, epidermal growth factor receptor inhibition and antitumor activity, 12–14, 233–236

Combination Cancer Therapy: Modulators and Potentiators (Cancer Drug Discovery and Development)

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development) (Cancer Drug Discovery and Development)

DNA Repair in Cancer Therapy (Cancer Drug Discovery and Development)

Checkpoint Responses in Cancer Therapy (Cancer Drug Discovery and Development)

Fluoropyrimidines in Cancer Therapy (Cancer Drug Discovery and Development)

Camptothecins in Cancer Therapy (Cancer Drug Discovery and Development)

Deoxynucleoside Analogs in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development)

Regional Cancer Therapy (Cancer Drug Discovery and Development)

Chemoradiation in Cancer Therapy (Cancer Drug Discovery and Development)

Gene Therapy for Cancer (Cancer Drug Discovery and Development)

Regional Cancer Therapy. Cancer Drug Discovery and Development

Death Receptors in Cancer Therapy (Cancer Drug Discovery and Development)

Cancer Drug Resistance (Cancer Drug Discovery and Development)

DNA Topoisomerases and Cancer (Cancer Drug Discovery and Development)

Apoptosis, Senescence and Cancer (Cancer Drug Discovery and Development)

Stem Cells and Cancer (Cancer Drug Discovery and Development)

Genomics in Cancer Drug Discovery and Development

Nucleic Acid Therapeutics in Cancer (Cancer Drug Discovery and Development)

Targets for Cancer Chemotherapy (Cancer Drug Discovery and Development)

Chemokine Receptors in Cancer (Cancer Drug Discovery and Development)

Tumor Models in Cancer Research (Cancer Drug Discovery and Development)

Biomarkers in Breast Cancer (Cancer Drug Discovery and Development)

Immunotherapy of Cancer (Cancer Drug Discovery and Development)

Cancer Drug Design and Discovery

Anticancer Drug Development Guide (Cancer Drug Discovery and Development)

Principles of Anticancer Drug Development (Cancer Drug Discovery and Development)

Nitric Oxide (NO) and Cancer: Prognosis, Prevention, and Therapy (Cancer Drug Discovery and Development)

Transforming Growth Factor-Beta in Cancer Therapy, Volume II: Cancer Treatment and Therapy (Cancer Drug Discovery and Development)

Combination Cancer Therapy: Modulators and Potentiators (Cancer Drug Discovery and Development)

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development) (Cancer Drug Discovery and Development)

DNA Repair in Cancer Therapy (Cancer Drug Discovery and Development)

Checkpoint Responses in Cancer Therapy (Cancer Drug Discovery and Development)

Fluoropyrimidines in Cancer Therapy (Cancer Drug Discovery and Development)

Camptothecins in Cancer Therapy (Cancer Drug Discovery and Development)

Deoxynucleoside Analogs in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Farnesyltransferase Inhibitors in Cancer Therapy (Cancer Drug Discovery and Development)

Antiangiogenic Agents in Cancer Therapy (Cancer Drug Discovery and Development)

Regional Cancer Therapy (Cancer Drug Discovery and Development)

Chemoradiation in Cancer Therapy (Cancer Drug Discovery and Development)

Gene Therapy for Cancer (Cancer Drug Discovery and Development)

Regional Cancer Therapy. Cancer Drug Discovery and Development

Death Receptors in Cancer Therapy (Cancer Drug Discovery and Development)

Cancer Drug Resistance (Cancer Drug Discovery and Development)

DNA Topoisomerases and Cancer (Cancer Drug Discovery and Development)

Apoptosis, Senescence and Cancer (Cancer Drug Discovery and Development)

Stem Cells and Cancer (Cancer Drug Discovery and Development)

Genomics in Cancer Drug Discovery and Development

Nucleic Acid Therapeutics in Cancer (Cancer Drug Discovery and Development)

Targets for Cancer Chemotherapy (Cancer Drug Discovery and Development)

Chemokine Receptors in Cancer (Cancer Drug Discovery and Development)

Tumor Models in Cancer Research (Cancer Drug Discovery and Development)

Biomarkers in Breast Cancer (Cancer Drug Discovery and Development)

Immunotherapy of Cancer (Cancer Drug Discovery and Development)

Cancer Drug Design and Discovery

Anticancer Drug Development Guide (Cancer Drug Discovery and Development)

Principles of Anticancer Drug Development (Cancer Drug Discovery and Development)

Nitric Oxide (NO) and Cancer: Prognosis, Prevention, and Therapy (Cancer Drug Discovery and Development)

Transforming Growth Factor-Beta in Cancer Therapy, Volume II: Cancer Treatment and Therapy (Cancer Drug Discovery and Development)

Recommend Documents