Analytical Profiles of Drug Substances and Excipients Volume 24

Analytical Profiles of Drug Substances

and Excipients

EDITORIAL BOARD

Abdullah A. Al-Badr Alekha K. Dash

Larry D. Kissinger David J. Mauo

Klaus Florey

Christopher T. Riley

Lee T. Grady

Timothy J. Wozniak

Dominic P. Ip

Analytical Profiles of Drug Substances and Excipients Volume 24 edited by

Harry G. Brittain Ohmeda Pharmaceutical Products Division, Inc. 100 Mountain Avenue Murray Hill, New Jersey 07974

Founding Editor:

Klaus Florey

ACADEMIC PRESS San Diego

New York

Boston London Sydney Tokyo Toronto

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@

Copyright ie 1996 by ACADEMIC PRESS, INC. All Rights Reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopy, recording, or any information storage and retrieval system, without permission in writing from the publisher.

Academic Press, Inc. A Division of Harcourt Brace & Company 525 B Street, Suite 1900, San Diego, California 92101-4495

United Kingdom Edition published by Academic Press Limited 24-28 Oval Road, London NW 1 7DX

International Standard Serial Number: 1075-6280 International Standard Book Number: 0-12-260824-0 PRINTED RJ THE UNITED STATES OF AMERICA 96 97 9 8 9 9 00 01 BC 9 8 7 6 5

4

3 2

1

CONTENTS AfJiriations of Ediforsand Coaiributors

vii

Preface

xi

1

1.

Carbenoxolone sodium S. Pindado, 0.1.Corrigan, and C.M 0 'Driscoll

2.

Clarithromycin LI. Salem

45

3.

Crospovidone E. S. Barabas and C.M. Adeyeye

87

4.

Fluvoxamine Maleate N.H. Foda, MA. Radwan, and O.A. A1 Deeb

165

5.

Gadoteridol K. Kumar, M F. Tweedle, and H G. Brittain

209

6.

Guar Gum K. Yu, D. Wong, J Parasrampuria, and D. Friend

243

7.

Mafenide Acetate A.K. Dash and S. Saha

277

8.

Maltodextrin MJO Mollan, Jr., andM Celik

3 07

V

CONTENTS

v1

9.

Nalmefene Hydrochloride H G. Brittain

35 1

10.

Polyvinyl Alcohol D. Wong and J. Parasrampuria

397

11.

Sertraline hydrochloride B.M Johnson and P.-T. L. Chang

443

12.

Solasodine G. Indrayanto, A. Syahrani, R. Sondakh, and M. H. Santosa

487

13.

Starch A. W. Newman, R.L. Mueller, I.M. Vitez, C.C. Kiesnowski, D.E.Bugay, W.P. Findlay, and C. Rodriguez

523

14.

Tobramycin A. K. Dash

579

Cumulative Index

615

AFFILIATIONS OF EDITORS AND CONTRIBUTORS Christiunuh M Adeyeye, Department of Pharmacy, Duquesne University, Pittsburgh, PA 15282 Abdulluh A. Al-Budr, College of Pharmacy, King Saud University, P.O. Box 2457, Riyadh 11451, Saudi Arabia Omur A. A1 Deeb, Department of Pharmaceutical Chemistry, College of Pharmacy, King Saud University, P.O. Box 22452, Riyadh 11459, Saudi Arabia Eugene S. Burabus, ISP Corporation, 1361 Alps Road, Wayne, NJ 07470 Hurry G. Brittuin, Ohmeda Pharmaceutical Products Division, Inc., 100 Mountain Avenue, Murray Hill, NJ 07974 David E. Buguy, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903 Metin celik, College of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 08855 Pei-Tei L. Chung, Central Research, Pfizer Inc., Groton, CT 06340 Owen I. Corrigun, Department of Pharmaceutics, University of Dublin, Trinity College, Ireland Alekhu K. Dash,, School of Pharmacy, Department of Pharmaceutical Sciences, Creighton University, 2500 California Plaza, Omaha, Nebraska 68178

W Paul Findluy, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903 Klaus Florey, 151 Loomis Court, Princeton, NJ 08540

Nugwu H. Fodu, Department of Pharmaceutics, College of Pharmacy, King Saud University, P.O. Box 22452, Riyadh 11459, Saudi Arabia David Friend, Cibus Pharmaceutical, Inc., 200 D Twin Dolphin Drive, Redwood City, CA 94065 Lee T. Grudy, The United States Pharmacopeia, 12601 Twinbrook Parkway, Rockville, MD 20852 vi i

...

Vlll

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Gunawan Indrayanto, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia Dominic P. Ip, Merck, Sharp, and Dohme, Building 78-210, West Point, PA 19486 Bruce M. Johnson, Central Research, Pfizer Inc., Groton, CT 06340 Christopher C. Kiesnowski, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903

Lurry D. Kissinger, The Upjohn Company, 7171 Portage Road, Kalamazoo, M149001 Krishan Kumar, Bracco Research USA, P.O. Box 5225, Princeton, NJ 08520 David J Mazzo, Department of Analytical & Physical Chemistry, RhBnePoulenc Rorer Recherche-Developpement,20, avenue Raymone Aron, 92 165 Antony Cedex, France Matthew J Mollan, Jr., College of Pharmacy, Rutgers, the State University of New Jersey, Piscataway, NJ 08855 Ronald L. Mueller, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903 Ann W. Newman, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903 Caitriona M. 0 'Driscoll, Department of Pharmaceutics, University of Dublin, Trinity College, Ireland Jugdish Parasrampuria, Cibus Pharmaceutical, Inc., 200 D Twin Dolphin Drive, Redwood City, CA 94065 Silvia Pindudo, National Pharmaceuticaland Biotechnology Center, Dublin, Ireland Muhasen A . Radwan, Department of Clinical Pharmacy, College of Pharmacy, King Saud University, P.O. Box 22452, Riyadh 11459, Saudi Arabia Christopher T Riley, Room 106, Building 353, Experimental Station, DuPont Merck Pharmaceutical Company, P.O. Box 80400, Wilmington, DE 19880-0400

AFFILIATIONS OF EDITORS AND CONTRIBUTORS

ix

Christine Rodriguez, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903 Shankar Saha, Department of Biomedical Sciences, Creighton University, 2500 California Plaza, Omaha, Nebraska 68 178 Isum I. Salem, Department of Pharmacy and Pharmaceutical Technology, University of Granada, 18071- Granada, Spain Mulja H Santosa, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia

Robby Sondakh, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia Achmud Syahruni, Laboratory of Pharmaceutical Biotechnology, Faculty of Pharmacy, Airlangga University, Surabaya, Indonesia Michael Tweedle, Bracco Research USA, P.O. Box 5225, Princeton, NJ 08520

Imre M Vitez, Bristol-Myers Squibb Pharmaceutical Research Institute, One Squibb Drive, New Brunswick, NJ 08903 David Wong, Cibus Pharmaceutical, Inc., 200 D Twin Dolphin Drive, Redwood City, CA 94065 Timothy J. Wozniak,Eli Lilly and Company, Lilly Corporate Center, MC769, Indianapolis, IN 46285 Karen Yu, Cibus Pharmaceutical, Inc., 200 D Twin Dolphin Drive, Redwood City, CA 94065

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The profiling of drug substances as to their physical and analytical characteristics remains as important today as it was when the Analytical Profiles series was first initiated. The compilation of concise summaries of physical and chemical data, analytical methods, routes of compound preparation, degradation pathways, and the like, is a vital function to both academia and industry. It is certainly fair to say that workers in the field require access to current state-of-the-art data, and the Analytical Profiles series has always provided information of the highest quality. For this reason, profiles of older compounds are updated whenever a sufficient body of new information becomes available. The series mission was expanded some time ago to include profiles of excipient materials, reflecting the developing situation that these materials are coming under a degree of scrutiny which is approaching that associated with drug compounds. These highly detailed compilations of excipient properties and analytical methods have been well received by workers in the field, and such profiles will continue to be sought. Perceptive readers will note that 1995 passed without publication of an Analytical Profiles volume, a situation caused by an insufficient number of chapter submissions. An increasingly ominous fact of our professional life is that scientists seem to have less time available for scholarly contributions, a phenomenon which may be related to the current trend of consolidation and downsizing. If this trend continues, more innovation in analytical profiling must take place. For instance, should a prospective author feel unable to complete an entire chapter, then the submitted portion will be accepted and the editor will find additional authors to complete the work. The initial submission can consist of either the physical characteristics section or the analytical methodology section. As always, a complete list of available drug and excipient candidates is available from the editor. We look forward to hearing from new and established authors, and to working with the pharmaceutical community on the Analytical Profiles of Drug Substances and Excipients. Harry G. Brittain xi

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CARBENOXOLONE SODIUM

Silvia Pindado"', Owen I. C~rrigan'~)

and Caitriona M. O'Driscoll*(2)

(1) National Pharmaceutical Biotechnology Center Dublin, Ireland.

(2) University of Dublin Department of Pharmaceutics School of Pharmacy, Trinity College

* Author for correspondence ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

1

Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

SILVIA PINDADO ET AL.

2

1.

Introduction

2.

Description 2.1 2.1.1 2.1.2 2.1.3 2.2 2.2.1 2.2.2 2.3 2.4 2.5 2.6

Nomenclature Chemical Name Nonproprietary Names Proprietary Names Formulae Empirical Structural Molecular Weight Appearance Official Compendia Other Compendia

3.

Synthesis

4.

Physical Properties 4.1 Spectroscopy 4.1.1 Ultraviolet Spectroscopy 4.1.2 Infiared Spectroscopy 4.1.3 Mass Spectrometry 4.1.4 Nuclear Magnetic Resonance (IH, I3C) Spectrometry X-Ray Diffraction 4.2 Optical Rotation 4.3 Thermal Methods of Analysis 4.4 4.4.1 Melting Point 4.4.2 Differential Scanning Calorimetry 4.4.3 Thermogravimetric Analysis Hygroscopicity 4.5 Dissociation Constants 4.6 Solubility 4.7 Partition Coefficients 4.8

CARBENOXOLONE SODIUM

5.

Methods of Analysis 5.1 Elemental Analysis 5.2 Identification 5.3 Titrimetric Analysis 5.4 Ultraviolet Spectrophotometry 5.5 ChromatographicMethods of Analysis 5.5.1 Thin Layer Chromatography 5.5.2 Gas Chromatography 5.5.3 High Performance Liquid Chromatography 5.6 Radioimmunoassay 5.7 Radioactive Labeling

6,

Stability

7.

Pharmacokinetics 7.1 Absorption 7.2 Distribution 7.3 Metabolism 7.4 Excretion

8.

Pharmacology 8.1 Therapeutic Indications and Uses 8.3 Toxicity and Side-Effects

9.

References

10.

Acknowledgements

3

SILVIA PINDAW ET AL.

4

1.

INTRODUCTION

Carbenoxolone is a triterpenoid, the ester of 18p-glycyrrhetic (enoxolone) acid with succinic acid. The di-sodium salt, identified as carbenoxolone sodium, is used in the treatment of gastric and duodenal ulcers (Doll et al., 1962; Pinder et al., 1976).

2. DESCRIPTION 2.1

Nomenclature

2.1.1 Chemical Names A.

Disodium 3~-(3-carboxylatopropionyloxy)1 1-oxo-olean- 12-en30-oate (B. P. 1993). Disodium salt of 3~-(3-carboxypropionyloxy)-11-oxo-olean-12-en30-oic acid (Martindale, 1993; The Pharmaceutical Codex, 1979).

B.

3-(3-Carboxy-1-oxopropoxy)-11-oxoolean-l2-en-29-oic acid. 3~-Hydroxy-ll-oxoolean-12-en-30-oic acid hydrogen succinate. 3P-Hydroxy- 1 1-0xoolean-12-en-30-oic acid 3-hemisuccinate. 3-O-(f3-carboxypropionyl)-1 1-oxo- 18p-olean-12-en-30-oic acid (Merck Index, 1989).

2-Carboxy-ethylpropionyl glycyrrhetinic acid (British patent, 1960).

2.1.2 Nonproprietary Names A.

Carbenoxolone Sodium, Disodium Enoxolone Succinate (BANM, USAN, rINNM) (Martindale, 1993).

5

CARBENOXOLONE SODIUM

B.

Carbenoxolone, Glycyrrhetinic Acid Hydrogen Succinate, Glycyrrhetic Acid Hydrogen Succinate, 18P-GlycyrrheticAcid Hydrogen Succinate (Merck Index, 1989; Clarke, 1986)

2.1.3 Proprietary Names Biogastrone, Bioplex, Bioral, Carbosan, Duogastrone, Gastrausil, Megast, Pyrogastrone, Sanodin, Ulcus-Tablinen (Martindale, 1993). 2.1.4 Chemical Abstracts Service (CAS) Registry Numbers 7421-40-1 5697-56-3

Carbenoxolone Sodium Carbenoxolone

2.2 Formulae

2.2.1 Empirical Carbenoxolone Sodium Carbenoxolone

2.2.2 Structural Carbenoxolone Sodium

Carbenoxolone

SILVIA PINDADO ET AL.

6

2.3 Molecular Weight 614.7 570.74

Carbenoxolone Sodium Carbenoxolone

2.4 Appearance Carbenoxolone sodium is a white or pale-cream colored, hygroscopic powder, with a slightly sweet taste followed by a persistent soapy aftertaste. It tends to adsorb to glass.

2.5 OMicial Compendia A monograph on Carbenoxolone Sodium is included in the British Pharmacopoeia, (1 993), and in the Chinese Pharmacopeia, (1985).

2.6 Other Compendia Carbenoxolone sodium is included in the Pharmaceutical Codex (1979), and in Martindale (1993). Carbenoxolone is included in the Merck Index (1989). Clarke (1986) gives a usehl summary of physical and chemical data.

3. SYNTHESIS. Carbenoxolone is synthesized from glycyrrhetinic acid, the aglycone of glycyrrhizic acid (Figure I), which may be obtained from liquorice root. Carbenoxolone is prepared by refluxing an organic acid with glycyrrhetinicacid in an organic solvent, or by the action of an acid anhydride in pyridine solution. The sodium salt is prepared by neutralization with an aqueous solution of sodium hydroxide (British patent, 1960; U.S. patent, 1962). The carbenoxolone free acid used in the studies conducted for this monograph was prepared from carbenoxolone sodium, B.P., by precipitation in hydrochloric acid. The precipitate was washed with water and dried to a constant weight at 105OC.

CARBENOXOLONESODIUM

I

OH

OH

Glycyrrhizic Acid

Glycyrrhetinic Acid

Figure 1.

> Synthesis of carbenoxolone.

Carbenoxolone

SILVIA PINDADO ET AL.

8

4. PHYSICAL PROPERTIES 4.1 Spectroscopy 4.1.1 Ultraviolet Spectroscopy

The ultraviolet spectrum of carbenoxolone sodium (0.005% w/v) is shown in Figure 2. The spectrum was obtained using a Shimadzu (UV-160) UVNIS spectrophotometerand I-cm quartz cells. The spectrum, as obtained in the range of 230 to 350 nm in a 1:1 v/v mixture of methanol and 0.02M sodium carbonate, exhibits a single maximum at 256 nm. The absorbance at this maximum is about 1.O. An E 1% of 200 has been reported in this solvent mixture (Pharmaceutical Codex, 1979). In aqueous acid and aqueous alkali, dual wavelength maxima have been reported at 248 nm and 257 nm, with E 1% values of 172 each (Clarke, 1986).

0.891

A

/

0.713 0.535 .

/ Y

0.356 0.178

0.000~

'

.

.

8

250

.

I

300 Wavelength (nm)

Figure 2. Ultraviolet spectrum of carbenoxolone sodium.

350

CARBENOXOLONE SODIUM

4.1.2 Infrared Spectroscopy The infrared absorption spectnun of carbenoxolone sodium and carbenoxolone are shown in Figures 3 and 4. The spectra were recorded with a Nicolet 5ZDX FT-IR spectrophotometer, from compressed potassium bromide disks. Structural assignments for some of the characteristic absorption bands in the spectra are listed in Table 1.

Table I. Infrared assignments for carbenoxolone sodium and carbenoxolone.

Peak Maximum (cm-')

Assignment

Carbenoxolone sodium 1720 1680 1550

C=O stretch (ester) C=O stretch (ketone) C=O stretch (carboxylate)

Carbenoxolone 1730 1710 1650

C=O stretch (ester) C=O stretch (carboxylate) C=O stretch (ketone)

9

SILVIA PINDADO ET AL

20

2000 1800 1600 1400 1200 1000 800

Wavenumber

Figure 3.

Infrared spectrum of carbenoxolone sodium.

600

CARBENOXOLONE SODIUM

2000 1800 1600 1400 1200 1000 800 Wavenumber

Figure 4.

Infrared spectrum of carbenoxolone.

11

600

12

SLVIA PINDADO ET AL.

4.1.3 Mass Spectrometry The fast atom bombardment (FAB) mass spectrum of carbenoxolone sodium, shown in Figure 5 , was recorded using a V.G. analytical 70e mass spectrometer and a FAE3 probe, with glycerol as the matrix. The intensities are calculated relative to the base peak at m/z 115. The spectrum shows an (M+H)+ peak at m/z 615 (relative intensity 3.56%), a peak at m/z 637 (5.53%) corresponding to (M+Na)+, and a further peak at m/z 659 (0.47%) corresponding to (M+2Na-H)+. Major peaks were detected at m/z (%) 571 (5.69), 137 (62), 115 (loo), 63 (53), 41 (63). The electron impact (EI) mass spectrum of carbenoxolone (Figure 6) was also obtained, by electron-impact at 70 eV and 200OC, using a Finnigan MAT Quadrupole mass spectrometer and a direct insertion probe. The molecular ion (M+) at m/z (%) 570 (4.16%) was observed. Major peaks were detected at d z (%) 303 (64), 262 (62), 135 (loo), 101 (64), 95 (61).

x10.00

- : 1

'I

14

12

1o!

59 1

!

659

Figure 5.

FAB mass spectrum of carbenoxolone sodium.

8.4E6 7.4E6 6.5E6 5.6E6 4.6E6 -3.7E6 -2.886 . I .9E6 -9.3E5

CARBENOXOLONE SODIUM

100.0

50.0

m

Figure 6. Electron impact (EI) mass spectrum of carbenoxolone.

4.1.4 Nuclear Magnetic Resonance (IH, 136) Spectrometry The NMR spectra of carbenoxolone sodium and carbenoxolone were obtained using a Bruker MSL 300. A. Carbenoxolone sodium The 1H-NMR spectrum for carbenoxolone sodium (Figure 7) was obtained at a frequency of 300.13 MHz, in deuterated water. The solution contained 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TSP) as the internal standard. A 2D H-H COSY spectrum was also obtained (Figure 8).

13

18688. 0.

14

SILVIA PINDADO ET AL.

Figure 7. Proton nuclear magnetic resonance spectrum of carbenoxolone sodium.

CARBENOXOLONE SODIUM

135

100.0

15

18688. 0.

50.0

Figure 6. Electron impact (EI) mass spectrum of carbenoxolone.

4.1.4 Nuclear Magnetic Resonance (lH, 13C) Spectrometry The NMR spectra of carbenoxolone sodium and carbenoxolone were obtained using a Bruker MSL 300. A. Carbenoxolone sodium

The 1H-NMR spectrum for carbenoxolone sodium (Figure 7) was obtained at a frequency of 300.13 MHz, in deuterated water. The solution contained 3-(trimethylsilyl) propionic-2,2,3,3-d4 acid, sodium salt (TSP) as the internal standard. A 2D H-H COSY spectrum was also obtained (Figure 8).

SILVIA PINDADO ET AL.

16

t

(PPW 5

Figure 8. 2-DH-H COSY nuclear magnetic resonance spectrum of carbenoxolone sodium.

17

CARBENOXOLONESODIUM

,

'

I

~

'

"

200

150

200

1%

"

'

I

'

"

'

,

"

'

IOU

50

100

50

(PPd

Figure 9. Carbon-13 nuclear magnetic resonance spectrum of carbenoxolone sodium (top), with DEPT 135' (middle), DEPT 90' (bottom).

18

SILVIA PINDADO ET AL.

(PPm) J

i

n

L'

1

1

t-

2

1

t

2 r. i

!

t 1. t-

I-

I

r 3

j I

t-

Figure 10. 2-D 'H - 'jC COSY nuclear magnetic resonance spectrum of carbenoxolone sodium.

CARBENOXOLONESODIUM

19

I

Figure 11. Proton nuclear magnetic resonance spectrum of carbenoxolone.

20

SEVIA PINDADO ET AL.

Figure 12. Carbon-13 nuclear magnetic resonance spectrum of carbenoxolone, with DEPT 1350.

CARBENOXOLONE SODIUM

21

/lit

p,

,,, , ,

, , , , , , , , ,

(PPm) 5

4

Figure 13.2-D H-H COSY niwlear magnetic resonance spectrum of carbenoxolone.

I

I

L

1

SILVIA PINDADO ET AL.

22

i

(PPm)

100

80

60

Figure 14.2-D 'H- I3C COSY nuclear magnetic resonance spectrum of carbenoxolone.

CARBENOXOLONE SODIUM

23

The 13C-NMR spectrum, with DEPT (1350 and 900), for carbenoxolone sodium was obtained at a frequency of 75.468 MHz in deuterated water. In this case, TMDS was used as the internal standard (Figure 9). A 2D H-C COSY was also obtained (Figure lo).

B . Carbenoxolone The 1H-NMR (Figure 11) and the l3C-NMR (Figure 12) spectra for carbenoxolone were obtained in deuterated chloroform (CDC13), using tetramethylsilane (TMS) as internal standard and the same frequencies as for the salt. The 2D H-H COSY (Figure 13) and the 2D H-C COSY (Figure 14) were also obtained.

4.2 X-Ray Diffraction The powder x-ray diffraction patterns of carbenoxolone sodium and carbenoxolone were obtained on a Siemens D-500 x-ray diffractometer, using a Cu x-ray tube, at 40 kV and 40 mA (Figure 15). It is evident that carbenoxolone is much more crystalline in nature, since the powder pattern for carbenoxolone sodium is almost that of an amorphous solid. The uptake of water significantly affects the structure of carbenoxolone sodium, as is evident from a comparison of the diffraction patterns obtained before and after drying (Figure 16). These findings are consistent with the hygroscopic nature of the sodium salt.

4.3 Optical Rotation In a 1 % w/v solution of carbenoxolone sodium in equal volumes of methanol and 0.02M sodium carbonate, rotation values of +132O to +1400 are obtained (B.P., 1993). For carbenoxolone, the specific rotation [ a ] ~ ~ 0 is +1280 in chloroform, (Merck Index, 1989).

SILVk PINDADO ET AL.

24

hJ

earbenoxolone

carbenoxolone sodium

0

5

10

15

20

25

Two - Theta (Degrees)

Figure 15.

X-ray powder diffraction patterns of carbenoxoIone sodium and carbenoxolone.

30

35

CARBENOXOLONE SODIUM

rigure 16.

X-ray powder diffraction patterns of carbenoxolone sodium before and after drying.

25

26

SILVIA PINDADO ET AL.

4.4 Thermal Methods of Analysis

4.4.1 Melting Point. Melting points for the carbenoxolone reported in the literature are in the temperature range of 291-2940 (Clarke, 1989; Merck Index, 1989). In the case of carbenoxolone sodium, melting with degradation occurred in the range of 290° to 300OC.

4.4.2 Differential Scanning Calorimetry (DSC) The DSC thermograms for carbenoxolone sodium and carbenoxolone were obtained using a Mettler DSC 20 TA 3000 system, at a scan speed of 1OoC/min. With both compounds, an initial peak was obtained at approximately 1OOOC which is attributed to the loss of water. Degradation of carbenoxolone sodium tended to occur from 260 to 3OO0C, while carbenoxolone melted below 300OC.

4.4.3 Thermogravimetric (TG) Analysis The TG thermogram of carbenoxolone sodium was obtained using a TG50 Thermobalance on the Mettler TA 3000 system, at a scan speed of 100C/min. An initial weight loss was noted at approximately 1OOOC which corresponded with loss of water. A further drop in weight occurred between 290-300OC, which was consistent with the events noted in the DSC studies. The acid form, carbenoxolone, also showed weight loss above 3OOOC indicating that it also would degrade at elevated temperatures.

4.5 Hygroscopicity

Carbenoxolone sodium is a very hygroscopic powder, so the moisture content should not exceed 4% w/w, as determined by Karl-Fisher titration (B.P. 1993; The Pharmaceutical Codex, 1979).

CARBENOXOLONE SODIUM

21

4.6 Dissociation constants Large differences in the values for the dissociation constants of carbenoxolone have been reported. These are summarized in Table 2.

Table 2. Dissociation constants (pKa) of carbenoxolone Method of determination

Reference

PKal 6.7

PKa PKQ 7.1

not stated

4.18 4.38

5.56 5.11

Partition Solubility

Downer et al.,(1970); Clarke (1986) Blanchard et al., (1988)

4.7 Solubility

Carbenoxolone sodium is soluble in 6 parts of water and in 30 parts of alcohol, and is practically insoluble in chloroform and in ether. A 10% wlv solution in water has a pH of 8.0 to 9.2 (Clarke, 1986; Martindale, 1993). At 24 and 37OC and pH 2, the intrinsic solubility of carbenoxolone was reported to be 1.16 and 1.63 x 10-5 Myrespectively, (Blanchard et al., 1988). A table of estimated solubilities for carbenoxolone in the pH range 4.0 to 6.5 has also been presented (Blanchard et al., 1990). The solubility of carbenoxolone sodium in the pH range 5.6 to 7.5 was determined, and the results are shown in Figure 17.

4.8 Partition Coefficients The distribution coefficients for carbenoxolone at 24 OC between chloroform and aqueous buffers have been reported as 2 (PH 7.4) and greater than 100 (PH 1.O). The distribution coefficients between n-octanol and aqueous buffers have been reported as 9 (PH 7.4) and exceeding 100 (PH 1.O) (Downer et al., 1970).

SILVIA PINDADO ET AL.

28

1

loo

x

5

7

6

8

PH Figure 17. pH solubility profile of carbenoxolone.

Apparent partition coefficients between n-octanol and a 0.1 M citratephosphate buffer at 37OC, and the fraction ionized for carbenoxolone, have been determined at various pH values (Bridges et al., 1976). The results are shown in Table 3, with the fiaction unionized being calculated assuming a pKa of 6.7 for carbenoxolone. Blanchard et al., (1988) determined the partitioning of carbenoxolone using tritium labeled drug. In this study, the apparent partition coefficients (APC) at different pH values (2.6 to 7.6), in an n-octanol/aqueous buffer system at 24 OC, were used to assess the pKa values. In the pH range studied, the APC decreased from over 600 to below 50. The true partition coefficient was reported as 643.8. The APC was independent of initial carbenoxolone concentration between pH 3 to 7, implying that carbenoxolone does not self-associate in the n-octanol or aqueous phases.

CARBENOXOLONE SODIUM

29

Table 3. Comparison of the apparent partition coefficients between octanol and buffer, and the fraction of carbenoxolone ionized at various pH values (Bridges et al., 1976). PH

Apparent partition coefficients (octanolhuffer) 14 27 60 214 350 484 679 908

8.0 7.4 7.1 6.8 6.5 6.2 5.6 5.0

Fraction unionized

0.05 0.17 0.29 0.44 0.61 0.76 0.93 0.98

5. METHODS OF ANALYSIS.

5.1 Elemental Analysis

Carbon Hydrogen Oxygen Sodium

Carbenoxolone Sodium (%) 66.43 7.87 18.22 7.48

Carbenoxolone

(%I

71.55 8.83 19.62

SILVIA PINDADO ET AL.

30

5.2 Identification The B.P. (1993) outlines four methods of identification for carbenoxolone sodium:

(A) The characteristic light absorption, as described in the UV Spectrophotometry section 4.1. The E 1% (1 cm) value at 256 nm is quoted as 199.

(B) 0.1 g is dissolved in 5 mL of water, made just acidic with 2M hydrochloric acid, stirred well, and filtered. The residue is washed with water until the washings are no longer acidic, and is then dried to constant weight at 105OC. The infrared absorption spectrum of this residue must be equivalent with the spectrum of authentic carbenoxolone, as outlined in section 4.2. (C) Color test: 5 mg of sample is mixed with 50 mg of resorcinol and 2 mL of sulfuric acid (80%). The mixture is heated at 2000 for 10 minutes, cooled, poured into 200 mL of water, and made barely alkaline with 5M sodium hydroxide. The product should exhibit an intense green fluorescence.

(D) The material must yield the reactions characteristic of sodium salts.

5.3 Titrimetric Analysis Carbenoxolone sodium may be titrimetrically assayed by non-aqueous titration, as described in the B.P. 1993. The salt is dissolved in water, acidified, extracted into chloroform, evaporated to dryness, and finally reconstituted in dimethylformamide. Tetrabutylammonium hydroxide is used as the titrant and thymol solution as the indicator.

CARBENOXOLONESODIUM

31

5.4 Ultraviolet Spectrophotometry Coleman and Parke (1963) described a method for the determination of pglycirrhetic acid (enoxolone) and its readily-hydrolysableesters (including carbenoxolone), in biological fluids. The material containing the glycyrrhetic acid or its esters is hydrolyzed with ethanolic sodium hydroxide, the glycyrrhetic acid is extracted fiom the acidified hydrolysate, submitted to two dimensional, thin-layer chromatography on alumina, eluted with ethanol, and estimated spectrophotometricallyat 248 nm. The UV absorption of carbenoxolone sodium in methanol and methanolic 0.1M NaOH were similar, with the reported wavelength maxima being 252 and 253.5. For these bands, the E 1%, 1 cm, values were 174 and 176, respectively. In aqueous solution, however, wavelength maximum shifted to 260 nm, and the E 1%, 1 cm, value was 172 (Kracmar et al., 1990). It was suggested that this information could be used to analyze formulations containing carbenoxolone.

5.5 Chromatographic Methods of Analysis 5.5.1 Thin Layer Chromatography Downer et al., (1970) have described a thin-layer chromatographic (TLC) system for identifying carbenoxolone and its metabolites once these are excreted in human bile following oral administration of the drug. Thinlayer plates (0.25 mm) of fluorescent silica gel HF 254 were used and developed in a solvent system containing: acetic acid-lY2-dichloroethanen-butanol-water (4 : 4 : 1 : 1 by volume). Carbenoxolone (Rfvalue = 0.95) was detected by a characteristic quenching of the background fluorescence when viewed under ultraviolet light (Chromatolite lamp). Clarke (1986) outlined a further TLC method for carbenoxolone sodium. The method used thin-layer plates of silica gel G (0.25 mm), and a choice of three mobile phases was given. These were ch1oroform:acetone (4:1); ethyl acetate:methanol:strongammonia solution (8: 1 0 3 ; and ethyl acetate. The Rfvalues for carbenoxolonewere 0.07,0.0, and 0.17 in each

32

SILVIA PINDAW ET AL.

of the respective systems, following visualization with acidified potassium permanganate solution.

The B.P. (1993) uses a TLC method to separate carbenoxolone sodium and to identify the presence of any related substances. In this method, silica gel F254 plates are used. The mobile phase contains ethyl acetate, methanol, water, and 13.5M ammonia (60:20: I 1:1 by volume). After removal of the plate, it is allowed to dry in air and is examined under ultraviolet light (254 nm). Alternatively, visualization may be performed by spraying with a 1.5%w/v solution of vanillin in sulfuric acid (60%) and heating at 105OC for 10-15 minutes.

5.52 Gas Chromatography A gas-liquid chromatographic procedure for the determination of carbenoxolone in human serum has been described (Rhodes and Wright, 1974). Chromatography of methylated derivatives was performed on glass columns containing 1% OV- 1 (dimethylsilicone gum) on a solid support of Gas-Chrom Q (100- 120 mesh) at a temperature of 265OC. The 18 a-isomer of carbenoxolone was used as the internal standard, and detection was by flame ionization. The method showed a greater than +11% coefficient of variation for all values determined, and the sensitivity was 5 mg/mL in serum. The specificity of the method was established by combining the gas-liquid chromatography analysis with thin-layer chromatography of prepared standards and serum from treated volunteers.

5.5.3 High Performance Liquid Chromatography Sanofi Winthrop, England, have developed a high performance liquid chromatographic (HPLC) method for quantifying carbenoxolne sodium in Pyrogastrone tablets. The column used was Spherisorb Hexyl5 mm (12.5 cm x 0.45 cm i. d.), the mobile phase was methanol (75% v/v), water (25% v/v), ammonium acetate (1% wh), and glacial acetic acid (0.1% v/v). The flow rate was 2 mL per minute, and detection was by UV absorption at 254 nm. Samples were prepared in 75:25 methanol water containing 1 to 2.5%

CARBENOXOLONE SODIUM

33

orthophosphoric acid (50% v/v). The retention time was approximately 1.5 minutes. The sensitivity reported for this method was 0.08 aufs.

5.6 Radioimmunoassay Peskar et al. (1 976)developed a radioimmunoassay for carbenoxolone. [3H]carbenoxolonewas synthesized by reduction of 3-keto-enoxolone with sodium borotritiide, followed by succinoylation of the resultant [3H]-enoxolone with succinic anhydride. For preparation of the antigen, carbenoxolone was conjugated to bovine serum albumin using the carbodiimide method described by Goodfiiend et al. (1964). The production of antisera against carbenoxolone was described, and their specificity and use for a radioimmunoassay were reviewed, The sensitivity of the method in serum was 1 ng/mL.

5.7 Radioactive Labeling Iveson et al. (1971) prepared [carboxypropionyl-1,4-14C2]-carbenoxolone, (0.1mCi/g). This derivative was used by Bridges et al. (1 976)to assess the gastrointestinal absorption of carbenoxolone in the rat. Samples were counted in either a dioxane based scintillator or in a Tritox 100-toluene (I :2by volume) scintillator containing 1% w/v butyl PPD. Radioactivity was measured by a scintillation spectrometer, and the counting efficiency determined using [14C]toluene as internal standard. Blanchard et al. (1988;1990)used [3H]carbenoxolone sodium, specific activity 6.9mCi/mg and labeled at C-3, to study the physicochemical properties and the absorption of carbenoxolone in the rat. Samples were counted using a liquid scintillation counter.

6. STABILITY Carbenoxolone is a triterpenoid, the ester of 18 p-glycyrrhetic (enoxolone) acid with succinic acid. It is stable in neutral and acid solution, but is hydrolyzed by alkalis into P-glycyrrhetic acid plus succinate (Parke, 1972).

34

SILVIA PINDADO ET AL.

7. PHARMACOKINETICS 7.1 Absorption Carbenoxolone is rapidly absorbed following oral administration of an aqueous solution of the sodium salt to patients, attaining high blood concentrations (Parke et al., 1972 ). When tablets of the drug were administered to man on an empty stomach, an initial plasma maximum occurred at 1-2 hours and another maximum at 3-6 hours after dosage (Parke et af., 1972; Downer et al., 1970 ). It has been suggested that the second peak is probably due to enterohepatic circulation of the biliary-excreted conjugates. Baron et a/. (1975), however, found no evidence for this pattern of absorption, and have reported a single absorption peak. When carbenoxolone was administered orally to patients subsequent to the administration of an alkaline buffer mixture, the drug did not appear in the blood plasma until the gastric acidity was restored, and the stomach contents attained a pH value of less than two (where less than 0.002% of carbenoxolone is ionized). Since carbenoxolone is a weak acid and in its non-ionized form is highly lipid-soluble, it was suggested that the major site for absorption was the stomach (Downer et af.,1970). The rapid absorption has been associated with the high affinity of the drug for proteins, for although it is sparingly soluble in acidic aqueous media (the stomach) the concentration of unbound drug in the plasma is so low as to be undetectable. The high plasma protein binding of this drug may therefore act to accelerate gastric absorption (Parke et af.,1972). In contrast, Bridges et al., (1976) studied the absorption of ['4C]carbenoxolone from inverted rat ileum in-vitro, and from rat stomach and illeum in-situ, and obtained greater absorption at pH values where the ionized form predominates. Iveson et al., (1966; 1971) reported that carbenoxolone was largely hydrolyzed to P-glycyrrhetic acid before absorption in the rat. In contrast, Downer et al., (1970) reported that in man carbenoxolone was absorbed largely unchanged. Bridges et af.,(1976) obtained no evidence of metabolism in the rat during absorption in either the stomach or the intestine. Bridges et al., (1976) observed an extensive tissue binding of carbenoxolone to the inverted rat ileum in-vitro and suggested that the

CARBENOXOLONE SODIUM

35

high percentage of carbenoxolone accumulated in the tissue was not entirely due to binding to tissue proteins and lipids, but also due to precipitation in, or adsorption to, the gut sac epithelium. Tissue binding to the ileum, in-situ, was not dependent on pH, except below pH 5.0, when extensive tissue accumulation of carbenoxoloneoccurred because of its low solubility. Tissue binding to the stomach increased markedly with decrease of pH from 7.4 to 6.5, and at pH 6.5 was 80 times greater than binding to the intestine. Contrary to the pH-partition hypothesis Bridges et al., (1 976) reported that carbenoxolone was absorbed from the intestine, and perhaps also from the stomach, at a rate 3.8 times faster when ionized than in its unionized form. The absorption of carbenoxolone was reevaluated by Blanchard et al., (1990) using an in-situ rat intestinal perfusion technique, in which disappearance from the intestinal lumen, binding to the perfixed jejunal segment, and appearance in the mesenteric (jejunal) vein were measured. The effect of the degree of ionization on these processes was examined by employing perfusion solutions of pH 4.0,4.4, 5.0, and 6.5. Tissue binding was observed to be independent of pH. There was a rank order correlation of the transfer rate of carbenoxolonewith the degree of ionization, which indicated that carbenoxolone was absorbed faster in its ionized form. This observation appeared to support the previously work of Bridges et al., (1976). However, Blanchard et al., (1990) have suggested a likely explanation for this unusual behavior is that at the low pH values some carbenoxolone precipitates out of solution during the perfusion experiments, thereby reducing the driving force for diffusion across the intestinal wall. Alternatively, ion-pairing of carbenoxolone with sodium ion present in the pH 6.5 buffer may occur. Food has been reported to delay the initial absorption phase of carbenoxolone in patients with peptic ulcer when given as a single dose, however, on subsequent accumulation of the drug after repeated administration over 3 to 7 days, no food effect was observed. Concurrent antacid administration, in these patients, did not significantly affect carbenoxolone absorption (Baron et al., 1975). Carbenoxolne is normally given in the form of tablets for gastric ulcer. It is also available as a "position-release"capsule for duodenal ulcer, and this formulation is designed to rupture near the pylorus as a result of gaseous distention and deformation by peristaltic abrasion and to deliver the drug

36

SILVIA PINDADO ET AL.

into the duodenum. In this form carbenoxolone is as readily absorbed as when administered by tablets, although it is not certain as to which area of the gastro-intestinal tract is involved and that the time to rupture may vary (Lindup et al., 1970).

7.2 Distribution Binding to plasma proteins can influence the distribution, pharmacological properties and excretion of drugs, particularly if the drug is very highly bound (Meyer and Guttman, 1968). During absorption studies Downer et ui.,( 1 970) noted high blood concentrations for carbenoxolone. This suggested that most of the drug was in the circulating blood, and indicated a high degree of binding of the drug to the plasma proteins. Parke (1 972) studied the binding of the drug in-vitro to whole heparinized plasma using an ultrafiltration technique involving centrifugation. At therapeutic plasma levels (10-100 mg/mL), the drug was more than 99.9% bound to plasma proteins of the male and female rat, dog, monkey and man. Using molecular sieve chromatography on Sephadex G200 [carboxypropionyl-14C1,4]-carbenoxolone (1 00 mg/mL) in human blood plasma was associated with the globulins (17%) and the remainder (83%) was associated with albumin. Other in-vitro and in-vivo experiments, using the radioactively-labelleddrug in kinetic studies and in fluorescence determinations, have shown that carbenoxolone binds to human serum albumin at two different classes of binding sites, with apparent association constants of 107 and 3 x 106, respectively. This binding gives rise to a pronounced conformational change in the albumin which appears to enhance the binding of carbenoxolone still further. Due to the high degree of binding of carbenoxolone to the plasma proteins, the drug is absorbed from the gastrointestinal tract, is conjugated in the liver and is excreted in the bile with very little appearing in the urine (Parke, 1972). Studies with [14C]-carbenoxolone administered to rats have confirmed that the drug is almost entirely located in the gastrointestinal tract, the liver and the blood plasma. Experiments in which [ 14C]-carbenoxolonewas administered intraperitoneally to rats have shown that the radioactive drug and conjugates migrate back into the gastrointestinal tract, in particular into the gastric mucosa. This suggested that there may be proteins present in the stomach mucosa which have a

CARBENOXOLONE SODIUM

37

special affinity for carbenoxolone. No significant distribution of [14C]carbenoxolone into the kidney, body fat, brain, musculature or tissues, other than that previously mentioned, was detected. A comparative study of healthy adults and geriatric patients suggested that protein binding of carbenoxolone was reduced in the elderly, and was associated with lower plasma albumin concentrations (Hayes et ul., 1977). The mean clearance, plasma half-life and volumes of distribution of carbenoxolonewere 4.72 mLkg.hour, 16.3 hours, and 0.105 L1 kg, respectively, in the healthy adult. This may be compared to the values of 3.28 mlkgohour, 22.9 hours, and 0.098 Lkg, respectively, which had been obtained in elderly patients. It has been suggested that these factors may contribute to the higher incidence of carbenoxolone side-effects in the elderly. Lower plasma half-lives for carbenoxolone, varying between 5.6 and 10 hours, have also been reported (Thorton et uf.,1980).

7.3 Metabolism Metabolism of carbenoxolone appears to be species dependent. Following oral administration to the rat, carbenoxolone was reported to be hydrolyzed in the gastrointestinal tract to the glucuronide and sulfate conjugates of pglycyrrhetic acid and succinate prior to absorption (Iveson et al., 1971; Parke, 1972). However, Bridges et al., (1976) found no evidence of metabolism during absorption in the rat. In man, the ester linkage appeared to be stable and carbenoxolone was absorbed largely unchanged (Downer et al., 1970). Following absorption, the drug was transported, bound to plasma proteins, to the liver and was conjugated with glucuronic acid and excreted in the bile (Parke, 1972).

7.4 Excretion

When [14C]-carbenoxolone was administered to man, 70-80% of the radioactivity was excreted in feces, 0.2-1% in urine, and 12-20% was excreted in expired air as 14CO2. The radioactivity present in feces occurred as carbenoxolone and represented the biliary excretion of carbenoxolone-30glucuronide, subsequently hydrolyzed in the intestine by the gut microflora, rather than non-absorbed drug. The 14CO2 present in the expired air

38

SILVIA PINDADO ET AL.

represented the extent of hydrolysis of [ 4C]-carbenoxolone into P-glycyrretic acid plus [ 4C]-succinate, since the latter is rapidly and completely oxidized to 14CO2. A small amount of the radioactivity excreted in the urine was present as urea and the remainder was likely to be derived from [14C]succinate for no terpenoid compounds were detected (Parke, et al., 1972; Downer et al., 1970).

*

Following oral administration of [ 14C]-carbenoxoloneto rats, 60-75% of the radioactivity was excreted as 14 C02 in the expired air, 12-35% in the feces as carbenoxolone originating from bile, and 2% in the urine (Parke, 1972).

8. PHARMACOLOGY 8.1 Therapeutic Indications and Uses. Carbenoxolone promotes ulcer healing and prevents ulcer relapse (Langman, 1980). The anti-ulcer activity is well established in both gastric and duodenal ulcer patients (Cooke, et al., 1980), however, the precise mechanism of action remains unclear. Several mechanisms of action have been proposed including an increased level of prostaglandins, particularly E2, (Peskar, 1980; RaskMadsen et al., 1983). Minuz et al., (1984) reported an increase in prostaglandin E2 activity following rectal administration,therefore suggesting that carbenoxolone also has a systemic action. Further reported actions of carbenoxolone which may contribute to its anti-ulcer effect include stimulation of mucus secretion (Goodier et al., 1967; Dean, 1968) and secretion of HCO3- into the unstirred mucus gel layer coating the gastric epithelium (Allan and Gamer, 1980) ,promotion of mucosal cell proliferation (Van Huis and Kramer, 198l), inhibition of mucosal cell exfoliation (Domschke et al, 1977) and inhibition of peptic activity (Henman, 1970). Pinder et al. (1 976) have reviewed the pharmacological properties of carbenoxolone and the therapeutic efficacy of the drug in peptic ulcer disease. In addition to the formulations used for oral administration, including tablets for gastric ulcers and "position-release'' capsules for duodenal ulcer, carbenoxolone is also used as a gel or mouthwash in the treatment of mouth ulcers. Topical carbenoxolone has been used to treat orofacial herpes simplex infections (Poswillo and Roberts, 1981). In-vitro, it has been shown to have

CARBENOXOLONE SODIUM

39

antiviral activity against various DNA and RNA viruses (Dargan and SubakSharpe, 1986). Treatment with carbenoxolone sodium solution every 4 hours as a mouthwash and gargle produced symptom relief and healing of oropharyngeal ulceration, associated with herpes simplex virus, in HIVinfected patients (Poswillo, 1990). Aphthous ulceration, which has been linked with varicella zoster virus, has also been successfully treated with carbenoxolone (Poswillo and Partridge, 1984). Carbenoxolone also has anti-inflammatoryactivity (Finney and Somers, 1958; Khan and Sullivan, 1968). When administered parenterally this activity was almost one third that of hydrocortisone and it is reduced by adrenalectomy (Sullivan, 1972).

8.2 Toxicity and Side-Effects.

Carbenoxolone sodium is a drug of relatively low acute toxicity in animals: the LD50 intravenously in mice is about 200 mgkg and the LD50 orally in rats is about 3.0 gkg. It produces necrosis if injected in concentrations above I %. Chronic toxicity testing in animals, using doses up to 40 times the therapeutic dose, produced no toxicity. Reproductive, teratogenic and carcinogenic tests on carbenoxolone in animals showed no significant adverse effects (Sullivan, 1972). Carbenoxole sodium has mineralocorticoid-likeeffects and may produce sodium and water retention and hypokalaemia (Porter, 1970). This may cause or exacerbate hypertension, cardiac failure, weight gain, oedema, alkalosis, and muscle weakness and damage (Davies et al., 1974; Dickinson and Swaminathan, 1978; Ganguli and Mohamed, 1980).

9. REFERENCES

Allan, A., and Garner, A. (1980). Gut, 2,249-262. Baron, J. H., Gribble, R. J. N., Rhodes, C., and Wright, P. A. (1975). In Fourth Symposium on Carbenoxolone Sodium, Avery Jones, F., and Parke, D. V. (Eds). Butterworths, London. pp. 115-128.

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Blanchard, J., Tang, L.M., and Earle, M. E. (1990). J. Pharm. Sci. 29,411414. Blanchard, J., Boyle, J. O., and Van Wagenen, S. (1988). J. Pharm. Sci. 22, 548-552. Bridges, J. W., Houston, J. B., Humphrey, M. J., Lindup, W. E., Parke, D. V., Schillingford, J. S., and Upshall, D. G. (1976). J. Pharm. Pharmac. 28, 117-126. British Patent, (1960), No. 843,133. British Pharmacopoeia, ( 1 993). British Pharmacopeial Commision London, HMSO, p. 1 10. Chinese Pharmacopoeia, (1985). Pharmacopeial Commision, Ministry of Hygiene of the People's Republic of China, Beijing. p. 56-57. Clarke, E.G.C. (1 986). Isolation and Identification of Drugs, 2nd Edn. The Pharmaceutical Press, London. p. 430-43 1. Coleman, T. J., and Parke, D. V. (1963). J. Pharm. Pharmac. 15,841-845. Cooke, P. J., Vincent-Brown, A., Lewis, S. I., Perks, S., Jewell, D. P., and Reed, P.I. (1980). Scand. J. Gastroenterol, L5 (Suppl. 65), 93-96. Dargan, D. J., and Subak-Sharpe, J. H. (1986). J. Antimicrob. Chemother. 18 (Suppi. W), 185-200. Davies, G. J., Rhodes, J., and Calcraft, B. J. (1974). Br. Med. J. 3,400402. Dean, A. C. B. (1968). In A Symposium on Carbenoxolone Sodium, Robson, J. M. and Sullivan, F. M. (Eds). Butterworths, London. p. 33-46. Dickinson, R. J., and Swaminathan, R. (1978). Postgrad. Med. J. 54,836837.

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Doll, R., Hill, I. D., Hutton, C., and Underwood, D. J. (1 962). Lancet 2, 793-796. Domschke, W., Domschke, S., Hagel, J., Demling, L., and Croft, D. N. (1 977). Gut s , 817-820. Downer, H. D., Galloway, R. W., Honvich, L., and Parke, D. V. (1970).J. Pharm. Pharmacol. 22,479-487. Finney, R. S. H., and Somers, G. F. (1958).J. Pharm. Pharmacol. U, 613620. Ganguli, P. C., and Deen Mohamed, S.(1980).Scand. J. Gastroenterol. 15 (Suppl. 65), 63-69. Goodfriend, T. L., Levine, L., and Fasman, G. D. (1964).Science 144, 1344-1346. Goodier, T.E.W., Horwich, L., and Galloway, R. W., (1967).Gut S, 544547. Hayes, M. J., Sprackling, M., and Langman, M. J. S.(1977).Gut U, 1054-1058. Henman, F. D. (1970).Gut 11,344-351. Iveson, P., Lindup, W. E., Parke, D. V., and Williams, R. T. (1971). Xenobiotica 1,79-95. Iveson, P., Parke, D. V., and Williams, R. T., (1966).Biochem. J. UN, 28p. Khan, M. H., and Sullivan, F. M. (1 968).In A Symposium on CarbenoxoloneSodium, Robson, J. M. and Sullivan, F. M. (Eds). Butterworths, London, pp. 5-14. Kracmar, J., Kracmarova, J., Bokovikova, T. N., Ciciro, V. E., Nesterova, G. A. Suranova, A. V. Truis, N. V. (1 990). Pharmazie 45,912-916.

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Langman, M. J. S. (1 980). In Advunces in Ulcer Diseuse, Holtermuller, K. H., and Malagelada, J. R. (Eds). Excerpta Medica, Amsterdam, pp. 406-4 1 5. Lindup, W. E., Parke, D. V., and Colin-Jones, D. (1970). Gut U, 555-588. Martindale. The Extra Pharmacopoeia, (1 993). 30th Edn. The Pharmaceutical Press, London, pp. 873-874. Merck Index, (1989). 1 lth Edn. pp. 1801. Meyer, M. C., and Guttman, D. E. (1968). J. Pharm. Sci. 52,895-918. Minuz, P., Cavallini, G., Angelini, G. P., Lechi, A., Brocco, G., Riela, A., Scuro, L. A., and Velo, G. P. (1984). Pharmacol. Res. Commun. Ifi, 875-883. Parke, D. V. (1 972). In Curbenoxolone in Gustroenterologv, Avery Jones, F., and Sullivan, F. M. (Eds). Butterworths. London, pp. 19-32. Peskar, B. M. (1980). Scand. J. Gastroenterol. 15(Suppl. 65), 109-112. Peskar, B. M., Peskar, B. A., and Turner, J. C. (1976). J. Pharm. Pharmac. 2, 720-721. Pinder, R. M.,Brogden, R. N., Sawyer, P. R., Speight, T. M., Spencer, R. and Avery, G. S. (1976). Drugs 11,245-307. Porter, G. A., (1 970). In Curbenoxolone Sodium, Baron J. H., and Sullivan, F. M. (Eds). Butterworths. London, pp. 33-47. Poswillo, D. E., and Partridge, M. (1984). Br. Dent. J. B, 55-57. Poswillo, D. E. and Roberts, G. J. (1981). Lancet hi, 143-144. Poswillo, D.E. (1 990). Lancet

8 13.

Rask-Madsen, J., Bukhave, K., Madsen, P.E.R., and Bekker, C. (1983). Eur. J. Clin. Invest. 11,351-356.

CARBENOXOLONE SODIUM

Rhodes, C., and Wright, P. A. (1974). J. Pharm. Pharmac. X,894-898. Sullivan, F. M. (1 972). In Carbenoxolone in Gastroenterology,Avery Jones, F., and Sullivan, F. M. (Eds). Buttenvorths, London, pp. 318. The Pharmaceutical Codex, (1979). 1lth Edn. The Pharmaceutical Press London, pp. 138-139. Thornton, P. C., Papouchado, M., and Reed, P.I. (1980). Scand. J. Gastroenterol. 1_5 (Suppl. 65), 35-38. U. S. Patent, (1962). No. 3,070,623. Van Huis, G . A., and Kramer, M. F. (1981). Gut 22,782-787.

10. ACKNOWLEDGEMENTS The authors wish to thank Dr. J. O'Brien (NMR Unit, Trinity College Dublin), Dr. P. Caplan (Mass Spectrometry Unit, University College Dublin), Dr. M. Meegan (Department of Pharmaceutical Chemistry, Trinity College Dublin) for their help and assistance, as well as Mr. J. Steel and Mr. D. Proctor (Sanofi Winthrop, Newcastle Upon Tyne, England) for the supply of carbenoxolone sodium and for information on HPLC.

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CLARITHROMYCIN

Isam Ismail Salem

Department of Pharmacy and Pharmaceutical Technology University of Granada

1 807 1 -Granada Spain

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIF'IENTS-VOLUME 24

45

Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

ISAM ISMAIL SALEM

46

Contents 1.

Introduction

2.

Description 2.1. Structural and Molecular Formulae, Molecular Weight 2.2. Nomenclature 2.2.1. Generic Names 2.2.2. Chemical Name 2.2.3. Chemical Abstracts Number 2.2.4. Trade Names 2.2.5. Other Names, Abbreviations, and Drug Codes 2.3. Color, Appearance, and Odor

3.

Synthesis

4.

Physical Properties 4.1 Powder X-Ray Diffiaction 4.2 Thermal Methods of Analysis 4.2.1 Thermogravimetric Analysis 4.2.2 Differential S c d g Calorimetry 4.3 Solubility 4.4 pHRange 4.5 Ultraviolet Absorbance Spectrum 4.6 Infrared Spectrum 4.7 Nuclear Magnetic Resonance Spectra 4.7.1 'H-NMR Spectrum 4.7.2 13C-NMRSpectrum 4.8 MassSpectrum

5.

Methods of Analysis 5.1 Elemental Analysis 5.2 Identification 5.3 Thin Layer Chromatography 5.4 Structural Details 5.5 High Performance Liquid Chromatography 5.6 Microbiological Analysis

CLARITHROMYCIN

6.

Stability

7.

Pharmacokinetics 7.1 Adsorption 7.2 Bioavailability 7.3 Distribution 7.4 Elimination

8.

Pharmacology 8.1 Mechanism of Action 8.2 Toxicity

9.

References

1.

INTRODUCTION

41

Clarithromycin is a new semi-syntheticantimicrobial 14-membered macrolide exhibiting a broad in vitro antibacterial spectrum. Structurally, it differs from erythromycin only in the substitution of an 0-methyl group for the hydroxyl group at position six of the lactone [11, with increased tissue or cellular penetration [2]. It has a more favorable pharmacokineticsprofile, than erythromycin, which allows twice-daily administration and a possible increase in compliance [3]. To improve the spectrum of activity and decrease the disadvantages of erythromycin, a new generation of macrolide compounds has been developed. These include azithromycin, clarithromycin,roxithromycin, dirithromycin, micocamycin and rokitamycin. Azithromycin and clarithromycin have been approved recently by the Food and Drug Administration (Oct. 1991). Clarithromycin appears to have more activity against Mycoplasma pneumoniae and Chlamydia trachomatis [4-91. Furthermore, clarithromycin

ISAM ISMAIL SALEM

48

(in combination with its microbiologically active metabolite, 14-hydroxyclarithromycin) has shown an additive or even synergistic activity against Haemophilus injluenzae, a species that often is resistant of intermediate susceptibility to erythromycin [101. The 14-hydroxy-clarithromycinitself is twice as active as the parent compound. The effect of combining clarithromycin with a variety of other drugs for the treatment and prevention of disseminated M avium infection in patients with AIDS is under investigation [l l-141. In addition, it has demonstrated activity in vitro and in clinical infections against staphylococci, streptococci, Haemophilus species, Campylobacter species, Mycoplasma species, Chlamydia species, Mycobacteria species, and Neisseria gonorrhoeae It has demonstrated activity, superior to that of erythromycin, against Legionella pneumophilia; and is active against anaerobes [ 15-171. Clarithromycin was discovered and patented by Taisho Pharmaceutical Co. Ltd. Japan (Watanabe et al., Eur. Pat. Appl. Ep 41,355 (CL. C07H17/08),09 Dec 1981; JP Appl. 80/75,258,04 June 1980, 18; US Pat. 4,331.803), and is being marketed by Abbott Laboratories.

2.

DESCRIPTION

2.1.

Structural and Molecular Formulae, Molecular Weight Molecular Formula: C38H69N013 Molecular Weight: 747.96

CLARITHROMYCIN

2.2.

49

Nomenclature 2.2.1. Generic Names Clarithromycin (BAN,USAN, rINN); clarithromycin (DCF); claritromicina (DCIT)

2.2.2. Chemical Name (2R,3S,4S,5R,6R,8R,1OR, 11R, 12S,13R)-3-(2,6-Dideoxy-3C,3-o-dimethyl-ct-~-ribo-hexopyranosyloxy)11,12dihydroxy-6-methoxy-2,4,6,8,10,12-hexamethy1-9-oxo-5-

(3,4,6-trideoxy-3-dimethylarnino-P-~-xylohexopyranosy1oxy)pentadecan-13-olide. This structure is shown in Figure 1. 2.2.3. Chemical Abstracts Number CAS-81103-11-9.

2.2.4. Trade Names Clarithromycin is marke-zd by Abbott under the proprie uy names, "Biaxin USA", "Klacid Switz",and "Klaricid U P .

2.2.5. Other Names, Abbreviations, or Drug Codes 6-o-Methylerythromycin;A-56268; Abbott-56268; TE-031; Erythromycin, 6-0-methyL.

2.3. Color, Appearance, and Odor White to off-white crystalline odorless powder. Colorless needles are obtained when the compound is crystallized from a mixture of 1:2 chlorofoddiisopropyl ether. During the synthesis of

so

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Figure 1.

Structure of clarithromycin.

CLARITHROMYCIN

51

clarithromycin, crystals were obtained from ethanol as orthorhombic needles, for which [aID= -90.4"at 24°C (c=l .O, CHC13solution) [11.

3.

SYNTHESIS

The original synthesis of A-56268(TE-031) was performed by Watanabe et al. in 1981. Clarithromycin was then obtained by methylating 0,N-dibenzyloxycarbonyl-des-N-methylerythromycin A with CH31, deblocking, and subsequent N-methylation with CH20. The preparation of clarithromycinwas reported by Morimoto et al. in 1985 [11. 2'-0,3'-N-Bis(benzyloxycarbonyl)-N-dimethyl-e~omycin A was methylated with CH31and NaH in dimethyl sulfoxide-tetrahydrofuran, and the mixture was chromatographed on a silica gel column. One of the products obtained after the separation was hydrogenated with Pd-black in ethanol in the presence of sodium acetate-acetic acid buffer, which was followed by reductive methylation with formaldehyde and hydrogen. The substance was recrystallized from chloroform-isopropyl ether to give a mixture of clarithromycin and 6,ll -di-o-methylerythromycin A [ 11.

The selective o-methylation of the C-6 hydroxyl group of erythromycin A was achieved by Watanabe et al. in 1990 [18], using erythromycin 9-oxime derivatives as the starting materials to obtain clarithromycin. To improve the synthesis method, Watanabe et al. [19] reported a new method for the preparation of clarithromycin via the erythromycin A quaternary ammonium salt derivative. The reactions involved in this synthetic pathway are shown in Figure 2, and introduce three advantages. First, the protection of the oxime and desosamine moieties is accomplished by the use of benzyl bromide and sodium hydride in one pot. Second, the removal of the three benzyl groups could be carried out using the CTH method. Finally, the high selectivity of the methylation at the C-6hydroxyl

= Benyc)

Figure 2.

Preparation of clarithromycin via the erythromycin A quaternary ammonium salt derivative.

CLARITHROMYCIN

53

group is sufficiently maintained. Clarithromycin was obtained in 53% overall yield from erythromycin A 9-oxime. Although in a previous synthesis [181, all the intermediateswere obtained with good crystalline properties and clarithromycin could be obtained in high purity, a large amount of benzyl chloroformate was required at the step where the benzyloxycarbonyl (Cbz) groups were introduced. The use of this reagent was a distinct drawback owing to the severe irritating action and toxicity of benzyl chloroformate. To resolve the handling difficulty and the problem of the elimination of benzyl groups by hydrogenation during the synthesis via erythromycinA quaternary ammonium salt derivative [19], Watanabe et al. [20] described a facile synthesis of clarithromycin via 2’4lylethers of erythromycinA derivatives (Figure 3). Using this synthetic pathway, it was possible to prepare clarithromycinin a 48% yield from erythromycin A 9-oxime without requiring the purification of each intermediate.

4.

PHYSICAL PROPERTIES

4.1.

Powder X-RayDiffraction

The x-ray powder diffkaction pattern of clarithromycin powder sample was obtained using a Philips diffkactometer system (model PW1710). The pattern was obtained using nickel filtered copper radiation (h=1 S405 A), and is shown in Figure 4. A full data summary is provided in Table I.

To date, only one polymorph or pseudopolymorph of clarithromycin has been detected.

2.

Figure 3.

Synthesis of clarithromycinvia the 2'-silylethers of erythromycin A derivatives.

le

28

40

Scattering Angle (degrees 2-8) Figure 4.

X-ray powder diffraction pattern of clarithromycin.

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Table I Crystallographic Data Deduced from the X-Ray Powder Pattern of Clarithromycin Scattering Angle (degrees 20) 8.60 9.56 10.92 11.56 11.96 12.48 13.28 13.84 14.12 15.24 16.60 17.00 17.44 18.20 18.44 19.12 20.00 20.60 21.44 21.64 22.32 23.20 25.00

d-spacing

Relative Intensity

(14) 10.273 9.2434 8.0951 7.6483 7.3934 7.0865 6.6613 6.3930 6.2669 5.8088 5.3358 5.2111 5.0806 4.8702 4.8073 4.6378 4.4357 4.3079 4.1409 4.1031 3.9796 3.8306 3S588

67.4 100.0 60.6 61.4 26.7 13.1 8.4 19.6 25.8 41.2 18.8 29.1 35.0 19.4 18.0 49.0 17.9 22.6 10.4 11.2 25.1 16.1 16.5

CLARITHROMYCIN

4.2.

57

Thermal Methods of Analysis 4.2.1. Thermogravimetric(TG)Analysis

TG thermograms were obtained using a Shimazu TGA 50H thermogravimetric analyzer, simultaneously connected to a Fisons Instruments Thermolab mass detector and a Nicolet TGA interface MagnaIR 550. The system was calibrated using the latent heat of melting of Indium. The experiments were carried out in flowing nitrogen or air (20 mL/min) at different heating rates (from 1O"C/min to 2OoC/min). The sample sizes used ranged between 6-8 mg, and were analyzed over a temperature range of 30°C to 650°C. Mass and IR spectrums of the gases produced during the analysis were recorded. The TG thermogram, and its first derivative, are shown in Figure 5 for a 7.196 mg sample of clarithromycin contained in an alumina cell. The TG studies indicated the loss of 91.O% at temperature values above 300°C. 4.2.2. Differential Scanning Calorimetry (DSC) The thermal behavior of clarithromycin was further examined by DSC, using a Shimazu DSC-50 differential scanning calorimeter. The system was calibrated with a high purity sample (5 mg) of Indium. Clarithromycin samples of 5-6 mg were run at a scanning rate of 5"C/min, over a temperature range of 30 to 400°C. Changes caused by fusion-cooling processes also were studied, and the peak transition and enthalpies of hsion were determined for all samples. DSC curves of clarithromycin showed one endothermicpeak of fusion, having a peak maximum of 225°C. When examined by hot-stage microscopy, the melting of the solid was observed to take place at the same temperature value. The DSC thermogram shown in Figure 6 shows a single, sharp, melting endotherm with an onset temperature of 222°C. Integration of the melting endotherm permitted an estimation of the enthalpy of fusion (AH) for clarithromycin as -41.29 J/g.

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58

8.00. I

6.00

-0.00

-2.00

4.00.

2-00. 321.09 'C

0.OOJ

I

I

--4.00 I

I

Temperature ("C)

Figure 5.

Thermogravimetryprofile (and its first derivative) of clarithromycin.

4.13

CLARITHROMYCIN

59

0.8' a7a6a5a4-

224.9 oc 1

im

1

1

"

I

I

I

150

I

. I

2al

I

-.

1

1

I

I

I

250

I

.

,

a

300

1

.

I

,

I

350

I

,

' 1

400

Temperature ("C)

Figure 6.

DSC thermogram of clarithromycin, obtained at a heating rate of S"C/min.

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ISAM ISMAIL SALEM

As noted, the exothermic decomposition peak has an onset temperature at 295"C, and a peak temperature at 320°C. This finding, and the data obtained from the IR and MS gas analysis, reafltirmed that the compound can undergo melting without simultaneous decomposition.

4.3.

Solubility

Clarithromycin is soluble in acetone, and is slightly soluble in methanol and in ethanol. It is practically insoluble in water. The solubility of clarithromycinhas been studied in different solvents, as were the effects of pH (ranging from 2.4 to 8.4) and buffer concentrations. A series of 0.05 M; 0.1 M phosphate buffer - water solutions were prepared at pH 2.4,5.4,7.4, and 8. An excess of clarithromycin was added to each medium, which were then shaken for 24 hours at 25°C. Once equilibrium was reached, the samples were centrifuged at 10,000 rpm for five minutes. The Supernatantswere clarified by filtration through a 0.45 pm membrane, and analyzed by HPLC (method described in section 5.5). All assays were conducted in triplicate. At lower pH values, it was found that the solubility of clarithromycin exhibited a slight buffer salt effect, which was most pronounced at high pH values (Figure 7). The solubility of clarithromycin was significantly increased at lower pH values, while the solubility was significantly increased when different concentrations of methanol (more than 80% v/v) were added to the stock solutions.

4.4.

pHRange

Clarithromycin is a basic substance, and the pH of its aqueous solutions is therefore dependent on the solute concentration. This behavior is depicted in Figures 8 and 9, which illustrate the relation of solution pH and the concentration of clarithromycin in media consisting of 80:20 v/v methanol-water and in pure water, respectively.

61

CLARITHROMYCIN

0 Water

14

C

-*-

I a r i rn t 9

r

12

* Buffer 0.1M

10

8

m

6

m L

4

0

Buffer 0.05M

2 0 pH = 2.4

Figure 7.

pH = 5.4

pH =7.4

pH = 8.4

Effect of pH and salt concentration on the solubility of clarithromycin.

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62

PH

0.0

0.5

1 .o

1.5

Concentration (mg/mL)

Figure 8.

Effect of clarithromycinconcentration on the apparent pH of 80:20 (v/v) methanol-water.

CLARITHROMYCIN

63

1

8.5 0.0

0.1

0.2

0.3

0.4

0.5

Concentration (mg/mL)

Figure 9.

Effect of clarithromycinconcentration on the pH of water.

64

4.5.

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Ultraviolet Absorbance Spectrum

The UV spectrum of clarithromycin was obtained using a PerkinElmer Lambda 5 UVNIS spectrophotometer. The spectra were scanned from 190 to 400 nm at 60 d m i n (2 nm spectral slit width), with the solutions being contained in 1 cm quartz cells. Solution concentrations of 2 mg/mL were used, and the data were obtained in methanol; chloroform, or methanol-water mixtures. Typical spectra of clarithromycin dissolved in methanol and in chloroform are shown in Figure 10. In methanolic solution spectral maxima were observed at 21 1 and 288 nm, while peaks at 240 and 288 nm were detected with chloroform as the solvent.

4.6.

Infrared Spectrum

The infrared spectrum of clarithromycin, obtained in a KBr pellet, is shown in Figure 1 I . The spectral peaks have been assigned to various molecular vibrations, and these are contained in Table 11.

4.7.

Nuclear Magnetic Resonance Spectra 4.7.1. 'H NMR Spectrum

The one-dimensional proton 'H NMR spectrum of 50 mg/mL clarithrornycin dissolved in CDCl, is shown in Figure 12. This spectrum was recorded on a General Electric QE-300 N M R system, and was internally referenced to TMS. Table I11 lists the 'H Nh4R spectral assignments of clarithromycin in CDCl,.

4.7.2. 13CNMR Spectrum Figure 13 shows the one-dimensional I3C N M R spectrum of clarithromycin dissolved in CDCl,. This spectrum was also recorded on the QE-300 N M R system at a solute concentration of 50 mg/mL. The spectrum

CLARITHROMYCIN

.

I

190.0

65

300.0

400.0

Wavelength (nm)

Figure 10.

W absorption spectra of clarithromycin in methanol and in chloroform.

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4000

3500

3000

xm

Figure 1 1.

2000

1800

1600

-,im

1200

1000 800

Wavenumber (cm )

Infrared spectrum of clarithromycin.

600

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Table I1 Infiared Spectral Assignments for Clarithromycin

Energy (wavenumbers)

Assignment

1690

uC4 (Ketone carbonyl)

1730

Lactone carbonyl

1420

(N-CH,)

2780-3000

Alkane stretching peaks

3450

Hydrogen bonds between OH groups

1000-1200

-C-0-C- stretch

1340-1400

CH, groups

I

-ri

CLARITHROMYCIN

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Table I11 Proton Nuclear Magnetic Resonance ('H-NMR) Assignments for Clarithromycin Chemical Shift 0.842 1.133 2.282 3.038 3.20 1 3.330 3.676 3.763 3.782 4.449 4.934 5.064

Number of Proton (Multiplicity) 4

1

S S S

dd S

d d dd d dd dd

Assignment 14-CH3 6-CH3 N(CH3)2 6-OCH3 2'-H 3"-OCH, 5-H 11-H 3-H 1'-H 1 "-H 13-H

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was recorded at 24°C and internally referenced to TMS. The 13CNMR spectral assignments are contained in Table IV. 4.8.

Mass spectrum

Mass spectra of clarithromycin were recorded on an HewlettPackard model 5988-A mass spectrometer. The CI mass spectrum, acquired with 70 eV chemical ionization, is shown in Figure 14. The LSIMS mass spectrum (Figure 15) of clarithromycin was obtained using the VG-70 SE system, using 3-nitrobenzyl alcohol as a matrix. The molecular ion (M-H) was observed at 748 d z , and some characteristic peaks are noted at d z values of 158,590, and 116.

5.

METHODS OF ANALYSIS

5.1.

Elemental Analysis

The following table shows the data calculated and found for the elemental analysis of clarithromycin. 0

carbon hydrogen nitrogen oxygen 5.2.

cal 61.02 9.30 1.87 27.81

%LEQud 60.57 9.13 1.82 28.48

Identification

Clarithromycin may be identified on the basis of its characteristic infrared absorption spectrum O(Br pellet method), as described in section 4.6.

r

a a 0

-cv

0

-4-

G

-a

C

-m

0

7

.o

E

h c4

. L (

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Table IV Carbon Nuclear Magnetic Resonance (I3C-NMR) Assignments for Clarithromycin. Carbon number

Chemical Shift @Pm)

Carbon number

Chemical Shift (Ppm)

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22

175.900 45.021 77.934 39.261 80.710 78.361 39.360 45.184 22 1.ooo 37.152 69.009 74.214 76.571 20.950 10.550 15.910 8.990 19.698 17.945 12.237 15.941 50.579

1' 2' 3'

102.789 70.940 65.501 28.530 68.710 2 1.440 40.2 12 96.045 34.835 72.629 77.414 65.750 18.649 21.423 49.43 1

4' 5' 6' 7,s' 1" 2** 3 4" 5 6" 7" 8" 'I

I'

CLARITHROMYCIN

50

Figure 14.

100 150 200 250 300 350 400 450 500 550 600 650 700 750 800 a / Z

The chemical ionization mass spectrum of clarithromycin.

73

Figure 15.

The LSIMS spectrum of clarithromycin, obtained using 3-nitrobenzyl alcohol as the matrix.

CLARITHROMYCIN

5.3.

15

Thin Layer Chromatography

A thin layer chromatographymethod was developed and used by Morimoto et al. [11 during the synthesis of clarithromycin. The samples were applied to TLC silanized silica gel plates, and the plates developed in 2:3 phosphate buffer (0.1 M, pH 7)-Methanol. In this system, the Rfvalue of clarithromycin was found to be 0.42.

5.4.

Structural Details

The molecular structure of clarithromycin is similar to that of erythromycin A, and to that of (14R)-14-hydroxy-6-o-methylerythromycin A [2 11. The absolute configuration of the asymmetric centers in clarithromycin was determined by Iwasaki and Sugawara in 1993 [213. 5.5.

High Performance Liquid Chromatography (HPLC)

A HPLC method for clarithromycinwas developed in the author’s laboratory, based on UV detection at 2 10 nm. For the present method, the analytical apparatus consisted of an LC-6A high-pressure pump and a Shimadzu SPD-6A variable-wavelengthdetector. Injections were made by SIL-1A loop (20 pL) injector. A prepacked 30 cm x 3.9 mm ID pbondapak CI8Waters column was used, with the back pressures ranging between 1800 and 2000 psi. The mobile phase consisted of 65% methanol and 35% (v/v) 0.05 M monobasic sodium phosphate. The pH of the buffer component was adjusted to 4.0 using orthophosphoric acid, and a flow rate of 1.O mL/min was used for all work. Owing to the inadequate solubility of clarithromycin in the mobile phase, serial dilutions of the drug were made by first dissolving clarithromycin in methanol. Samples were subsequently diluted to the desired volume with mobile phase. The standard solutions were injected into the HPLC system five times, and average values deduced from the mean of the five measurements. The calibration curve covered the concentration range of 0.005 1 mg/mL to 1.08 mg/mL, and was found to be linear with a correlation coefficient equal to 0.9960 (r2=99.20 %). The

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precision of this assay was found to characterized by a relative standard deviation of 1.72%, and the limit of detection was deduced as 0.04 pg/mL. A typical chromatogram obtained using this method is shown in Figure 16. Numerous HPLC procedures for clarithromycin have been reported by other authors to identify and quantify the drug in biological samples [22, 231, and as methods for the analysis of clarithromycin and related compounds of the synthesis [20]. A method suitable for the determination of clarithromycin in body fluids was developed, which makes use of a Nucleosil Cs (5 pm, 250 mm x 4.6 mm ID) column and a mobile phase of 39:9:52 acetonitrile-methanol(0.04 M) NaH2P04 [24]. The pH of the medium was adjusted to 6.8 with NaOH. The flow rate was set at 1.2-1.4 mL/min. Electrochemical detection was used to monitor the analysis, with the potential of the screening electrode being set at M.5 V and the working electrode at +0.78 f 0.04 V. A quantification limit of 10.03 p g / d in plasma was established, and a relative standard deviation of less than 5% was obtained.

Clarithromycin was extracted using the following procedure. 0.5 mL aliquots of plasma are transferred to clean tubes. Approximately 750 ng of internal standard (erythromycin A 9-o-methyoxime, dissolved in 1:1 acetonitrile/ water) is added to each tube, along with 0.2 mL of 0.1 M sodium carbonate solution and 3 mL of 1:1 ethyl acetate-hexane. The samples are then vigorously vortexed for 1 minute, and centrifuged at 800 g for 5 min. The organic layer is transferred to a suitable container and evaporated to dryness at 45 "C. The residue is dissolved in 200-400 pL of 50% acetonitrile/ water, and 20 pL portions were injected into the HPLC system. A similar extraction procedure was used with urine samples. The simultaneous detemination of clarithromycin and related products was realized by Morgan et al. [25]. The mobile phase consisted of 4 8 5 2 v/v acetonitrile-KH2P04(0.33 M), with the pH being adjusted to between 5.3 and 5.5. A flow rate of 1.0 mL/min was used, and a sample size of 50 pL was found to be appropriate. A Cis Column (5 pm, 250 x 4.6 mm ID) was used, which was heated to 50°C. The detection wavelength chosen

Figure 16.

Typical HPLC chromatogram of clarithromychdissolved in methanol-watermixtures.

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was 205 nm, with the detector sensitivity being set at 0.03 AUFS. The percent of all known compounds was obtained as the area percent, and most identified species were detectable at the 0.1% level.

5.6.

MicrobiologicalAnalysis

Serum and tissue concentrations of clarithromycin were measured by the agar dif3kion method with Sarcina Iutea ATCC 934 1 as the test microorganism, and with antibiotic medium No. 11 (heart infusion agar) 126,271. A validated bioassay method for clarithromycin has also been described by Fernandes [28]. The latter assay consists of the use of Micrococcus luteus ATCC 9341 as the indicator organism.

6.

STABILITY

Clarithromycin is stable under normal storage conditions. It should be stored in tight containers, protected &om light. It is more stable to the effects of acid than is erythromycin A, owing to the presence of the 6-0methyl group which blocks the formation of the 6,9;9,12-spiroketal derivatives responsible for the gastrointestinal imtation associated with erythromycin use. Although, clarithromycin gradually loses its antibacterial activity in dilute HCl solution [l], its increased acid stability leads to improved intracellular bioactivity . The HPLC method described in section 5.5 has been used to test the stability of aqueous and hydroalcoholic solutions of clarithromycin prepared during the solubility study. No degradation products were observed in these samples when they were maintained at 4°C for 20 days.

CLARITHROMYCIN

7.

PHARMACOKINETICS

7.1.

Adsorption

79

Clarithromycin is stable in gastric acid, and is rapidly absorbed from the gastrointestinal tract regardless of when it is taken. Food intake before dosing slightly delays both the onset of absorption and slightly retards the formation of the 14-hydroxy clarithromycin antibacterial active metabolite. It actually appears that the bioavailability of clarithromycin is improved by its administration with food. [29-3 11. This suggests that clarithromycin can be taken orally (in tablet form or in suspension) without concern for timing in relation to meals. 7.2.

Bioavailability

The absolute bioavailability of clarithromycin, after oral administration, has been reported to be approximately 55 % [30]. It has a long serum half-life (4.9 hours), and exhibits peak serum concentrations of 2.51 p g l d within two hours after administration of a single 500 mg dose in a fasting, healthy subject [(32-341. The rapid first-pass metabolism of clarithromycin leads to the formation of its active metabolite (14-hydroxy clarithromycin), which also reaches a peak serum concentration of 2.1 p g / d within two hours after administrating a single 500 mg dose [32-341. Steady-state amounts (1 pg/mL) of clarithromycin and the 14hydroxy metabolite are reached after 2-3 days of administering a 250 dose every 12 hours. For both compounds, the steady-state peak plasma concentrations in children (following 7.5 mgkg every 12 hours of drug product suspension) were 3-7 and 1-2 p g / d , respectively [33]. 7.3.

Distribution

Clarithromycin and the 14-hydroxy metabolite are widely distributed into most body tissues, and reach especially high concentrationsin the lung.

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Tissue concentrations exceed those of serum and because of high intracellular concentration ,negligible accumulation is observed [35-371. The protein binding of ciarithromycin in vitro is low, and 14-hydroxy protein binding decreases with increasing serum drug concentration. 7.4.

Elimination

Clarithromycin is largely metabolized in the liver, by the hepatic cytochrome P-450 enzymes. The major metabolic pathway is by hydroxylation at the 14 position, and by oxidative N-demethylation [38]. Clarithromycin and its principal metabolites are excreted in feces via bile, in urine by renal and nonrenal mechanisms. Between 20-30% of the dose is excreted in this way as the unchanged drug [38]. Clarithromycin follows a one-compartment, open pharmacokinetic model, and its elimination seems to follow nonlinear dose-dependentpharmacokinetics [38-39].

8.

PHARMACOLOGY

8.1.

Mechanism of action

Like the rest of the macrolide group, clarithromycinexerts its antibacterial action by binding to the 50s ribosomal subunit of susceptible organisms and by inhibiting protein synthesis through translocation of aminoacyl transfer-RNA [40]. The site of action of clarithromycin seems to be the same as that of erythromycin. Clarithromycin, like other sixteen-membered macrolides, is a poor inducer of mRNA and does not itself cause activation of the methylase enzyme. It thereby retains activity against inducible bacteria in the absence of a strong inducer [30]. The activity of clarithromycin is equivalent to between two and fourfold that of erythromycin against all isolated tested microorganisms. Unlike erythromycin, it generates in vivo an active metabolite (14-hydroxy clarithromycin),which by itself often exhibits more activity against bacteria

CLARITHROMYCIN

81

than does erythromycin. The combmation of clarithromycinand its metabolite yields a synergistic effect [4,11,13]. The compound has another major advantage over erythromycin, its activity against Mycobucterium Avium [41] and A4 leprae [2]. Unlike penicillin or cephalosporin antibiotics, the uptake of clarithromycinby human neutrophils is high, leading to higher concentration of this drug in human macrophages, lymphocytes and polymorphonuclear leukocytes. It thereby displays major activity against intracellular microorganisms, such as S. uureus or Legionellu. [2,43]. The more potent anti-inflammatory effects exhibited by clarithromycinmay enhance its clinical efficacy. It has been demonstrated that clarithromycin inhibits the production of interleukin-1 (IL-1)by murine peritoneal macrophages, lymphocyte proliferation, and lymphocyte transformation of murine spleen cells at low concentrations [44]. 8.2.

Toxicity

No toxicity was described during clinical trials, and clarithromycin has proven to be well tolerated. The most common adverse effects have been mild-to-moderate GI irritation. Hepatotoxicity occurred in all animal species tested at doses two times greater than the maximum human daily dose. Renal tubular degeneration occurred in rats,dogs, and monkeys at doses 3-8 times greater than the maximum human daily dose. Corneal opacity and lymphoid depletion in dogs occurred after the administrationof 3 to 12 times the maximum human daily dose, respectively. Clarithromycincauses teratogenic effects in laboratory animals. No data are available in pregnant women; so it should not be used during pregnancy, unless no alternative therapy is appropriate [45].

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9.

REFERENCES

1.

Morimoto, S., Takahashi, Y., Watanabe, Y. and Omura, S. (1984). J Antibiot. X ?187.

2.

Anderson, R., Joone, G. and van Rensburg, E.J. (1 988). J. Antimicrob. Chemother. 22,923.

3.

Physicians G e m . (1995). 5* Ed., Riverside, CT, p. 463.

4.

Perronne, C., Gikas, A., Truffot-Pernot, C. (1991).Antimicrob. Agents Chemother. 35,1356.

5.

Femandes, P.B. (1987). Antimicrob. News. 4,25.

6.

Mor, N., Vanderkolk, J., Mezo, N. and Heifets. (1994).Antimicrob. Agents Chemother. 38,2738.

7.

Hoppe, J.E. and Eichhorn, A. (1989). Eur. J. Microbiol. Infect. Dis. 8,653.

8.

Rastogi, N. and Goh, K.S. (1 992). Antimicrob. Agents Chemother. 3,2841.

9.

Gorzynski, E.A., Gutman, S.I. and Allen, W (1989).Antimicrob. Agents Chemother. 23,591.

10.

Olsson-Liljequist, B. and Hoffman, B.M. (1991). J. Antimicrob. Chemother. 22, Suppl. A, 11.

11.

Masur, H. (1993). N. Engl. J Med. 2 2 , 8 9 8 .

12.

Fattorini, L., Li, B., Piersimoni, C. (1995). Antimicrob. Agents Chemother. 19,680.

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13.

Furney, S.K., Skinner, P.S., Farrer, J. and Orme, I.M. (1995). Antimicrob. Agents Chemother.22, 786.

14.

Perronne, C., Gikas, A., Truffot-Pernot, C. (1990). Antimicrob. Agents Chemother.34,1508.

15.

Rolston, K., Gooch, G. and Ho, D. (1989). J Antimicrob. Chemother.23, 455.

16.

Hardy, D.J., Hensey, D.M., Beyer, J.M. (1988). Antimicrob.Agents Chemother.2 , 1 7 10.

17.

Sefton, A.M., Maskell, J.P., Yong, F.J. (1988). Eur. J Clin. Microbiol. Infect. Dis. 2,798.

18.

Watanabe, Y., Adashi, T., Asaka, T. (1 990). Heterocycles 3, 2 121.

19.

Watanabe, Y., Kashimura, M., Asaka, T. (1993). HeterocycZes.Xi, 243.

20.

Watanabe, Y., Adashi, T., Asaka, T. (1993). J Antibiotics.%, 1163.

21.

Iwasaki, H. and Sugawara, Y. (1993). Acta Cryst. 49,1227.

22.

Sundberg, L. and Cederberg, A. (1 994). J Antimicrob. Chemother.

33,299.

23.

Ohtake, T., Ogura, K., Iwatate, C. and Suwa, T. Chemotherapy (1988). Xi, 192.

24.

Chu, S., Senello, L. and Sonders, R. (1991). J Chromutogr.521, 199.

25.

Morgan, D., Cugier, P., Marello, B. (1990). J Chromatogr.m, 351.

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26.

Fraschini, F., Scaglione, F., Pintucci, G. (1991). J Antimicrob. Chemother. ;?z, Suppl. 4,61.

27.

Nagate, T., Sugita, K., Miyachi, M. (1988). chemotherapy. X, 170.

28.

Fernandes, P.B., Hardy, D.J., McDaniel, D. (1989). Antimicrob. Agents Chemother.11,153 1.

29.

Bahal, N. and Nahata, M.C. (1992). Ann. Phurmacother. &46.

30.

Neu, H.C. (1991). J Antimicrob. Chemother.22, Suppl. A, 1.

31.

Davey, P.G. (1991).J Hosp. Infect.B,Suppl. A, 29.

32.

Peters, D.H. and Clissold, S.P. (1992). Drugs. 44, 117.

33.

Drug Facts and Comparisons (1995). Facts and Comparisons. St. Louis, MO, p. 2003.

34.

Fraschini, F., Scaglione, F., Pintucci, G. (1991). 31St Interscience Conference on Antimicrobial Agents and Chemotherapy (1991). Chicago. Abstract 51 2.

35.

Fraschini, F., Scaglione, F., Pintucci, G. (1991). J Antimicrob. Chemother. 25, Suppl. A, 73.

36.

Kohno, Y., Ohta, K., Suwa, T. and Suga, T. (I 990). Antimicrob. Agents Chemother.34,562.

37.

Kohno, Y., Yoshida, H., Suwa, T. and Suga, T. (1990).J Antimicrob. Chemother.26,503.

38.

Ferrero, J.L., Bopp, B.A., Marsh, K.C. (1990). Drug Metabol. and Dispos. Ls,441.

CLARITHROMYCIN

85

39.

Saito, A., Shimada, J., Ohmori, M. (1 988). Chemotherapy.36, Supp. 3. 576.

40.

Petska, S. “Inhibitors of protein synthesis”, in: Molecular mechanism of protein biosynthesis (1977). Weissbach, H. and Petska, S. eds. New York, p. 467.

41.

Dautzenberg, B., Truffot, C., Legris, S.(1991). Am. Rev. Respir. Dis. 144,564.

42.

Franzblau, S.G. and Hastings, R.C. (1988). Antimicrob. Agents Chemother.32,1758.

43.

Femandes, P.B., Bailer, R., Swanson, R. (1986). Antimicrob. Agents Chemother.32,865.

44.

Takeshita, K., Yamagishi, I., Harada, M. (1989). Drugs Ex-. Clin. Res. k5,527.

45.

AMA Drug Evaluations (1994). American Medical Association. USA, p. 1450.

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CROSPOVIDONE

Eugene S. Barabas' and Christianah M. Adeyeye2

(1) ISP Corporation 1 3 6 1 Alps Road Wayne, NJ 07470

(2) Department of Pharmacy Duquesne University Pittsburgh, PA 15282

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

87

Copyright Q 1996 by Academic Press. Inc. All rights of reproductionin any form reserved.

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EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

Contents 1.

Introduction 1 .I Structure 1.2 Nomenclature 1.3 Polymerization

2.

Methods of Preparation 2.1 Official Methods 2.1.1 Preparation without Added Crosslinking Agent 2.1.2 Preparation with Crosslinking Agent

3.

Physical Properties 3,l Description of the Polymer 3.2 Glass Transition Temperature 3.3 Hygroscopicity

4.

Primary Uses of Crospovidone 4.1 Pharmaceutical Applications 4.1.1 Tablet Disintegrant 4.1.2 Tablet Binder 4.1.3 Miscellaneous Pharmaceutical Uses 4.2 Medical Applications 4.3 Uses in Production of Alcoholic and Non-Alcoholic Beverages 4.3.1 Stabilization of Beer 4.3.2 Stabilization of Wine 4.3.3 Stabilization of Other Beverages and Liquids 4.4 Miscellaneous Other Uses 4.4.1 Isolation and Stabilization of Enzymes 4.4.2 Use in Agriculture 4.4.3 Use in Analytical Chemistry 4.4.4 Use in Catalysis

5.

Health and Safety 5.1 Acute Toxicity 5.2 Subacute Toxicity 5.2.1 28-Day Feeding Study in Rats 5.2.2 90-Day Feeding Study in Rats 5.2.3 28-Day Feeding Study in Dogs

CROSPOVIDONE

5.3 5.4 5.5 5.6

5.2.4 180-Day Feeding Study in Rats Teratogenicity Pharmacokinetics Skin and Mucous Membrane Tolerance Pharmacology

6.

Compliance with Pharmacopoeia1and Food Regulations Identification Tests 6.1 6.1.1 Reaction with Iodine 6.1.2 Infrared Spectrum Compendia1Testing 6.2 6.2.1 Water Content 6.2.2 Nitrogen Content 6.2.3 pH 6.2.4 Non-Volatile, Water Soluble Content 6.2.5 Heavy Metals 6.2.6 Residue on Ignition 6.2.7 Vinyl Pyrrolidone Content Other Characteristics 6.3 6.3.1 Soluble Poly(VinylPyrro1idone) 6.3.2 Arsenic 6.3.3 Zinc 6.3.4 N,N'-Divinylimidazolidone 6.3.5 Peroxides 6.3.6 Loss on Drying 6.3.7 Surface Area 6.3.8 Particle Size Distribution 6.3.9 Bulk Density 6.3.10 Flow Properties Microbial Limit Tests 6.4

7.

Interactions of Crospovidone with Drug Substances

8.

References

89

90

1.

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

Introduction

Crospovidone is the insoluble form of polyvinylpyrrolidone, and its use in the pharmaceutical industry as a tablet excipient (tablet disintegrant and binder) has been widely documented. It is medically used for the treatment of some intestinal disorders, as solubilizing excipient to improve the bioavailability of drugs (such as steroids), and as germicides in wound treatment. It is also commonly used as clarifier in alcoholic and nonalcoholic beverages. 1.1

Structure

Crospovidone is produced by the proliferous polymerization of vinylpyrrolidone monomer: HZC-

" : 7 ' --

Polymerization

ti&

H2C

\/=O

I

CH =CH2

\/=O CH-CH2

j n

The earliest observation of spontaneous "popcorn" polymer formation had been made with dimethylbutadiene by Kondakov [I]. Later Staudinger and Huseman found a similar phenomenon with the styrenedivinylbenzene system [2]. It was, however, Breitenbach and his coworkers who found that numerous other monomers [3] (including vinylpyrrolidinone [4])were also capable of proliferous polymerization. His work contributed significantly to the elucidation of the mechanism of this unique reaction. The product of this polymerization is a densely crosslinked structure insoluble within the system in which it is made. It has a very low degree of swelling and consists of a very voluminous structure which contains many voids. The polymer has a white, opaque appearance, quite different from the normal crosslinked polymer of vinylpyrrolidinone. Because of the formation of the "popped" structure, this voluminous polymer is also referred to as a "popcorn" polymer. The structure of "popcorn" PVP resembles a polymer foam, with its void space

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91

not being formed by a gas or vapor but instead developed by the polymerization process itself. An unusual property of this polymer type is that when it is brought in contact with more monomer, it can transform the latter into a polymer of the same ''popcorn" structure. In the absence of the proliferating seed, the monomer would turn into a normal soluble polymer.

In the case of the vinylpyrrolidone "popcorn" polymer, the product is formed through a complex mechanism in which both the copolymerization with certain in situ formed crosslinking agents and the physical entanglement of newly forming polymer chains contributes to the development of the "popcorn" structure. 1.2

Nomenclature

Chemical Abstract Services Registration number: 9003-39-8 Chemical Abstract Name: Crosslinked homopolymer of 1-ethenyl-2pyrrolidinone Crosslinked poly(vinylpyrro1idinone) has been known under a variety of names. Some of those have been used as "approved names" by the regulatory authorities of different countries. The commonly used names include: Crospovidone Crosslinked Polyvidone Crosslinked homopolymer of 1-ethenyl-2-pyrrolidone Insoluble crosslinked homopolymer of N-vinyl-2 pyrrolidone Insoluble PVP Polyvinylpolypyrrolidone(PVPP) PolyvinylpyrrolidonumInsolubilis Crosslinked poly(vinylpyrrolidinone), beside being available in technical grades with different specifications, is sold as a pharmaceutical grade conforming to the requirements of various national and international Pharmacopoeias, as well as to the demands of national and international food regulatory authorities.

92

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

Pharmaceutical grades are marketed under the trade name of Kollidon CL by BASF (Badische Anilin and Soda Fabrik A.G.) and Polyplasdone XL by GAF Corporation (recently changed to ISP - International Specialty Products Corporation). The products used by the beverage industry are sold under the trade names Divergan (BASF) and Polyclar (ISP) [5,6].

1.3

Polymerization

The proliferous polymerization takes place through a free radical mechanism, although the presence of a free radical initiator is not always necessary to this type of polymerization. The radicals may also develop through the rupture of polymer chains with the combined actions of polymerization and swelling [7], which form a great number of active sites. As a consequence, growing chains are initiated at different sites of the polymer chains at fixed positions, which form independently growing centers unable to interact with each other. These new chains, which also contain the in situ formed or deliberately added bifunctional crosslinking monomers, get entangled with chains already formed. The overall result of these reactions is a system having a high crosslink density. The monomer is absorbed by the swelling of the polymer network and converted to a part of the network by polymerization. By the continuous repetition of this process, the existing polymer chains first become strained due to the swelling, and then rupture. This creates new free-radical sites which react with more monomer molecules, producing new growing chains. The straining of the polymer structure can be observed with the help of polarizing microscopy [8]. The degree of optical anisotropy depends upon the chemical character of the popcorn polymer [9]. While the presence of divinyl compounds is not indispensable for the formation of popcorn polymer [lo], they often play an important role in the development of the structure. Higher concentrations of divinyl compounds produces a higher degree of crosslink density and yields higher gel strengths. On the other hand, it also produces greater number of pendant double bonds on the polymer, which leads to greater number of growing chains and increases the tendency for chain splitting. The unusual mechanicochemical part of the mechanism of the proliferous polymerization was proven very convincingly by Breitenbach and Dwovak

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[l 11. These workers used a dimethacrylate Schiff-base as a crosslinking agent. The swelling of the polymer made in this fashion was very low, and showed the usual anisotropy to the polarizing microscope. After the chemical crosslinking was destroyed by the addition of 0.1M dichloroacetic acid, most of the optical anisotropy disappeared while the polymer remained insoluble and only the degree of swelling increased slightly. These results clearly indicated the presence of chain entanglement, as well as the fact that there was some increase in the swelling. This showed that the crosslinking agent was also responsible for the formation of the structure, at least to some degree. The number of fiee radicals formed during the splitting of C-C bonds, together with the formation (or addition) of bifunctional monomers, are mainly responsible for determining the rate of growth. This usually follows an exponential law, with a linear dependence between the logarithm of the weight of polymer (w) and the time of growth (t): kw

=

W

-

and

dw f dt kt

wo e

This is, however, an ideal law, and is exactly obeyed only when the newly formed "popcorn" polymer has constant growth capability and the medium remains unchanged during the process. Generally these conditions exist only approximately. Pravednikov and Medvedev studied the course of the proliferous polymerization using I4C-labeledpopcorn seed which was added to unlabelled monomer [ 131. These workers found that at the end of the polymerization, the original labeled seed material was quite uniformly distributed throughout the polymer. In order to achieve the nearly uniform distribution of the seed, a great number of C-C bonds had to be ruptured during the polymerization, thus creating a large number of free radicals. The free radicals formed in the course of the bond splitting must be largely responsible for the high rate of polymerization. It had been found that the reaction medium exerts a great influence on the course of the reaction [141. For instance, a styrene-p-divinylbenne system containing 30 vol. % methanol shows evidence of proliferous

94

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

polymerization in about half of the time compared to a reaction without methanol. This effect was attributed to the dimension of the polymer coils and its dependence upon the nature of the solvent, with a reduction in coil dimensions favoring popcorn polymer formation. It is assumed that an optimum range of coil dimensions exists, which can be achieved by adding a good soivent to the monomer-polymer system for which the polymer is completely insoluble in the monomer. In the case of Nvinylpyrrolidinone, this good solvent is water or (to a degree) methanol. Unlike most other systems, poly(vinylpyrro1idinone) is soluble in its monomer. It is therefore probable that the addition of water causes only dilution, and a reduction of chain segment density at the same conversion 1141.

2.

Methods of Preparation

The PVP "popcorn" polymer may be prepared by two different methods. In the first method (I), vinylpyrrolidinone is heated at temperatures exceeding 100°C in the presence of an alkali metal hydroxide and a small amount of water [ 151. The presence of water has been shown to be an important factor in the formation of the "popcorn", and also influences the rate and induction period of the polymerization. This effect is most probably due to the swelling of the polymer coils to the dimensions which favor "popcorn" polymer formation [16]. Beside the physical entanglement, a certain degree of chemical crosslinking must also be responsible for the densely crosslinked structure of the vinylpyrrolidinone "popcorn" polymer. It has been shown by pyrolysis gas chromatography that during the process, 1vinyl-3-ethylidenepyrrolidinone YEP) and ethylidene-bis-3-(N-vinylpyrrolidinone (EVP) are formed. The amount of these unsaturated compounds was found to be 1.5% and 0.1%, respectively [ 171. Structures for these two compounds are provided on the following page.

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95

H H2C-C=C-CH, HzC,~,C=O I I I CH=CH2

1 -Vinyl-3-Ethylidenepyrrolidinone

The formation of EVP is possible due to the two phase system that comes into being because of the high concentration of NaOH which is used in the system. The aqueous phase contains the caustic and part of the vinylpyrrolidone, and under strong agitation turns to small droplets in the organic phase. The organic phase consists of the rest of the vinylpyrrolidone and any EVP (which has a very low water solubility of 2 mg/mL). As the reaction progresses, the water layer becomes the continuous phase so any EVP forming in the process is protected from the effect of the caustic. One mole of vinylpyrrolidone monomer and one mole vinylpyrrolidone carbanion (from the water phase) form an anionic adduct. The adduct then splits to a thermodynamicallymore stable bifunctional compound and the 2-pyrrolidone anion. In the strongly basic environment, the bifunctional compound isomerizes to EVP, while the 2-pyrrolidone ion hydrolyzes to 4-aminobutyrate [181. The proposed reaction mechanism is shown as Scheme I. In the second method (11) of production, an aqueous solution of vinylpyrrolidinone and a small amount of a bifunctional monomer is heated at temperatures exceeding 100°C [191. N-N y -divinylethylene urea

96

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

Step 1.

&+

NaOH

-0 N

i/

d

VP Monomer

h

VP Carbanion

d i/

4AB

EVP

Reaction Scheme I

CROSPOVIDONE

97

and similar acid amides carrying two unsaturated groups are suitable bifunctionals for this polymerization [21]. Polymers made by the two synthetic methods have been shown to exhibit identical infrared spectra, which are also similar to that of linear PVP. This finding can be attributed to the following: a) The large crosslink density is partly due to physical entanglements, so the number of covalent linkages is smaller than usual b) The structure of the bifunctional crosslinking agent is very similar to that of vinylpyrrolidone. Any slight difference in the structure is not sufficient for differentiation. The corresponding infiared spectra are shown in Figure 1. These "popcorn'' polymers made with 1.6% of the two aforementioned bifunctionals are much more densely crosslinked than are polymers obtained by the copolymerization of vinylpyrrolidinone with much larger amounts of crosslinking agents and using a free radical initiator. Consequently, the "popcorn" polymers show considerably lower swelling characteristics than does PVP crosslinked with conventional crosslinking agents. These correlation is shown in Figure 2. The difference between "popcorn" and conventionally crosslinked polymers can be demonstrated by studying their respective glass transition (Tg) temperatures. The Tg of linear PVP K-90 is 175"C, and the Tg of "popcorn" PVP is only 1520°Chigher (approximately 195°C). These "popcorn" polymers which contain about 1.6% of in situ formed crosslinking agents have a gel-volume of 5 mL/g. On the other hand, a conventionally crosslinked polymer made with 1.6 mol-% crosslinking agent, which has a Tg similar to that of the "popcorn" polymers (195°C) and a gel volume of 42 mL/g. The more than eight-fold increase indicates the existence of a more loosely crosslinked structure. If one uses a tenfold higher amount (1 6 mol-%) of bifunctional crosslinking agent in order to increase the crosslink density of the conventionally prepared polymer, the gel volume will be 12 mL/g, but the Tg will be as high as 270°C. This is 95°C higher than that of the linear PVP polymers [ 171. This correlation is shown in Table 1.

98

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

Table 1 Glass Transition Temperatures of Annealed Linear and Crosslinked PVP [ 191 Sample

Ta ("C)

povidone

175

crospovidone according to method I

190

crospovidone according to method I I

195

copolymer from VP with 1.6 mol-% bifunctional monomer

195

copolymer from VP with 16 mol-% bifunctional monomer

270

The Tg of crystalline polymers is higher than that of the amorphous ones of the same composition. However, X-ray scattering studies failed to show the presence of any crystalline domains in the structure of amorphous "popcorn" polymerized vinylpyrrolidinone.

2.1

Official Methods of Preparation

At the present time there are two official methods for the preparation of crospovidone. One of the methods was developed by GAF (now ISP) Corporation and consists of a mechanicochemical sequence of reactions. In this sequence, the network structure is developed without the addition of crosslinking agents, through the inclusion of compounds having double hctionalities which are developed in situ and take part in the polymerization. The other method was developed by Badische Anilin and Soda Fabrik A.G. BASF, and utilizes a unique crosslinking agent (divinylimidazolidone) whose chemical structure is similar to that of the vinylpyrrolidone monomer.

99

CROSPOVIDONE

m‘1700’ wo

’

wo’ Ilw ’ womm d i s

Energy (wavenumbers)

Figure 1. Infrared spectra of linear PVP and crospovidone [171, prepared according to the two reaction schemes of section 2.1.

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

30

20.

lo-

-

1

5

1

10

15

I

20

Concentration, Bifunctional Monomer (mol-%)

Figure 2.

Sedimentation volume of AIBN-initiated PVP and “popcorn”-PVP in water [ 171.

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101

2.1.1 Preparation (I), without added Crosslinking Agent A mixture consisting of 200g of N-vinylpyrrolidone and 2g of sodium hydroxide flakes is heated for 3 hours under total reflux in a distilling flask at reduced pressure (100 mm Hg). At this time the temperature rises from 145-156°C to 190"C, and the refluxing monomer gradually converts to a white solid. After cooling, the polymer is slurried with water to wash out the caustic and any unconverted monomer. The slurry is filtered, and dried at 50-60°C in vucuo [ 151. An alternative procedure exists where a reaction mixture consisting of vinylpyrrolidone monomer at a concentration of about 70-90% by weight in an aqueous strongly basic solution (containing about 0.3-1.5 % base) is heated to a temperature of about 130-170°C under an inert gas atmosphere. The temperature is kept at the reaction temperature for a sufficiently long time so as to create in situ crosslinking. After that, the basic reaction mixture is diluted with distilled water to a vinylpyrrolidone concentration of about 5-30 %. The reaction is continued at about 100°C to form a white crosslinked polymer of low swell volume [20]. 2.2.1 Preparation (11), with Crosslinking Agent [21,22] In a vessel having a capacity of 500 parts by volume and equipped with a thermometer and reflux condenser, a mixture is prepared consisting of 100 parts of vinylpyrrolidinone, 100 parts of distilled water, 1 part of N,N'divinylimidazolidinone, and one bare-metal iron packing element (such as a Pall ring, 15 x 15 mm). About 0.005 % of dibenzoylperoxide (based on the vinylpyrrolidinone content) is added, and the mixture heated to 35°C. After approximately 90 minutes small white polymer seeds are seen on the surface of the packing element and these seeds grow visibly. The growing mass soon projects above the level of the liquid and eventually fills the entire volume of the vessel. During polymerization, the reaction mass heats to its boiling point of 102°C. Vaporized water is condensed in the reflux condenser and flows back into the vessel. The period between the appearance of the first polymer seed and the point at which the entire volume of the vessel is full of white, crumbly polymer mass is about 15 minutes and takes place after consumption of all of the

I02

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

liquid phase. Boiling slows down soon afterwards and eventually stops completely. The reaction product is removed from the vessel, washed three times with distilled water to remove soluble portions, and dried in a vacuum oven at 80°C. The yield is 90 parts of a pure, white granular crumbly polymer, which is sparingly swellable in water, but completely insoluble in the usual organic solvents (such as hydrocarbons, alcohols, ethers, ketones, organic halogen compounds, and organic nitrogen compounds. The product is non-fusible and decomposes above 300°C.

3.

Physical Properties

3.1

Description of the Polymer

Crosslinked poly(viny1pyrroiidinone) is a white to off-white free-flowing powder. It is practically odorless, although sometimes it may exhibit a faint (but not objectionable) odor, It may have a slightly salt-like taste. The material is hygroscopic and should be kept in tightly sealed container. Because of its crosslinked structure, the polymer is insoluble in water and all ordinary solvents. However, it will swell when in contact with water, as well as with some organic solvents. Because of its insolubility, the molecular weight of the polymer is indeterminate. 3.2

Glass Transition Temperature

The glass transition temperature (Tg) of crospovidone varies with the method of preparation, and whether the polymer co-exists with the, vinylpyrrolidone monomer. A summary of glass transition temperature data was presented in Table 1. 3.3

Hygroscopicity

Because of its hygroscopic nature, reference standard material must be kept away from atmospheric humidity and is to be dried at 105°C for one hour before its use.

CROSPOVIDONE

4.

Primary Uses of Crosslinked PVP

4.1

Pharmaceutical Applications

103

The primary pharmaceutical application for crospovidone is that of a tablet disintegrant, although it can also function as a tablet binder. In order for the polymer to be useful as a pharmaceutical excipient, grades of material need to possess the following properties [36]: a) high swelling capacity b) high capillary activity c) high hydration capacity d) low bulk density e) large specific surface area rapid uptake and high moisture absorption f) g) complete insolubility in water no tendency to form gel on contact with water h) i) high binding characteristics j) effectiveness in tablet disintegration k) accelerated drug dissolution rates 1) good shelf stability In addition, the polymer must be chemically and biologically inert, as well as non-reactive with the other ingredients of the formulation. Furthermore, it must be non-toxic and non-irritating, either when administered orally or when applied externally. 4.1.1 Tablet Disintegrant Crospovidone, because of its highly hydrophilic character, rapid moisture sorption, and good swelling properties, is widely used as a tablet disintegrant [35]. The specific surface area of the polymer is reasonably large (1.25 m2/g), so it has very high capillary activity and hydration capacity. As a consequence of these properties, water is rapidly drawn into the tablet. The water uptake stretches out the folded molecular chains lying between the crosslinks, causing an instant expansion of the polymer. The increase of volume creates an internal pressure exceeding that of the tablet strength, and results in fast disintegration of the tablet body [36].

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EUGENE S.BARABAS AND CHRISTIANAH M. ADEYEYE

According to Huttenrauch and co-workers, the mechanism of disintegration is very complex [37]. The efficiency of a disintegrating agent requires a low water solubility, strong hydration capacity, good plastic deformability, and high capillary activity [38,39]. Kornblum and Stoopak, who were the first to report on the use of PVPP as a potential high performance tablet disintegrant concluded that a large specific surface area and substantial hygroscopicity are also required to assure fast and complete disintegration [ 3 5 ] . More recent studies carried out by List and M u m indicated that neither capillarity nor the heat of adsorption were responsible for the degree of disintegration, but that the pressure developed during the swelling within the system is the decisive factor [40]. This internal pressure is developed through the quick expansion of the system caused by the absorbed water, implying that the rate of water sorption at the early stages was particularly important. This matter was studied by Gissinger and Stamm. through a comparison of Crospovidone with other disintegrants [41]. The comparative degree of water uptake by some commercially available tablet disintegrants after one minute of contact [42] is shown in Figure 3. Rudnic and co-workers also studied the mechanism of disintegration [43], and concluded that while the magnitude of the force produced by the swelling has an underlying relevance to disintegrant action, the rate of the growth of that force must also have a strong influence on the disintegration process. They proposed that the rate of swelling is a function of the rate by which the force is increasing, and can be expressed by :

dF __ dt

dV - K * dt

where dF/dt is the rate of the force development, dV/dt is the rate of swelling, and K is a constant for any given formulation at constant porosity. If the porosity is high, then the physical properties of the disintegrant (surface area, density, etc.) will be the determining factors. If K is small, dV/dt is influenced mainly by water absorption [43].

CROSPOVIDONE

25

Figure 3 .

--

105

AC-01-SOL

Comparative water uptake of disintegrants after I minute of exposure [44].

106

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

If the force grows slowly, then the elasticity of the tablet matrix will be allowed to adjust to the stress without a consequent structural change. If however, the force develops rapidly, the matrix will not be able to adjust and the will structure rupture. The capacity of the disintegrant to sorb water and swell as a result of the absorption can be evaluated an apparatus designed by Nogami and coworkers [42]. A low degree of water solubility, or even complete insolubility in that medium, is one of the most important prerequisites for a well-performing disintegrant. If a polymer intended for use as a disintegrant has any solubility in the medium or has a loosely crosslinked structure, the initial stages of the water interaction will yield an intractable coating on the tablet. This can partially or completely block the small pores of the tablet, hindering or even completely stopping water penetration into the narrow channels. Any slight dissolution of the polymer will result in a solution of increased viscosity. The higher the molecular weight of the dissolved polymer, the higher will be the viscosity of the resulting medium. The viscosity increase will slow down the absorption of the dilute polymer solution, resulting in slow or prematurely-ended disintegration. The fact that crospovidone is completely insoluble in water and in other solvents eliminates the conditions for slow or uneven disintegration. Scanning electron micrographs show that popcorn polymerized crospovidone consist of an amorphous structure having no crystalline domains. The solid consists of microspherical particles, 5-10 pm in diameter, fused into agglomerates of 350-400 pm. This sponge-like structure allows quick and free penetration of water, with a consequent expansion of volume. The swollen network will shrink when dried, and will also expand again on re-wetting. Owing to the nature of their formation, tablets are not expected to swell isotropically. Khan and Rhodes have determined the swelling ratio of tablets, and found that the Ratio Value (the change in thickness to the change in diameter) was higher than one [45]. Studies by Bronnsack showed that a higher tablet hardness was generally detrimental to its disintegration [46]. Crospovidone, however, did not lead to this type of behavior. The change in disintegration time of tablets made

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with crospovidone showed only minimal differences as a function of tabletting pressure. Khan and Rooke reported that while the relationship between compressional pressure and dissolution efficiency depended upon the type of disintegrant, tablets made with crospovidone and dicalcium phosphate dihydrate produced an increase in dissolution efficiency with an increased tabletting pressure [47]. With tablets containing crospovidone and lactose, the maximum dissolution efficiency was obtained when the compressional pressure was between 1000-2000kg/cm. In another report, Gordon et al. observed that there was no significant correlation between changes in tablet hardness and dissolution after storage at elevated temperature and no substantial swelling at the same storage condition [29]. The crospovidone was incorporated extragranularly, intragranularly, or by even distribution into the wet granulated tablet formulation. This would imply that the mode of incorporation of the disintegrant, or the method of manufacture of tablets, may play a significant role in the rate of water uptake and resulting tablet performance [29]. Jovanovic and co-workers showed that intragranularly incorporated crospovidone was a more effective disintegrant for antacid tablets than when it was added extragranularly [55]. Rudnic and co-workers found that an increase in the mean particle size enhanced disintegration (and also powder flow and dissolution), but that tablet hardness and friability were slightly better from finer grades [48]. Studies by List and Muazzam also showed that swelling pressure and disintegrationtime were particle size dependent [49]. The swelling characteristics of crospovidone were studied by Wan and Prasad using a video recording technique [50]. The Ferret diameters of the swollen particles were 40- 120pm. The large differences between the projected area and perimeter diameters observed in the dry state were absent following hydration because swelling resulted in smoothing of the particle edge texture. Ringart and Guyot-Hermann found that for crospovidone (and also for other disintegrants consisting of rounded particles), the most effective concentration could be calculated using:

X

=

0.32 J d r / d 2 [ ( D i /

0 2

+

- 11 Di/Di,J

1 ox

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

where d, and d, are the densities of the disintegrant and drug respectively, D1 and D2 are the average diameters determined by microscopy, and Di, is the diameter of the disintegrant in the disintegration medium [51]. Hennig and Schubert compared crospovidone with other starch-based disintegrants, and found that former was the most effective with respect to disintegration time and compression strength [52]. As reported by Gordon and Chowhan, the polymer hygroscopicity can directly affect its ability to act as a disintegrant [23]. These workers observed a decrease in disintegrant efficiency and dissolution rates of directly compressed tablets, resulting from the composite hygroscopicity of the tablet formulation [53].

According to Wan and Prasad, the presence of other excipients can influence the water uptake, so it is sometimes difficult to correlate water uptake with decreased disintegration times [27]. The amount of granulating fluid (water) containing crospovidone and the presence of other excipients can also influence the water uptake [26]. When crospovidone was omitted fiom a sulfanilamide formulation (but containing 2% methylcellulose as a binder), the water uptake was high and the disintegration time was short. In the presence of 2.5%crospovidone, the same observations were noted, but when the amount of granulating liquid was increased longer a disintegration time was noticed. This was attributed to a decrease in the water uptake. Film formation and a more even distribution of methylcellulose were reported to be responsible for these differences in water penetration [26]. Van Kamp and co-workers studied the effect of water-uptake in tablet disintegration, and determined that crospovidone showed the highest penetration rate among the disintegrantstested [54]. The water uptake of this excipient was approximately five times that of its own weight. Johnson et al. found that the solubility and hygroscopicity of crospovidone could affect disintegration efficiencies, and reported that the greater the overall hygroscopicity and solubility of a naproxen tablet formulation containing different disintegrants, the greater the decrease in disintegrant efficiency [34].

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Crospovidone used in a sulfadiazine tablet formulation prepared at ambient temperature and humidity was reported to increase the bioavailability of the drug. When the same formulation was prepared under 100% relative humidity, the disintegration time increased ,the dissolution rate decreased, all with a consequent decreased urinary excretion [28]. Moisture sorption of tabletted phenobarbitone sodium formulations and tensile strength of the tablets were correlated in the work of Malamataris and Dimitriou [25]. Formulations containing 36% w/w of the drug and exposed to 93% relative humidity showed the greatest tendency for moisture uptake and the minimum tensile strength. Van Kamp and coworkers found that the crushing strength, disintegration, and dissolution properties of tablets made by wet granulation with 20% potato starch as the disintegrant could be markedly improved when the starch was replaced by a much smaller amount (4%) of crospovidone [56]. Phadke and Anderson carried out studies on the wet granulation of powder blends of acetaminophen and crospovidone, using hydroxypropyl methylcellulose (HPMC) as the binder, and found that an increase in the level of crospovidone led to an increase in the amount of fines in the particle distribution of the dried granules [57]. At the same time, an inverse ratio was found between the amount of crospovidone in the blend and the bulk density of the formula. These studies indicated that the interference in the hydration of HPMC and the increase in the total surface area were attributable to the presence of crospovidone [57]. Wan and Prasad also found that the use of crospovidone led to increased disintegration times when the molecular weight of the binder (methyl cellulose) was increased, in spite of higher degree of water uptake [50]. Obviously, the hydrophilicity of the binder plays a crucial role in influencing disintegration time. Wan and Choong found that the disintegration and dissolution times of the tablet were functions of the water penetration [58]. Differences in dissolution times were due mainly to the absorptive power of the binder (starch), with the porous capillary network in the tablet exerting only a secondary importance. In this respect crospovidone was effective by also reducing the hydrophobic property of the lubricant.

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Using infrared spectroscopy, Casahourisat and co-workers found that the interaction between excipients and drug influenced the disintegration time, but not the drug dissolution rate [59]. Any correlation between dissolution rate and compression force was found to depend upon the chemical composition of the drugs tested. Petroczki found that when crospovidone used as an adjuvant for the tabletting of sulfamidine, salicylamide, bisubsalcyilate, and terpin hydrate, very fast disintegration was obtained [60]. This finding permitted the use of these active ingredients in tablets, while previously they could only be formulated as suspensions. Experiments conducted by Esteve and coworkers showed that the dissolution kinetics of phenylbutazone tabletted with crospovidone were first order, and that the dissolution rate was independent of the tablet hardness [61]. Researchers at Sandoz A.G. found that tablets containing a griseofulvin-polyethyleneglycol dispersion and formulated with crospovidone dissolved very fast [62]. Similar tablets made with other disintegrants (such as alginic acid, sodium starch glycolate, or cornstarch) dispersed the fungicide much slower. In the studies of Miseta and co-workers, the release of poorly compressible phenylbutazone was affected by the use of various disintegrants, and crospovidone produced the best drug release [63]. Desai et al. reported that the use of crospovidone improved the dissolution stability of hydrochlorothiazide (HCTZ) capsules when compared to other disintegrants such as Explotab or corn starch [64]. This was thought to be associated with the moisture scavenging ability of the polymer, which prevented the formation of traces levels of formaldehyde (a hydrolysis product of HCTZ) in the presence of excipient-related moisture. Without crospovidone, the generated formaldehyde would interact with the gelatin capsule shell and the corn starch, resulting in the formation of less soluble compounds and a consequently decreased dissolution rate. Other investigations in which crospovidone was found to be effective as a tablet disintegrant include the work of Sakr et al. [65], Gordon et al. [66], Baykara et al. [67], Jovanovic ef al. [68), Botzolakis and Augsburger [69], van Kamp et al. [70], Wan and Lai [71], and Liu et al. [72].

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4.1.2 Tablet Binder Due to its good flow properties and plastic deformability, crospovidone has good binding properties. These properties enhance the performance of the polymer in spray, dry, and wet granulations. The polymer can also be used for the direct tabletting of a variety of drugs (such as sulfadiazine, phenacetin, phenazone, or pancreatin) without the need for granulation [73,74]. The good compatibility of crospovidone with many organic and inorganic active ingredients (and other excipients) makes this polymer suitable for use in all types of dosage forms [43]. Gillard found that crospovidone was an effective tablet binder when used at concentration levels of 5-20% together with lactose [75]. 4.1.3 Miscellaneous Pharmaceutical Uses Fast dissolving pharmaceutical preparations (e.g.,indomethacin) can be made by formulating solid dispersions of the drug in crospovidone. The preparation can be made by suspending crospovidone in a solution of the drug dissolved in a low boiling solvent, followed by the evaporation of the solvent [76]. Crospovidone has also been used in the formulation of solid dispersions of furosemide with the goal of improving the dissolution [77]. During comparisons of various excipients (such as PVP, croscarmellose sodium, or PH- 101 microcrystalline cellulose), crospovidone was less sensitive to the presence of other additives. However, the effect of other excipients depended on their levels and on the drug concentration. It has been found that when a poorly soluble drug is mixed with a water swellable, crosslinked polymer, after vacuum drying the product the dissolution rate of the drug increased considerably. For example, griseofulvin and crospovidone were vacuum dried after standing for 24 hours in methylene chloride, and then exhibited a significant increase in the dissolution rate [78]. High energy co-grinding of 6-methylene-rosta-1,4-diene-3,17-dione (an aromatase inhibitor only slightly soluble in water) with crospovidone gave a product exhibiting increased wettability and dissolution rate. As determined by thermal analysis and x-ray diffraction studies, the crystallinity of the drug decreased simultaneously [79]. In another system where a drug (FCE-24304) was co-ground with crospovidone, it was

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found that the high-energy milling resulted in a significant improvement of the dissolution rate of the formulation [80]. A dry emulsion of griseofulvin was prepared by using crospovidone as a solid support and a 1:4 combination of polysorbate 80 and sorbitan monooleate as an emulsifier. The emulsion containing 55% (by weight) of emulsifier had excellent physical stability 18 11. A formulation having an increased dissolution rate was prepared by dryblending the active drug substance with a water-swellable, hydrophilic, crosslinked polymer (such as crospovidone) in a ballmill for an extended period of time (2 hours) at 70 rpm. Tablets made with the usual excipients showed good disintegration characteristics, and noticeably increased rates of dissolution [82]. Sustained release oral formulations were successfully made for active substances whose solubility was known to be dependent upon pH. These oral formulations consisted of a weakly basic drug (such as dypyridamole, cinnarizine, or ketanserin), a water swellable polymer (crospovidone), and a gastro-resistant polymer (e.g., a cellulose derivative or acrylic polymer) within a hydrophilic or lipophilic matrix. It was found that the formulation released the drug at the same rate in both gastric and enteric environments [83]. The presence of crospovidone in suppositories was found to increase the dissolution rate and absorption of antipyretic analgesics, such as acetaminophen [841. A crosslinked mixture of poly(vinylpyrro1idone) and poly(viny1 pyridine oxide) was found to be an effective hydrogel matrix for the sustained release of drugs [85]. A Japanese patent application has been filed for a drug-treated surgical bandage made from radiation crosslinked poly(vinylpyrro1idone) [86]. Crospovidone is used in the manufacturing of a medical tape, where the topical device consists of a mixture of eperisone or tolperisone (or their salts), crospovidone, and a base carrier. An adhesive formulation was prepared by mixing the drug combination with crospovidone, and then adding this combination to a solution of 2-ethylhexylacrylate and 2ethylhexylmethacrylate. The stirring of this mixture was continued until a homogenous dispersion suitable to coat the base carrier was obtained [87].

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Crospovidone was used in forming a multilayer sustained release tablet which placed ephedrine in two phases within the system. About 50% of the drug was released from one layer in about 20-30 minutes, while the remainder was released more slowly over several (10-20) hours [88]. The combination of nifedipine with crospovidone in a sustained release formulation changed the physical form of the drug from crystalline to amorphous. The combination of the drug with the crosslinked, hydrophilic polymer, increased the water solubility of former significantly [89]. Crospovidone has also been recommended also for the stabilization of pharmaceutical suspensions [90]. A combination of infrared spectroscopic and thermal analysis studies was used to prove that oxamniquine and praziquantel do not interact physicochemically, either with each other or with crospovidone. This finding proved the feasibility of making a combination of the two in a solid dosage form [91]. Crospovidone is used in the preparation of a two-phase composite, conductive, pressure-sensitive adhesive. The continuous phase of the hydrophilic adhesive is a solid state pressure-sensitive compound, ionically-conductive regardless of the amount of water present in the phase. The discontinuous phase is made up of domains of a hydrophobic, pressure-sensitive adhesive, which enhance adhesion to mammalian skin. In a typical preparation, crospovidone was swollen in glycerin and an aqueous KC1 solution, and then mixed with Robond 60 acrylic latex. The mixture was coated on a polyester backing pretreated with E-1700 Ag ink. The adhesive properties of the tape were good, and the average skin impedance on human subjects is reported to be 165 kQ [92].

4.2

Medical Applications

The non-toxic character, high complexing ability, and lack of solubility makes crospovidone suitable for a variety of medical applications, and has been tested successfully both in human and in veterinary medicine [93]. It was found that crospovidone is beneficial for the treatment of infectious or chronic diarrhea caused by food poisoning, change of diet, the or excessive use of laxatives and diarrhea following the use of antibiotics. It is recommended also for external use in the form of ointments, such as in

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the treatment and cicatrization of sores caused by varicose ulcers [94]. A patent was received also for a composition of tannin and crospovidone that was found to be effective for the treatment of diarrhea and wounds [95]. Medroxyprogesteron acetate is known to be an effective anticancer drug in the treatment of breast cancer and endometrial cancer, but is characterized by very low bioavailability. It was found now that this property could be significantly enhanced by the addition of crospovidone to formulations. A study was carried out on 22 female breast cancer and endometrial cancer patients, with the new oral formulation being administered twice daily in 200 mg sachets. This treatment regime was compared to the standard formulation, consisting of a 500 mg Farlutal tablet administered twice daily. The bioavailability of the novel combination averaged 3 1/2 times higher than that of the standard tablet [96]. The bioavailability of medroxyprogesterone acetate (MPA) was studied with the participation of 26 female breast cancer patients. In the randomized crossover study, the MPA formulation (using a 200 mg sachet in which MPA had been loaded in crospovidone) was compared to the 500 mg standard tablet. The relative bioavailability of the MPA-crospovidone formulation was approximately three times superior to that of the standard formulation. This discovery might have important clinical implications for the treatment of hormone-sensitive cancer [97]. An iodine complex can be made by dry-tumbling crospovidone with elemental iodine. These complexes are efficient germicides and disinfectants, and can be used as antiseptic dusting powders, as rubber glove antiseptics for physicians and nurses, as foot powders, and for skin treatments of pets and farm animals [98]. Blood, blood derivatives, other body tissues, fluids and cells intended for transfusion or transplantation can be disinfected by combinations containing iodine, hydrogen peroxide and a carrier (such as crospovidone), which react with the germicide. The preparation kills pathogenic microbes without affecting the utility of the tissues, fluids, or cells [99]. Crospovidone, in combination with karaya gum, was found to be effective in the treatment of chronic constipation without organic cause [ 1001. This combination was recommended also as topical digestive agent for the

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treatment of chronic colonic diseases, such as colitis [loll, gastritis and diverticulitis [1021. Crospovidone was also recommended for use in the preparation of hemodialysis membranes for artificial kidney machines. It was suggested that these membranes may eliminate blood clotting problems [ 103,104]. Crosslinked poly(vinylpyrro1idone)was found to be suitable for the fabrication of hydrogel contact lenses [1051. It has been found that cotton dust and cotton stems contain naturally occurring components which precipitate P-lipoprotein and y-globulin (mostly IgG) in a non-immunologic manner. Sera of textile workers and human controls gave similar reaction with these extracts. Treatment with crospovidone eliminated the pseudo-immune reaction, thus making the study of the pathogenesis of byssinosis possible [ 1061. Intestinal contents and urine excreted through an artificial outlet in the body (colostomy devices) are treated with crospovidone to facilitate their handling [1071. Crospovidone was found to rapidly and efficiently absorb bilirubin. Adsorption of bilirubin onto crospovidone reaches the saturation point in a few minutes [ 1081. Crospovidone was used for the stabilization of prostaglandin (especially of the PGE type), with the system being plausible in a variety of dosage forms [ 1091. A patent was obtained by the Yamanouchi Pharmaceutical Company for stable formulations containing prostaglandin E [1lo]. A solution of nifepidin and povidone was absorbed onto crospovidone and then dried. The resulting powder was gelled with water, and tabletted to yield a sustained release system [l 1I]. Crospovidone is used for the preparation of adhesives intended for use on oral mucosa. The adhesive consists of poly(methacry1ic acid) or alginic acid (or their pharmaceutically acceptable salts), and crospovidone at a level of 550% of the total polymer. The polymers made with such composition are excellent in their adhesiveness and water resistance. The preparations can be used for the transmucosal delivery of saliva-mediated sustained release of drugs, as well as for the protection of injury and diseases in the oral cavity [112].

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4.3

EUGENE S. BARABAS AND CHRISTIANAH M . ADEYEYE

Uses of Crospovidone in the Production of Alcoholic and NonAlcoholic Beverages

As does PVP, PVPP forms complexes with a wide variety of natural products, some of which can be found in various vegetable beverages (both alcoholic and non-alcoholic). Since "popcorn" PVP is completely insoluble. its complexes are equally insoluble and thereby removable from beverages (such as beer, wine, vinegar, and fruit juices).

The current FDA regulation covering crospovidone reads as [ 1 131: "The food additive poly(vinylpyrro1idinone) may be safely used in accordance with the following prescribed conditions: a)

The additive is a homopolymer of the purified vinylpyrrolidinone catalytically produced under conditions producing polymerization and crosslinking such that an insoluble polymer is produced.

b)

The food additive is so processed that when the finished polymer is refluxed for three hours with water, five percent acetic acid, and 50 percent alcohol, no more than 50 parts per million of extractables is obtained with each solvent. It is used or intended for use as a clarifying agent in beverages and vinegar, followed by removal with filtration."

One of the most important uses of PVPP is the colloidal stabilization of beverages made from raw agricultural ingredients. These materials contain proteins and phenolic compounds, which during preparation and storage polymerize and form species which react with the proteins to form polymeric complexes of limited solubility. Smaller molecular weight complexes form colloidal particles whose solubility is temperature dependent. These are soluble in the medium at ambient temperature, but become insoluble at lower temperatures (known as the "chill haze"). If, however, the molecular weight of the polyphenol protein complex is high, a so-called "permanent haze" is developed which is visible even at room temperature.

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4.3.1 Stabilization of Beer Beer is a unique and complex food, and also a very sensitive colloid system. It is made through a multistep process which converts agricultural raw materials into the beverage through a series of biochemical reactions. Beer has been known almost throughout the history of mankind and its purity and wholesomeness has always been a concern. As early as 1516 in "Reinheitsgebot", the ruling prince of Bavaria proclaimed that, ?here shall upon threat of withdrawal of the brewing charter, for every beer taken and used no other material except barley, hops and water". While this combination allows the brewing of a good product, at that time it could not be taken into consideration that both malted barley and hops contain a variety of natural chemical ingredients which can and do react with each other. Some of the reaction products have been found to be objectionable. One of the problems is related to the stability of the beer. Beer contains as much as 150 mg/L of phenolic compounds, both as monophenols and polyphenols [ 1141. These compounds are collectively denoted as "flavonoids", and contain condensed rings systems of the following general type:

Three different types of polyphenols belong to this group, and differ in the oxidation state of Ring B:

QUERCmN

CYANIDIN

CATECHIN

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EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

Polyphenols that belong to the cyanidin group differ in the number of phenolic hydroxyls on Ring C. Beside cyanidin, pelargonidin and delphinidine also belong to this class of polyphenols:

do. -

-

OH PELARGONIDIN

CYANIDIN

DELPHlNlDlN

and are called anthocyanidins. Anthocyanidins can be transformed into red pigments by heating in dilute HCl [1 151. The phenolic ingredients of the beer are either monomeric or polymeric. It is difficult to differentiate between these, but usually members of the first group have molecular weights smaller than 1000, while the molecular weight of the polymeric polyphenols exceeds 1000. The compounds belonging to the two groups can usually be separated by paper chromatography [ 1 161. These polymerized polyphenols are erroneously referred to as tannins. Natural tannins are compounds of intricate structure, one of which is based on aromatic hydroxy acids (such as gallic acid or hexahydrodiphenic acid [117]), while the other type has flavonoid building blocks (such as catechins or anticyanogens [ 1 181). These polyphenols are quite susceptible to oxidation, although their reaction with air is very slow. On the other hand, they rapidly oxidize with oxydase enzymes. These are copper-containing proteins which can be found in plant tissues. The oxidation products (orthoquinones) are very reactive, and easily undergo condensation reactions with proteins, yielding darkcolored polymers [119]. The polymerization of polyphenols can be illustrated by the example of the so called "Beer Constituent #12" (derived through the dehydration of catechin and flavanols) which through the effect of slow oxidation and fast acid catalysis undergoes this reaction:

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The fact that B is only the dimer of A is illustrative of the complicated structure of polymeric polyphenols [ 1201. The other components of beer are proteins, the large part of which forms complexes with polyphenols. The solubility of these complexes depends upon their molecular weight and the temperature of the system. Kringstad and Damm claim that this complex has to be in an oxidized state to form a haze, or alternatively the polyphenols have to go through an oxidative polymerization to become reactive with proteins and form a haze [121]. Since the haze comes into being by the reaction of proteins with oxidized polyphenols, colloidal stability of the beer may be enhanced by removing either one or both of the ingredients of this reaction. Various methods had been suggested to achieve this goal, which can be grouped into four categories [122]: a) Preventing the oxidation either by running the whole process under anaerobic conditions, or by the addition of reducing agents or antioxidants (such as ascorbic acid, sodium tetrathionate, etc.). b) Accelerated haze formation by the addition of haze forming agents, such as tannic acid. c) Elimination of the proteins, achieved, for instance, by the addition of proteolytic enzymes (such as papain). This approach has several disadvantages, such as the enzyme

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d)

remaining in the beer, the foam stability being reduced, and the body of the beer suffering. Removal of polyphenols and polyphenol-protein complexes. The adsorbents used first were of the polyamide type (such as nylon 66, nylon 1 1, or perlon), which form complexes with polyphenols in a way similar to proteins [123]. However, the other type of adsorbents ((poly(vinylpyrro1idinone) and "popcorn" polymerized PVPP) were superior, with PVPP having the added advantage of complete insolubility. The use of PVPP for this application was first proposed by McFarlane and Bayle [123].

Chillproofing beer by the adsorption method gives a product which is more resistant to oxidation [ 1251. The treatment with PVPP removes the tannin precursors which are originally inactive in haze formation but which can condense during storage to active haze forming tannins when in contact with oxygen [126]. As it has been described before, silica gel removes most of the proteins from beer, while PVPP is effective in binding polyphenols. Recently it was found [ 1271 that a mixture of silica gel and PVPP prepared in an 18% H,SO, solution of the former, which after thorough washing, drying, and milling, gave a product which was excellent in clarifying beer. The filtration properties of PVPP could be further improved by irradiating the crosslinked polymer with a 5 megarad dose of electron beam [128]. While the amount of polymer to be used, as well as the contact time necessary for the successful removal of the polyphenols, depends upon the nature and quality of the brewing materials. In production, the use level is generally 8-20 ghectoliter for a 24 hour contact time.

4.3.2

Stabilization of Wine

In the winemaking process, the must is fermented, and then the wine is aged. During this time the dissolved and dispersed proteinaceous and polyphenolic substances create a disturbance in the equilibrium of the wine colloid system and appear in the form of haze. The degree of haze

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formation depends upon the concentration of these substances. The haze formation is the most serious with red table wines and dessert wines, whose production methods usually result in the most complete extraction of polyphenolic substances fiom the fibrous parts of the grape. On aging, these hazeforming substances settle out and can be removed to a degree by filtration or decantation. Nevertheless, the wine treated this way remains sensitive to temperature changes and oxidation [1291. When exposed to air, the flavonoid polyphenols of wine can react with oxygen, either through non-enzymatic or enzymatic routes to form quinoids and semiquinone radicals. These can further react to form brown polymeric pigments, which are responsible for the so called "browning" which harms the flavor, aroma, and color of wine. They come mostly from the skins, seeds and stems of the grape. Since these parts of the h i t differ greatly from grape to grape, different wines show different tendency for browning [1301. For instance, white wines generally have about 50 mg gallic acid equivalent (GAE) per liter, but this value can be as high as 2500 GAE per liter in the case of red wines, which are fermented with the skins [131].

As a consequence of oxidation, the wine may develop a harsh taste and strong discoloration. In order to diminish the degree of oxidation, sulfur dioxide or ascorbic acid (or both) are sometimes added to the wine. These materials, however, remain in the wine and affect its wholesomeness and natural character. Other materials, such as charcoal, bentonite, or nylon 66, had also been tried with some success for wine stabilization. However, it was shown that PVPP was superior in preventing haze formation [ 132,1331. Silica gel preferentially adsorbs higher molecular weight proteins and bentonite binds proteins of lower molecular weight, but their adsorptive performance on phenolic compounds is poor and nonspecific. PVPP, on the other hand, is highly specific with a strong and selective adsorbing action on tannins, leucoanthocyanins,and anthocyanins [ 132,1341. Some researchers reported good results with the combination of PVPP and protein-adsorbing compounds. Drboglav and co-workers used PVPP with bentonite and &Fe(CN),, and claimed to achieve excellent stabilization by decreasing both phenolic and proteinaceous materials [ 1351.

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Lyubchenko and co-workers clarified must and wine with a combination of PVPP, gelatin, and silicon dioxide [ 1361. Aivazov and co-workers found that by adding PVPP to hot bottled wine, color stability of at least 36 months could be achieved [ 1371. McKissock found that PVPP not only prevented the formation of brown pigments, but also that this adsorbent could remove any already formed discoloration [ 1381. Vojnovic tested various adsorbents in forced browning studies and determined that PVPP was the most effective in preventing browning [ 1391. In the studies conducted by Farkas and Ruzickova PVPP did not only control browning and remove tannins, but also eliminated the unpleasant taste of oxidized wine [1401. Since it was known that oxidation could seriously damage the quality and the saleability of wine, the industry applied various technical innovations to avoid it. While the use of colder fermentation and shielding the wine from air reduced the browning problem, another phenomenon known as “pinking” could not be avoided by these precautions. Pinking in white wines is most probably caused by the conversion of flavenes to red flavylium salts through reaction with oxygen. Flavenes can be formed by the slow dehydration of leucoanthocyanidins, which turn to brown dyes when oxidized. However, in the absence of oxygen, flavenes can accumulate in the wine, and during the boiling stage exposure to oxygen turn the flavenes to red flavylium salts [141]. While PVPP is not necessarily the only adsorbent useful with wines, Simpson and co-workers showed that PVPP was more effective in preventing pinking than either activated carbon or casein [ 1423. The adsorption of phenolic ingredients was found to be dependent upon the concentration of PVPP in the system and of the amount of hydroxyl groups on the phenols. The adsorption takes place by means of hydrogen bonding, and is fast and it is usually complete in less than 10 minutes (even at temperatures as low as 3°C. On the other hand, the reaction rate is only very slightly dependent upon temperature. For instance, a temperature increase from 3°C to 27°C brings about only a 10% increase in the rate of adsorption [143]. There are several other important features that are associated with the use of PVPP. The compound has no affinity towards the aroma substances

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present in the wine, so the taste of the wine does not suffer as a consequence of the treatment. Use of PVPP produces a dense and compact precipitate which increases the rate of filtration. Most importantly, PVPP can be completely removed from the wine. The use of PVPP is particularly advantageous in the treatment of sherry wines, where sulfur dioxide could not be used due to its effect on the yeast at levels above 3 ppm. PVPP, however, can be used in sufficiently high amounts to assure the stability of color without affecting the yeast. PVPP can be used to prevent browning or pinking reactions at use-levels of 24-72 ghectoliter. It can be used also to brighten the color and improve the flavor of red wines at a use-level of 6-12 gl hectoliter [ 1313. Heavy metal cations (particularly of iron, copper, zinc, tin, and cadmium) may cause a metallic taste, undesirable color changes, or haziness in the wine. Formerly, these cations were removed by the addition of potassium hexacyanoferrate or calcium physiate. A patent proposes the treatment of wine with a popcorn polymer consisting of N-vinylpyrrolidinone, and/or vinylimidazol with N,N'divinylethyleneureaas crosslinker [ 1441. It is suggested that treatment with these popcorn polymers eliminates the toxicological and operational drawbacks of the other methods used for the removal of heavy metal contaminants. The diminution of heavy metals was found to depend upon the dosage level and the contact time. The pH also influences the amount of heavy metals retained by the system, although maleic acid and lactic acid were found to have no effect of the performance of crospovidone in this particular application [1451.

4.3.3 Stabilization of Other Beverages and Natural Liquids

Juices: Although the treatment of juices with PVPP is less extensive than that discussed for beer and wine, its use has been studied by various researchers. PVPP produced good color stability and citric acid recovery with elderberry, black currant and raspberry juices, and it was found that moist, swollen PVPP was more effective than the dry adsorbent [146]. Redelinghuys received a patent for removing bitter and astringent proanthocyanidins from juices [1471. Lejeuene and Pourrat obtained betanin with 98% purity by passing beet juice through Dowex 50-X2 (H+) columns and through a PVPP column [ 1481. Hums and co-workers showed that apple juice could be stabilized by PVPP, and that the

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adsorbent could be regenerated by treatment with dilute sodium hydroxide [149]. PVPP reduced the naringin content of grapefruit juice by 78% and it slimonin content by 17.5% [ 1501. This adsorbent has been used successfully also for the stabilization of cider { 1513. 0.1% tea solution could be made polyphenol-free by treating it at 5°C with 0.4% PVPP for less than one day [1521.

&:

Coffee: Coffee causing no dyspepsia was produced by mixing a standard coffee extract with PVPP, which removed the phenolic and gastric juicestimulating components. After the addition of antiacid materials, the solution was freeze-dried [153]. PVPP was found to be an effective filtering material to remove mutagens from coffee extracts [ 1541. VineEa: Fermented vinegar is made from wine, so its stability problems are similar to those of wine. PVPP was found to be a suitable and preferred adsorbent for the removal of haze developed during the manufacturing process [15 51. 4.4

Miscellaneous Other Uses

Beside the stabilization of alcoholic beverages and other natural liquids, crosslinked poly(vinylpyrro1idinone) finds numerous applications in the food industry, in agricultural processes, and in a variety of other uses. These uses utilize the complete insolubility, chemical inertness, and total lack of toxicity characteristic of crospovidone. 4.4.1

Isolation and Stabilization of Enzymes

The presence of phenolic compounds in plant tissues complicates the extraction of enzymes from them. In the intact plant the enzyme and the tissue are separated from each other, however, when the material is broken up, reactions begin between the enzymes and the phenolic compounds. The products of these reactions are quinones and tannin-type compounds, which further react with the enzyme proteins. The enzymes modified this way are either inactive or substantially altered [156]. To separate the enzymes at the required purity and activity, it is necessary to remove the phenolic compounds fiom the system. PVPP is eminently suitable for this

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purpose because it has strong affinity towards vegetable tannins and can complex them to water insoluble entities through hydrogen bonding 11571. In the isolation of enzymes from apples Jones and co-workers used various grades of PVP (PVPP among them) to prevent the inhibition of the activity of mitochondrial preparations and certain soluble enzymes [ 1581. It was found that the effect of PVP (and PVPP) on the activity of various enzymes of the mitochondria (such as malic dehydrogenase, pyruvic carboxylase, and phenolase) was concentration dependent, and 1% was the optimal concentration for all enzymes. Walker and Hulme found that when mitochondria were incubated in the presence of various amounts of PVP (or PVPP), the degree of oxygen uptake inhibition increased up to a PVP concentration of 1%. Thus, 1% PVP or PVPP brought maximal inhibition of mitochondrial phenolase and also maximum activity of mitochondrial dehydrogenase [159]. Gustavson found that the complexes formed by PVP and PVPP with vegetable tannins could be split by high concentrations (5-8 M) of urea or sodium dioctyl sulfosuccinate detergent, which partially reactivated the PVPP-inhibited mitochondria [1601. Sanderson obtained 5-dehydroshikimate reductase by grinding frozen fresh shoots of the tea plant with PVPP and acid washed sand in 0.1M sodium phosphate buffer. Maximum activity was obtained with 0.6 g of PVPP per 1 g fresh weight of tissue [161]. Loomis and Battaille studied the isolation of active enzymes in peppermint and other monoterpene producing plants, using PVPP to remove the inhibitory phenolic compounds [1621. The extraction procedure yielded mevalonic kinase and phosphomevalonic kinase, and was carried out by grinding the fresh tissue with PVPP in liquid nitrogen. After the addition of buffer and sodium ascorbate, the thawed solution was purified by Sephadex G-50 gel filtration. Kaiser and Lewis reported that in the plants of nitrate-fed Heliunthus Annuus, the nitrate reductase activity is restricted to the roots of the plant [1631. Using an improved extraction technique consisting of a medium containing 2%casein and 1.5 g PVPP per each gram of material, the leaves showed a far greater nitrate reductase activity than did the roots. It was found also that with the addition of casein and PVPP, the glutamine synthetase activity increased in both leaves and roots. The extraction of proteins from plant tissues is rendered difficult by the presence of phenols

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EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

and phenoloxidase enzymes. In the extraction of Citrus madurensis Ramamurthy and Luddens used 2-mercaptoethanol as buffer [ 1641. This compound was added to the fresh leaf and root tissue before homogenization. When PVPP was added to the homogenized system, the activity and stability of glutamate synthetase and glutamicoxalacetate transaminase were noticeably increased. Fernandez reported that by treating raw and cooked water extracts of Phaesolus Vulgaris beans with PVPP, the inhibition due to trypsin inhibitor and polyphenolics could be separated with a good degree of reliability ( ~ 0 . 9 3 [)1651. Lastra reported the use of PVPP as a support medium of immobilized enzymes [ 1663.

4.4.2 Use in Agriculture PVPP was tested successfully in the determination of anthocyanidine glucosides from grapes and anthocyanins from various other plants. The crosslinked polymer was used to separate raspberry, rhubarb and strawberry anthocyanins [ 1671. Wilson and coworkers used PVPP columns for the preliminary isolation of chlorogenic acids from the whole extracts of plant tissues. This step separated the acid from other constituents that might interfere with its gas chromatographic analysis and eliminated the losses of phenolic compounds which usually occur when solvent extraction or similar procedures are used [ 1681. Generally, PVPP column eluted with methanol was found to afford an effective and rapid purification step of plant hormone containing extracts [ 1691. Aoki and coworkers determined chlorogenic acid in apple flesh, and also used PVPP column chromatography with ultraviolet absorption of the methanol extract at 320 nm [ 1701. Budini and coworkers developed a sensitive, high performance liquid chromatographic method for the determination of indole-3-acetic acid in the berries of Nitis vinifera using PVPP for the removal of interfering phenolic compounds. The use of PVPP and determination on a silica-A column gave a 97% recovery with a standard deviation of less than 5% [171]. Bjorsten and coworkers showed that apple-allergens are probably proteins, and that they can be extracted in an active form only if reactions with phenolic compounds present in the apple are inhibited. This was accomplished by incorporating PVPP in the extraction medium [ 1721.

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PVPP was used successfully for the purification of leaf nucleotides and nucleosides prior to chromatography. Passing extracts of the leaves of alfalfa, cotton, grape, and orange through a PVPP column removed 5991% of the substances that absorbed at 230 nm and 93 to 97 % of substances that absorbed at 320 nm. Nucleotides and nucleosides passed rapidly through the column, while the plant phenols were retained [ 1731. Soluble phenols were determined in experimental tobacco materials that were produced by practices that altered the concentration of these compounds. The estimation of phenolic compounds was based on the extent of hydrogen bonding of phenols to PVPP [1741. An efficient fertilizer can be obtained by adding PVPP to animal excrement, and fermenting the mixture under aerobic conditions. Due to the highly absorptive nature of PVPP, the water content of the excrement can be reduced by 25% [175]. The phytohormones in Scots pine (Pinus sylvestris) and spruce (Picea abies) can usually be extracted only with great losses of the active ingredients. Chromatography on PVPP-Sephadex LH 20 columns allows remarkable purification, and the collection of indol-3-acetic acid in one fraction. The other fraction contains phenylacetic acid and most of the known CI9giberellins [176]. PVPP was used for the removal of phenolic compounds in forage digestibility studies. The removal appeared to increase the digestibility of cellulose and protein in alfalfa. After treatment with Polyclar, the digestion process increased the amount of the McDougall-buffer soluble phenolic compounds. These materials were insoluble prior to indigestion [1771. Clifford, while investigating the chlorogenic acids of green coffee beans separated as many as 48 compounds belonging to this overall class. By using thin layer chromatography and a PVPP-calcium sulfate adsorbent system, ferrloylquinic acids (monohydroxyphenols),caffeoylquinic acids (dihydroxyphenols), and dicaffeoylquinic acids (tetrahydroxy-phenols) were separated. The butanone-methylphenylketone-5O%acetic acid (5:5:4 by volume) solvent system was found to give the best separation. The system which used several structure-specificlocating agents was proposed as a structure-diagnostic aid [1781.

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Crospovidone has been proposed for use as a disintegrant in granules containing water-soluble pesticides. The active compounds are rapidly dissolved from such formulations in flooded rice paddies, and disperse readily [ 1791. A water-soluble antioxidant was prepared from black tea leaves by extracting these with water, and then fractionating the extract using chromatography. One part of tea leaves was treated with 29 parts of water at 50 psi pressure and 175OC for 75 minutes. After that, the solution was filtered and fractionated on a crospovidone column (other similar column fillers, such as Sephadex G25M are also suitable). The antioxidant fractions which are suitable for food applications are clear, and contain a faint tea odor [1 S O ] .

4.4.3 Use in Analytical Chemistry Crospovidone is a useful material for a variety of applications in the field of analytical chemistry. The most important among these is that of chromatography (both column and thin layer), in which the polymer was found to be an efficient solid phase with good separation properties. The origin of its separation ability lies in its strong hydrogen bonding character. PVPP was found suitable for separating nucleic acids components from the salts used during their isolation. The salts pass through the PVPP phase without interaction, while the bases and nucleosides are retained [ 18 13. PVPP was found to be effective for the separation of aromatic amino acids (phenylalanine, tyrosine, and tryptophan), with excellent resolution of the three components [ 1821. PVPP was successfully used for the separation of cytosine and thymine. The separation is carried out at pH 3.5 where thymine is hydrogen bonded to the pyrrolidone carbonyl, or at pH 10.3 where the NH-group of thymine is ionized, disrupting the hydrogen bonding [183]. Nucleotide derivatives can be separated on PVPP columns and eluted with water in the order of nucleotides, pyrimidines, and purines [1841. PVPP was found to be effective as a stationary phase in the chromatographic separation of aromatic hydrocarbons, where the order of elution is determined by the number of condensed rings. The method is

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being used for the characterizationof products from coal liquefaction processes [1851. Good results were achieved by using PVPP as a coating for thin layer chromatography plates. Bach Marles and coworkers obtained a patent for this application, focusing on the analysis of corticosteroids, fat -soluble dyes, bactericides, and flavonoids [1861. This method was used for the chromatographic analysis of paramethasone and other hydroxy cortcosteroids, as well as for the accurate determination of other epimers [ 1871. A mixture of corticosteroids (hydrocortisoneacetate, prednisolone acetate, fluorocortisone acetate, fluoroprednisoloneacetate, paramethasone acetate, and betamethasone acetate) were separated by thin layer chromatography, using PVPP as the stationary phase and 1:1 methanolchloroform as the mobile phase [1881. A patent was issued for a novel determination of reduced Vitamin C using 2,6-dichlorophenolindophenolsupported on PVPP. The reagent mixture may be coated on adhesive tape, or may be kneaded onto a filter, for formation of the active compound [ 1891. A PVPP substrate, coated with immobilized 8-quinolinol, was used for the preconcentration of cadmium, zinc, lead, copper, iron, manganese, nickel, cobalt, and chromium salts from seawater prior to their measurement by electrothermal atomic absorption spectrometry [1901. Nakamura and coworkers developed a method for the quantitative determination of nitrates and nitrites in red wine. In this method, the red pigments and sulfur dioxide that hindered the determination of nitrates and nitrites were eliminated by passing the wine through a PVPP column and adding triethanolamine [1911. The colorimetric determination of the tartrate content of red wines by the vanadate method could not be performed without first decolorizing the wine. PVPP was found to be the most effective decolorizing medium for that purpose [1921. Quarmby studied the separation of phenolic acids and flavonoids by TLC, using a combination of PVPP and Celite as the adsorbent. He studied the effect of particle size of the plate coating components, the composition of the solvent system and the detecting agents. The Rf values of 18 flavonoids and related compounds were determined, each in three solvent systems. It was found that by the use of formic acid as the solvent in one

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EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

direction offers the advantage of carrying much of the non-phenolic components of the extracts with the solvent front where they cannot interfere with the chromatographic separation [ 1931. Olson and Samuelson studied the column chromatography of aromatic compounds containing hydroxyl and carboxylic acid substituents, and efficient separation was achieved with 0.001M hydrochloric acid as the eluent. This eluent was found to be more suitable for quantitative evaluation than water, since acid groups gave broader elution peaks in water. With substances containing several proton donating groups, improved results were obtained with 5M acetic acid as the diluent. The volume distribution coefficients were calculated from peak elution volumes. These were determined in a large number of experiments, with single solutes and with mixtures of several solutes using phenol as a marker. The degree of sorption was found to increase in a direct manner with the number of proton-donating groups on the solutes. It was also found that compounds which contain oxygen atoms in suitable distance can act as intramolecular hydrogen acceptors. Phenyl substituted alcohols eluted much earlier than phenols [ 1941. Carpenter, Siggia and Carter investigated the separation and concentration of phenolic materials on PVPP. Infrared studies conducted on the samples after equilibration showed differences indicative of hydrogen bonding. The hydroxyl stretching mode of phenol shifted from 3600 cm-' to 3200 cm cm-' when adsorbed on PVPP. The carbonyl band of PVPP also shifted from 1690 cm cm-' to 1670 cm cm-'. The maximum uptake was dependent upon the pH of the phenol solution, and in certain cases (e.g. catechols) it was narrow, but in other cases (e.g.naphtols) it was quite wide. As would be expected above pH 10, no adsorption took place due to the formation of phenolate anions. The percent uptake increased with the number of hydroxyl groups. The order of uptake for simple phenols was phloroglucinol> resorcinol > phenol; and pyrogallol > catechol > phenol. However, the correlation between uptake and -OH groups was not found to be linear. The extent of aromaticity also influenced the uptake. For instance, naphthol was removed more vigorously then phenols, owing to the attraction between the carbonyl group and the aromatic n-electron system. The pKa value did not seem to influence the uptake, as shown by the observation that adsorbed phenolics could be separated fiom the

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column by treating with 4M urea. The recovery was nearly quantitative, although a large excess of urea was required since the hydrogen bonding between urea and pyrrolidone is weaker than that between the latter and phenols [195]. PVPP was found to bind flavanoids rapidly, with the binding efficiency being found to be directly proportional to the number of hydroxyl groups attached to the flavanoid molecule. It was determined that for flavanoids of different types, but containing identical number of hydroxyl groups, flavones bind better than isoflavones, which then bind better than flavonones and dihydroxyflavonones [ 1961. Ianniello reported a sensitive method for the determination of phloroglucinol in aqueous solution by square wave voltametric detection. The carbon paste electrode used for the analysis was prepared by exposing graphite and vinylpyrrolidinone to an argon RF plasma discharge. SEM studies showed the existence of a uniform 1 pm film on the surface of the graphite. The film was insoluble, indicating a crosslinked structure, and the IR spectrum of the film was similar to that of PVPP [ 1971. Very small amounts of the growth regulator diaminozide could be determined through its adsorption onto PVPP. The adsorption capacity was calculated to be 16.2 mg per gram of adsorbent. Since the surface area of the diaminozide molecule (deduced using CPR space filling models) was estimated to be 56.2 A. This suggests the existence of a multilayer adsorption of diaminozide on crospovidone, which was bought about by hydrogen bonding [1981. X-ray photoelectron spectroscopy was used to identify the location of griseofulvin loaded within a crospovidone matrix [210]. The obtained spectral data was related to the drug loading mechanism, and the resulting properties of the crospovidone-griseoflvin system. It was concluded that the drug loading mechanism took place via intramolecular diffusion.

4.4.4

Use in Catalysis

A rhodium-based heterogeneous catalyst system was prepared by stirring PVPP with rhodium acetate in a solvent medium consisting of methanol,

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EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

acetic acid, and methyl iodide. After agitating the mixture at 190°C for 2 hours, a solid product was obtained which contained 1.062% rhodium. This catalyst system is usehl for carbonylation reactions, such as that of methanol conversion to acetic acid. It is capable of withstanding the carbonylation temperature of 150°C [1991.

5.

Health and Safety

Since the most important applications of crospovidone are in the pharmaceutical and medical fields, as well as in various areas of the food industry, a lack of biological activity and a chemical inertness are indispensable properties for the material. Furthermore, it must not interact with other ingredients of the system any more than that of other widely used excipients. 5.1

Acute Toxicity

Rats and mice were fed 5 g k g (body weight) crospovidone, and no abnormal symptoms or deaths were observed after 24,48, or 72 hours [201]. These studies may be summarized as follows.

5.2

Subacute Toxicity

A variety of subacute toxicity studies have been performed with crospovidone, which serve to further demonstrate the safety profile of this material [201]. 5.2.1

28-Day Feeding Study in Rats

Five groups of 40 rats each were dosed for 4 weeks at 0, 1,2.5,5, or 10% crospovidone in their feed, followed by 2 weeks of no crospovidone in the diet. At the 10% level, a slight bodyweight decrease was observed. Clinical chemistry and hematology, necropsy results, and other observations showed that there were no differences from the control animals. Crospovidone was not deposited in the small intestine mucosa or in the mesenteric lymph nodes.

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5.2.2 90-Day Feeding Study in Rats Three groups of 40 Wistar strain rats were fed diets for 90 days at 0,2, or 10% crospovidone. No compound-related effects were noted in behavior, appearance, food consumption, feed efficacy, bodyweight gain, clinical studies (hematology, clinical biochemistry and urinalysis), necropsy or histopathology results. 5.2.3 28-Day Feeding Study in Dogs Three groups of 6 beagle dogs were dosed with crospovidone at 1000, 2000, or 5050 mgkg bodyweight for 4 weeks. No compound-related effects were noted in food and water consumption, hematology, clinical chemistry, urinalysis and histopathology. No accumulation of crospovidone was found in the liver, kidneys or mesenteric lymph nodes. 5.2.4 180-Day Feeding Study in Dogs Three groups of 8 beagle dogs were dosed with crospovidone at 300, 1200, or 4888 mgkg bodyweight for 26 weeks. No compound-related effects were noted in behavior, food and water consumption, growth, hematology, clinical chemistry or urinalysis results, electrocardiography,opthamology, auditory tests, gross or histopathology. No crospovidone deposition or storage was found in the liver, kidneys or mesenteric lymph nodes. 5.3

Teratogenicity

Three groups of 26 pregnant SPF rats were dosed with crospovidone by gavage (at levels of 0,1000, or 3000 mgkg bodyweight) from day-6 to day- 15, and sacrificed on day-20 of gestation. One group was left untreated. No changes in fetal length were observed, and there were no significant clinical symptoms, maternal deaths, and no compound-related necropsy findings for the dams. Conception rates and live and dead implantations were not affected. The fetuses displayed no compoundrelated skeletal or visceral abnormalities. Two additional groups of 12 pregnant SPF rats received the same dose levels (1000 or 3000 mgkg) from day-15 to day-21 post-parturition. Weight gain, mortality, delivery time and litter size of the test animals

I34

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

were comparable to those of the control. No macroscopic compoundrelated changes were observed in the mothers. Pups of treated animals showed similar mortality, bodyweight and behavior as did the control. Autopsies revealed no compound-related skeletal or visceral abnormalities.

5.4

Pharmacokinetics

Studies on I4C-labeledcrospovidone in rats showed almost no absorption or gastrointestinal accumulation of orally dosed crospovidone and no biliary excretion [202]. Six Sprague-Dawley rats received an oral dose of 250 mg 14C-labeled crospovidone by gastric intubation. The crospovidone used had been treated to remove polymers having molecular weights less than 10,000 daltons. Approximately 0.128% of the ''C-crospovidone was excreted in the urine, most being excreted within the first 24 hours. SO-99% of the I4C was recovered in the feces within the 12-24 hour period. Less than 0.1% of the I4C was recovered in the carcass, most being in the G.I. tract. There was no evidence of preferential binding of the I4C to any organ or tissue.

5.5

Skin and Mucous Membrane Tolerance

The studies carried on rabbits indicated no evidence of irritation in either short action ( 1 -1 5 minute) or long action (26 hour) exposures. The polymer was applied on the skin of the rabbit as a 50% aqueous suspension. Eye irritation studies indicated the existence of very slight irritation that was not stronger than that obtained with using talc under the same conditions.

5.6

Clinical Studies (Pharmacology)

Long-term clinical studies showed that crospovidone remains unabsorbed in the gastrointestinal tract [201]. Therefore, no inherent pharmacological action would be expected from crospovidone when this excipient is used

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in medical or pharmacological applications, particularly when it is used in tablets at the common usage levels of 20-80 mg per tablet.

6.

Compliance with Pharmacopoeias and Food Regulations

As shown in Table 2, the various pharmacopoeiasand food regulations differ only in some minor details. Even these slight variations should be removed during the harmonization process currently taking place.

6.1

Identification Tests

All pharmacopoeias and food regulations use the same methods to identify crospovidone. These are the reaction with iodine, and the characteristic infrared spectrum. Non-crosslinked, water soluble polyvinylpyrrolidinone gives a deep red color when reacted with iodine. The reaction product of the crosslinked polymer is colorless. The absorption peaks of the infrared spectrum are equivalent to those of linear poly(vinylpyrro1idone).

6.1.1 Reaction with Iodine 1 gram of sample is suspended in 10 mL of distilled water, to which 0.1 mL of 0.1N iodine solution is added. The suspension is shaken for 30 seconds, and then 1 mL of starch solution is added. On shaking the solution, no blue coloration should develop.

6.1.2 Infrared Spectrum Crospovidone can be identified by its characterizationinfixed absorption spectrum. The spectral peaks characteristic of the polymer containing the pyrrolidone moiety, are the triplet at 1495 cm-I, 1463 cm-', and 1420 cm-l The results of the test conducted on an unknown must be compared to those obtained using an authentic Reference Standard. Both the test specimen and standard must be pre-dried in similar fashion, and analyzed on the same instrument using the same settings.

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Table 2 ldentification, SDecifications and Other Characteristics Eur.

I

Spectrum ,

FCC

ation

JECFA IR Spectrum, Iodination 6.0

11-12.8

11-12.8

11-12.8 5-8

~

1.5

I

0.00 1

---

1.5

0.001

0.001

0.4

~

0.1

I

I

0.1

o.oos**

I

-

0.1

I I

--

0.0003

0.00025

0.002

0.04

5.0

1

1

-

--

* There are no specifications for this value, but current production characteristically contains less than 0.005%. * * When used in clarifying agent for beverages and vinegar.

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The test specimen and the Reference Standard are dried under vacuum at 105°C for 1 hour, and then compressed in a potassium bromide pellet. A typical infrared spectrum is shown in Figure 4.

6.2

Cornpendial Testing

The compendia1testing discussed in this section is that required by most or all of the pharmacopoeias on behalf of their respective regulatory agencies.

6.2.1 Water Content The water content of crospovidone is determined using Karl Fischer titration. Measurement of this quantity is important in that water may come from the reaction medium in which the polymer is prepared, or it may be absorbed from the atmosphere. Since crospovidone is very hygroscopic, the amount of water absorbed from the environment can be considerable, and will depend upon the relative humidity to which the polymer is exposed. Water is to be considered as a reactive entity which can influence the chemical behavior of the polymer, and act as a plasticizer capable of affecting its performance a in pharmaceutical application. The method suitable for determination of the water content of crospovidone is the same as previously published for povidone [reference 203, page 5861. However, since crospovidone is insoluble in the reaction medium, the sample matrix must be stirred continually during the titration. 6.2.2 Nitrogen Content The crosslinked polymer consists entirely of vinylpyrrolidinone monomer units, which each contain 12.6% nitrogen. The nitrogen content thus serves as a method to for the assay of crospovidone. The method suitable for determination of the nitrogen content of crospovidone is the same as previously described for povidone [reference203, page 5931.

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EUGENE S . BARABAS AND CHRISTIANAH M. ADEYEYE

1

I

I

9500

3000

a00

I ZOO0

I

I

lSQ0

1000

Energy (wavenumbers)

Figure 4. Infrared spectnun of crospovidone (courtesy ISP Corporation).

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6.2.3 pH

The pH of a crospovidone solution is determined using potentiometric means, following the procedure previously described {reference203, page 5901. Since crospovidone is insoluble, it must be kept in suspension during the determination. 6.2.4 Non-Volatile, Water Soluble Content

The non-volatile, water soluble content of crospovidone consists of residual uncrosslinked poly(vinylpyrrolidinone), any reaction by-products, and adventitious contaminants. Because of their solubility and possible lack of chemical or biological inertness, these may cause adverse effects during the use of crospovidone. The pharmacopoeias and food regulations limit the allowable concentration of these substances at 1.5%. To perform the test, 25.0 g of crospovidone is transferred to a 400-mL beaker, to which 200 mL of distilled water is added and the suspension stirred for 1 hour. The suspension is transferred to a 250-mL volumetric flask, rinsed with sufficient water for transfer, and diluted to volume with water. The bulk of the solids are allowed to settle overnight, but the settling time must not be allowed to exceed 24 hours. Use a volumetric pipette to transfer 100 mL of the supernatant liquid to a pressure filtration cell. Filter about 70 mL of the supernatant through a 0.45 pm membrane filter, protected against clogging through the use of a 3 pm membrane prefilter. Transfer exactly 50.0 mL of the filtered solution to a tared 100mL beaker, evaporate to dryness, and dry at 110°C for 3 hours. The nonvolatile content is calculated using: % Water soluble

=

Wt. of residue in beaker

* 5 * 100

25

6.2.5 Heavy Metals

This test evaluates the content of common metallic impurities (silver, arsenic, bismuth, cadmium, copper, mercury, and lead) that yield insoluble, colored precipitates when reacted with hydrogen sulfide. The heavy metal content is expressed in terms of lead equivalents, and should

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EUGENE S. BARABAS AND CHRISTIANAH M.ADEYEYE

not exceed 10 ppm for pharmaceutical applications. The method for determination of heavy metal content of crospovidone is the same as previously described for povidone [reference 203, page 5971. 6.2.6 Residue on Ignition

The residue on ignition test measures the total amount of non-volatile substances, whether these are soluble or insoluble. Generally, the ash content of a crospovidone sample reflects the amount of residual salt contained in the material. The method suitable for determination of the residue on ignition of crospovidone is the same as previously described for povidone [reference 203, page 5961, must not exceed 0.4%.

6.2.7 Vinyl Pyrrolidone Content The content of N-vinyl-2-pyrrolidone monomer is determined following the methanol extraction of this species from a polymer sample. The methanolic solution is analyzed by reversed-phase HPLC, using a basedeactivated C8 column and quantified by UV detection at 235 nm. The soluble PVP is washed from the column inlet using an automated backflushing technique. The method is applicable for samples for which the monomer levels ranging from 0.4 to 100 ppm. The mobile phase is prepared by pipetting 200 mL of HPLC grade methanol into a 1000-mL volumetric flask. The contents are diluted to volume with HPLC grade water and mixed completely. The mobile phase should be filter and degassed before use. The working standard solution is prepared at a concentration of 1000 ppm by accurately weighing 0.1 gram of N-vinyl-2-pyrrolidone in a 100-mL volumetric flask, and diluting to volume with mobile phase. Additional standard solutions having concentration values of 100 ppm, 10 ppm, and 1 ppm are prepared from the working standard solution by appropriate dilutions. The sample solution is prepared by accurately weighing 2.0 grams of crospovidone in a 8-dram vial, and pipetting 20 mL of HPLC grade methanol into the vial containing the sample. The vial contents are placed on an automatic shaking apparatus, and shaken for one hour at a speed of

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130 cycles per minute. After this time period, the vial is removed from the shaker, and the supernatant is filtered through an Xydex Autovial into an HPLC autosampler vial. The HPLC system should be equilibrated by passing mobile phase through the column and detector for at least one hour before analyzing the standard and samples. The analysis is conducted using an injection volume of 20 pL, a flow rate of 1 mL/minute, and a detection wavelength of 235 nm at 0.01 AUFS. A typical run time is 10 minutes for standards alone without column backflushing, or 60 minutes for samples and column backflushing. During typical work, a 10 ppm standard is injected in triplicate every 6-10 samples to monitor system performance. The concentration of N-vinyl-2-pyrrolidonemonomer is calculated using: ppm VP

7.0 0 (Peak Area sample) (RspmsSactor) (grams sample) where the response factor is obtained using: =

Response factor

6.3

=

(Standard conce(Peak Area standard)

Other Characteristics

There are certain requirements are not required by the various pharmacopoeias, but are limited by certain food regulations. The methods to determine compliance with these regulations are described in this section.

6.3.1 Determination of Soluble Poly(Viny1 Pyrrolidone) It is presumed that during the preparation of crospovidone all of the polymer chains become part of an insoluble, crosslinked structure. It remains possible that some chains remain uncrosslinked at the completion of the reaction, and these polymer chains would be water or solvent soluble. Although poly(vinylpyrro1idone)is non-toxic, in certain uses the presence of soluble PVP is undesirable.

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Soluble PVP can be determined using a turbidimetric procedure. The sample is prepared by mixing 60 grams of sample and 600 mL of distilled water in a 1000-mL beaker, and heating the stirred mixture at 90- 1OO°C for 3 hours. At the end of this period, the mixture is cooled and centrifuged at 2500 rpm for 1 hour. The liquid is decanted, and sufficient water is added to restore the volume to 600 mL. At this point 25 grams of Dicalite 2 15 and 25 grams Hyflow Super Cel are thoroughly mixed, and 5 grams of the mixture is mixed with 100 mL of the sample solution. A filter bed is prepared by moistening a piece of filter paper (#42) in a 100 mm Buchner funnel, to which about 20 grams of the Super Cel mixture slurried in water is added. The drained water is discarded, and the sample slurry is then filtered. 50 mL of the filtrate is transferred to a 150 mL beaker, 10 mL of 7 1% perchloric acid is added, and the solution is stirred. The NTU Units on the turbidimeter must be read within one minute after the addition of the perchloric acid.

A blank solution is prepared by slurrying 100 mL of distilled water with 4 grams of the Super Cel mixture, and the mixture is filtered as just described. 0.25 mL of 0.1% K-30 solution is added to 50 mL of the filtrate, followed by 10 mL of 71% perchloric acid. This solution is stirred, and its turbidity measured. The soluble poly(viny1 pyrrolidone) in the sample is estimated using the relation: A * 50 B where A is the sample reading and B is the standard reading. This analytical procedure simulates the conditions used in breweries during the production of beer.

6.3.2 Arsenic The World Health Organization has set a limit where the maximum allowable limit of arsenic in crospovidone is 3 mg per kilogram. The method for determination of arsenic content in crospovidone is the same as previously described for povidone [reference 203, page 61 81.

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6.3.3 Zinc All of the major pharmacopoeias have no limit on zinc content, but the JECFA regulations state that zinc cannot exceed 2.5 mg per kilogram. If necessary, zinc can be determined using a spectrophotometricmethod. The most commonly used method follows the USP general method <25 1>, that makes use of the dithizone reagent.

6.3.4 Free N,N'-divinylimidazolidone In some methods used to prepare crospovidone,N,N'-divinylimidazolidone is used as a crosslinking agent. In those cases, the determination of any residual bifunctional monomer is required. This is performed using gas chromatography. An internal standard is prepared by dissolving 200 mg of N-(3methoxypropy1)-pyrrolidone or 300 mg of E-caprolactam (accurately weighed), and dissolving in 100 mL of isopropanol. The sample solution is prepared by accurately weighing 2-2.5 grams of polymer into a 50-mL Erlenmeyer flask, to which is added 1.O mL of the internal standard solution. 25 mL of acetone are added, the mixture is shaken for 4 hours, whereupon the supernatant solution is analyzed. A calibration solution is prepared by accurately weighing 25 mg of N,N'divinylimidazolidone into a 100-mL volumetric flask, and diluting to volume with isopropanol. 2.0 mL of this solution is pipetted into a 50-mL volumetric flask, and diluted to volume with acetone. 2 mL of this solution is transferred to a 25-mL volumetric flask, 1 mL of the internal standard solution is added, and the contents diluted to volume with acetone. The gas chromatography system uses a 1-meter column of 2 mm inner diameter, packed with 5% KOH and 15% polypropylene glycol 2025 supported on 45-60 mesh kieselguhr or 45-60 mesh Celite 545 NAW. An oven temperature of 170°C and an injector temperature of 250°C are appropriate. Detection is made by thermal conductivity, at a temperature is 250°C. The carrier gas is helium (flow rate of 38 mL/min), and injection volumes of 1 pL are used.

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

144

The calibration factor is calculated using: D As, f = W WSf AD where: WD = amount of N,N'-divinylimidazolidonetaken (mg) Wst = amount of internal standard taken (mg) Ast = peak area of internal standard AD = peak area for N,N-divinylimidazolidone The N,N'-divinylimidazolidonecontent is calculated using:

where: CD =

f A,

= =

W,, = A,, = W, =

concentration of N,N-divinylimidazolidone(mgkg) calibration factor peak area for N,N'-divinylimidazolidone amount of internal standard added (mg) peak area of internal standard amount of specimen taken (g)

6.3.5 Peroxides Ti(IV) is known to form a colored complex with H202 and unhindered hy droperoxides, and these complexes may be spectrophotometrically determined through their absorption at 405 nm. The peroxide content of the sample is measured after 30 minute contact time with reagent, and concentrations established by comparison to an external calibration curve of hydrogen peroxide standard solutions. The method is suitable for the determination of hydrogen peroxide and hydroperoxides in crosslinked PVP at levels ranging from 10 ppm to 1000 ppm. The Ti(1V) reagent is prepared by dissolving 2.0 g of TiOSO, in 1 liter of distilled water, which contains 25 mL of concentrated H2S04.

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The stock standard solution is prepared by weighing 3.30 g of 30% H202 into a 100-mL volumetric flask, and diluting to volume with distilled water. This solution should be standardized by KMn04 titration, following the usual procedure. A 100 ppm working standard is prepared by then pipetting 1 mL of the diluted H202 to 100 mL. H202calibration standards are prepared by pipetting 0,2,4,10, and 20 mL of the working standard into 1 00-mL volumetric flasks, adding 20 mL of the Ti(1V) reagent to each flask, and diluting to volume with distilled water. These solutions correspond to 0,0.2,0.4, 1.O, and 2.0 mg H202 standards, respectively. The sample is prepared by accurately weighing 2.00 g of polymer into a 100-mL volumetric flask, adding 50 mL of distilled water, and suspending by inversion. 20 mL of the Ti(1V) reagent is added, and the contents diluted to volume with distilled water. The solution is allowed to stand for 30 minutes, with occasional mixing during the contact time. The suspension is allowed to settle, and then a portion of the supernatant liquid is filtered through a 0.45 pm teflon filter. This filtered solution is in the sample solution. After the spectrophotometer is zeroed at 405 nm, the absorbance of the standard and sample solutions is recorded against the blank solution. A calibration curve is constructed, where the absorbance measured at 405 nm is plotted against the mg H202 in the standard solutions. A linear response is obtained, which is fitted using least-squares regression. The peroxide content in the sample is calculated using: (A - b) 0 1000 ppm H~ozin sample = m o w where: = absorbance of the sample solution A = y-intercept of the calibration curve b = slope of the calibration curve m W = sample weight, g

6.3.6

Loss on Drying

Besides water and monomeric vinylpyrrolidone, crospovidone may contain other volatile contaminants associated with the monomer, such as aldehydes, diols, imides, and their reaction products. The method suitable

146

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

for determination of loss on drying is the same as previously described for povidone [reference 203, page 6211. 6.3.7 Surface Area

The sorption of water by crospovidone is crucial to its disintegrant function, and the rate of sorption is certainly affected by the surface area of the solid. The most widely used procedure for the determination of the surface area of a powdered material is the nitrogen adsorption method developed by Brunauer, Emmett and Teller, the B.E.T. method [204]. The method suitable for determination of crospovidone surface area is the same as previously described for povidone [reference 203, page 6221.

6.3.8 Particle Sue Distribution The particle size distribution of crospovidone is important to its use in pharmaceutical technology, with different particle sizes being utilized to suit the needs of the formulator. The particle size distribution may be determined by analytical sieving, optical microscopy, light-scattering, or zone-sensing methods, although sieving is the most commonly used method. The sieving procedure involves the mechanical distribution of samples through sieves of successively smaller openings, followed by determination of the percent of the sample retained on each sieve. Since crospovidone is sensitive toward environmental factors, these must be standardized if reproducible results are to be obtained. Temperatures between 70 and 85"F, and relative humidities between 45 and 48% are considered to be acceptable ambient conditions. The method suitable for determination of particle size distribution of crospovidone is the same as previously described for povidone [reference 203, page 6281. 6.3.9 Bulk Density

The bulk density of a powder is defined as the ratio of its mass to the volume it occupies, and is normally expressed in units of g/cm3 (g/mL). The bulk density of a powder differs from the absolute density inasmuch

CROSPOVIDONE

147

as the bulk density includes the contribution of the interstitial voids as well as the volume actually occupied by the solid portion of the particles. Knowledge of the bulk density is important primarily due to equipment considerations during manufacturing, handling, and storage, but is also important to considerations of product uniformity related to differences among the densities of the formulation constituents. The "poured" bulk density is determined by slowly pouring a weighed amount of powder into a graduated cylinder, and determining the volume occupied by the solid. The "tapped" bulk density is obtained by tapping the cylinder in a predetermined fashion until the powder volume remains constant [205]. The method suitable for determination of crospovidone densities is the same as previously described for povidone [reference 203, page 6271. 6.3.10 Flow Properties Pharmaceutical powders may be characterized as being free flowing or cohesive ( i e . , non-free flowing), or anything in between [206]. Good flow properties assures efficient mixing and transport of the powder, which is necessary for the production of uniform tablets. The flow rate is influenced by factors which affect the free movement of the particles, such as their intrinsic adhesive properties, the electrostatic forces which develop as a consequence of the friction between moving particles, and any adsorbed moisture. Knowledge of the nature of moisture contained within a powder is essential, since it can dissipate electrostatic forces, while at the same time forming bridges among the particles. Since crospovidone is hygroscopic, the possibility always exists that it might change its flow properties during industrial manipulations. For these reasons, careful control of the powder conditions is required in order to obtain reliable flow measurements. The method suitable for determination of the powder flowability of crospovidone is the same as previously described for povidone [reference 203, page 6351. 6.4

Microbial Limit Tests

Owing to its use as a pharmaceutical excipient, crospovidone, the material must be free of both gram-positive and gram-negative bacteria, as well as

148

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

yeasts and molds [207]. Owing to its cross-linked structure and insolubility, the polymer can foster bacterial growth when wet or exposed to high humidity conditions.

To ascertain the validity of the corresponding tests, it first must be shown that crospovidone is free of microorganisms which can inhibit or prevent the growth of test cultures. Crospovidone should not contain more than 100 aerobic bacteria or yeasts or fungi per gram of polymer, and must be completely free of Escherichia coli, Pseumonas aeruginosa, Staphylococcus aureus (no bacteria per gram of crospovidone), and SaZrnonella (no bacteria per 10 grams of crospovidone). The method suitable for determination of microbial content of crospovidone is the same as previously described for povidone [reference 203, page 6481.

7.

Interactions of Crospovidone with Drug Substances

Crospovidone is generally a fairly inert excipient, and the literature does not contain a large number of papers describing interactions between the excipient and various drugs. Crospovidone has been reported to be incompatible with some antineoplastic agents (mitonafide and amondide) when stored at conditions of 45°C and 45% or 72 YOrelative humidity. The interaction was attributed to alterations in the water uptake capacity of the polymer [25],and was detected by differential scanning calorimetry. Study of a possible incompatibility of famotidine and excipients such as povidone, crospovidone, and talc also revealed interaction of the drug with crospovidone [30]. Interaction between crospovidone and some non-steroidal antiinflammatory drugs has also been documented. Botha and Lotter observed an interaction between naproxen and crospovidone in a tablet formulation [ 3 11, and also found an interaction between ketoprofen and crospovidone [32]. Depending on the particle size of ketorolac, interactions may occur that decrease the dissolution rate, enhanced by the presence of magnesium stearate [33]. The interaction observed with some drugs (such as amonafide) is due to moisture uptake [24].

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149

An interaction between famotidine and excipients (including

crospovidone) studied using HPLC. The existence of an interaction between the active and crospovidone was found to affect the chromatography of famotidine [30]. The effect of crospovidone and other excipients, such as hydroxyethylcelluloseand f.3-cyclodextrinon the stability of mefloquine tablets was studied using thin layer chromatography. The TLC system consisted of precoated silica gel, and the developing solvents were toluene, ethanol, ammonium hydroxide, and isopropanol. No degradation of mefloquine was observed which could be attributed to crospovidone, indicating the inertness of this excipient [208]. Differential scanning calorimetry (DSC) was used to study the possible interaction between naproxen and crospovidone [3 11. A 1:1 physical mixture of these compounds revealed the existence of a second, but smaller, endotherm, having an onset temperature of 153°C. The presence of an additional thermal feature not associated with either pure component was taken to indicate the existence of an interaction between the two [311. DSC has also been used to study the possible interaction between crospovidone and ketoprofen or ibuprofen, and it was concluded that there indeed was an interaction between the excipient and these drug compounds [32]. Idrayanto et al. also used DSC to study the interaction between famotidine and crospovidone as part of the HPLC studies [30]. In contrast, Botha and Lotter were unable to detect an interaction between crospovidone and oxyprenolol hydrochloride [209].

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8.

References

1.

J. Kondrakow, J. Pract. Chem. 64,109 (1901).

2.

H. Staudinger and E. Huseman, Ber. dt. Chem. Ges. f&16 18 (1935).

3.

J.W. Breitenbach, Poly. J. 6, 119 (1974).

4.

H.F. Kauffmann and J.W. Breitenbach, Angew. Makromol. Chem., 45,167 (1975).

5.

E.S. Barabas, “N-Vinylamide Polymers”, in the Encyclopedia of Polymer Science and Technology ,Volume 17, Second edition, John Wiley and Sons, New York, p. 212,1989.

6.

V. Buhler, Kollidon, BASF Aktiengesellschaft, Feinchemie, D6700, Ludwigshafen, p. 129 (1992).

7.

J.W. Breitenbach and H. Salek, Mubomol. Chem. 1Q8,225 (1960).

8.

J.W. Breitenbach, A. Dreisinger and E. Tomschik, Monutsh. Chem. %, 807 (1963).

9.

J.W. Breitenbach and H. Burgmann, Monatsh. Chem. eZ,1399 (1966).

10.

J.W. Breitenbach and H. Axmann, in Polymerization Kinetics and Technology, Advances in Chemistry Series. No. 128. Chapter 7. N.A.J. Platzer, ed., American Chemical Society, Washington D.C. 1973. p.110.

11.

J.W. Breitenbach and H. Dworak, J. Poly. Sci. 4,1328 (1966).

12.

L.M. Welch et al., Ind. Eng. Chem. 39,826 (1947).

13.

A.N. Pravednikov and. S.S. Medvedev, Dokl. Akad. Nauk SSSR, N,461 (1955).

151

CROSPOVIDONE

14.

J.W. Breitenbach and H.F. Kaufhann, Makromol. Chem., 125, 2597 (1974).

15.

F. Grosser, U.S. Patent 2,938,017.

16.

E.V. Hort, F. Grosser and A. Schwarz, U.S. Patent 3,294,765.

17.

F. Haaf, A. Senner and F. Straub, Poly. Journal, U , 143 (1985).

18.

S. Tseng and P. Wolf, Private Communication, ISP Co. (1993).

19.

A. Senner, F. Straub and C.H. Tschang, in Proceedings of the International Symposium on Povidone. G.A. Digenis and J. Ansell, eds. University of Kentucky, College of Pharmacy. Lexington, KY, 1983, p. 20.

20.

J.S. Shi and S.Y. Tseng, US. Patent 5,286,826 (Feb. 15, 1994).

21.

W. Denzinger et al., U.S. Patent 4,451,582 (May 29, 1984).

22.

E. Hof€inann and K. Herrle, U.S. Patent 3,759,880 (Sept. 18, 1973).

23.

M. S. Gordon and Z. T Chowhan, J Pharm. Sci., 26,907 (1987).

24.

A. I. Torres and M. A. Camacho, Eur. J Pharm. and Biopharm. &,41 (1994).

25.

S . Malamataris and A. Dimitriou, J. Pharm. Pharmacol., 42, 158 (1990):

26.

L.S. Wan and K. P. Prasad, Pharm. Znd.,

27.

L.S. Wan and K. P. Prasad, Znt. J. Pharm 3 , 1 4 7 (1989).

28.

E. Grafet al, Pharm. Znd.,B, 291 (1986).

29.

M. S. Gordon et al, Znt. J. Pharm. E, 119 (1993).

fl, 105 (1989).

152

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

a,91 1 (1994).

30.

G. Idrayanto et al., Drug Dev. Ind. Pharm.,

31.

S. A. Botha and A. P. Lotter, Drug. Dev. Ind. Pharm., 16,673 (1 990).

32.

S. A. Botha and A. P. Lotter, Drug. Dev. Ind. Pharm., fi,415 (1989).

33.

Z. T. Chowhan and L. H. Chi, Pharm. Technol., g(Apr.), 28 (1985).

34.

J. R. Johnson et al., J. Pharm. Sci.,84,469 (1991).

35.

S.S. Kornblum and S.B. Stoopak, J. Pharm. Sci.

36.

Anon. Polyplasdone XL, GAF Bulletin, 2302-099R 1 (1982).

37.

R. Huttenrauch, Pharmazie, 26,645 (1971).

38.

R. Huttenrauch and J. Jacob, Pharmazie, 3 , 2 9 3 , 5 6 3 (1971).

39.

R. Huttenrauch and U. Schmeis, Pharmazie, 25,699 (1970).

40.

P.H. List and V.A. M u m , Pharm. Ind., 41,459 (1979).

41.

D. Gissinger and A. Stamm, Pharm. Ind. 42,189 (1980).

42.

H. Nogami et al., Chem. Pharm. Bull. U , 1450 (1969).

43.

E.M. Rudnic et al., Drug. Develop. Ind. Pharm. 8, 87 (1982).

44.

A.H. Bronnsack, in Proceedings of the International Symposium on Povidone, G.A. Digenis and J. Ansell, eds. Univ. of Kentucky, College of Pharmacy, Lexington, KY, 1983 p.471

45.

K.A. Khan and C.T. Rhodes, Can. J. Pharm. Sci.,8,l (1973).

46.

A.H. Bronnsack, Pharm. Ind. 40, 1255 (1982).

a,43 (1973).

CROSPOVIDONE

153

47.

K.A. Khan and D.I. Rooke, J. Pharmacol., 28,633 (1976).

48.

E.M. Rudnic et al., Drug. Develop. Ind. Pharm. 6,291 (1980).

49.

P.H. List and V.A. Muazzam, Pharm. Ind. & 1075 I,(1979).

50.

L.S.C. Wan and P.P. Prasad, Acta Pharm. Technol. 36,20 (1990).

51.

J. Ringart and A.M. Guyot-Hermann, Drug. Develop. Ind. Pharm. 14,2321 (1988).

52.

D. Hennig and E. Schubert, Pharmazie, 42, 725 (1987).

53.

M.S. Gordon and Z.T. Chowhan, J. Pharm. Sci., 26,907 (1987).

54.

H.V. Van Kamp et al., Pharm. Acta Helv.,

55.

M. Jovanovic et al., Pharmazie, 43, 727 (1988).

56.

H.V. Van Kamp, G.K. Bolhuis, and C.F. Lerk, Pharm. Weekbl.,5,

a,22 (1986).

165 (1983). 57.

D.S. Phadke and N.R. Anderson, Drug. Develop. Ind. Pharm. 16, 983 (1990).

a,50 (1986).

58.

L.S.C. Wan and Y.L. Choong, Pharm. Acta Helv.,

59.

L. Casahoursat et al., Bull. Tech. Gattefosse Rep. My33 (1987).

60.

M. Petrbczki, Gydgyszeriszet, 23,88,(1979).

61.

J. Esteve et al., Congr. NUC.Farm. Ind.[ActaJ, I,373 (1978).

62.

A.G. Sandoz, Austrian Patent 348,132 (Jan. 25, 1979).

63.

M. Miseta et al., Acta Pharm. Hung., Z , 4 5 (1987).

64.

D. S. Desai et al., Int. J Pharm.llQ, 257-265 (1994).

154

EUGENE S . BARABAS AND CHRISTIANAH M. ADEYEYE

65.

A. Sakr et al., Pharm. Ind., 55,953-957 (1993).

66.

M. S. Gordon et al., J. Pharm. Sci,82,220 (1993).

67.

T. Baykara et al., Drug Dev. Ind. Pharm., iz,2359 (1991).

68.

M.D. Jovanovic et al., Farm-Obz, 58,221 (1989).

69.

J. E. Botzolakis and L.L. Augsburger, Drug Dev. Ind. Pharm., 14, 29 (1988).

70.

H. V. van Kamp et al., Pharm- Weekbl-Sci., 5, 165 (1 983).

71.

L. S.C. Wan and W. F. Lai, STP Pharma Sciences, 4,336 (1994).

72.

C. H. Liu et al., Drug Dev. Ind. Pharm.,

73.

E. Graf et al., Pharm. Znd., 43, 576 (1981).

74.

E. Graf et al., Pharm. Ind., 44,3 17 (1982).

75.

J. Gillard, Acta Pharm. Tech., 2 , 4 (1980).

76.

H. Imainuni, N. Nambu, and T. Nagai, Chem. Pharm. BUZZ.,2, 25 10 (1983).

77.

J. Akbuga, Pharm. Ind., 53,857, 1991.

78.

M.L. Lovrecich, Eur Pat. Appl. EP 364,944 (Apr. 25, 1990).

79.

A. Martini, C. Torricelli and R. De Ponti, Int. J. Pharm. 25,141

a,1911 (1994).

(1991). 80.

A. Martini et al., Int. J. Pharm., Z5, 141 (1991).

81.

S.P. Wyas, C.P. Jain, A. Kaushik and V.K. Dixit, Pharmazie, 42, 463 (1992).

CROSFQVIDONE

155

82.

U. Conte, A. La Manna and P. Giunchedi, Eur. Pat Appl. EP 468,392 (Jan. 29, 1992).

83.

U. Conte and P. Giunchedi, PCT Int. Appl. WO 93 00,889 (Jul. 8, 1991).

84.

A. Palmieri, T. Danson and W. Grob, Drug. Dev. Ind. Pharm. 9, 421 (1983).

85.

V.N. Harsici, Biomaterials (En@ Transl.), 2,3 (1981).

86.

H. Maeda et al., Japan Tokyo Koho JP 7807493 (March 18,1978).

87.

M. Azuma, T. Nezama, M. Yoshida, and H. Fujimori, Eur. Pat. Appl. EP 454,089 (Oct. 30, 1991).

88.

U. Conte, A. La Manna and L. Maggi, PCT Int. Appl. WO 94 06,416 (Mar. 3 1, 1994).

89.

M. It0 et al., Jpn. Kokai Tokyo Koho, JP 05,306,225 (Nov. 19, 1933).

90.

V. Buehler, Ger. Offen DE 3,0056,635 (Aug. 27, 1981).

91.

R.J. Prankerd and S.M. Ahmed, J Pharm. Pharmacol. 44,259 (1992).

92.

T.P. Dietz, R.A. Asmus and R.Uy, PCT Int. Appl. WO 93 09,713 (May 27,1993).

93.

F. Kutas et al., Hungary Teljes HU 17691 (Feb. 28, 1980).

94.

A. Queuille and R. Larde, U S . Patent 3,725,541 (Apr. 3, 1973).

95.

K. Laemmerhirt et al., Ger. Offen. DE 3,622,896 (Jan. 21, 1988).

96.

M.C. Etienne et al., J Pharm. Sci. 8Q, 1130, (1991).

156

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

97.

M.B. Kjaer, P. Jacobsen, D.M.F. Edwards, and M. StrolinBenedetti, Anti-Cancer Drugs, 4,437 (1993).

98.

F. Grosser and J. Susko, U.S. Patent 3,136,755 (June 9, 1864).

99.

E. Shanbrom, PCT Int. Appl. WO 04 00,23 1 (Jan.6, 1994).

100.

J.P. Gendre, Gaz. Med. FR (France), M, 122 (1983).

101.

J. Guerre and M. Neuman, Med. Chir. Dig.(France), 8,679 (1979).

102.

R.E. Jeanpierre et al., Med. Chir. Dig. (France), 6/7,499 (1977).

103.

M. Luttinger and C.W. Cooper, J. Biomed. Mat. Res., 1,67 (1967).

104.

R.A. Markle, R.D. Falb, and R.I.Leininger, Rubber Plastics Age, 45,880 (1 964).

105.

S.I. Gordon, Ger. Offen DE 2,751,453 (Jan. 6, 1978).

106.

S.A. Katz et al., Chest, D , 4 t h Suppl., 45 S. (1981).

107.

Chemische Fabrik Stockhausen and Cie, Ger. Offen 2,706,135 (Aug. 17, 1978).

108.

D.S. Henning, G.R. Brown and E. St-Pierre, J. Znt. Artif Organs 5,373 (1982).

109.

B. Streuff et al., Ger. Offen DE 3,304,867 (Aug. 16, 1984).

110.

Yamanouchi Pharmaceutical Co., Jpn. Kokai Tokyo Koho, JP 84 76017 (Apr. 28,1984).

111.

S. Mulligan and R.T. Sparks, S. African Patent IA 87 00738 (Sept. 30, 1987).

112.

K. Yoneto, Jpn Kokai Tokyo Koho JP 051,155,760 (Jan. 22, 1993).

CROSPOVIDONE

113.

157

Federal Drug and Cosmetic Act, Title 21, Chapter 1, Part 121, 1110 (1963).

114.

Anon., “Polyclar AT Stabilizer in Brewing”, GAF Bulletin 2302082 (1974).

115.

W.D. McFarlane, Tech. Quart. of the Master Brewers Assoc. of Amer. 5,87 (19683.

116.

J.W. Gramshaw, Tech. Quart. of the Master Brewers Assoc. of Amer. 6,239 (1969).

117.

R. Armitage et al., J. Chem. SOC.,1842 (1961).

118.

M.J. Betts et al., Chem. Comm., 1110 (1967).

119.

H.S. Mason, Advances in Enzymolog;~, _14, 105 (1 965).

120.

J.W. Gramshaw, Tech. Quart. of the Master Brewers Assoc. of Amer. 2,167 (1970).

121.

H. Kringstad and E. Damm, Proc. Eur, Brew. Conj Stockholm, 128 (1965).

122.

M. Dadic and J.G. Lavale, J. Am, SOC.Brew. Chem., 41, 141 (1983).

123.

W.D. McFarlane, U.S. Patent 2,088,550 (Sept. 7, 1954).

124.

W.D. McFarlane and P.D. Bayle, Proc. Eur. Brew. Conv. Vienna, 278 (1967).

125.

W.D. McFarlane, K.D. Thompson, and R.H. Garrat, J Am. SOC. Brew. Chem., 102 (1963).

126.

K. Steiner and H.R. Stocker, Proc. Eur. Brew. Conv. Madrid, 407 (1968)

158

127.

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

C.W. Chi and M.E. Winyal, Ger. Offen DE 3,302,258 (Aug. 11, 1983).

128.

J. Daugherty et al., U.S. Patent 4,792,400 (Aug. 20, 1988).

129.

V. L. Singleton and P. Esau, Phenolic Substances in Grape and Wine and Their Significance, Academic Press, New York, 1969.

130.

V.L. Singleton Tech. Quart. of the Master Brewers Assoc. of Amer., 4,245 (1967).

131.

Anon., “Polyclar AT Stabilizer in Winemaking”, GAF Bulletin, 9653-003 (1975).

132.

F. Drawert and G.P. Lauhus, Weinbau, Nos. 27-28 (1971).

133.

N.M. Pavlenko et al., Puti Povysh. Stabil nosti Vin i Vinomaterialov, 73 (1 982).

134.

A. Caputi Jr. and R.G. Peterson, Amer. J. Enology and Viticulture, &, 9, (1965).

135.

E.S. Drboglav et al., Pishch. Prom. St. Ser. 1,11 (1980).

136.

1.1. Lyubchenko et al., Vinodel. Vinograd. SSSR, 12 (1982).

137.

A.A. Aivazov et al., Lozar. Vinar. 32,13 (1983).

138.

A. McKissock, Food Technol. New Zealand, 12 (1966).

139.

V. Vojnovic, Kem. Ind. (Belgrad), 32, LL5 (1983).

140.

J. Farkas and A. Ruzickova, Vinohrad (Bratislava), 23, 134 (1985).

141.

V. L. Singleton, in Advances in Food Research, Supplement 111. 143 (1972).

142.

R.F. Simpson, G.C. Miller, and G.L. Orr, Food Technol. Aust. 34, 44 (1982).

CROSPOVIDONE

159

143.

A. Caputi Jr., T. Brown and M. Veda, presented at the Ann. Meeting of the Am. SOC.Enol., San Francisco, June 26, 1969.

144.

J. Detering, A. Sanner, and B. Fussnegger, U.S. Patent 5,094,867 (March 19,1992).

145.

B. Fussnegger, R. Maurer. and J. Detering, Wein-Wiss, 47, 8 (1992).

146.

M. Ugrinovits, Mitt. Geb. Lebensmittelunters. Hyg. ll,484 (1971).

147.

H.J.P. Redelinghuys, So. African Patent 8 2,505,023 (June 23, 1983).

148.

B. Lejeunne and S . Pourrat, Ann. Pharm. Fr., 22,463 (1981).

149.

N. Hums et al., Fluess. Obst., 41,238 (1980).

150.

G. Bohun and J.F. Drilleau, BIOS 2, 13 (1976).

151.

M.O. Nisperos and G.L. Robertson, Phillip. Agric., 65,275 (1982).

152.

S. Kozai and T. Hoshina, Chagyo, Kenkyu Hokoku,No.52,36 (1980).

153.

C. Fuehrer and R. Franke. Ger. Offen DE 2,72 1,117 (Nov. 23, (1978).

154.

Advance Kaihatsu Kenkyusho K.K., Jpn. Kokai 8 420,128 (Feb. 1, 1984).

155.

Anon., “Polyclar L, a new Ciarifling and Stabilizing Agent for Vinegar”, GAF Bulletin AP-143 (1963).

156.

D.S. Bendall et al., in Enzyme Chemistry of Phenolic Compounds, J.G. Pridham, ed, Pergamon, London, 1963, p. 7.

157.

K.H. Gustavson, Leder, M,27 (1963).

I60

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

158.

J.D. Jones, A.C. Hulme and L.S.C. Wooltorton, Phytochemistry 3, 210 (1964).

159.

J.R.L. Walker and A.C. Hulme, Phytochemistry, 4,677 (1 965).

160.

K.H. Gustavson, Svensk. Kern. Tidskr., 66,359 (1959).

161.

G.W. Sanderson, Biochem. J. 28,248 (1966).

162.

W.D. Loomis and J. Battaille, Phytochemistry, 5, 423 (1966).

163.

J.J. Kaiser and O.A.M. Lewis, Plant Soil, 31, 127 (1984).

164.

S. Ramamurthy and P . Ludders, Gartenbauwissenschafr, 46, 162 (1981).

165.

R.J. Fernandez, J. Agric. Food Chem., 32,734 (1982).

166.

M.C. Lastra and M. Ortega, Ann. R. Acad. Farm., 2,123 (1985).

167.

R.A. Wrolstad and B.J. Struthers, J. Chromatogr. B , 4 0 (1971).

168.

J.L. Wilson, W.J. Dunlap, and D.S.H. Wender, J. Chromatogr. 35, 329 (1968).

169.

D.M.A. Mousdale and M. Knee, J. Chromatogr. U , 3 9 (1979).

170.

S. Aoki, Y. Yohagi and T. Tamura, Nippon Shokugin Kogyo 1 10 ( 1 984). Gakkaishi, 2,

171.

R. Budin et al., Microchem. J., 22, 365 (1982).

172.

F. Bjorksten et al., Allergy, Z, 671 (1980).

173.

G. Guinn, Anal Biochem., 54,276 (1973).

174.

R.A. Anderson, T.C. Tso and J.F. Chaplin, J. Agric. Food Chem., X,89 (1979).

CROSPOVIDONE

161

175.

Naga-0 Soda K.K., Jpn. Kokai 58 208,189 (Dec. 3,1983).

176.

S. Sandberg, A. Dunberg and P.C. Oden, Physiol. Plant, =,2 19, (1987).

177.

J.H.G. Jung and G.C. Fahey, J. Agric. Food Chem. 29,817, (1987).

178.

N.M. Clifford, J. Chromatogr.,94,261 (1974).

179.

T. Shimada and S. Shiboyama, Jpn Kokai Tokyo Koho JP 06,186,228 (Jul. 8, 1994).

180.

R.S.C. So, Eur. Pat. Appl. EP 547,370 (June 23, 1993).

181.

T.M. Dougherty and A.I. Schepartz, J. Chromatogr. 411,299 (1969).

182.

T.M. Dougherty and A.I. Schepartz,J. Chromatogr.42,415 (1969).

183.

T.M. Dougherty and A.I. Schepartz,J. Chromatogr., (1969).

184.

J.L. Lerner, T.M. Dougherty, and A.I. Schepartz, J. Chromatogr. 3 , 4 5 3 (1969).

185.

G. Goldstein, J. Chromatogr. (Amsterdam), D,61 (1976).

186.

J. Bach Marles et al., Spanish Patent 378,422 (June 16, 1972).

187.

J. Moreno Dalmau, J. Pla-Delfina and A. Ojeda del Pozo, J. Chromatogr. 28,165 (1973).

188.

J.M. Marcos, J. Moreno Dalmau and J. Pla-Delfina, Cienc. Ind Farm., 9,92 (1977).

189.

Norin Suisansho Shokabin Sogo Kenkyu Socho, Jpn. Kokai 57 154056 (March 20,1984).

a,397

162

EUGENE S. BARABAS AND CHRISTIANAH M. ADEYEYE

190.

S.N. Willie, R.E. Sturgeon and S.S. Berman, Anal. Chim. Acta m , 5 9 (1983).

191.

M. Nakamura, M. Hashimoto, and H. Kondo, Fakuokashi Eisei Shikenshoho, 2,60, (198 1).

192.

L.R. Mattick and C. Rice, Amer. J. Enol. Vitic., 22,297 (1981).

193.

C. Quarmby, J. Chromatogr. 2 , 5 2 (1968).

194.

L. Oisson and 0. Samuelson, Chromatographia, U, 135 (1977).

195.

A. Carpenter, S. Siggia and S. Carter, Anal. Chem., 48,222 (1976).

196.

L.W. Doner, G. Becard and P.L. Irwin, J. Agric. Food Chem., U , 753 (1993).

197.

R. M. Ianniello, Anal. Lett., 25, 125 (1992).

198.

R.M. Ianniello, Anal. Chim Acta, Le3,8 1 (1987).

199.

M.D. Scales, J.R. Warner and P.G. Torrance, U.S. Pat. 5,281,359 (Jan. 25, 1994).

200.

F. Fusiak, K.H. C h u g , and T. Bohrer, US Pat. 5,268,117 (Dec. 7, 1993).

201.

Anon., “PVPP Summary of Toxicity Information to JEFCA”. ISP, (1994).

202.

Anon., Kollidon CL. BASF Technical Data Sheet, M.5650 (1977).

203.

C.M. Adeyeye and E.S. Barabas, “Polyvinylpyrrolidone”, in Analytical Profiles of Drug Substances and Excipients. Vol. 22, H. G. Brittain, ed., Academic Press, Inc., 1993.

204.

S. Brunauer, P.H. Emmett and E. Teller, J. Am. Chem. SOC.612,309 (1938).

CROSPOVIDONE

205.

Drug Standards, 2 , 2 2 (1 952).

206.

R.L. Carr Jr., Chem. Eng., Feb. Issue, 69 (1965).

207.

United State Pharmacopoeia, XXII Revision. Microbial Limit Tests. U.S. Pharmacopoeial Convention, Inc., p. 1619.

208.

S. Kitamura et al., Int. J. Pharm., 141,127 (1994).

209.

S. A. Botha and A. P. Lotter, Drug Dev. Ind. Pharm., fi, 1843 (1 989).

210.

F. Carli and F. Garbassi, J. Pharm. Sci.,14,963 (1985).

163

Acknowledgements

The authors wish to express their gratitude to Messrs. John F. Tancredi and Louis Blecher, and Drs. Robert M. Ianniello and Chi-San Wu for their strong support and valuable advice. Special thanks are due to Dr. Edward G. Malawer for the sound observations and helpful suggestions with regard to methods and content. Mr. Ira Naznitsky’s kind help in collecting the literature pertinent to the subject is gratefully acknowledged. The authors with to express their gratitude to Dr. Harry G. Brittain, whose careful examination and thoughtful suggestions built important improvements into the manuscript. Finally, the authors would like to thank Suzanne Currie and Susan Thomas for their contribution in typing the manuscript.

This Page Intentionally Left Blank

FLUVOXAMINE MALEATE

Nagwa H. Fodal, Mahasen A. Radwan2, and Omar A. A1 Deeb3

(1) Department of Pharmaceutics (2) Department of Clinical Pharmacy (3) Departments of Pharmaceutical Chemistry College of Pharmacy King Saud University (1,2) University Center for Women Students P.O. Box 22452, Riyadh 11459 Kingdom of Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

165

Copyright Q 1996 by Academic F’ress. Inc. All rights of reproduction in any form x ~ ~ e d .

NAGWA H. FODA ET AL.

166

CONTENTS 1.

Description 1.1 Nomenclature 1.1.1 Chemical Name 1.1.2 Proprietary Names 1.2 Formulae 1.2.1 Empirical 1.2.2 CAS Registry Number 1.2.3 Structural 1.3 Molecular Weight 1.4 Appearance 1.5 Uses and Applications

2.

Methods of Preparation

3.

Physical Properties 3.1 Polymorphism 3.2 Powder X-ray Diffraction Pattern 3.3 Thermal Methods of Analysis 3.3.1 Melting Behavior 3.3.2 Differential Scanning Calorimetry 3.4 Hygroscopicity 3.5 Solubility Characteristics 3.6 Partition Coefficient 3.7 Ionization Constant 3.8 Spectroscopy 3.8.1 Ultraviolet Spectroscopy 3.8.2 Vibrational Spectroscopy 3.8.3 Nuclear Magnetic Resonance Spectra 3.8.3.1 IH-NMR Spectrum 3.8.3.2 13C-NMR Spectrum 3.8.4 Mass Spectrometry

4.

Methods of Analysis 4.1 Elemental Analysis 4.2 Spectrophotometric Methods of Analysis 4.3 Polarographic Methods of Determination 4.4 Chromatographic Methods of Analysis 4.4.1 Thin-Layer Chromatography

FLUVOXAMINE MALEATE

4.4.2 Gas Chromatography 4.4.3 High Performance Liquid Chromatography 5.

Stability 5.1 Incompatibilities with Functional Groups

6.

Drug Metabolism and Pharmacokinetics 6.1 Absorption 6.2 Distribution 6.3 Elimination 6.3.1 Metabolism 6.3.2 Excretion

7.

Adverse Reactions

8.

Drug Interactions

9.

References

167

NAGWA H.FODA ET AL.

168

1.

Description

1.1

Nomenclature

1.1.1 Chemical Name [I]

5-Methoxy-l-[4-(trifluoromethyl)-phenyl]-l-pentanone0-(2-aminoethyl) oxime maleate 5-methoxy-4'-(trifluoromethyl)valerophenone (E)-O-(2-aminoethyl) oxime maleate 1.1.2 Proprietary Names

Dumirox, Faverin, Fevarin, Floxyfral, Maveral. 1.2

Formulae

1.2.1 Empirical:

C19H25F3N206

1.2.2 CAS Registry Number:

6 1718-82-9

1.2.3 Structure

CHCOOH

II

CHCOO1.3

Molecular Weight: 434.4

1.4

Appearance

When obtained from acetonitrile, fluvoxamine is a white crystalline powder [ 11.

FLUVOXAMINE MALEATE

1.5

169

Uses and Applications

Fluvoxamine is an antidepressant. It selectively inhibits the re-uptake of serotonine, but has relatively little effect on noradrenaline re-uptake. Fluvoxamine is reported to cause fewer antimuscarinic side-effects than do the tricylic antidepressants, but its mode of action is not fully understood. In the treatment of depression, fluvoxamine is given orally as the maleate salt, in doses of 100 to 200 mg daily. In some patients, doses of 300 mg daily may be required. It is recommended that daily doses exceeding 100 mg should be given in divided doses, in the manner of many other antidepressants. Fluvoxamine has been investigated in obsessivecompulsive disorders with reports of some benefit [2].

2.

Method of Preparation

Welle and Claassen [3] postulated three methods in their patent for the synthesis of fluvoxamine maleate. The current manufacturing method is that described in Scheme 1. A mixture of 5-methoxy-4’-trifluoromethyl valerophenone and 2-minooxyethylamine dihydrochloride is refluxed in absolute ethanol, using pyridine as an acid scavenger. The maleate salt is prepared by the addition of an equimolar quantity of maleic acid to a solution of fluvoxamine in absolute ethanol, which is then heated until a clear solution is obtained. The ethanol is removed, and the residue recrystallized from acetonitrile.

3.

Physical Properties

3.1

Polymorphism

Differential scanning calorimetry (DSC), infrared spectroscopy, and x-ray powder diffraction have been used to reveal the presence or absence of polymorphism [4]. Fluvoxamine maleate was recrystallized from PEG 4000,6000,20000, Tween 60, Tween 80, and PVP (as 1% and 10% for each). The x-ray powder patterns showed that all obtained materials were crystalline. The DSC and powder diffraction results indicated the existence of polymorphism but, the IR spectra were less clear.

n

CHCOOH

Scheme 1 The Method of Fluvoxamine Maleate Synthesis

FUJVOXAMINE MALEATE

3.2

171

X-ray Powder Diffraction Pattern

The x-ray powder diffraction pattern of crystalline fluvoxamine maleate was obtained using a Phillips x-ray diffraction spectrogoniometer, equipped with PW 1730/10 generator. Radiation was provided by a copper target (Cu anode 2000 W, 1 = 1.5418A) high intensity x-ray tube operated to 40 kV and 35 mA. The monochromator was a curved single crystal unit (PW 1752/00), and the divergence and receiving slits were 1 and 0.1, respectively. The scanning speed of the goniometer (PW 1050/81) used was 0.02 degrees 2 9 per second. The goniometer was aligned before all work using a silicone sample. Figure 1 illustrates the obtained powder pattern of fluvoxamine maleate. The crystallographic parameters data are listed in Table 1.

3.3

Thermal Methods of Analysis

3.3.1 Melting Behavior The melting range of fluvoxamine maleate is 120 - 121.5OC [11.

3.3.2 Differential Scanning Calorimetry The differential scanning calorimetry thermogram for fluvoxamine maleate is shown in Figure 2, and was obtained using a differential scanning calorimeter Perkin-Elmer model DSC-4 calibrated with indium (99.999%). The thermogram was obtained using a heating rate of 1OoC/minute, under a nitrogen atmosphere.

A single sharp endotherm was observed, having an onset of 119.4OC and a maximum at 121.6 OC. This endotherm is assigned to the melting of the compound, and is characterizedby a AH value of 26.42 cal/gm. 3.4

Hygroscopicity

At 90% relative humidity, the water uptake at ambient temperature is less than 1%.

60 Figure 1 X - Ray Powder Diffraction Pattern of Fluvoxamine Maleate

FLUVOXAMINE MALEATE

Table 1 X-ray Powder Diffraction Data of Fluvoxamine Maleate

173

0.00. 40.00 1

1

1

x)o.oo

I

1

160.00

1

1

228.00

TEMPRATURE ( C >

Figure 2 Thermal Curve of Fluvoxamine Maleate

I

1

2 80.00

1

1

340.00

EUVOXAMINE MALEATE

3.5

175

Solubility Characteristics

Fluvoxamine maleate is sparingly soluble in water, fieely soluble in ethanol and chloroform, and practically insoluble in ether.

3.6

Partition Coefficient

The following partition coefficients have been obtained:

0.4 0.1 50

3.7

n-heptanelwater dichloromethane/water (PH 1) dichloromethane/water (PH 12)

Ionization Constant

Fluvoxamine maleate contains only a single ionizable group, whose pKa has been found to be 8.7.

3.8

Spectroscopy

3.8.1 Ultraviolet Spectroscopy The UV spectrum of the protonated form of fluvoxamine maleate was obtained in 0.1 N HCl, being scanned from 190-400 nm using Pye Unicam model PU 8850 UVNIS specptrophotometer. In this form, the compound exhibits the characteristic UV spectrum shown in Figure 3a, characterized by an absorption maxima at 237.9 nm. The E-1% value was found to be 301. To obtain the UV spectrum of the deprotonated form of fluvoxamine maleate, a sample was dissolved in IN alcoholic KOH. The W spectrum obtained in this medium is found in Figure 3b, where the absorption maximum was observed to shift to 256.2 nm. The intensity of the absorption was also found to decrease, being characterized by a E- 1% value of 230.

176

NAGWA H.FODA ET AL.

Wavelength (nm)

Figure 3a.

Ultraviolet absorption spectrum of fluvoxamine maleate in 0.1 N HC1.

FLUVOXAMINE MALEATE

I

200

240

280

177

-

3 20

Wavelength (nm)

Figure 3b.

Ultraviolet absorption spectrum of fluvoxamine maleate in 1 N alcoholic KOH.

178

NAGWA H. FODA ET AL.

3.8.2 Vibrational Spectroscopy [5]

The infrared absorption spectrum of fluvoxamine maleate, obtained in a KBr disc, is shown in Figure 4. The spectrum was recorded on a PerkinElmer model 1760X infrared spectrometer. The major observed bands and their assignments are found in Table 2.

3.8.3 Nuclear Magnetic Resonance Spectra [5]

Both the proton nuclear magnetic resonance ( H-NMR) and carbon nuclear magnetic resonance (13C-NMR) spectra of fluvoxamine have been obtained in CDC13, using TMS as the internal standard. The assignments for both the 1H-NMR and 13C-NMR spectra make use of the following numbering scheme: 3

1 2

4

I

8

C CH2CH&H&Ht OCHI

IJ

7

NOCH26H2NH2 3.8.3.1

lH-NMR Spectrum [5]

The ambient temperature 300 M H z 1H-NMR spectrum of fluvoxamine was obtained on a Varian XL-300 NMR spectrometer. The spectrum itself is shown in Figure 5, and a summary of the chemical shifts and spectral assignments is provided in Table 3. 3.8.3.2

13C-NMR Spectrum [5]

The ambient temperature 75 MHz 13C-NMR spectrum of fluvoxamine was obtained on a Varian XL-300 NMR spectrometer. The spectrum is shown in Figure 6, and a summary of the chemical shifts and spectral assignments is provided in Table 4. The 13C signal multiplicities were determined by APT and DEPT experiments. Evidence in support of the 1H and 13C spectral assignments was obtained from two-dimensional correlation spectroscopy (COSY) experiments. The

-

c.

Y

d

a t-

20-

WAVENUMBER (CM-')

Figure 4 Infrared Spectrum of Fluvoxamine Maleate, KBr Disc

NAGWA H.FODA ET AL.

I80

Table 2 Vibrational Spectral Assignments of Fluvoxamine Maleate

Assignment

Band Frequency (cm-1)

3200 - 2740 (3road)

Asymmetrical and symmetrical NH3 stretching; OH stretching ~~

~

_____

3015 - 3005 (Weak)

Two bands for aryl and olefinic C-H stretching

2950 - 2880 (Weak)

Two bands for asymmetrical and symmetrical aliphatic C-H stretching

C=O stretching (-OH)

1700 (Weak)

_____

~~

1640 (Weak)

C=N stretching

1605 - 1450 (Broad)

Asymmetrical aliphatic C-H bending, aromatic and olefinic C=C stretching; COO- stretching, and NH3 bending

1365 (Medium)

Symmetrical aliphatic C-H bending,

1335 (Strong)

C-0 stretching of carboxylic acid _____

I

C-F stretching Two bands for ether C-0 stretching 950 - 650 ~

Multiple bands due to the 1,4-disubstituted benzene ring

:: n

n

1

Figure 5

1 H-NMRSpectrum of Fluvoxamine in CDC13 from TMS.

1xz

NAGWA H. FODA ET AL.

Table 3 Chemical Shifts and Spectral Assignments of the 1H NMR Spectrum of Fluvoxamine Maleate

Chemical Shift (6; ppm)

Multiplicity

Assignment

Number of Protons

1.60-1.62

m

C4-H, C3-H

4

2.70

s (broad)

-NH2

2

2.79

t

C2-H

2

3.04

t

C8-H

2

3.30

S

C6-H

3

3.37

t

C5-H

2

4.24

t

C7-H

2

7.61

d

C3',5'/H

2

7.72

d

C2',6'/H

2

singlet triplet

d: m:

>

~~

s: t:

doublet multiplet

Figure 6

C-NMR Spectrum of Fluvoxamine in CDCI3 from TMS

NAGWA H. FODA ET AL.

Table 4 Chemical Shifts and Spectral Assignments of the 1% NMR Spectrum of Fluvoxamine Maleate

I

t

-

Chemical Shift

1

Assignment

(6; PPm) I

~~

~

23.09 26.08 29.49

I

c-3 c-2 c-4

41.47

58.53 -~

72.15 ~~

76.18

c-7

125.28 - 125.43

CF3, C-4', C-3'/5'

126.49 - 126.50

C-2'/6'

139.07

c-1'

157.48

c-1

185

FLUVOXAMINEMALEATE

proton-proton (HH-COSY) and carbon - proton (CH-COSY or HETCOR) results are shown in Figures 7 and 8, respectively.

3.8.4 Mass Spectrometry The 70 eV electron impact (EI) mass spectrum of fluvoxamine maleate is shown in Figure 9, and was recorded on a Finnigan Mat model 5 100 series GCMS system. The molecular ion peak of fluvoxamine at a m/z of 3 18 is not detectable in the EI spectrum. The chemical ionization (CI) mass spectrum of fluvoxamine maleate is shown in Figure 10, and was obtained on a VG ZAB-HF mass spectrometer equipped with an Ion Tech Saddle-Field FAB gun and a standard VG source. The gun was operated at 8 KeV. Fluvoxamine maleate yielded a very prominent [M+H]+ ion (base peak) at m/z 3 19 in the CI spectrum, whereas the molecular ion at m/z 3 18 was not observed. Assignment of the possible structures of the various fragments and their relative intensities under EI conditions are listed in Table 5.

4.

Methods of Analysis

4.1

Elemental Analysis

The calculated elemental content of fluvoxamine [ 11 and fluvoxamine maleate are given in Table 6. Table 6. I

Summary of Elemental Analyses

I

Element

Fluvoxamine (w/wYo)

Fluvoxamine maleate (w/w Yo)

EK% 56.59

17.90

I

0

I

10.05

52.53 5.80 13.12 6.49 22.10

e I

7

1

I

I

6

5

4

I

3

0

0

o

a I

2

F2 (PPM)

Figure 7

HH - COSY Spectrum of Fluvoxamine.

I c-I

I

I

I

I

I

1

d0

140

140

140

1dO

1iO

id0

9b

8b

7b

6b

5b

F2 (PPM)

Figure 8

-

The Assigned CN COSY (HETCOR) Specuum of Fluvoxaminc.

i0

30

2

100 .o

226

1

50.C I

9

03 M

'1

216

55

95

Mi2

50

160

125

150

E

260

Figure 9 Electron Impact Mass Spectrum of Fluvoxamine Maleate

-P 250

3 3

Figure 10 Chemical Ionization Mass Spectrum of Fluvoxamine Maleate

190

NAGWA H.FODA ET AL.

Table 5 . Fragmentation assignments of the mass spectrum of Fluvoxamine Maleate

FLUVOXAMINE MALEATE

Table 5 Fragmentation assignments of the mass spectrum of Fluvoxamine Maleate (Continued) Fragment Assignment

-

F3C--(3-fH

dH=CH CH,CH20 CH3

191

192

NAGWA H. FODA ET AL.

Table 5 Fragmentation assignments of the mass spectrum of Fluvoxamine Maleate (Continued) d z (%)

Fragment Assignment

FLUVOXAMINE MALEATE

Table 5 Fragmentation assignments of the mass spectrum of Fluvoxamine Maleate (Continued) d z (%)

71 (87)

Fragment Assignment + CH,=CH CH, O=CH, +

69( 3 )

CF3

55(22)

CHz CH,Ch=CH,

45(77)

C H 2 = 6 CH3

31(3)

CH,= 6 H

c

193

1 94

4.2

NAGWA H.FODA ET AL.

Spectrophotometric Methods of Analysis

‘Two spectrophotometric assay methods have been described for the quantitation of fluvoxamine in tablets. The first method is based on formation of a charge-transfer complex with chloranil [6]. This complexation with chloranil substantially enhanced the weak UV absorption of fluvoxamine, and permitted measurement of the absorbance at 347 nm. The second method utilized the same complexation technique [7], but the kinetics of the complexation reaction were studied at 30 and 40OC. The reaction rate constant, and the relevant thermodynamic activation parameters, have been calculated.

4.3

Polarographic Methods of Determination

A polarographic method has been developed for the determination of fluvoxamine maleate in tablets [8]. The drug was extracted from tablets with Britton-Robinson buffer solution (pH 7.4). The polarographic cell was equipped with a Ag-AgC1 reference electrode, a Pt auxiliary electrode, and a dropping mercury working electrode. The supporting electrolyte was 0.1 M acetate buffer (pH 3.7), and the drop time was 0.6 seconds, with a scanning rate of -500 mV/min.

Tuncel et al. (9) studied the polarographic behavior and the optimum polarographic conditions for the determination of fluvoxamine using direct current, differential pulse, and superimposed amplitude pulse [9]. The technique was applied to a pharmaceutical dosage form.

4.4

Chromatographic Methods of Analysis

4.4.1

Thin-Layer Chromatography

A method for the determination of fluvoxamine in human plasma has been described [lo]. Fluvoxamine was extracted by heptane/2-propanol, and after evaporation of the organic layer, the residue was dissolved in 0.1 M NaHC03 and 0.1 % 4-chloro-7-nitrobenzofurazan.The derivative was separated by TLC using silica gel 60, with CHC13/ethyl acetate (25: 1) as

FLUVOXAMINE MALEATE

195

the mobile phase. The derivative was fluorimetrically detected at 546 nm, using an excitation wavelength of 434 nm.

4.4.2 Gas Chromatography Fluvoxamine was determined in human plasma by electron capture gas chromatography, with its structural analogue (clovoxamine) being used as the internal standard. The method required derivatization and multiple extraction steps [l 11. Dawling et al. presented a gas chromatographic method, which uses a single extraction step and nitrogen-phosphorus detection. The method is characterized by lower sensitivity [12].

4.4.3 High Performance Liquid Chromatography A number of HPLC systems have been reported, which are suitable for the identification and separation of fluvoxamine maleate have been reported [13-201. A summary of these systems is given in Tables 7 and 8. Keating et al. developed an assay procedure which involved the HPLC measurement (with electrochemical detection) of plasma serotonin levels as an indirect index of the in vivo activity of fluvoxamine [131. Haertter et al. described an automated, column switching, HPLC determination of fluvoxamine in plasma [141. The method involved solid phase extraction, which was characterized by a recovery of 97-100%. Linearity in analyte response was achieved fiom 25 to 1000 p g h , and the detection limit was 10 ng/mL. Foglia et al. determined fluvoxamine in human plasma by HPLC with ultra-violet detection at 2 15 nm, and obtained a detection limit of 25-400 ng/mL at a with relative standard deviation of 3.2-9.7% [16]. A sensitive one-step extraction procedure for the column liquidchromatographic determination of fluvoxamine in human and rat plasma was described by Van-der-Meersch-Mougeot V et al. [ 151. In this method, more than 99% of the mobile phase was organic in nature, and superior sensitivity was obtained (0.5 ng/mL could be detected). The within-day relative standard deviation was 1.8 to 5.6%, and the between-days RSD was 14.4%. In addition, there was no interference from 28 other drugs.

No

-1 2

Table 7. HPLC Assays for the Analysis of Fluvoxamine Using UV or Electrochemical Detections

Flow

Rate

1

Stationary Phase

Mobile Phase

Detector

Hypersil ODS Column 0.2 ml/L of sodium heptane sulfmate in 0.1 M - phosphate it + 0.6V vs a (15 x 0.46 cm) buffer (PH 5.5): methanol (17:3) 4glAgCl ref.. :lectrode

pH 6.8- MeOH-ACN- 10 mM 1.5 Hypersil MOS C8 (10 p)& (25 x 0.46 k phosphate buffer an) Nucleosil 100 CN (188:378:235) (5 pn)

Remarks

G

Nc

-

serotonin as an index of in viva activity of fluvoxamine

13

214 nm

Column switching and on line injection of plasma samples

14

Detection limit 0.5 ng/d. No 15 interference from other 28 COadministered drugs 16 Determination in human plasma

3

1

(15x 0.39 an)Resolve MeOH-ACN-THF-H20spherical silica (5 pan) diethylamine (9859:100:20:20:1)

254 nm

4

1

(12 x 0.46 an) Nucleosil C8 (5 pn)

16 mM-KH2P04 buffer @H 2.5)-ACN (16:9)

215 nm

5

1.5

phndapak-Cl8, (30 X 0.39 cm) (10 pn)

CH3CN-0.01 M CH3COONa buffer adjustcd to pH 3.5 with CH3COOH

240 nm

Evaluation in tablets

6

0.05 M ammonium acetate 0.8 (10 x 0.8 cm) c18 and ACN 40% V/V Bondapack (1 0 pm)

240 nm

Determination in tablets

7

--

17

Table 8. HPLC Assays for the Analysis of Fluvoxamine Involving Derivatization. Flow

Rate mllmin

Stationary Phase

Mobile Phase

(25 x 0.46 cm) Supelcosil LC-18- ACN:1O m M DB (5 pm)- guard cartridge (1.5 x k phosphate pH7.2 0.32 cm) New Guard RP-8 (7 pm: (17:3)

Detector

F1uorimetric

-

I

Remarks

Ref

Derivatization by 40 mMNaHC03, dansyl chloride & acetone

18

No -

0.5

(15 x 0.31 cm) Lichrosorb RP-18 ACN-2.5 mM imidizole Chemiluminescent Naphthalene-and anthracene 19 (5 pm) or Lichrosorb Si 60 (5 pm buffer of pH 7 (3: 1) at 418 nm 2,34aldehyde as pre-column (25 x 0.31 cm) labeling reagnet for primary amines.

1.5

(12.5 x 0.46 cm)Hypersil ODS at 3OoC

45 to 65% ACN gradieni FluOrimetric elution over 10 min.

1

(25 x 0.46 cm)Zorbax SIL (7 pm) & a precolumn (5 cm x 4.6 nun) Lichrosorb Si 100 (30 pm)

MeOH-propan2-01 (125:l)

c

s

I

F1uorimetric at nm at 455 nm)

Detection limit 1.5 ng/ml in plasma (0.1M Na2C03 & 10 ml dansyl chloride solutn 10 mg/ml in acetone).

20

Derivatization by 0.1M NaHC03 & 4chloro-7nitiobenzofurazan solution human plasma.

10

I

-

I98

NAGWA H. FODA ET AL.

Naphthalene and anthra~ene-2~3-dialdehyde have been used as pre-column labeling reagents (reacting at the primary amine site) in the reversed-phase and normal-phase liquid chromatography with peroxyoxalate chemiluminescence detection [ 191. This assay methods requires that special care be employed during the derivatization reaction owing to the nature of the steps involved and the precautions which need to be taken. The method has not been applied to determinations in body fluids. Schweitzer et al. described the fluorimetric determination of fluvoxamine or clovoxamine in human plasma after thin-layer chromatographic or normal phase HPLC separation [ 101. The method involved derivatization after extraction, and was characterized by a detection limit of 1-200 ng/mL and a recovery of about 99%.

A HPLC method for the determination of fluvoxamine in human plasma was developed by Pommery and Lhermitte [20]. Linearity in the method was attained in the range of 10-400 ng/mL, with a detection limit of 1.5 ng/mL. These workers found no interference from 20 other drugs. The method recovery was 62 to 77%, with this low recovery possibly being due to the many steps involved in extraction and derivatization. Hagga. et al. used HPLC and charge-transfer complexation methods to evaluate fluvoxamine maleate in tablets [7]. They obtained linearity was over the range of 3- 100 pg/mL, with a detection limit of 0.15 pg/mL. The recovery of fluvoxamine from tablets exceeded 99%. Foda has described an HPLC assay for the determination of fluvoxamine maleate in tablets [ 171. Linearity was obtained was over the range from 0.5 to 12 pg/mL, and the fluvoxamine recovery from tablets was 100%.

5.

Stability

5.1

Incompatibilities with Functional Groups

The physicochemical compatibility between fluvoxamine maleate and a number of tablet and capsule excipients were investigated by differential scanning calorimetry [21]. Fluvoxamine maleate was found to be fully compatible with starch, polyvinylpyrollidone, triglycerides, microfine

FLUVOXAMINE MALEATE

199

cellulose, and microcrystalline cellulose. The drug was found not to be compatible with stearic acid, magnesium stearate, lactose, or sodium carboxymethyl-cellulose.

6.

Drug Metabolism and Pharmacokinetics

Fluvoxamine is a second generation antidepressant, characterized by a potent, selective, and inhibitory activity on neuronal serotonin (5hyroxytryptamine, 5HT) re-uptake. In addition to its lack of effects on other monoamine re-uptake mechanisms, fluvoxamine has little or no effect on the neuronal function of other monoamines, and has a low affinity for receptors of a variety of neurotransmitters. The drug is structurally unrelated to the tricyclic group of antidepressants. For the treatment of depression or obsessive-compulsivedisorder, the usual daily dose is 50 to 300 mg administered once daily, or in divided doses [22]. The administration is usually started at 50 mg/day, and is slowly increased up to 300 mg/day in an effort to improve the patient tolerance to the nausea and vomiting which are associated with the initiation of fluvoxamine therapy [23-251.

6.1

Absorption

Fluvoxamine is almost completely absorbed (but relatively slowly) from the gastrointestinal tract [26]. After the administration of single oral doses of rapidly dissolving formulations, the peak plasma concentration (Cm,) is usually observed within 2-8 hours. For enteric coated tablets (the commercially available dosage form), the Cm, may be observed at 4 to 12 hours after administration. Food did not interfere with fluvoxamine absorption after administration of rapidly dissolving formulations [27]. This finding is not necessarily applicable to the enteric-coated dosage form, since the absorption of drugs fiom this dosage form is delayed by food intake [28]. 6.2

Distribution

Approximately 77% of fluvoxamine is bound to human plasma proteins at plasma concentrations up to 1 mg/L [22,29]. This implies that plasma

NAGWA H. FODA ET AL.

200

protein binding interactions of any clinical relevance are unlikely to occur with this drug 1301. After intravenous administration of radiolabelled fluvoxamine to rats, the radioactivity distributes rapidly and is found in higher concentration in most organs than found in plasma [22]. Since fluvoxamine has not been intravenously administered to humans, its apparent volume of distribution is estimated after oral administration as ViF (where V = apparent volume of distribution, and F = bioavailability). The ViF factor varies between 10 and 20 L/kg [29].

6.3

Elimination

The elimination of fluvoxamine after a single oral dose follows a biexponential decline [26]. Following the administration of enteric-coated fluvoxamine tablets, the mean elimination half-life (t1/2 p) was 16.9 hr in healthy volunteers, and 23.2 hr in depressed patients [31]. The mean elimination half-life was 22 hr after multiple dosing (50 mg twice daily for 28 days), which may be compared to 19 hr after single dosing (50 mg) in the same subjects. There is no apparent accumulation of fluvoxamine in plasma after multiple-dose therapy. The area under the plasma concentration-time curves (AUC) was similar after single and multipledose therapy. The AUC tended to be longer following multiple oral-dose as compared to single-dose administration [32]. This implies non-linearity in fluvoxamine disposition after higher single dose (exceeding 100 mgj or multiple-dose therapy.

6.3.1 Metabolism Following the administration of a single dose of radiolabelled fluvoxamine, at least 11 metabolites are known to accumulate in human urine. Nine of the metabolites have been identified by mass spectrometry, and account for 85% of the total urinary radioactivity [33]. The metabolic pathways of fluvoxamine in humans are depicted in Scheme 2. The major (65%) fluvoxamine metabolite is produced by oxidative demethylation of the aliphatic methoxy group. Lesser amounts of other metabolites are produced by degradation at the primary amino group (1 5%j, at both the methoxy and m i n e groups (20%), or by the removal of the entire ethanol amhe group (10%). Two of the primary metabolites (Compounds I and I1 of Scheme 2) do not possess any psychotropic

F , C O F-CH,-CHz-CH,-CH2-O-CH, N \

-

0 CH,-Mz-NH,

F,C~-CH,-CH~-CH~C-OH N \

V

\c~;-c~-cH,-cH,-cH,-o-cH, N

O-CHrM@

vi

F,C~$-CH;-CH~CH~~-OH N

‘0- CHfC-fH 0

Scheme 2 The Metabolic Pathways of Fluvoxamine Maleate

va

‘OH

NAGWA H. FODA ET AL.

202

Table 9 Drugs Reported to Interact with Fluvoxamine

Reference

Drug

of Interaction

Effect of the Interaction

Inhibition of hepatic oxidation

Decrease C1. Increase t 1/2and plasma concentration

44-45

Inhibition of hepatic metabolism

Increase plasma concentration

46

Propranolol

NJA

Increase plasma concentration

22

Theophylline

Competitive inhibition of hepatic metabolism

Decrease C1. Increase plasma concentration

47

Warfarin

NJA

Increase plasma concentration

22

(brom azepam, alprazolam)

I

~~~~~~

I----

FLUVOXAMINE MALEATE

8.

203

Drug Interactions

The recent reports of drug interaction observed during administration of fluvoxamine to healthy volunteers and patients with CNS disorders are summarized in Table 9. The incidences of toxicity were found to increase as an outcome of these drug interactions. It should be mentioned that fluvoxamine affects only the pharmacokinetics of benzodiazepines eliminated by hepatic oxidation [44-451.

9.

References

1.

The Merck Index. 1lthed. Merck and Co. Inc., Rahway, New Jersey, 1989, p. 659.

2.

Martindale - The Extra Pharmacopoeia. 30 ed. J.E.F. Reynolds, ed. The Pharmaceutical Press, London, 1994, p. 1450.

3.

H.B.A. Welle V. and Claassen, US Patent 4,085,225, April 18, 1978.

4.

O.M. Al-Gohary, N.H. Foda, and F. El Shafie, Identification of polymorphs of fluvoxamine maleate, Die Pharmazie, 49,592-594 (1994).

5.

O.A. A1 Deeb, N.H. Foda, and M.A. Radwan, unpublished results.

6.

A.A. A1 Haider, M.E.M. Hagga, M.E. Alawady, and E.A. GadKariem, Spectrophotometricdetermination of fluvoxamine in tablets based on charge-transfer complex with chloranil, Anal. Lett., 26,887-901 (1993).

7.

M.E.M. Hagga, A.A. A1 Haider, H.A. Al-Kahmees, M.E. Alawady, and E.A. Gad-Kariem, Evaluation of fluvoxamine maleate in tablets by high performance liquid chromatogrphic and chargetransfer complexation methods, Saudi Pharm. Journal, 1,70-75 ( 1 993).

th

204

NAGWA H. FODA ET AL.

8.

K. Albert, Polarographic determination of fluvoxamine maleate in tablets, Pz-win, 3, 59-61 (1990).

9.

M.Tuncel, G . Altiokka, and Z. Atkosar, The polarographic determination of fluvoxamine maleate, Anal, Lett., 27, 1 135-1 145 ( 1 994).

10.

C. Schweitzer, H. Span, and E. Mutschler, Fluorimetric determination of fluvoxamine or clovoxamine in human plasma after thin-layer chromatographic or high performance liquidchromatographic separation, J Chromatgr. Biomed. Appl. 55 (J Chromatogr.,382) 405-41 1 (1986).

11.

H.E. Hurst, D.R. Jones, C.H. Jarboe, and H. DeBree, Determination of clovoxamine concentration in human plasma by electron capture gas chromatography, Clin. Chem. 27, 1210-12 12 ( 1 98 1).

12.

S . Dawling, N. Ward, and E.G. Essex, Rapid measurement of basic drugs in blood applied to clinical forensic toxicology, Ann. Clin. Biochem., 27,473-477 (1990).

13.

J. Keating, L. Dratxu, M. Lader, and R.A. Sherwood, Measurement of plasma serotonin by high-performance liquid chromatography with electrochemical detection as an index of the in vivo activity of fluvoxamine, J. Chromatogr. Biomed. Appl., 126 (J Chromatogr.,615) 237-242 ( 1 993).

14.

S. Haertter, H. Wetzel, and C. Hiemke, Automated determination of fluvoxamine in plasma by column switching high performance liquid chromatography, Clin. Chem., 38,2082-2086 (1992).

15.

V. Van-der-Meersch-Mougeot and B. Diquet, Sensitive one-step extraction procedure for column liquid-chromatogrphic determination of fluvoxamine in human and rat plasma, J. Chromatogr. Biomed. Appl., 105 (J Chromatogr.,567) 441 -449 (1991).

FLUVOXAMINE MALEATE

205

16.

J.P. Foglia, L.A. Birder, and J.M. Perel, Determination of fluvoxamine in human plasma by high-performance liquid chromatographywith ultra-violet detection, J. Chrornatogr. Biomed. Appl., 87,(J. Chromatgr., 495) 295-302(1989).

17.

N.H. Foda, High performacne liquid chromatogrphic determination of fluvoxamine maleate in tablets. J. Liquid. Chromatgr., 18,1591-1601(1995).

18.

R.H. Pullen and A.A. Fatmi, Determination of fluvoxamine in human plasma by high performance liquid chromatographywith fluorescence detection, J. Chromatgr. Biomed. Appl. 112,(J. Chrornatogr.,574) 101-107(1992).

19.

P.J.M. Kwakman, H. Koelewijn, J. Kool, U.A.T. Brikman, and G.J. De-Jong, Naphthalene and anthracene-2,3-dialdehydeas precolumn labelling reagents for primary mines using reversed and normal-phase liquid chromatography with peroxyoxalate chemiluminescencedetection, J. Chromatgr.,511, 155-166 (1 990).

20.

J. Pommery and M. Lhermitte, High-performance liquidchromatographic determination of fluvoxamine in human plasma, Biomed. Chrornatogr.,3, 177-179 (1 989).

21.

N.H. Foda, Compatibility study between fluvoxamine maleate, mebeverine hydrochloride and tablet excipients using differential 73-79(1992). scanning calorimetry, Egypt J. Pharm. Sci.,

a,

22.

P. Benfield and A. Ward, Fluvoxamine: a review of its pharmacodynamic and pharmacokineticproperties, and therapeutic efficacy in depressive illness, Drugs, 32,313-334(1986).

23.

L. Conti, L. Dell’Osso, and F. Re F, Fluvoxamine maleate: double-blind clinical trial vs. placebo in hospitalized depressed patients. Curr. Ther. Res., 43,468-479(1988).

24.

A.J. Martin, V.M. Tebbs, and J.J. Ashford, Affective disorders in general practice. Treatment of 6000 patients with fluvoxamine, Pharmatherapeutica,5,40-49 (1 987).

206

NAGWA H.FODA ET AL.

25.

W.K. Goodman, L.H. Price, and S.A. Rasmussen, Efficacy of fluvoxamine in obsessive-compulsive disorder: a double-blind comparison with placebo, Arch. Gen. Psychiatry, 46,36-44 (1989).

26.

H. DeBree, J.B. Van der Schoot, and L.C. Post, Fluvoxamine maleate: disposition in man, Eur. J. Drug Metab. Pharmacokinet., 8, 175-179 (1983).

27.

J. Van Harten, P. Van Bemmel, and M.R. Dobrinska MR, Bioavailability of fluvoxamine given with and without food, Bioparm. Drug Dispos., 12,571-575 (1991).

28.

E. Perucca, Routes of drug administration., Med. Int., 101,42294234 (1 992).

29.

D.P. Doogan, Fluvoxamine as an antidepressant drug, Neuropharmacologv, 19, 1215-1216 (1980).

30.

J. Van Harten, Comparative pharmacokinetics of selective serotonin re-uptake inhibitors, Clin. Pharmacokinet., 24,203-220 (1993).

31.

U.A. Siddiqui, S.K. Chakravarti, and D.K. Jesinger, The tolerance and antidepressive activity of fluvoxamine as a single dose compared to a twice daily dose, Curr. Med. Res. Op., 9,68 1-690 (1985).

32.

M.H. De Vries, M. Raghoebar, and I S . Mathlener, Single and multiple oral dose fluvoxamine kinetics in young and elderly subjects, Ther. Drug Monitor., 14,493-498 (1992).

33.

H. Overmars, P.M. Scherpenisse, and L.C. Post, Fluvoxamine maleate: metabolism in man. Eur. J. Drug Metabol. Pharmacokinet., 8,269-280 (1983).

34.

V. Claassen, Review of the animal pharmacology and pharmacokinetics of fluvoxamine, Br. J. Clin. Pharmacol., 15 (3 SUPPI.),3498-355s (1983).

FLUVOXAMINE MALEATE

207

35.

H.M. Ruijten, H. DeBree, and J.M. Borst, Fluvoxamine: metabolic fate in animals, Drug Metab. Disp., 12, 82-92 (1984).

36.

S. Wright, S. Dawling, and J.J. Ashford, Excretion of fluvoxamine in breast milk, Br. J. Clin. Pharmacol., 31,209 (1991).

37.

W. Wagner, B. Plekkenpol, and T.E. Gray, Review of fluvoxamine safety database, Drugs, 43 (Suppl2), 48-54 (1992).

38.

J.P. Ottervanger, P.M.L.A Van Den Bemtt, and G.H.P. de Koning, Risk of bleeding during treatment with fluoxetine (Prozac) or fluvoxamine (Fevarin), Ned Tijdschr Geneeskd, 137,259-26 1 (1993).

39.

M.I. Wilde, G.L. Polsker, and P. Benfielf, Fluvoxamine: An update review of its pharmacology, and therapeutic use in depressive illness, Drugs, 46, 895-924 (1993).

40.

M.A. Jenike, S. Hyman, and L. Baer, A controlled trial of fluvoxamine in obsessive -compulsive disorder: implication for a serotonergic theory, Am. J. Psychiatry, 147, 1209-1215 (1990).

41.

W.K. Goodman, L.H. Price, and P.L. Delgado, Specificity of of serotonin reuptake inhibitors in the treatment of obsessivecompulsive disorder, Arch. Gen. Psychiatry, 47, 577-585 (1990).

42.

W.K. Goodman, M.J. KO&, and M. Liebowitz, Efficacy of fluvoxamine in obsessive-compulsive disorder; a second multicentre study, Poster presented at the 18* Collegium International Neuro-Psychopharmacologicum;Nice, France; June 28-July 1, 1992.

43.

G.K. Mallya, K. White, and C. Waternaux, Short-and long-term treatment of obsessive-compulsive disorder with fluvoxamine, Ann. Clin. Psychiatry, 4, 77-80 (1992).

NAGWA H. FODA ET AL.

208

44.

J. Van Harten, R.L. Holland, and K. Wesnes, Influence of multiple-dose administration of fluvoxamine on the pharmacokinetics of benzodiazepine bromazepam and lorazepam: a randomised, cross-over study, Eur. Neuropsychopharmacol., 2, 381 (1992).

45.

J.C. Fleishaker and L.K. Hulst, Effect of fluvoxamine on the pharmacokinetics and pharmacodynamics of alprazolamin healthy volunteers, Pharm. Res. 9 (Supplement), S-295 (1 992).

46.

E. Spina, A. Avenoso, and A.M. Pollicino, Carbamazepine coadministration with fluoxetine or fluvoxamine, Ther. Drug Monitor., 15,247-50 (1993).

47.

A.H. Thomson, E.M. McGovern, and P. Bennie, Interaction between fluvoxamine and theophylline, Pharm. J., 249, 137 (1 992).

Acknowledgements

The authors gratefully acknowledge the assistance of Dr. Faiyza El Shafie. The skillful technical assistance of Mr. Abdel Rahman AlGhdeer is highly appreciated.

GADOTERIDOL

Krishan Kumar,' Michael Tweedle,' and Harry G. Brittain2

(1) Bracco Research USA P.O. Box 5225 Princeton, NJ 08520

(2) Ohmeda, Inc. Pharmaceutical Products Division 100 Montain Avenue Murray Hill, NJ 07974

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

209

Copyright 0 1996 by Academic Press, Inc. All nghrs of reproduction in any form reserved.

210

NAGWA H. FODA ET AL.

Description Name, Formula, and Molecular Weight 1.1 1.2 Appearance 1.3 History 2. Synthesis 3. Physical Properties Infrared Spectrum 3.1 NMR Spectrum 3.2 Mass Spectrum 3.3 Ultraviolet Spectrum 3.4 Luminescence Spectrum 3.5 Optical Activity 3.6 CrystallographicProperties 3.1 Relaxivity and Water of Hydration 3.8 Thermal Analysis 3.9 3.10 Hygroscopicity 3.1 1 Microscopic Characterization 3.12 Complex Formation Constant 3.13 Kinetic Inertness 3.14 Partition Coefficients 3.15 Solubility 3.16 Conductivity and Osmolality 3.17 Viscosity and Specific Gravity 4. Methods of Analysis 4.1 Elemental 4.2 Spectrophotometric 4.3 Chromatographic 4.3.1 Thin-Layer 4.3.2 High Performance Liquid 5. Stability 5.1 Solid State Stability 5.2 Solution Stability 5.3 Stability in the Presence of Endogenously Available Ions 5.4 In Vivo Stability 5.5 Stability in Biological Fluids 6. Biological Studies 6.1 Tissue Distribution in Mice 6.2 Pharmacokinetics 6.3 Toxicity 7. Acknowledgements 8. References 1.

FLUVOXAMINE MALEATE

1.

Description

1.1

Name. Formula. and Molecular Weight

211

Gadoteridol (Gd(HP-D03A) is a nonionic contrast agent for magnetic resonance imaging (MRI). The commercial product (ProHance) is available as a 0.5 M sterile clear colorless to slightly yellow aqueous solution in vials, for intravenous injection. The systematic chemical name for Gadoteridol is 10-(2-hydroxypropy1)-1,4,7,10-tetraazacyc1ododecane1,4,7-triaceticacid, monogadolinium salt. The Chemical Abstracts identification number is CAS-120066-54-8. The structural formula is:

c 17H29N407Gd

Anhydrous M.W. 558.69

The elemental composition corresponding to the anhydrous form is C 36.55%, H 5.23%, N 10.03%, 0 20.05, and Gd 28.15%. Each mL of ProHanceRcontains 279.3 mg gadoteridol, 0.23 mg CalteridolRcalcium (calcium salt of calcium complex of HP-D03A), 1.21 mg of Trizma (as Tris acetate buffer) and water for injection. ProHance contains no antimicrobial preservative. 1.2

Apearance

Gadoteridol is a white to off-white crystalline solid, obtained as aggregate clumps of fine needle-like microcrystals. The compound has no inherent odor. 1.3

History

Gadoteridol, unformulated raw material, was designed as a nonionic and hydrophilic Gd3+chelate species, intended for use as a contrast agent in Magnetic Resonance Imaging (MRI) [ 1-31. The contrast-enhancedMRI images are used to differentiate diseased from normal tissue, and to identify specific disease states (such as infarcts, abscesses, and tumors).

212

NAGWA H. FODA ET AL.

Agents of this type have been found to be effective as water-proton relaxation catalysts in both aqueous solution and in human serum, and their use greatly improves the quality of data available in such studies [ 1-31. The relaxivity of gadoteridol is comparable to that of the ionic agents, Gd(DTPA)'- and Gd(D0TA)-.

2.

Synthesis

The synthesis of the free ligand and its Gd3+ complex gadoteridol, is published [4,5] (scheme given below). The procedure involves protection of one nitrogen of the macrocyle, 1,4,7,10-tetraazacyclodoecane,by forming a novel intermediate, 1,4,7,10-tetraazacyclododecane-1carboxyaldehyde. Alkylation of this protected macrocycle by tbutylbromoacetate in basic media and subsequent hydrolysis by sulfuric acid yielded D03A. D03A was reacted with propylene oxide at pH 12 to produce HP-D03A. Excess propylene oxide was removed under vacuum. The crude material was purified by cation exchange, anion exchange, and PVP resins. HP-D03A was reacted with gadolinium oxide in water to produce Gd(HP-D03A).

FLUVOXAMINE MALEATE

3.

Physical Properties

3.1

Infrared Suectrum

213

The infrared absorption spectrum of gadoteridol was obtained in a KBr disc, and is shown in Figure 1. One distinctive carbonyl band was observed at 1613 cm-', and is assigned to the three unresolved, equivalent carboxylate groups of the ligand. The numerous bands noted in the fingerprint region can be assigned on the basis of their band energies, and a summary of the assignments is found in Table I.

Energy (ern-')

Figure 1. Infrared absorption spectrum of gadoteridol, as obtained in a KBr disc. Table I. Band Positions and Assignments for the Vibrational Transitions of Gadoteridol Frequency (l/cm) 1085 1323 1384 1458 1477 1613 1635 2858 2975

Assignment Secondary hydroxyl group stretching mode Symmetric carbonyl stretching mode of the carboxylate groups Combination band of the carbonyl stretching (carboxylate groups) and the symmetric methyl deformation modes Methylene asymmetric deformation Methylene symmetric deformation Asymmetric carbonyl stretching mode of the carboxylate groups Hydroxyl bending mode Methylene stretching mode Asymmetric methyl stretching mode

NAGWA H.FODA ET AL.

214

3.2

Nuclear Magnetic Resonance Spectrum

Owing to the high paramagnetism associated with the Gd" ion in Gadoteridol, NMR spectra of gadoteridol cannot be obtained. However, the 'H and 13C NMR spectra of the free ligand, HP-D03A, have been obtained. The 'H NMR spectrum was obtained at 400 MHz in D20 solution, and is shown in Figure 2. This spectrum was internally referenced to the HOD peak at 4.65 ppm. The protons of the C1 methyl group were observed to resonate as a doublet at 1.13 ppm. The multiplet peak at 4.08 ppm was assigned to the C2 proton, and the remaining methylene protons resonate as the broad, partially overlapped multiplets observed between 2.9 and 3.7 ppm.

c... . . ~ i C b c m i i Shift (ppm)

Figure 2. 'H NMR spectrum of HP-D03A, obtained at 400 MHz in D20 solution, and internally referenced to the HOD peak at 4.65 PPm. The 13C NMR spectrum was obtained at 270 MHz, and is found in Figure 3. This spectrum was externally referenced to p-dioxane at 67.6 ppm. The C 1 carbon resonates at 2 1.6 ppm, while the C2 carbon was observed at 64.4 ppm. The carbonyl groups (C 13, C 15, and C 17 yield the peaks noted at 172.9 and 174.6. The 12 methylene carbons yield the partially overlapped resonances noted at 49.8,50.7,51.2,56.2,56.4, and 60.5 ppm.

FLUVOXAMINE MALEATE

215

Figure 3. 13C NMR spectrum of HP-D03A, obtained at 270 MHz, and externally referenced to p-dioxane at 67.6 ppm. 3.3

Mass Spectra

Gadoteridol has been characterized using both positive and negative ion fast atom bombardment (FAB), with the compound being dissolved in water and thioglycerol and sputtered by 8 keV xenon atoms. These data are illustrated in Figures 4 and 5, where it should be noted that the characteristic isotope pattern for gadolinium is evident in all the spectra. The relative natural abundances of gadolinium isotopes are 152Gd (0.2%), 154Gd(2.2%), 155Gd (14.8%), 156Gd (20.5%), 157Gd (15.7%), 158Gd (24.8%), and 160Gd (21.8%). The (M+H)+ ion (where M represents the 158Gd gadoteridol species) occurs at 560' in the positive ion spectrum, while the corresponding (M-

H)-ion is present at 558- in the negative ion spectrum. The remaining peaks are due to the thioglycerol used in the solvent system.

Figure 4. Positive ion, fast atom bombardment (FAB), mass spectrum of gadoteridol.

NAGWA H.FODA ET AL.

216

;

u_-

(00

alr

Figure 5 . Negative ion, fast atom bombardment (FAB), mass spectrum of gadoteridol. 3.4

Ultraviolet S-pectrum

The full UV spectrum obtained for gadoteridol (aqueous solution) is shown in Figure 6. The observed spectrum consists of a number of sharp transitions, each of which can be assigned on the basis of known spectral behavior [6]. The assignments are fully described in Table 11. Most of the absorption bands are too weak to be useful for analytical purposes, but the 8S--> 6P band system (270 - 280 nm) is sufficiently intense to permit its use. The distinctive triplet of absorption bands noted in this region can be used as an identity test for Gd3' in gadoteridol or its formulation. The most intense component of the triplet is observed at 274 nm, and its molar absorptivity is approximately 2.5 M-1cm-1.

1

f-

Wavelength (am)

Figure 6. Complete UV absorption spectrum of a 55 mM aqueous solution of gadoteridol.

FXUVOXAMINE MALEATE

217

Table II. Absorption Bands Observed for Gd3+in Gadoteridol

--->6D5/2

3.5

32,785

305.0

35,840

279.0

35,970

278.0

36,300

275.5

36,365

275.0

36,630

273.0

36,630

273.0

39,685

252.0

40,325

248.0

40,650

246.0

40,815

245.0

40,985

244.0

Luminescence Spectrum

Gd3+can be excited through the 8S--> 6P band system, and metal-centered luminescence can be observed. The luminescence spectrum of an aqueous gadoteridol solution is shown in Figure 7. The strongest emission band is observed at 3 1 1 nm, and corresponds to the 6P7/2 --> 8S7/2 transition [7].

w~veiengtb Cnm)

Figure 7. Luminescence spectrum of a 55 mM aqueous solution of gadoteridol. An excitation wavelength of 274 nm was used to obtain the spectrum.

218

NAGWA H. FODA ET AL.

The intensity of this luminescence is sufficient that it has been used as the basis of a variety of analytical methods. 3.6

Optical Activity

Gadoteridol contains one center of dissymmetry, and is therefore capable of being resolved into two enantiomers. The (S)-enantiomer is identified as SQ-33236, while the (R)-enantiomeris identified as SQ-34208. Gadoteridol is marketed as the racemic mixture of these, and therefore exhibits no optical activity. The circular dichroism (CD) spectra of SQ-33236 and SQ-34208 were obtained [8], and are shown in Figure 8. Owing to the very small molar absorptivity values, it was not possible to obtain the CD spectra within the intrinsic Gd” absorption bands. The CD spectra shown in Figure 8 correspond to the chirality within the carboxylate absorption bands of the chiral ligand, with the CD peaks being observed at 193 nm. As would be anticipated, the CD spectra of the enantiomers were mirror images of each other. The absolute value of the circular dichroism (A&) was found to be 0.146 M-lcm-l, while the absolute value of the molar ellipticity ([el) was found to be 481 degrees-cm2/decimole.

.P

,a

x..

W-rsl-ltb

-

zy



Figure 8. Circular dichroism spectra of the enantiomers of gadoteridol. Shown are data for SQ-33236 (the (S)-enantiomer) and SQ34208 (the (R)-enantiomer). 3.7

Crystalloeraphic ProDerties

The powder x-ray diffraction pattern of the 3.5-hydrate form of gadoteridol was obtained using a copper source (1.54060 A) [9], and is

FUJVOXAMINE MALEATE

219

reproduced in Figure 9. A total of 36 peaks were detected at scattering angles between 2 and 32 degrees 2-8. The two most diagnostic scattering peaks suitable for identification were observed at 8.7 degrees 2-8 (dspacing of 10.20A) and at 10.1 degrees 2-8 (8.75 A), although a very diagnostic series of scattering peaks was noted between 12 and 16 degrees 2-8. A summary of scattering angles, d-spacings, and relative intensities associated with the most intense peaks is found in Table III.

Figure 9. Powder x-ray diffraction pattern of gadoteridol. Gadoteridol does not appear to exhibit either polymorphism or pseudopolymorphismwhen crystallized from water/isopropanol mixtures. The anhydrous compound exhibited a similar powder pattern to that shown in Figure 9 for the 3.5-hydrate. When exposed to humidity values between 15 and 70%,gadoteridol exhibits a constant powder pattern. The structure of gadoteridol was determined by single crystal x-ray diffraction analysis [lo]. The space group was found to be P212121, with cell constants of a = 16.974 A, b = 25.45 A, and c = 11.247 A. The asymmetric unit was found to contain two crystallographically independent ennea-coordinate gadolinium complexes, and five partially occupied sites of hydration. These two structures are illustrated in Figure 10. In each molecule, the macrocyclic ring Gd(HP-D03A) adopts a quadrangular [3333] conformation, in which the four nitrogen and four oxygen donor atoms are coordinated to the central Gd3+ion. The ninth apical site is occupied by a water molecule in the capped square antiprism arrangement, and this finding has been confirmed using spectroscopic methods [ 113. The nitrogens are coplanar within experimental error, as are

220

NAGWA H.FODA ET AL.

Table 111. Powder X-ray Diffraction Data Obtained for Gadoteridol: Scattering Angles, D-spacings, and Relative Intensities Scattering Angle (degrees2-8) 8.6255 10.0800 10.9975 1 1.7625 12.5575 13.0775 13.5200 14.0475 14.8175 15.7750 16.7525 18.9275 19.2025 20.1950 22.6450 24.7500 25.1200 25.9600 26.3400 26.4200 26.9025 27.2375 27.8875 28.1000 28.3950 28.7800 29.6400 30.4200 30.8625 31.8000

D-spacing

(A, 10.2468 8.7682 8.0387 7.5175 7.0433 6.7644 6.5440 6.2994 5.9738 5.6133 5.2879 4.6849 4.6184 4.3936 3.9235 3.5943 3.5422 3.4295 3.3809 3.3708 3.3 114 3.2715 3.1967 3.1730 3.1407 3.0995 3.01 15 2.9361 2.8950 2.81 17

Relative Int. (IAomax) 100.00 97.75 17.58 9.7 1 43.19 37.44 14.84 51.37 28.36 12.74 7.40 9.89 18.30 18.06 7.40 31.14 8.03 12.74 11.75 10.61 30.52 36.75 19.78 23.19 17.82 24.86 22.38 8.68 11.36 13.56

FLUVOXAMINE MALEATE

22 I

the four coordinated oxygen atoms of the ligand arms. The Gd3+ion lies between these parallel planes, 1.61 %, above the nitrogen plane and 0.75 8, below the oxygen plane. The coordinated water molecule lies 1.72 8, above the oxygen plane, and an extensive hydrogen-bonded network joins the complexes with the water of hydration. It is of interest that the two independent complexes in the asymmetric unit have distereomeric conformation.

Figure 10. Independent complexes in the asymmetric unit of gadoteridol, as determined by single crystal x-ray diffraction.

3.8

Relaxivitv and Water of Hvdration

The catalysis of the relaxation rate of water protons of tissues is governed by a second-order rate constant called relaxivity (rl). The T i relaxivity, '1, of a paramagnetic metal ion was determined by the measurements of relaxation times of several Gd3+solutions at different concentrations of GdL at 20 MHz. The slope of a plot of l/T, vs. [Gd3+]gave the measured relaxivity, 2or1 . The 20r1 value for Gadoteridol is 3.7 mM-1s-1 in water [2]. The number of coordinated waters (Q) on corresponding Tb(II1)

NAGWA H. FODA ET AL.

222

chelates were determined by a literature procedure and the calculated value is 1.3 [ 111.

20 F

. t r

15

c

10

5

0 0

2

6

4

l o J [Gd (HP-D03A)],

M

Figure 11. Plot of 1R1 vs. [Gd(HP-D03A)]. Taken from ref. 2. 3.9

Thermal Analvsis

A typical differential scanning calorimetric thermogram of gadoteridol

was obtained at heating rate of 10"C/min [9], and is shown in Figure 12. The compound exhibits a poorly defined dehydration endotherm around loO°C, and a much sharper dehydration endotherm around 17OoC. The low temperature thermal event is undoubtedly associated with the loss of the network water, while the high-temperature endotherm is certainly due to the removal of the water coordinated directly to the Gd3+ion. Even when run under the same temperature ramping conditions as differential scanning calorimetry, the thermogravimetric analysis of gadoteridol does not differentiate between the two types of water bound in the crystalline solid [9]. As evident in Figure 13, the compound exhibits a gradual weight loss, which is only complete by 175OC. The anhydrous compound formed above this temperature exhibits no tendency for thermal decomposition, even when heated up to 3OOoC.

FLUVOXAMINE MALEATE

223

-0.4

75

125

175

275

Temperature (“C) Figure 12.

Differential scanning calorimetry of gadoteridol

-

h

100

Mass Loss (%)

9610.9% 92.

90

Figure 13.

Thermogravimetric analysis of gadoteridol.

NAGWA H. FODA ETAL.

224

Re1uIvo Humidity <%)

Figure 14.

3.1 1

Moisture uptake of gadoteridol. as a function of external relative humidity.

Microscopic Characterization

As evident in Figure 15, the component crystals of gadoteridol are orthorhombic in nature, and are normally long and rod-like in appearance [ 121.

The long crystal axis is often terminated in well-defined bi-capped faces.

3.10

Hveroscopicity

Gadoteridol samples were exposed to various relative humidity conditions (over a series of saturated salt solutions), after which the water content was determined using thermogravimetry 181. The water sorption isotherms are shown in Figure 14, where it is evident that a region of constant moisture uptake exists between relative humidity values of 15 to 70%. Averaging all the weight losses within these humidity values for both samples yielded an average weight loss of 10.17%. The calculated water content for a 3.5-hydrate species would be 10.14%, indicating that 7 waters of hydration are shared between two gadoteridol molecules. This conclusion is exactly what was deduced from the single crystal x-ray diffraction studies [ 101.

FLUVOXAMINE MALEATE

Figure 15.

Scanning electron photomicrograph of gadoteridol, obtained at (a) 150x and (b) 500x magnification.

225

NAGWA H.FODA ET AL.

226

3.12

Complex Formation Constants

Gadoteridol is formed by the complexation of Gd3' with the chelating ligand, HP-D03A. Both the ligand protonation constants of HP-D03A and the complex stability constant of gadoteridol have been determined [ 101. The ligand protonation constant (log K at 25.W.l0C and p= 0.1 (TMAC1) were determined from pH-potentiometric titrations and the values are: 11.96,9.24,4.43, and 3.48. The stability constant of Gadoteridol was determined by a spectrophotometric titration using Arsenazo-I11as the indicator. The stability constant of gadoteriodol is logK = 23.8, which is one of the highest formation constants measured for a lanthanide chelate species. A knowledge of the conditional stability constant at pH 7.4 is useful in the context of physiological conditions. The stability constant is reduced considerably due to the competition between hydrogen ions and the metal ion for the ligand (Fig. 16). A calculated

2

4

6

8

10

12

PH Figure 16. Effect of pH on the Stability Constant of Gd(HP-D03A). Taken from ref. 2. 3.13

Kinetic Inertness

Gadoteridol is kinetically very inert under physiological conditions [ 131. However, at lower pH, the chelate slowly equilibrates into free Gd3+ and the free ligand. For example, the half-lives of the chelate are 30 and 3 h at pH 2 and 1, respectively. A plot of k b s d vs. [H+] is shown in Fig 17.

FLUVOXAMINE MALEATE

30

227

t

20

10

0

I

I

0.4

0.8

1.2

W+l, M Plot of k b s d vs. [H+] for acid-assisted dissociation of Gd(HP-D03A). Reprinted with permission from ref. 13. Copyright 1993American Chemical Society. Another method to verify the kinetic inertness of a metal chelate is to study the exchange of metal ion with its metal chelate, The isotopeexchange reaction (1) was studied [ 141 and no exchange at pH 7.4 and 3.8 was seen within 14 days [14]. Low pH conditions are required to observe the acid-assisted exchange reaction of Gd(HP-D03A). Figure 17.

IS3Gd3+ + Gd(HP-D03A) <=====> '53Gd(HP-D03A)+ Gd3+ 3.14 Partition Coefficients

(1)

The partition coefficients of gadoteridol in butanoVwater and octanoVwater were determined by the traditional shake-flask method. The values of log P are: -3.68 in octanoVwater and -1.98 in butanouwater [ 151. In both instances, the aqueous phase was buffered to pH 7. The negative values obtained for the compound are consistent with the interpretation that gadoteridol is highly hydrophilic. 3.15

Solubility

Gadoteridol exhibits a wide range of solubilities in various solvents [8]. Gadoteridol was found to be freely soluble in water and methanol, soluble in isopropanol, and much less soluble in a variety of other neat solvents. The data are summarized in Table IV. The solution pH of aqueous gadoteridol solutions was not found to vary strongly with concentration. Over the range of 5 - 20 mg/mL, an average solution pH of 6.4 was observed [8].

228

NAGWA H. FODA ET AL.

Table IV. Solubility of Gadoteridol in Various Solvents Solvent Water Methanol Isopropanol Dimethylformamide Acetonitrile Methylene chloride Ethyl acetate Acetone Hexane Toluene 3.16

mg/mL 737 119 41 10.1 6.1 5.2 0.5 0.4 0.2 0.3

USP Definition Freely soluble Freely soluble Soluble Sparingly soluble Slightly soluble Slightly soluble Very slightly soluble Very slightly soluble Very slightly soluble Very slightly soluble

Conductivitv and Osmolalitv

The conductivity of gadoteridol solutions was measured to demonstrate the nonionic nature of the compound. The molar conductivity (measured in units of micro Siemens) was found to be 0.8, a value which is far below the ranges noted for either 1: 1 or 1:2 electrolyte species [ 161. The negligible conductivity indicates that gadoteridol does not dissociate into ions upon dissolution, and that it remains nonionic in aqueous solutions. The lack of ionic character was further studied through osmolality measurements. A 0.5 M solution of gadoteridol was found to exhibit an osmolality of only 0.6 Osmolkg H20 [ 161, which clearly places the compound in the nonionic category. 3.17

Viscositv and Specific Gravity

The viscosity of a formulated 0.5 M solution of Gadoteridol was measured by a rolling- ball method with the use of a PAR-200 microviscometer. The measured viscosity values [ 161 of the product are 2.0 and 1.3 CPat 20OC and 37OC, respectively. The viscosity at body temperature, 37OC, is very close to the viscosity of blood, which is 1.32 cP. The specific gravity of the formulated product is 1.140. 4.

Methods of Analysis

4.I

Elemental Analysis

The racemic mixture of the R- and S-isomer was synthesized by the literature procedure as given above. The elemental analysis was satisfactory as given below [ 101.

FLUVOXAMINE MALEATE

229

Analysis: Found (calculated) for C17H29N407Gd.1.3H20: C, 34.86 (35.07); H, 5.58 (5.48); and N, 9.77 (9.63) 4.2

Spectrophotometry

A qualitative and quantitative spectroscopic method for the identification of Gd" in Gadoteridol was developed. The characteristic UV absorbance spectra consists of a triplet of bands at 274,276, and 279 nm. This pattern of bands was used for qualitative identification of gadoteridol. The absorbance of the 274 nm peak (EE 2.5 M-1cm-l) was used to qualitatively evaluate the concentration of the product. 4.3

Atomic Absorbance Spectrometry

Gadoteridol samples were analyzed for the presence of trace metal ion concentrations, (e.g. Zn, Mn, Fe, Cu, and Cr) by Flame Atomic Absorption Spectrometry [17]. Figure 18 and 19 show correlation curves for Cu, Fe, Mn, and Cr and Zn, respectively. No trace metals were detected in the samples. The limits of detection (given in parenthesis as pg/mL) of these trace metals were as follows: Cu (2.5), Cr (5), Fe(2.5), Zn( 1.O), and Mn( 1).

Concentration, p g / d Figure 18. Correlation curves for Cu, Fe, and Mn

Concentration, pg/mL Figure 19. Correlation curves for Cr and Zn.

Total calcium in ProHance, was determined by a Flame Atomic Absorption Spectrometric method [17]. Figure 20 shows the calcium absorbance as a function of concentration from 0 to 2.5 pg/ml. A correlation coefficient of 0.99994 indicates good linearity over this range.

NAGWA H.FODA ET AL.

230

Figure 21 shows addition curve, i.e., response of assay concentration vs. added calcium concentration in the formulation. From this curve the concentration of total calcium was calculated as 30.3H.3 pg/mL (n=5), which can be compared with the theoretical calcium concentration in the formulation of 30.06 pg/mL.

[Calcium], @nL

I

I

10

20

7

1

Cohc. of Ca Added, p g / d

Figure 20. Plot of Calcium absorbance vs. concentration of Ca2+. Figure 2 1. Addition Curve for the determination of Ca2+.

4.4

Chromatopraphic 4.4.1

Thin-Layer Chromatomauhy

Thin-layer chromatography was evaluated to determine free Gd3+ in gadoteridol by the tracer technique [18]. The method is valid to determine free Gd3+ at the 0.1% level. In the method gadoteridol was first radiolabeled with 153Gd3+ by either the exchange method or by the truetracer radiolabeling method. Free gadolinium was precipitated as Gd(P0,) by addition of KH2PO4 (KspGd(Po4)= Then the sample was applied to an ITLC (Instant Thin Layer chromatography) plate and developed in 50% aqueous methanol containing 10% ammonium acetate. Free Gd3+ as Gd(P04), was found at the origin while gadoteridol moved near the solvent front. Mass transfer and poor recovery made this method unacceptable for determination of low concentrations of free Gd3+ in Gadoteridol.

FLUVOXAMINEMALEATE

23 1

4.4.2 High-Performance Liquid Chromatoeraphv 4.4.2.1

Determination of free Gd3+ in Padoteridol

Free Gd” in the sample of Gadoteridol was determined by complexing free Gd3+with EDTA, followed by separation of Gd(EDTA)-(rt = 2.9 min) from Gd(HP-D03A) (rt =10.8 min ) by an HPLC method [19]. This method utilizes a mobile phase consisting of 10 mM EDTA and 50 mM Tris acetate at pH 7.4 with 2%acetonitrile as an organic modifier. A C18 reversed-phase Nucleosil column was used. A fluorescence detection method with excitation and emission wavelengths at 274 and 3 11 nm was used. Using this method one can determine very low concentrations of free Gd3+as well as Gd(D03A) (rt = 5.5) and Gd(D0TA)- (rt = 4.6 min) as other possible impurities (Fig. 22). The limit of detection were determined as follows: 0.00066 m g / d of free Gd”, which correspond to 0.002% of Gd(HP-D03A) in the formulation. Similarly, the limit of detection for Gd(D0TA)- and Gd(D03A) were determined as 0.005% and 0.017%, respectively. In a research sample, the percentages of Gd3+,Gd(DOTA), and Gd(D03A) were determined as <0.002%, <0.004%, and 0.07%, respectively. 4.4.2.2

Determination of the Free Ligand bv HPLC

The determination of the free ligand, HP-D03A in Gadoteridol, involves complexation of HP-D03A by Cu(II), and subsequent UV detection (at 280 nm) of the copper complex. The linearity, reproducibility, and recovery of the method were found to be suitable for analysis [20]. The method utilizes a Hamilton PRP-X100, anion exchange column (15 x 0.41cm), mobile phase: 98.8%buffer (10 mM EDTA and 50 mM Tris acetate, pH 7.4), 1% acetonitrile, and 0.2% THF, UV-Vis detector at 280 nm and flow rate of 1.0 mUmin. In the method, copper acetate, in tris acetate buffer (no EDTA), is first mixed with Gadoteridol solution so that free ligand reacts with Cu(I1) to form Cu(HP-D03A). Unreacted Cu(I1) is then reacted with the EDTA (which is in excess) in the mobile phase. Excess Cu(II) in the form of Cu(EDTA)- and Free ligand in the form Cu(HP-D03A) are separated by HPLC with retention times of 4.5 and 2.3 min, respedtively (Fig. 23). As low as 0.002% (w/w) free ligand can be determined with good precision.

NAGWA H.FODA ET AL.

232

Cd (DOTA

21.031. 000

I

.

212

I 4.25

1,. 2

.

SQ 30626

I

6.35

.

I

8.50

.

I

10.53

.

I

12.76

.

I

.

14.52

RT in minutes

Fig. 22.

HPLC chromatogram to determine the concentration of free Gd3+ in a sample of Gadoteridol.

Fig. 23.

HPLC chromatogram of determination of HP-D03A in Gadoteridol.

4.4.2.3

Potencv Determination

An HPLC method of satisfactory linearity and reproducibility has been developed to obtain the identity, the characteristic retention time of the analyte, and potency of Gd(HP-D03A) in either gadoteridol or

17.01

FLUVOXAMINE MALEATE

233

ProHance. The HPLC conditions used are the same as used for the determination of free Gd3’in Gadoteridol. The fluorescence detection method with excitation and emission wavelengths at 274 and 3 11 nm, respectively, was used. First a linearity or calibration curve, peak area vs. [Gd(HP-D03A)], is established. The concentration of the unknown solution is calculated from the slope of calibration curve and peak area of unknown solution of gadoteridol. With the use of this method the concentration of ProHance was determined as 0.505+0.005 M. 4.4.2.4

Determination of Complexed Calcium in the Formulation

The HPLC method to determine total complexed calcium, or the concentration of Ca[Ca(HP-D03A)]2 ,involves the reaction of Ca[Ca(HPD03A)]2 with Cu2+ to form Cu(HP-DO3A). followed by separation of Cu(HP-D03A) from the unreacted Cu2+ . In the procedure, the reaction of known concentrations of CuC12 and Ca[Ca(HP-D03A)]2 is completed in 10 mM Tris.HC1 buffer at pH 4.0 in 1 h. Separation of Cu(HP-D03A) formed and unreacted CuC12 is accomplished by an HPLC method (Fig. 24). From the calibration curves of standards and the peak area of Cu(HP-D03A) formed in the unknowns, one can determine the concentration of Ca[Ca(HP-D03A)]2. The conditions of separation are: PLRP-S (15 x 0.46 cm) column, 99: 1 buffer:acetonirile (buffer being 50 mM NH4H2PO4 at pH 4.0, 1.O mL/min flow rate, and 270 nm UVNis detector. 161.56

h

lU.01

II

1..

0.00

1.2S

RT in minutes

2.50

3.16

5.01

6.26

Z.52

8.17

Figure 24. Determination of Ca” as Ca[Ca(HP-D03A)], in ProHance.

NAGWA H. FODA ET AL.

233

4.4.2.5

Determination of Tris in the Formulation

The concentration of Tris [Tris (hydroxymethyl)aminomethane]buffer in ProHance was determined using ion-pair reversed-phase HPLC with refractive index detection. The conditions used were: Partisil-5-ODs-3 (25~0.46cm) column, 50% aqueous methanol containing 7 mM Dodecanesulfonate at pH 5.0,1.O a m i n flow rate, and Shimadzu RID-6 refractive index detector. The concentration of Tris in the formulation was within 10051% of the concentration claimed on the packaging insert [21]. 4.4.3 Ion Chromatopraphv Simultaneous determination of sodium and potassium in gadoteridol were accomplished by an ion chromatography method [22]. The method employs a 25 cm Dionex HPLC-CS3 cation exchange column and an eluant of mixture of 12 mM HC1 and 0.5 mh4 D,L-2,3-diaminopropionic acid monohydrochloride (DAP.HC1). Detection was carried out by conductivity with chemical suppression. Since Gadoteridol is a neutral molecule, it gives a weak conductivity response and is not retained on the analytical column. Peak responses of sodium and potassium were found to be linear in the range of 0.21 to 10.9 ppm (w/v) (with correlation coefficient =0.999548) and 0.20 to 20.1 ppm (with correlation coefficient =0.999548), respectively (Fig. 25 and 26). The limit of detection (LOD) and minimum quantifiable limit (MQL) are: 0.01% and 0.05% (w/w), respectively. Samples of gadoteridol were analyzed and the percentages of Na+ and K+ were c 0.1 and c 0.05, respectively, for the sample in which KOH was used for pH adjustment.

Figure 25. Correlation of peak area with "a+].

FLUVOXAMINE MALEATE

235

I Figure 26. Correlation of peak area with [K+]. 5. Stability

5.1

Solid State Stability

The effect of temperature and humidity (-2OOC, 5OC, 22OC/80% relative humidity, 33OC and 40OC/75% relative humidity) was studied on the bulk material for a long period of time. The samples were analyzed, at a regular interval for 18 months, for appearance, color, and moisture by Karl Fischer titration, free Gd3+ level, potency and impurity index by HPLC and TLC. No significant changes in potency or in impurity levels were observed by either HPLC or TLC. These results indicate that Gadoteridol is reasonably stable to heat and heat/moisture conditions. The effect of light on the bulk material was also studied. The samples were stored in open petri dishes under 400 and 900 foot candles of fluorescent light and short wave UV light. Batches exposed to 400 and 900 foot candles of fluorescent light for a period of two months showed reasonable stability. However, batches showed some light sensitivity between two and three months under these conditions. All the batches exposed to short wave light showed degradation within the first month as indicated by decreased potency, increased free gadolinium levels and increased HPLC and TLC impurity levels. Thus, solid gadoteridol samples should not be exposed to light over extended periods of time [23].

NAGWA H. FODA ET AL.

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5.2

Solution Stabilitv

The stability and potency of gadoteridol was determined over a pH range of 6 to 9 by validating HPLC methods. Time dependent determinations of free HP-D03A ligand, free Gd%on, and the concentration of gadoteridol showed no signs of any dissociation or decomposition indicating that the product is stable in solution in this pH range within 22 days [24]. 5.3

Stabilitv in the Presence of Endogenouslv Available Ions

The extent of reaction of Gadoteridol in vitro with Cu2+ and Zn2+ in the presence of phosphate was determined 1251. Due to the presence of macrocycle in the chelate, Gadoteridol proved to be inert, while other agents reacted with Cu2+ and Zn2+ in the presence of 66 mM phosphate (Fig. 27) PO*”-

GdL

+ M <------>Gd + ML

where M = C u o r Zn

DTPA-BMA

Fig. 27.

5.4

DTPA

DOTA

HP-D03A

Reaction of Cu2’and Zn 2+ with Some Gd3+ chelate in the presence of phosphate in 30 min. First bar is for Cu(I1) and second for Zn (11).

In Vivo Stability

In vivo stability of Gadoteridol was measured in terms of residual free Gd3+ in mice post 14 days intravenous administration of radiolabeled Gadoteridol [26,27]. Consistent with greater kinetic inertia, Gadoteridol leaves very low (
100

-4 u

237

FLUVOXAMINE MALEATE

-

0.1% free Gd

Gd ( EDTA)

10

$

ei

2

Gd(NP-DOBA) Gd(DTPA-BMA)

1 -

ap

c

g

3

0.1

-

0.01

I

Gd (D03A)

I

Gd ( DTPA) Gd( DOTA) Gd (HP-DOJA)

I

1

I

I

I

5 rnin

60

I d

7d

14d

rnin

Time Post InJectlon

Fig. 28 5.5

Plot of % ID versus time for some Gd3+ chelates. Stabilitv in Biological Fluids

Measurement of relaxivity of Gadoeridol in rat serum was made as a function of time. Several concentrations of Gadoteridol were incubated at 37OC with rat serum. No change in the relaxivity over a period of 60 min suggested that the chelate does not have any reaction with biological components of rat serum. This also suggested that there is no significant dissociation of the product. [28}

6. Biological Studies 6.1

Tissue Distribution in Mice

The tissue distribution of Gadoteridol was studied following iv administration of 0.1 to 0.25 mmovkg 153Gd-labeled Gadoteridol to unanesthetized mice [26]. In one hour, approximately 90% material was excreted from mice. Greater than 97% of Gadoteridol was excreted into urine within 1 day. The primary route of excretion was found to be renal. 6.2

Pharmacokinetics

Eighteen normal male volunteers were studied at doses of 0.025-0.3 m o l l k g intravenous gadoteridol in phase I study, to determine safety and pharmacokinetics. Mean distribution half-life was 0.2 h and mean eliminatjon half-life was 1.6h [ 161.

NAGWA H.FODA ET AL.

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6.3

Toxicity

In mice, the acute intravenous LD50 was 11-14 mmol/kg, and in rats the minimal lethal dose was 10 mmolkg. In two week studies, no serious effects were observed in mice given 3 mmol/kg or in dogs given 1.5 mmol/kg daily. In vitro, 50 mh4 formulated gadoteridol showed no tendency to hemolyze human erythrocytes [ 161.

7. Acknowledgements The authors wish to thank the coworkers at Bristol-Myers Squibb Co. and Bracco Research USA, who allowed us to cite their unpublished work.

FXUVOXAMINE MALEATE

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8. References 1. R. B. Lauffer, "ParamagneticMetal Complexes as Water Proton Relaxation Agents for NMR Imaging: Theory and Design", Chem. Rev. 87,90 (1987). 2. K. Kumar and M. F. Tweedle, "MacrocyclicPolyamino Carboxylate Complexes of Lanthanides as Magnetic Resonance Imaging Contrast Agents", 65,515 (1993). 3. M. F. Tweedle, "Relaxation Agents in NMR Imaging" in Lanthanide Probe in Life. Chemical, and Earth Sciences, J. -C. G. Bunzli, G. R. Choppin, editors, Elsevier Publishing Co. Amsterdam, the Netherlands, 1989. 4. M. F. Tweedle, G. T. Gaughan, and J. H. Hagan, 1-Substituted -1, 4,7 -Tris Carboxymethyl - 1,4,7 -Tetraazacyclododecane and Analogs" US Patent # 4,885 363. 5. D. D. Dischino, J. F. Delaney, J. E. Emswiler, G. T. Gaughan, J. S. Prasad, S. K. Srivastava, and M. F. Tweedle, Synthesis of Non-Ionic Gadolinium Chelates Useful as Contrast Agents for Magnetic Resonance Imaging", Inorg. Chem. 30, 1265 (1991).

6. C.A. Morrison and R.P. Leavitt, "SpectroscopicProperties of Triply Ionized Lanthanides in Transparent Host Crystals", chapter 46 in the Handbook on the Physics and Chemistry of Rare Earths, K.A. Gschneider and L. Eyring, eds, North-Holland Pub., Amsterdam, 1982. 7. G. Stein and E. Wurzberg, "Energy Gap Law In the Solvent Isotope Effect on Radiationless Transitions of Rare Earth Ions", J. Chem. Phvs. 62,208 (1975). 8. H.G. Brittain, Unpublished Work.

9. S.J. Bogdanowich, Unpublished Work.

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10. K. Kumar, C. A. Chang, L. C. Francesconi, D. D. Dischino, M. Malley, J. Gougoutas, and M. F. Tweedle,"Synthesis, Stability, and Structure of Gadolinium(1II) and Yttrium(II1) Macrocyclic Poly(amino carboxylates)", Inorg. Chem. 33, 3567 (1994).

1 l.X. Zhang, C. A. Chang, H. G. Brittain, J. M. Garrison, J. Telser, and M. F. Tweedle, "pH Dependenceof Relaxivities and Hydration Numbers of Gadolinium (III) Complexes of Macrocyclic Aminocarboxylates", Inorg. Chem. 3 1,5597(1992). 12. J. DeVincentis, Private Communication.

13. K. Kumar, C.A. Chang, and M. F. Tweedle, "Equilibrium and Kinetic Studies of Some Lanthanide Complexes of Macrocyclic Polyamino carboxylates", 1norg.Chem. 32,587 ( 1993). 14. K. Kumar, A. Chang, and K. Sukumaran, Unpublished Work. 15. K. Kumar, K. Sukumaran, S. Taylor, C. A. Chang, A. D. Nunn, and M. F. Tweedle, "Partition Coefficients (log P), and HPLC Capacity Factors (k') of Some Gd" Complexes of Linear and Macrocyclic Polymino Carboxylates", J. Liquid Chromatomaphy, 17,3735, 1994. 16. M. F. Tweedle and V. M. Runge, "Gadoteridol" Drugs of the Future, 17, 187, 1992.

17.S.S.Black, N. S. Lewen, and P. S. Valatin, Unpublished Work. 18. K. Kumar, K. Sukumaran, C. Allen Chang, M. F. Tweedle, and W. C. Ekkelman, "True Tarcer Radiolabeling of Gadolinium Complex of 10(2- Hydroxypropy1)- 1,4,7,1O-tetraazacyclododecane1,4,7triacetic acid (HP-D03A)" J. Labelled Compounds and Radiopharmaceuticals 33,473(1993). 19. J. Hagan, S. Taylor, and M. Tweedle, "Fluorescence Detection of Gadolinium Chelates Separated by Reversed-Phase High-Performance Liquid Chromatography", Anal. Chem. 60,514(1988).

20. D. B. Whigan and A. Schuster, Unpublished Work. 21. S. M. Riseman, Unpublished Work.

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241

22. K. M. Varrichio, Unpublished Work. 23. S . Srivastava, Unpublished Work. 24. K. Kumar and C. A. Chang, Unpublished Work. 25. M. F. Tweedle, J.J. Hagan, K. Kumar, S . Mantha, and C. A. Chang, Reactions of Gadolinium Chelates in the Presence of Endogenously Available Ions”, Mag. Res. Imag. 9,409 (1991). 26. P. W. Wedeking, K. Kumar, and M. F. Tweedle, “Dissociation of Gadolinium Chelates in Mice: Relationship to Chemical Characterstics”, Mag. Res. Imag. 10, 641 (1992). 27. M. F. Tweedle, “Physicochemical Properties of Gadoteridol and Other Magnetic Resonance Imaging Contrast Agents”, Invest. Radiology, 27, S2, (1992) 28. Z. Ugwuneri and K. Kumar, Unpublished Report.

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GUAR GUM

Karen Yu, David Wong, Jagdish Parasrampuria, and David Friend

Cibus Pharmaceutical, Inc. 200 D Twin Dolphin Drive

Redwood City, CA 94065

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

243

Copyright 0 1996 by Academic Press. Inc. All rights of reproductionin any form reserved.

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1.

INTRODUCTION 1.1 History, Sources, and Manufacturing 1.2 Chemistry and Grades

2.

DESCRIPTION Name, Formula, and Molecular Weight 2.1 Appearance, Color, Odor, and Taste 2.2

3.

PHYSICAL PROPERTIES 3.1 Solubility 3.2 Surface Properties 3.3 Hydration 3.4 Viscosity 3.5 Crystallographic Characteristics Effect on the Crystallization of Water 3.6 3.7 Films

4.

METHOD OF ANALYSIS 4.1. Colorimetric Methods 4.2 Nuclear Magnetic Resonance 4.3 Chromatography 4.3.1 High Performance Liquid Chromatography 4.3.2 Gas Chromatogaphy

5.

STABILITY 5. I Thermal Degradation 5.2 Flow Degradation 5.3 Degradation by Irradiation 5.4 Electrolytes 5.5 Biodegradation 5.5.1 Microbial Growth 5.5.2 Colonic

6.

APPLICATIONS 6.1 Pharmaceutical 6.2 Chromatographic 6.3 Food Industry

7.

PHARMACOLOGICAL EFFECT AND TOXICITY

GUAR GUM

1.

INTRODUCTION

1.1

History, Sources, and Manufacturing

245

Guar gum is derived from the ground endosperm of the guar plant, Cyamopsis tetragonolobus, of the legumnosae family. This hardy, drought resistant plant has been cultivated for human and animal consumption in India and Pakistan, and was introduced to the semiarid regions of the southwestern United States in the early part of this century. The component which imparts its physical characteristicsis the galactomannan content. Galactomannans are water-soluble polysaccharides common to several types of legumes, including carob, guar, lotus pedunculatus, lucerne (Medicagosativa var. wairau), red clover (Trifoliumpratense var. montgomery),Sophorajaponica, and soybean (Glycine m a ) [l]. The use of carob goes as far back as the ancient Egyptians who prepared strips with which they bound their mummies using carob paste. Guar itself is derived from the Indian language Gua-ahar where ‘gau’ means cow and ‘ahar’ means food [2]. The commercial use of guar gum emerged as World War I1 brought about a shortage of carob, also known as locust bean gum, to the textile and paper industries in the United States. In commercial processing of guar gum, a variety of methods are utilized to efficiently separate the guar endosperm from the husk and germ. Methods may include multistage grinding, flame treatment, or wet-milling. The separated endosperm is ground to finer particle sizes and sold as guar gum. Variations in processing techniques affect the viscosity, rate of hydration and dispersion properties of the guar product. Guar gum was first put to use in the paper and textile industries. It served to improve paper strength and formation during bonding in the paper industry, and as a thickener in textiles. Since its approval as a food additive, this low-cost polysaccharide thickener has been extensively used in the food industry. Aside from thickening, guar gum is also used as a viscosity modifier, a binder of free water, a suspending agent stabilizer and a dietary fiber source. It can be found in such food products as ice cream, processed cheeses, cake mixes, salad dressings, meat products, and pet foods. In the pharmaceutical and cosmetic industries, guar gum has been utilized as a disintegrant and binder in tablet formulations, and as a thickener in lotions and creams. Other areas of industrial applications

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include mining, tobacco, explosives, water treatment, and petroleum drilling.

1.2

Chemistry and Grades

Guar gum is a galactomannan consisting of one galactose on every other mannose unit, yielding a ratio of approximately 2: 1 (mannose:galactose). As a natural product, guar gum contains a variety of impurities including: moisture, protein, acid insoluble residue, ether extractable fat, and ash. Typically, guar gum may contain 80% galactomannan, 12% water, 5% protein, 2% acid insoluble residue or crude fiber, 0.7% ash, 0.7% fat, a trace of heavy metals, zero arsenic, and zero lead. The amounts can differ from crop to crop. A variety of guar gum grades are available from several manufacturers and suppliers. Each supplier markets their products by certain brand names, such as Supercol' (from Aqualon) or Jaguar@(from Rhone-Poulenc). Guar gum may be produced as pharmaceutical, food or industrial grades, and may range in particle size from coarse to fine.

2.

DESCRIPTION

2.1

Name, Formula, and Molecular Weight

Guar gum is a polysaccharide consisting of a straight chain of Dmannopyranose units joined by p(1+4) linkages with a side-branching unit of a single D-galactopyranose unit joined to every other mannose unit by a-(1--A) linkages. The structure of guar g u m is shown in Figure 1. The side chain of galactose accounts for its cold water hydration as well as its hydrogen-bonding activity. It hydrates and swells rapidly in cold water forming a viscous colloidal dispersion or sol. Manufacturers of guar gum products state that the molecular weight of guar gum, on average, can range from 1 to 2 million. The literature has not come to the same conclusion, with some stating that the molecular weight falls around 250,000, while others report it to be in the millions [3]. A problem that arises is the lack of a sufficient standard for use in molecular weight

GUAR GUM

247

determination. Additionally, the molecular weights can vary from crop to crop, season to season.

Figure 1.

2.2

Segment of a guar gum molecule. The polysaccharide backbone is composed of Dmannopyranose units, while the pendant groups are D-galactopyranose units

Appearance, Color, Odor, and Taste

Guar gum is obtained as a granular powder, which can be off-white to very lightly yellow or green in color It is nearly odorless with a bland taste.

3.

PHYSICAL PROPERTIES

3.1

Solubility

Guar gum will disperse and swell almost completely in cold or hot water to form a viscous sol or gel. It is slightly soluble in water, but not in organic solvents. A variety of organic compounds are often used in the purification of guar, particularly when required for analytical purposes [4,51.

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3.2

Surface Properties

Both crude and purified guar gums are surface active, and have been shown to reduce the surface tension of water to 55 Nlm [ 6 ] . Since both crude and pure guar differ mainly in the protein content, the surface activity must be due to a non-protein component. The effect of concentration on the surface tension of water is illustrated in Figure 2. Galactomannans can be used as emulsifiers as well as stabilizers, where they can contribute to steric stabilization and to the depletion stabilization of oil-in-water emulsions.

3.3

Hydration

The hydration rate and optimum viscosity of guar is strongly affected by the galactomannan content, the size and structure of the polymer molecules, and the particle size distribution [7]. The rate of hydration is also affected by the presence of other hydrophilic substances, the availability of water, variations in temperature and pH, and mechanical agitation. The maximum hydration rate of guar takes place at pH 8.0, while the minimum is noted at 3.5. Guar solutions are usually stable over a pH range of 3.5 to 9.0, but other parameters such as temperature, ionic strength, and additional solutes may effect the overall stability [2]. Following ingestion, the type of meal eaten, the degree of mixing with food, and the degree of pre- and postprandial hydration can strongly affect the viscosity development in the stomach and small intestine [7].

3.4

Viscosity

The viscosity of aqueous solutions of guar gum is much higher than that of many common water-soluble polymers [8-1 11. It shows very pronounced shear thinning [7], namely a decrease in solution viscosity with an increased rate of movement. Solutions of guar are non-Newtonian in nature, exhibiting pseudoplastic behavior [ 121, where the viscosity varies inversely with temperature [2]. Hydrodynamic properties of various galactomannans have been reported by Sharman, et al. [ 13, who made measurements of viscosity as a function of shear rate, sedimentation, and diffusion coefficient on aqueous solutions of galactomannans. Average molecular weight calculations were made from sedimentation and

GUAR GUM

70 60

50

40

30 0.01

0.10

Log C (grldl)

Figure 2. Surface tension of locust bean gum (LBG) and guar gum solution at 25°C.

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KAREN YU ET AL.

250

diffusion coefficients using the Svedberg equation. The Mark-Houwink relationship between intrinsic viscosity and average molecular weight were found to hold for the galactomannans despite differences in mannose-to-galactose ratios. The dependence of intrinsic viscosity on average molecular weight is shown in Figure 3. Anincrease in the polymer concentration yields an increase in the viscosity of a solution to a greater extent than it decreases the diffusivity [ 131. The viscosity can also increase with increasing molecular weights of guar gum. Diffusion of bovine serum albumin was shown to decrease in the presence of increasing molecular weights of guar gum [14]. The viscosity of a guar solution can be altered to that of a gel-like structure by the addition of other gums (ie.,locust bean gum and carrageenan) [ 151. It has been recommended that when the replacement of guar gum is required in a formulation, a proportionate amount should be used based upon viscosity rather than on a dry weight to weight replacement [16].

3.5

Crystallographic Characteristics

X-ray diffraction has been used to study of the chain conformation and three-dimensional packing schemes of galactomannans [ 171. The unit cells of guar gum are orthorhombic, consisting of two chain segments of opposite polarity per unit cell. X-Ray diffraction data has also shown that the Q dimension of the unit cell is highly sensitive to relative humidity and galactose substitution [ 181. The measured dZoospacing corresponds to the crystallographic plane in which cohesion is maintained when water of hydration is added. Figure 4 demonstrates the change in the d,,, spacings of three galactomannans as a h c t i o n of relative humidity.

3.6

Effect on the Crystallization of Water

Guar has been shown to retard the rate of crystallization of water at -3°C [ 191. This particular characteristic forms the basis of the use of guar gum in the ice cream industry. 3.7

Films

Solutions of guar gum have been cast to form films which are brittle in nature [20].

GUAR GUM

25 1

1.5

1 .o

0.5

0.0

I

).5

I

I

I

4.0

4.5

5.0

I

5.5

I

6.0

log (b)

Figure 3.

The dependence of intrinsic viscosity, [q], on the average molecular weight, Mww.

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KAREN YU ET AL.

17

16

15

-

5.

14

C .-m 0

m

(r

rn

13

12

TG 11

10

I

10

I

20

1

30

I

40

I

50

I

60

I

70

I

80

1

90

Relative Humidity (%)

Figure 4.

Change in the dzoospacing of guar gum (GG), locust bean gum (LBG), and tara gum(TG) as a function of relative humidity.

GUAR GUM

253

4. METHOD OF ANALYSIS 4.1

Colorimetric Methods

Methods for determination of galactomannan content typically involve an extraction and purification step, followed by a gravimetric or colorimetric analysis. The crude gum is not homogenous, but rather consists of a mixture of galactomannansvarying in the mannose/galactoseratio and in the molecular weight [4]. Different extraction techniques will often result in fractions of varying galactomannan content, where the amount of galactomannan can be determined by a phenol-sulfuric acid assay [21]. Identification of individual sugars can be carried out by incubation with 0.5MH2S04for 8 hours at 100°C, followed by neutralization and analysis via gas-liquid chromatography.

4.2

Nuclear Magnetic Resonance

Pulsed field gradient spin-echo NMR was used to study the diffusional properties of water in guar solutions [22]. The diffusion coeficient of water in a non-gelling guar system was found to be independent of the polymer concentration and the same as that in water alone. Table 1 gives diffusion coefficients measured in guar solutions. NMR has also been used to study the interaction of guar and borate ions, a system characterized by the formation of strong gels [23,24].

4.3

Chromatography

4.3.1 High Performance Liquid Chromatography HPLC has been used to separate the galactose and manose components of guar seeds (4). A representative chromatograph is given in Figure 5. Another application of chromatography is that of size exclusion to determine the molecular weight of guar (3). Galactomannansbehave as a homologous series differing only in molecular weight (1). HPLC has also been used to evaluate the interaction of guar gum with food components such as ascorbic acid, niacin, caffeine and phenylalanine (25). H-bonding was found to occur with the -OH, -NH, or -COOH groups of all these compounds except caffeine, resulting in an exothermic absorption

KAREN YU ET AL.

254

Figure 5 .

Separation of galactose, mannose and myo-inositol (internal standard) by HPLC. The sample consisted of guar seed with free sugars removed.

255

GUAR GUM

process. Caffeine interacts in an endothermic absorption proposed to be related to hydrophobic and or electrostatic forces.

Table 1 Diffusion Coefficients Measured in Guar Solutions Guar Concentration (% w/w)

I

Diffusion Coefficient m2 s-')

2.5

2.34

3.0

2.34

3.5

2.42

4.0

2.27

4.5

2.39

5.0

2.23

4.3.2 Gas Chromatogaphy Gas chromatography has been used to determine the polysaccharides in gums (including guar gum), found in food products after hydrolysis and derivatization [26]. Foods such as dairy products, salad dressings, and meat sauces have been tested. The process involves hydrolysis with trifluoroacetic acid and conversion of the products to an aldonitrile acetate derivative. Monosaccharide patterns of hydrolyzed gums are shown in Figure 6.

5.

STABILITY

5.1

Thermal Degradation

As shown in Figure 7, Arrhenius plots of guar data at different concentrations show different rate constants, with the rate constants of

KAREN YU ET AL.

256

h

I

P Y

0

I

I

I0

20

Figure 6 .

.

I

I

30

I

I

I

i;

40

I0

20

30

40

I

Typical monosaccharide patterns, obtained from the GC analysis of hydrolyzed gums. Xylitol was used as the internal standard.

GUAR GUM

251

-1 3

-* C

-14

k

-15

0

\ I 0

0

\

-1 6

-1 7

2.6

2.7 103 x

Figure 7.

2.8

2.9

in( ~ - 1 )

Arrhenius plots for guar gum data: filled square=0.9%; filled circle=0.8%; open circle=0.7%; open triangle=0.6% and filled trangle=O.5 %.

KAREN YU ET AL.

258

guar gum depolymerization being dependent on galactomannan concentration. The temperature dependence has been shown to become weaker with increasing gaiactomannan concentration [271. In addition, the viscosity of guar solutions will vary inversely with temperature [2].

5.2

Flow Degradation

Shearing of guar solutions causes depolymerization to take place, leading to a decrease in the intrinsic viscosity and in the molecular weight [27]. This effect is illustrated in Figure 8. Depolymerization will also increase with growing wall shear stress. Table 2 gives molecular weight and intrinsic viscosity data for heat treated guar, highly and lightly sheared guar, and untreated guar 1273.

Table 2. Mofecular weight and intrinsic viscosity data

Molecular Weight (dmol)

Intrinsic Viscosity (dlk)

Native Guar

700,000

11.51

Heat Treated Guar (0.5%)

160,000

5.87

Highly Sheared Guar (0.8%)

450,000

6.7

Lightly Sheared Guar (0.8%)

5.3

9.4

Degradation by Irradiation

Gamma irradiation of guar powder has been shown to cause a decrease in viscosity of guar solution at room temperature and at 80°C for 1 hour [28]. It was proposed that low doses of irradiation lead to a decrease of polymer aggregation in solution, while a high dose of irradiation yielded

GUAR GUM

259

10 9

8

7 b

5!6 X

.-

b

W

5

Y

4 a

3

i

2 1

30

Figure 8.

u 40

50

60

70

The effect of the applied shear stress on the apparent rate constant of guar depolymerization. Data are shown for 0.5% (filled squares) and 0.8% (filled circles).

KAREN YU ET AL.

260

degradation by hydrolysis. Figure 9 illustrates the effects of gamma irradiation on guar gum viscosity.

5.4

Electrolytes

Because guar is non-ionic, it has good electrolyte compatibility. However, compounds such as borax, chromium, zirconium, calcium, and aluminum will precipitate guar [2]. Further applications of the insolubilized guar are not practical, since the pH and concentrations required are suitable for human consumption. The stability of a guar solution may be improved by adding sequestering agents such as citric, tartaric, or othophosphoric acids at 0.25 to 0.5%.

5.5

Biodegradation

5.5.1

Microbial Growth

Being a natural product, guar gum contains a resident microbial population that is monitored by manufacturers. At 25"C, the viscosity of guar gum will decrease significantly over 1 week. Food grade preservatives such as sodium benzoate, methyl and propyl parabens help to maintain molecular integrity [2].

5.5.2 Colonic The viscosity of a guar gum solution is reduced by 75% and the pH will fall significantly over 40 minutes when incubated with a homongenate of feces. Short chain fatty acids are generated, along with gases such as hydrogen, methane, and carbon dioxide [29,30]. Figure 10 shows the change in viscosity of guar as a function of incubation time [30]. These findings imply that guar gum can be fermented by fecal bacteria, which explains the flatulence that can occur after ingestion of a large amount of guar gum. The related compound xanthan gum (which possesses a similar p-( 1-+4) linked glucose backbone as guar), is not affected by fecal enzymes because of dense side chains which prevent access of the enzymes to its backbone 1301.

GUAR GUM

u 0.6

-

0.4

-

0.2

-

n

0

f

Figure 9.

261

I

Effect of gamma irradiation on the viscosity of a 1% guar gum solution: dispersion prepared at room temperature (open squares) and dispersion prepared at 80°C (open circle). The values were obtained 1h after preparation.

I

KAREN YU ET AL.

262

"1

nr

Control

?Qmirn

inltld

tat

ZIh

tat

Figure 10.

Cantml 30im Initial test

2lh M

Median initial control value, and intial and final test values for viscosity of the n=6 polysaccharides. The dashed line represents the viscosity of water measured by this system (2.0 mPa.s).

GUAR GUM

263

Heat-sterilized feces do not change the viscosity and pH of guar gum solutions. If bacteria-free filtrate is added to guar, the viscosity can decrease drastically, indicating the presence of an extracellular enzyme that can reduce the viscosity of guar gum [29,313. Table 3 summarizes the changes in pH and hydrogen concentrations produced upon incubation with feces at 37°C for 21 hours. Example strains of Bacteroides known to digest guar gum include B. fiagilis, B. ovatus, B. variabilis, B. uniformis, B. distasonis, and B. thetaiotaomicron [3 11.

Table 3 Changes in pH and hydrogen concentration produced by guar incubation with feces at 37OC for 21 hours

Components guar gum alone control untreated feces control untreated feces withguargum filtered feces with guar gum autoclaved feces with guar gum (extracted from reference 29)

6.

APPLICATIONS

6.1

Pharmaceutical Applications

PH initial 8.60 8.12 8.14 8.39 8.75

final 8.29 6.91 6.18 8.00 8.60

H, conc. (mL/L) 0.0 26.8 63.8 0.0 0.0

In the pharmaceutical industry, guar gum is used primarily as a disintegrating and binding agent in compressed tablets. As a disintegrant, guar gum has been found to be superior to some common disintegrants such as corn starch, celluloses, algins, and magnesium aluminium silicate [32]. From studies that have been conducted to evaluate the use of guar, it has been shown that an increase in guar can lead to decreased disintegrating ability [33], while elevated temperatures can increase binding characteristics [34]. Guar gum as a 1.5% solution in wet

264

KARENYU ETAL.

granulation is an effective binder [35], while at 10% in a base formulation it is a sufficient disintegrant [36]. At 3% and 5%, guar gum was shown to retard drug release better than other excipients such as acacia, Carbopol 934, sodium carboxymethylcellulose, and Explotab [37]. Although guar gum has been used as binder, its actual effect on the hardness of tablets is still unclear [35]. Particle size can affect disintegration, with finer particle sizes having greater disintegrating capabilities [38]. The reason for the superiority of guar as a disintegrant may be related to its strong affinity for water, with the finer grade guar being a more effective disintegrant due to a better distribution within the tablet. This coincides with the finding that disintegration correlates with water absorption rates [32]. Detracting from its usefulness as a disintegrant, however, is the tendency of guar to decrease the friability of tablets [34]. Guar gum has also been used to thicken liquid suspensions of over-the-counter antacid products, as well as various lotions and creams. Some research has also been conducted regarding the use of guar in gels for ophthalmic and skin infection preparations [39]. Many natural gums have been considered in sustained release formulations [40] and as bioadhesives [41]. Guar has been studied during the development of controlled release dosage forms [42-491, where it has been shown to increase the dissolution rate of water-insoluble drugs [8,9]. One study suggested that the release of drug from tablets is governed by the degree of branching in the guar gum [45]. One aspect of guar usage in controlled release formulations is its ability to act as a gel-former to retard drug release from tablets [46,47,49]. However, under certain conditions, the drug release can be accelerated by increasing the amount of guar gum [48]. It was found that the gel layer formed can depend proportionally on the ionic strength [42]. The gel layer formed by guar is not as thick as that of other water-soluble polymers such as arabic gum, sodium alginate, carrageenan, locust bean gum, tragacanth, pectin, methylcellulose, and hydroxpropylcellulose [8,9,11]. For sustained release Solbutamol, guar gum alone would not retard drug release satisfactorily and a certain amount of hydroxypropyl methylcellulose (HPMC) had to be added to the tablet for better results [44]. HPMC in combination with guar gum or karaya gum exhibited a potential for the two gums as release retarding material [50]. The dissolution profile (rotating basket assembly at 100 rpm) of isoniazid from karaya gum, guar gum, and hydroxypropyl methylcellulose is shown in Figure 11. Additionally, Bhalla found that combinations of HPMC, guar gum and ethylcellulose could provide tablets

GUAR GUM

265

80 -

Y

60-

3

-a

(II

2

.(II -

40-

CI

i

3

20 -

0

2

4

6

8

10

12

Time [h]

Figure 1 1

In vitro release of isoniazid from various formulations. The polymer used in Formulation A was guar gum, that of B was Karaya gum, and that in C was hydroxypropyl methylcellulose. The drug:polymer ratio was 1:1.5, and 1% talc and 2% magnesium stearate was added to each formulation.

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with suitable characteristics and appropriate drug release profiles for ketoprofen [Sl].

6.2

Chromatographic Applications

Guar gum has been used as an absorbent for enantiomer separation, cross-linked as a gel-filtration media for biopolymers, and as an ionexchanger-base [5]. As an enantiomer separator, guar purification with methanol and derivatization with quinine has been performed. The pair of cis -OH groups on each anhydroushexose unit confers strong dual Hbonding interaction between guar gum and other molecules. This property provides for weak chiral discrimination and suggests a premise for controlled release of bioactive materials. The addition of quinine enhances the resolution as a chiral selector.

6.3

FOOD INDUSTRY

One of the major users of guar gum is the food industry, whereit serves many purposes [2]. Guar efficiently modifies the behavior of water, acts as a stabilizer, minimizes friction and aids in processing and palatability of foods, and is a viscosity aid in the control of crystal size in saturated sugar solutions. Some of guar’s most common uses in foods are summarized in Table 4. With frozen desserts, guar imparts smoothness to ice cream by promoting small ice crystal development during freezing. Guar also contributes to the body of ice cream, improving the eating quality. In cottage cheeses, guar gum promotes curd integrity by friction reduction or lubricity which aids in processing. It also controls free water in finished products to promote good storage characteristics. As found in cheese products, guar has numerous applications. In cold packed cheese, guar eliminates weeping and creates a more uniform texture and flavor by controlling moisture and migration. The advantages of guar use in pet foods and meat products are the prevention of migration during storage, the maintenance of a homogenous state, and a reduced tendency for product voids in the can. Guar gum also creates a gloss or sheen to pet foods, as well as facilitates the removal from the can by reducing friction. In baked goods, guar imparts softness and yields a more moist product, allowing easier removal from molds and reducing crumbling. In packaged dry mixes, such as cake mixes, guar thickens and controls viscosity, as

GUAR GUM

261

well as improves mouthfeel, prolongs shelf life due to improved moisture retention, and permits freezing of finished cakes. Finally, in beverages and condiments, guar improves mouth feel, facilitates homogeneous dispersions, has a bland flavor, has appropriate hydration rate an viscosity, and is cost effective

Table 4

Food Applications of Guar Products

Examples

Dairy

ice cream, ice milk, sherbet, ices, low fat soft serve, milk shakes, cottae cheese, dressing, processed cheese, cheese dips

Pet foods

dry and canned pet foods

Baked goods

cakes, cake icing, cheese cake, pizza

Packaged dry mixes

cake mix, salad dressing mix, breadings, instant soups, instant snacks, instant cereal

Condiments

barbecue sauce, cocktail mix, relishes, taco sauce

Beverages

juices, nectar, syrups

7.

PHARMACOLOGICAL EFFECT AND TOXICITY

Guar gum has the ability to reduce the postprandial rise in blood glucose and lower cholesterol levels, especially in patients with Type I diabetes [52-601. There is a minimum or threshold amount of guar that is needed

268

KAREN YU ET AL.

for reducing the postprandial blood glucose rise, although larger doses do not show additional effects [61,62]. The higher the initial cholesterol level, the greater the reduction in cholesterol [63]. A similar effect of gum on the absorption of hydroxyproline has also been observed [64]. The exact mechanism for the hypocholesterolemic effect of guar gum is unknown, but there are three basic explanations. First, guar is believed to form a extremely viscous intra-luminal gel which act as a barrier to glucose or cholesterol diffusion, causing a delay in absorption [61,63,65701. At the same time, guar gum may reduce the gastric emptying rate [71-761. Ingestion of 30 g of guar was shown to increase the transit time by 5 1% in dogs [77]. A second explanation is the formation of a coat or a protective layer on the epithelial surface of the intestine by guar gum, slowing down the intestinal bulk phase diffusion [55,78,79]. This mechanism has also been used to explain the suggested use of guar gum as an anti-ulcer agent. These first two explanations are commonly accepted because an increase in elimination of bile acids and steroids in stool is observed in guar studies [53,64,80,81]. Additionally, the retardation effect on glucose absorption disappears when guar gum viscosity is destroyed by hydrolysis [61]. The final explanation involves the induction of the activity of HMG CoA reductase [82] and hepatic cholesterol-7-ahydroxylase [83] which act to reduce serum cholesterol levels.

Various formulations and methods of administration are available including powders, granules, capsules, biscuits and crispbread [52,55,60,84-86]. The selection of a formulation is important because it can affect patient compliance with regards to long-term utilization of guar. The effective dose of guar needed for a significant reduction in plasma total cholesterol anci LDL cholesterol is as high as 15 grams daily or 6 grams three times daily [54,55]. For better control of cholesterol in patients with type IIa hyperlipidemia, guar gum needs to be combined with a pharmacologic agent [87,88]. In addition to the above pharmacological effects, guar gum has been shown to reduce fecal mutagenicity. This is because guar gum contains a short chain fatty acid which produces butyrate. Butyrate is an antiproliferative and differentiating agent in cell culture lines [SS].

GUAR GUM

269

Long term use of guar gum is reported to have no adverse effects [90], but ingestion of large amounts of guar may lead to early satiety, bloating, flatulence, increased stool frequency [53], and a decrease in its hypocholesterolemic effect [59]. After guar gum is enzymatically degraded to low molecular weight galactomannan,it becomes a carbon source for intestinal bacteria such as Ruminococcus, Bifdobacterium and Bacteroides [91]. This accounts for the apparent increase in flatulence upon ingestion [92]. One study showed that intake of 20 grams of guar gum a day increased the fecal weight by 20%, explained by the increase water-holding capacity by the gel formation of guar [93]. In addition to

the increased fecal weight, guar gum dilutes large intestinal contents and can thus speed up transit times [89]. The viscous gel formation of guar gum may also interfere in the absorption of other pharmacologic agents and micronutients [84]. Finally, alcohol should be avoided during ingestion of guar gum, since it is a non-solvent of guar [84].

KAREN YU ET AL.

270

8.

REFERENCES

1.

W. R. Sharman, E. L. Richards, and G. N. Malcolm, Biopolymers, 17 (1978), 2817-2833.

2.

C. T. Herald, in “Food Hydrocolloids”, Vol. I11 (1982), M. Glickman (ed.), CRC Press Inc., pp. 171-184.

3.

H.G. Barth and D. A. Smith, J. Chromatogr., 206 (1981), 410-415.

4.

R. W. Hansen, S. M. Bymes, and A. D. Johnson, J. Sci. Food Agric., 59 (1 992), 4 19-421.

5.

R. Mathur, S. Bohra, C. K. Narang, and N. K. Mathur, J. Liq. Chrornatogr., 15 (1992), 573-584.

6.

N. Garti and D. R. Reichman, Food Hydrocoll. 8 (1994), 155-173.

7.

P. R. Ellis, E. R. Morris, and A. G. Low, Diabetic Med., 3 (1986), 490-491.

8.

T. Takai, K. Takayama, N. Nambu, and T. Nagai, Chem. Pharm. Bull., 32 (1984), 1936-1941.

9.

K. Takayama, N. Nambu, and T. Nagai, Chem. Pharm. Bull., 30 (1982), 673-678.

10.

K. Takayama, N. Nambu, and T. Nagai, Chem. Pharm. Bull., 30 (1982), 3013-3016.

11.

K. Takayama, N. Nambu, and T. Nagai, Chem. Pharm. Bull., 30 (1982), 3 70 1-37 10.

12.

N. Sarisuta and E. L. Parrott, Drug Dev. Ind. Pharm., 8 (1982), 605-616.

13.

N. Sarisuta and E. L. Parrott, Drug Dev. Ind. Pharm., 9 ( I 983), 861-875.

GUAR GUM

27 1

14.

P. Roger, C. Hoebler, T. Ficadiere, and P. Colonna, Food Hydrocoll., 5 (1991), 145-146.

15.

E. Costell, M. H. Damasio, L. Izquierdo, and L. Duran, Food Hydrocoll., 6 (1992), 275-284.

16.

I. A. Schlakman and A. J. Bartilucci, Drug Standards, 25 (1957), 149-154.

17.

K. Veluraja and E. D. T. Atkins, Carbohydr. Res., 183 (1988), 13 1-134.

18.

R. H. Marchessault, A. Buleon, Y. Deslandes, and T. Goto, J. Colloid Interface Sci., 71 (1979), 375-382.

19.

E. R. Budiaman and 0. Fennema, J. Dairy Sci., 70 (1987), 547554.

20.

P. Parashar, S.C. Datt, A. Jain, and U. D. N. Bajpai, Polymer Testing, 11 (1992), 185-192.

21.

M. Dubois, K. A. Gilles, J. K. Hamilton, P. A. Rebers, and F. Smith, Anal. Chem., 28 (1956), 350-356.

22.

E. Brosia, A. D’ubaldo, and B. Verzegnassi, Cell. Mol. Biol.,40 (1994), 569-573.

23.

0.Noble and F. R. Taravel, Carbohydr. Res., 166 (1987), 1-11.

24.

0. Noble and F. R. Taravel, Carbohydr. Res., 184 (1988), 236-243.

25.

A. L. Khurana and C . T. Ho, J. Liq.Chromatog.,13 (1990), 15591569.

26.

J. F. Lawrence, and J. R. Iyengar, J. Chromatogr.,350 (1985), 237-244.

27.

T. D. Bradley, A. Ball, S. E. Harding , and J. R. Mitchell, Carbohydrate Polymers, 10 (1989), 205-214.

272

KAREN YU ET AL.

28.

K. King and R. Graz, Food Hydrocoll., 6 (1993), 559-569.

29.

J. Tomlin, N. W. Read, C. A. Edwards, and B. I. Duerden, Br. J Nutr., 55 ( 1 986) 48 1-486.

30.

J. Tomlin, J. S. Taylor, and N. W. Read, Nutr.Rep. Int., 39 (1989), 121-135.

31.

J. Tomlin, C. A. Edwards, B. I. Duerden, and N. W. Read, Proc. Nutr. SOC.,45 (1986), 105A.

32.

A. M. Sakr and H. M. Elsabbagh, Pharm. Ind., 37 (1975), 457-459.

33.

E. M. Rudnic., C. T. Rhodes, J. F. Bavitz, and J. B. Schwartz, Drug Dev. Ind. Pharm., 7 (1981), 347-358.

34.

M. 2. Iqbal, M. Amin, and N. A. Muzaffar, J. Pharmacy, 1 (1979), 17-34.

35.

L. E. Eatherton, P. E. Platz, and F. P. Cosgrove, Drug Standards, 23 (1955), 42-47.

36.

W. Feinstein and A. J. Bartilucci, J. Pharm. Sci., 55 (1966), 332334.

37.

H. M. Elsabbagh, A. M. Sakr, and S. E. Abd-Elhadi, Pharmazie, 33 ( 1 978), 730-73 1 .

38.

A. M. Sakr and H. M. Elasbbagh, Pharm. Ind., 38 (1976), 732-734.

39.

N. Udupa and L. Bhat, Drug Dev. Ind. Pharm., 18 (1992), 21972205.

40.

S. K. Baveja, K. V. Ranga, and J. Arora, Znd. J. Pharm. Sci., 50 (1987), 89-92.

41.

M. A. Longer and J. R. Robinson, Pharmacy Int., May (1986), 114-117.

GUAR GUM

273

42.

P. J. Waaler, M. Anderson, C. Graffner, and B. W. Muller, Acta Pharm. Nord., 4 (1992), 167-170.

43.

H. E. Huber, and G. L. Christenson, J Pharm. Sci., 57 (1968), 164166.

44.

H. L. Bhalla, and Y. D. Sanzgiri, Indian J Pharm. Sci., 49 (1987), 22-25.

45.

S. K. Baveja, K. V. Ranga Rao, and J. Arora, Ind. J. Pharm. Sci., 30B (1991), 133-137.

46.

H. L. Bhalla and S . 0. Gulati, Indian Drugs, 24 (1987), 338-342.

47.

P. J. Waaler, C. Graffner, and B. W. Muller, Acta Pharm. Nord., 4 (1992), 9-16.

48.

P. J. Waaler, K. Arnesen, C. Graffner, and B. W. Muller, Acta Pharm. Nord., 4 (1992), 291-296.

49.

P. J. Waaler, C. Graffner, and B. W. Muller, Int. J Pharm., 87 (1922), 229-237.

50.

N. K. Jain, K. Kutkarni, and N. Talwar, Pharmazie, 47 (1992), 277-278.

51.

H. L. Bhalla and A. A. Shah, Indian Drugs, 28 (1991), 420-422.

52.

A. R. Khan, G. Y. Khan, A. Mitchel, and M. A. Qadeer, Am. J Clin. Nutr., 34 (198 l), 2446-2449.

53.

M. E. McIvor, C. C. Cummings, M. A. Van Duyn, T. A. Leo, S. Margolis, K. M. Behall, J. E. Michnowski, and A. I. Mendeloff, Atherosclerosis, 60 (1986), 7- 13.

54.

G. A. Spiller, J. W. Farqunar, J. E. Gates, and S . F. Nichols, Arterio. Throm., 11 (1991), 1204-1208.

55.

H. R. Superko, W. L. Haskell, L. Sawrey-Kubicek, and J. W. Farquhar, Am. J. Cardiol.,62 (1988), 51-55.

214

KAREN YU ET AL.

56.

L. A. Simons, S. Gayst, Balasubramaniam, and J. Ruys, Atherosclerosis, 45 (1982), 101- 108.

57.

J. W. Anderson, A. E. Jones, and S. Riddell-Mason, J. Nutr., 124 (1994), 78-83.

58.

H. Vorinen-Markkola, M. Sinisalo, and V. A. Koivisto, Am. J. Clin. Nutr., 56 (1992), 1056-1060.

59.

A. Aro, M. Uusitupa, E. Voutilainen, and T. Korhonen, Am. J. Clin.Nutr., 39 (1984), 9 11-9 16.

60.

D. J. A. Jenkins, D. Reynoids, B. Slavin, A. R. Leeds, A. L. Jenkins, and E. M. Jepson, Am. J. Clin. Nutv., 33 (1980), 575-581.

61.

I. Torsdottir, M. Alpsten, H. Anderson, and S. Einarsson, J. Nutr., 119 (1989), 1925-1931.

62.

R. C. Spiller, Pharmac. Ther., 62 (1994), 407-427

63.

L. M. Morgan, T. J. Goulder, D. Tsiolakis, V. Marks, and K. G. M. M. Alberti, Diabetologia, 17 (1979), 85-89.

64.

J. M. Gee, N. A. Blackburn., and I. T. Johnson, Br. J. Nutr., 50 (1 983), 2 15-224.

65.

I. T. Johnson and J. M. Gee, Gut, 22 (1981), 398.

66.

F. G. Robers, A. G. Low, S. Young, H. A. Smith, and P. R. Ellis, Proc. Nutr. SOC., 50 (1991), 72A.

67.

N. A. Blackbum, J. S. Redfern, H. Jarjis, A. M. Holgate, I. Hannings, J. H. B. Scarpello, I. T. Johnson, andN. W. Read, Clin. Sci., 66 (1984), 329-336.

68.

D. J. A. Jenkins, T. M. S. Wolever, A. R. Leeds, M. A. Gassull, P. Haisman, J. Dilawari, D. V. Goff, G. L. Metz, and K. G. M. M. Alberti, British Med. J., 1 (1978), 1392-1394.

GUAR GUM

215

69.

T. M. S. Wolever, D. J. A. Jenkins, R. Nineham, and K. G. M. M. Alberti, Br. J. Nutr., 41 (1979), 505-510.

70.

T. K. Ray, K. M. Mansell, L. C. Knight, L. S. Malmud, 0. E. Owen, and G. Boden, Am. J. Clin. Nutr., 37 (1983), 376-381.

71.

V. Tagliaferro, M. Cassader, C. Bozzo, E. Pisu, A. Bruno, S. Marena, P. Cavallo-Perin, L. Cravero, and G. Pagano, Diabete Metab., 11 (1985), 380-385.

72.

C. J. Leclere, M. Champ, J. Boillot, G. M. Guille, G. Lecannu, C. Molis, F. Bornet, M. Krempf, J. Delort-Laval, and J. P. Galmiche, Am. J. Clin. Nutr. (Suppl.), 59 (1994), 7768.

73.

E. Harju, Chirurgia Intaliana, 37 (1985), 139-147.

74.

S. Holt, R. C. Heading, D. C. Carter, L. F. Prescott, and P. Tothill, Lancet, 24 (1979), 636-639.

75.

S. J. French and N. W. Read, Am. J. Clin. Nutr., 59 (1994), 87-91.

76.

H. Kasper, C. Eilles, C. Reiners, and J. Schrezenmeir, Hepatogastroenterol, 32 (1985),69-71.

77.

L. Bueno, F. Praddaude, J. Fioramonti, and Y. Ruckebusch, Gastroenterology, 80 (198 l), 701-707.

78.

C. D. Jensen, G. A. Spiller, J. E. Gates, A. F. Miller, and J. H. Whittam,J. Am. Coll. Nutr., 12 (1993), 147-154.

79.

B. S. Katoch, J. S. Chawla, and A. Rekib, Indian Vet. J., 48 (1971), 142-146.

80.

P. Ebeling, H. Yki-Jarvlnen, A. Aro, E. Helve, M. Sinisalo, and V. A. Koivisto, Am. J. Clin. Nutr., 48 (1988), 98-103.

81.

U. Smith and G. Holm, Atherosclerosis, 45 (1982), 1-10.

82.

C. Moundras, S. R. Behr, C. Demigne, A. Mezur, and C. Remesy, J. Nutr., 124 (1994), 2179-2188.

276

KAREN YU ET AL.

83.

P. D. Overton, N. Furlonger, J. M. Beety, J. Chakraborty, J. A. Tredger, and L. M. Morgan, Br. J Nutr., 72 (1994), 385-395.

84.

P. A. Todd, P. Benfield, and K. L. Goa, Drugs, 39 (1990), 917928.

85.

P. R. Ellis, T. Kamalanathan, F. M. Dawoud, R. N. Strange, andT. P. Coultate, Eur. J. Clin. Nutr., 42 (1988), 425-435.

86.

H. S. Fuessl, G. Williams, T. E. Adrian, and S . R. Bloom, Diabetic Med., 4 (1987), 463-468.

87.

D. J. A. Jenkins, A. R. Leeds, B. Slavin, J. Mann, and E. M. Jepson, Am. J. Clin. Nutr., 32 (1 979), 16-18.

88.

H. R. Superko, Curdio. Rev. Rep., 6 (1985), 1253-1265.

89.

S. A. Bingham, Proc. Nutr. Soc., 49 (1990), 153-171.

90.

M. E. Mclvor, C. C. Cummings, and A. I. Mendeloff, Am. J. Clin. Nutr., 41 (1985), 891-894.

91.

T. Okubo, N. Ishihara, H. Takahashi, T. Fujisawa, M. Kim, T. Yamamoto, and T. Mitsuoka, Biosci. Biotech. Biochem., 58 (1994), 1364-1369.

92.

T. M. S. Wolever, P. T. Wal, P. Spadafora, and P. Robb, Am. J. Clin. Nutr., 55 (1 992), 7 19-722.

93.

H. Takahashi, N. Wako,T. Okubo, N. Ishihara, J. Yamanaka, and T. Yamamoto, 3: Nutr. Sci. Vitaminol., 40 (1994), 251-259.

MAFENIDE ACETATE

Alekha K. Dash1 and Shankar Saha2

(1) Department of Pharmaceutical and Administrative Sciences

(2) Department of Biomedical Sciences Creighton University Omaha, NE 68178

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

277

Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

ALEKHA K. DASH AND SHANKAR SAHA

278

CONTENTS 1.

History and Therapeutic Properties

2.

Description 2.1 Nomenclature Formula, Molecular weight, and Structure 2.2 2.3 Elemental'Composition 2.4 Appearance, Color, and Odor 2.5 Pharmaceutical Dosage Form

3.

Synthesis

4.

Physical properties 4.1 Infiared spectrum 1H Nuclear Magnetic Resonance Spectrum 4.2 13C Nuclear Magnetic Resonance Spectrum 4.3 4.4 Ultraviolet Absorption Spectrum 4.5 MassSpectrum 4.6 Thermal Analysis 4.7 Melting Point 4.8 Solubility and Partition coefficient 4.9 X-Ray Powder Diffraction

5.

Methods of Analysis 5.1 IdentificationTests 5.2 Spectrophotometry 5.3 Chromatographic Methods 5.3.1 Thin-Layer Chromatography 5.3.2 High Performance Liquid Chromatography

6.

Stability

7.

Pharmacokinetics

8.

Toxicity

9.

Acknowledgments

10.

References

MAFENIDE ACETATE

1.

279

History and Therapeutic Properties

Mafenide is a synthetic anti-infective, which is closely related to sulfonamides in its chemical composition. It differs structurally from the sulfonamides in that it contains a methylene group between the benzene ring and the amino nitrogen of the basic sulfonamide structure. Mafenide is normally obtained as the acetate salt.

In 1939, Klarer [ 11 first applied for a German patent for mafenide acetate. The drug is commercially available in a water-miscible cream formulation. This sulfonamide is not inactivated by p-aminobenzoic acid, or by pus and serum. It is effective when applied topically in the prevention and treatment of bacterial infection associated with second and third-degree burns. Mafenide acetate has a broad spectrum of antibacterial activity and is effective against both Gram-positive and Gram-negative organisms, as well as clostradia [2]. It is also reported to be active against Pseudomona aeruginosa [3], but has no activity against fungi and viruses. The action of mafenide is primarily bacteriostatic. Unlike other sulfonamides, it does not compete with the essential bacterial metabolite, p-aminobenzoic acid. This suggests that its activity may be due to either analogous competition with another metabolite, or to an entirely different mechanism [2]. Even though the exact mechanism of the antibacterial activity of mafenide is not yet known, Harrison et al. [4] have shown that a marked inhibition of protein synthesis occurs at the drug concentrations which are used in the treatment of burn wounds.

2.

Description

2.1

Nomenclature Chemical Name:

Benzenesulfonamide, 4-(aminomethyl)monoacetate a-amino-p-toluenesulfonamidemonoacetate

Genekic Name:

Mafenide Acetate

280

ALEKHA K. DASH AND SHANKAR SAHA

Trade Names:

14-Homosulfanilamide, Maphenide, Marfanil, Mesudrin, Mesudin, Sulfamylon, Homosulfamine, Ambamide, Neofamid, Septicid, Emilene, Homonal, Paramenyl.

CAS Registry Number:

2.2

13009-99-9

Formula and Molecular Weight

C ~ H ~ O N ~ O ~ S . C ~(MW H ~=O246.29) ~ Structure:

CH2NH2

2.3

Elemental Composition

The elemental composition of mafenide acetate, based on the theoretical composition of C7H1oN202S.C2H402, is calculated as: C = 43.89% H = 5.68% 0 = 25.98% N = 11.37% S = 13.02%

2.4.

Appearance, Color, and Odor

Mafenide acetate is obtained as a white, crystalline powder from alcohol.

MAFENIDE ACETATE

2.5.

28 1

Pharmaceutical Dosage Form

Mafenide acetate is available only as a cream formulation containing 11.2% w/w of mafenide acetate. The excipients present in this formulation are methylparaben, propylparaben, sodium bisulfate, and disodium edetate.

3.

Synthesis

Several syntheses of mafenide acetate have been reported in the literature [5- 101. Miller et al. have synthesized mafenide acetate starting frompcyanobenzenesulfonamide[5]. This compound was dissolved in ethanolic hydrochloric acid, and hydrogenated using Pdcharcoal as a catalyst to yield the hydrochloride salt of mafenide. The hydrochloride salt was then dissolved in water, and ammonia added. The liberated base was collected, and reacted with acetic acid to give mafenide acetate. This procedure has been illustrated in Scheme 1. Bergeim and Baker [7] have also synthesized mafenide following a procedure similar to that described by Miller et al. [6]. Angyal and Jenkin [101were able to synthesize mafenide in a four step procedure, characterized by an overall yield of 11%. Commercially availablep-toluene sulfonyl chloride was first chlorinated using a stream of chlorine at 160°C for 10 hours, to yieldp-chloromethylbenzene sulfonyl chloride in 23% yield. Amination of this chlorinated product in alcoholic ammonia gave the correspondingp-chloromethyl benzene sulfonamide in 86% yield. The conversion of this compound to its quaternary hexaminium salt was carried out in chloroform at 96%yield. Finally, desaltation of the salt in refluxing alcohol and concentrated hydrochloric acid gave mafenide an 85% yield from step 3. This procedure has been illustrated in Scheme 2.

282

ALEKHA K.DASH AND SHANKAR SAHA

Scheme 1 Synthesis of mafnide acetate, according to the procedure of Miller, Sprague, Kissing, and McBurney [5]

MAFENIDE ACETATE

283

Scheme 2 Synthesis of mafnide acetate, according to the procedure of Angyal and Jenkin [lo]

CH2C1

23%

SOZCl

HC1 ______)

EtOH, 85%

86%

SO2CI

284

ALEKHA K. DASH AND SHANKAR SAHA

4.

Physical properties

4.1.

Infrared Spectrum

The infrared spectrum of mafenide acetate was obtained in a potassium bromide disk (0.5% w/w) using FTIR methodology, and is shown in Figure 1 [ 1 13. The band assignments are found in Table 1.

4.2.

*H Nuclear Magnetic Resonance Spectrum

The 200 mHz proton nuclear magnetic resonance spectrum of mafenide acetate was obtained at room temperature using deuterated methanol as the solvent [ 111, and is shown in Figure 2. The chemical shifts, multiplicities, and the peak assignments of the characteristic proton resonances are summarized in Table 2 (relative to trimethylsilane), and make use of this atomic numbering system:

4.3.

l3C Nuclear Magnetic Resonance Spectrum

The 200 mHz 1% nuclear magnetic resonance spectrum of mafenide acetate was obtained in deuterated methanol at room temperature [ 1 11, and is shown in Figure 3. The chemical shifts and assignments for the carbon resonances are given in Table 3.

I

4000

I

-0

1

I

I

I

I

1

3000

2800

2000

i600

too0

600

WAVENUMBER

(cm-

Figure 1. Infrared Spectrum of Mafenide Acetate

286

ALEKHA K. DASH AND SHANKAR SAHA

Table 1 Infrared Spectral Assignments for Mafenide Acetate

Energy (cm-’)

Assignment

3550-3000

N-H stretching (s, br)

1542, 1519

N-H bend (scissoring)

1413

CH, (scissoring)

1319

C-N stretching

1143

Sulfonamide group

1090

N-H wagging

(br) = Broad (s) = Strong intensity

..................................

9.0

8.5

8.0

7.5

7.0

6.5

I - - -

6.0

5.5

5 -I-"

Figure 2. Proton Magnetic Resonance Spectrum of Mafenide Acetate in Deuterated Methanol

288

ALEKHA K. DASH AND SHANKAR SAHA

Table 2

'H NMR Spectral Assignments for Mafenide Acetate

Chemical Shift

Multiplicities

Number of

Assignments

protons

(p.m.)

8.0

2

Aromatic protons (475)

2

7.55

Aromatic protons (Z6)

2

4.12

Amino protons connected to sulfoxide

1.55

S

3

methyl protons of acetate

d = doublet, s = singlet

Figure 3. I3C NMR Spectrum of Mafenide Acetate in Deuterated Methanol

ALEKHA K. DASH AND SHANKAR SAHA

Table 3 13CN M R Chemical Shift Assignments for Mafenide Acetate

Chemical shift

Assignments

(P.rn.1

143, 140

QuaternaryAromatic Carbons (1%4)

131

Aromatic Carbons

(33 129

Aromatic Carbons (2,6)

MAFENIDE ACETATE

4.4.

29 1

Ultraviolet Absorption Spectrum

The ultraviolet absorption spectrum of mafenide acetate was obtained at a concentration of 1 mg/mL in both water and in methanol [1I], and the resulting spectra are shown in Figure 4. The absorption maxima found between 220-267 nm demonstrates the presence of an aromatic ring in the molecule. The bathochromic shift in absorption maximum noted on passing fiom methanol to water is consistent with the usual trend where more polar solvents move n+n* bands to longer wavelengths. The solvent trend therefore provides a strong assignment for the nature of the observed band as being associated with the aromatic chromophore.

4.5.

Mass Spectrum

Using a gas chromatographic interface, mafenide acetate was introduced into a Finnigan INCOS-SOB quadrupole mass spectrometer, and the mass spectrum obtained using electron impact (electron energy of 70 eV and a source temperature of 18OOC) [111. The spectrum obtained using this procedure is shown in Figure 5. A (M-59) molecular ion peak (at m/e = 187) was observed, and other fragment assignments are given in Table 4.

4.6.

Thermal Analysis

The differential scanning calorimetry @SC) thermogram of mafenide acetate is shown in Figure 6a. The sample was heated under a nitrogen purge from 30-250°C in a nonhermetically crimped aluminum pan at a rate of 10°C/minin a Shimadzu model DSC-50 thermal analysis system. Thermogravimetric(TG) analysis of mafenide acetate was conducted using a Shimadzu model TGA-50 system, and the TG thermogram obtained on a sample heated at a rate of 1O"C/min is shown in Figure 6b. DSC and TG thermograms of mafenide samples heated at 5"C/min are shown in Figure 7, while the DSC thermogram of mafenide acetate heated from 30-250°C in a hermetically crimped pan (at a rate of 10°C/min)is shown in Figure 8.

292

ALEKHA K. DASH A N D SHANKAR SAHA

0) U C

m

20

In

n

a

260.0

Wavelength (nm)

308.8

a

U C

tu

f0 v)

a

a

3ee .0

288.8

Wavelength (nm)

Figure 4. Uttraviolet Spectra of Mafenide Acetate; (a) in Water and (b) in Methanol

1

1 187

89

122

1

48

68

88

141

,~,

356

l h 128 148 168 108 288 228 248 268 288 388 328 340It368 p

-

Figure 5. Mass Spectrum of Mafenide Acetate Electron Impact

294

ALEKHA K. DASH AND SHANKAR SAHA

Table 4 Mass Spectral Data for Mafenide Acetate

Fragment Ion

mle

P~(SO~NH~)CH~NHSN+

187

PhCH2NH

106

PhCH'

89

Ph

77

MAFENIDE ACETATE

osc mY/n 1.0

295

TG4

-

x 130 30 130.30

'

//--

0330 03.30

0.3 .B3 53 .B3.53

75 30 70.30 -1.0

60.30 63 30

-2.ou

I

3.50

jO.3C I

t

I

200.30

130.30

Temperature ("C)

Figure 6. (a) Differential scanning calorimetry, and (b) thermogravimetry thermograms of mafenide acetate, obtained at a heating rate of 10"Clminute.

A

296

ALEKHA K. DASH A N D SHANKAR S A W

DSC

TGA

10.30

1.30

1.33

1.30

1.90

1.30

3.30

200.00

100 .30

Temperature ("C)

Figure 7. (a) Differential scanning calorimetry, and (b) thermogravimetry thennograms of mafenide acetate, obtained at a heating rate of 5"Clminute.

MAFENIDE ACETATE

297

f

1.30.

,

3.30.

-1 00.

-2.90.

-.:.do 4 ‘

1

I

,

I

I

,

*

I

Temperature (“C)

Figure 8.

Differential scanning calorimetry thermogram of mafenide acetate, obtained at a heating rate of 10OClminute in a hermetically sealed pan.

I

298

ALEKHA K. DASH AND SHANKAR SAHA

The DSC thermogram consisted of two endothermic peaks within the range of 160-200°C. Thermomicroscopicstudies confirmed that the first endothermic feature (at 169°C)was due to compound melting. Thermomicroscopic studies conducted on sample immersed in silicone oil did not show any liberation of any bubbles, which indicates that any weight loss which accompanied sample heating was not due to a dehydration mechanism.

4.7.

Melting point

The reported melting range of mafenide acetate is 151- 152"C[121. In the present work, the melting point of mafenide acetate was observed to take place at approximately 167°C [I I].

4.8.

Solubility and Partition Coefficient

Mafenide acetate has been reported to be freely soluble in water and methanol [ 131. Using the partition coefficient computational program produced by Advanced Chemistry Development, Inc., (Toronto, Canada), it was would be -0.80 k 0.23. This value is consistent with deduced that log the observed solubility data where the compound is freely soluble in polar media, but apparently not soluble in non-polar media.

4.9.

X-ray Diffraction

Mafenide acetate was determined to be quite crystalline, yielding the powder diffraction pattern shown in Figure 9 [ 111. The powder pattern was obtained using a wide angle x-ray dieactometer (model X D S 2000, Scintag). The calculated d-spacings for the dieaction patterns are provided in Table 5 .

v

0

I n m

0

m

N

In

0

N

5

0

r(

ln

ai

Figure 9. X-Ray Diffraction Pattern of Mafenide Acetate

ALEKHA K. DASH AND SHANKAR SAHA

300

Table 5

P wd

X-Ray Diffraction Data for Mafenide Acetat

?

Relative Peak

d-spacing

Intensity

No

(A)

(%)

1

5.92

32.8

2

5.67

100

3

4.96

35.3

4

4.87

48.0

5

3.84

44.8

6

3.75

43.8

7

3.68

39.7

8

3.21

27.8

9

3.12

26.8

10

2.77

25.9

11

2.68

58.2

12

2.42

34.3

13

2.38

21.4

14

2.30

76.1

MAFENIDE ACETATE

5.

Methods of Analysis

5.1

Identification

301

5.1.1 Infrared absorption: The infrared spectrum of mafenide acetate, obtained in a potassium bromide disk, can be used for identification of the drug substance. The compound will exhibit peaks at 1592,13 16,1299, 1151, 1089, and 897 wavenumbers [141. 5.1.2 Thin-Layer Chromatography: Mafenide acetate can also be identified by a thin layer chromatographic method, which is conducted according to the method described in section 5.3.1 of this profile. The Rf value of the compound under investigation must correspond with that of the USP standard run under the identical conditions 5.1.3 Color Test: Mafenide acetate produces a violet color reaction when reacted by the Koppanyi-Zwikker test [ 141. The sample is dissolved in 1 mL of ethanol. Addition of one drop of a 1% wlv solution of cobalt nitrate in ethanol, followed by 10 pL of pyrrolidine with slow agitation, produces the violet color.

5.2

Spectrophotometry

Ten mL of a 2 mg/mL mafenide solution in water is transferred into a 100mL volumetric flask containing 1 mL of 1N HC1, and diluted to volume with water. Mafenide acetate reference standard solution (200 pg/mL) is prepared by dissolving USP reference standard mafenide acetate in 0.01N HCl. The amount of mafenide acetate in the unknown solution is quantitated by measuring absorbances of the unknown and standard solutions at 267 nm [131. 5.3

Chromatographic Methods

5.3.1 Thin-Layer Chromatography The purity of mafenide in pharmaceutical formulations can be quantitated by the TLC method described in the USP [13]. A 50 mg/mL mafenide

302,

ALEKHA K. DASH AND SHANKAR SAHA

acetate solution is prepared in methanol, and 5 pL of this solution is applied to a chromatographic plate coated at 0.25 mm with a silica gel mixture. The chromatogram is developed in a chamber containing a mixture of ethyl acetate, methanol and isopropyl m i n e (77:20:3 v/v). The plates are air dried, and examined under short-wavelength ultraviolet light. A TLC method has also been reported by Harrison et al. [4], and was used to determine the pharmacokinetics of mafenide acetate in rats. One microliter of the sample was applied to a plate coated at 250-pm with silica gel, and separated by ether: isopropylamine:methanol(94:3:3 v/v). The spots were identified by their fluorescence after excitation at 254 nm.

Steyn has reported the use of a TLC method for the analysis of mafenide in biological fluids, which uses a small volume of deproteinating serum in the method [ 151. The supernatant is spotted on the plates, and fluorescamine is reacted with the spots to produce a more intense fluorescence. The relative intensities of the unknown fluorescence and standards fluorescence are used to calculate the concentration of mafenide in the unknown sample.

5.3.2 High Performance Liquid Chromatography Dash and Harrison have reported an ion-pair chromatographic method for the analysis of mafenide acetate in pharmaceutical formulations [ 161. This method used a C column, and the detection is based on the UV absorbance at 270 nm. The mobile phase consists of 65:35 v/v methanolphosphate buffer @H 5.0), and also contains 2 mM of 1-heptane sulfonic acid as an ion-pairing agent. The mobile phase flow rate was maintained at 1 mL/min.

6.

Stability

Commercially available mafenide acetate cream should be stored in tight, light resistant containers, and kept away from excessive heat [ 131.

MAFENIDE ACETATE

7.

303

Pharmacokinetics

Mafenide acetate is unsuitable for systemic antibacterial therapy because of its rapid inactivation in blood. Harrison et al. have reported the absorption and metabolism of mafenide acetate in Sprague-Dawleyrats [4]. The absorption of the drug, when applied to burns, is greatly reduced by the layer of lipid and protein normally present on the rat skin. Removal of this layer produced a 16-fold increase in the peak concentration within one hour. The drug reached the subcutaneous tissue muscle (2-3 mm from the site of application) within 30 minutes. Following absorption, mafenide acetate is rapidly metabolized to p carboxybenzene sulfonamide, and is excreted in the urine [171. This metabolite has no antibacterial activity and is a weak carbonic anhydrase inhibitor. Organ clearance has been reported to be logarithmic in rats, with a 10 minute half-life for kidney tissue. Approximately 97% of the drug is excreted in the urine within 24 hours. Mafenide and its metabolite inhibit carbonic anhydrase, reduce the renal tubular buffering mechanism in maintaining normal body pH, systemic acidosis, increase bicarbonate excretion, and retention of chloride ion in the blood. Complete absence of ammonia in the urine may occur in the case of treated burn patients having large burn surfaces (usually over 30%). This may also lead to hyperventilation [181.

8.

Toxicity

The intravenous LD50 of mafenide acetate in the mouse is reported to be 1,580 mgkg but is 2,040 m a g in the case ofthe rat. No LD50 for oral administration has been reported. Monkeys tolerated single oral doses of 20 gkg mafenide cream with no evidence of toxicity. Both in rats and mice, symptoms of acute intoxication included respiratory depression, terminal clonic-tonic convulsions, and death due to respiratory arrest occurred within 1 to 5 minutes [19]. When 10 gikg dose of the cream formulation were applied daily to the shaved skin of rabbits over a 3 month period, no evidence of irritation was detected. Hematological studies did not show any abnormalities, and

304

ALEKHA K. DASH AND SHANKAR SAHA

slight elevation of blood glucose occurred occasionally with higher doses of mafenide acetate [181.

9.

Acknowledgment

The authors thank Mr. William Ihm (Forensic Laboratory, School of Medicine, Creighton University) for conducting the mass spectroscopic studies.

10.

References

1.

J. Klarer, German Patent 726, 386, applied for 27.1 (1939).

2.

R.J. Holt, R. Murphy, and P.J. O’Donnel, Br. J. Plast. Surg., 21, 106-110 (1968).

3.

R.E.M. Thompson, F.C. Path, E.W. Colley, M.C. Path, and G.J.G. Chinnock-Jones, Br. J. Plast. Surg., 22,207-209 (1969).

4.

H.N. Harrison, H.W. Bales, and F. Jacoby, J. Trauma, 12,986993 (1972).

5.

E. Miller, J.M. Sprague, L.W. Kissing, and L.F. McBurney, J. Am. Chem. SOC.,62,2099-2103 (1940).

6.

J. Klarer, U.S. Patent 2,288,531 (1942).

7.

F.H. Bergeim and W. Baker, J. Am. Chem. SOC.,66, 1455-1460 (1944).

8.

M. Kusaim and Z. Yakagaku, J. Pharm. SOC.Japan, 64,51 (1944).

9.

Z.F. Komokina, J. Appl. Chem. (USSR), 21,681-684 (1948).

MAFENIDE ACETATE

305

10.

S.J. Angyal and S.R. Jenken, Aust. J Sci. Res., 3A, 461-465 (1950).

11.

A.K. Dash and S. Saha, unpublished results.

12.

The Merck Index, 1 lthEdition, Merck and Co., Inc., Rahway, N.J., USA, 1989, p. 5523.

13.

The United States Pharmacopeia, 22nd Revision, United States Pharmaceutical Convention, Rockville, Maryland, 1989, pp. 784785.

14.

Clarke's Isolation and Identification of Drugs in Pharmaceutical,Body FIuids, and Post-mortem Material, A.C. Moffat, ed., 2nd Edition., The Pharmaceutical Press, London, 1988, pp. 716-717.

15.

J.M. Steyn, J: Chromatogr., 143,210-213 (1977).

16.

A.K. Dash and J.S. Harrision, J. Chromatogr., 708,83-88 (1995).

17.

J.A. Moncrief, Clin.Pharmacol. Therap., 10,439-448 (1969).

18.

Mafenide Acetate Cream, Drugs, 1,434-460 (1971).

19.

T.W. Skulan and J.O. Hope, Life Sci.,5,2279-2282 (1966).

This Page Intentionally Left Blank

MALTODEXTRIN

Matthew J. Mollan Jr. and Metin Celik

Pharmaceutical Compaction Research Laboratory College of Pharmacy Rutgers, The State University of New Jersey Piscataway, NJ 08855

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIF'IENTS-VOLUME 24

307

Copyright 0 19% by Academic Ress, Inc. All rights of reproduction in any form reserved.

MATTHEW J. MOLLAN JR.AND METlN CELIK

308

CONTENTS

1. Description

1.1 1.2 1.3 1.4

Name, Definition, Formula Appearance Carbohydrate Profile Uses and Applications

2. Physical Properties 2.1 Particle Morphology 2.2 Crystallographic Properties 2.3 Thermal Analysis 2.4 Particle Size Distribution 2.5 Surface Area 2.6 Mercury Poroshnetry 2.7 Density 2.8 Moisture 2.9 Powder Flow 2.10 Compaction 2.11 Viscosity

3. Methods of Analysis 3.1 Cornpendial Tests 4. Identification 4.1 Maltodextrin Saccharide Separations 4.2 Thin Layer Chromatography 4.3 Liquid Chromatography 4.4 Supercritical Fluid Chromatography 4.5 NMK Spectroscopy 5 . Acknowledgments

6. References

MALTODEXTRIN

309

1. Description 1.1

Name

Maltodextrins are also known as hydrolyzed cereal solids, and are starch conversion products which contain a relatively small amount of dextrose and maltose. The United States Pharmacopeia and National Formulary [l] definition of a maltodextrin is: Maltodextrin is a non-sweet, nutritive saccharide mixture of polymers that consist of D-glucose units, with a Dextrose Equivalent less than 20. It is prepared by the partial hydrolysis of a food grade starch with suitable acids andor enzymes. It may be physically modified to improve its physical and functional characteristics. The chemical Abstracts identification number for Maltodextrin is AS9050-36-6. Maltodextrin has a general formula of H( C6H1005 >"-OH and is composed of D-glucose units linked primarily by a-1-4 bonds. Maltodextrin is listed as generally recognized as safe (GRAS)for human consumption under 21CFR 184.1444. Maltodextrins have both Food Chemical Codex and National Formulary monographs. Starch conversion products with a dextrose equivalent values above twenty are referred to as corn syrup solids. Starch conversion products with only a trace amount of dextrose are known as dextrins. Starch conversion products having a dextrose equivalent value not substantially above twenty, and containing small amounts of dextrose are known as maltodextrins. Kanig [2] described the dextrose content of a maltodextrin as less than about 2.4% by weight, and the amount of maltose as less than about 9.0% by weight. The term Dextrose Equivalent Value, D.E., is defined as the reducing value of the hydrolysate material compared to the reducing value of an equal weight of dextrose expressed as a percent, dry basis.

MATTHEW J. MOLLAN JR. AND METlN CELIK

310

D.E.

=

Reducing Value of Hydrolysate Material x 100 Reducing Value of Dextrose

The dextrose equivalent value is an indicator of the degree of depolymerization of starch, and the D.E. value will influence the physical properties of the maltodextrin. The higher the D.E. value, then the greater the extent of starch hydrolysis.

1.2

Appearance

Maltodextrins are white to off white powders or granules. Maltodextrins are bland, odorless, with a low sweetness level. The materials are often physically processed to improve their handling characteristics. Maltodextrins are produced from starch, usually corn. The starch, which is almost pure carbohydrate, is cooked or pasted to open the granule and then hydrolyzed. Products can be made by hydrolyzing with acid or enzymes or with a combination of acid and enzymes. After the desired amount of hydrolysis has occurred, the reaction is stopped, the product is filtered to remove insoluble materials, then dried.[3]. The average molecular weight decreases as the dextrose equivalent value of the maltodextrin increases, but even at low D.E. values, it is much smaller than the original starch. This relative molecular weight difference between starch and the hydrolyzed sugars gives the maltodextrins a portion of their valuable functional properties for the food and pharmaceutical industry.

1.3

Carbohydrate Profile

Maltodextrins will have different carbohydrate profiles depending on their dextrose equivalent value. The carbohydrate profile of a maltodextrin has important effects for the physicochemical properties of the maltodextrin. For example, the low molecular weight components will influence sweetness, viscosity, and humectant properties, while the

MALTODEXTRIN

31 1

high molecular weight components will influence solubility and solution stability. Maltrin@M150 [4] maltodextrin Standard Specifications Dextrose Equivalent 13.0 - 17.0 Carbohydrate Profile (Dry Basis) Monosaccharides Disaccharides Trisaccharides Tetrasaccharides Pentasaccharides and Above

0.7% 4.5% 6.6% 5.3% 82.9%

Maltrin@M5 10 [4]maltodextrin Standard Specifications Dextrose Equivalent 9.0 - 12.0 Carbohydrate Profile (Dry Basis) Monosaccharides Disaccharides Trisaccharides Tetrasaccharides Pentasaccharides and Above

0.8% 2.9% 4.4% 3.8% 88. I %

Maltodextrin [5] Standard Specifications Dextrose Equivalent 12.1 Carbohydrate Profile (Dry Basis) Monosaccharides Disaccharides Trisaccharides Tetrasaccharides Pentasaccharides and Above

0.9% 2.5% 4.0% 3.4% 89.2%

1.4

Uses and Applications

Maltodextrin is generally used in chewable tablet formulations, however the modified forms can be used in oral tablet formulations. It is also used in tablet coating formulations. Some uses of maltodextrins which

312

MATTHEW J. MOLLAN JR. AND METIN CELIK

have been given patent protection are: US patent # 3873694 [2] for use in direct compression tabletting; South African patent # ZA 800209 A [6] for use in coating: and South African patent # ZA 5000209 A [7] for use in coating. 'Tablet formulations containing a significant percentage of high D.E. value maltodextrin must guard against moisture uptake at high humidities since the material is hygroscopic. Tablets containing a significant portion of maltodextrin have extended disintegration times in water, (greater than five minutes), which may influence dissolution behavior. Maltodextrins are used extensively in the food industry as a moisture conditioner, food plasticizer, crystallization inhibitor, stabilizer, carrier and bulking agent [8]. The material is often used pharmaceutically in solid dosage forms as a tablet fillerhinder excipient, but is usually physically processed by spray drying, fluidized bed agglomeration, and roller compaction to improve its physical characteristics. Maltodextrins are generally considered not to be susceptible to undergo the Maillard reaction, which leads to browning and discoloration. A paper by Schmidt and Brogmann [9] did find discoloration of ascorbic acidkodium bicarbonate effervescent tablets containing maltodextrin as a tablet binder. The found the intensity of discoloration increased with the degree of degradation of dextrins and maltodextrins, and was proportional to their dextrose equivalent value. Maltodextrins have been used as binding agents in wet granulation processes [lo], and as direct compression binding agents [ l 1, 12, 13, 141 for tablet formulations. They have also been used as stabilizers in solidstate emulsion systems [ 151.

2. Physical Properties The physical properties of maltodextrins are determined by their respective dextrose equivalent value, their saccharide profile, and by the method by which they were physically processed for use.

MALTODEXTRIN

2.1

313

Particle Morphology

Scanning electron photomicrographs of some maltodextrins are shown in Figures 1-5. The photomicrographsare at 1OOX, 400X, and 1200X magnification, from top to bottom, respectively. Figure 1 is of a roller compacted maltodextrin, (Experimental Maltodextrin, Edward Mendell Co.), and shows a very dense structure with many small particles adhering to the surface. Figure 2 is of a spray dried maltodextrin, (Maltrin@M5 10, Grain Processing Co.), and shows a relatively smooth surfaced material with a generally round shape. At higher magnifications, large pores are distinctly seen. Figure 3 is of a fluidized bed agglomerated maltodextrin, (Maltrin@M500, Grain Processing Co.), and appears as a very porous mass with many irregularities. Figures 4 and 5 are of the fluidized bed agglomerated maltodextrins, (Malta*Gran TG@,and Malta*Gran lo@,ZumbroAFP Inc.) and illustrate the smooth surface and large pores which exist from the fluidized bed agglomeration method.

2.2

Crystallographic Properties

The x-ray powder diffraction pattern of the maltodextrins which had SEM photomicrographstaken are shown in Figures 6-10. The samples all behaved similarly, and were considered amorphous materials according to x-ray powder diffraction.

2.3

Thermal Methods of Analysis

Differential scanning calorimetry thermograms of maltodextrins did not show any significant events such as glass transitions or melting endotherms in the temperature range examined, 25OC to 225OC. A slight endotherm was seen from approximately 6OoCto 110°C due to desorption of unbound water [161. Thermogravimetricweight loss for maltodextrins was found to have a total volatile content with a range of 7.0%-7.4%, and the individual

FIGURE 1: Scanning Electron Photoinicrograph ot Experimental Maltodextrin 314

FIGURE 2: Scanning Electron Photomicrograph of Maltrin M510

315

FIGURE 3: Scanning Electron Photomicrograph of Maltrin M500

316

FIGURE 4: Scanning Electron Photomicrograph of Malta*Gran TG

317

FlGURE 5: Scanning Electron Photomicrograph of Malta*Gran 10

MALTODEXTRIN

319

2.00,

i .ao. 1.60.

1.40'

1.20. 1.00-

0.80.

0.60

0.40'

0.20'

0.0

10.0

5.0

t5.0

20.0

30.0

25.0

FIGURE 6 : Powder X-Ray Diffraction Pattern of Experimental Maltodextrin

1.80.

1.60'

1.40'

I

i .OO'

8p%

0.80.

/Ifr

0.60'

*w

0.40-

0.20-

.

/ / F

._,..-

,__

-

1

,

-

- I- ,

-

.

I

-

FIGURE 7: Powder X-Ray Diffraction Pattern of Maltrin M510

MATTHEW J. MOLLAN JR. AND METIN CELlK

320

i.aa. 1

60-

0

a01

i 0 60i

FIGURE 8: Powder X-Ray Diffraction Pattern of Maltrin M500

1 40

I

FIGURE 9: Powder X-Ray Diffraction Pattern of Malta*Gran TG

MALTODEXTRIN

321

x io= 2.00

l._/

1.60'

1.40'

1.20-

0.0

5.0

10.0

15.0

20.0

25.0

FIGURE 10: Powder X-Ray Diffraction Pattern of Malta*Gran 10

90.0

122

MATTHEW J. MOLLAN JR.AND METIN CELIK

results are shown in Table 1. All samples behaved similarly, and no bound waters were discernable.

2.4

Particle Size Distribution

The particle size distributions of maltodextrins available vary considerably, depending on the method of processing the material. Very fine grades of maltodextrin powder are available, however the particle sizes of the physically processed maltodextrins are much larger. The particle size distribution, by sieve analysis, of several physically processed maltodextrins are shown in Figure 1 1. The fluidized bed agglomerated maltodextrins had the largest particle size because they are created from spray dried maltodextrin which is then agglomerated with water in a fluidized bed process.

2.5

Surface Area

The surface areas of the maltodextrins was recorded by a krypton gas 3point BET method [ 171. The surface area analysis results are shown in Table 2. The spray dried and fluidized bed agglomerated maltodextrins had similar surface areas, while the roller compacted maltodextrin had a much larger surface area. These results were consistent with the observations made by electron microscopy, which illustrated the smooth surfaces of the spray dried and fluidized bed agglomerated maltodextrins, while showing the rough surface of the roller compacted maltodextrin.

2.6

Mercury Porosimetry

The measurement of pore volume and distribution of maltodextrins was performed by the method of Ritter and Drake [ 18). Figures 12 and 13 are from the results of the incremental amount of mercury intruded into the maltodextrin powder vs pore diameter. Figure 12 shows the fluidized bed agglomerated maltodextrins to have similar profiles, with Malta*Gran 1Om having a larger number of pores due to its larger mean particle size. The spray dried maltodextrin had a smaller mean pore size than the fluidized bed agglomerated maltodextrins. Figure 13 is the

Maltodextrin

Processing Method

Total Volitile Content from TG (%)

Experimental Maltodextrin

roller compaction

7.0

Maltrin" M 5 10

spray drying

7.2

Maltrin" M500

fluidized bed agglomeration

7.4

Malta'Gran TG"

fluidized bed agglomeration

7.2

Malta 'Gran 10"

fluidized bed agglomeration

7.0

TABLE 1: Thermogravimetric Weight Loss of the Maltodextrins

M A T H E W J. MOLLAN JR. AND METIN CELIK

323

100

90

,

0

ir!

e

30

c4

20 10

0

I

0

I

'

/

'

I

100 200 300 400

'

I

'

I

I

/

'

l

500 600 700 800

PARTICLE SIZE (microns)

FIGURE 11: Particle Size Distribution of the Maltodextrins 0 = Experimental Maltodextrin = Maltrin M510 0 = MaltrinM500 r = Malta*GranTG V = Malta*Gran 10

'

Maltodextrin

Processing Method

Surface Area (m2/g)

Experimenta I Maltodextrin

roller compaction

1.66

Maltrin" M 5 1 0

spray drying

0.12

Maltrin" M 5 0 0

fluidized bed agglomeration

0.12

Malta*Gran TG"

fluidized bed agglomeration

0.10

Malta'Gran 10"

fluidized bed agglomeration

0.14

TABLE 2: Surf;ice Area of the Maltodextrins

MATTHEW J. MOLLAN JR. AND METIN CELIK

326

1 1

0.0

Pore Diameter (um)

FIGURE 12: Mercury Porosimetry 0 = Experimental Maltodextrin = Maltrin M510 0 = Maltrin M500 V = Malta*Gran 10 0

321

MALTODEXTRIN

T t"

0.

a 1 V 0

1 U

m

e

0.

0.

.o Pore Diameter (um)

FIGURE 13: Mercury Porosimetry: log log scale 0 = Experimental Maltodextrin = Maltrin M510

0 0

= Maitrin M500

V = Malta*Gran 10

328

MATTHEW J. MOLLAN JR.AND METIN CELIK

same data plotted on a log-log scale and illustrates the roller compacted maltodextrins large number of small pores, which accounts for its large surface area as compared with spray dried or fluidized bed agglomerated maltodextrins.

2.7

Density

The densities of the maltodextrins are shown in Table 3 . True density was determined by helium pycnometry, while tap density (100 taps) and bulk density were determined in a 100 mL glass cylinder. The densities of the maltodextrins differ due to their method of processing. This is especially shown in the "true density" measurements, which does not measure internal or ''closed" pores, only external or open pores.

2.8

Moisture

The moisture content of the maltodextrins depends on the relative humidity of the surrounding atmosphere, and maltodextrins can sorb and desorb significant amounts of water. Figure 14 illustrates the sorption isotherm of the maltodextrins examined. They exhibited very similar profiles to each other despite differences in their method of processing. The maltodextrin powders began to "gel", and formed a pliable mass when stored at conditions above 75% relative humidity. The United States Pharmacopeia and National Formulary states that maltodextrin should be stored in tight containers or well closed containers at a temperature not exceeding 3OoC and a relative humidity not exceeding 50% [ 13. Hygroscopicity and D.E. value generally correlate, with the degree of hygroscopicity increasing with the increase in D.E. value.

2.9

Powder Flow

Flow parameters measured for the maltodextrins included: flow through and orifice, angle of repose, and percent compressibility [19]. The results are shown in Table 4. The maltodextrins tested were all free flowing, and the gravimetric method of describing powder flow showed the greatest difference between the samples due to density

Maltodextrin

Bulk Density (g/ml)

Tap Density (g/mll

True Density (g/ml)

Experimental Maltodextrin

.57

.65

1.503

Maltrin M510

.50

.56

1.425

Maltrin M500

.27

.32 I

I

1.410 I

Malta *Gran TG

.38

.44

1.424

Malta *Gran 10

.29

.35

1.417

TABLE 3: Densities of the Maltodextrins

330

MATTHEW J. MOLLAN JR. AND METIN CELIK

50

-

45

-

0

10

20

30

40

50

60

70

80

90

100

ZELATIVE HUMIDITY ( X i

FIGURE 14: Sorption Isotherm of the Maltodextrins 0 = Experimental Maltodextrin = Maltrin M510 0 = Maltrin M500 v = Malta*Gran TG V = Malta*Gran 10

Maltodextrin

TABLE 4: Flow Paraineters of the Maltodextrins

332

MATTHEW J. MOLLAN JR. AND METIN CELIK

considerations, i.e. high density materials generally tend to flow better than low density materials. Other very fine grade maltodextrin powders often will not flow, and this one of the primary reasons for physically processing maltodextrins for pharmaceutical use.

2.10

Compaction

Compression of a maltodextrin powder mass in a die causes the formation of a compact. Compaction of maltodextrin powder, (with 0.5% magnesium stearate added as a lubricant) into a tablet was performed at several pressures and the results are shown in Figure 15. The maltodextrins form relatively strong tablets at modest compressional forces, and can be considered to have good binding ability.

2.1 1

Viscosity

Maltodextrins exhibit Newtonian viscosity, and decrease in viscosity as they are heated. They generally exhibit low viscosity at modest solids content. Kenyon and Anderson [3] presented data of viscosity of maltodextrin solid solutions at varied percent solids. They stated that maltodextrins will demonstrate good solubility in the following solids range: 5 D.E. = 30-45%; 10 D.E. = 45-55%; 15 D.E. = 5045%; and 20 D.E. = 60-75%.

3. Methods of Analysis 3.1

Compendia1 Tests

According to the United States Pharmacopeia and National Formulary [ 11, maltodextrin is tested according to/for: microbial limits, pH, loss on drying, residue on ignition, heavy metals, protein, sulfur dioxide, and dextrose equivalent.

M ALTODEXTRIN

275 250

0 w 175 d

0 150 Lr,

0 125

i5 z

2

100

CA

75 50 25 0

0

50 100 150 200 250 300 350 400 450

APPLIED PRESSURE (ma) FIGURE 15: Tablet Crushing Force vs Applied Pressure of the Maltodextrins 0 = Experimental Maltodextrin 0 = Maltrin M510 0 = Maltrin M500 v = Malta*GranTG V = Malta*Gran 10

333

334

MAlTHEW J. MOLLAN JR. AND METIN CELIK

. . .

Microbial hmlts. b e t h o d <6 1 >] It meets the requirements of the tests for absence of Salmonella species and Escherichia coli.

g& [method <79 1 >] The pH is between 4.0 and 7.0, in a 1 in 5 solution in carbon dioxide-free water. Loss ondrv- i n5. lmethod <73 I>] Dry at 105°C for 2 hours in a forced-air oven: it loses not more than 6.0%of its weight. Residue on ignition: [method <281>] The residue on ignition is not more than 0.5%. Heavy Metals: [method I1 <23 Not more than 5 ppm heavy metals can be present Protein: Transfer about 10 g of Maltodextrin, accurately weighed, to an 800-mL Kjeldahl flask, and add 10 g of anhydrous potassium sulfate or sodium sulfate, 300 mg of copper selenite or mercuric oxide, and 60 mL of sulfuric acid. Gently heat the mixture, keeping the flask inclined at about a 45' angle, and after frothing has ceased, boil briskly until the solution has remained clear for about I hour. Cool, add 30 mL of water, mix, and cool again. Cautiously pour about 75 mL (or enough to make the mixture strongly alkaline) of sodium hydroxide solution (2 in 5) down the inside of the flask so that it forms a layer under the acid solution, and then add a few pieces of granular zinc. Immediately connect the flask to a distillation apparatus consisting of a Kjeldahl connecting bulb and a condenser, the delivery tube of which extends well beneath the surface of an accurately measured excess of 0.1 N sulfuric acid contained in a 50mL flask. Gently rotate the contents of the Kjeldahl flask to mix, and distill until all ammonia has passed into the absorbing acid solution (about 250 mL of distillate). To the receiving flask add 0.25 mL of methyl red-methylene blue TS, and titrate the excess acid with 0.1 N sodium hydroxide. Perform a blank determination, substituting pur.: sucrose or dextrose for the test specimen, and make any necessary correction. Each mL of 0.1 N sulfuric acid consumed is equivalent to

MALTODEXTRIN

335

1.401 mg of nitrogen (N). Calculate the percentage of N in the specimen taken, and then calculate the percentage of protein by multiplying the percentage of N by 6.25. The limit is 0.1%.

Sulfur dioxide Hydrogen peroxide solution: Dilute 30 percent hydrogen peroxide with water to obtain a 3% solution. Just before use, add 3 drops of methyl red TS, and neutralize to a yellow endpoint with 0.01 N sodium hydroxide. Do not exceed the endpoint.

Nitrogen: Use high-purity nitrogen, with a flow regulator that will maintain a flow of 200 f 10 mL per minute. Guard against the presence of oxygen by passing the nitrogen through a scrubber, such as alkaline pyrogallol, prepared as follows. Add 4.5 g of pyrogallol to a gas-washing bottle, purge the bottle with nitrogen for 3 minutes, and add a solution containing 85 mL of water and 65 g of potassium hydroxide, while maintaining an atmosphere of nitrogen in the bottle. Apparatus: The apparatus (see Figure I ) is designed to effect the selective transfer of sulfur dioxide from the specimen in boiling aqueous hydrochloric acid to the Hydrogen peroxide solution. The backpressure is limited to the unavoidable pressure due to the height of the Hydrogen peroxide solution above the tip of the bubbler, F. Keeping the backpressure as low as possible reduces the likelihood that sulfur dioxide will be lost through leaks. Preboil vinyl and silicone tubing. Apply a thin film of stopcock grease to the sealing surfaces of all the joints except the joint between the separatory funnel and the flask, and clamp the joints to ensure tightness. The separatory funnel, B, has a capacity of 100 mL or greater. The inlet adapter, A, with a hose connector provides a means of applying headpressure over the solution. [Note: A pressure-equalizing dropping funnel is not recommended because condensate, which may contain sulfur dioxide, is deposited in the funnel and the side arm.] The roundboom flask, C, is a 1000-mL flask with three 24/40 tapered joints. The gas inlet tube, D, is long enough to permit introduction of the nitrogen within 2.5 cm of the bottom of the flask. The Allihn condenser, E, has a jacket length of 300 mm. The bubbler, F, (see Figure 11) is fabricated

336

MATTHEW J. MOLLAN JR. AND METIN CELIK

FIGURE I. Apparatus for Sulfur Dioxide Test (reproduced with permission of USP)

MALTODEXTRIN

331

from glass according to the dimensions given in Figure 11. The Hydrogen peroxide solution is contained in a vessel, G, having an inside diameter of about 2.5 cm and a depth of about 18 cm. Circulate coolant, such as a mixture of water and methanol (4:1) maintained at 5', to chill the condenser. Procedure: Position the Apparatus in a heating mantle controlled by a powerregulating device. Add 400 mL of water to the flask. Close the stopcock of the separatory funnel, and add 90 mL of 4 N hydrochloric acid o the separatory funnel. Begin the flow of nitrogen at a rate of 200 f 10 mL per minute. Start the condenser coolant flow. Add 30 mL of Hydrogen peroxide solution to vessel, G. After 15 minutes, remove the separatory funnel, and transfer a mixture of 50.0 g of Maltodextrin, accurately weighed, and 100 mL. of alcohol solution (5 in 100). Apply stopcock grease to the outer joint of the separatory funnel, return the separatory funnel to the tapered joint flask, and concomitantly resume the nitrogen flow. Apply headpressure above the hydrochloric acid solution in the separatory funnel with a rubber bulb equipped with a valve. Open the stopcock of the separatory b e 1 to permit the hydrochloric acid solution to flow into the flask. Continue to maintain sufficient pressure above the hydrochloric acid solution to force it into the flask. [Note: The stopcock may be temporarily closed, if necessary, to pump up the pressure.] to guard against the escape of sulfur dioxide into the separatory funnel, close the stopcock before the last few mL of hydrochloric acid drain out. Apply power to the heating mantle sufficient to cause about 85 drops of reflux per minute. After refluxing for 1.75 hours, remove vessel G, add 3 drops of methyl red TS, and titrate the contents with 0.01 N sodium hydroxide VS, using a 10-mL burette with an overflow tube and a hose connection to a carbon dioxide-absorbing tube, to a yellow endpoint that persists for a least 20 seconds. Perform a blank determination, and make any necessary correction (see Titrimetry <23 l>). Calculate the quantity, in pg, of SO, in each g of the Maltodextrin taken by the formula:

in which 32.03 is the milliequivalent weight of sulfur dioxide, V is the volume, in mL, of titrant consumed, N is the normality of the titrant, and

338

MATTHEW J. MOLLAN JR.AND METIN CELIK

FIGURE 11. Bubbler (F) for Sulfur Dioxide Apparatus (reproduced with permission of USP)

MALTODEXTRIN

339

W is the weight, in g, of Maltodextrin taken. Not more than 40 pg of sulfur dioxide per gram of Maltodextrin can be found (.004%).

Dextrose Eauivalent; Standard solution: Dissolve an accurately weighed quantity of USP Dextrose RS in water, and dilute quantitatively with water to obtain a solution having a known concentration of about 10 mg per mL. Test solution: Transfer about 5 g of Maltodextrin, accurately weighed, with the aid of hot water to a 100-mL volumetric flask, cool, add water to volume, and mix. Procedure: Transfer 25.0-mL portions of alkaline cupric tartrate TS to each of two boiling flasks. Bring the contents of one flask to boiling within about 2 minutes while titrating with Standard solution to within 0.5 mL of the anticipated endpoint. Boil gently for 2 minutes. Continue to boil gently, add 2 drops of methylene blue solution (1 in loo), and complete the titration within 1 minute by adding the Standard solution dropwise or in small increments until the blue color disappears, determined by viewing against a white background in daylight or under equivalent illumination. If more than 0.5 mL of the titrant was required after the addition of the indicator, repeat the titration, adding the necessary volume of titrant before adding the indicator. Bring the contents of the second flask to boiling, and similarly titrate with Test solution. Calculate the Dextrose equivalent, on the dried basis, by the formula: [ 1OO/( 1 - 0.01A)](C,/C,)(V,N,),

in which A is the percentage Loss on drying of the Maltodextrin taken, C, is the concentration, in mg per mL, of Maltodextrin in the Test solution, C, is the concentration, in mg per mL, of USP Dextrose RS in the Standard solution, and V, and V, are the titrated volumes, in mL, of the Test solution and the Standard solution, respectively. The Dextrose equivalent is not more than 20. [Note: This is a limit test. For

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MATTHEW J. MOLLAN JR. AND METIN CELIK

maltodextrins with lower reducing values, other procedures may give other results.]

4. Identification 41

Maltodextrin Saccharide Separations

Maltodextrins can be considered non to weakly UV absorbing compounds. Maltodextrins are often characterized by their D.E. values, yet the physicochemical properties of the maltodextrins are dependent on their overall saccharide profile present in the final hydrolysate. Various liquid chromatographic separation techniques for the separation of oligosaccharides, including maltodextrins, have been recently reviewed by Chums [20]. Typical phase systems applied are: (a) a reversed acetonitrile + water gradient on aminopropyl- or cyanopropyl-modified silica; (b) an aqueous mobile phase on octadecyl-modified silica; (c) an acidic (PH 5 2) aqueous mobile phase on microparticulate cationexchange resins such as an Aminex HPX-22H phase [21]; and (d) anion exchange chromatography using an alkaline (PH 2 13) aqueous mobile phase and a sodium acetate gradient [22]. The separation of saccharides on the basis of chain length ( DP = degree of polymerization) in which DP 1 is glucose, DP2 is disaccharides, etc., becomes increasingly difficult above DPlO because of the extremely low levels which are found in maltodextrins.

4.2

Thin Layer Chromatography (TLC)

Classical TLC is of limited use in attempting to separate and identify simple saccharides in maltodextrins. Covacevich and Richards in 1976 [23J used thin layer chromatography in the continuous mode at 30 2" using 20 x 20 cm precoated plates. The solvent used was n-propanolnitromethane-water (50:20:30). The compounds were detected by spraying the plate with 50% sulfuric acid followed by heating at 110°C for 30 minutes. They were able to separate isomaltodextrins up to DP9.

*

MALTODEXTRIN

341

Bosch-Reig et al. [24] used monodimensional TLC to separate a aqueous solution of maltodextrins in several biological fluids. They used cellulose plates with two different eluents: (a) n-butanoYethanol/water (3:2:2), and (b) pyridine/ethyl acetate/acetic acidwater (5:5: 1:3). The maltodextrins were located with silver nitrate reagent. They were able to separate maltodextrins from DP2 to DP7 components. More recently, the use of high performance thin layer chromatography,HPTLC, has been used successfully in the analysis of maltodextrins. Vajda and Pick [25] used HPTLC to separate maltodextrin hydrolyzate on HPTLC silica plate. They used an eluent of 60% acetonitrile: 10% isopropanol; 15% aqueous ammonia 0.3%: 15% aqueous potassium chloride 0.67M, with a membrane pressure of 5 bars, flow rate of 0.04 mL/min, and a development distance of 170 mm. They separated up to DP14, but were unable to separate glucose and fructose.

4.3

Liquid Chromatography (LC)

High resolution techniques are essential when performing carbohydrate analysis, since carbohydrates have a number of isomers and homologs that structurally resemble one another. Since maltodextrins do not exhibit characteristic absorbances in useful regions of the UV spectrum, derivatization is often used. Post column labeling has the advantage of direct injection of intact samples, but has the limitation of sensitivity of detection due to poor yields of derivatives. Precolumn labeling is often used because high yields of derivatives can be easily achieved. If an m i n e type of column is used in HPLC analysis of maltodextrins, then DPl is the saccharide which will elute first. Increases in chain length then cause increases in elution time. If a resin based column is used, then the high molecular weight material, high DP, will elute initially and the DP1 will elute last. Scobell, Brobst, and Steele [26] described an automated liquid chromatographic system for quantitative analysis of carbohydrate mixtures. In 1981, Scobell and Brobst [27] discussed problems in separating oligosaccharides, and specifically when using cation-exchange resins that the loss of resolution for higher oligosaccharides is probably due to the unique helical structure of a-1-4 linked corn-derived oligosaccharides. They also used clean-up procedures prior to analysis,

342

MATTHEW J. MOLLAN JR. AND METIN CELIK

since salts, acids, soluble proteins, and particulate matter will interfere with the chromatographic analysis. They found that the use of silver form resins provided superior analysis to that of equivalent resins in the calcium form. Cheetham, Sirimanne, and Day 2281 used a C,,-bonded silica column with water as an eluent at a flow rate of 1 .O mL/min. They were able to separate maltodextrins to a DP6, although pairing was seen above DP3. The pairs of peaks was attributed to the a and b anomers of the oligosaccharide. They then performed a sodium borohydride reduction which replaced each pair of peaks with a single peak. This peak was then taken to be due to the corresponding oligosaccharide alditol. Warthesen [29] separated maltodextrins by an HPLC column (HPX-42A from Bio-Rad laboratories) with two precolumns, cation exchanging and anion exchanging, and used water as the mobile phase. Detection was by differential refractive index (RI) detector. With a flow rate of 0.5 ml/min, the chromatographic analysis was completed for a maltodextrin in 21 minutes and gave resolution of DPl to DPlO. The lowest detectable level for each saccharide was about 2 pg injected or 0.1% expressed as a dry weight basis. He overcame the problems of insolubility of some carbohydrate material, and nonlinearity of the large molecular weight peak, by using external standards for quantitation instead of area normalization. Brooks and Griffin 1301 examined corn syrup solids and maltodextrins and characterized the water soluble saccharides. The DP 1 - 10 saccharide components were separated by using a plastic cartridge C18 Resolve column compressed in a radial compression module (Waters Associates) with water as the mobile phase and detection by a differential refractometer. Pairs of peaks were obtained for most components between DP3 and DPIO. The peaks were attributed to the a and p anomers of the saccharides. An external glucose standard was used to obtain percent composition. They also determined overall molecular weight profiles by High Performance Size Exclusion Chromatography (HPSEC) with E-HighA and E-500 pBondage1 silica gel permeation columns (Waters Associates) connected in series. Water containing 0.15M NaCl was used as the mobile phase. The HPSEC data showed

MALTODEXTRIN

343

that as the D.E. of the hydrolysate increased, the amount of soluble high molecular weight saccharides decreased. McGinnis, Prince and Lowrimore [313 used reverse-phase HPLC with a refractive index detector. A reverse phase column was used since it has wide availability, high capacity, and can use water as an eluent. Adjustment of the polarity of the carbohydrate was needed since their is very little interaction between the carbohydrate and the column. They found the best column for separation of a mixture of maltodextrins was with a ODs-2 packed with CIS(Whatman c-18). The retention times on the column depended on the type of sugar unit, linkage, the anomeric configuration, and the molecular weight. Honda et al. [32] recently found that 1-phenyl-3-methyl-5-pyrazolone reacts with reducing carbohydrates almost quantitatively under mild conditions to yield strongly UV-absorbing and electrochemically sensitive derivatives. A homologous series of isomaltodextrins was separated with precolumn labeling. Niessen et al. [33] in 1992 used on-line liquid chromatography/mass spectrometry (LC/MS) for the analysis of maltodextrins, and could detect oligomers up to a DP 10. Their methodology used a mobile phase of 50 mmol/L ammonium acetate, with gradient elution with 0-50% methanol and the use of a octadecyl column. Peak splitting occurred at higher DP values due to separation of the two anomeric forms of the sugar. LC/thermosprayMS analysis of maltodextrin was performed with 1O4 M aqueous sodium acetate as the mobile phase and 20 mg injection with an octadecyl column. The higher DP value oligomers were more difficult to detect due to the fact that the weight percentage decreases with increasing DP value.

4.4

Supercritical Fluid Chromatography (SFC)

The new technique of supercritical fluid chromatography (SFC) uses a highly compressed gas as the mobile phase, with carbon dioxide as the most widely used mobile phase in SFC. Since carbon dioxide is nonpolar, polar solutes require derivatization to enhance miscibility. Lafosse et al. [34] in 1992 used an evaporative light scattering detector (ELSD)

344

MATTHEW J. MOLLAN JR. AND METIN CELIK

with both HPLC (to separate maltodextrin components) and SFC (to separate sugars). Maltodextrin HPLC analysis was performed by separation on octadecyl-bonded silica with a water-methanol gradient and detection by ELSD.

4.5

N M R Spectrametry

The use of water-elimination Fourier transform nuclear magnetic resonance to determine the degree of derivatization of maltodextrin with acrylic acid glycidyl ester at alkaline pH was described by Lepisto, et al. [3 51. They used a JEOL JNM-FX 100 FT-NMR spectrometer at 100 MHz and were able to determine the number of double bonds in acryloylated maltodextrins. Radosta ana Schierbaum [36]used NMR relaxation times (both TI and T2) for starch polysaccharides in solution to characterize bound and free water. German et al. 1371used Proton Pulse NMR to study water mobility in solutions of maltodextrins and on the sol-gel transition mode. They found that maltodextrin gels have a micro-inhomogeneous structure, and were able to describe the maltodextrin gelation process by percolation theory. McIntyre and Vogel [38] used two dimensional NMR to obtain the complete assignment of the overlapping proton NMR spectrum of starches, as well as maltodextrins, in D 2 0 solutions at 25'C. MoraGutierrez and Baianu [39] used solid-state I3CNMR techniques, and found significant differences between the spectra of corn and potato maltodextrins.

5. Acknowledgments We would like to acknowledge A. Newman, G. Young, T. Cortina, and H. Brittain of Bristol-Myers Squibb for their work for the TG, x-ray powder diffraction, and surface area data. We would also like to thank the Edward Mendell Company for their generous support of (MM) throughout the research project.

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6. References 1) United States Pharmacopeia and National Formulary, NF XVII, Sixth Supplement, Rockville, MD, pp. 2962-2963 (1 994). 2) Kanig, J., "Direct Compression Tabletting Composition and Pharmaceutical Tablet Produced There From", U.S. patent 3873694, March 25,1975. 3) Kenyon, M.M. and Anderson, R.J., "Maltodextrins and LowDextrose-Equivalence Corn Syrup Solids", Chapter two in ACS Symp. Ser., pp. 7-1 1 (1988).

m,

4) Maltrin@Maltodextrin, Product Data Technical Bulletin, Grain Processing Corporation, Muscatine, Iowa. 5 ) Wartman, A.; Hagberg, C.; and Eliason, M., "Refractive Index-Dry

Substance Relationships for Commercial Corn Syrups", J. Chem. Eng. Data, 2, 459-468 (1976). 6) Porter, S.C., and Woulicki, E.J., "Maltodextrin Coating", South African patent ZA8S00209A, August 28,1985. 7) Porter, S.C., and Waznicki, E.J., "Maltodextrin Coating", South African patent ZA5000209A, August 28,1985. 8) Lloyd, N.E., and Nelson, W.J., Starch: Chemistry and TecSecond Edition, R.J. Whistler, eds., Academic Press, London, p. 611 (1984). 9) Schmidt, P., and Brogman, B., "Effervescent Tablets: Choice of a New Binder for Ascorbic Acid", Acta Pharm. Technol., 34. 2226, (1988). 10) Visavarungroj, N., and Remon, J.P., "Evaluation of Maltodextrin as a Binding Agent", Drug Dev. Ind. Pharm., 18,1691-1700 (1992).

MATTHEW J. MOLLAN JR.AND METIN CELIK

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11 ) Parrott, E.L., "Comparative Evaluation of a New Direct Compression Excipient, Soludex 15", Drug Dev. Ind. Pharm., B, 561-583 (1989).

12) Papadimitriou, E.; Efentakis, M.; and Choulis, N.H., "Evaluation of Maltodextrins as Excipients for Direct Compression Tablets and their Influence on the Rate of Dissolution", Int. J Pharm., 86, 131-136 (1992). 13) Mollan, M.J., and Celik, M., "Characterization of Directly

Compressible Maltodextrins Manufactured by Three Different Processes", Drug Dev.Ind. Pharm., le , 2335-2358 (1993). 14) Munox-Ruiz,A.; Mondero Perales, M.C.; Velasco Antequera, M.V.,

and Jimenez-Castellanos, M.R., "Physical and Rheological Properties of Raw Materials", S. T.P. Pharma Sci., I, 307-3 12 (1993).

15) Myers$., and Shively, M.L., "Solid-state Emulsions: The Effects of Maltodextrin on Microcrystalline Aging", Pharm. Res., 14, 1389-1391 (1993). 16) Mollan, M.J.,

. . of a Roller C o w t e d Maltodextrin

for Direct Corny- * , PhD thesis, Rutgers, The State University of New Jersey, May 1993. 17) Brunauer, S.; Emmett, P.; and Teller, E., "Adsorption of Gases, Multimolecular Layers", J. Am. Chem. Soc., 612,309-316 (1938). 18) Ritter, H.C., and Drake, L.C., "Pore-Size Distribution in Porous

Materials. Pressure Porosimetry and Determination of Complete Macropore Distribution", Ind. Eng. Chem. Anal. Ed. , 12, 782786 (1945). 19) Carr, R., "Evaluating Flow Properties of Solids", Chem. Eng., a(2), 163-168 (1965).

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20) Churms, S.C., "Recent Developments in the Chromatographic Analysis of Carbohydrates", J. Chromatography, 5M, 555-583 (1990). 2 1) Hicks, K.B., and Hotchkiss, A.T., "High-Performance Liquid

Chromatography of Plant-Derived Oligosaccharides on a New Cation-Exchange Resin Stationary Phase: HPX-22H", JChromatography, 441, 382-386 (1988). 22) Lee, Y .C., "High-Performance Anion-Exchange Chromatography 151-162, for Carbohydrate Analysis", Anal. Biochem., .l.@ (1990). 23) Covacevich, M.T., and Richards, G.N., Tontinuous Quantitative

Thin-Layer Chromatography of Oligosaccharides", J. Chromatography, 129, 420-425 (1976). 24) Bosch-Reig, F.; Marcote, M.J.; Minana, M.D.; and Cabello, M.L.,

"Separation and Identification of Sugars and Maltodextrins by Thin Layer Chromatography: Application to Biological Fluids and Human Milk", Talanta, 3,1493-1498 (1992). 25) Vajda, J. and Pick, J., "Separation of some mono-, di-, tri-, and oligosaccharides", 2ndProc. Int. Conf. Biochem. Sep., J. Pick and J. Vajda, eds., 191-197 (1988). 26) Scobell, H.D.; Brobst, K.M.; and Steele, E.M., "Automated Liquid

Chomatographic System for Analysis of Carbohydrate Mixtures", Cereal Chem., 54, 905-917 (1977). 27) Scobell, H.D., and Brobst, K.M., "Rapid High-Resolution

Separation of Oligosaccharides on Silver Form Cation Exchange Resins", J. Chromatography, U ,5 1-64 (198 1). 28) Cheetham, N.W.H.; Sirimanne, P.; and Day, R.W., "High

Performance Liquid Chromatography Separation of Carbohydrate Oligomers", J. Chromatography, 222,439-444 (198 1).

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29) Warthesen, J.J., "Analysis of Saccharides in Low Dextrose Equivalent Starch Hydrolysates using High Performance Liquid Chromatography", Cereal Chem., 194-195 (1984).

a,

30) Brooks, J.R., and Griffin, V.K., "Saccharide Analysis of Corn(maize) Syrup Solids and Maltodextrins Using High Performance Liquid Chromatography", Cereal Chem., 255 (1987).

a,253-

3 1) McGinnis, G.D.; Prince, S.; and Lowrimore, J., "The Use of Reverse-Phase Columns for Separation of Unsubstituted Carbohydrates", J. Carbohydrate Chem., 5, 83-97 (1 986). 32) Honda, S.; Akao, E.; Suzuki, S.; Okuda, M.; Kakehi, K.; and Nakamura, J., "High Performance Liquid Chromatography of Reducing Carbohydrates as Strongly Ultraviolet Absorbing and Electrochemically Sensitive 3-methyl- 1-phenyl-5-pyrazolone Derivatives'', Anal. Biochem., .€.@, 35 1-357 (1989). 3 3 ) Niessen, W .M.A.; Van der Hoeven, R.A.M.; Van der Greef, J., Schols, H.A., and Voragen, A.G.J., "Online Liquid Chromatography/Thermospray Mass Spectrometry in the Analysis of Oligosaccharides", Rapid Commun. Mass Spectrom., $, 197-202 (1992). 34) Lafosse, M.; Elfakir, C.; Morin-Allory, L.; and Dreux, M., "Advantages of Evaporative Light Scattering Detection in Pharmaceutical Analysis by High Performance Liquid Chromatography and Supercritical Fluid Chromatography", J High Resolution Chromatogr., 15, 3 12-3 18 (1992).

35) Lepisto, M.; Artursson, P.; Edman, P.; Laakso, T.; and Sjoholm, I., "Determination of the Degree of Derivatization of Acryloylated Polysaccharides by Fourier Transform Proton NMR Spectroscopy", Anal. Biochem., U, 132-135 (1983). 36) Radosta, S. and Schierbaum, F., "Polymer-Water Interaction of Maltodextrins Part II.", Starch, 428-430 (1989).

a,

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37) German, M.L., Blumenfeld, A.C., Yuryev, V.P., and Tolstoguzov, V.B., "AnNMR Study of Structure Formation in Maltodextrin Systems", Carbohydrate Polymers, U, 139-146 (1989). 38) McIntyre, D.D. and Vogel, H.J., "Two-Dimensional Nuclear Magnetic Resonance Studies of Starch and Starch Products", Starch, 42, 287-293 (1990). 39) Mora-Gutierrez, A. and Baianu, I.C., "Carbon-13 Nuclear Magnetic Resonance Studies of Chemically Modified Waxy Maize Starch, Corn Syrups, and Maltodextrins. Comparisons with Potato Starch and Potato Maltodextrins", J. Agric. Food Chem., 22, 1057-1062 (1991).

This Page Intentionally Left Blank

NALMEFENE HYDROCHLORIDE

Harry G. Brittain

Ohmeda Inc. Pharmaceutical Products Division 100 Mountain Avenue

Murray Hill, NJ 07974

ANALYTICALPROFILES OF DRUG SUBSTANCES 351 AND EXCIPIENTS-VOLUME 24

Copyright 0 1996 by Academic Press, Inc. All rights of reproductionin any form reserved.

352

HARRY G. BRI'ITAIN

CONTENTS 1.

Description 1.1 Nomenclature 1.2 Formulae 1.2.1 Empirical 1.2.2 Molecular Weight 1.2.3 S t r u c ~ 1.3 Appearance 1.4 Uses and Applications

2.

Methods of Preparation

3.

Physical Properties Particle Morphology 3.1 Crystallographic Properties 3.2 3.2.1 Polymorphism 3.2.2 X-Ray Powder Diffraction Patterns 3.2.3 Single Crystal Structure Optical Activity 3.3 3.3.1 Optical Rotation Thermal Methods of analysis 3.4 3.4.1 Melting Behavior 3.4.2 Differential Scanning Calorimetry 3.4.3 Thermogavimetric Analysis Hygroscopicity 3.5 Solubility Characteristics 3.6 3.6.1 Solution pH 3.6.2 Partition Coefficient Ionization Constants 3.7 Spectroscopy 3.8 3.8.1 UVNIS Spectroscopy 3.8.2 Vibrational Spectroscopy Nuclear Magnetic Resonance Spectrometry 3.9 3.9.1 'H-NMR 3.9.2 13C-NMR 3.10 Mass Spectrometry

NALMEFENE HYDROCHLORIDE

4.

Methods of Analysis 4.1 Identification 4.2 Elemental Analysis 4.3 Titrimetric Analysis 4.4 SpectrophotometricMethods of Analysis 4.5 ChromatographicMethods of Analysis 4.5.1 Thin Layer Chromatography 4.5.2 High Performance Liquid Chromatography Determination in Body Fluids and Tissues 4.6

5.

Stability 5.1 Solid-state Stability 5.2 Solution-Phase Stability

6.

Acknowledgements

7.

References

353

354

HARRY G . BRITTAIN

1.

Description

1.1

Nomenclature

Chemical Name:

17-(cyclopropylmethyl)-4,5-a-epoxy-6methy~enemorphinan-3,14-diol hydrochloride 6-desoxy -6-meth y lenenaltrexone TM

Proprietary Names: Revex

Chemical Abstracts Number: 58895-64-0 1.2

Formulae

1.2.1 Empirical:

C21H2503N.HCI

1.2.2 Molecular Weight: 375.9

1.2.3 Structural

1.3

Appearance

Nalmefene hydrochloride is obtained as a white to off-white crystalline powder.

NALMEFENE HYDROCHLORIDE

1.4

355

Uses and Applications

Nalmefene hydrochloride is a pure narcotic antagonist, an analog of naltrexone, and is structural1 similar to naloxone. It has been developed as TM a parented solution (Revex ) for acute reversal of unwanted or excessive opioid effects, particularly opioid-inducedrespiratory depression. The need for opioid reversal occurs following opioid-based anesthetic techniques, or opioid overdoses. Opioid agonists and antagonists are a well established group of drugs which find extensive clinical use. Morphine and naloxone are, respectively, the classical opioid agonist and antagonist, with their activity being mediated through opioid receptors. Opioid receptor antagonists, such as nalmefene and naloxone, inhibit the pharmacological effects of administered opioid agonists and also the endogenous opioid systems. The clinical usefulness of the opioid antagonists comes from their ability to promptly (and selectively) reverse the effects of opioid agonists, including central nervous and respiratory system depression. Nalmefene is an opioid antagonist structurally similar to naloxone. Its mode of action is thought to be by competitive affinity to opioid receptor sites. The pharmacological actions of nalmefene are similar to those of naloxone, but appear to be more potent and have a longer duration of action [1,2]. RevexTMis presented in ampules, having concentrations of either 0.1 mg/mL or 1 mg/mL. Owing to its longer duration of action, the drug is administered as a single bolus dose administration. This presents a clear advantage over naloxone, which must be administered as a continuous intravenous infusion.

2.

Methods of Preparation

As illustrated in Scheme 1, the methods used for the preparation of nalmefene are based on a Wittig reaction carried out on naltrexone. In the original synthesis [3], Corey’s modified reagent [4,5] was used to obtain the 6-methylene derivative of naltrexone. A solution of methylenetriphenylphosphorane is prepared from sodium hydride and methyltriphenylphosphonium bromide in dimethyl sulfoxide. Naltrexone also dissolved in

HARRY G. BRITTAIN

356

Scheme 1.

Literature routes to nalmefene.

NH

Oxycodone

N-Ccyclopropylmethylnoroxycodone 0

1

0-DemethylaWn

Naltrexone

Noroxycodone

?&

H

NH

Noroxymotphone

Nalmefene

~

NALMEFENE HYDROCHLORIDE

357

DMSO is added, and the reaction mixture stirred at 55-60°C under a positive pressure of nitrogen for 18 hours. Work-up of the product, and chromatographicpurification, yielded nalmefene free base in 83% yield [3]. Attempts to scale-up the original synthetic method were not successfbl, so an improved method for the production of nalmefene was developed [6]. Using an ethereal solvent and an alkoxide base to prepare the methylenetriphenylphosphoranereagent, only slightly in excess of three moles of methyltriphenylphosphoniumbromide is required, and not the 60 moles as required by the original process. In addition, the alkoxides employed are significantly easier and safer to handle than sodium hydride, making it possible to perform the reaction on a larger scale [6]. Once the nalmefene free base is obtained, it is converted into the hydrochloride salt. This last step permits the recrystallizationof the product from water, and results in the formation of a highly purified drug substance. When obtained via the aqueous crystallization route, the material invariably consists of a monohydrate crystal phase.

3.

Physical Properties

3.1

Particle Morphology

As shown in Figure 1, crystals of nalmefene hydrochloridemonohydrate form as thin,tabular plates. The shortness of the long axis appears to be a result of crystal breaking, which yields the plate-like appearance of the crystals. The largest particles are generally 25-50 pm in diameter, but typical samples contain crystal debris as small as 10 pm in diameter. Most large crystals were found to exhibit birefiingence, indicating the existence of appreciable crystallinity. The thinness of the crystals was such that only first-order birefiingence could be observed. In addition, the crystals exhibited parallel extinction, which would place them in either a orthorhombic or monoclinic crystal system.

358

HARRY G . BRI'TTAIN

Figure 1. Optical microscopy of naimefene hydrochloride, obtained at a magnification of 200x.

NALMEFENE HYDROCHLORIDE

3.2

359

CrystallographicProperties

3.2.1 Polymorphism Upon crystallization from water, nalmefene hydrochloride is obtained as the monohydrate crystal phase. The water of hydration is tenaciously held, and is maintained through rigorous drying conditions (18 hours at 12OoC over phosphorous pentoxide). A stable ethanolic solvate can be obtained fiom samples recrystallized fiom ethanol. This material is metastable with respect to atmospheric moisture, and will sorb water upon standing. The ethanolic solvate may be desolvated under vacuum to yield an anhydrate species, but that species is highly unstable with respective toward atmospheric moisture and is not readily handled.

3.2.2 X-Ray Powder Diffraction Patterns X-ray powder diffraction data were obtained by means of a Philips model APDl700 automated powder diffractometer, using a copper source. The patterns were scanned between 10 and 30 degrees 2-8, at a scan rate of 0.015 degrees 2-0 per minute. All nalmefene hydrochloride materials were found to be very crystalline in nature, and did not yield a detectable halo due to the presence of amorphous material, The powder pattern obtained for the monohydrate phase is shown in Figure 2, while the associated table of scattering angles, d-spacings, and relative intensities is provided in Table I. The powder pattern of the ethanolic solvate is shown in Figure 3, and its summary of crystallographic data is found in Table 11. 3.2.3

Single Crystal Structure

Difficulty was obtained in obtaining a single crystal of the monohydrate phase whose quality was suitable for diffraction work, but the appropriate crystals of the ethanol solvate were readily produced. A clear parallelepiped crystal, having approximate dimensions of 0.25 x 0.20 x 0.40 mm, was mounted on a glass fiber, and the measurements made on a Rigaku AFC5S

r

l

Q Q Q In

l

l

Q Q

m w

l

1

0 0

m m

1

1

€

1

1

0

m

In

N

N

.Q

d

.m

rl

.In

4 0 8 0

1

r(

1

Q

Q 0 (rJ

1

Figure 2. X-ray powder diffraction pattern of the monohydratephase of nalmefene hydrochloride.

l

0

m m

9

NALMEFENE HYDROCHLORIDE

361

Table I CrystallographicData Associated with the X-ray Powder Diffraction Pattern of the Monohydrate Phase of Nalmefene Hydrochloride Scattering Angle (degrees 2-0)

d-Spacing

(4

(%I

11.405 11.960 12.505 13.585 14.090 15.055 15.925 16.355 17.715 18.110 18.585

7.7714 7.3938 7.0728 6.5 128 6.2805 5.8801 5.5607 5.4155 5.0027 4.8944 4.7704

100.0 4.2 4.4 6.2 17.0 3.3 4.1 2.3 47.9 16.0 4.3

Relative Intensity

0

0

v

Q Q

0 Q

N

0 ID

0 0

0

rl

0 Q

m

Figure 3. X-ray powder diffraction pattern of the ethanol solvate of nalmefene hydrochloride.

0

!n

Q

8

0

Q

9

NALMEFENE HYDROCHLORIDE

363

Table I1 Crystallographic Data Associated with the X-ray Powder Difiaction Pattern of the Ethanol Solvate of Nalmefene Hydrochloride

HARRY G . BRllTAIN

36.4

difhctometer with graphite monochromated Cu K a radiation and a 12 kW rotating anode generator. The data were collected at a temperature of - 160°C using the w-28 scan technique. The compound was found to crystallize in the monoclinic crystal system, and the space group was determined to be P21. The unit cell was characterized by the following dimensions: a b

-

C

=

p

=

V

=

=

7.977 (2) A 17.499 (2) 8, 17.348 (6) A 90.05 (3) O 2283 (2) A3

As shown in Figure 4, the asymmetric unit is composed of two protonated nalmefene molecules, two chloride ions, and two ethanol solvent molecules. All bond lengths and bond angles were found to be reasonable, and the deduced structure confiied the absolute configuration of the nalmefene unit. A complex hydrogen bonding system was detected, which involved the chloride ions, the hydroxyl oxygens, the protonated nitrogens, and the solvent hydroxyl groups. One view of the crystal packing is provided in Figure 5.

3.3

Optical Activity

As illustrated in Scheme 1, morphine is the starting material for nalmefene, and is a naturally derived material of known stereochemistry [5]. The o d y stereospecific reaction involved in the synthesis is the introduction of the 14hydroxyl function by means of oxidation. Due to conformational constraints in the molecule, the introduction of the hydroxyl h c t i o n proceeds with retention of configuration. No configurational inversion at a dissymmetric center is possible during the other steps of the synthesis. 3.3.1

Optical Rotation

The specific rotation of nalmefene hydrochloride, determined at 589 nm (sodium D-line) and a temperature of 25"C, is -164.8", calculated on an anhydrous basis.

NALMEFENE HYDROCHLORIDE

365

Figure 4. View of the asymmetric unit deduced for the ethanol solvate of nalmefene hydrochloride.

OCL1

HARRY G . BRlTTAIN

366

Figure 5 . Packing of nalmefene units in the ethanol solvate structure.

0

0

0

0

NALMEFENE HYDROCHLORIDE

3.4

Thermal Methods of analysis

3.4.1

Melting Behavior

367

The melting behavior of nalmefene hydrochloride was studied using hotstage microscopy. The dehydration of the crystals is visually observed over the range of 92403°C. The crystals are noted to melt to a clear liquid within the 180-185°C temperature range, but the melt discolors when the heating is continued beyond 200°C. This finding demonstrates that the compound melts with decomposition. 3.4.2 Differential Scanning Calorimetry A typical DSC thermogram of nalmefene hydrochloride, obtained in a closed pan and at a heating rate of 2"C/min, is shown in Figure 6. On the basis of the hot-stage microscopic findings, the large endotherm observed at 113°C is assigned to the dehydration process, and the small endotherm observed at 179°C is assigned to the melting transition. That the melting process is associated with compound decomposition is evidenced by the drastic change in the thermogram baseline noted after the melting endotherm. Integration of the peak areas associated with each transition yielded enthalpy changes of 150 mJ/mg for the dehydration endotherm and 11 r d m g for the melting endotherm. 3.4.3

Thermogravimetric Analysis

The TG analysis of nalmefene hydrochloride consists of two regions. The first of these is complete by approximately 113"C, and reflects the loss of lattice water. In ~ p i c alots, l the magnitude of this weight loss is 4.8%, which is consistent with the anticipated water content of the monohydrate phase. The second weight loss begins at 200"C, and is indicative of the oxidative decomposition of the compound. 3.5

Hygroscopicity

The monohydrate phase of nalmefene hydrochloride is essentially nonhygroscopic, and can only sorb up to 1% adventitious moisture. Material

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Figure 6. Differential scanning calorimetry thermogram of nalmefene hydrochloride.

200-

2 0

-200.

-400-

-800 -800'

45

95

145

Temperature ("C)

195

NALMEFENE HYDROCHLORIDE

369

which has been artificiallydried is very hygroscopic, and will quickly adsorb enough water to produce the monohydrate phase. 3.6

Solubility Characteristics

In its protonated form, nalmefene hydrochloride is very soluble in water, but becomes essentially insoluble once the solution pH is raised sufficiently so as to deprotonate the compound. The hydrochloride salt also exhibits a wide range of solubilitiesin non-aqueous media, being highly soluble in polar solvents and insoluble in non-polar solvents. All of the solubility data are summarized in Table 111. 3.6.1. Solution pH The equilibrium pH of a 0.10 M solution of nalmefene hydrochloride was determined to be 5.5, while the equilibrium pH of a 0.05 M solution was found to be 5.9. 3.6.2 Partition Coefficient As the solubility data in Table I11 indicate, nalmefene hydrochloride is a very hydrophilic substance. Its octanoVwater partition coefficient was found to be 0.075 (log Po,w= -1.125), which is consistent with its hydrophilic character.

3.7

Ionization Constant

Nalmefene hydrochloride contains a single ionizable group. Using an aqueous titration method, the pKa of the compound was determined to be 7.63. 3.8

Spectroscopy

3.8.1 WMS Spectroscopy The ultraviolet absorption spectrum of nalmefene hydrochloride is shown in Figure 7. The various aliphatic groups yield a reasonably intense absorption maximum observed at 2 11 nm, which is characterized by a molar absorptivity of 1977 L*mol/cm. This band is accompanied by a shoulder at approximately 230 nm (molar absorptivity of 778 L*moVcm). Finally, the

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Solubility Characteristics of Nalmefene Hydrochloride

I I

Solvent System

Solubility (mg/mL)

aqueous, pH = 2.25

128

aqueous, pH = 5.7 1

I

131 ~~~

I I

aqueous, pH = 6.15

133

aqueous, pH = 6.25

124

aqueous, pH = 7.85

1.09

aqueous, pH = 8.5

0.18

aqueous, pH = 9.15

0.09

aqueous, pH = 10.4

0.23

methanol

319

ethanol acetonitrile

I I

86.2 1.07

acetone

0.23

chloroform

0.13

NALMEFBNE HYDROCHLORIDE

37 1

Figure 7. Ultraviolet absorption spectrum of nalmefene hydrochloride, obtained in aqueous methanol at a solute concentration of 0.4 mg/mL.

2.00

1so

1.00

0.50

-W-\*

I

220

240

260

Wavelength (nm)

280

300

HARRY G . BRl3TAIN

372

phenolic functional group is responsible for the weaker absorption band located at 285 nm, which is characterized by a molar absorptivity of 194 L*moVcm. 3.8.2

Vibrationai Spectroscopy

The infia red absorption spectrum of nalmefene hydrochloride was obtained in a IU3r pellet, and a typical spectrum is shown in Figure 8. A summary of the band assignments is provided in Table IV. 3.9

Nuclear Magnetic Resonance Spectrometry

Nuclear magnetic resonance studies were conducted on nalmefene hydrochloride using a JEOL GSX-270 spectrometer, operating at 270.05 MHz ('H-NMR) or at 67.8 MHz (I3C-NMR). Spectra were obtained in DMSO-d6 at room temperature, and the resonance assignments make use of the following numbering scheme:

19

H

3.9.1

'H-NMR Spectrum

The one-dimensional 'H-NMR spectrum of nalmefene hydrochloride is shown in Figure 9. The assignments for the observed resonance bands are provided in Table V, with additionaljustification being obtained using twodimensional proton-proton correlated spectroscopy (COSY, Figure lo), and two-dimensional proton-carbon correlated spectroscopy (HETCOR, Figure 1 1). As there is severe overlap of resonances in certain instances, a range of

NALMEFENE HYDROCHLORIDE

Figure 8. Infrared absorption spectrum of nalmefene hydrochloride, obtained in a KBr pellet.

Energy (wavenumbers)

373

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374

Table IV Infrared Absorption Spectral Assignments of Nalmefene Hydrochloride

F

Transition Energy r r )

1

Assignment

1

799,814

I

=CH (aromatic)

r

r[--

904

-CH,

942,1031,1116

C-0 (furangroup)

1170

13%

I

I

3200-3600

I

0-H (phenolic group) C=C stretch

1505,1619 1638

C-OH (tertiary hydroxyl group)

I

I

aromatic ring modes

I

-OH and/or NH stretch

I

Figure 9. One-dimensional 'H-NMR spectrum of nalmefene hydrochloride.

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Table V Assignments of the Resonance Bands Observed in the ‘H-NMR Spectrum of Nalmefene Hydrochloride

I I I I I

1 I I I I I

I

I

Chemical Shift

Proton

OPm)

1

6.495 (d)

2

6.63 (d)

5

5.02 (s)

7, 7’

8,s’ 9 10’10’ 15, 15’

16, 16’ 17, 17’ 18

19,20 21,21’ 22 23 24

I I I I

1 I I I I

I I

I

2.02,2.52 (br) 1.15, 1.71 (br) 3.82 (br, d) 2.93’3.26 (br) 1.39,2.53 (br) 2.45, 3.02 (br) 2.86,3.19 (br) 1.03 (m) 0.34 - 0.59 (m) 4.77, 5.13 (s) 9.3 (s) 6.5 (s)

8.88 (br)

NALMEFENE HYDROCHLORIDE

Figure 10. Two-dimensional proton-proton correlated spectroscopy (COSY) of nalmefene hydrochloride.

,

P 8

317

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378

Figure 1 1. Two-dimensional proton-carbon correlated spectroscopy (HETCOR) of nalmefene hydrochloride.

-

L

V

t

I

0

NALMEFENE HYDROCHLORIDE

379

chemical shifts has been reported for selected protons. The -OH and NH proton resonances were assigned by long-range coupling. 3.9.2

13

C-NMR Spectrum

The one-dimensional I3C -NMR spectrum of nalmefene hydrochloride is shown in Figure 12. The assignments for the observed resonance bands are provided in Table VI, with additionaljustification being obtained using twodimensional proton-carbon correlated spectroscopy (HETCOR, Figure 11). Quaternary carbons were assigned by searching similar fragments on the Sadtler database. Because of the severe overlap in the proton spectrum, and the lack of resolution in the HETCOR experiment, the assignments for C7 and C20 are not unequivocal. 3.10

Mass Spectrometry

The mass spectrum of nalmefene hydrochloride was obtained using a VGTrio 2 mass spectrometer, operating in the GC-MS (EI+) mode. The spectrum is shown in Figure 13, and the assignments are given in Table VII :

Table VII Interpretation of the EI Mass Spectrum of Nalmefene Hydrochloride

I

d Z

Relative Intensity

Fragment

339

100

[MI+ - HCI

298

21.3

284

14.8

242

I

17.3

110

28.2

55

65.9

In Table VII, M denotes the nalmefene unit, C21H2503N.

I

1

Figure 12. One-dimensional 13C-NMR spectrum of nalmefene hydrochloride.

NALMEFENE HYDROCHLORIDE

38 1

Table VI Assignments of the Resonance Bands Observed in the 13C-NMRSpectrum of Nalmefene Hydrochloride Carbon

Chemical Shift (PP@

1

1 19.08

2

117.57

3

140.76

4

143.50

5

87.16

6

145.04

7

26.79

8

3 1.33

9

61.35

10

22.97

11

120.64

12

129.15

13

46.56

14

70.35

15

26.95

16

46.73

17

56.58

18

5.73

19

2.58 or 5.12

20

2.58 or 5.12

21

111.13

382

HARRY G . BRITTAIN

Figure 13. Electron-impact mass spectrum of nalmefene hydrochloride.

NALMEFENE HYDROCHLORIDE

4.

Methods of Analysis

4.1

Identification

383

The id en ti^ of nalmefene hydrochloride drug substance is most conveniently established using infrared absorption spectroscopy. Approximately 1 mg of nalmefene hydrochloride is thoroughly mixed with approximately 300 mg of potassium bromide, the mixture ground until homogeneous, and a pellet compressed from the mixture. The IR spectrum is scanned between 400 and 4000 wavenumbers, and should exhibit maxima only at the same energies as does the authentic standard (Figure 8). An alternative method is to dissolve the drug substance in a small quantity of methanol, and mix the solution with the solid potassium bromide. Afier complete removal of the solvent, the pellet may be compressed. A second identity test for nalmefene hydrochloride has been developed, which uses thin-layer chromatography methodology. This test will be discussed in section 4.5.1. 4.2

Elemental Analysis

The determination of the elemental analysis of nalmefene hydrochloride presents no unusual problems for the analyst. Carbon and hydrogen analyses can performed by the combustion technique, while the nitrogen analysis is performed using the Dumas method. Typical results are presented in Table VIII.

Table VIII Elemental Analysis of Nalmefene Hydrochloride

I

% %H H

% %C C

I theoretical 1 I experimental* I

67.10 67.3 67.311

*corrected for water content

I I

6.97 7.04

I I

%N

% % c1

3.73

9.43

3.63

9.39 1-9 .391

HARRY G . BRITTAW

384

4.3

Titrimetric Analysis

The assay value of nalmefene hydrochloride is established using aqueous titration methodology. Approximately 125 mg of sample are accurately weighed into a titration vessel, to which 125 mL of glacial acetic acid and 10 mL of 6% mercuric acetate solution (in acetic acid). The sample is titrated potientiometrically with 0.02 N perchloric acid (in acetic acid) using glasdcalomel electrodes. The purity of the drug substance is calculated using: N V (0.3759) (100) assay W {l-”/H~O} where N

=

v

=

W

=

YOH,O

=

0.3759 =

4.4

normality of perchloric acid titrant volume of titrant consumed massofsampletaken decimal fiaction of water in the sample milliequivalent weight of nalmefene hydrochloride

SpectrophotometricMethods of Analysis

The magnitude of the ultraviolet absorbance at 285 is used to differentiate between the 0.01 mg/mL and 0.1 mdmL formulations of RevexTM.

4.5

Chromatographic Methods of Analysis

4.5.1 Thin-Layer Chromatography A TLC method has been developed for nalmefene hydrochloride which is cased on the use of silica gel 60 F254 (Merck) as the adsorbent. The solvent system is cyclohexane/chlorofoddiethylarnine (10:75:15). The sample is prepared by weighing 10 mg of drug substance, and dissolving in 1 mL of methanol. 10 pL of this solution is applied to the plate, and dried using a nitrogen flow. The plate is allowed to develop to a height of approximately

NALMEFENE HYDROCHLORIDE

385

12 cm, and then allowed to dry. The spot is viewed using short-wave UV (254 nm). A typical thin-layer chromatogram is shown in Figure 14, where the relative retention (Rf) for nalmefene is 0.41, the Rf for naltrexone is 0.28, and the Rf for bisnalmefene (the sole degradant product) is 0.07. The criteria used when the TLC method is used for identification purposes is met if the retention time of the sample is identical to that of an authentic nalmefene hydrochloride standard.

4.5.2 High Performance Liquid Chromatography An isocratic, stability-indicating, HPLC method has been developed to determine the impurity profile of nalmefene hydrochloride, and the assay

content in the drug products. The analytical separation is effected using a PrimsphereTMCIScolumn, and uses a mobile phase consisting of 20:80 acetonitrile:O.O5M phosphate buffer. The buffer contains 0.2% triethylamine, and the pH is adjusted to 4.2 with 85% phosphoric acid. Using a mobile-phase flow rate of 1.O mL/min, the analyte detection is effected on the basis of the UV absorbance at 2 10 nm. Sample and standard solutions are prepared at concentrations of 0.01 mg/mL, and the method may be used to assay the content of either the 1.O or 0.1 mg/mL presentations of RevexTM, as long as aliquots of the neat formulation are diluted to 0.01 mg/mL prior to analysis. When the method is used to obtain nalmefene concentrations (expressed as the free base in units of mg/mL) within drug product formulations, the assay value is calculated using:

=

AStd

339.4

W (l.O-%H*O) P

A&m

conc.(mg/mL)

X

X

100

375.9

where: = average peak area of nalmefene in the sample solution

Asam AStd

=

average peak area of the nalmefene standard solution

386

HARRY G. BRITTAIN

Figure 14. Thin-layer chromatogram of nalmefene hydrochloride. Moving across the chromatogram from left to right, the spot at furthest left is that of a 1 mg sample, and next is the standard at the same concentration. The next two spots (at the same Rf values) are the standard spotted at concentrations of 5 pg and 2 pg, respectively. The next two spots correspond to naltrexone (5 pg) and bis-nalmefene ( 5 pg).

NALMEFENE HYDROCHLORIDE

387

W

=

mass of nalmefene hydrochloride standard taken (mg)

%H,O

=

the water content of the standard, expressed as a fraction

=

Conversion factor, from nalmefene hydrochloride salt to the free base

=

Purity factor of the nalmefene hydrochloride reference standard, expressed as a fraction

339.4f379.5 P

The most important use of the analytical method is in the determination of the impurity profile of nalmefene hydrochloride. As evident in Figure 15, excellent separation of nalmefene from its process impurities (naltrexone and A7-nalmefene)and sole degradant (bis-nalmefene) is obtained. Structures of these related substances are provided in Figure 16.

4.6

Determination in Body Fluids and Tissues

The first method reported method for the determination of nalmefene in human plasma used HPLC and electrochemical detection [6]. Following extraction of the drug substance at pH 9, the extract was chromatographed on a reversed-phase C 18 column, and detected using a glassy carbon electrode detector. The method sensitivity was reported to be 3 ng/mL, after extraction of a 1 mL sample of plasma. A specific radioimmunoassay has been developed for the quantitation of nalmefene in human plasma [7]. Since nalmefene and naltrexone were found to cross-react equally well with a rabbit antiserum to an albumin conjugate of naltrexone-6-(o-carboxymethyl)oxime,it was possible to use fHI-naltrexone as the radioligand. Plasma samples were mixed with pH 9 borate buffer, extracted into ether, and the organic phase dried prior to reconstitution in the assay buffer. When extracting 0.5 mL of plasma, a limit of sensitivity equal to 0.2 ng/mL of nalmefene was obtained. A HPLC method suitable for the measurement of nalmefene in human plasma after either oral or intravenous administration has also been described [S]. The drug is extracted from plasma using a solid-phase cyanopropyl column, and eluted with a 60% (v/v) acetonitrile in dilute

388

HARRY G . BRITTAIN

Figure 15. HPLC chromatogram of nalmefene hydrochloride spiked with 7 naltrexone, A -nalmefene, and bis-nalmefene.

NALMEFENE HYDROCHLORIDE

389

Figure 16. Structures of the nalmefene process impurities (naltrexone and 7 A -nalmefene) and the sole degradant (bis-nalmefene).

A'-NdwCene

HO

Ndtrexone

HARRY G . BRIl’TAIN

390

sodium pentanesulfonic acid solution. The concentrated and filtered eluent is injected onto a HPLC system, which uses a phenyl column to effect the separation and a mobile phase consisting of 30% (v/v) acetonitrile in dilute sodium pentanesulfonic acid solution. The analyte species are detected by electrochemical means, using a dual-electrode system. A signal-to-noise ratio of 4.5 was reported for nalmefene when a 1 ng/mL spiked plasma sample was analyzed.

5.

Stability

5.1

Solid-state Stability

Nalmefene hydrochloride has been found to be extremely stable when stored at 15-25°C in well closed containers, and protected from both light and moisture. Some bulk drug substance has been stored at room temperature for up to seven years with no detectable change in compound purity.

5.2

Solution-Phase Stability

Using the HPLC method described in section 4.5.2, the stability of nalmefene hydrochloride in both the 0.1 mg/mL and 0.0 1 mg/mL formulations has been studied at a variety of temperatures for periods up to 36 months [ 1 11. The trends in nalmefene potency obtained during the course of these studies are illustrated in Figure 17, while the formation trend of the sole degradant (2,2’-bisnalmefene) is found in Figure 18. The stability data were found to be interpretable using first-order kinetics, and essentially comparable rate constants were calculated for both the potency loss and the formation of 2,2’-bisnalmefene. Applying the Arrhenius equation to these data, a rate constant of 0.000441 month-’ was deduced for the reactions taking place at 25°C. When this value for the first-order rate constant was substituted into the integrated rate expression, it was predicted that the drug product would not exceed its impurity specification (not more than 2.5% 2,2’-bisnalmefene can be present at the end of the product shelf life) for approximately 4.8 years [l 11.

NALMEFENE HYDROCHLORIDE

391

Figure 17. Trends in product potency observed during the stability study of the 1.O mg/mL RevexTMformulation (2 mL fill in a 2-mL ampule) at 4,30,40,55, and 75°C.

100

A r s

U

90

0

t

0

e

80

70

I

5

15

25

Time (months)

35

392

HARRY G . BRITTAIN

Figure 18. Formation of 2,2’-bisnalmefene observed during the stability study of the 1 .O mg/mL RevexTMformulation (2 mL fill in a 2mL ampule) at 4,30, and 40°C.

2.5

1.5

30°C

7

0.5 4°C

5

15

25

Time (months)

35

NALMEFENE HYDROCHLORIDE

393

In a subsequent study, the short-term stability of RevexTMwas determined in a number of diluents commonly employed for intravenous use [ 121. Dilutions of RevexTMwere prepared separately in 0.9% sodium chloride injection, 0.45% sodium chloride injection, 5% dextrose injection, 5% dextrose and 0.45% sodium chloride injection, lactated Ringer's injection, 5% dextrose and lactated Ringer's injection, and 5% sodium bicarbonate injection. Each admixture was stored at 4"C, room temperature (21"C), and 40"C, with samples being tested after storage at each temperature for 0,24,48, and 72 hours. The results of this study are summarized in Table IX. Defining stability as the retention of at least 95% of the initial drug concentration at the end of the storage period, it was concluded that the diluted solutions of RevexTMwere uniformly stable for up to 72 hours in all of the injectable solutions maintained at either 4"C, 21"C, or 40°C.

6.

Acknowledgements

Special appreciation is given to those who contributed toward the work described in this analytical profile, especially Linda Lafferty, Petranna Bousserski, Glenn Diegnan, Ralph Lessor, Satish Pejaver, Camille Small, Satya Murthy, Ashok Krishnaswami, Kamalesh Johri, George Owoo, Patrice Rafalko, and Martin Gall.

394

HARRY G. BRTITAIN

Table IX Stability of RevexTMin Various Injectable Solutions

Solution

Storage condition

Percent Remaining

Percent Remaining

Percent Remaining

5% Dextrose and Lactated Ringer's 5% Sodium Bicarbonate

4uc RT 4OoC 4uc RT 4OoC

101.0 99.0 100.5 99.5 100 100.5

99.5 98.0 99.5 99.5 99.5 99.5

99.0 98.5 100.0 101.0 100.0 99.0

NALMEFENE HYDROCHLORIDE

395

7.

References

1.

D.H. Dyson, T. Doherty, G.I. Anderson, and W.N. McDonell, Veterinary Surg., 19,398-403 (1990).

2.

T.J. Gal and C.A. DiFazio, Anesthesiology, 64, 175-180 (1986).

3.

E.F. Hahn, J. Fishman, and R.D. Heilman, J. Med. Chem., 18,259262 (1975).

4.

E.J. Corey and M. Chaykovsky, J. Am. Chem. SOC.,87,1345-1349 (1 965).

5.

R. Greenwald, M. Chaykovsky, and E.J. Corey, J. Org. Chem., 28, 1128-1129 (1963).

6.

P.C. Meltzer and J.W. Coe, U.S. Patent 4,535,157 (1985).

7.

W. Klyne and J. Buckingham, Allas of Stereochemistry, 2ndedn., volume 1, Oxford University Press, New York, 1978, p. 141.

8.

J. Hsiao and R. Dixon, Res. Comm. Chem. Path. Pharm., 42,449454 (1983).

9.

R. Dixon, J. Hsiao, W. T d e , E. Hahn, and R. Tuttle, J. Pharm. Sci., 73, 1645-1646 (1984).

10.

J.Z. Chou, H. Albeck, and M.J. Kreek, J. Chrom., 613,359-364 (1 993).

11.

H.G. Brittain, L. Lafferty, P. Bousserski, G. Diegnan, R. Lessor, C. Small, and S. Pejaver, PDA J. Pharm. Sci. Tech., in press.

12.

S.S. Murthy and H.G. Brittain, J. Pharm. Biomed. Anal., submitted.

This Page Intentionally Left Blank

POLYVINYL ALCOHOL

David Wong and Jagdish Parasrampuria

CIBUS Pharmaceutical, Inc.

200 D Twin Dolphin Drive

Redwood City, CA 94065

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPENTS-VOLUME 24

397

Copyright 0 1996 by Academic Press, Inc. All rights of reproductionin any fom reserved.

398

DAVID WONG AND JAGDISH PARASRAMPURIA

1.

Introduction

2.

Description

3.

Sources and Manufacturing

4.

Physical Properties solubility 4.1 Viscosity 4.2 Moisture Sorption 4.3 Permeation of Polyvinyl Alcohol Films 4.4 Glass Transition 4.5 Crystallinity and Preparation of Polyvinyl Alcohol 4.6 Hydrogels 4.7 Surface Properties of Polyvinyl Alcohol Hydrogels Optical Properties of Polyvinyl Alcohol Hydrogels 4.8 Mechanical Strength of Polyvinyl Alcohol Hydrogels 4.9 4.10 Water Content and Swelling Properties of Polyvinyl Alcohol Hydrogels 4.1 1 Protein Absorption by Polyvinyl Alcohol Hydrogels

5.

Methods of Analysis 5.1 Cornpendial Methods 5.1.1 pH 5.1.2 Loss on Drying 5.1.3 Residue on Ignition 5.1.4 Viscosity 5.1.5 Water-Insoluble Substances 5.1.6 Degree of Hydrolysis 5.2 Other Analysis Studies

6.

Stability

7.

Applications

8.

Pharmacological Effects and Toxicity

9.

References

POLYVINYL ALCOHOL

1.

399

Introduction

Polyvinyl alcohol (CAS number 9002-89-5, and often abbreviated as PVA) was first commercially used in Germany in the 1920's, and since then has been applied in various areas such as textiles, paper, adhesives, cements, and films [5]. During the last 10 years, great advances have been made in medical and pharmaceutical technologies, and polyvinyl alcohol application methods have also undergone significant changes. Polyvinyl alcohol has been an article in the United States Pharmacopoeia, and the material specifications are those listed in the official monograph ~71.

2.

Description

As illustrated in Figure 1, polyvinyl alcohol is a polyhydric alcohol containing secondary hydroxyl groups on alternate carbon atoms.

Figure 1.

Structure of polyvinyl alcohol.

Polyvinyl alcohol is usually not chemically modified, nor is it crosslinked. However, for special purposes a polymer can be obtained through crosslinking by difunctional or polyfunctional reagents. Polymerization and alcoholysis are well-controlled during the preparation of different grades of polyvinyl alcohol, yielding materials characterized by different viscosities and percentages of hydrolysis. The "percentage hydrolyzed grades" are determined by the time used for alcoholysis, and

400

DAVID WONG AND JAGDISH PARASRAMPURIA

the "viscosity grades" are controlled by the time used for polymerization of vinyl acetate. "Chain-breakers" are occasionally added to assist in controlling the molecular weight (viscosity grades) of polyvinyl alcohol. Complete alcoholysis will lead to a "fully hydrolyzed grade" [4,51. Examples of different commercially available grades of polyvinyl alcohol are listed in Table 1.

3.

Sources and Manufacturing

The raw material for polyvinyl alcohol is vinyl acetate. Vinyl acetate is first polymerized to polyvinyl acetate by conventional techniques such as bulk, solution, or emulsion polymerization. Polyvinyl acetate is then dissolved in an organic solvent (such as mixed methanol and methyl acetate) and hydrolyzed so that the pendant acetate groups are replaced by pendant hydroxy groups. The polyvinyl alcohol precipitates out of the reaction medium because of its low solubility, and is then filtered, washed, dried, and packaged [5].

4.

Physical Properties

Polyvinyl alcohol is an odorless, white, granular powder, that decomposes when heated to about 200°C [63,64]. It dissolves in water, forming an acidic solution [3]. A summary of the important physical properties is provided in Table 2.

4.1

Solubility

Polyvinyl alcohol is soluble in water, and in most mixtures consisting of water and a water-miscible organic solvent (such as glycerin, ethylene glycol, or dimethyl sulfoxide) [ 11. The compound has a high solubility in water and mixed solvents, but can form lumps that require a long time to dissolve completely. Therefore, polyvinyl alcohol should be dissolved according to the following procedure:

POLYVINYL ALCOHOL

Table 1 Some Standard Grades of Polyvinyl Alcohol [3]

401

402

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 2 Physical Properties of Polyvinyl Alcohol [3]

I

I

1

Appearance

White to cream granular powder

Bulk Density

40 1bs/ft3 ~~

Specific Gravity (solid)

1.27 - 1.31

Specific Gravity (1 0% solution at 25°C)

1.02

Thermal Stability

Gradual discoloration at about 100°C; darkens rapidly above 150°C; rapid decomposition above 200°C

Refractive Index (film) at 20°C

1.55

Thermal Conductivity

0.2 W/(m.K)

Electrical Resistivity

(3.1-3.8) X 107 ohm.cm

Specific Heat

1.5 J/(g.K)

Melting Point (unplasticized)

230°C for fully hydrolyzed grades; 180-190°C for partially hydrolyzed grades 75 - 85°C

i

Stability (solid)

Stable when protected from moisture

Flammability

Burns similar to paper

Stability to Sunlight

Excellent

POLYVINYL ALCOHOL

403

The solid is first dispersed and agitated in cold water. When all particles are wet, then the solution temperature is raised to 85-96°C until all particles dissolve [ 3 ] . The exact temperature for preparing complete solutions depends on the polyvinyl alcohol concentration and its grade. The solubility of polyvinyl alcohol is primarily a function of its hydrolysis percentage and viscosity, properties derived from its molecular weight. For example, 87-89 % hydrolyzed polyvinyl alcohol is soluble in both cold and hot water. Polyvinyl alcohol hydrolyzed over 89% dissolves only in hot water, while 75 to 80% hydrolyzed polyvinyl alcohol is soluble only in cold water [4].In addition to its percentage of hydrolysis, tacticity can also influence the solubility. In water, the atactic isomer is more soluble than is the isotactic isomer [16]. The maximum amount of solids recommended for solutions prepared using conventional high-speed mixers are summarized in Table 3.

4.2

Viscosity

The most important single feature of polyvinyl alcohol is its water-binding capacity, which is commonly expressed as viscosity. These measurements can be performed using a Brookfield or Haake viscometer, since polyvinyl alcohol dissolves in water to give a viscous solution. The viscosity of this solution depends on the temperature, molecular weight, and concentration of the solute [ 3 ] . Table 4 shows the relationship between viscosity and polyvinyl alcohol concentrations at various temperatures.

4.3

Moisture Sorption

When polyvinyl alcohol films are used in controlled-release dosage forms, the permeability of the film to moisture, water, and drug becomes important. Films can be obtained simply by heating a dispersion of polyvinyl alcohol in water at 60°C for two hours, and then drying at room temperature. Untreated polyvinyl alcohol films are readily permeable to water and drugs, reducing its usefulness in controlled-release dosage forms. To reduce the permeability, cross-linking or increasing crystallinity via heating can be applied [131. Thus, studies of permeation

404

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 3 Maximum Recommended Solid Amounts for Polyvinyl Alcohol Solutions (3)

Percent Hydrolysis

Viscosity, cps (4

Maximum Recommended Solid

Super Hydrolyzed (99.3%)

28 - 32 62-72

10% 7%

Fully Hydrolyzed (98.0 - 98.8%)

5.5 - 6.6 28.5 - 32.5 62 - 72

20% 10% 7%

Intermediate Hydrolyzed (95.5 - 97.5%)

14 - 17 27 -31

15% 10%

Partially Hydrolyzed (87 - 89%)

3.5 - 4.5 5.2 - 6.2 23 - 27 45 - 55

30% 20% 10% 7%

(a) 4% aqueous solutions, 20°C

POLYVINYL ALCOHOL

405

Table 4

Kinematic Viscosity Determination of Polyvinyl Alcohol Solutions (W40/140, Wacker Chemical Company); n=6 191

Polyvinyl Alcohol Concentration (%)

Kinematic Viscosity at 33°C (mm2s-1)

Kinematic Viscosity at 37°C (llldS-1)

0

0.80 20.01

0.72 50.01

1

1.86 20.01

1.70 50.02

3.5

17.32 k1.10

13.22 L0.59

5

56.95 L1.91

46.23 L0.44

406

DAVID WONG AND JAGDISH PARASRAMPURIA

and absorption of polyvinyl alcohol films are important in the design of formulations/dosage forms [ 11- 131. Three principal forms of water are recognized in films, with the water being designated as either tightly bound, moderately bound or free. Tightly bound water is taken as the water bound within the crystalline phase of the polymer, and can be determined by an intense extraction procedure using vacuum over P202 for a week [8]. Moderately bound water is envisioned as the water adsorbed to hydroxyl groups in the amorphous phase of a polymer. The amount of moderately bound water can be determined by using thermogravimetric analysis (Figure 2). Figure 2 shows a transition from 30°C to 130"C, with a 10% original film weight loss, and a sharper transition at about 260°C with a further 70% weight loss. The first transition corresponds to loss of moisture, while the second transition represents film decomposition and a further loss of moisture. Finally, the free water is that which resides in a bulk or condensed form on the surface or within voids of films [8]. This can be determined by the mass balance of the total moisture content obtained under static conditions (WA), moderately bound (WM)), and tightly bound water contents (WT):

Moisture content data for a typical polyvinyl alcohol film at 25°C are shown in Table 5.

4.4

Permeation of Polyvinyl Alcohol Films

Water-soluble additives (such as citric acid or urea) have been found to lower the moisture diffusion through polyvinyl alcohol films, but also increase their solubility coefficients without changing their permeability coefficients. Usually, 5-10 wt. YOare the estimated limits of compatibility with the film-former for the additives [ 1 11. Heat treatment of polyvinyl alcohol has been used to increase its crystallinity, which also reduces the water solubility, equilibrium water content, and degree of swelling [ 13,18,19]. Without heat treatment, polyvinyl alcohol films dissolve slowly in water. However, increasing the heating time at i00"C decreases the membrane permeability to methylene

POLYVINYL ALCOHOL

Figure 2.

407

Thermogravimetric analysis of a polyvinyl alcohol film [S].

Temperature ("C)

408

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 5 Moisture Content Data for a Typical Polyvinyl Alcohol Film at 25OC [8]

Condition

Moisture Content

@> ~~~

Just after casting

9.65 i 0 . 2 8

Storage at 75% relative humidity for one week

23.24 L0.5 1

Tightly bound water

7.84 f 0.12

Moderately bound water

10.10 5 0.4

Free water

5.30

POLYVINYL ALCOHOL

409

blue, as has been illustrated in Figure 3. Similarly, drug release from polyvinyl alcohol films is slowed by increasing the time of heat treatment (Figure 4).

4.5

Glass Transition

The differential scanning calorimetry (DSC) and thermomechanical analysis (TMA) themograms of a polyvinyl alcohol film containing no additives are shown in Figure 5 [lo]. Three transitions are present in the DSC thermogram: a glass transition (Tg) at about 70°C, an unknown endotherm (Tu) at 12O-15O0C,and a melting endotherm (Tm) between 150-215°C. Three transitions were also found in the penetration mode of the TMA thermogram: two transitions indicating polymer softening (Ts 1 and Ts2) and another (Tm) related to melting/degradationof polyvinyl alcohol. However, Tg transitions were not visible in the expansion mode of the TMA thermogram. The effect of citric acid and urea on the Tg, Ts, and Tm of polyvinyl alcohol films has been reported [lo]. Urea decreases Tg, Ts, and Tm to a slightly larger extent than does citric acid.

4.6

Crystallinity and Preparation of Polyvinyl Alcohol Hydrogels

Polyvinyl alcohol is reported to be semicrystalline [lo]. Incorporating additives such as urea and citric acid into the material changes its crystallinity. The percent change in crystallinity is usually obtained by comparing the corresponding heat of fusion obtained for pure polyvinyl alcohol with that of material containing additives. A typical data set is provided in Table 6 . Polyvinyl alcohol hydrogels can be obtained by increasing the degree of crystallinity. A semicrystalline or densified polyvinyl alcohol structure can be enhanced by either cooling a solution consisting of water and a water-miscible solvent, or by exposing an aqueous polyvinyl alcohol solution to several freeze-thaw cycles. Typical data obtained for this process are shown in Table 7 [l , 56-58]. The structure of hydrogels formed by cooling a polyvinyl alcohol solution is porous, in which the pore diameter can be varied by changing the concentration of the solvent used. Scanning electron micrographs of a polyvinyl alcohol hydrogel prepared by exposing solutions to several freeze-thaw cycles are shown in

410

DAVID WONG AND JAGDISH PARASRAMPURIA

Figure 3 .

Effects of heating time at 100°C on polyvinyl alcohol membrane permeability (P=l/Rm) to methylene blue at 37°C. Error bars represent the standard deviations [13].

0-1 r I t I I I I I I 0 20 40 60 80 100 120 140 160 Heating h e a t IOO'C, hours

POLYVINYL ALCOHOL

Figure 4.

41 1

Release profiles of sulphathiazole from polyvinyl alcohol films subjected to different periods of heat treatment at 160°C. Time periods of 1 hour (V), 2 hours (A), 3 hours (0)and 4 hours (0) are shown.

412

Figure 5 .

DAVID WONG AND JAGDISH PARASRAMPURIA

Differential scanning calorimetry and thermomechanical analysis thermograms of polyvinyl alcohol film [ 101.

Temperature “C

POLYVINYL ALCOHOL

413

Table 6 Heats of Fusion and Percent Change in Crystallinity of Polyvinyl Alcohol Films Containing Citric Acid and Urea [lo]

Additive

Citric Acid

Urea

Percent Crystallinity Change

Content (wt %)

Percent Crystallinity Change

0

30.9

0.0

30.9

0.0

1

32.1

+3.8

27.7

-10.3

3

29.1

-5.8

26.2

-15.2

28.2

-8.7

22.5

-27.3

25.4

-17.8

19.4

-37.2

DAVID WONG AND JAGDISH PARASRAMPURIA

414

Table 7 Heat of Fusion and Degree of Crystallinity of Freeze-thawed Polyvinyl Alcohol Gels after Five Freeze-thaw Cycles [57]

Thawing Time at 25°C (hours)

Degree of Crystallinity, Dry Basis

Heat of Fusion (Jk)

Degree of Crystallinity, Swollen Basis (%)

47.45 O

5.5

I

1

29.34

I

34.2

5.4

21.1

3.6

5.1

9.7

POLYVINYL ALCOHOL

415

Figure 6. Other ways to prepare polyvinyl alcohol hydrogels include the cross-linking of polyvinyl alcohol by a mixture of aldehydes, or by exposing polyvinyl alcohol to an electron beam or gamma-rays [6]. A proposed structure for polyvinyl alcohol cross-linkages is shown in Figure 7 [61. 4.7

Surface Properties of Polyvinyl Alcohol Hydrogels

Alkylation of a polyvinyl alcohol hydrogel surface increases the percent transmittance of the infrared absorption at 3300 wavenumbers associated with the hydroxyl group, but decreases the absorbance of the urethane peaks at 1690 wavenumbers (Figure 8) [2]. Such alkylation can be quantitatively analyzed by using x-ray photoelectron spectroscopy, with typical data being shown in Figure 9 [2]. The degree of surface irregularity of hydrogels can be observed easily by scanning electron microscopy (Figure 10) [11.

4.8

Optical Properties of Polyvinyl Alcohol Hydrogels

The transmittance of light through a polyvinyl alcohol hydrogel increases with increasing concentration of dimethyl sulfoxide (DMSO) used in the solvent mixtures associated with its preparation (Figure 11).

4.9

Mechanical Strength of Polyvinyl Alcohol Hydrogels

The mechanical properties (such as tensile strength and elongation) of polyvinyl alcohol hydrogels prepared by freeze-thaw cycles or the cooling method are dependent on the composition of their solvent mixtures, polyvinyl alcohol concentrations, and crystalline polyvinyl alcohol content. These relationships have been illustrated in Figures 12 and 13. Table 8 compares the physical properties of polyvinyl alcohol hydrogels prepared from a 20% solution in a mixture of water and DMSO (20%/80%) with the properties of hydrogels made of other polymers. The elongation at the breaking point of the polyvinyl alcohol hydrogels was found to be approximately three times that obtained for the other materials.

416

Figure 6.

DAVID WONG AND JAGDISH PARASRAMPURIA

Scanning electron micrographs for polyvinyl alcohol gels prepared without and with pluronic block polymer L-62 [ S S ] . Images are shown for L-62 concentrations of (a) 0% and (b) 1.O%, where the scale bar equates to 10 pm.

POLYVINYL ALCOHOL

Figure 7.

Schematic representation of the swelling of a semicrystalline polyvinyl alcohol network [6].

417

118

DAVID WONG AND JAGDISH PARASRAMPURIA

FTIWATR spectra of (a) polyvinyl alcohol, (b) polyvinyl alcohol after 4 hours of reaction in dimethyl formamide, and (c) polyvinyl alcohol after 9 hours of reaction in dimethyl sulfoxide [2].

Figure 8.

If 4

94 74

54

34 14

t15

97 79

61

50 43

Wave number

POLYVINYL ALCOHOL

Figure 9.

419

High-resolutionx-ray photoelectron spectra obtained for the carbon 1s region of polyvinyl alcohol after 4 hours of reaction in dimethyl formamide (upper trace) and polyvinyl alcohol after 9 hours of reaction in dimethyl sulfoxide (lower trace) [2].

278.3

2983 8iflding €rtergy (eV)

420

Figure 10.

DAVID WONG AND JAGDISH PARASRAMPURIA

Scanning electron micrographsof polyvinyl alcohol hydrogels obtained from water and dimethyl sulfoxide mixed solutions of polyvinyl alcohol. The ratios of water:DMSO are (A) 100:0, (B) 80:20, (C) 60:40, (D) 40:60, (E) 20:80, and (F) 0:lOO. The scale bar equates to 5

Pm P I .

POLYVINYL ALCOHOL

Figure 11.

42 1

Measurements of the light transmittance of polyvinyl alcohol hydrogels at different concentrations of dimethyl sulfoxide [ 13.

Q

0

E

E tJa

C

a

L

V

V

t

P .-.

0

20

40

60

ao 100

Concentration of OMSO in water (w/w%)

422

DAVID WONG AND JAGDISH PARASRAMPURIA

Figure 12.

40

Relationship between the tensile strength (0)and elongation (0)of polyvinyl alcohol hydrogels and their concentration of polyvinyl alcohol (water:dimethyl sulfoxide ratio = 2090) [l].

m

- 400 a

00

I

I

10

t

I

I

20

P V A concentratton ( % )

200

0 30

POLYVINYL ALCOHOL

Figure 13.

423

Variation of the tensile modulus of semicrystalline polyvinyl alcohol hydrogels with the crystalline polyvinyl alcohol content (density = 2.66 X mole/cm3) [6].

"E -' I '

I

I '

I

> 11 U

I

'

I

'

e -

>r

{9c

5t20

CRYSTALLINE

PVA

30 CONTENT ('I*)

1

I

-I

324

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 8 Physical Properties of Hydrogels Prepared Using Various Polymers [11

Polyvinyl Alcohol

Polyhydroxyethyl Methacry late

Copolymer of Methylmethycrylate and N-vinyl Pyrrolidone

(%I

78

38

78

Tensile strength (kgkm2)

47

Physical property

Water content

Elongation

("/I

500

Light transmittance (?%)a

99- 100

99-100

99- 100

Oxygen permeability b

44

10

46

------t

(a) 550 nm, 0.2 mm thick in water (b) 10-1 1 cm3 (STP) crn2/cm3 s mm Hg (35°C)

POLYVINYL ALCOHOL

425

The mechanical strength of cross-linked polyvinyl alcohol hydrogel has been found to be proportional to the content of cross-linking agents, such as magnesium chloride, formaldehyde, or glutaraldehyde [ 191. Table 9 shows that the effect of inversely increasing cross-linking agents parallels the effect on the compression modulus and cross-link density. As the water content decreases by increasing glutaraldehyde concentration, the gel becomes stronger with higher compression moduli and cross-link densities. 4.10

Water Content and Swelling Properties of Polyvinyl Alcohol Hydrogels

Polyvinyl alcohol hydrogels do not dissolve in water, but will swell in volume upon contact with bulk water. After polyvinyl alcohol films or hydrogels are swollen in buffers for a predetermined period, their water content can be calculated using: water content

where:

=

Ww Wd

Ww-Wd WW = =

weight of swollen film weight of dry film

The weight of dry film can be found by drying films/gels for 48 hours, with the temperature being gradually increased from 40 to 70°C. In most cases, water does not influence the crystalline regions upon swelling of polyvinyl alcohol hydrogels [ 6 ] . The swelling behaviors of most hydrogels can be characterized using similar calculations. The effect of ionic strength on the swelling behavior of heparin- polyvinyl alcohol hydrogel is shown in Table 10 [36]. It was found that the hydrogels shrank as the ionic strength of the medium was increased. This phenomenon can be explained by the screening effect of salt on the charges within the gel. 4.1 1

Protein Adsorption by Polyvinyl Alcohol Hydrogels

The study of protein adsorption by polyvinyl alcohol hydrogels is important, since these hydrogels are used as contact lenses and artificial tissues. In Figure 14, the effect of alkylation on the amount of albumin

426

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 9 Physical Characteristics of Selected Polyvinyl Alcohol Hydrogels [191

Formulation

Compression Modulus (kg/cm2)

10% polyvinyl alcohol; 0.5% glutaraldehyde

7.8 k 0.14

Cross-link Density (gram moie/c&)

1.41

x 10-4

Water Content (%, w/w)

74.7 & 1.0

10% polyvinyl

alcohol; 2% glutaraldehyde

68.9 k 3.1

POLYVINYL ALCOHOL

427

Table 10 Shrinkage of Heparin-PolyvinylAlcohol Gel after Incubation in Solutions of Varying Ionic Strengths [36]

Relative Weight of Gel (n=3) ~~~

0

1 .oo L 0.01

0.15

1.05 k 0.06

0.50

0.99 f.0.08

1.o

0.89 k 0.04

2.0

0.76 2 0.04

3.O

0.68 k 0.01

All solutions contain 0.01M sodium phosphate

428

DAVID WONG AND JAGDISH PARASRAMPURIA

Figure 14.

Surface concentration of albumin on polyvinyl alcohol, solvent-treated polyvinyl alcohol, and alkylated polyvinyl alcohol after 1 hour adsorption from a 9.4 pM solution of albumin. Results are presented as the mean k SD for the indicated number of tubes; each tube value being a mean of several segments [2].

POLYVINYL ALCOHOL

429

adsorbed to the polyvinyl alcohol surface is illustrated [2]. It was found that the degree of adsorption increased with increasing alkyl chain length. The adsorption rates of three different proteins are shown in Table 11, where it may be seen that polyvinyl alcohol hydrogel adsorbed the least amount of protein [l].

5.

Methods of Analysis

5.1

Compendia1 Methods

According to the United States Pharmacopeia [7], polyvinyl alcohol is a synthetic resin, for which the number of repeating units (the n factor in Figure 1) lies between 500 and 5000. It is prepared by the 85-89% hydrolysis of vinyl acetate. 5.1.1 pH The pH is determined using USP general test <791>, and should be between 5.0 and 8.0 for a 1 in 25 solution. 5.1.2 Loss on Drying When the solid is dried at 110°C to constant weight, is cannot lose more than 5.0% of its weight. The details of the method are given in USP general test <73 1>. 5.1.3 Residue on Ignition When tested according to USP general test <281>,the residue on ignition cannot be more than 2.0%. 5.1.4 Viscosity After determining the loss on drying, weigh a quantity of undried polyvinyl alcohol, equivalent to 6.00 g on the dried basis. Over a period of seconds, transfer the test specimen with continuous slow stirring to about 140 mL of water contained in a suitable tared flask. When the

430

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 11 Protein Adsorption Rates of Hydrogels [l]

Hydrogel

Water Content (%)

Immunoglobulin G (pg/cm2)

Bovine Serum Albumin (vdcm2)

Polyvinyl Alcohol

78

0.074

0.005

0.195

Polyhydroxyethyl Methacrylate

40

0.271

0.037

0.230

78

0.889

0.169

4.991

Copolymer of Methylmethycrylate and N-vinyl Pyrrolidone

POLYVINYL ALCOHOL

43 1

specimen is well-wetted, increase the rate of stirring, avoiding mixing in excess air. Heat the mixture to 90"C, and maintain the temperature for about 5 minutes. After that, discontinue the hearing, and continue stirring for 1 hour. Add enough water to bring the mixture up to a total weight of 150 g, and resume stirring to obtain a homogeneous solution. Filter the solution through a tared 100-mesh screen into a 250-mL conical flask, cool to about 15"C, mix, and measure the viscosity according to USP general test <911>.

5.1.5 Water-Insoluble Substances Wash the tared 100-mesh screen used in the test for viscosity with two 25mL portions of water, and dry at 110°C for 1 hour. Not more than 6.4 mg (0.1%) of water-insoluble substances can be found.

5.1.6. Degree of Hydrolysis Transfer about 1 gram of polyvinyl alcohol, previously dried at 110°C to constant weight and accurately weighed, to a wide-mouth, 250-mL conical flask fitted by means of a suitable glass joint to a reflex condenser. Add 35 mL of dilute methanol (3 in 5), and mix gently to assure complete wetting of the solid. Add 3 drops of phenolphthalein test solution, and add 0.2 N hydrochloric acid or 0.2 N sodium hydroxide (if necessary) to neutralize. Then add 25.0 mL of 0.2 N sodium hydroxide VS, and reflux gently on a hot plate for 1 hour. Wash the condenser with 10 mL of water, collecting the washings in the flask, cool, and titrate with 0.2 N hydrochloric acid VS. Concomitantly perform a blank determination in the same manner, using the same quantity of 0.2 N sodium hydroxide VS. Calculation of the saponijication value. Calculate the saponificationvalue by the formula:

[(B - A) N 56.1 11 / W where B and A are the volumes (in mL) of 0.2 N hydrochloric acid VS consumed in the titration of the blank and test preparation, respectively. N is the exact normality of the hydrochloric acid solution, W is the weight (in grams) of the portion of sample taken, and 56.1 1 is the molecular weight of potassium hydroxide.

432

DAVID WONG AND JAGDISH PARASRAMPURIA

Culculution of the degree of hydrolysis. Calculate the degree of hydrolysis, expresses as a percentage of hydrolysis of polyvinyl acetate, by the formula: 100 - [7.84 S / (100 - 0.075 S)] in which S is the saponification value of the sample taken. The degree of hydrolysis should be within 85% and 89%.

5.2

Other Analysis Studies

Filter paper treated with potassium iodide and iodine solutions has been used to measure polyvinyl alcohol concentrations in waste water over a concentration range of 1000 to 20000 ppm [5]. Polyvinyl alcohol can be reacted with boric acid to yield a green complex which can be used to detect small amounts of the compound in polyvinyl chloride resins [ 5 ] . The molecular weight of polyvinyl alcohol is determined by gel permeation chromatography [2 I]. Turbidity is used to assay polyvinyl alcohol in biological media [5]. Combinations of various thermal techniques, such as differential scanning calorimetry, thermomechanical analysis, and thermogravimetric analysis have been demonstrated to assess the glass transition, softening, melting, plasticization, and crystallinity and moisture content of polyvinyl alcohol [8, lo]. Compression modulus, tensile strength and elongation are commonly employed to assess the physical strength of polyvinyl alcohol hydrogels and films. X-ray diffraction and UVNIS absorption spectra have been used to characterize the effect of tacticity of polyvinyl alcohol on its complexion with iodine [ 141. Linear dichroism techniques and polarized photoacoustic, absorption, and fluorescence spectra were used to study the orientation of pigments in stretched polyvinyl alcohol films [26,15]. Nuclear magnetic resonance (NMR) has been used to investigate the structural consistency of the polyvinyl alcohol copolymer [2 11. Fourier transform infrared spectroscopy coupled with attenuated total reflectance optics, x-ray photoelectron spectroscopy, and scanning electron microscopy were shown to be useful in characterizing the surface properties of polyvinyl alcohol hydrogels [ 1,2].

POLYVINYL ALCOHOL

6.

433

Stability

Polyvinyl alcohol degrades slowly at 1OO'C, but the degradation takes place rapidly at 200°C [63,64]. The polymer is considered to be biodegradable because of its solubility in water [ 161. The degradation process initiates with water diffusing into the polyvinyl alcohol matrix, followed by swelling, fragmentation, and dissolution. An apparent first-order kinetics is observed for the hydrolysis of polyvinyl alcohol. The hydrolysis mechanism has been found to be that of specific hydrogen- and hydroxyl-ion catalysis, and the pH of optimal stability is 4.75 [17]. Polyvinyl alcohol also undergoes esterification and the other reactions of a compound with secondary hydroxyl groups. At high concentrations, it is incompatible with sulfates and phosphates [63].

7.

Applications

Polyvinyl alcohol is considered an emulsifyingagent, a suspending agent, and an emollient in pharmaceutical science [60,63-651. Consequently, it is employed as a lubricant and protectant in various liquid preparations, such as topical lotions, suspensions, creams, ophthalmic solutions, artificial tears, vaginal emulsions, aerosol foams, and decongestants [65,60]. Other applications include oral tablets and film-coated tablets, as well as films for transdernial controlled release [60,49]. Recently, drug-containing polyvinyl alcohol microparticles have been prepared by emulsion, spray-drying, spray-desolvation,or spray polycondensation [53,54]. When a sparingly water-soluble drug is embedded in polyvinyl alcohol microspheres, its dissolution rate is enhanced by increasing its wettability and its total surface area[69]. Such formulations require a fine dispersion of drug particles in the polyvinyl alcohol matrix, When a water-soluble drug is embedded in polyvinyl alcohol particles, its release is sustained by the viscous gel formed at the microsphere surface [20,29]. If a cross-linking agent is involved in the preparation, drug release will be inversely proportional to the amount of cross-linking agents or the dose of initiating UV irradiation.

434

DAVID WONG AND JAGDISH PARASRAMPURIA

Polyvinyl alcohol is suitable for various applications because of its high film strength, flexibility, toughness, abrasion resistance, and adhesive strength. In the United States in 1976, polyvinyl alcohol was used mainly in textile warp-sizing and finishing, adhesives, polymerization, and papersizing and coating [64,5,55]. It provides a high weaving efficiency at low levels, and stiffness for textile warp size and finish. Polyvinyl alcohol, combined with polyvinyl acetate and starch, is used as an adhesive in bag making, carton sealing, tube winding, and solid-board lamination. It also facilitates polymerization in polyvinyl acetate emulsions for adhesive uses. Polyvinyl alcohol is also used in grease-resistant paper coating and binding for pigments in certain paper coatings. Miscellaneous other applications include building products, packaging, chemicals, cosmetics, ceramics, steel, automotive safety glass, and materials binding [16]. Polyvinyl alcohol finds application in medical technology and surgery. Polyvinyl alcohol sutures have demonstrated low tissue reaction and loss of strength. Polyvinyl alcohol is sometimes cross-linked into hydrogels for special uses in artificial tissues, contact lenses, dressing material, and embolisms. These materials show low protein absorption, mild tissue reaction, and advantageous physical properties. However, they must be washed thoroughly in a sterile solution before use since they are prepared in formalin. In some cases, a special functional group is added to polyvinyl alcohol hydrogels for some special functions, such as thrombin inactivation. Some examples of polyvinyl alcohol applications in medical areas are listed in Table 12.

8.

Pharmacological Effects and Toxicity

Polyvinyl alcohol is not classified as a hazardous material according to the American Standard for Precautionary Labeling of Hazardous Industrial Chemicals [ANSI 2129.1- 19761, However, some studies show that polyvinyl alcohol sponges cause local calcification, fibrosis, and sarcomas [5,16,32]. Repeated injections of concentrated polyvinyl alcohol solutions or suspensions can produce toxicities such as glomerulonephritis and portal hypertension [61,62]. The oral toxicity rate and ecological information for polyvinyl alcohol is listed in Table 13.

435

POLYVINYL ALCOHOL

Table 12 Examples of Polyvinyl Alcohol Applications in Medical and Pharmaceutical Technologies

Application

I Artificial tears and wetting- agents for contact lenses Coat for implanted intraocular lenses Dressing material for donor sites and partial thickness skin-loss bums Intraluminal intervascular splints to replace sutures for anastomosis Daily-wear contact lenses Artificial tissues Heparin-polyvinyl alcohol hydrogel Intravaginal contraceptive barriers Remover of low-density lipoprotein Particles used in embolization therapy Glucose responsive insulin-release system Disposable rubber-like elastic electrode for the electroretinogram Material for magnetic resonance imaging (MRI) for soft tissue Protective material for unfixed cryostat sections Mounting medium for preserving fecal material, ova, and parasites Medium for embedding hemoglobin and myoglobin for XI ray studies Material for freeze-fracture technique Medium for electrophoretic size separation of particles Material for inducing disease states of animals to serve as models Protectants against fluid-mechanical injury of freelysuspended cells Supporter for immobilizing biocatalysts

Reference(s)

I 31,15,23,38 _ . ,

I

28 50 25 1

32-34 2, 36 24 27 39,40-46,68 52 47

u 22

_ _ ~

37 48 61, 62 66 67

I

436

DAVID WONG AND JAGDISH PARASRAMPURIA

Table 13 Toxicological and Ecological Information on Airvol-205 Polyvinyl Alcohol

I

1

Acute Toxicity

Polyvinyl Alcohol Dose

Oral LD50 (rat)

23,854 mg/kg

Dermal LD50 (rabbit)

I

>7490 mgkg

Inhalation L C ~ O (rat)

I

64,000 p p d 4 h

I

8300 m g L 96 h

LC 5 0 daphnia magna

r

I

Environmental Fate

I Chemical Oxygen Demand (COD) I ~~

~~

1800 mg/g

1

Biochemical Oxygen Demand

I

BOD5 = 0 - 5%; BOD = 100%

I

I

Biodegradability

I

>90% (Zehn-Wellens Test)

I

POLYVINYL ALCOHOL

437

9.

REFERENCES

1.

M. Kita, Y. Ogura, Y. Honda, S. H. Hyon, W. Cha, and, Y. Ikada, Graefe's Arch. Clin. Exp. Ophthulmol., 228, 533-537 (1990).

2.

I. Strzinar, and M. V. Sefton, J. Biomed. Mat. Res., 26, 577-592 (1992).

3.

Air Products, Airvol@ Polyvinyl Alcohol, 1-12 (1993).

4.

T . W. Modi, Handbook of Water-soluble gums & resins, In Davidson, ed., 1980, McGram-Hill Book Company, pp., 20-1 to 2031.

5.

IARC Monographs on the Evaluation of the Carcinogenic Risk of Chemicals to Humans, 19,341-366 (1979).

6.

N. A. Peppas, and E. W. Merrill, J. Biomed. Muter. Res., 11, 423434 (1977).

7.

U. S. Pharmacopeia National Formulary, United Pharmacopeid Convention, Inc., 23, 1288 (1995).

8.

A. 0. Okhamafe, and P. York, J. Pharm. Pharmacol., 37, 385-390 (1984).

9.

C. G. Wilson, 0. Olejnik, and J. G. Hardy, J. Pharm. Phamcol., 35, 451-454 (1983).

10.

A. 0.Okhamafe, and P. York, J. Pharm. Sci., 77, 438-443 (1988).

11.

A. 0. Okhamafe, and H. U. Iwebor, Phurmuzie, 42, 611-613 (1987).

12.

M. J. Reimers, M. J. Cibulsky, and T. A. Barbari, J. Polymer Sci.: Part B: Polymer Physics, 31, 537 - 543 (1993).

13.

P. R. Byron, and R. N. Dalby, J. Pharm. Sci., 76, 65-67 (1987).

14.

Y. Choi, and K. Miyasaka, J. Applied Polymer Sci., 48, 313-317 (1993).

15.

L. J. Juszczak, B. A, Zilinskas, N. E. Geacintov, J. Breton, and K . Sauer, Biochimica et Biophysica Acta, 1058, 363-373 (1991).

16.

D. K. Gilding, Biocompatibilityof Clinical Implant, In D. F. William ed., 1981, CRC Press, Florida, 11, pp., 209-232.

States

438

DAVID WONG AND JAGDISH PARASRAMPURIA

17.

N. Alkhalili, and B. J. Meakin, J. Pharm. Pharmacol., 29, 35P ( 1 977).

18.

L. S. C. Wan, and L. Y. Lim, Drug Dev. & Indust. Pharm., 18, 1895-1905 (1992).

19.

P. K. Watler, C. H. Cholakis, and M. V. Sefton, Biomaterials, 9, 150-154 (1988).

20.

B. C. Thanoo, M. C. Sunny, and A. Jayakrishnan, J. Phurm. Pharmcol., 45, 16-20 (1993).

21.

D. Miller, and N. A. Peppas, Biomaterials, 7 , 329-339 (1986).

22.

I. Mano, H. Goshima, M. Nambu, and M. Iio, Magnetic Resonance in Medicine, 3, 921-926 (1986).

23,

D. J. Nelson, and R. L. Farris, Arch. Ophthalmol., 106, 484487 (1 988).

24.

M. Chvapil, T. A. Chvapil, J. A. Owen, M. Kantor, J. B. Ulreich, and C. Eskelson, J. Biomedical Materials Res., 13, 1-3 ( 1979).

25.

K. H. Niijima, Y. Yonekawa, H. Handa, and W. Taki, J. Neurosurg., 67, 579-583 (1987).

26.

D. Frackowiak, L. Lorrain, D. Wrobel, and R. M. Leblanc, Biochem. & Biophy. Res. Comm., 126, 254-261 (1985).

27.

N. Maaskant, A. Bantjes, and H. J. M. Kempen, Atherosclerosis, 62, 159-166 (1986).

28.

R. J. Olson, H. Kolodner, K. S. Morgan, H. Escapini, D. Sevel, and H. E. Kaufman, Arch. Ophthalmol., 98, 1840-1842 (1980).

29.

L. B. Wingard Jr., T. R. Tritton, and K. A. Egler, Cancer Res., 45, 3529-3536 (1985).

30.

Seidel, J., Pediatrics, 66, 132-134 (1980).

31.

D. A. Benedetto, D. 0. Shah, and H. E. Kaufman, Annals of Ophthal.,April, 537-442 (1978).

32.

P. H. B. Bolton-Maggs, and R. W. Motson, Thorax, 34, 561562 (1979).

POLYVINYL ALCOHOL

439

33.

M. A. Aboulghar, and Y. El-Kateb, J. Egypt. Med. Assoc., 61, 127-134 (1978).

34.

K. Tamura, H. Mizuno, K. Okada, H. Katoh, S . Hitomi, and T. Teramatsu, Biomat., Med. Dev., Art. Org., 13 (3&4), 133-152 (1985 - 86).

35.

T. Y. Teng, and H. W. Huang, Biochimica et Biophysica Acta, 874, 13-18 (1986).

36.

M. F. A. Goosen, and M. V. Sefton, J. Biomedical Materials Res., 17, 359-373 (1983).

37.

D. Brown, J. Microscopy, 121, 283-287 (1981).

38.

Zaki, P. Fitzgerald, J. G. Hardy, and C. G. Wilson, Pharm. Pharmacol., 38,463-466 (1986).

39.

I. A. Szwarc, C. H. Carrasco, and W. Richli, AJR, 146, 591-592 (1986).

40.

K. C. Wright, J. H. Anderson, C. Gianturco, S . Wallace, and V. P. Chuang, Radiology, 142, 351-354 (1982).

41.

S. Kusano, M. Koichiro, H. Ohuchi, 0. Motohashi, and H. Atari, Investigative Radiology, 22, 388-392 (1987).

42.

J. A. Belis, and J. A. Horton, Urology, 19, 224-227 (1982).

43.

C. R. Jack, G. Forbes, M. K. Dewanjee, M. L. Brown, and F . Earnest IV, AJNR, 6, 595-597 (1985).

44.

J. I. Ausman, L. H. Gold, S . M. Tadavarthy, K. Amplatz, and S . N . Chou, J. Neurosurg., 47, 119-125 (1977).

45.

A. Berenstein and D. A. Graeb, Radiology, 145, 846 (1982).

46.

Sadi, H. I., J. Comp. Path., 87, 503-513 (1977).

47.

Y. Honda, N. Nao-i, S. Kim, E. Sakaue, and M. Nambu, Documenta Ophthalmologica, 63,205-207 (1986).

48.

T. Guszczynski, M. M. Garner, M. Deml, and A. Chrambach, Applied and Theoretical Electrophoresis, 2, 151-157 (1991).

49.

Inactive Ingredients for Currently Marketed Products, The Interphaim Group of Companies, Buffalo Grove, 116 (199 1).

40

DAVID WONG AND JAGDISH PARASRAMPURIA

50.

J. W. L. Davies, Burns, 10, 94-103 (1984).

51.

R. G. Butcher, Histochemical Journal, 14, 165-168 (1982).

52.

S. Kitano, Y. Koyama, K. Kataoka, T. Okano, and Y. Sakurai, J . Controlled Release, 19, 162-170 (1992).

53.

C. Voellmy, P. Speiser, and M. Soliva, J. Pharm. Sci., 66, 631-634 ( 1 977).

54.

T. Y. Ting, I. Gonda, and E. M. Gipps, Pharm. Res., 9, 1330-1335 ( 1 992).

55.

0. Augustine, and P. York, J. Pharm. Phurmacol., 37, 492-493 ( I 985).

56.

N. A. Peppas, and S. R. Stauffer, J. Controlled Release, 16, 305310 (1991).

57.

N. A. Peppas, and J. E. Scott, .I. Controlled Release, 18, 95-100 (1992).

58.

A. Takamura, F. Ishii, and H. Hidaka, J. Controlled Release, 20,

21-28 (1992). 59.

Material Safety Data Sheet (Airvol# 205 PV Alcohol), Air Products and Chemicals, Inc.

60.

In A. R. Gennaro ed., Remington’s Pharmaceutical Sciences, 1990, Marck and Co., Inc., 18th edition, p., 1307.

61.

F. A. Burgener, 0. H. Gutierrez, and G. A. Logsdon, Radiology, 143, 379-385 (1982).

62.

J. E. Riviere, G. L. Coppoc, W. W. Carlton, and E. J. Hinsman, Am J Vet. Res., 41, 502-505 (1980).

63.

In S. Hickok ed., Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, 1994,2nd Edition, pp., 383-384.

64.

In S. Budavari ed., The Merck Index, Merck & Co., Inc., 1989, 1lth edition, p., 1208.

65.

In J. E. F. Reynolds ed., Martindale The Extra Pharmacopoeia, The Pharmaceutical Press, 1993, p., 1031.

66.

J. D. Michaels and E. T. Papoutsakis, J. Biotechnol., 19, 241-257 (1 99 1).

POLYVINYLALCOHOL

441

67.

H. Ichijo, J. Nagasawa, and A. Yamauchi, J. Biotechnol., 14, 169178 (1990).

68.

J. J. Kepes, and W. L. Yarde, The Am. J. Surg. Path., 6 , 709-711 (1995).

69.

D. Wong, B. Nguyen, and R. Bodmeier, Pharm. Res. Supp., 10, S276 (1993).

This Page Intentionally Left Blank

SERTRALINEHYDROCHLORIDE

Bruce M. Johnson and Pei-Tei L. Chang

Central Research Pfizer Inc Groton, CT 06340

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

443

Copyright 0 1996 by Academic Press, Inc. All rights of reproductionin any form reserved.

w4

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

SERTRALXNE HYDROCHLORIDE 1. Introduction 2. Description 2.1. Structural Formula 2.2. Molecular Formula and Molecular Weight 2.3. Nomenclature 2.4. Laboratory Codes 3. Synthesis 4. Physico-Chemical Properties 4.1. Appearance, Color, Odor 4.2. Melting range 4.3. Solubility 4.4. Hygroscopicity 4.5. Ultraviolet Spectra 4.6. Infrared Spectra 4.7. Proton Nuclear Magnetic Resonance Spectra 4.8. Carbon- 13 Nuclear Magnetic Resonance Spectra 4.9. Mass Spectra 4.10. Optical Rotation 4.11. pH andpKa 4.12. Single Crystal X-Ray 4.1 3. Polymorphism 5. Methods of Analysis 5.1. Elemental Analysis 5.2, Ionic Chlorine 5.3. Identification 5.4. Thin Layer Chromatography 5.5, Ultraviolet Spectroscopy 5.6. Potentiometric Titration 5.7. High Performance Liquid Chromatography 5.8. Gas Liquid Chromatography 5.9. Biological Fluids 6 . Stability 7. Pharmacokinetics and Metabolism 7.1. Systemic Bioavailability 7.2. Metabolism

SERTRALINE HYDROCHLORIDE

1.

445

introduction Sertraline hydrochloride is an antidepressant for oral administration. It is chemically unrelated to tricyclic, tetracyclic, or other available antidepressant agents. It is a novel inhibitor of serotonin reuptake in the brain'. The mechanism of action of sertraline is presumed to be linked to its inhibition of CNS neuronal uptake of serotonin (5HT).Studies at clinically relevant doses in man have demonstrated that sertraline blocks the uptake of serotonin into human platelets. Studies in animals also suggest that sertraline is a potent and selective inhibitor of neuronal serotonin reuptake and has only very weak effects on norepinephrine and dopamine neuronal reuptake. In vifrostudies have shown that sertraline has no significant affinity for adrenergic (alpha 1, alpha 2, beta), cholinergic, GABA, dopaminergic, histaminergic, and serotonergic (5HT1A, SHTlB, 5HT2)or benzodiazepine receptors; antagonism of such receptors has been hypothesized to be associated with various anticholinergic, sedative, and cardiovascular effects for other psychotropic drugs. The chronic administration of sertraline was found in animals to downregulate brain norepinephrine receptors, as has been observed with other clinically effective antidepressants. Sertraline does not inhibit monoamine oxidase.*

2.

Description

2.1.

Structural Formula

Sertraline is the S-cis enantiomer of a disubstituted tetrahydronaphthalene. The structural formula of sertraline hydrochloride is given below:

446

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

H-N-CH,

I

mHCI

Cl

Sertraline Hydrochloride 2.2.

Molecular Formula and Molecular Weight Molecular Formula

2.3.

Sertraline

c1 7H 17Nc12

Molecular Weight 306.2

Sertraline hydrochloride

Cl7H@C13

342.7

Nomenclature

Chemical Names: 1. ( 1S-cis)-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-Nmethyl-1-naphthalenaminehydrochloride (CAS-79559-97-0) Preferred use name.

2. cis-( lS,4S)-N-rnethyl-4-(3,4-dichlorophenyl)-1,2,3,4tetrahydro- 1-naphthalenamine hydrochloride (J. Med. Chern. 1984, 27(11), 1508) 3. cis-( 1S)-N-methyl-4-(3,4-dichlorophenyl)-1,2,3,4tetrahydro- 1-naphthalenamine hydrochloride (US Patent 4,536318)

SERTRALINE HYDROCHLORIDE

441

4. 1-naphthalenamine,4-(3,4-dichlorophenyl)-1,2,3,4tetrahydro-N-methyl, hydrochloride, ( 1S-cis)- (USAN chemical name 1) 5. (lS,4S)-4-(3,4-dichlorophenyl)-1,2,3,4-tetrahydro-Nmethyl- 1-naphthylamine hydrochloride (USAN chemical name 2)

2.4.

B.A.N.:

Sertraline hydrochloride

U.S. A.N. :

Sertraline hydrochloride

I.N.N.:

Sertraline hydrochloride

Proprietary or Trade Name:

Zolofim (USA) LustralTM(UK)

Laboratory Codes Sertraline has the Pfizer code CP-5 1,974 Sertraline hydrochloride has the Pfizer code CP-5 1,974-01

Synthesis The pure drug substance is manufactured from commonly available intermediates using a four step process. The structurally significant starting material is 4-(3,4-dichlorophenyl)-3,4-dihydro-1(2H)n a p h t h a l e n ~ n e which ~ s ~ ~is~reacted ~ ~ ~ ~with ~ methylamine to form the imine. The imine, (N-[4-(3,4-dichlorophenyl)-3,4-dihydro1(2H)-naphthalenylidenelmethanamine), is reduced by catalytic hydrogenation to a pair of racemic diastereomers. The racemic cis isomers are separated from the racemic trans isomers by selective crystallization of the hydrochloride salts. The racemic cis enantiomers are then resolved with D(-)-mandelic acid. In the final step the mandelate counterion is replaced by chloride to give the finished drug substance, sertraline hydrochloride.’* lo Crystallization conditions in the final step determine the polymorphic form produced.

BRUCE M.JOHNSON AND PEI-TEI L. CHANG

448

4.

Physico-Chemical Properties

4.1.

Appearance, Color, Odor

Sertraline hydrochloride is a white to off-white, crystalline powder having no odor. It is an irritant, contact with skin and eyes should be avoided.

4.2.

Melting range

Determination of the melting point by the capillary method in a Buchi 510 melting point apparatus with a heating rate of 1°C per minute on Form I shows the onset of change at about 160 to 180°C with movement of the sample and a slight color change. At about 218°C a partial melt is observed but the sample does not become clear until a temperature of 245 to 250°C is reached. This is consistent with the DSC and hot stage microscopic observations.

4.3.

Solubility

4.3.1. Aqueous Solubility The solubility of a saturated solution of sertraline hydrochloride (Form I) in distilled water at room temperature is 3.8 mg/mL. The pH of this saturated solution is 5.3. Solubility in water is pH dependent, as the data in the following table shows. These data were determined by preparing a saturated solution in distilled water, with pH adjustments made by addition of sodium hydroxide and/or hydrochloric acid, followed by appropriate filtering, diluting, and assay by HPLC.

SERTRALINE HYDROCHLORIDE

DH

1 .o 2.0 2.9 4.2 6.0 6.2 6.3 6.5 7.2 7.7 8.8 9.8 10.2 11.1 12.1

449

Solubility (mg/mL) 0.89 2.98 3.88 4.07 4.46 4.93 4.93 3.32 0.72 0.21 0.02 0.006

0.004 0.004 CO.004

4.3.2. Solubility in Other Solvents The approximate solubility of sertraline hydrochloride in selected solvents at room temperature is given below. These data were obtained at room temperature, with saturated solutions that were appropriately filtered, diluted, and assayed by HPLC. Solvent Aqueous 0.1 N HC1 Aqueous 0.1 N NaOH Ethanol Isopropyl alcohol Chloroform Acetone N,N-dimethy1formamide Dimethylsulfoxide Ethyl acetate Acetonitrile Methanolic 0.1 N HC1 Chloroform/methanol, 1: 1

Solubility (mg/mL,) 0.5 1 0.002 15.7 4.3 110 1.1 88 147 0.20 0.85 47 134

450

4.4.

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

Hygroscopicity

The hygroscopicity of sertraline hydrochloride (Form I) was determined by exposing a sample for one week to an atmosphere of 37"C/75% relative humidity, and a sample to room temperature/88% relative humidity. Karl Fischer titration was used to determine water content. The results showed water contents of
4.5.

Ultraviolet Spectra

The ultraviolet absorption spectra of sertraline hydrochloride were recorded in methanol, methanolic 0.01 N hydrochloric acid, and methanolic 0.01 N sodium hydroxide. These spectra are presented as Figures 1,2, and 3. Absorptivity data for sertraline hydrochloride are given below: Solvent 0.01 N HCYMethanol

&-nm

265 273 28 1

Absorptivity 2.58 3.04 1.64

Methanol

265 273 28 1

2.61 3.06 1.64

0.01 N NaOWMethanol

265 273 28 1

1.92 2.26 1.60

SERTRALINE HYDROCHLORIDE

45 1

The table below demonstrates that the absorptivity and hremain effectively unchanged in solutions of differing acid strengths. Acid Strength 0.005 N 0.01 N 0.05 N 0.10 N 0.50 N

Lax

273 273 273 273 273

Absorptivity 3.03 3.04 3.1 1 3.03 3.04

Sertraline hydrochloride also adheres to Beer's Law at this wavelength, as evidenced by the following data: Concentration (mg/mL) 0.101 0.201 0.302 0.402 0.503 0.603 0.704 0.804 0.905 1.01 avg. k std. dev.

Lax 273 273 273 273 273 273 273 273 273 273

Absorptivity 2.99 2.97 3.04 2.99 2.99 3.01 2.96 2.97 2.95 2.88 2.98 f 0.04

BRUCE M.JOHNSON AND PEI-TEI L.CHANG

452

A b S

0

r b a

n C

e

Figure 1. Ultraviolet Spectrum of Sertraline HC1 in Methanol

i

2.0

A b S 0

r b a

n C

Wawkngth (nm)

Figure 2. Ultraviolet Spectrum of Sertraline HCl in Methanolic HCl

SERTRALINEHYDROCHLORIDE

453

Wavelength (nm)

Figure 3. Ultraviolet Spectrum of Sertraline HCl in Methanolic NaOH

Infrared Spectra The infrared spectra of sertraline hydrochloride (Form I) as a potassium bromide disc and as a Nujol mull were recorded on a Nicolet 5 10 FTIR spectrophotometer; the spectra in KBr and Nujol are virtually identical except for the Nujol-related bands. The spectra are presented in Figure 4. Assignments of absorption maxima are given below. 4.6.

Band (approx. cm-1)

Assignment

3100 - 3000 (w)*

Aromatic C-H stretching vibrations

3000 - 2800 (m)*

Aliphatic C-H stretching vibrations

27 10 - 2500 (m)

NH+ stretching vibration

2500 - 2450 (m)

NH+ stretching vibration

454

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

1585 (m) 1560 (m)

Aromatic C=C skeletal in-plane vibrations C-H stretching vibration; aromatic H

1470 - 1450 (s)*

Aliphatic C-H deformations, N-CH, stretching vibration

1400 (s), 1430 (m)

Asymmetric CH3 deformation

1375 (m)

Symmetric CH3 deformation

1340 (m) 1215 (m)

Aromatic C-H in-plane deformations

1135 (s)

Ahphatic secondary amine

1060 (m) 1030 (m) 1015 (m) 955 (m) 930 (m) 920 (m)

overtones and aromatic C-H in-plane deformations

825 (s) 800 (s) 790 (s) 760 (s) 710 (m) 700 (s) 670 (s)

aromatic C-H out-of-plane deformations; C-Cl stretching vibrations

* (w)=weak intensity; (m)=medium intensity; (s)=strong intensity

The dlfferent polymorphs of sertraline hydrochloride yield different infrared spectra. Polymorphism is discussed more fully in section 4.13.

80 70 60

x T r a

n S

m

50

40

30

I

t t

20

a

n

C

10

a 0

-10

-20

-30 4000

U 2500

Figure 4. Infrared Spectra of Sertraline HCl in Nujol and KBr

m00

1500

1000

500

456

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

4.7.

Proton Nuclear Magnetic Resonance Spectra

The 300 MHz proton nuclear magnetic resonance spectrum of sertraline hydrochloride reference standard was recorded using a Briicker AM300 NMR instrument. The solvent was deuterated dimethylsulfoxide. Proton assignments are listed below, and the spectrum is reproduced as Figure 5. a

b

CI

Chemical Shift 1.9 - 2.4 ppm 2.6 ppm 4.1 - 4.2 ppm 4.4 - 4.5 ppm

6.7 - 6.8 ppm 7.2 - 7.4 ppm 7.35 - 7.5 ppm 7.5 - 7.65 ppm 7.7 ppm 7.75 - 8.0 ppm 9.5 - 9.8 ppm

Md tiDlicitv

# of Protons Assignment

broad, complex multiplet singlet broad, complex multiplet broad multiplet multiplet mu1tiplet multiplet doublet doublet multiplet broad singlet

4

d, e

3 1

a f

1 1 2 1 1 1 1 2

C

j k, 1 1

h g m b

451

SERTRALINE HYDROCHLORIDE

The peaks at 2.5 and 3.4 ppm are solvent related.

*

n

,a

.:.

.5

L.

u

,a

I .

u

*.

&,

<.

a.

u

u

u

.

.*

.r

,>

Figure 5. Proton NMR Spectrum of Sertraline HCl in DMSO-d6

4.8.

Carbon-13 Nuclear Magnetic Resonance Spectra

The carbon- 13 nuclear magnetic resonance spectrum of sertraline hydrochloride, Figure 6, was recorded using a Briicker WM250 instrument. The solvent was deuterated dimethylsulfoxide. Assignments have been allocated to the spectral lines where possible by reference to calculated shifts" and to information gained from low power noise and/or selective decoupling.

458

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

Cl

Chemical Shift.ppm 22.9 26.6 30.3 44.3 54.9 126.4 128.9 129.1 129.5 129.7 130.2 130.5 130.9 131.0 139.9 146.6

Assignment d C

a e b 0

i n

j P h,q (by intensity) g 1 f

The multiplet at 40 ppm is due to the solvent. Both the proton and carbon- 13 nuclear magnetic resonance spectra are consistent with the structure for sertraline hydrochloride.

I

I

180

170

160

150

'l

I

140

-

I-

130

' ~I

120

l

110

90

100

80

PPm Figure 6. Carbon-13 NMR Spectrum of Sertraline HCl in DMSO

70

60

50

40

30

1

20

10

460

4.9.

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

Mass Spectra

The mass spectrum of sertraline hydrochloride was recorded using a Finnigan 45 10 mass spectrometer in the direct exposure electron impact mode. Instrument conditions are summarized below: Inlet system Accelerating voltage Electron ionization Ion source temperature Resolving power Calibration

Direct insertion probe 1.8 kV 70 eV 160°C lo00 perfluorotributylamine (FC-43) (nominal mass 614)

A representative spectrum is shown in Figure 7. Structure assignments for a number of the fragments are provided in Figure 8. These assignments were confmed by high resolution mass spectrometry, and the structures for the chlorine-containing fragments are consistent with the observed isotope ratios.I2 A typical spectrum (for peaks with relative intensity >6.0%) is summarized in the table below. The data are consistent with the structure for sertraline hydrochloride.

SERTRALINEHYDROCHLORIDE

46 1

Electron Impact Mass Spectrum of Sertraline Hydrochloride Mass vs Intensity (relative intensity >6.0%) Relative Intensity d Z 304 279 278 277 276 275 274 264 262 248 242 241 240 239 238 214 213 212 205 204 203 202 201 200 199 191 190 189 178 176 165 164 163 162 161 160

6.5 6.1 20.0 20.5 84.7 20.6 100.0 21.1 33.2 8.4 6.8 11.6 7.7 28.5 7.4 6.9 11.2 16.7 6.4 16.7 18.0 22.1 7.0 7.1 8.1 6.3 11.1 11.7 25.2 12.4 13.6 6.4 15.8 8.0 47.6 10.3

m/z 159 158 145 144 143 138.5 133 132 131 130 129 128 127 121.5 120.5 118 117 116 115 113 109 103 102 101 95 91 90 89 88 87 77 75 70 63 57 51

Relative Intensitv 99.4 10.4 19.6 18.8 7.6 8.9 44.7 44.2 11.7 22.4 33.2 30.6 14.3 13.0 24.8 18.4 14.7 35.3 36.4 7.9 12.3 67.6 31.5 20.3 14.4 23.8 6.2 15.2 8.8 8.8 9.4 8.3 15.8 7.5 6.3 6.4

462

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

Figure 7. Mass Spectrum of Sertraline HCl

SERTRALINE HYDROCHLORIDE

/It

463

+N

HN

’

I+ \

QC1

Vz c1

m/z 159

274

C1

Figure 8. Mass Spectrometric Fragmentation of Sertraline Hydrochloride

464

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

4.10. Optical Rotation Optical purity is determined by measuring the rotation of a 1% solution of sertraline hydrochloride in methanolic 0.05 N hydrochloric acid. The specific rotation found for the reference standard was 40.2".

4.11. pH and pKa The pKa of sertraline hydrochloride as determined by potentiometric titration in ethano1:water (1: 1, v/v) was found to be 8.5. The pKa determined in methano1:water (4060, v/v) was 8.6. Titration of sertraline hydrochloride in water was carried out in the presence of sodium chloride and a measured excess of HCl. Titration with NaOH provided a curve that was evaluated by the method of Clarke and C a h ~ n ' ~ The . pKa in water calculated by this method was 9.48 f 0.04.

4.12. Single Crystal X-Ray Perhaps the single best confirmatory evidence for the structure of the sertraline hydrochloride (Form I) molecule is provided by single crystal x-ray. A single crystal of sertraline hydrochloride was grown and its structure determined on a Nicolet R3M-m x-ray spectrometer. The resultant structure as determined is reproduced in Figure 9, which confirms the structure of sertraline hydrochloride.

SERTRALINE HYDROCHLORIDE

Single Crystal X-Ray Crystallographic Analysis of Sertraline Hydrochloride Crystal Parameters Crystal size, mm

0.11xo.11 x0.12

Cell Dimensions

a = 8.004(5) 8, b = 8.372(5) 8, c = 25.21(2) 8, a = 90.0" p = 90.0" x = 90.0" V = 1689.3(6) 8,3

Space group

~12121 Orthorhombic

Moleculedunit cell

4

Density observed, g/cm3

1.37

Density calculated, g/cm3

1.354

Linear absorption coefficient

49.48

465

466

BRUCE M . JOHNSON AND PEI-TEI L. CHANG

CI

Figure 9. Single Crystal X-Ray Structure of Sertraline HCl (Form I)

SERTRALINE HYDROCHLORIDE

461

4.13. Polymorphism Five crystalline polymorphic forms of sertraline hydrochloride have been isolated and ~haracterized.'~ Four of the polymorphs have been examined by infrared absorption spectroscopy, X-ray powder diffraction, single crystal X-ray, differential scanning calorimetry (DSC), hot stage optical microscopy, and aqueous solubility studies. Only a few crystals of the fifth polymorph have been isolated and the only characterization data available are from single crystal X-ray studies and infrared spectra. The polymorphs of sertraline hydrochloride have been designated as Forms I, 11, 111, IV, and V. Form I represents the lower melting polymorph which may crystallize from an acidic solution of the compound in isopropyl alcohol, hexane, or ethyl acetate depending on temperature and rate of crystallization. Forms 11 and IV are metastable polymorphs which can be isolated by rapid crystallization of sertraline hydrochloride from various solvents (e.g., methanol, ethyl acetate, acetonitrile); however, slow crystallizations and granulations yield polymorph Form I. Form 111 is generated from Forms I, 11, or IV by heating the solid to temperatures above about 180°C. Granulating either Form 11, 111, or IV in isopropanol, ethyl acetate, or hexane at 40 to 60°C causes conversion to Form I. Form V is produced by sublimation of Form I onto a cold finger condenser. Form I is the thermodynamically most stable polymorph at room temperature. The following information summarizes observations concerning the characterization, stability, and thermal behavior of sertraline hydrochloride polymorphs. 4.13.1. Characterization Data Infrared Absorption Spectrophotometq The infrared absorption spectra of Forms I, 11, 111, IV,and V are different from one another (Figure 10). The spectra are obtained using KBr pellets to suspend the sample during

468

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

measurement. Figure 10 displays only the region of 1650 to 400 cm-1. The region from 4000 to 1650 cm-1 is not useful for comparing polymorphic differences. Qualitative comparison of the infrared spectra of Form I to Forms 11, 111, IV, and V show the following differences: a. At approximately 740 to 750 cm-l and 1075 to 1085 cm-1 Forms 11, 111, IV, and V exhibit absorption bands of significantly higher relative intensity. b. At approximately 780 cm-l Forms 11, 111, and IV exhibit major peaks. Forms I and V exhibit a major peak at approximately 790 cm-1with only a shoulder at about 780 cm-1. c. Forms 11, 111, IV, and V show absorption bands at approximately 870 and 520 to 540 cm-1 while no absorption is observed for Form I at these wavelengths. d. Form 11 shows a strong absorption band and Forms 111, IV, and V a weak band at about 640 cm-l, while a barely detectable absorption is observed for Form I.

e. Differences for Forms 11, 111, and IV are also observed in wavelength and relative intensity of the absorption bands in the region of 800 to 850 cm-1 compared to Forms I and V. Single Crystal X-Ray Analysis Absolute structure determination of Forms I, 11, HI, IV, and V by single crystai X-ray analysis shows that the polymorphic forms differ in rotational conformation at the methylamino and the dichlorophenyl positions. Forms 111and IV are similar rotationally but differ in their space group. Using these configurations the crystal packing diagrams are constructed and the crystal densities are calculated. Form I gives a density of 1.354 gkc, Form I1 gives a density of 1.314 g/cc, Form 111gives a density of 1.3 13 g/cc,

z T I

a n S

m I

t t

a n C 8

-120

4

Y 1600

1500

1400

1300

1200

1100

1mo

900

800

700

600

Figure 10. Infrared Spectra of Sertraline HCl Polymorphs (Wavelength range 1650 to 400 cm-1)

500

470

BRUCE M. JOHNSON AND PEI-TEIL. CHANG

Form IV gives a density of 1.349 g/cc, and Form V gives a density of 1.308 g/cc. This result supports Form I as the thermodynamically stable form at room temperature since the most dense crystal at a given temperature is considered to be the most thermodynamically stabiei5. Using these single crystal data it is possible to calculate the theoretical powder diffraction patterns. The theoretical patterns shown in Figure 1 1 for Forms I, II, III, and IV match the observed patterns very closely. X-Ray Powder Diffraction The four polymorphs of sertraline hydrochloride for which samples are available give distinctive X-ray powder diffraction patterns. Each form has diffractions at unique values of 28 that are diagnostic: Form1

7.1, 12.7, 14.1, 15.3, 15.7,21.2,23.4, and26.3

Form11

5.4, 10.8, 14.6, 16.3, 18.1, 19.0,20.3,21.8, 24.4, and 27.3

FormIII

14.3, 15.5, 17.4, and 19.6

Form IV

15.6,22.4,25.4,28.9,31.9,and 32.1

In these powder diffraction patterns of the pure polymorphs, each of the unique peaks is greater than about 10%of the normalized intensity. In normal practice the 28 values remain constant but the relative intensities may vary somewhat due to particle size effects. Figure 1 1 shows the powder diffraction patterns calculated from the single crystal data. The pattern for Polymorph V is also included here and shows a distinctive pattern as well.

SERTRALINE HYDROCHLORIDE

47 1

POLYHORPH I

Figure 11. Calculated X-Ray Powder Diffraction Patterns

4.13.2. Relative Stability of Sertraline Hydrochloride Polymorphs Interconversion Experiments

Forms 11and TV have been made by rapid crystallization out of solvents and Form 111has been made by heating a sample of Form I at about 180°C for a period of several hours. When mixtures of Form I and Forms 11, 111, or IV were granulated in isopropyl alcohol at 50°C, the resulting solid was shown to be Form I. Similar behavior has been observed in other solvents. In an experiment viewed under the microscope at room temperature, a mixture of Forms I, 11, and 111was placed on a slide

412

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

and covered with a cover slip. Isopropanol saturated with sertraline hydrochloride was added at the edge of the cover slip and the sample was kept wet with solvent throughout the observation. Crystals of Form III disappeared fairly rapidly. Crystals of Form II shrunk in size slowly while the Form I crystals increased in size. In a similar experiment with Forms I and IV,Form IV dissolved slowly while Form I grew. These observations are consistent with the greater thermodynamic stability of Form I. Solubility The room temperature aqueous solubility of the four polymorphs is essentially equivalent at about 4 m u d . Polymorphs 11,III, and IV appear to be slightly more soluble than Form I, further indicating that Form I is the most stable at room temperature. 4.13.3. Thermal Behavior

Differential Scanning Calorimetry CDSC) The DSC thermograms for Forms I - IV are shown in Figures 12, 13, 14, and 15. The features in the thermograms may be interpreted as follows:

Form I shows an endotherm with an onset temperature of about 219°C resulting from the melting of Form I. An exotherm immediately following the melting endotherm of Form I appears at about 225°C caused by partial crystallization of the melt to Form III, and a second endotherm appears with an onset at about 246°C resulting from the melting of Form III. Rapid thermal decomposition takes over after the final melt of Form III causing the baseline to increase rapidly and become erratic in appearance. On rare occasions, the DSC of Form I shows only a single melting endotherm with an onset temperature of about 219°C. This can occur when the sample does not have sufficient time or seed to

SERTRALINE HYDROCHLORIDE

413

recrystallize as Form 111prior to reaching the Form 111melting temperature.

In the thermograms obtained for Forms 11and IV,a very small endotherm may be observed with an onset temperature at about 180°C which corresponds to a solid-solid transition to Form 111. This event is followed by melting of Form 111at about 246°C. The thermogram obtained for Form 111 shows a single endotherm at about 246°C. Above the melting temperature of 246" C thermal decomposition increases rapidly. The thermal events discussed above demonstrate that Form 111undergoes only a single melting process at 246°C followed by increased thermal decomposition after the melt has occurred. Thermogravimetry and hot stage optical microscopy reveal that all four forms undergo slight decomposition and sublimation at temperatures above about 160°C. Prolonged heating studies on the Form Worm 111 conversion in the solid state have shown that below 160°C Form I is the preferred polymorph. Above 180°C Form I converts to Form 111in the solid state. This conversion takes place at varying rates in the DSC experiment depending on sample characteristics such as particle size, previous handling, or the presence of Form III seed. Thus, in some instances the first endotherm in Form I may show a smaller endotherm with a lower onset temperature as a result of solid state conversion of Form I to Form 111during heating. The solid state conversion is a very low energy transition and fails to give a reliably detectable DSC event. A sample of Form I spiked with 3% of Form 111(Figure 16) shows an endotherm for the solid state transition and the absence of the Form I melting endotherm. The Form 111endotherm appears at the expected temperature of about 246°C. Under the conditions of this DSC experiment the solid state transition is fast enough to cause complete conversion to Form 111before the Form I melt temperature is reached. Similar

BRUCE M.JOHNSON AND PEI-TEI L.CHANG

474 01

Figure 12. DSC Thermogram of Sertraline HCl Form I)

u

I V

c

m

aa

Figure 13. DSC Thermogram of Sertraline HCl (Form II)

SERTRALINE HYDROCHLORIDE

415

I

Figure 14. DSC Thermogram of Sertraline HCl (Form III)

Figure 15. DSC Thennogram of Sertraline HCl (Form IV)

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

476

LOO

U LSD -I

s

0.w

Figure 16. DSC Thermogram of Sertraline HCl (Form I containing 3% of Form 111) behavior was observed in two other samples of Form I spiked with 5% of Form I1 or 5% of Form IV.

In a related experiment, small amounts of each of Forms I, 11, and IV were heated to 185°C for 15 minutes. The infrared spectra of each sample confmed that they had converted to Form 111. Hot Stage Optical Microscopy Microscopic observation of a Form I sample heated on a hol stage is consistent with the observed DSC behavior. At about 150 to 180°C the sample begins to move about and appears to "pop". This may be caused by the onset of decomposition, solvent release, or sublimation. Partial melting may be observed at about 210 to 220°C with the formation of some new crystals (Form 111) as the temperature is raised to about 235°C. (In some instances, a solidsolid transition to Form I11 is observed at temperatures above about

SERTRALINE HYDROCHLORIDE

411

180°C.) At about 245 to 250°C the newly formed crystals melt completely. The melt solidifies very slowly upon cooling and usually remains as a yellowish glass. This suggests that significant decomposition has taken place. In mixtures of Form I11 in Form I, the first melt at 210 to 220°C does not cause all of the sample to liquefy, a small number of crystals may remain and increase in size as Form 111crystallizes around them. Holding the temperature at about 235°C permits the melt to recrystallize to Form III. Cooling this melt to room temperature and subsequent reheating does not show the Form I melt at 210 to 220°C. This suggests that the conversion to Form 111is not rapidly reversible under these conditions. Microscopic observation of Forms 11or IV on the hot stage shows an apparent solidkolid transition at about 180°C without evidence of melting. Continued heating gives a complete melt at about 245°C. The temperature region of 180 to 230°C appears also to cause decomposition and sublimation. Microscopic observation of Form 111 shows sample movement similar to Form I at about 160°C followed by melting at about 245 to 250°C. No other thermal events are seen and the observation is consistent with the DSC behavior. When samples are heated in silicone oil the appearance of gas bubbles is observed starting at around 150 to 180°C suggesting the onset of decomposition. The yellowish color of the melt above 250°C results from chemical decomposition of the sample. This phenomenon occurs for all four polymorphic forms.

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

47%

4.13.4. Literature Precedent Polymorphs of sertraline hydrochloride behave similarly to those of gepirone hydrochloride which has been characterized and discussed in the 1iteraturel6. Both compounds have a polymorph which is most thermodynamically stable at room temperature which can go through an endothermic melt and subsequently recrystallize as a higher melting polymorph.

5.

Methods of Analysis 5.1.

Elemental Analysis

The elemental composition of sertraline hydrochloride was determined by a series of microanalytical determinations. A typical set of analyses is shown below.

Assay % carbon % hydrogen % nitrogen % chlorine, ionic % chlorine, total

Total (C,H,N,Cl)

59.54 5.30 3.85 10.24 30.54 99.23

59.58 5.29 4.09 10.35 31.04 100.00

The results for percentage carbon, hydrogen, nitrogen, and chlorine for sertraline hydrochloride are consistent with the theoretical values for the formula C17HIgNC13. 5.2.

Ionic Chlorine

The ionic chlorine content of sertraline hydrochloride may be determined by potentiometric titration with a standard silver nitrate solution, which precipitates the chloride ion as AgC1. Titration is carried out by dissolving the sertraline hydrochloride in ethanovwater (1: 1, v/v) and titrating using a silver measuring electrode and a Ag/ AgCl reference electrode. The theoretical ionic

479

SERTRALINE HYDROCHLORIDE

chloride content of sertraline hydrochloride is 10.35%;the amount found for the reference standard lot was 10.24%.

5.3.

Identification

Sertraline hydrochloride is identified in the bulk form using the infrared spectrum, the TLC retention, and the HPLC retention by comparison with a working standard. In dosage forms the TLC retention and the HPLC retention are used for confirming identity of drug substance.

5.4.

Thin Layer Chromatography

Thin layer chromatography is used as a technique for establishing the identity of sertraline and for examining for the presence of process related substances and other potential impurities. The following systems have been used with silica gel 60 F-254 (Merck Darmstadt) plates. Solvent System

Rf

Detection

Ethyl acetate/Methanol/Ammonium acetate 160:40:2 v/v

0.55

1

Chloroform

0.00

2

Chloroform/Methanol/ammoniumacetate 12050:15 v/v

0.91

2

Detection Systems 1 2

5.5.

Dragendorff's Reagent Ultraviolet light - 254 nm

Ultraviolet Spectroscopy

Assay for sertraline content of drug substance and drug products may be carried out using ultraviolet spectrophotometry.

BRtiCE M. JOHNSON AND PEI-TEI L. CHANG

480

Sertraline solutions are prepared in 0.01 N methanolic hydrochloric acid at a concentration of approximately 0.05 mg/mL. Absorbance measurements are taken at 273 nm and compared to the absorbance of a well characterized standard.

5.6.

Potentiometric Titration

Being a hydrochloride salt, sertraline hydrochloride has an acidic proton which can be titrated with 0.5 N aqueous sodium hydroxide in the solvent mixture ethanovwater, 1:1, v/v. When 98.67 mg of sertraline hydrochloride reference standard was titrated in 20 mL of the solvent mixture, a single inflection point was observed. This was attributed to the single acidic proton located on the arnine nitrogen of the sertraline hydrochloride molecule. The neutralization equivalent (molecular weight) calculated for the single inflection point was 345.5. Based on this result, an assay of 89.3% sertraline was calculated for the reference standard lot, which is 99.9% of theory (89.4%). This titrimetric result confirms the stoichiometry of sertraline hydrochloride and provides an equivalent weight value consistent with the structure. A representative curve for this titrimetric procedure is included in Figure 17.

5.7.

High Performance Liquid Chromatography

Purity assay of sertraline hydrochloride is done using HPLC by the external standard method by comparing chromatographic response with a well characterized standard. An isocratic reverse phase system using a C-18 column and UV detection at 254 nm is suitable for both drug substance and formulated products. Column: Detector: Mobile Phase: Buffer:

Waters Nova-Pak C- 18 UV at 254 nm

Acetonitrile/MethanoYSuffer 45: 15:40 v/v 0.05 M Acetic acid/O.OZ M Triethylamine (aqueous)

SERTRALINE HYDROCHLORIDE

481

PH 9 1 2 3 4 6 6 7 8 P 1 9 1 1 1 2 1 1 1 4 1 1 1 1 l l 1 1 1 1 l l l l 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1

0 -

0.10

0.20

0.30

-

-

0.40 mL

-

0.60

-

0.60

-

0.70

-

0.80

-

Figure 17. Potentiometric Titration of Sertraline HCl in EthanoVWater (1:l)

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

4x2

Metabolic studies have used HPLC to measure sertraline and its metabolites in urine, bile, and feces using radiolabeled drug17 and a radioactivity detector and/or a variable wavelength UV detector’*. Two different mobile phases were reported. Column: Waters C - 18 pBondapak Berthold radioactivity monitor or UV Detector: Mobile Phase 1 : Acetonitrile/SO mM Sodium phosphate (pH 4.5) 5050 v/v Mobile Phase 2: AcetoniUilelSO mM Ammonium acetate (pH 5.0) 33167v/v

Another HPLC method has been reported for measuring sertralirie and desmethylsertraline in mouse cerebral cortex using an internal standard method”.

Column: Detector: Mobile Phase:

5.8.

Versapack C- 18 UV at 235 nm Acetonitrile/0.25 M Potassium phosphate (pH 2.7) 30:70 v/v

Gas Liquid Chromatography

Gas liquid chromatography is used to separate sertraline from its process related substances, in particular the dechlorinated homologs. Hydrochloride salts are converted to their free base form by extraction of a basified solution into methylene chloride prior to injection. The following system has been used for the drug substance. Column:

Column Temp: Injector Temp: Detector Temp: Detector:

5% OV-17 on Chromosorb W (acid washed and DMCS treated) packed in a 7 ft. x 0.25 in. OD glass column 225°C 250°C 300°C Flame ionization

SERTRALINE HYDROCHLORIDE

483

Gas chromatography has also been used to measure sertraline levels in biological fluids. The drug is extracted from plasma and derivatized with trifluoroacetic anhydride. The derivative is chromatographed using either a mass spectrometric detector2' or a 63Ni electron capture detector21. GCMS Method Column: 3% Silar 1OC on Gas Chrom Q (80 - 100 mesh) packed in a 0.8 m x 2 mm ID glass column Column Temp: 255°C Injector Temp: 200°C Transfer Temp: 290°C Detector: Mass spectrometer Electron Capture Method Column: SE-54,0.25 pm film thickness, 12 m x 0.32 mm ID capillary column Column Temp: 165°C for 0.5 min, 20"C/min to 21OoC, hold for 12 min Injector Temp: 260°C Detector Temp: 300°C Detector: 63Ni Electron Capture

5.9.

Biological Fluids

The levels of sertraline and its major metabolites are measured in biological fluids by HPLC, GC/EC, and GCMS using the methods described above. Metabolism studies have resulted in the identification of N-desmethylsertraline, sertraline carbamoyl-0glucuronide, N-hydroxysertraline, a ketone [4-S-(3,4dichlorophenyl)-3,4-dihydro-1(2H)-naphthalenone], and the ahydroxyketone [4-S-(3,4-dichlorophenyl)-3,4-dihydro-2-R,Shydroxy-1(2H)-naphthalenone] as well as the glucuronides of Nhydroxysertraline and the a-hydroxyketone2*.

BRUCE M. JOHNSON AND PEI-TEI L. CHANG

484

6.

Stability The stability of sertraline hydrochloride has been studied under extreme challenge conditions as well as the more traditional pharmaceutical challenge conditions. The extreme conditions included exposing excess drug substance to refluxing water for 3 hours, refluxing 5 N hydrochloric acid for 3 hours, refluxing 5 N NaOH for 3 hours, or 10% hydrogen peroxide for 6 hours. The more traditional stability challenge conditions of 3 months at 50°C, 12 months at 37"C, 60 months at 30°C, or 3 months in a light cabinet were used with different packaging systems. Careful examination of the resulting samples revealed no significant degradation under any of the conditions used in these studies. The only trace degradation product identified was the ketone [4-S-(3,4dichlorophenyl)-3,4-dihydro-1(2H)-naphthalenone] which has also been identified as a metabolite. Sertraline hydrochloride appears to be a very stable compound under a variety of challenge conditions.

7.

Pharmacokinetics and Metabolism 7.1.

Systemic Bioavailability

In man, following oral once-daily dosing of sertraline hydrochloride (Form I) over the range of 50 to 200 mg for 14 days, mean peak plasma concentrations (C,) of sertraline occurred between 4.5 to 8.4hours postdosing. The average terminal elimination half-life of plasma sertraline is about 26 hours. Based on this pharmacokinetic parameter, steady-state sertraline plasma levels should be achieved after approximately one week of oncedaily dosing. Linear dose-proportional pharmacokinetics were demonstrated in a single dose study in which the C, and area under the plasma concentration time curve (AUC) of sertraline were proportional to dose over a range of 50 to 200 mg. Consistent with the terminal elimination half-life, there is an approximately two-fold accumulation, compared to a single dose, of sertraline with repeated dosing over a 50 to 200 mg dose range. The single dose bioavailability of sertraline tablets is approximately equal to an equivalent dose of solution23.

SERTRALINE HYDROCHLORIDE

485

The effects of food on the bioavailability of sertraline were studied in subjects administered a single dose with and without food. AUC was slightly increased when drug was administered with food but the C, was 25% greater, while the time to reach peak plasma concentration decreased from 8 hours post-dosing to 5.5 hours. 7.2.

Metabolism

Sertraline undergoes extensive first pass metabolism. The principal initial pathway of metabolism for sertraline is Ndemethylation. N-desmethylsertraline has a plasma terminal elimination half-life of 62 to 104 hours. Both in vitro biochemical and in viva pharmacological testing have shown Ndesmethylsertraline to be substantially less active than sertraline. Both sertraline and N-desmethylsertraline undergo oxidative deamination and subsequent reduction, hydroxylation, and glucuronide conjugation. In a study of radiolabeled sertraline involving two healthy male subjects, sertraline accounted for less than 5% of the plasma radioactivity. About 40-45% of the administered radioactivity was recovered in urine in 9 days. unchanged sertraline was not detectable in the urine. For the same period, about 40-45% of the administered radioactivity was accounted for in feces, including 12-14%unchanged sertralineZ4. Desmethylsertraline exhibits time-related, dose dependent and Cmin, with about a 5-9 increases in AUC (0-24 hour), C, fold increase in these pharmacokinetic parameters between day 1 andday 14.

Acknowlegement The authors wish to thank our many Pfizer colleagues in the Central Research laboratories in both Groton, CT, USA and Sandwich, Kent, UK who have contributed to the development of sertraline hydrochloride and to the information presented in this overview.

486

BRUCE M.JOHNSON AND PEI-TEI L. CHANG

References 'Koe, B. K.; Weissman, A.; Welch, W. M.; Browne, R. G. J. Exp. Ther. 1983,226, 686. 2Package Insert, Zoloft*, Pfizer Inc, Jan. 1992 3Quallich, G. J.; Williams, M. T. U.S. Patent 4 777 288, 1988. 'Quallich, G. J.; Williams, M. T. U.S. Patent 4 839 104, 1989. 5Williams, M. T.; Quallich, G. J. Chemistry and Industry 1990,21, 315. 6Adrian, G. P. U.S. Patent 5 019 655,1991. 'Quallich, G. J.; Williams, M. T. European Patent 0 295 050 A l , 1988. 'Quallich, G . J.; Williams, M. T.; Friedmann, R. C. J. Org. Chem. 1990,55,4971. w e l c h , W. M.; Kraska, A. R.; Sarges, R.; Koe,B. K. J. Med. Chem. 1984,27, 1508. '()Welch, Jr., W. M.; Harbert, C. A.; Koe, B. K.; Kraska, A. R. U.S. Patent 4 536 518, 1985. "Ewing, D. F. Organic Magnetic Resonance 1979, 12(9), 499. 12 Sharp, T. R.; Horan, G. J.; Day, S.V.O. Proceedings of the 41st ASMS Conference 01 Mass Spectrometry and Allied Topics, San Francisco, California, 1993. I3Clarke, F. H.; Cahoon, N. M. J. Pharm. Sci. 1987,8,611. '*Sysko, R. J.; Allen, D. M. J. US.Patent 5 248 699, 1993. "Haleblian, J.; McCrone, W. J. Pharm. Sci. 1969,58(8), 911. %ehme, R. J.; Brooke, D.; Farney, R. F.; Kensler, T. T. J. Pharm. Sci. 1985, 74(10), 1041. "Welch, W. M.; Vivieros, D. M. J. Labelled Compd. Radiopkam. 1987, 24, 987. 18 Tremaine, L. M.; Stroh, J. G.; Ronfeld, R. A. Drug Metab. Dispos. 1989, 17,58. 'weiner, H. L.; Kramer, H. K.; Reith, M. E. A. J. Chromatog. 1990,527,467. 2%ouda, H. 6.; Ronfeld, R. A.; Weidler, D. J. J. Chromatog. 1987,417, 197. "Tremaine, L. M.; Joerg, E. A. J. Chromatog. 1989,496,423. 2?remaine, L. M.; Welch, W. M.; Ronfeld, R. A. Drug Metab. Dispos. 1989, 17, 542. 23PackageInsert, Zolofi@,Pfizer Inc, Jan. 1992 24 Package Insert, Zoloft@, Pfizer Inc, Jan. 1992

SOLASODINE

Gunawan Indrayanto, Achmad Syahrani, Robby Sondakh, and Mulja H. Santosa

Laboratory of Pharmaceutical Biotechnology Faculty of Pharmacy Airlangga University Surabaya, Indonesia

ANALYTlCAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

487

Copyright 0 1996 by Academic Press, lnc. All rights of reproduction in any form reserved.

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GUNAWAN INDRAYANTO ET AL.

1. DESCRIPTION 1.1. Nomenclature 1.1.1. Chemical names 1.1.2. Synonym 1.2. Formula 1.2.1. Empirical 1.2.2. structural 1.2.3. CAS Registry No. 1.3. Molecular weight 1.4. Elementary composition 13.Appearance 1.6. Occurence 1.7. Use

2. PHYSICAL PROPERTIES 2.1. Melting point 2.2. Specific rotation 2.3. Solubility 2.4. Dissociation constant 2.5. Thermal Analysis 2.6. Spectm Properties of solasodine 2.6.1. Ultra Vioiet Spectrum 2.6.2. Absorbance Reflectance Spectrum 2.6.3. Near I n h Red Spectrum 2.6.4. Infra Red Spectrum 2.6.5. 'H-Nuc~EKMagnetic Resonance Spectrum 2.6.6. '3C-Nuclear Magnetic Resonance Spectrum 2.6.7. Mass Spectrum

3. ISOLATION OF SOLASODINE 4. BIOSYNTHESIS OF SOLASODINE

5. CHEMICAL CONVERSION OF SOLASODINE TO STEROID HORMONES

6. BIOTRAWFORMATION OF SOLASODINE

SOLASODINE

489

7. METHOD OF EXTRACTION AND QUANTITATNE ANALYSIS OF SOLASODINE

7.1. Method of extraction 7.2. Method of quantitative analysis 7.2.1. Titration 7.2.2. Colorimetric 7.2.3. Potentiometric 7.2.4. Gas chromatography 7.2.5. High Performance Liquid Chromatography 7.2.6. Radioimmunoassay 7.2.7. Thin Layer Chromatography - Densitometric 8. PHARMACOLOGICAL AND BIOLOGICAL ACTIVITIES OF SOLASODINE AND ITS GLYCOSIDES

GUNAWAN INDRAYANTO ET AL.

490

1. DESCRlPTION 1 . 1 . Nomenclature 1.1.1. Chemical names

Spirosol-5-en-3P-01; solasod-5en-3P-ol 1.1.2. Synonym Solancarpidine; Solanidhe-s; Purapuridine 1.2. Formula 1.2.1. Empirical C27H4,No,

1.2.2. smctural

1.2.3. CAS Registry No. 126-17-0 1.3. Molecular weight

413,6 1.4. Elementary composition C : 78.40 96;

H : 10.48 96; N : 3.39 96; 0 : 7.74 96

1.5. Appearance A fine white odorless crystalline powder; hexagonal plates crystal of solasodine can be recrystallized from methanol or by sublimation in high vacuum

SOLASODINE

49 1

1.6. Occurence Solasodine was accumulated in the species of Genus Solanum. Solasodine was produced commercially mostly from Solanwn laciniatwn, Solanum khasianwn and Solanwn marnmoswn (1,2,3). In Solanum plants, solasodine was found mostly as diglycosides, triglycosides and tetraglycosides (4). The sugar moiety of the important glycosides are shown in the table 1. Table 1. Sugar moiety of solasodine glycosides Glycosides J3+alamarginc

I

Sugar moiety Rhamnose 01 (1-> 4) - Glucose p (1-> 3) - R

p-Solasonine Glucose p (1-> 3)

- Galactose p (1->

>

I

3) - R

Rhamnose a (1-> 2)

Glucose p (1-> 3)

Solamargine Rhamnose a (1-> 4)

-R

Rhamnose a (1-> 2) >&lactose

Solasonine Glucose Solaradixine

p (I-> 3) - R

p (1-> 3)

Rharnnose a (1-> 2) >Galactose G~UCOS~ p (1-> 2 G I U C Op~(1-> 3)

p (1-> 3)-R

R = Solasodine

1.7. Use Solasodine is used as raw material for producing steroid hormone in pharmaceutical industry. 2. PHYSICAL PROPERTIES 2.1. Melting point 200 - 202O c (5) 2.2. Specific rotation [ aD ] at 25OC (c = 0.14 in methanol) = -98' ; [ aD ] =-113' (in chloroform) (5)

GUNAWAN INDRAYANTO ET AL.

391

2.3. Solubility The solubility data of solasodine are listed in table 2 Table 2. Solasodine solubility in various solvents

Benzene Methanol Ethanol 95 % Acetone n-Hexane Water Ether

Freely soluble. Freely soluble.

9.5 5.0 3.5 <1.0 <1.0 Practically insoluble'

* Data from Merck Index (5) 2.4. Dissociation constant pK, in 60 % ethanol was 6.31 (6). 2.5. Thermal Analysis The DSC (differential scanning calorimetry) thermogram of solasodine was obtained on a Shimadzu DT-30 Thermal Analyzer. The instrument was calibrated with indium standard. The heating rate was 10°C/min. The thermogram is presented in figure 1. The DSC curve revealed an endothermic residual moisture peak on a single sharp endothermic melting peak (200OC) of solasodine. 2.6. Spectral Properties of solasodine 2.6.1. Ultra Violet Spectrum The U V (ultra violet) spectrum of solasodine in methanol (ca. 40 ppm) was obtained on a Hewlett Packard 8452A photo diode array spectrophotometer. It exhibits a maximum at 206 nm. The UV spectrum and its first order derivative spectrum are presented in figure 2A and 2B. 2.6.2. Absorbance Reflectance Spectrum The visible absorbance reflectance spectrum of solasodine spotted on Kieselgel GF254 precoated plate (EMerck), eluted with chloroform:

Figure 1. DSC thermogram of solasodine (Sigma).

GUNAWAN INDRAYANTO ET AL.

194

I

0.72649,

i

I

I A

A

%z 0.43589-

2c:

20.29059m

<

I

0.14530/

I 200

\

nm,

2.50

300

350

400

B 0.06829-

-w 0.03927 >

pO.OIa26 U

Figure 2. UV spectrum of solasodine (Sigma)in methanol (A) and its fxst order derivative spectrum (B).

2.000

1.600

* sr

1.200

3 8

0.800

0.400

81

O.Oo0

- 0.200 -

I

400

,

.

. . , . 450

.

.

.

I

500

.

,

I

.

I

550

.

.

.

-

1

600

.

I

-. 650

K1 nm

Figure 3. Visible absorbance ref'lectantce spectrum of solasodine (Sigma) on Kieselgel GF254 precoated plate (Merck). BL: base line.

GUNAWAN INDRAYANTO ET AL.

496

methano1:diethyl amine (20:2:0.5) and Visualized by anise aldehyde-sulphuric acid (7)spray reagent (10OoC,10 minutes), is shown in figure 3. It shows a maximum at 385 nm. The spectrum was recorded on a Shimadzu CS-930TLC Scanner. 2.6.3. Near Infra Red Spectrum The NIR (near infra red spectrum) of 0.5 % solasodine in CCl, was recorded by using a Shimadzu 365 spectrophotometer. The N I R spectrum of solasodine is shown in figure 4. The Xmx data are presented in table 3. Table 3. NIR characteristics of solasodine Amax (nm)

Assignment

1413

OH stretching

1527

NH stretching

2309 2352

CH stretching

2.6.4. Infra Red Spectrum The IR (infra red) spectrum of solasodine as KBr disc (1.5mg/ 100 mg) was recorded on a Jasco 5300 FT IR spectrophotometer. The IR spectrum of solasodine is shown in figure 5. The characteristic bands of solasodine are given in the table 4. Table 4. IR characteristic bands of solasodine ~~

~

Frequencies (crn-') 3455 3362 2843,2967 1690 1675 1451, 1344 1379 1060, 1239

I

Assignment Stretching vibration of OH Stretching vibration of NH Stretching vibration of CH steroid skeleton Stretching vibration of C = C bond Deformation vibration of NH Deformation vibration of methyl Deformation vibration of methylene Stretching vibration of C-QC

9"

0

c 5 ABSORBANCE

'0 Q

0 F!

3

-

100.00

%T

0.001.. 4600.0

1

.

4000.0

.

.

.

I

3000.0

.

.

. .

I

2000.0

(cm-')

I

1ooo.o

Figure 5. IR spectrum of solasodine (Sigma) in KBr disc.

400.0

SOLASODINE

499

2.6.5. 'H-Nuclear Magnetic Resonance Spectrum The 'H-NMR spectrum of solasodine (Figure 6A and 6B) were determined in CDCl, at 90 MHz employing a Hitachi R-1900 FT NMR using TMS as internal standard. The chemical shift values are presented in Table 5. Table 5. 'H-NMR chemical shifts of solasodine Chemical shift (6 ppm)

Proton assignment

Pyridine - D5

CDCI,

Me-18 Me-19 Me-2 1 Me-27 H-6 H-3a H-16 H-26a, p H ~ u P,

0.82 (s) 1.03 (s) 0.98 (s) 0.88 (s) 5.32 (br.d) 3.50 (m) 4.27 (4;6.5,6.5, 6.5 I+) 2.57-2.65 (m; not resolved) 2.21-2.30 (m;not resolved)

1.05 (s) 0.91 (s) 1.13 (s) 0.84 (s)

3.80 (m) 4.43 (4;6.7, 6.7, 6.7 Hz)

By using two-dimensional NMR technique (COSY,one bond, long range 'H-I3C chemical shift correlation and NOESY),obtained on a Bruker AMX-500 NMR spectrometer (500MHz), Puri et al (8) could elucidated all the proton chemical shifts of solasodine. The data are presented in the table 6.

5x

....

-.. 5.00

. . , , 4.00

..,. ._.. 3.00

' . . . ' I . . . ' -

2.00

Figure 6A. Proton NMR spectruni of solasodine (Sigma) in CDCh

'.OO

PPM

501

CUNAWAN INDRAYANTO ET AL.

502

Table 6. 'H-NMR chemical shifts of solasodine (6 ppm, CDCl,)* _e_l

Proton assignment -

l-a

1-P 22-9

3 4-a 4-P 6 7* 7-P 8 9 11 12-a 12-P 14

Chemical shift

1.oo 1.80 1.78 1.40 3.42 2.24 2.16 5.23 1.48 1.97 1.58 0.90 1.48; 1.41+ 1.10 1.68 1.02

1 Proton assignment 151543 16 17 Me-18 Me-19 20 Me-2 1 23 24-

24-P 25 2626-P Me-27

Chemical shift 1.95 1.25 4.20 1.65 0.74 0.95 1.81 0.88 1.56 1.55 1.35 1.48 2.58 2.52 0.77

~.

+) not resolved

*)

Data from Puri et al. (8)

2.6.6. '3C-Nuclear Magnetic Resonance Spectrum The broad band decoupling 13C-NMR spectrum (Figure 7) and the J modulation spin echo 13C-NMR spectrum (Figure 3) with 1/J = 7 p sec of solasodine in CDCl were recorded on a Hitachi R-1900 FT NMR (22.6MHz) using TMZ as internal standard. The spectral assignments are presented in Table 7. The data are identical with previously reported spectrum (9, 10).

TMS

J! L L

Figure 7. Broad band decoupled 13C-NMR spectrum of solasodine (Sigma) in CDC13.

PPM

J

CH,CH3

C,CHz

1

TMS

CDCh

50.00

Figure 8. J modulation spin echo (1/J = 7 ItSec) I3C-NMR spectrum of solasodine (Sigma) in CDC13.

00.00

PPM

505

SOLASODINE

Table 7. 13C-NMR chemical shifts of solasodine Carbon No.

1

-~

Chemical shifts

Chemical shifts

Carbon No.

(6 P P d

(6 P P d

1

37.3

15

32.1

2

31.6

16

78.7

3

71.6

17

62.8

4

42.3

5

140.7

19

19.4

6

121.3

20

41.3

7

32.1

21

15.3

8

31.4

22

98.1

50.1

23

34.1

10

36.7

24

30.3

11

20.9

25

31.4

12

39.9

26

47.7

13

40.5

27

19.3

14

56.5

9 ~~

I

18

I

16.4

~~

2.6.7. Mass Spectrum The MS (mass spectrum) of solasodine presented in Figure 9 and Figure 10. were obtained by electron impact (EI) and chemical ionization (CI) using methane as reagent on a Jeol JMS-DX-303 Mass Spectrometer. The ionizing electron beam energy was at 70 eV. The main fragments of solasodine are given in the table 8.

GUNAWAN INDRAYANTO ET AL.

506

Table 8. The main fragments of solasodine Species

El

CI

M+ + 29

442 (8)

M+ + 1

414 (55)

M+ + 1 - 18(H,O)

396 (58)

M+ W

413 (27) -

15(CH,)

398 (3)

M + - 28(C2H,)

385 (13)

C,H2,NO+

271 (2)

a*

138 (70)

138 (10)

b’

114 (100)

114 (27)

* see figure 1 1 The fragments of m/z 138 and 114 are characteristic for spirosolane ring of solasodine (11, 12). The EI spectrum of solasodine is identical with previously published spectrum (13).

MIIZ

Figure 9. EI-MSspectrum of solasodine (Sigma)

RELATIVE ABUNDANCE

SOLASODINE

509

+

4.

O

mlz 138

?

3

m/z 114

Figure 11. Characteristic fragmentation of solasodine.

510

GUNAWAN INDRAYANTO ET AL.

3. ISOLATION OF SOLASODINE In our laboratory, solasodine was isolated from fruits of SoZunum wrightii with the following method, as reported by Indrayanto et al.(14).

Fresh ripe fruits (3 kg) were cut into small pieces and homogenized in a blender with 4 times methanol-acetic acid 3 % until slurry was formed. The slurry was refluxed for 30 minutes then filtered. Each 50 ml of filtrate was treated with 5 ml HC1 conc. and hydrolyzed by refluxing for 3 hours on boiling water bath. After neutralized with 10 %. NaOH, the alkaloids was extracted with chloroform. The chloroform extract was evaporated in vacuo to yield a dark brown semisolid mass (22 g),then purified through a column of A1 0, with chloroform as the eluting solvent. After evaporating the cgloroform, the residue was chromatographed on silica gel 60 with benzene:acetone (15: 1) as eluting solvent. Recrystallization with methanol gave 3.63 g pure solasodine (TLC, UV,TR, MS). 4. BIOSYNTHESIS OF SOLASODINE

The biosynthetic pathway of solasodine followed the general pathway of steroid biosynthesis, starting from acetylcoenzym A via the in termediates mevalonic acid, squalene, cycloartenol and cholesterol. The nitrogen atom was introduced through simple replacement of the terminal hydroxy group by an amino group (15). In solanidine the donor molecule was an amino acid arginine (16). The main steps of the pathway (partially hypothetical) of cholesterol to solasodine in are presented in figure 12 (15,17,18). The figure shows that the formation of ring E can precede or after the formation of ring F.

5. CHEMICAL CONVERSION OF SOLASODINE TO STEROID HORMONES In order to obtain steroid hormone like materials, ring E and F of solasodine attached to C16 and C 17 first must be removed. Solasodine can be degraded into 16-dehydropregnenoloneacetate (16-DPA), which is an excellent starting material for the preparation of most type of steroid hormone (Marker degradation). In figure 13 the conversion of solasodine to some steroid hormone is presented (19,20).

SOLASODINE

511

Fmesyl pyrophosphate

Mevalonic acid

Acetyl-CoA

HO

#-@ Squalcne

Cycloartenol

Dormantino1

Dormanunonc

4 22, 26-Epimino-Ssholesten-3n,16R-diol

no

26-Am:no-160-hydroxycholer~rol

1

Verazine I

HO

26-A iiiiiiodihydrodiosgenin

-

Etiulinr

1

\ & ."-OH n " ^ * ' .NO &\ % Solasodine I40

Teinemine

Figure 12. The biosynthetic pathway of solasodine

512

GUNAWAN INDRAYANTO ET AL

J

Figure 13. Chemical conversion of solasodifie to steroid hormone.

SOLASODINE

513

6. BIOTRANSFORMATION OF SOLASODINE

Patel et al. (10) reported recently that the fungus Cunninghumella ekguns could transform solasodine into solasod-5ene- 38, 7BIB-di01, solasod-5ene-38,7adioland 38- hydroxysolasod-5-en-7ae. In contrast, incubation of solasodine with fungus PenicilZiumpanrlum gave solasod4ene-3a1e and the 6-methylsalicylicacid salt of solasodine. By using Mycobucteriumphlei solasodine could be transformed to 4-androstene-3,17dioneand 1,4-androstadiene-3,17dione(21). Shulz et al. (22) reported that in leaves extract of Solmum luciniutum, solasodine converted to its glucoside by a solasodine glucosyltranferase. 7. METHOD OF EXTRACTION AND QUANTITATIVE ANALYSIS OF SOLASODINE 7.1. Method of extraction For the extraction of solasodine as glycoalkaloid, the plant or cell culture materials could be extracted with methanol ( 23), ethanol 95 % (24,25), methano1:chloroform (2: 1) (26), 3 - 5 % aqueous acetic acid (27,28), 5 % acetic acid in methanol (29), 2 % acetic acid in 90 % ethanol (30) or 2 % oxalic acid (31). To determine the aglycone (solasodine) the extracts must be hydrolyzed by 1N HC1, neutralized with NaOH or ammonia, then extracted with organic solvent (chloroform). To determine solasodine, the biomass also could be directly hydrolyzed with 1N HCl (32,33) or 2N HCl in methanol (34). For separating non polar components (eg. free sterols) , the biomass was extracted with chloroform before hydrolized (35). To minimize the decomposition of solasodine into solasodiene, Segal et al. ( 36) recommended using of non aqueous low boiling point alcohols and acid concentration not exceeding 1N. By using two phase system consisting carbon tetrachloride and HCl as hydrolized mixture, Van Gelder (17) could prevent the degradation of the steroid alkaloid. Oven drying above 100°C of the plant materials also lead to solasodine loss (37). 7.2. Method of quantitative analysis 7.2.1 Titration Method of Eldridge and Hockridge (26)

51.2

GUNAWAN INDRAYANTO ET AL.

To determine the glycoalkaloid content in Solanum prycamhum, the dried berries was extracted with methanol :chloroform (2: l), then the extract was mixed with 0.8 % Na,SO,, shaken in a separatory funnel and ailowed to settle overnight. The methanol phase was collected, dried, and dissolved in 2N H SO,. This solution was heated for 2 hours and made basic with 4fi NaOH. The aglycone was extracted 3 times with benzene. After evaporating the residue was dissolved in methanol and titrated with a solution of 0.067 % bromophenol blue and 10 % phenol in absolute methanol, against a blank of methanol. Method of Valovich (31) After extraction of the glycoalkaloid with 2’% oxalic acid, hydrolyzed with HQ, the hydrolysate was added a 50 % solution of caustic soda to bring the pH to 9.5 for precipitating solasodine. The solasodine was extracted with neutral chloroform, and the chloroform solution was titrated with O.OO5N solution of n-toluene-sulphonic acid in chloroform, with 0.1 % solution of dimethyl yellow as indicator. 7.2.2. Colorimetric Method of Birner (25) Finely powdered materials was refluxed with 95 % ethanol for 30 minutes, filtered, and the ethanol extract was collected. After evaporating, the residue was hydrolyzed with 1N HCI,then neutralized with 1N NaOH. The aglycone was comphed with methyl orange and the colored complex extracted into chlorohrm and determined colonmetrically at 430 nm. Method of Khdagi et al. (24) In this method, solasodine was dissolved in 95 % ethanol, after addition of phosphate buffer pH 7.5, a solution of 0.1 % bromothymol blue in alcohol 50 %! was added to the mixture. The bromothymol blue-solasodine complex was extracted using benzene, and measured on spectrophotometer at 400 nm before 45 minutes. Method of Nigra et al. (38). Firstly the hydrolyzed sample was alkalinized with 60 % NaOH then solasodine was extracted with chloroform. After addition of 2. 1i4M of brornothymol blue in borax buffer pH 6.8 and mixed in a vortex-mixer,

SOLASODINE

515

the aqueous phase was taken out, added solution of methanolic 0.01N NaOH to the chloroform phase, then measured at 610 nm. 7.2.3. Potentiometric Method of Telek (27) The steroid glycoalkaloids were extracted from freshly harvested fruits with 2 % acetic acid and methanol. After hydrolysis, the common aglycone solasodine was extracted in benzene. An aliquot was mixed with an equal volume of acetone and titrated potentiometrically with 0.005 N perchloric acid in dioxane, using glass and silver electrodes for determination. 7.2.4. Gas chromatography

Methof of Carle (34) In this method, the freeze dried biomass was hydrolyzed using 2N HCl in methanol. After neutralization with ammonia 25 % , solasodine was extracted by chloroform. An aliquot of the chloroform phase was injected to a GC with the following condition, column glass (1/8 inch x 6 ft) packed with 3 % SE-30 (Gas Chrom Q, 100-120 mesh); FID and injector temperature were 30OoC; column temperature was isothermal at 250OC. By this condition solasodine and diosgenin/tigogenin were separated and quantitatively determined from Solmum plant materials. Method of Indrayanto (39) By using a glass column ( 2m x 2mm id.) packed with 3 % OV-1 on Gas Chrom Q ( 100-120 mesh); FID and injector temperature 30OoC; oven temperature was programmed from 200 to 280°C, S°C/minute, solasodine, hecogenin, diosgenin and sterols (cholesterol, campesterol, stigmasterol, sitosterol and isofucosterol) could be good separated. Method of Van Gelder (17) To prevent steroid alkaloid, degradation, van Gelder was used two phase system of carbon tetrachloride and HC1 to hydrolyze the plant materials. For analyzing the steroid alkaloids two systems of GC were used by the author. System 1 : glass column (lm x 2mm id.) packed with 10 % SE-30 on Chromosorb W HP (80-100 mesh); maximum operating temperature were 325OC (isothermal), 35OoC (programmed); FID temperature 350OC;

516

GUNAWAN INDRAYANTO ET AL.

injector temperature 325OC. System 2 : fused capillary column of 50m x 0.22 mm id., coated with CP-Sil 5 (film thickness 0.12 pm or CP-Sil 19 (film thickness 0.19 pm); oven temperature were 30O0C (isothermal), 325OC (programmed); R D and injector temperature were as the same as system 1. With both systems, solasodine, solanidine, demissidine, tomatidine, solasodiene and solanthrene could be separated. By using two detectors (FID and NPD), the identity of 21 steroid alkaloids can be elucidated by retention indexes and NPD/FID response ratio.

7.2.5. High Performance Liquid Chromatography Some of HPLC methods in the determination of solasodine or its glycosides that have been published are listed in table 9. By using combination of HPLC and thermospray mass spectrometry (LC-MS), the structure of the steroid alkaloid glycosides could be elucidated. This method seems well suited for a screening of a complex mixtures for a certain steroidal alkaloids (40). 7.2.6. Radioimm unoassay Weiler et al. (46) have reported a rapid, spesific and sensitive RIA for the determination of solasodine in vegetative and generative parts of Soiunwn luciniatwn. The antiserum added did not react at all with sterols, solanidanes and spirostanols, but showed strong reactivity with tomatidine and solasodine glycoalkaloids. The assay allowed the detection as little as 0.7 ng solasodine glycosides. 7.2.7. Thin Layer Chromatography - Densitometric Some of the published methods of Thin layer chromatography (TLC) of solasodine are listed in table 10. In our laboratory the solasodine content of in the vitro cultures of S o l a w spp. was determined by densitometric method. Firstly the dried biomass was extracted with chloroform 3 times on a vortex mixer to remove the non polar components (eg. free sterols), then hydrolised with 2N HCl in methanol (2 hours, 75'C), After neutralised with 10N NaOH, solasodine was taken out by chloroform. The chloroform extract was spotted on precoated Kieselgel G 60 F254 (Merck), and chloroform : methanol : diethylamine (20:2:0.5) was used as eluent. The solasodine

M

w 240,

sifidcacidradal

w

I acebnibae:wster(77.5:22.5);pH4.0

sample

a

Kasslbrataetrl. (21)

205nm

-,17 Qne, 1,4 adrostadecle3,17dare,~@EO&IO

W205nm

sdssodne,-ne

Vogd ad.(23)

WMom

acet#libile :Pic eS :TEA (83:17 :0.1); pH 2.8

W205Nll

:0.1 MThkmer(9: 1)

w 210 nm IWWI6 Hurler(41)

Hurleretal.(42)

W 213 nm WMSn pBondapakC18

I metranol:O.OlMTriskner(75:25)

a-,B-,wlvcosidea daaodm

Table 9. HPLC methods for determining solasodine and its glycosides

I nHaxas:EW

(1:l) (1:l) C W : M H (23:2) clt2az:MsoH (9:l) ctt2c12:AceQne (4: 1) nHaxas :h b l e (1 : 1) CHzClz :&OH :AmUc acid(= : 13: 2) nHaxas:EtOH

PqNoj

Me0H:CHQ (1:9)

Table 10. TLC methods for Analysing of solasodine

SOLASODINE

519

spots was detected by anise aldehyde-H2S04reagent (lOO°C, 5-10 minutes). Quantitation was done by measurmg at the maximum absorbance reflectance (385 nm). The determination of solasodine was made by calculation with a calibration graph obtained using solasodine (Sigma) as external standard on the same TLC plate. With this method linearity of solasodine was achieved from 0.4 to 1.6 pg/spot (r=0.998, n=7, V =2.1 %); LOD (limit of detection)= 0.11 pg/sjmt; LOQ (limit of q1,?&titation)=0.31 pg/spot; 'mean accuracy @ f SD) with standar addition method was 98.92 f 2.35 %, n = 14; RSD of precision determination was 1.87 % (n=10). 8. PHARMACOLOGICAL AND BIOLOGICAL ACTMTIES OF SOLASODINE AND ITS GLYCOSIDES As early as 1965 Kupchan et al. (53) demonstrated a tumorinhibiting activity of the glycoside 13 solamargine. Cham et al. (54) showed t!!at glycoalkaloid solasonine, solamargine and another mixture of glycosides containing aglycone solasodine, had on antineoplastic activity against Sarcoma 180 in mice. Solasodine inhibited the growth some fungal strains, although its activity was less compared to solafloridine and verazine (55). Some steroidal alkaloids including solasodine showed inhibitory effect on the enzymatic conversion of dihydrolanosterol into cholesterol (56). The steroidal glycoalkaloid solasonine and solamargine showed a membrane-disrupting properties of phosphatydylcholinekholesterol liposomes at concentration > 50 pM (57). A slight acetylcholinesteraseinhibitory activity of solasonine and solamargine was also reported (58).

Recently Frohne (59) reported that solasodine had a cortisone like effect such as anti phlogistic or reduction of blood vessel permeability. Solasodine also can prevent of anaphylactic shock in guinea pig. In a clinical trial a dose of 1 mg solasodine citrate which was given 2 times a day showed cardiotonic effect. This clinical trial also showed that solasodine gave a desensitization effect especially in patient with a rheumatoid polyarthritis.

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GUNAWAN INDRAYANTO ET AL.

References 1. Telek,L., Delphin,H., Cabanilias,E., (1977) Economic Botany 3 1: 120-128. 2. Nigra,H.M., Alvarez,M.A., Giulietti,A.M., (1990) Plant Cell Tissue and Organ Cultures 21: 55-60. 3. Sudiarto, Chairani,F., Rosita, S.M., Wahid,P. (1985) Jurnal Litbang Pertanian 4: 71-76. 4. Macek, T.E., (1989) Solanum aviculare Forst., Solanum laciniatum Ait. (poroporo): In vitro cultures and the production of solasodine, in :Bajaj,Y.P.S. (ed.)Biotechnology in Agriculture and Forestry Vol. 7, Springer Verlag, Berlin Heidelberg, pp.444-467. 5. The Merck Index, 11thEd.(1989),Merck & CO., Inc, p. 520. 6. Bentley, K.W. and Kirby, G.W. (1972) Elucidation of Organic structure by physical and chemical methods, 2nd ed., Interscience, p. 565. 7. Europaiesches Arzneibuch,Band I, I1 (1976), Wissenschaftliche Verlagsgesselschaft MBH Stuttgart Goviverlag GMBH, Frankfurt. 8. Puri, R., Wong, T.C., Puri, R.K. (1993) Magnetic Resonance in Chemistry 31:278-282. 9. Radeglia, R., Adam, G . , Ripperger, H. (1977)'Tetrahedron Letter 11:903-906. 10. Patel, A.V., Blunden, G., Crabb, T.,4. (1994) Phytochemistry 35: 125-133. 11. Budzikiewicz, H., Wilson, J.M.. Djerassi, C. (1962) Monatshefte fiir Chemie 93: 1033-1041. 12. Budzikiewicz, H., Djerassi, C., William, D.H., (1964) Structure elucidation of natural products by Mass Spectrometri, Vol 11, Holden Day Inc., San Fransisco, hndon, Amsterdam, pp. 110-120. 13. Budzikiewicz, H. (1964) Tetrahedron 20: 2276. 14. Indrayanto, G., Tutuk Budiarti, Soebahagiono, Emma, Sutarjadi (1979) The solasodine content of Solanm grandiflonun, paper presented in UNESCO Regional Seminar on Medicinal Plants Bangkok, Thailand. 15. Heftmann, E. (1983) Phytochemistry 22: 1843-1860. 16. Kaneko, K., Tanaka, M.W., Mitsuhashi (1976) Phytochemistry 15: 1391. 17. Van Gelder, W.M.J.(1989), PhD thesis, Agriculture University Wageningen

SOLASODINE

52 1

18. Tschesche, R. and Brennecke, H.R. (1980) Phytochemistry 19: 1449-1451. 19. Wall, M.E. (1986) Status of raw materials for production of oral contraseptives in Indonesia, Paper presented in the Workshop of Biotechnology of Steroid Compounds as Contraceptives and drugs, Jakarta, Indonesia. 20. Sebek ,O.K. ,(1986) Production of steroid compound by fermentation, paper presented in the Workshop of Biotechnology of Steroid Compounds, Jakarta, Indonesia. 21. Kartasubrata,J., Loyniwati, Jarnilah, Karossi, A.T. (1993) Indonesian Journal of Applied Chemistry 3: 61-68. 22. Schulz,D., Eilert,U., Ehmke, A. (1993) Planta Media Supplement 59: A649. 23. Vogel,H., Jatisatienr, A., Bauer, R. (1990) Angew. Botanik 64: 393-400. 24. Khafagi, S.M., Amin, S.W., Hassanin, R. (1970) Planta Medica 21: 139-141. 25. Birner, J. (1969) Journal of Pharmaceutical Sciences 58: 259. 26. Eldridge, A.C., and Hockridge, M.E. (1983) Journal Agric. Food Chemistry 31: 1218-1220. 27. Telek, L. (1977) Journal of Pharmaceutical Sciences 66: 699-702 28. Cham, B.E. and Wilson, L. (1987) Planta Medica 59-61. 29. Hunter, I.R., Heftmann, E. (1983) Journal of Liquid Chromatography 6 ~281-289. 30. Chowdhury, A.R., Prasas, R.N., Uddin. A. (1979) Quart. Journal of Crude Drug Research 17 : 137-138. 31. Valovich, N.A. (1965) Meditzinskaya Promyshlennost USSR 19: 4548. 32. Ehmke, A. and Eilert, U. (1993) Sulunwn dulcumuru L.: Accumulation of steroidal alkaloids in the Plants and in different in vitro cultures, in : Bajaj, Y.P.S. (ed.) Biotechnology and Forestry, Vol. 21, Medicinal and Aromatic Plants VI, Springer Verlag, Berlin Heidelberg, pp.339-349. 33. Chandler, S., Dodds, J. (1983) Plant Cell Reports 2: 69-72. 34. Carle, R. (1979) Ph D thesis, University of Tuebingen. 35. Indrayanto, G., Studiawan, H., Noor Cholies (1994) Phytochemical Analysis 5 : 24-26. 36. Segal, R., Breuer, A., Milo-Goldzweig, I. (1978) Journal of Pharmaceutical Sciences 67: 1169-1170.

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37. Crabe, P.G., Fryer, C., (1982) Journal of Pharmaceutical Sciences 71: 1356-1362. 38. Nigra, H.M., Caso, O.H., Giulietti, A.M., (1987) Plant Cell Reports 6: 135-137. 39. Indrayanto, G., (1983) Ph D thesis, University of Tuebingen. 40. Ehmke. A., Schiebel, H.M., McDowell, M. (1987) Pharmaceutisch Weekblad Scientific Edition 9: 232. 41. Heftmann, E., Hunter, I. R. (1979) Journal of Chromatography 165: 283-299. 42. Hunter, I.R., Walden, M.K., Heftmann, E. (1980) Journal of Chromatography 198: 363-366. 43. Crabbe, P.G. and Fryer, C. (1980) Journal of Chromatography 187: 87- 100. 44. Indrayanto, G, Utami, W., Santosa, M.H., Syahrani, A., Diosgenin, Analytical . Profile of Drug Substances and in: Brittain, H.G.(4) Excipients Vol. 23., Academic Press, in Press. 45. Ehmke, A. and Eilert, U. (1986) Plant Cell Reports 5: 31-34. 46. Weiler. E.W., Kriiger, H., Zenk, M.H. (1980) Planta Medica 39: 112-124. 47. Hunter, I.R., Walden, M.K., Wagner, J.R., Heftmann, E. (1976) Journal of Chromatography 118 :259-262. 48. Puri, R.K. and Bhatnagar, J.K. (1975) Phytochemistry 14: 2096. 49. Willuhn, G. (1966) Planta Medica 14: 408-419. 50. Willuhn, G. and Kun-anake, A. (1970) Planta Medica 18:354-359 51. Kadkade, P.G. and Rolz, C. (1979, Phytochemistry 16:1128 52. Kartasubrata, J., Fitri, T.Y., Halomoan, V.A., Lotulung, P., Buchori, Karossi, A.T., (1993), Annales Bogorienses 2: 12-15 53. Kupchan, S.M., Barboitis, S.J., Know, J.R., Lau Cam, C.A. (1965) Science 150: 1827-1828. 54. Cham, B.E., Giliver. M., Wilson, L. (1987) Planta Medica 34-36. 55. Kusano, G., Takahashi. A . , Sugiyama, K., Nozoe, S . (1987) Chemical Pharmaceutical Bulletin 35: 4862-4867. 56. Kusano, G., Takahashi, A., Nozoe, S., Sonoda, Y.,Sato, Y. (1987) Cheinical Pharmaceutical Bulletin 35: 4321-4323. 57. Roddick, J.G., Rijnenberg. A.L., Weisenberg, M. (1990) Phytochemistry 29: 1513-1518. 58. Roddick, J.G. (1989) Phytocheinistry 28: 2631-2634. 59. Frohne, D. (1993) Zeitschrift fur Phytotherapia 14: 337-342.

STARCH

Ann W. Newman, Ronald L. Mueller, Imre M. Vitez, Chris C.

Kiesnowski, David E. Bugay, W. Paul Findlay, Chris Rodriguez

Bristol-Myers Squibb Pharmaceutical Research Institute One Squibb Drive New Brunswick, NJ 08903

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

523

Copyright 0 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.

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ANN W. NEWMAN ET AL.

CONTENTS

I. Description 1.1 Name, Formula, and Molecular Weight 1.2 Types of Starch 1.3 Appearance 1.4 General Chemical Properties 1.5 Uses and Applications 2. Method of Preparation 3. Starch Derivatives 4. Physical Properties 4.1 Structural Information 4.2 Infrared Spectroscopy 4.3 Raman Spectroscopy 4.4 Nuclear Magnetic Resonance Spectroscopy 4.5 Thermal Properties 4.6 Moisture Content 4.7 Particle Morphology 4.8 Optical Microscopy 4.9 Particle Size Measurements 4.10 Surface Area Measurements 4.1 1 Bulk Powder Properties 4.12 Hygroscopicity

5 . Methods of Analysis 5.1 Compendia1 Tests 5.2 Chromatography 6. Excipient Studies 6.1 Drng Compatibility 6.2 Tableting 6.3 Disintegration

7. References

STARCH

525

I. Description

1.1 Name. Formula. and Molecular Weight Starch is a polymeric material with a molecular formula of (C6H1005)n, where n ranges from 300 to 1000 [l]. Common starches contain two types of D-glucopyranose polymers called amylose and amylopectin. Amylose is a linear polymer of a-D-glucopyranosyl units linked (1-4) as shown in Figure la. These molecules can be comprised of 100 to over 1000 glucose units [2]. Amylopectin is a branched polymer of a-Dglucopyranosyl units containing (1-4) linear linkages and (1‘ 6 ) linkages at the branch points, as shown in Figure 1b. This polymer is three or more times larger than amylose [2]. Most naturally occurring starches contain approximately 30% amylose, however, specific starches and their properties are determined by the size and amount of each type of polymer molecule present in the material. The starch granules are formed by the attractive forces between the polymeric molecules. The linear portions tend to associate into micelles which bind the molecules together to form a somewhat ordered structure. Models of this structure have been proposed [3], and it is known that the structure is rigid and insoluble in water. The Chemical Abstracts identification number is CAS-9005-25-8.

1.2 T p e s of Starch Starch can be derived from a number of natural sources, including those listed in Table I. It is found in various parts of the plants and several extraction methods are used to isolate the material. The most common type of starch used in the pharmaceutical industry is corn, although studies with other forms have been performed [4-91.

ANN W. NEWMAN ETAL.

536

Figure 1.

Structure of (a) a linear amylose starch molecule, and (b) a branched amylopectin starch molecule.

0-

OH

r

0-

OH

OH

n

-

521

STARCH

Table I. Sources and Characteristics of Various Starches [ 101

Starch Type

Extracted From

Granule Shape

Granule Size (Pm)

Corn (Maize)

Seed

Round or polygonal

5 -25

Tapioca

Root

Round or oval

2 - 25

Potato

Root

Egg-shaped

15 - 100

Wheat

Seed

Round or elliptical

2 - 1 0 or 20 - 35

Sago

Stem

Oval or egg-shaped

20 - 60

Arrowroot

Root

Oval

15 - 70

Rice

Seed

Polygonal

3-8

Barley

Seed

Round or elliptical

2 - 6 or

Waxy sorghum

Seed

Round or polygonal

6-30

Sweet potato

Root

Polygonal

10 - 25

Waxy maize

Seed

Round or polygonal

5 - 25

20 - 35

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ANN W. NEWMAN ET AL.

A number of starch modifications also exist and are used in pharmaceutical applications [ 11. Pregelatinized or compressible starch has been chemically or mechanically processed to rupture all or part of the granules in water. It is then dried to yield an excipient material suitable for direct-compression formulations. Sterilizable maize starch contains magnesium oxide (not greater than 2.2%) and has been chemically or physically treated to prevent gelatinization upon exposure to moisture or steam sterilization. Soluble starch results when potato or maize starch has been chemically treated to destroy the gelatinizing ability of starch. 1.3 ApDearance Starch is a fine white powder which is odorless and tasteless. It is composed of very small spherical or elliptical granules. The botanical origin of the starch material will determine the granule shape and size, and these characteristics are summarized in Table I. When the granules are analyzed microscopically, a distinct cleft called the hilum is observed, which is considered the origin of granule growth. 1.4 General Chemical Properties Starch is insoluble in alcohol, most solvents, and cold water. Alkaline solutions, however, will degrade starch and its polysaccharide components [lo]. Starch is relatively resistant to carbohydrases other than a-amylose

[ I 13. When starch is suspended in water and heated to a critical point called the gelatinization temperature, water will penetrate the granules and swell them to produce a viscous mass. With the rising temperature, the hydrogen bonds that hold the micellar structural units and the water molecules in an aggregated state tend to dissociate. The dissociated water molecules are then able to penetrate the weakened starch structure and gradually hydrate the many hydroxyl groups along the length of the starch molecule. Gelatinization temperatures vary from starch to starch, but range from 60 to75"C [lo]. Starch granules will lose their characteristic shape as gelatinization proceeds.

STARCH

529

The reaction of starch with iodine is a common identity test for starch. A dilute solution of iodine stains starch a blue to bluish red color. It is believed that the amylose portion complexes with iodine by forming a helix around it [lo]. This blue color has been used both as a qualitative and quantitative test for starch in various systems. 1.5 Uses and ApD1ication.s Starch is used widely in the pharmaceutical industry because, among its other properties, it is readily available, inexpensive, white, and inert. It has been described as a tabledcapsule diluent, tablet disintegrant, and glidant [l]. The function of starch can depend on how it is incorporated into the formulation. Starch will function as a disintegrant when it is added in the dry state prior to adding a lubricant. It may exhibit both binding and disintegrant properties when it is incorporated either as a paste or dry before granulation with other agents [ 121. 2. Method of Preparation The corn kernel is made up of the tip cap, the pericarp or hull, the germ or the embryo, and the endosperm. The endosperm contains the starch granules embedded in protein cell walls. Corn kernels have two distinct regions of endosperm. The floury region in the grain center has loosely packed, rounded starch granules with a low protein content. The horny region of the endosperm at the grain edges contains angular granules with a high protein content. The granules are removed from the kernel during starch preparation. The most common preparation of starch is wet milling although dry milling is also performed [1I]. The corn is screened to remove any cob, sand or other unwanted material and aspiration is used to remove the light dust and chaff. In a process called steeping, the material is placed in a vat and the kernels are softened for milling using water, heat, and sulfur

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ANN W. NEWMAN ET AL.

dioxide. The steeped corn is coarsely ground in an attrition mill to break loose the germ which is then removed in a cyclone separator based on its density. The resulting aqueous slurry is milled a second time to release the starch granules. The kernel suspension containing starch, gluten, and fibers is passed through a concave screen to remove the fibers. The starchgluten suspension is then concentrated by centrifugation to reduce soluble material and to separate the gluten based on its density. The starch is concentrated again and washed numerous times using the cyclone separator. The starch suspension may be dried (unmodified corn starch), gelatinized and dried, or modified by chemical or physical means. After this processing. corn starch is a white powder with a pale yellow tint and bledching is required to achieve absolute whiteness. 3. Starch Derivatives Starches undergo many reactions characteristic of alcohols because of the many hydroxyl groups present in the structure. Modification of the Dglucopyranosol units can occur by oxidation, esterification, etherification, or hydrolysis. The resulting starch derivatives are defined by a number of factors such as plant source, prior treatment (acid-catalyzed hydrolysis or dextrinization), aniyloseiamylopectin ratio or content, molecular weight distribution or degree of polymerization, type of derivative (ester, ether, oxidized). nature of the substituent group. and physical form (granular, pregelatinized) [ 131.

'I he degree of substitution (DS) is a common method of characterizing starch derivatives and is a measure of the average number of hydroxyl groups on each D-glucopyranosyl unit. It is expressed as the moles of substitucnt per D-glucopyranosyl unit and the maximum DS is 3 since thrct! hydroxyl groups are available in the unit for substitution. Most conimercially produced derivatives have a DS less than 0.2. The molar substitution is used when the substituent group reacts further with the reagent to form a polymeric substituent. It is defined as the level of substitution in terms of mole of monomeric units (in the polymeric substituent) per mole of D-glucopyranosyl unit and can be larger than 3.

STARCH

53 1

Manufacture of starch derivatives includes treatment of aqueous slurries and the dry starch. For slurries, a 35-45% aqueous suspension at pH 7 to 12 and temperatures ranging up to 60°C are commonly used. Conditions are adjusted to prevent gelatinization and to allow recovery of the starch derivative in granular form by filtration or centrifugation. For dry material, the starch is treated with the required reagents by dry blending, spraying the reagents onto the starch granules or filter cake, or by suspending the starch in a reagent solution and then recovering the starch. The treated powders are then heated to temperatures up to 150°C to yield granular products with DS values up to one. Many variations on starch derivatives exist and they have been exploited in a number of ways in the food, paper, textile, and adhesive industries. More detailed discussions of starch derivatives are available [11,13,14]. Some common starch derivatives are listed below.

Hydroxyalkyl Starch Ethers such as hydroxyethyl and hydroxypropy1 Starch Phosphates such as starch monophosphates and starch phosphate diesters Cationic Starches such as the tertiary and quaternary aminoalkyl ethers Oxidized Starches made by introducing carbonyl and carboxyl groups Starch Acetates Cross-Linked Starches Acid-Modijied Starches 4. Physical Properties

The physical properties of unmodified and pregelatinized corn starch from three vendors were characterized and the materials analyzed are summarized in Table 11. Examples of unmodified corn starch are STA-Rx and Purity 2 1. Starch 1500 is a sample of partially pregelatinized starch and Starch 1551 represents fully pregelatinized starch. Starch 1500 LM is a partially pregelatinized starch with a low moisture content.

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ANN W. NEWMAN ET AL.

Table 11. Starch Materials Analyzed for Physical Properties

Starch Type

Vendor

Trade Name

Total Volatile Content (%)

tinmodified corn

Staley

STA-Rx

10.9

Unmodified Corn

National

Purity 21

10.2

Pregelatinized

National

1551

8.3

Pregelatinized

Colorcon

1500

9.4

PregelatinizedLow Moisture

Colorcon

1500 LM

6.5

STARCH

533

4.1 Structural Information Starch is a semicrystalline polymer. The linear amylose molecules are amorphous in nature, but the branched amylopectin portion has been reported as partially crystalline. It is believed that the crystalline regions in the starch granule are interspersed in a continuous amorphous phase [3,13,15]. X-ray diffraction studies have shown that starch exists in three crystal forms designated A, B, and C. These forms are dependent on the botanical source of the starch. Pattern A is observed for cereal grain starches, whereas pattern B is characteristic of tuber, fruit, and stem starches. Pattern C is intermediate between the A and B patterns and has been attributed to mixtures of A and B type crystallites [151. The A type pattern is commonly observed for corn starch. Single crystal x-ray diffraction data for the crystalline portion of A type starch has been reported [16], and it was found to crystallize in a monoclinic lattice with a = 2.124 nm, b= 1.172 nm, c=l.069 nm and y=123.5 '. The unit cell contains 12 glucose residues located in two lefthanded, parallel-stranded double helices packed in a parallel fashion. Four water molecules are located between these helices. Intramolecular hydrogen bonding was not observed and the interstrand stabilization in the type hydrogen bonds. double helix is attributed to O(2)...0(6) It has been reported that the B type starch also contains chains arranged in double helices [ 171. The currently accepted hexagonal unit cell has dimensions of a=b=l.85 nm and c= 1.04 nm. The A and B structures differ in crystal packing of the chains and in moisture content. Powder x-ray diffraction patterns for representative unmodified and pregelatinized corn starches are given in Figure 2. The unmodified corn starch was found to have some crystalline character as evidenced by the broad peaks present. The pattern is indicative of a semicrystalline material and is similar to the A type pattern, but a definite determination of the form is difficult based on the quality of the pattern. The pregelatinized

ANN W. NEWMAN ET AL.

534

X-ray powder diffraction patterns of unmodified (upper trace) and pregelatinized (lower trace) corn starch

Figure 2.

m m

Ln N

Q

N

v) r(

0

rl

Ln

1

1

m - m m ~ Q + Q D N f r l

1

1

1

1

m m

m

9

f

rl

rl

1

~

1

Q

N

rl

1

m

1

Q

m

4

1

1

m m QD

1

1

Q

~

9

1

1

m r 1p

1

1

Q

n

N

Q

1

-

m s Q

m

o

STARCH

535

Starch 1551 material exhibits a broad amorphous halo in the powder pattern indicating that the majority of the sample is amorphous material. This type of pattern was expected since the processing of pregelatinized starch destroys the crystalline portions of the granule. 4.2 Infrared Spectroscopy Diffuse reflectance (DR) infrared (IR) spectra were acquired for unmodified, partially pregelatinized, fully pregelatinized, and partially pregelatinized (low moisture) corn starch. Identical IR spectra were measured for the unmodified, partially pregelatinized, and fully pregelatinized starch samples and are represented in Figure 3. The partially pregelatinized (low moisture) starch sample displayed slight shifts in the absorbance bands assigned to the glycosidic COC and coupled CO stretch vibrational modes. The IR spectral band assignments are presented in Table I11 [IS]. The measured absorbance bands are consistent with the structure of starch. 4.3 Raman Spectroscopy Raman spectra were acquired for unmodified, partially pregelatinized, fully pregelatinized, and partially pregelatinized (low moisture) corn starch. Identical Raman spectra were measured for the unmodified, partially pregelatinized, and fully pregelatinized starch samples and are represented in Figure 4. The Raman spectral band assignments are presented in Table IV [191. The measured absorbance bands are consistent with the structure of starch. 4.4 Nuclear Magnetic Resonance Spectroscopv Solid-state 13Ccross polarizatiodmagic angle spinning (CPMAS) nuclear magnetic resonance (NMR) spectra were acquired for unmodified, partially pregelatinized, fully pregelatinized, and partially pregelatinized (low moisture) corn starch. Identical I3C spectra were measured for each and are represented in Figure 5.

5%

Figure 3

ANN W. NEWMAN ET AL.

Infrared spectra of ( a ) unmodified corn starch, STA-Rx, (b) partially pregelatinized corn starch, Starch 1500, (c) fully pregelatinized corn starch, Starch 1551, and (d) partially pregelatinized corn starch with low moisture content, Starch 1500 LM

T

i

STARCH

Table 111. Diffuse Reflectance IR Spectral Assignments Wavenumber fern-')

Structural Assignment

-3350

OH stretch

2929,2898

CH stretch

1452

CH, bend

1417,1335, 1302

CH bend

1364,1240,1206

OH in-plane bend

1148 (1 166 for LM starch)

glycosidic COC asymmetric stretch

1076 (1065 for LM starch), 1012

coupled CO stretch, CC stretch, and OH bend

930

ring vibration

862

C,-group vibration

764

ring breathing vibration

709,643,606,576,524

low frequency ring vibrations

537

538

Figure 4.

ANN W. NEWMAN ET AL.

Raman spectra of (a) unmodified corn starch, STA-Rx, (b) partially pregelatinized corn starch, Starch 1500, (c) fully pregelatinized corn starch, Starch 1551, and (d) partially pregelatinized corn starch with low moisture content, Starch 1500 LM.

STARCH

Table IV. Raman Spectral Assignments Raman shift (cm-')

Structural Assignment

-3365

OH stretch

2932,2907

CH stretch

1459

CH,, CH, deformation

1380

CH, symmetric deformation

1335

CH deformation (?)

1123,1081,1051

CC stretch

938,855,577,479,440

ring and lattice vibrations

539

ANN W. NEWMAN ET AL.

540

Figure 5 .

Solid-state 13CCP/MAS nuclear magnetic resonance spectra of (a) unmodified corn starch, STA-Rx, (b) partially pregelatinized corn starch, Starch 1500, (c) fully pregelatinized corn starch, Starch 1551, and (d) partially pregelatinized corn starch with low moisture content, Starch 1500 LM.

c

c

STARCH

54 1

Table V. Solid-state I3CNMR Spectral Assignment

NMR Chemical Shift (ppm)

Structural Assignment"

61.9

C6

72.2

c4

81.5

C2, C3, and C5

101.2

c1

~~

"Carbon numbering scheme based upon Figure 6.

Figure 6.

13

Numbering system of the starch sub-unit used for the CNMR assignments.

OH

542

ANN W. NEWMAN ET AL

The resonance positions and structural assignments for the solid-state 13C NMR spectrum of starch are presented in Table V. Although the CP/MAS technique provides a “liquid-like” NMR spectrum, the broad nature of the carbon resonances prevented spectral resolution of all the carbon signals at a Larmor frequency of 62.89 MHZ. Solution-phase, ‘H NMR studies in DMSO-d, have also been performed on amylose and model compounds 1201. Spectral assignments and intramolecular hydrogen bonding suggest that the same conformation is perpetuated along the amylose chain. 4.5 Thermal Properties Thermal analysis has also been used to characterize the structure of starch. A melting endotherm due to the crystalline portions of starch has been studied [21], but it is not clearly resolved in all samples due to the small amount of crystalline material present in the samples. The transition is also dependent on the sample preparation and moisture content of the material. The melting point of starch has been calculated using an equation developed by Florey [22] for polymeric systems. Using this approach, the melting point of the pure polymer has been reported as 168“C [2 11. Using the same equation and hot stage data, a melting point of 210°C has also been reported [23,24]. The glass transition and gelatinization temperatures have also been studied for starch materials [25,26]. Studies of the glass transition in wheat starch have been performed using differential scanning calorimetry (DSC) [25]. This thermal event was found to be dependent on the moisture content of the preparation and was ill-defined below 13% moisture. When observed, it occurred at approximately 40°C. The onset of melting was also dependent on the moisture content of the samples in this study and was found to range from approximately 60 to 80°C. Gelatinization studies of starch have also used DSC [26]. Concentrated starcWwater suspensions produced a well-defined endotherm under suitable conditions which could then be integrated to obtain the heat of gelatinization for various starches.

STARCH

543

A representative DSC curve collected for corn starch with a low moisture content (9%) is given in Figure 7. The sample was analyzed as received in a hermetically sealed pan with a pinhole to allow for controlled pressure release. A broad endotherm is observed for the sample over a temperature range of 50-150°C. The thermogravimetric (TG) curve in the overlay shows a weight loss in the same temperature range as the endothermic transition, therefore, it was designated as a dehydration. A similar curve was also observed for the pregelatinized starch samples. The glass transition for the sample was not expected to be clear due to the low moisture content of the samples. The dehydration predominated with the sample preparation used and the melt endotherm was not observed for the corn starch sample.

To obtain information on the glass transition in pregelatinized starch, a sample was equilibrated in a 90% relative humidity chamber for 19 days. The volatile content of the sample was 17% after equilibration. The sample was then placed in a hermetically sealed pan and analyzed. The glass transition was evident at approximately 53 "C, as shown in Figure 8. It is evident that the sample preparation is a critical factor in obtaining specific information using DSC. 4.6 Moisture Content The moisture content of starch has been determined using a number of techniques including Karl Fisher measurements [27], thermogravimetry (TG) [28], loss on drying (LOD) [l], and NMR spectroscopy [28]. The National Formulary (NF) and British Pharmacopeia (BP) specify that moisture contents should be less than 14, 15, or 20%, depending on the botanical source of the material. The moisture contents of the commercial corn starch lots were measured using TG analysis and are given in Table 11. The unmodified corn starch samples had moisture levels around 1O%, which is similar to that reported for similar types of samples [13. The amount of water in the pregelatinized samples was slightly lower than that of the unmodified corn starch samples, ranging fiom approximately 8 to 9%. The low moisture

ANN W. NEWMAN ETAL

544

Differential scanning calorimetry thermogram (lower trace) and thermogravimetry profile (upper trace) of unmodified corn starch.

Figure 7.

9

y: 0

In

4

9 r

a Y

n U

3 Y

i?

r

0

P

10G

2;

-

3

E n

O

E

:+

. 10 I

0 10

I

N 10

0

STARCH

545

Differential scanning calorimetry thermogram of pregelatinized starch showing the glass transition.

Figure 8.

0 m r

m

t

v) N

/ *

I

9

0

m

0

I

0

I

?J 9 ‘y

5Jh

ANN W. NEWMAN ET AL.

pregelatinized starch did exhibit a significantly lower moisture content of 6.5%. 4.7 Particle MorpholoFy A number of excellent reviews on the microscopy of starches have been published [ 10, 29-32]. Areas covered in these references include granule morphology, granule size, gelatinization temperatures, and staining. Our discussion will focus on granule morphology using scanning electron microscopy.

As summarized in Table I for a number of starches, the granule shape and size is characteristic of the botanical origin and can be used to identify the materials. It has been reported that the floury granules, as found for potato and tapioca starches, tend to be larger and more regular in shape. Descriptive terms used for these types of granules include round, elliptical, or oval. Horny starches, such corn and rice, are usually described as polygonal because of the angular sides of the granules caused by the close packing of the granules in the kernel. Starches are found as individual granules, but aggregated materials are also observed and are attributed to the drying conditions. Extensive heat and moisture during drying will produce a slight gelatinization of the surface of the granule and cause the granules to adhere together to form the aggregates.

Electron microscopy has been useful in the morphological study of starch grunules 133-351. Scanning electron microscopy (SEM) exhibits good depth of field and gives detailed information about the surface of dry granules. The low magnification SEM micrograph in Figure 9a shows the uniform particle size and shape for STA-Ku. The polygonal and round shape of' the unmodified corn starch is illustrated in Figure 9b at a higher magnification. The surfaces of the granules are relatively smooth and pores are not evident at this magnification. An aggregate showing some fusion of the granulc surfaces of unmodified corn starch is shown in Figure 1 0. The pregelatinized starch sample exhibits an entirely different morphology, as shown in Figure 1 1. The particles are irregular and show large pores for the majority of particles. The pregelatinization process has

STARCH

Figure 9a. Scanning electron micrograph of unmodified corn starch granules, taken at a magnification of 270x.

541

548

ANN W. NEWMAN ET AL.

Figure 9b. Scanning electron micrograph of unmodified corn starch granules, taken at a magnification of 2700x.

STARCH

549

Figure 10. Scanning electron micrograph of an aggregate found in an unmoMied corn starch sample, taken at a magnification of 1500~.

SS(1

ANN W. NEWMAN ET AL

Figure 1 1. Scanning electron micrograph of pregelatinized Starch 155 1, taken at a magnification of I OOx.

STARCH

55 1

ruptured the granules resulting in a new particle morphology. SEM has also been used to investigate the properties of starch in a tablet [36]. It was found that starch possessed elastic properties during compression, with maize and rice starch being more elastic than potato. As the applied pressure was increased, deformation of the starch granules was reported and could predominate at very high pressures. It was also concluded that starch particles do not fuse together and were nearly always surrounded by a space which contributed to the disintegration properties.

4.8Optical Microscopv Optical microscopy has been a powerful tool in the study of starch materials and common starches have been readily identified using this technique. It has been suggested that starch be examined as a 0.2-0.3% suspension in water or glycerol to obtain the best images [30,3 11. A polarizing microscope also gives information about the starch granules. When unmodified starch granules are observed using polarized light, two dark lines intersecting at the hilum will form a cross or a V-shape. The shape of the cross can be used to help identify the type of starch. One explanation for this feature suggests that the density and distribution of moisture throughout the granule are not uniform and the hilum contains more moisture than the other regions [lo]. As the granules dry,stresses are formed within the granule resulting in the bright regions observed under the polarized light. When the starch swells or is gelatinized, the cross is no longer visible with the polarizing microscope [32]. The absence of this cross is a simple and accurate determination of the presence of gelatinized granules in a starch sample. An example of an unmodified corn starch suspended in water is given in Figure 12a. The polygonal and round shape is evident for the granules, but less detail is obtained when compared to the SEM micrographs. The sample was also analyzed under crossed polarizers, and the crosses are clearly observed for the granules, as shown in Figure 12b. When partially pregelatinized starch 1500 is suspended in water and observed under the optical microscope, very few details of the particles are discerned, as

552

ANN W. NEWMAN E T A L

Figure 12a Optical micrograph of unmodified corn starch, using ordinary illumination at a magnification of 400x

STARCH

553

Figure 12b. Optical micrograph of unmodified corn starch, using crossed polarizers at a magnification of 400x.

55-1

Figure 13,

ANN W. NEWMAN ETAL.

Optical micrograph of pregelatinized Starch 1500, obtained using ordinary illumination at a magnification of 200x.

STARCH

555

Figure 13b. Optical micrograph of pregelatinized Starch 1500, obtained using crossed polarizers at a magnification of 200x.

556

ANN W. NEWMAN ET AL.

shown in Figure 13a. The transparency of the particles is due to the refractive index similarities between the sample and the solvent. The irregular shape of the particles is evident, however, and a few intact granules may be present. Figure 13b is a micrograph of this sample under crossed polarizers. It is evident that the majority of the sample was pregelatinized, however, some intact granules exhibiting crosses are also visible in the lower left quadrant. 4.9 Particle Size Measurements

Particle size can affect the disintegration, flow, handling, and tableting properties of these materials. Sieving is a common method for obtaining specific size fractions for granulation and disintegration studies [37-401. Other studies characterize the particle size distribution of the materials as received from the vendors to investigate possible variations in the properties of the excipient. A wide range of granule sizes, spanning from 2 to 150 pm, has been reported for various starches [lo]. The size of the granule is generally expressed as the length of its longest axis in microns [3 11. The results of a number of measurements can be expressed either as a range or as an average size. Starches such as rice show a relatively uniform distribution, therefore, an average size is appropriate. Other starches, such as maize, show a wide distribution of sizes and a range would more accurately describe the granule size. Rye and wheat starches are known to exhibit bimodal distributions of very large and very small granules. A number of methods for determining the particle size of various starches have been used. For bulk powder analysis, sieving is employed for large amounts of material [ 13. One of the most common methods for particle size determination is optical microscopy 141,423 because it gives a direct measurement of the individual particles. Automated systems have been used to examine the particle sizes of starch materials with good results [43,44]. Laser light scattering analysis has also been utilized to measure the size of dry particles (particles in air) and suspensions (particles in liquid) [45]. This technique is suitable in many cases, but since it is not a

STARCH

557

direct measure of the particles, the data was checked using SEM. It was concluded that laser light scattering analysis was dependent on the model used to fit the data and better reproducibility was obtained with samples suspended in liquid. The particle size distributions of starch samples from the various vendors were collected using optical microscopy and an image analysis system. This type of measurement was suitable for the fine particles in the sample but large aggregates would not have been included. The mean particle sizes are summarized in Table VI. A measure of 95% of the particles is also given in Table VI to assess the number of large particles in the sample. The mean particle size exists in a narrow range from 5.7 x 9.4 to 12.8 x 18.9pm. The different processing of the various starches does not appear to have substantially changed the mean particle size of the fines. The distributions are shown graphically in Figure 14 for the particle length range 0-30 pm since this range included most of the particles measured. The distribution of the particle length is similar for the five starch samples, with the majority of the particles having a length between 4 and 20 pm. STA-Rx and Starch 1551 appear to have more particles in the range 2 to 10 pm, whereas Starch 1500 and 1500 LM appear to have more particles in the 10 to 20 pm range. 4.10 Surface Area Measurements Surface area measurements of starches have been obtained by air permeametry [9,39] or nitrogen adsorption [46]. Relatively low surface areas ranging from approximately 0.1 to 0.5 m2/ghave been reported [9], however, values as high as 3 m2/ghave also been observed [46]. Surface area measurements on unmodified and pregelatinized corn starch samples were obtained using a five-point nitrogen BET analysis after outgassing the samples at room temperature under vacuum. The values are listed in Table VI. The surface areas range from 0.18 to 0.36 m2/g which is in agreement with literature values reported for similar materials. The low surface areas can be partially explained by the low porosity of the materials as seen in the SEM photos.

ANN W. NEWMAN ET AL.

558

Table VI. Particle Size and Surface Area Data for Starch Lots

Starch Type

Mean Particle Size (pm)

95% Less-Than Value (pm> *

Surface Area (m2/g)

STA-RX

8.4 x 11.1

17.3 x 22.6

0.35

Purity 21

10.6 x 15.0

25.4 x 35.7

0.36

1551

5.7 x 9.4

15.1 x 24.7

0.18

1500

12.8 x 18.9

31.1 x 46.2

0.26

1500 LM

10.1 x 14.8

19.6 x 28.9

0.24

*

Particle size for which 95% of the measured particles were smaller than.

Figure 14.

STARCH

Particle size distribution of commercial starches.

0'2-82

8Z-92

PZ-ZZ

zz-oz OZ-81 81-91

91-91

P 1-2 1

ZL-01 01-8

8-9 9-P P-Z

559

560

ANN W. NEWMAN ET AL.

4.1 1 Bulk Powder Properties Bulk powder properties are important in understanding the handling properties of an excipient or a granulated product. A number of studies have investigated the bulk powder properties of starch [9,42,47] and granulations made with starch [6,12,48]. Common parameters measured are bulk and tap density. From these values the compressibility can be calculated using the following equation:

100 x

(tap density - bulk density)

tap density

= %

compressibility

(1)

A classification system to evaluate the flow properties of powders has been introduced by Can: [49,50]. A flowable powder is defined as freeflowing and will tend to flow steadily and consistently, whereas, a floodable powder will exhibit an unstable, discontinuous, and gushing type of flow. The parameters in Carr's system include angle of repose, angle of spatula, compressibility, cohesion, and dispersibility. Based on these parameters, flowability and floodability indices are calculated to determine the handling properties of bulk solids.

Various starches have been characterized using C a d s system and the Hosokawa powder characteristics tester [47]. To compare the bulk powder properties of unmodified and pregelatinized corn starches from the various vendors, the same instrumentation and procedures were used. The results are summarized in Table VII. The parameters used to obtain the flowability index are compressibility (from bulk and tap density), angle of repose, and angle of spatula. The values obtained for these tests were similar to those previously reported for similar materials [47]. The flowability indices of 3 1 and 36 for the two unmodified starches are similar and indicate the materials to be very

STARCH

56 1

poorly flowing powders. The pregelatinized starches exhibited flowability indices in a narrow range of 52-54 indicating that these materials would also have poor flow properties. The floodability indices were evaluated using the angle of fall, angle of difference, and dispersibility measurements. The floodability indices for all five starches ranged from 62 to 86. Values in this range are indicative of floodable material which will exhibit difficult handling properties for the bulk powder. Previous data on similar materials reported poor to borderline flow characteristics using this system [47]. The mass flow rate data are also given in Table VII for comparison. The relatively low mass flow rates ranging from 0.04 to 1.OO g/sec demonstrate the poor flow properties of these materials. The unmodified starch samples exhibited the lowest flowability indices and the lowest mass flow rates. The small particle size is largely responsible for the floodable properties of these materials. 4.12 Hygroscopicity Starch has been classified as a moderately hygroscopic material [5 13. Water sorption studies have been conducted using static methods (saturated salt solutions in closed chambers) [5 1-53], modified inverse frontal gas chromatography [54], as well as other techniques [27,55]. The isotherms are typically type I1 curves exhibiting hysteresis (an amount of sorbed moisture remaining during desorption) [5 11. Hysteresis is usually attributed to ink bottle pores in which the water cannot escape the pore due to the constricted neck. In the case of starch, the hysteresis has been attributed to intra- and intermolecular hydrogen bonding of water with the hydroxyl groups of the starch molecule [51,55,56]. The extent of hydration and swelling depends on the accessibility of the hydroxyl groups in the starch to the water [55]. It has been suggested that the amorphous regions are responsible for the reversible swelling of starch upon the adsorption of water [131.

S62

ANN W. NEWMAN ET AL.

Table VII. Bulk Powder Data for Starch Lots

Property

STA-Rx

Purity 21

Starch 1551

Starch 1500

Starch 1500 LM

1.54

1.54

1.55

1.54

Bulk density (g/mL)

0.46

0.55

0.46

0.60

0.63

Tap density (gimL)

0.79

0.83

0.65

0.87

0.86

Compressiblity

42

34

30

31

27

Angle of Repose (deg.)

62

62

43

36

41

Angle of Spatula (deg.)

84

77

64

72

63

Dispersibility

7

6

8

6

4

Cohesion

4

5

15

12

12

Angle of Fall (deg.)

48

54

28

28

25

(3.)

563

STARCH

Table VII. Bulk Powder Data for Starch Lots (continued)

Property

STA-Rx

Purity21

Starch 1551

Starch 1500

Starch 1500 LM

Angle of Difference (deg.1

14

8

15

9

16

Flowability Index

31

36

52

54

53

Flowability performance

very poor

verypoor

poor

poor

poor

Floodability Index

68

62

72

72

86

Floodability performance

floodable floodable

Mass Flow Rate (g/sec)

0.04

0.06

floodable floodable

very floodable

0.53

1.oo

0.83

ANN W. NEWMAN ET AL.

.%-I

The amount of water sorbed by a solid can be expressed in a number of' ways. Many investigators report the amount of water sorbed, but the amount of water initially in the sample is not taken into account. The calculation of percent uptake relative to the dry weight of the sample normalizes samples to the same initial point and makes data from various samples comparable. The percent sorbed relative to dry weight is calculated from equation 2.

where:

W, = weight of sample at equilibration W, = original weight of sample A = percent moisture in original sample

A second method of reporting data is the equilibrium moisture content (EMC) [ 1,571 which is calculated using the following equation:

EMC

=

P

x 100 +

100

where [ Wo x

P =

wo -

1- A

100

P o x

*B X I

1-

00

A

(4)

100

B = weight change at equilibrium 'The hygroscopicity of the commercial starches was investigated with an automated moisture balance system in the range 0 to 90% RH [%I. EMC sorption values were calculated from the raw data at each relative

STARCH

565

humidity and are summarized in Table VIII. Representative curves are given in Figures 15 and 16. The sorption curves for the various starch samples are similar. The Type I1 isotherm and the hysteresis are evident for all the samples and the weight percent sorbed at 90%RH is fairly consistent at about 20%. The unmodified corn starch data are shown in Figure 15 for the Purity 2 1 sample. It is apparent that unmodified corn starch readily sorbs moisture along the entire range up to 90%RH. The isotherm for the pregelatinized starch in Figure 16 is similar, but the magnitude of the hysteresis is larger in the range 30-80%RH. These sorption curves are in agreement with data reported for similar types of samples [ 11. 5. Methods of Analysis 5.1 Compendia1 Tests The National Formulary [59] contains the following assays for starch: Botanic characteristics: Corn starch is described as polygonal, rounded, or spheroidal granules up to about 35 pm in size and usually having a circular or several rayed central cleft. Identification: A smooth mixture of starch and cold water is made. Boiling water is added and the mixture is boiled gently for 2 minutes. When the product is cooled, the product is a translucent, whitish jelly. In a second test, a water slurry is colored reddish violet to deep blue by iodine test solution (TS). Microbial limits: Following test method <61>, the sample meets the requirements for the absence of Salmonella species and Escherichia coli. pH: A slurry of starch and water is prepared in a nonmetallic container and agitated continuously for 5 minutes. The pH is measured immediately to the nearest 0.1 unit. For corn starch, a

566

Figure 15

ANN W. NEWMAN ET AL.

Moisture isotherm for unmodified corn starch, purity 2 1 .

0 - m 0

Figure 16. 1551.

STARCH

Moisture isotherm for pregelatinized corn starch, Starch ~.

5 67

ANN W.NEWMAN ET AL.

568

Table VIII. Sorption EMC Values at Various Relative Humidities

Relative Humidity

STA-Rx

Purity 21

Starch 1551

Starch 1500

Starch 1500 LM

1

3.4

4.4

3.6

3.5

3.6

10

6.0

6.3

5.6

5.4

5.4

20

8.1

8.3

7.3

7.5

6.9

30

9.5

9.6

8.6

9.0

7.9

40

10.7

10.8

9.7

10.1

9.1

50

11.9

11.9

10.8

11.2

10.7

60

13.1

13.2

12.2

12.1

12.3

70

14.6

14.9

14.1

13.2

14.1

80

16.8

16.9

16.8

15.5

16.2

90

19.9

20.1

21.5

19.2

20.5

(%I

STARCH

569

value between 4.5 and 7.0 should be obtained.

Loss on drying: Following the general test method <73 1>, the sample is dried at 120°C for 4 hours. It should not lose more than 14.0% of its weight. Residue on ignition: Following test method <281>, the weight of the residue can not exceed 0.5% on a 2 g sample. Iron: The residue obtained in the test for Residue on ignition is dissolved in hydrochloric acid with gentle heating, diluted with water, and mixed. This sample is diluted with water and tested for iron according to test <241>. The limit is 0.002%. Oxidizing substances: Starch and water are swirled for 5 minutes and decanted into a centrifuge tube. It is centrifuged to clarify. The clear supernatant is transferred and glacial acetic acid and potassium iodide are added. The sample is swirled and allowed to stand in the dark for 25-30 minutes. Starch TS is added and the solution is titrated with sodium thiosulfate volumetric solution (VS) until the starch-iodine color disappears. Not more than 1.4 mL of sodium thiosulfate is required. Sulfur dioxide: Starch and water are mixed until a smooth suspension is obtained. The solution is filtered. Starch TS is added to the clear filtrate and the solution is titrated with iodine to the first permanent blue color. Not more than 2.7 mL is consumed. Organic volatile impurities: The sample should meet the requirements for Method N in general test <467>.

5.2 Chromatomaphv High-pressure liquid chromatography (HPLC) methods have been used by the food [60] and paper [61] industries to analyze for starch. One method uses acid hydrolysis of the starch to detect glucose as the starch

570

ANN W. NEWMAN ET AL.

degradation product [60]. Good agreement was obtained with titrimetric data. A second method converts the starch to dextrose using enzymes and subsequent HPLC analysis [61]. The chromatographic method was found to be more reproducible than a spectrophotometric method in quantifying starch. 6. Excipient Studies

6.1 Drug Compatibilitv Starch is a relatively inert material and interactions with active drug substances do not occur often. Excipient compatibility studies of starch and various active drugs have been performed using thermal methods of analysis. As an example, starch has been found to be compatible with erythromycin [62], cephalexin [63], and acetylcysteine [64] using this method of excipient screening. 6.2 Tableting Various properties of starch have been studied to better understand its role in the granulation and compaction processes. It has been reported that starches deform mostly by plastic flow, but this was found to be dependent on the particle size, size distribution, and particle shape [38]. Characterization of wet granulated formulations made with starches from various vendors have shown no physical differences, yet, the compaction and ejection forces were found to be different for the formulations [48]. The effect of heat formation during compression has also been investigated for various starches and excipients [65,66]. The effect of the binder and tablet geometry have been related to the hardness and tensile strength of starch tablets [67]. Studies have shown that the particle size of an excipient may also affect various tablet properties [38,39,41,68]. A decrease in the particle size of compressible starch has resulted in a decrease [68] and an increase [38] in tablet strengths.

STARCH

57 1

The tabletting properties of starch in various formulations with active drug substances have also been reported [4,37,69]. Various starches (maize, rice, potato, wheat) were studied for use in double compressed tablets of aspirin and it was found that corn starch exhibited the best disintegration properties while rice exhibited the worst [4]. The compression speed used to tablet aspirin and compressible starch showed weaker and more porous tablets at high compression speeds [37]. A comparison of compressible starch with regular starch in an aspirin formulation resulted in better flow and compressibility properties for the compressible starch, as expected from the properties of the two materials [69]. 6.3 Disintegation As discussed previously, starch is used in many formulations for its disintegrationproperties. The mechanism for the disintegration properties of starch has been studied and various conclusions have been published [70-731. The most common explanation for the disintegration properties is the swelling of the starch granules when exposed to water and it has been proposed that amylose is the component responsible for the disintegration properties of starch due to swelling [69]. Measurements of the granules to quantify the amount of swelling have been performed using various methods [56,74]. A second mechanism was proposed that suggested the disintegrating action of starch in tablets is due to capillary action rather than swelling [70]. A third mechanism has been proposed based on the particle-particle repulsion forces between the tablet constituents when in contact with water and the hydrophilic nature of starch [72].

When comparing various starches as tablet adjuvants, it was found that disintegration time for the tablets was independent of the compressional force, but was dependent on the type of starch [5,42] and the dissolution method [5]. A study of compression forces showed that starch tablets had a decrease in disintegration with an increase in tablet hardness [73]. Particle size has also been correlated with the disintegration times of tablets containing starch [39], and it has been suggested that tight controls on particle size can minimize batch failures [44].

572

ANN W. NEWMAN ET AL.

7. References 1.

Handbook of Pharmaceutical Excipients, American Pharmaceutical Association, Washington, 1986, pp. 289-293.

2.

McGraw-Hill Encyclopedia of Science and Technology, McGrawHill Book Company, New York, Vol. 17, 1987, pp. 326-328.

3.

D. R. Lineback, J Jpn. SOC.Starch Sci., 33(1):80-88 (1986).

4.

M. A. F. Gadalla, M. H. Abd El-Hameed, A. A. Ismail, Drug. Dev. Znd. Pharm., 15(3):427-446 (1 989).

5.

T. W. Underwood, S. E. Cadwallader, J Pharm. Sci., 61(2) 239243 (1972).

6.

R. N. Nasipuri, F. 0. Kuforji, Pharm-Ind., 43(10):1037-1041 (1981).

7.

T. Ishizaka, H. Honda, M. Koishi, J Pharm. Pharmacol., 45:770774 (1993).

8.

H. Yoshizawa, M. Koishi, J Pharm. Pharmacol., 42:673-678 (1990).

9.

C. E. Bos, G. K. Bolhuis, H. Van Doome, C. F. Lerk, PharmWeekhl-Sci-Ed, 9:274-282 (1987).

10.

R. W. Kerr, ed., Chemistry and Industry of Starch, Academic Press, Inc., New York, 1950.

1I .

R. L. Whistler and J. R. Daniel, Starch. In: Kirk-Othmer Encyclopedia of Chemical Technology, 3rd ed. (M. Grayson, ed.). John-Wiley and Sons, New York, Vol. 21, 1983, pp. 492- 507.

STARCH

573

12.

J. B. Schwartz, E. T. Martin, E. J. Dehner, J. Pharm. Sci., 64(2):328-332 (1975).

13.

A. D. French. In: Starch: Chemistry and Technology, 2nd ed. (R.L. Whistler, J. N. BeMiller, and E. F. Paschall, eds.), Academic Press, New York, pp. 183-247 (1984).

14.

0. B. Wurzburg. In: Handbook of Food Additives (T. E. Furia, ed.), The Chemical Rubber Company, Boca Raton, FL, Vol. 1, 1972, pp. 361-395.

15.

W. Banks, C. T. Greenwood, Starch and its Components, John Wiley and Sons, New York, 1975.

16.

A. Imberty, H. Chanzy, S. Perez, J. Mol. Biol., 201:365-378 (1988).

17.

A. Imberty, A. Buleon, V. Tran, S. Perez, Staerke, 43(10):375-384 (1991).

18.

B. Casu and M. Reggiani, Staerke, 7:218-229 (1966).

19.

F. R. Dollish, W. G. Fateley, F. T. Bentley. In: Characteristic Raman Frequencies of Organic Compounds, John Wiley & Sons, Inc., New York, 1974.

20.

M. St-Jacquies, P. R. Sundararajan, K. J. Taylor, and R. H. Marchessault, J. Am. Chem. SOC.,98( 15):4386-4391 (1976).

21.

J. W. Donovan, Biopolymers, 18:263-265 (1979).

22.

P. J. Florey, i;inciples of Polymer Chemistry, Cornell University Press, Ithaca, NY, 1953, chapter 13.

23.

J. Lelievre, J. Appl.Polym. Sci. , 18:293-296 (1 973).

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ANN W. NEWMAN ET AL.

24.

J. Lelievre, Polymer, 17:854-858 (1976).

25.

K. J. Zeleznak, R. C. Hoseny, Cereal Chem., 64(2):121-124 (1987).

26.

D. J. Stevens, G. A. H. Elton, Staerke, 23(1):8-11 (1971).

27.

M. Nduele, A. Ludwig, M. Van Ooteghem, S. T.P. Pharma Sci., 3(5):362-368 (1993).

28.

M. Tomessetti, L. Campanella, T. Aureli, Thermochimica Acta, 143:15-26 (1989).

29.

0. A. Sjostrom, Ind. Eng. Chem., 28(1):63-74 (1936).

30.

T. J. Schoch, E. C. Maywald,Anal. Chem., 28(3):382-387 (1956).

31.

G. E. Moss, The Microscopy of Starch. In: Examination and Analysis of Starch and Starch Products (J. A. Radley, ed.), Applied Science Publishers, Ltd, London, 1976, pp. 1-32.

32.

T. J. Schoch, E. C. Maywald, Industrial Microscopy of Starches. In: Starch: Chemistry and Technology, 2nd ed. (R.L. Whistler, J. N. BeMiller, and E. F. Paschall, eds.), Academic Press, New York, pp. 637-647 (1984).

33.

D. J. Gallant, Electron Microscopy of Starch and Starch Products. In: Examination and Analysis of Starch and Starch Products (J. A. Radley, ed.), Applied Science Publishers, Ltd, London, 1976, pp. 33-59.

34.

W. C. Mussulman, J. A. Wagoner, Cereal Chem., 45(2): 162-17 I (1968).

35.

R. F. Shangraw, J. W. Wallace, F. M. Bowers, Pharm. Tech., Oct., 44-60 (1981).

STARCH

515

36.

H. Hess, Pharm. Tech., June, 54-68 (1987).

37.

G. D. Cook, M. P. Summers, J Pharm. Pharmacol., 42:462-467 (1990).

38.

A. McKenna, D. F. McCafferty, J. Pharm. Pharmacol., 34:347351 (1982).

39.

A. J. Smallenbroek, G. K. Bolhuis, C. F. Lerk, Pharm. Weekblad, 11611048-1051(1981).

40.

C. E. Bos, H. Vromans, C. F. Lerk, Znt. J. Pharm., 67:39-49 (1991).

41.

P. Paronen, M. Juslin, J. Pharm. Pharmacol., 35:627-635 (1983).

42.

M. Juslin, P. Kahela, P. Paronen, 1. Turakka, Acta. Pharm. Fenn., 83-93 (1981).

43.

K. P. P. Prasad, L. S. C. Wan, Pharm. Res., 4(6):504-508 (1987).

44.

H. G. Brittian, C. J. Sachs, K. Fiorelli, Pharm. Tech., Oct., 38-52 (1991).

45.

P. Merkku, J. Yliruusi, E. Kristoffersson, Acta Pharm. Nord., 4(4):265-270 (1992).

46.

N. N. Hellman, E. H. Melvin, J Am. Chern. SOC.,74:348-350 (1952).

47.

B. Vennat, S. Gross, A. Pourrat, H. Pourrat, Drug Dev.Znd Pharm., 19(11):1357-1368 (1993).

48.

M. K. Kottke, H.-R. Chueh, C. T. Rhodes, Drug Dev. Ind. Pharm., 18(20):2207-2223(1992).

ANN W. NEWMAN ET AL.

576

49.

R. L. Carr, Chem. Eng. 72:163-168(1965).

50.

R. L. Carr, Chem. Eng. 7369-72(1965).

51.

D. Faroongsamg, G. E. Peck, Drug Dev. Ind. Pharm., 20(5):779798 (1994).

52.

L. Sair, W. R. Fetzer, Ind. Eng. Chem., 36:205-208(1944).

53.

S. Malamataris, P. Goidas, A. Dimiriou, Znt. J. Pharm., 6851-60 (1 99I).

54.

S. W. Paik. S. G. Gilbert, J. Chrom., 351:417-423(1986).

55.

B. Das, R. K. Sethi, S. L. Chopra, Isr. J. Chem., 10:963-965

(1972). 56.

S. E. Wurster, G. E. Peck, D.O. Kildsig, Drug Dev. Znd. Pharm.,8(3):343-354(1982).

57.

J. C. Callahan, G. W. Cleary, M. Elefant, K. Kaplan, T. Kensler, R. A. Nash, Drug Dev. Ind. Pharm., 8(3):355-369(1982).

58.

VTI Moisture Balance System, VTI Corporation, Hialeah, FL.

59.

The National Formulary, NF 18,United States Pharmacopeial Convention, Inc, Rockville, MD, 1995,pp. 2309-2310.

60.

N. P. Boley and M. J. S. Burn, Food Chem., 36:45-51 (1990).

61.

N.Rirosel-Boettcher, Tuppi J., 76(3):207-208(1993).

62.

H. H. El-Shattawy, D. 0. Kildsig, G. E. Peck, Drug. Dev. Znd. Pharm., 8(6):937-947 (1982).

STARCH

511

63.

H. H. El-Shattawy, D. 0. Kildsig, G. E. Peck, Drug. Dev. Ind. Pharm., 8(6):897-909 (1982).

64.

J. Kerc, S. Srcic, U. Urleb, A. Kanalec, B. Kofler, J. Smid-Korbar, J Pharm. Pharmacol., 445 15-5 18 (1992).

65.

N. A. Armstrong, A. M. Gough, I. L. Baker, Int. J. Pharm. Tech. andprod. MJi.., 6(2):13-15 (1985).

66.

D. E. Wurster, J. R. Creekmore, Drug. Dev. Ind. Pharm., 12(10):1511-1528 (1986).

67.

R.-C. Hwang, E. L. Parrot, Drug Dev. Ind. Pharm., 19(5):507-519 (1993).

68.

G. Alderborn, C. Nystrom, Acta Pharm Suec., 19:381-390 (1982).

69.

K. S. Manudhane, A. M. Contractor, H. Y. Kim, R. F. Shangraw, J. Pharm. Sci., 58(5):616-620 (1969).

70.

L. C. Curlin, J Am. Pharm. Assn., Sci.Ed., 44(1):16 (1955).

71.

N. R. Patel, R. E. Hopponen,J Pharm. Sci.,55(10):1065-1068 (1966).

72.

A. M. Guyot-Hermann, Drug Dev. Ind. Pharm., 7(2):155-177 (1981).

73.

P. M. Hil1,J Pharm. Sci., 65(11):1694-1697 (1976).

74.

N. N. Hellman, T. F. Boesch, E. H. Melvin, J. Amer. Chem. SOC. 74:348-350 (1952).

This Page Intentionally Left Blank

TOBRAMYCIN

Alekha K. Dash

Department of Pharmaceutical Sciences School of Pharmacy and Allied Health Professions Creighton University Omaha, NE 68 178

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS-VOLUME 24

579

ALEKHA K. DASH

580

TABLE OF CONTENTS

1.

History and Therapeutic Properties

2.

Description 2.1 Nomenclature 2.1.1 Chemical Name 2.1.2 Generic Name 2.1.3 Trade Names 2.1.4 CAS Registry Number 2.2 Formula and Molecular Weight 2.3 Elemental Composition 2.4 Appearance, Color and Order 2.5 Pharmaceutical Dosage Forms

3.

Synthesis

4.

Physical Properties 4.1 Infrared Spectrum 4.2

H Nuclear Magnetic Resonance Spectrum

4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10

3C Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Thermal Behavior Melting Point Solubility X-Ray Powder Diffraction Patterns Dissociation Constants

TOBRAMYCIN

5.

Methods of Analysis 5.1 Identification Tests 5.2 Spectrophotometric 5.3 Chromatographic 5.3.1 Thin Layer Chromatography 5.3.2 High Pressure Liquid Chromatography 5.3.3 Gas Chromatography 5.4 Biological 5.4.1 Microbiological Assay 5.4.2 Radioimmuno Assay 5.4.3 RadioenzymaticAssay 5.4.4 Fluorescence Polarization Immunoassay 5.4.5 Fluorescence Immunoassay

6.

Stability, Degradation and Incompatibility

7.

Pharmacokinetics

8.

Toxicity

9.

Acknowledgments

10.

References

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ALEKHA K. DASH

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1.

History and Therapeutic properties

In 1957, investigators at Lilly Research Laboratories first isolated a streptomyces species from soil samples collected in Hermosillo (Sonora, Mexico). Seven antibiotic factors were isolated from Streptomyces tenebrarius (ATCC 17920) [ 11, and factor 6 was subsequently designated as tobramycin [2]. Tobramycin is an aminoglycoside antibiotic, exhibits bactericidal activity against a broad spectrum of bacteria, and is only active on actively multiplying bacteria. It inhibits protein synthesis, possibly on the 30s subunits of the bacterial ribosomes [3]. Indications of this drug include sepsis, urinary tract infections, infections of the skin, soft tissue infections, respiratory tract infections, erc. It is especially useful in treatment of infections due to Pseudomonas and indolepositive Proteus.

2.

Description

2.1

Nomenclature 2.1.1 Chemical Name

0-3-amino-3 -deoxy-a-D-glucopyranosy1-(1-6)-0- [2,6-diamino2,3,6-trideoxy-a-D-ribo-hexopyranosyl-( 1-4)]-2-deoxy-Dstreptamine.

0-3-amino-3-deoxy-a-D-glucopyranosyl-( 1-4)-0-[2,6-diamino2,3,6-trideoxy-a-D-ribo-hexopyranosyl-( 1-6)]-2-deoxy-Lstreptamine. 2.1.2 Generic Name

Tobramycin

2.1.3 Trade Names Distobram, Gernebcin, Obramycin, Nebcin, Tobradistin, Tobralex, Tobramaxin, and Tobrex.

583

TOBRAMYCIN

2.1.4

CAS Registry Number

32986-56-4 2.2

2.3

Formula and Molecular Weight Free base

C I 8H37N509

Sulfate salt

(C18H37N509)2 5 H2S04 MW = 1425.4

MW=467.45

Elemental Composition The theoretical elemental composition of tobramycin, based on the molecular formula C18H37N509, is: C 46.24% , H 7.98%, N 14.98%, 0 30.80% [4]. Elemental analysis of tobramycin monohydrate has been reported by Koch et al. [5] as: Calculated, C: 44.52%; H: 8.10%; N: 14.43%. C: 44.39%; H: 8.15%; N: 14.07%. Found,

2.4

Appearance, Color and Odor

Tobramycin is obtained as a crystalline, white to off-white, hygroscopic powder.

ALEKHA K. DASH

5x4

2.5

Pharmaceutical Dosage Forms

Tobramycin is available in the following dosage forms: tobramycin injection, tobramycin sulfate injection, tobramycin ophthalmic ointment and tobramycin ophthalmic solution.

3.

Synthesis

Takagi et al. [6] have synthesized tobramycin from kanamycin B. Kanamycin B was converted to penta-N-ethoxycarbonyl-4",6"-0cyclohexylidene-2"-benzoylkanamycinB. This compound (1 mol equivalent) was then treated with excess of p-toluenesulfonyl chloride (5 mol equivalent) in pyridine at 25°C overnight to give the 3'-O-tosyl derivative as the major product. Iodination of 3'-O-tosyl derivative (mp 149-150°C) was achieved after reacting with sodium iodide in dimethylformamide (4.9 g NaI in 10 mL of DMF) at 100°C to produce a unstable 3I-iodide derivative. This unstable derivative was subsequently hydrogenated with Raney nickel and hydrogen in dioxane to give 2"-0benzoyl-4",6"-O-cyclohexylidene-3'-deoxy-penta-Nethoxycarbonylkanamycin (mp 2485250°C). This compound was successively treated with hot 4N barium hydroxide and 50% v/v acetic acid at 80OC to give crude 3'-deoxykanamycin B. This material was purified by chromatography and recrystallized as a monohydrate.

4.

Physical Properties

4.1.

Infrared Spectrum

The infrared spectrum of tobramycin is shown in Figure 1. The spectrum was obtained in potassium bromide disk (0.5% w/w) using a FTIR (model 1600,Perkin-Elmer spectrophotometer. Assignments of the characteristic bands in the spectrum are listed in Table 1 [7].

c

.d

ALEKHA K. DASH

586

Table 1

Infrared Spectral Assignments for Tobramycin

Energy (cm-1)

Assignment

3400 - 3200

N-H stretching (s, br) 0-H stretching (s, br)

2910

Aliphatic C-H stretching (m)

1588

N-H bending (s)

1461

CH2 scissoring (m)

1377, 1349

0-H inplane bending vibration (m)

1032

C-N stretching ( s ) C - 0 stretching (s)

(br) = Broad (m) = Medium intensity (s) = Strong intensity

TOBRAMYCIN

4.2

587

'H Nuclear Magnetic Resonance Spectrum

The 200-MHz proton nuclear magnetic resonance spectrum of tobramycin was obtained on a Bruker AM 200 NMR spectrometer, and is shown in Figure 2. The spectrum was recorded at an ambient temperature. Deuterated water (D20) was used as the solvent, and tetramethylsilane was the reference standard [7].] The chemical shifts, multiplicities and peak assignments of characteristic protons are given in Table 2, and these were found to be close to the reported values [5].

4.3

13C Nuclear Magnetic Resonance Spectrum

The 15.08 MHz 13C Nuclear Magnetic Resonance spectrum of 0.3-0.5 M aqueous solution of tobramycin were recorded in aqueous solution, containing 5% dioxane as an internal standard. The nmr instrument consisted of a Varian Associates DP-60 magnet working at 14 kG with an external 19F lock. The samples were spun in 13-mm 0.d. tubes [8]. Chemical shifts and structural assignments are outlined in Table 3, and are based on the number system given above.

Figure 2.

Proton Magnetic Resonance Spectrum of Tobramycin free base in D7O.

589

TOBRAMYCIN

Table 2

N M R Spectral Assignments for Tobramycin Chemical Shift @Pm>

Multiplicities

Number of protons

Assignment

6.25 6.05

d d

1 1

Anomeric Protons (1' and 1")

4.15-4.85

m

10

Protons on carbon bonded to hydroxyl group or ether linkage

3.85-4.15

m

6

Protons on carbon bonded to amino groups

2.9-3.17

m

2 (es)

Methylene group in a six membered ring

2.55

9

1 (ax)

Methylene group in a six membered ring

2.25

9

1 (ax>

Methylene group in a six membered ring

d = doublet m = multiplet q = quartet

eq = equatorial ax = axial

ALEKHA K.DASH

590

Table 3

C NMR Spectral Assignments for Tobramycin [81 Chemical Shift @Pm>

Assignments

99.2 49.5 34.7 65.9 73.1 41.5 50.2 35.5 49.0 86.0 74.4 87.8 99.1 71.6 54.2 69.2 71.9 60.2

c-1' c-2' c-3' c-4' c-5' C-6' c-1 c-2 c-3 c-4

c-5 C-6 c-1" c-2" c-3" c-4" c-5" C-6"

TOBRAMYCIN

4.4

59 1

Ultraviolet Spectrum

-

Owing to its saturated ring system and lack of suitable chromophores, tobramycin does not exhibit any significant absorption between 230 and 360 [91.

4.5

Mass Spectrum

A Finnigan INCOS-SOB quadrupole mass spectrometer linked to a HewlettPackard gas chromatograph using electron impact at an electron energy of 70 eV and a source temperature of 180°C was used to study the mass spectrum and fragmentation behavior of tobramycin. Unfortunately, no useful mass spectra were obtained, as had been reported [lo].

4.6

Thermal Behavior

The Differential Scanning Calorimetry (DSC) thermogram of tobramycin base is shown in Figure 3. The sample was heated from 30 - 25OoC in a nonhermetically crimped aluminum pan at a rate of 1O"C/min on a DuPont model 950 thermal analysis system. The first endothermic peak was attributed to compound dehydration, and was followed by the melting of the metastable form at 164OC. The metastable forms recrystallizes to the stable form as evidenced by the exotherm at 197SoC, and finally melts at 217OC [111. Thermogravimetric (TG) analysis of the base was carried out on a DuPont model 95 1 thermogravimetric analyzer, and the resulting TG and differential TG thermograms are shown in Figure 4. It was concluded from this work that the commercially available sample consisted of the monohydrate phase containing some absorbed water [l 11.

4.7

Melting Point

The metastable form was observed to melt at approximately 164OC, while the stable form melts at approximately 2 17OC [111.

113.69-C

t63.91°C

216.78-C

I

50

1

100

I

I

150

200

Temperature ('13

Figure 3.

Differentialscanning calorimetry thermogram of Tobramycin free base.

PI

0.W

100

0.06

-

-u

0.04

5 bJ

C

98-

0

-..

:

VI

W w

0.02

; .LI

a.

u

0

960.00

-0.02

Figure 4.

(a) Thermogravimetricanalysis and (b) differential thermogravimetricanalysis thermograms of Tobramycin free base.

594

4.8

ALEKHA K. DASH

Solubility

'Tobramycin is freely soluble in water (1 in 1.5 parts), very slightly soluble in ethanol (1 in 2000 parts), and practically insoluble in chloroform and ether [9,12]. A 10% (w/v) solution of tobramycin in water has a pH of 9-1 1 [13].

4.9

X-Ray Powder Diffraction Pattern

The powder pattern data of tobramycin base was obtained using a wide angle X-Ray diffractometer (model D500, Siemens). The powder diffraction patterns of the two polymorphs are shown in Figure 5a and 5b. The calculated d-spacings for the diffraction patterns are provided in Table 4 [14].

4.10

Dissociation Constants

In one publication, three pKa values were reported for tobramycin as 6.7, 8.3. and 9.9 [15]. However, in another work four pKa values (6.2,7.4, 7.6, and 8.6) were reported by Raymond and Born [ 161.

5.

Methods of Analysis

5.1

Identification [13]

Tobramycin is identified by a thin layer chromatographic method, and the exact details of this procedure are described in the subsequent TLC discussion (section 5.3.1).

595

TOBRAMYCIN

6.0 8.0

10

12

14

$6

I8

20

22

24

28, degrees

Figure 5.

Powder x-ray diffiaction patterns of; (a) commercially available Tobramycin free base (Form I), and (b) Tobramycin free base heated to 208OC (Form 11).

ALEKHA K. DASH

SY6

Table 4 Powder X-Ray Diffraction Data for Tobramycin Diffraction pattern of Tobramycin base (Form I) Peak No

d-Spacing

(4

Relative Intensity

Diffraction pattern of Tobramycin base (Form 11)

Peak d-Spacing Relative (A) Intensity No

(%I 1

2 3 4 5 6 7 8 9 10 11 12 13 14 15

15.07 10.35 8.87 8.14 7.48 6.17 4.97 4.79 4.69 4.56 4.39 4.24 4.08 3.91 3.79

27 48 49 36 33 53 57 100 47 35 36 36 54 31 53

1 2 3 4 5 6 7 8 9 10 11 12

15.77 7.69 7.08 5.90 5.50 4.98 4.72 4.64 4.27 3.95 3.77 3.60

10 18 89 29 29 84 25 20 100 31 27 31

TOBRAMYCIN

5.2

591

Spectrophotometric Methods

A colorimetric method based on the reaction between tobramycin and copper sulfate has been developed for the quantification of this compound in injectable formulations [17]. A colorimetric method based on the reaction between tobramycin and 2,4-dinitrofluorobenzene(Sanger's reagent) has been reported for the quantification of tobramycin in topical formulations [181.

A spectrofluorimetric method for the determination of tobramycin in biological fluids using a fluorescent dihydro-lutidine derivative has also been reported [191. The fluorescent derivative is formed by condensation of the primary amino groups of tobramycin with acetyl-acetone and formaldehyde under acidic conditions (pH=2.4). Sampath and Robinson have reported a spectrophotometricmethod for the analysis of tobramycin and compared their method with the existing methods [20].

5.3

Chromatographic Methods

5.3.1 Thin Layer Chromatography [13]

Tobramycin solution is prepared in distilled water (0.6% w/v). A 3 pL portion of this solution is applied to a silica gel (0.25-mm layer) TLC plate. The chromatogram is developed by equilibrating the plate for 5.5 hours in a chromatographic chamber containing a mixture of methanol, ammonium hydroxide and chloroform (60:30:25; v/v/v). The plate is removed from the chamber and heated at 11O°C for 15 minutes. The spots are detected by spraying with a 1 in 100 solution of ninhydrin in a mixture of butyl alcohol and pyridine (100:1, v/v), and tobramycin is visualized as a pink spot.

5.3.2 High Performance Liquid Chromatography (HPLC)

The very low absorptivity of tobramycin in the UV and visible region does not permit its direct quantification at low concentrations. This problem can be solved by derivatizing this compound with a suitable absorbance-

598

ALEKHA K. DASH

enhancing or fluorescence-producing agent. This deficiency can be overcome through the use of either pre-column or post-column derivatization. Various HPLC methods using pre-column [21-261 and postcolumn [27,28] derivatization to quantify tobramycin in pharmaceuticals and biological fluids have been described and are summarized in Table 5 .

5.3.2

Gas Liquid Chromatography (GLC)

A CiLC method has been described by Mayhew et al. [29] for the assay of tobramycin in biological fluids. A silanized Pyrex column (2 m by 3 mm id) packed with 3% OV-101 coated onto 80-100 mesh Chromosorb WAW was utilized in this method. Nitrogen gas was used as a carrier. The injector and detector temperature were maintained at 272' and 287OC respectively, and a electron captured detector was used in this study.

5.4

Biological Methods

5.4.1

Microbiological assay

Various microbiological assay methods for the analysis of tobramycin have been described [24,30,3 11. The method developed by Maitra et al. [24] used an agar diffusion method using Bacillus subtilis (ATCC 6633) as a test organism. The organism was grown on seeded agar at 37°C for 16-18 hours. These microbiological assays are reliable and simple but they are time consuming and less specific.

5.4.2

Radioimmunoassay (RIA)

Radioimmunoassays have been developed for measuring tobramycin in biological fluids [32,33]. The RIA is based on the competition between 1251-tobramycinand unlabeled tobramycin in the sample to be analyzed for the antibody binding sites. Unbound 251-tobramycin is separated by centrifugation, and the radioactivity of the bound tobramycin is counted and the levels calculated from a standard curve. This method is highly sensitive and specific.

Table 5

HPLC Methods for the Analysis of Tobramycin Type of Derivatizing Derivatization Agent

Mobile Phase

Column

Pre-column

1-fluoro-2,4dinitrobenzene

Water:acetone:acetic acid (30:70:0.1; v/v/v) Flow = 3 mL/min

C18 30 cm x 3.9 mm

2,4,6-trinitrobenzenesulfonic acid

Acetonitri1e:phosphate buffer (70:30 v/v) Flow = 3 mL/min

CIS 30 c m x 4 mm

uv

1-fluoro-2,4dinitrobenzene

75% V/V (NH4)3P04 and 25% v/v Acetonitrile Flow = 2 mL/min

c 18 30cmx4mm

Pre-column

Pre-column

Pre-column

o-phthalaldehyde, Methano1:water (72:26) C18 mercaptoethanol and 0.005 EDTA 30 cm x 4 mm Flow = 1 mL/min

Detection Mode

uv

Sample Type

Referenc e

Biological fluids

21

22

(365 nm)

Biological fluids

uv

Formulations

23

Biological fluids

24

(365 nm)

(365 nm)

Fluorescence (360 nm EX 430 nm EM)

Fluorescence (360 nm EX 430 nm EM)

Biological fluids

25

CIS 25 cm x 4.6 mm

uv

Formulations

26

p-Bondpak C18 30 cm x 3.9 mm (Waters)

Fluorescence (340 nm EX 4 18 nm EM)

Biological fluids

27

Fluorescence (340 nm EX 418 nm EM)

Biological fluids

28

Pre-column

o-phthalaldehyde. mercaptoethanol

250 mL of 0.5 M Tris p-Bondpak C18 buffer + 10 mL of (Waters} triethylamine, and qs to I L with methanol Flow = 2 mL/min

Pre-column

2,4,6-trinitrobenzenesulfonic acid

Acetonitrile5OmM Phosphate buffer (62:38 v/v) Flow = 2.5 mL/min

Post-column

o-phthalaldehyde, mercaptoethanol

Sodium. sulfate (0.1 M), sodium pentasulfonate (0.02 M), and 17.4 mM acetic acid in 1 L of water Flow = 2 mL/min

Post-column

o-phthalaldehyde

water:methanol:acetic Lichosorb 5 RP acid (99.7:0.2:0.1 mole C8 (15 cm) %) containing 0.2 (Chrompack) moles of sodium sulfate and 0.02 moles of sodium pentane sulfate, Flow = 1 mL/min

(340 nm)

TOBRAMYCIN

5.4.3

60 1

Radioenzymatic assay

Radioenzymatic assays for the assay of tobramycin in biological fluids have been reported [34-361. The method involves the specific enzymatic transfer of a radioactive modifying group to the drug. These enzymes are present in organisms that carry resistant (R) factors which are responsible for the activation of the drug. The entire reaction process is stoichiometric, and the amount of radioactivity incorporated is proportional to the concentration of the antibiotic. These assays are simple, accurate and precise, but the need to work with radioactive material may pose a disadvantage for some clinical laboratories.

5.4.4

Fluorescence polarization immunoassay

Fluorescence polarization immunoassay (FPI) is a method that combines the principles of competitive protein binding with the principles of fluorescence polarization, and has also been utilized to determine the tobramycin concentration in serum [37,38].

5.4.5

Fluorescence immunoassay (FIA)

FIA uses the principle of competitive protein binding, and has been used to quantify tobramycin in biological samples [28, 39-41]. Competitive binding reactions are set up with fluorogenic tobramycin reagent, a limiting amount of antibody against the drug, and the serum sample to be analyzed. Tobramycin in the serum sample competes with the fluorogenic tobramycin reagent for the antibody binding sites. The unbound fluorogenic reagent is then hydrolyzed by P-galactosidase to produce the fluorescence which is detected as the observable parameter.

6.

Stability, degradation and incompatibility

Tobramycin solution in water at pH 1 to 11 was reported to be stable for several weeks at temperatures from 5 to 37OC, and could be autoclaved without loss of potency [12]. When aqueous tobramycin was adjusted to pH

602

ALEKHA K. DASH

1.2 with HC1 and autoclaved for 30 minutes at 120°C in sealed glass ampules, an extra peak was observed in the chromatogram. This was attributed to a possible degradation product [26]. Tobramycin is compatible with most available IV fluids, but is not compatible with heparin solution. In addition, it can interact chemically with p-lactam compounds of the penicillin, cephalosporin, and cephamycin family [ 3 ] . This interaction depends upon the concentration and pH of both tobramycin and f3-lactam compounds. Solutions of tobramycin sulfate and clindamycin phosphate have been reported to be unstable in dextrose injection [42].

The stability of tobramycin in 30 and 50% dextrose peritoneal dialysate concentrate (PDC) fluids have been reported [43]. This study indicated that if tobramycin is to be added to PDC fluids containing 50% wlv of dextrose, it should be used within 12 hours of admixture.

7. Pharmacokinetics 7.1 Absorption Tobramycin is not appreciably absorbed when taken orally, but does exert an antibacterial effect in the intestine. When applied to the skin, the drug is not absorbed to a degree sufficient to produce any therapeutic effects. There is no significant absorption of the drug from the bronchi and lungs after administration as an aerosol [44]. Studies in rabbits suggest that tobramycin is absorbed into the aqueous humor following topical installation of 3 mg/mL solution of the drug onto the eye and absorption is greatest when the cornea is abraded [45]. Owing to these adsorption characteristics, tobramycin should be administered either intramuscularly (IM) or intravenously (IV). Absorption after IM injection is rapid, with the peak serum concentration being achieved at 20-45 minutes. Senun concentrations of tobramycin following a single IM injection are given in Table 6 [46-511. Mean peak serum concentrations of tobramycin following various rates of IV injections are given in Table 7 [49,50,52,53].

603

TOBRAMYCIN

Table 6 Mean Peak Serum Concentration of Tobramycin Following Single Intramuscular Injections Dose

Serum concentrations

References

(I.ldmL) 50 mg/m2 100 mg 2.5 mgkg 100 mg 100 mg 40 mg 80 mg

4.6 5.1 7.1 3.8 5.2 2.4 3.7

46 47 48 49 50 51 51

ALEKHA K. DASH

604

Table 7 Mean Peak Serum Concentration of Tobramycin Following Intravenous Injections Rate of Injection

Dose

Peak serum levels (yg/mL)

References

1 hour infusion

1 mgkg

4.4

52

30-45 min

1 mg/kg

5.5

49

30-45 min

1.5 mgkg

6.0

49

1 hour infusion

100 mg

4.6

50

bolus injection (2.5-3 min)

80 mg

11.2

53

bolus injection (2.5-3 min)

1 mgkg

10.0

53

TOBRAMYCIN

7.2

605

Distribution

The distribution of tobramycin in human tissue and body fluids is summarized in Table 8 [54-601. The half-life of the drug in serum ranges between 1.6 and 3.5 hours in normal individuals. Ragamey et al. have reported the apparent volume of distribution (AVD) value for tobramycin to be 24.5 liters [50]. However, Simon et al. have reported the AVD value to be 16.9 liters [51].

7.3

Protein binding

Using equilibrium dialysis, Ramirez-Ronda et al. have reported that approximately 70% tobramycin is bound to plasma proteins at a concentration of 10 mg/mL or less [61]. However, Gorden et al. [62], and Neber et a!. [63] have reported that under conditions of physiological pH and temperature, no serum protein binding of tobramycin occurred at a concentration of 5 mg/mL. A similar effect was also reported by Ullmann et al. using steady state gel filtration and frontal analysis [64].

7.4

Excretion

Tobramycin is rapidly excreted unchanged in the urine after an IM or IV injection [65]. However, Pechere and Dugal[66] have suggested that 10% of the drug is eliminated by extrarenal mechanisms. The renal excretion of tobramycin takes place entirely by glomerular filtration [46,50]. The total plasma clearance of tobramycin from IV infusion studies have been reported to be 113.7 mL/min/1.73 m2 [50] and 87.9 mL/min/1.73 m2 [51]. The rate of recovery of tobramycin from urine over a 6 hour period is 60% and 8085% during the 24 hours after injection [67,68]. During the first 6 hours after a dose of 1 mgkg (given by infusion over a period of 1 hour), urinary concentrations between 70 and 300 mg/mL have been reported [52].

Table 8 Distribution of Tobramycin in Various Tissues and Body Fluids Tissue or other body fluids

Dose and Routes of administration

Breast milk

80 mg by

IM

Time elapsed

Concentration detected (pg/mL)

References

1 hour 8 hours

0.60 0.85

55

3 hours

1.2

56


57

54

Amniotic fluid

80 mg by IM

Cerebrospinal fluid

3-4.5 mgkg by IV

sputum

5 m g k g IM injection in three divided doses

1 to 3 hours

0.3

58

Bile

80 mg im

1 hour

1.4

59

Palatine tonsil samples

80 or 160 mg im

1 hour

0.5 to 1.3

60

TOBRAMYCIN

607

Table 9

LD50 of Tobramycin in Rat and Mouse [69]

Animal species

Route of Administration

LD50 (mg/kg)

Rat Rat Rat Rat Mouse Mouse Mouse Mouse Pig

intraperitoned subcutaneous intravenous intramuscular intraperitoneal subcutaneous intravenous intramuscular subcutaeous

1030 969 104 913

445 3 67 72.5 440 676

ALEKHA K.DASH

8.

Toxicity

The LD50 values determined for tobramycin in different animal species are given in Table 9 [69]. The LD50 for tobramycin is found to be 50-66% of that of gentamicin in mice, and 70% of that of rats [70,71]. Lethal and sublethal doses of tobramycin produce hyperactivity, decreased respiration and prostration in mice, rats and guinea pigs. No noticeable changes in behavior, appearance, or in hematological or biochemical values were noticed in dogs when 3.75 and 7.5 mgkg of tobramycin is administered for 90 days. There was evidence of slight cloudy swelling of proximal portions of nephrons after 30 mgkg dose of the drug for 90 days.

9.

Acknowledgments

The useful suggestions of Professors. Raj Suryanarayanan (University of Minnesota, College of Pharmacy) and Shanker L. Saha (Creighton University) are gratefully acknowledged.

10.

References

1.

C. E Higgens and R. E. Kastner, Antimicrob. Agents Chemother., 1967, 324-331 (1971).

2.

R. Q. Thompson and E. A. Presti, Antimicrob. Agents Chemother., 1967,332-340 (1971).

3.

H. C. Neu, J. Infect. Diseuse., 134 (Suppl.), S3-Sl9 (1976).

4.

The Merck Index, S. Budavari, ed., 1l* Edition, Merck and Co., Inc., Kahway, N.J., USA, 1989, p. 1499.

5.

K. F. Koch and J. A. Rhoades, Antimicrob. Agents. Chemother., 1970,309-313 (1971).

6.

Y. Takegi,T. Miyake, T. Tsuchiya, S. Umezaya and H. Umezaya, J Antibiotics., 26,403-406 (1973).

TOBRAMYCIN

609

7.

A. K. Dash, unpublished data.

8.

K.F Koch, J. A. Rhoades, E. W. Hagaman and E. Wenkert, J. Am. Chem. Soc., 96,3300-3305 (1974).

9.

Clarke's Isolation and Identification of Drugs in Pharmaceutical, Body Fluids and Post-mortem Material, A. C. Moffat, ed., 2nd Edition., The Pharmaceutical Press, London, 1988, p. 1027.

10.

T. Mills I11 and J. C. Roberson, Instrumental Data for Drug Analysis, 2"d Edition, Vol3, Elsevier, NY, 1987, pp. 2256-2257.

11.

A. K. Dash and R. Suryanarayanan, Pharm. Res., 8, 1159-1165 (1991).

12.

Martindale, The Extra Pharmacopoeia, J. E. F. Reynolds, ed., 29* Edition,The Pharmaceutical Press, London, England, 1989, pp. 326328.

13.

The United States Pharmacopeia, 22ndRevision, United States Pharmacopeial Convention ,Rockville, MD. 1989, p. 1381.

14.

A. K. Dash., Ph. D. Thesis, University of Minnesota, 1990, p. 183.

15.

A. Albert and E. P. Serjeant, The Determination of Ionization Constants, 3rdEdition, Chapman and Hall, NY. 1984, p 174.

16.

G. G. Raymond and J. L. Born, Drug Intel. Clin. Pharm., 20,683686 (1986).

17.

V. Das Gupta,J Clin. Pharm. Therapy, 13, 195-198 (1988).

18.

J. A. Ryan, J Pharm. Sci.,73,1301-1302 (1984).

19.

A. Csiba, J Pharm. Pharmacol., 31,115-116 (1979).

20.

S. Sampath and D. H. Robinson, J. Pharm. Sci., 79,428-431 (1990).

21.

D. M. Barends, C. L. Zwaan and A. Hulschoof, J. Chromatogr., 225, 417-426 (1981).

ALEKHA K. DASH

610

22.

P. M. Kabra, P.K. Bhatnagar, M. A. Nelson, J. H. Wall and L. J. Marton, Clin. Chem., 29,672-674(1983).

23)

N. A. Athanikar, R. W. Jurgens Jr., R. J. SturgeonL. A. Zober, P. P. Deluca and D. D. Papadimitriou, J. Parenteral Sci. Tech.,B, 125128 (1 983).

24)

S. K. Maitra, T. T. Yoshikawa, J. L. Hansen, M. C . Schotz, and L. B. Guze, Am. J. Clin.Pathol., 71,428-432(I 979).

25)

S.-E, Back, I. Nilsson-Ehle and P. Nilsson-Ehle, Clin.Chem., 25,1222-1225 (1979). A. K. Dash and R. Suryanarayanan, J. Pharm. Biomed. Anal., 9, 237-245(1991).

J. P. Anhalt and S. D. Brown, Clin.Chem., 24, 1940-1947(1978).

E. E. Stobberingh, A. W. Houben and C. P. A. van Boven, J. Clin. Microb., 15,795-801(1982). 29)

J. W. Mayhew and S. L. Gorbach, J. Chrornatogr., 151, 133-146

(1978). S. E. Edberg and A. Chu, Am. J. Med. Technol., 41,99-105(1975).

D. C. Shanson, C. J. Hince, and J. V. Daniels, J. Infect. Disease,l34 (Suppl), S104-SlO9(1976). 32)

K. Jones, J. Reynolds,T. 0. Oei and J. W. Smith, Clin. Chem., 25,

3 3)

A. Broughton, J. E. Strong, L. K. Pickering and G. P. Bodey, Antimicrob. Agents Chemother., 10,652-656(1 976).

34)

E. L,. Frank, S. Srinivasan, P. Labthavikul and H. C. Neu, J. Clin. Microb.. 13,93-96(1981).

1096-1098(1979).

TOBRAMYCIN

611

35)

R. P., McKnight and J. E. Willis, Clin. Chem., 27, 1256-1261 (1981).

36)

A. S. Lewis, G. Taylor, H. N. Rowe, H. 0. Williams and M. H. Lewis, Am. J. Hosp. Pharm., 44,568-571 (1987).

37)

TDX Fluorescence Polarization Immunoassay System; Abbott Laboratories, Diagnostics Divison, North Chicago, IL 60064.

3 8)

I. A. Cohen, J. M. DeKeyser and D. M. Hyder, Am. J. Hosp. Pharm., 42,605-609 (1985).

39)

Y. G. Tsay and R. J. Palmer, Clin Chem. Actu, 109, 151-157 (1981).

40)

H. T. Kames, J. C. Gudat, C. M. ODonnell and J. D. Wineforder, Clin. Chem., 27,249-252 (1981).

41)

M. Sheehan and G. Caron, Ther. Drug. Monit., 7,108-1 14 (1985).

42)

D. A. Marble, J. A. Bosso and R. J. Townsend, Drug Int. Clin. Pharm., 20,960-963 (1986).

43)

K. S. Nance and G. R. Matzke, Am. J Nephrol., 4,240-243 (1984).

44)

G. E. Hoff, P. 0. Schiotz and J. Paulsen, Scand. J Infict. Dis., 6 , 333-337 (1974).

45).

AHFS Drug Information 93, G. K. McEvoy ed., American Society of Hospital Pharmacists, MD, 1993, pp. 1695-1696.

46)

N. Horikoshi, M. Valdivieso and G. P. Bodey, Am. J Med. Sci., 266, 453-458 (1973).

47)

W. R. Lockwood and J. D. Bower, Antimicrob. Agents Chemother., 3,215-219 (1973). J. Klastersky, D. Daneau and V. DeMaereaer, Clin. Pharmacol. Ther., 14, 104-1 11 (1973).

612

ALEKHA K . DASH

49)

B. R. Meyers and S. Z. Hirschman, J. Clin. Pharmacol. New Drugs, 12,32 1-324 (1972).

50)

C. Regamey, R. C. Gordon and W. M. W. Kirby, Clin. Pharmacol. Ther., 4, 396-403 (1973).

51j

V. K. Simon, E. U. Mosinger and V. Malerczy, Antimicrob. Agents Chemotherapy, 3,444-450 (1973). H. Lode, P. Kernmerich and P. Koeppe, Antimicrob. Agents Chemotherapy, 8, 396-401 (1 975).

53)

B. C. Startford and S. Dixson, Lancet, 1, 378-379 (1974).

54)

Z. Takase, H. Shirauji, M. Uchida and T Sugawa, Chemotherapy, 23, 1378- (1 975).

55)

M. Uwaydah, S,. Bibi and S. Salman, J. Antimicrob. Chemother., 1, 429-437 (1975).

56)

R. Motomura, N. Nakajima and S. Miura., Chemotherapy, 23, 14031408 (1 975).

57)

A. B. Kaiser and Z. A. McGee, Clin. Res., 22,33A (1974).

58)

H. B. Hawley, R. Lewis, D. R. Swartz and D. W. Gump, Curr. Ther. Res., 16,414-423 (1974).

59)

K. Konomi,Y. Yamamoto, K. Egashira, Y. Kawauchi, T. Furusawa and T. Hisatsugu, Chemotherapy, 23,239 (1975). T. Iwasawa, Chemotherapy, 23, 1 168 (1 975). C. H. Ramirez-Ronda, K. Holmes and J. P. Sandford, J. Clin. Invest., 53,63A (1974).

62j

R. C. Gordon, C. Regamey and W. M. Kirby, Antimicrob. Agents Chemotherapy, 2 , 2 14-2 16 ( 1 972).

TOBRAMYCIN

63)

613

K. G. Naber, S. R. Westenfelder and P. 0. Madsen, Antimicrob. Agents Chemotherapy,3,469-473 (1973). U. Ulmann, J. Antimicrob. Chemotherapy,2,213-214 (1976).

H. Ymada, T. Ichihashi and H. Kinoshita, Chemotherapy, 23,900907 (1975).

J.-C. Pechere and R. Dugal, J. Infect, Dis., 134 (Suppl.), S118-S124 (1976). 67)

J. R. Black and R. S. Griffith, “Comparative pharmacology of tobramycin and gentamycin in adult volunteers”, in Tobramycin: selected proceedings from the Eighth International Congress of Chemotherapy, Athens, 1973, Excerpta Media, Amsterdam, 1974, pp. 24-30. G. Jaffe, B. R. Meyers and S . Z. Hirshman, Antimicrob. Agents Chemotherapy, 5,6 11-616, 1974. Material Safety Data Sheet for tobramycin, USP reference standard, United States Pharmacopeial Convention ,Rockville, MD. 1988, p. 3.

70)

F. DeRosa, U. Buoncristiani, P. Capitanucci and R. F. Frongillo, J. Int. Med. Res., 2, 100-114 (1974).

71)

J. S. Welles, J. L. Emmeson, W. R. Gibson, R. Nickander, N. V. Owen and R. C. Anderson, Toxicol.Appl. Pharmacol., 25,398-409 (1973).

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CUMULATIVE INDEX Bold numerals refer to volume numbers.

Acebutolol, 19, Acetaminophen, 3, 1; 14, 551 Acetazolamide, 22, 1 Acetohexamide, 1, 1; 2, 573; 21, 1 Allopurinol, 7, 1 Amantadine, 12, 1 Amikacin sulfate, 12, 37 Amiloride hydrochloride, 15, 1 Aminobenzoic acid, 22, 33 Aminoglutethimide, 15, 35 Aminophylline, 11, 1 Aminosalicylic acid, 10, 1 Amiodarone, 20, 1 Amitriptyline hydrochloride, 3, 127 Amobarbital, 19, 27 Amoddiaquine hydrochloride, 21, 43 Amoxicillin, 7, 19; 23, 1 AmphotericinB, 6, 1; 7, 502 Ampicillin, 2, 1; 4, 518 Apomorphine hydrochloride, 20, 121 Ascorbic acid, 11, 45 Aspirin, 8, 1 Astemizole, 20, 173 Atenolol, 13, 1 Atropine, 14, 325 Azathioprine, 10, 29 Azintamide, 18, 1 Aztreonam, 17, 1 Bacitracin, 9, 1 Baclofen, 14, 527 Bendroflumethiazide, 5, 1; 6, 597 Benperidol, 14, 245 Benzocaine, 12, 73 Benzyl benzoate, 10, 55 Betamethasone diproprionate, 6, 43 Bretylium tosylate, 9, 71 Bromazepam, 16, 1

Bromcriptine methanesulfonate, 8, 47 Bumetanide, 22, 107 Bupivacaine, 19, 59 Busulphan, 16, 53 Caffeine, 15, 71 Calcitriol, 8, 83 Camphor, 13, 27 Captopril, 11, 79 Carbamazepine, 9, 87 Carbenoxolone sodium, 24, 1 Cefaclor, 9, 107 Cefamandole nafate, 9, 125; 10, 729 Cefazolin, 4, 1 Cefotaxime, 11, 139 Cefoxitin sodium, 11, 169 Ceftazidime, 19, 95 Cefuroxime sodium, 20, 209 Celiprolol hydrochloride, 20, 237 Cephalexin, 4, 21 Cephalothin sodium, 1, 3 19 Cephradine, 5, 21 Chloral hydrate, 2, 85 Chlorambucil, 16, 85 Chloramphenicol, 4, 47; 15, 701 Chlordiazepoxide, 1, 15 Chlordiazepoxide hydrochloride, 1,39 Chloropheniramine maleate, 7, 43 Chloroquine, 13, 95 Chloroquine phosphate, 5, 61 Chlorothiazide, 18, 33 Chlorprothixene, 2, 63 Chlortetracyclinehydrochloride, 8, 101 Chlorthalidone, 14, 1 Chlorzoxazone, 16, 119 Cholecalciferol, 13, 655 Cimetidine, 13, 127; 17, 797 Cisplatin, 14, 77; 15, 796

h16

CUMULATIVE INDEX

Clarithromycin, 24, 45 Clidinium bromide, 2, 145 Clindamycin hydrochloride, 10, 75 Clioquinol, 18, 57 Clofazamine, 18, 91 Clofazimine, 21, 75 Clonazepam, 6, 61 Clonfibrate, 11, 197 Clonidine hydrochloride, 21, 109 Ciorazepate dipotassium, 4, 91 Clotrimazole, 11, 225 Cloxacillin sodium. 4, 113 Clozapine, 22, 145 Cocaine hydrochloride, 15, 151 Codeine phosphate, 10, 93 Colchicine, 10, 139 Crospovidone, 24, 87 Cyanocobalamin, 10, 183 Cyclandelate, 21, 149 Cyclizine, 6, 83; 7, 502 Cyclobenzaprine hydrochloride, 17, 4 1 Cycloserine, 1, 53; 18, 567 Cyclosporine, 16, 145 Cyclothiazide, 1, 65 Cypropheptadine, 9, 155 Dapsone, 5, 87 Dexamethasone, 2, 163; 4, 519 Diatrizoic acid, 4, 137; 5, 556 Diazepam, 1, 79; 4, 518 Dibenzepin hydrochloride, 9, 181 Dibucaine, 12, 105 Dibucaine hydrochloride, 12, 105 Diclofenac sodium, 19, 123 Didanosine, 22, 185 Diethylstifbestrol, 19, 145 Diflunisal, 14, 491 Digitoxin, 3, 149; 9, 207 Dihydroergotoxine methanesulfonate, 7, 8 1 Diltiazern hydrochloride, 23, 53 Dioctyl sodium sulfosuccinate, 2, 199 Diosgenin, 23, 101 Diperodon, 6, 99 Diphenhydramine hydrochloride, 3, 173

Diphenoxylate hydrochloride, 7, 149 Dipivefrin hydrochloride, 22, 229 Disopyramide phosphate, 13, 183 Disulfiram, 4, 168 Dobutamine hydrochloride, 8, 139 Dopamine hydrochloride, 11, 257 Doxorubicine, 9, 245 Droperidol, 7, 171 Echothiophate iodide, 3, 233 Econazole nitrate, 23, 127 Emetine hydrochloride, 10, 289 Enalapril maleate, 16, 207 Ephedrine hydrochloride, 15, 233 Epinephrine, 7, 193 Ergonovine maleate, 11, 273 Ergotamine tartrate, 6, 1 13 Erthromycin, 8, 159 Erthromycin estolate, 1, 101; 2, 573 Estradiol, 15, 283 Estradiol valerate, 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7, 23 1 Ethynodiol diacetate, 3, 253 Etomidate, 12, 191 Etopside, 18, 121 Fenoprofen calcium, 6, 161 Flecainide, 21, 169 Flucytosine, 5, 115 Fludrocortisone acetate, 3, 281 Flufenamic acid, 11, 313 Fluorouracil, 2, 221; 18, 599 Fluoxetine, 19, 193 Fluoxymesterone, 7, 25 1 Fluphenazine decanoate, 9,275; 10,730 Fluphenazine enanthate, 2,245; 4, 524 Fluphenazine hydrochloride, 2, 263; 4, 5 19 Flurazepam hydrochloride, 3. 307 Fluvoxamine maleate, 24, 165 Folic acid, 19, 221 Furosemide, 18, 153 Gadoteridol, 24, 209 Gentamicin sulfate, 9, 295; 10, 73 1 Glafenine, 21, 197

CUMULATIVE INDEX

Glibenclamide, 10, 337 Gluthethimide, 5, 139 Gramicidin, 8, 179 Griseofulvin, 8, 219; 9, 583 Guanabenz acetate, 15, 3 19 Guargum, 24, 243 Halcinonide, 8, 251 Haloperidol, 9, 341 Halothane, 1, 119; 2, 573; 14, 597 Heparin sodium, 12, 2 15 Heroin, 10, 357 Hexestrol, 11, 347 Hexetidine, 7, 277 Homatropine hydrobromide, 16, 245 Hydralazine hydrochloride, 8, 283 Hydrochlorothiazide, 10, 405 Hydrocortisone, 12, 277 Hydroflumethaizide, 7, 297 Hydroxyprogesteronecaproate, 4, 209 Hydroxyzine dihydrochloride, 7, 3 19 Hyoscyamine, 23, 155 Imipramine hydrochloride, 14, 37 Impenem, 17, 73 Indapamide, 23, 233 Indomethacin, 13, 21 1 Iodamide, 15, 337 Iodipamide, 2, 333 Iodoxamic acid, 20, 303 Iopamidol, 17, 115 Iopanoic acid, 14, 181 Iproniazid phosphate, 20, 337 Isocarboxazid, 2, 295 Isoniazide, 6, 183 Isopropamide, 2, 315; 12, 721 Isoproterenol, 14, 391 Isosorbide dinitrate, 4, 225; 5, 556 Ivermectin, 17, 155 Kanamycin sulfate, 6, 259 Ketamine, 6, 297 Ketoprofen, 10, 443 Ketotifen, 13, 239 Khellin, 9, 371 Lactic acid, 22, 263

617

Lactose, anhydrous, 20, 369 Leucovorin calcium, 8, 3 15 Levallorphan tartrate, 2, 339 Levarterenol bitartrate, 1, 149; 2,573; 11, 555 Levodopa, 5, 189 Levothyroxine sodium, 5, 225 Lidocaine, 14, 207; 15, 761 Lidocaine hydrochloride, 14, 207; 15, 761 Lincomycin, 23, 275 Lisinopril, 21, 233 Lithium carbonate, 15, 367 Lobeline hydrochloride, 19, 261 Lomefloxacin, 23, 327 Lomustine, 19, 315 Loperamide hydrochloride, 19, 341 Lorazepam, 9, 397 Lovastatin, 21, 277 Mafenide acetate, 24, 277 Maltodextrin, 24, 307 Maprotiline hydrochloride, 15, 393 Mebendazole, 16, 291 Mefloquine hydrochloride, 14, 157 Melphalan, 13, 265 Meperidine hydrochloride, 1, 175 Meprobamate, 1, 207; 4, 520; 11, 587 Mercaptopurine, 7, 343 Mestranol, 11, 375 Methadone hydrochloride, 3,365; 4, 520; 9, 601 Methaqualone, 4, 245 Methimazole, 8, 351 Methixen hydrochloride, 22, 3 17 Methocarbamol, 23, 377 Methotrexate, 5, 283 Methoxamine hydrochloride, 20, 399 Methoxsalen, 9, 427 Methylclothiazide, 5, 307 Methylphenidatehydrochloride, 10, 473 Methyprylon, 2, 363 Metipranolol, 19, 367 Metoclopramide hydrochloride, 16, 327 Metoprolol tartrate, 12, 325 Metronidazole, 5, 327 Mexiletine hydrochloride, 20, 433

618

CUMULATIVE INDEX

Minocycline, 6, 323 Minoxidil, 17, 185 Mitomycin C, 16, 361 Mitoxanthrone hydrochloride, 17, 22 1 Morphine, 17, 259 Moxalactam disodium, 13, 305 Nabilone, 10, 499 Nadolol, 9, 455; 10, 732 Naiidixic acid, 8, 371 Nalmefene hydrochloride, 24, 35 1 Nalorphine hydrobromide, 18, 195 Naloxone hydrochloride, 14, 453 Naphazoline hydrochloride, 21, 307 Naproxen, 21, 345 Natamycin, 10, 513; 23, 405 Neomycin, 8, 399 Neostigmine, 16, 403 Nicotinamide, 20, 475 Nifedipine, 18, 221 Nitrazepam, 9, 487 Nitrofurantoin, 5, 345 Nitroglycerin, 9, 5 19 Nizatidine, 19, 397 Norethindrone, 4, 268 Norfloxacin, 20, 557 Norgestrel, 4, 294 Nortriptyline hydrochloride, 1,233; 2, 573 Noscapine, 11, 407 Nystatin, 6, 341 Oxamniquine, 20, 60 1 Oxazepam, 3, 441 Oxyphenbutazone, 13, 333 Oxytocin, 10, 563 Papaverine hydrochloride, 17, 367 Penicillamine, 10, 601 Penicillin-G, benzothine, 11, 463 Penicillin-G, potassium, 15, 427 Penicillin-V, 1, 249; 17, 677 Pentazocine. 13, 361 Pergolide Mesylate, 21, 375 Phenazopyridine hydrochloride, 3, 465 Phenelzine sulfate, 2, 383 Phenformin hydrochloride, 4,3 19; 5, 429

Phenobarbital, 7, 359 Phenolphthalein, 20, 627 Phenoxymethyl penicillin potassium, 1, 249 Phenylbutazone, 11, 483 Phenylephrine hydrochloride, 3, 483 Phenylpropanolamine hydrochloride, 12,357 Phenytoin, 13, 417 Physostigmine salicylate, 18, 289 Phytonadione, 17, 449 Pilocarpine, 12, 385 Piperazine estrone sulfate, 5, 375 Pirenzepine dihydrochloride, 16, 445 Piroxicam, 15, 509 Polythiazide, 20, 665 Polyvinyl alcohol, 24, 397 Polyvinylpyrollidone, 22, 555 Povidone, 22, 555 Pralidoxine chloride, 17, 533 Prazosin hydrochloride, 18, 35 1 Prednisolone, 21, 415 Primidone, 2, 409; 17, 749 Probenecid, 10, 639 Procainamide hydrochloride, 4, 333 Procarbazine hydrochloride, 5, 403 Promethazine hydrochloride, 5, 429 Proparacaine hydrochloride, 6, 423 Propiomazine hydrochloride, 2, 439 Propoxyphene hydrochloride, 1,301; 4,520 Propylthiouracil, 6, 457 Pseudoephedrine hydrochloride, 8, 489 Pyrazinamide, 12, 433 Pyridoxine hydrochloride, 13, 447 Pyrimethamine, 12, 463 Quinidine sulfate, 12, 483 Quinine hydrochloride, 12, 547 Ranitidine, 15, 533 Reserpine, 4, 384; 5, 557; 13, 737 Riboflavin, 19, 429 Rifampin, 5, 467 Rutin, 12, 623 Saccharin, 13, 487 Salbutamol, 10, 665 Salicylamide, 13, 52 1

CUMULATIVE INDEX

Salicylic acid, 23, 427 Scopolamine hydrobromide, 19, 477 Secobarbital sodium, 1, 343 Sertraline hydrochloride, 24, 443 Silver sulfadiazine, 13, 553 Simvastatin, 22, 359 Sodium nitroprusside, 6,487; 15,781 Solasodine, 24, 487 Sotalol, 21, 501 Spironolactone, 4, 431; 18, 641 Starch, 24, 523 Streptomycin, 16, 507 Strychnine, 15, 563 Succinycholine chloride, 10, 691 Sulfacetamide, 23, 477 Sulfadiazine, 11, 523 Sulfadoxine, 17, 571 Sulfamethazine, 7, 401 Sulfamethoxazole, 2, 467; 4, 521 Sulfasalazine, 5, 5 15 Sulfathiazole, 22, 389 Sulfisoxazole, 2, 487 Sulfoxone sodium, 19, 553 Sulindac, 13, 573 Sulphamerazine, 6, 5 15 Sulpiride, 17, 607 Talc, 23, 517 Teniposide, 19, 575 Tenoxicam, 22, 431 Terazosin, 20, 693 Terbutaline sulfate, 19, 60 1 Terfenadine, 19, 627 Terpin hydrate, 14, 273 Testolactone, 5, 533 Testosterone enanthate, 4, 452 Tetracaine hydrochloride, 18, 379 Tetracycline hydrochloride, 13, 597 Theophylline, 4, 466 Thiabendazole, 16, 61 1 Thiamine hydrochloride, 18, 4 13 Thiamphenicol, 22, 461 Thiopental sodium, 21, 535 Thioridazine, 18, 459

619

Thioridazine hydrochloride, 18, 459 Thiostrepton, 7, 423 Thiothixene, 18, 527 Ticlopidine hydrochloride, 21, 573 Timolol maleate, 16, 641 Titanium dioxide, 21, 659 Tobramycin, 24, 579 a-Tocopheryl acetate, 3, 111 Tolazamide, 22, 489 Tolbutamide, 3, 513; 5, 557; 13, 719 Tolnaftate, 23, 549 Trazodone hydrochloride, 16, 693 Triamcinolone, 1,367; 2, 571; 4, 521; 11, 593 Triamcinolone acetonide, 1, 397; 2, 571; 4, 521; 7, 501; 11,615 Triamcinolone diacetate, 1, 423; 11, 651 Triamcinolone hexacetonide, 6, 579 Triamterene, 23, 579 Triclobisonium chloride, 2, 507 Trifluoperazinehydrochloride, 9, 543 Triflupromazinehydrochloride, 2,523; 4, 521; 5, 557 Trimethaphan camsylate, 3, 545 Trimethobenzamide hydrochloride, 2, 55 1 Trimethoprim, 7, 445 Trimipramine maleate, 12, 683 Trioxsalen, 10, 705 Tripelennamine hydrochloride, 14, 107 Triprolidine hydrochloride, 8, 509 Tropicamide, 3, 565 Tubocurarine chloride, 7, 477 Tybamate, 4, 494 Valproate sodium, 8, 529 Valproic acid, 8, 529 VerapamiI, 17, 643 Vidarabine, 15, 647 Vinblastine sulfate, 1, 443; 21, 61 1 Vincristine sulfate, 1, 463; 22, 517 VitaminD3, 13, 655 Warfarin, 14, 423 Xylometazoline hydrochloride, 14, 135 Yohimbine, 16, 731 Zidovudine, 20, 729 Zomepirac sodium, 15, 673

I S B N 0-12-260824-0