35107468 Analytical Profiles of Drug Substances and Excipients Vol 21 1992 ISBN 0122608216 9780122608216

Analytical Profiles of

Drug Substances and

Excipien t s Volume 21

Edited by

Harry G. Brittain Bristol-Myers Squibb

Pharmaceutical Research Institute New Brunswick, New Jersey

Founding Editor

Klaus Florey

ACADEMIC PRESS, INC. Harcourt Brace Jovanovich, Publishers

San Diego New York Boston London Sydney Tokyo Toronto

Abdullah A. Al-Badr

Gerald S. Brenner

Glenn A. Brewer

Harry G. Brittain

Klaus Florey

EDITORIAL BOARD

George A. Forcier

Lee T. Grady

David J. Mazzo

Thomas W. Rosanske

Timothy J. Wozniak

Academic Press Rapid Manuscript Reproduction

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Copyright 0 1992 by ACADEMIC PRESS, INC.

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AFFILIATIONS OF EDITORS AND CONTRIBUTORS

Mohummud A. Abounussf, Pharmaceutical Chemistry Department, College of Phar-

Abdul Furruh A. A. Ajfy, Department of Pharmacognosy, College of Pharmacy, King

Iqbal Ahmad, Pharmaceutical Chemistry Department, Faculty of Pharmacy, Univer-

Tuuqir Ahmud, Department of Pharmaceutical Chemistry, Faculty of Pharmacy, Uni-

Abdulluh A. Al-Budr, Pharmaceutical Chemistry Department, College of Pharmacy,

Fuhud J. Al-Shammury, Clinical Laboratory Sciences Department, College of Ap-

Silvia Alessi-Severini, Faculty of Pharmacy and Pharmaceutical Sciences, University

Syed Laik Ali, Zentrallaboratorium Deutscher Apotheker, 6236 Eschborn, Germany Adnun A. Budwun, The Jordanian Pharmaceutical Manufacturing Company, Naor,

Gary Burberu, Bristol-Myers Squibb, Pharmaceutical Research Institute, New

Gerald S. Brenner, Merck Sharp & Dohme Research Laboratories, West Point, Penn-

Glenn A. Brewer, Bristol-Myers Squibb, Pharmaceutical Research Institute, New

Hurry G . Brirruin, Bristol-Myers Squibb, Pharmaceutical Research Institute, New

Marvin A. Brooks, Merck Sharp & Dohme Research Laboratories, West Point, Penn-

Robert A . Curr, Faculty of Pharmacy and Pharmaceutical Sciences, University of

Owen 1. Corrigan, School of Pharmacy, University of Dublin, Dublin 4, Ireland Ronald T. Coutts, Faculty of Pharmacy and Pharmaceutical Sciences, University of

macy, King Saud University, Riyadh 11451, Saudi Arabia

Saud University, Riyadh 11451, Saudi Arabia

sity of Karachi, Karachi 75270, Pakistan

versity of Karachi, Karachi 75270, Pakistan

King Saud University, Riyadh 11451, Saudi Arabia

plied Medical Sciences, King Saud University, Riyadh 11433, Saudi Arabia

of Alberta, Edmonton, Alberta T6G 2N8, Canada

Jordan

Brunswick, New Jersey 08903

sylvania 19486



sylvania 19486

Alberta, Edmonton, Alberta T6G 2N8, Canada


vii

AFFILIATIONS OF EDITORS AND CONTRIBUTORS ... V l l l

Joseph D. DeMarco, Merck Sharp & Dohme Research Laboratories, West Point,

Joseph DeVincenfis, Bristol-Myers Squibb, Pharmaceutical Research Institute, New

Humeida A. El-Obeid, Pharmaceutical Chemistry Department, College of Pharmacy,

Dean K. Ellison, Merck Sharp & Dohme Research Laboratories, West Point, Penn-

Klaus Florey, Bristol-Myers Squibb Company, Lawrenceville, New Jersey 08543 George A. Forcier, Central Research Division, Pfizer, Inc., Groton, Connecticut

Robert T. Foster, Faculty of Pharmacy and Pharmaceutical Sciences, University of

Lee T. Grady, The United States Pharmacopeia, Rockville, Maryland 20852 Dominic I? Zp, Merck Sharp & Dohme Research Laboratories, West Point, Pennsyl-

vania 19486 Fakhreddin Jamafi, Faculty of Pharmacy and Pharmaceutical Sciences, University

of Alberta, Edmonton, AlbertaT6G 2N8, Canada Eric C. Jensen, Lilly Research Laboratories, Eli Lilly and Company, Indianapolis,

Indiana 46285 Michael J . KauJinan, Merck Sharp & Dohme Research Laboratories, West Point,

Pennsylvania 19486 David J. Mauo, Department of Analytical & Physical Chemistry, RhBnC-Poulenc

Rorer Recherche-Development ,92165 Antony Cedex, France Michael J . McLeish, School of Pharmaceutical Chemistry, Victorian College of Phar-

macy, Monash University, Parkville, Victoria 3052, Australia Mohammad Safeem Mian, Pharmaceutical Chemistry Department, College of Phar-

macy, King Saud University, Riyadh 11451, Saudi Arabia Neelofur Abduf Aziz Mian, Clinical Laboratory Sciences Department, College of

Applied Medical Sciences, and Department of Pharmacognosy, College of Pharmacy, King Saud University, Riyadh 11433, Saudi Arabia

Ann W. Newman, Bristol-Myers Squibb, Pharmaceutical Research Institute, New Brunswick, New Jersey 08903

Caifriona M . O’Driscoll, School of Pharmacy, University of Dublin, Dublin 4, Ire- land

Mahmoud A1 Omari, The Jordanian Pharmaceutical Manufacturing Company, Naor, Jordan

Franco M. Pasutto, Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, Alberta T6G 2N8, Canada

Thomas W Rosanske, Marion Merrell Dow, Inc., Kansas City, Kansas 64134 Charles M. Shearer, Wyeth-Ayerst Research, Rouses Point, New York 12979 Delores J. Sprankle, Lilly Research Laboratories, Eli Lilly and Company, Indianapo-

Pennsylvania 19486


King Saud University, Riyadh 11451, Saudi Arabia

sylvania 19486

06340


lis, Indiana 46285

AFFILIATIONS OF EDITORS AND CONTRIBUTORS ix

K . Usmanghani. Department of Pharmacognosy, Faculty of Pharmacy, University of

G. Michael Wall, Alcon Laboratories, Inc., Fort Worth, Texas 76134 Titnothy J. Wozniak, Eli Lilly and Company, Lilly Corporate Center, Indianapolis,

Muhammad B . Zughul, Department of Chemistry, Faculty of Science, University of

Karachi, Karachi 75270, Pakistan

Indiana 46285

Jordan, Amman, Jordan

PREFACE

The profiling of drug compounds as to their physical and analytical characteristics has been the focus of the preceding twenty volumes in the Analytical Profiles series, and the need for this information is as important today as it was when the 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. Under the editorship of Klaus Florey, the Analytical Profiles has met this need over its twenty year history.

With the publication of Volume 21, the editorship has been assumed by Harry Brittain. The focus of the chapters will remain unchanged, but the scope of the Analytical Projiles series has expanded to include profiles of excipient materials, and this has led to a modification of the series title. The series will henceforth be known as the Analytical Profiles of Drug Substances and Excipients. The first excipient profile (anhydrous lactose) appeared in Volume 20, and a profile on titanium dioxide is included in the present volume.

The success of the series will continue to be based on the contributions of the chapter authors, and on the quality of their work. We seek profiles of new drug compounds as they come to markets but we also wish to profile important older compounds that have escaped attention thus far. A complete list of available candidates can be obtained from the editor by any prospective author. 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 Editor

Klaus Florey Founding Editor

xi

ACETOHEXAMIDE

Abdullah A. Al-Badr and Humeida A. El-Obeid

Pharmaceutical Chemistry Department

College of Pharmacy

King Saud University

Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21 1

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form

2 ABDULLAH A. AL-BADR AND HUMEIDA A. EL-OBEID

C O N T E N T S

1. DESCRIPTION 1.1 Nomenclature

1.1.1 Chemical Names 1.1.2 Genermic Names 1.1.3 Trade Names

1.2.1 Empirical 1.2.2 Structural 1.2.3 GAS No.

1.3 Molecular Weight 1 .4 Elemental Composition 1 .5 Appearance

1.2 Formulae

2. PHYSICOCHEMICAL PROPERTIES 2.1 Melting Range 2.2 So lub i l i t y 2.3 Polymorphism 2 .4 Thermal Analysis 2.5 X-ray Powder D i f f rac t ion 2.6 Spectral Properties

2.6.1 Ul t rav io le t Spectrum 2.6.2 Infrared Spectrum 2.6.3 Proton Nuclear Magnetic Resonance (PMR)

Spectrum 2.6.4 lac-Nuclear Magnetic Resonance (‘SC-NMR)

Spectrum 2.6.5 Mass Spectra

3. SYNTHESIS

4. METHODS OF ANALYSIS 4.1 Ti t r imet r i c Methods

4.1.1 Nonaqueous 4.1.2 Gravimetric 4.1.3 Campleximetric

4.2 Spectrometric Methods 4.2.1 Colorimetric 4.2.2 U1 t rav io le t 4 .2 .3 Infrared 4.2.4 Fluormetr ic 4.2.5 Proton Magnetic Resonance

ACETOHEXAMIDE 3

4.3 Chromatographic Methods 4.3.1 Thin-Layer Chromatography (TLC) 4.3.2 Gas-Liquid Chromatography (GLC) 4.3.3 High-Performance Liquid Chromatography

(HPLC)

5. PHARMACOKINETICS 5.1 Introduction 5.2 Mechanism o f Action 5.3 Onset and Duration o f Action 5.4 Absorption 5.5 Distribution 5.6 Metabol ism 5.7 Excretion 5.8 Half-Life

ACKNOWLEDGEMENT

REFERENCES


ACETOHEXAMIDE

1. DESCRIPTION

1 - 1 Nomenclature

1.1.1 Chemical Names

4-Acetyl-N-[(cyclohexylamino)carbonyl]benzenesul-

l-[(pAcetylphenyl)sulfonyl]-3-cyclohexylurea. 3-Cyclohexyl-l-(pacetylphenylsulfonyl)urea. N-(pAcetyl benzyl sul fonyl l-N -cyclohexyl urea.

fonamide

1.1.2 Generic Names

Acetohexamide, Acetohexamidum

1 - 1.3 Trade Names

Cycl am1 de , Dime 1 i n , Dime1 o r , Dyme 1 or , Gamad i abet, Metaglucina, Ordimel, Tsiklamid.

1.2 Formulae

1.2.1. EmDlriCal

Ct sHzoNz04S

1.2.2 Structural

1.2.3 CAS No.

[968-81-01

1.3 Molecular Weight

324.42 (1)

1.4 Elemental ComDosltion

C 55.54%, H 6.21%, N 8.64%, 0 19.73%, S 9.89% (1).

ACETOHEXAMIDE 5

1.5 Armearance

2.

A whi te , c r y s t a l l i n e powder; odor less o r almost odorless (2).

PHYSICOCHEMICAL PROPERTIES

2.1 Melt ing Range

Crys ta ls from 90% aqueous ethanol m e l t between 188-190" (3 ) . Crystals from d i l u t e ethanol melt between 175-177 (4).

2.2 S o l u b i l i t y

Soluble i n pyr id ine, s l i g h t l y soluble i n alcohol and chloroform. Insoluble i n water and ether (1 ) .

2 .3 P01YmOrDh'ism

The l i t e r a t u r e reports ind icate t h a t acetohexamide e x i s t s as more than one polymorphic forms (5-15) . G i rgis-Takla and Chroneos (5) prepared acetohexamide polymorphs A and B by heat ing the drug ( 1 gm) w i t h g l a c i a l acet ic acid o r chloroform respectively, before c r y s t a l l i z a t i o n a t 1 0 5 ' and room t e m p e r a t u r e respectively. While acetohexamide polymorph A showed a melt ing range o f 180"-183', the acetohexamide polymorph B m e l t e d a t 1 8 3 ' - 1 8 5 " . D i f f e r e n t i a l s c a n n i n g calorimetry and I R spectroscopy showed t h a t c rys ta ls o f polymorph B were converted t o polymorph A by grinding. A c c o r d i n g l y , t h e s e r e s u l t s i n d i c a t e t h a t any i d e n t i f i c a t i o n t e s t u t i l i z i n g g r i n d i n g may f a i l to i d e n t i f y the two polymorphs. I n t h e i r phystco-chemical studies on the polymorphism o f acetohexamide, Kuroda e t a7 (6) obtained three polymorphs o f acetohexamide by r e c r y s t a l l i z a t i o n from d i f f e r e n t solvents. These are form I, form I 1 and CHC13-11. A l though t h e X-ray d i f f r a c t i o n pa t te rns , I R spect ra and d i f f e r e n t i a l scanning calorimeter curves o f the CHC13-I1 polymorph were ident ica l w i th those o f polymorph 11, the CHC13-I1 type contained a C H C l j molecule which could n o t be removed by normal drying condit ion. Polymorph CHC13-I1 seemed t o be unsuitable f o r medicinal use. Form I1 i s 1.2 times more soluble than form I.

6 ABDULLAH A. AL-BADR AND HUMElDA A. EL-OBEID

Burger ( 7 ) cha rac te r i zed t h e t h r e e polymorphic modi f icat ions o f acetohexamide by thermomicroscopy, d i f f e ren t i a l scanning calorimetry and I R spectroscopy. The s o l u b i l i t y behavior o f the three modifications o f the drug i n butanol and buffer solutions i s described and discussed i n r e l a t i o n t o thermodynamics and pharmacological parameters such as b ioava i l ab i l i t y from t a b l e t s and USP X I X d issoc ia t ion tes t . Muel ler and Lagas ( 8 ) have c o n f i r m e d t h e e x i s t e n c e and characterized two polymorphic forms o f acetohexamide using d i f f e ren t i a l scanning calorimetry, thermogravimetric analysis, scanning electron microscopy as we1 1 as I R , NMR and X-ray analysis. The study has pointed t o the unsu i tab i l i t y o f phosphate buf fer solut ion which i s sometimes prescribed f o r use i n the dissolut ion tes ts o f the drug since the s a l t o f the drug c rys ta l l i zes out during the test . I n another study (9) the same authors reported tha t form I decomposed during melting and form I1 melted a t 180" and then recrysta l l ized t o form I. A t a heating rate o f lO'/minute melting points o f 193.6" and 180.5" were found f o r forms I and 11, respectively. No morphological differences were observed between the two forms. I n s o l u b i l i t y and dissolut ion rate studies i n sodium potassium bu f fe r , potassium acetohexamide c r y s t a l l i z e d e x h i b i t i n g a lower s o l u b i l i t y than acetohexamide. I n t h i s respect, form I1 was transferred t o potassium acetohexamide more quickly than form I.

Yokoyama e t a7 (10) calculated the thermodynamic values o f forms I and I1 o f acetohexamide from s o l u b i l i t y measurements. The t rans i t lon temperature and the heat o f t rans i t ion were 154" and 230 cal/mole, respectively. It i s found that the polymorphic forms o f acetohexamide d i d no t a f f e c t i t s b i o a v a i l a b i l i t y when i n v i v o absorption studies o f form I & I1 were carr ied out i n beagle dogs. The p repara t i on o f f o u r c r y s t a l l i n e modifications o f acetohexamide was reported (11). Their thermograms, I R spec t ra , X-ray d i f f r a c t i o n and s o l u b i l i t y are also reported. Two o f the forms reverted t o the most stable form on storage i n solution.

Sol id dispersion o f acetohexamide was studied by Graf e t a7 (12-14) using d i f f e r e n t polymers and var ious r a t i o s . C o p r e c i p i t a t e s o f acetohexamide w i t h polyethylene g lyco l (PEG 6000) were prepared by the solvent method w i t h ethanol ( c r y s t a l l i n e form I) o r with chloroform (crystal 1 ine form 111). Phase diagrams

ACETOHEXAMIDE I

o f form I-PEG and form 111-PEG coprecipitates were o f the pe r i t ec t i c type and the molecular compounds were formed i n the r a t i o o f 1 mole o f acetohexamide t o 4 moles o f PEG. The e u t e c t l c temperature, e u t e c t i c composition and the end o f melting o f the two binary system were, however, d i f f e r e n t ( 1 2 ) . Both the s o l u b i l i t y and the solut ion ra te were increased by PEG. S i m i l a r r e s u l t s were o b t a i n e d by s u b s t i t u t i n g po ly (v iny lpy r ro1 idone) (PVP) f o r PEG ( 1 3 ) . Also, c o p r e c i p i t a t e s o f acetohexamide-PVP ( i n e thano l ) containing drug concentrations o f 60% or more showed the same X-ray d i f f rac t i on pattern as tha t o f form I. Increasing the PVP concentration (> 55%) d i d not show any crysta l behavior i n the X-ray analysis. I n another r e p o r t Graf e t a7 ( 1 4 ) descr ibed t h e methods o f preparat ion and the e f f e c t o f the solvents on the acetohexamide-PVP coprecipi tates. They were obtained from ethanol or chloroform by evaporating the solvent a t room temperature, under vacuum or by spray drying. Changing t h e so l ven t and/or i t s evapora t ion r a t e affected the polymorphic form, the c r y s t a l l i n i t y and the solut ion rate o f acetohexamide i n coprecipitates containing less than 70% PVP.

Kassem e t a7 (15) studied the enhancement o f the rate o f release o f acetohexamide from i t s t a b l e t s by the fo rmat ion o f s o l i d d i spe rs ions w i t h each o f f o u r water-sol uble pol ymers prepared i n d i f fe ren t r a t i 0s. The polymers were rated i n the order o f decreasing r e l e a s e r a t e s a s f o l l o w s : P E G 6 0 0 0 , P V P , hydroxypropylmethylcellulose, methylcellulose.

2.4 Thermal Analysis

The hea t o f f u s i o n and m e l t i n g p o i n t o f acetohexamide were done using DuPont TA 9900 on the DSC- u n i t a t a temperature range ind icated i n the thermogram (Figure 1). Sample i s done i n duplicate and the average o f the value i s reported as follows:

A H f = 63.7 kJ/mOle Pur i ty = 99.82% T m = 187.45 C

2.5 X-ray Powder D i f f rac t ion

The X-ray powder d i f f r a c t i o n pattern o f acetohexamide was determined using Ph i l ips f u l l automated X-ray d i f f r a c t i o n spectrogoniometer equipped wi th PW1730/10

PURITY v l . l A

F igu re 1. Thermal cu rve o f acetohexamide.

ACETOHEXAMIDE 9

J

Figure 2 . X-Ray powder d i f f r a c t i o n pat te rn of acetohexamide.


generator. Radiation was provided by a copper target (Cu anode 2000W, X = 1.5480 A), high In tens i ty X-ray tube operated a t 40 kV and 35 mA. The monochromator was a curved s ing le c r y s t a l one (PW1752/00). Divergance s l i t and t h e r e c e i v i n g s l i t were 1 and 0.1 respect ive ly . The scanning speed o f the gonlometer (PW1050/81) used was 0.02 2 8 per second. The instrument i s combined w i t h P h i l i p s PM8210 p r i n t i n g recorder w i t h bo th analogue recorder and d i g i t a l p r i n t e r . The goniometer was a l igned using s i l i c o n sample before use.

The X-ray pattern o f acetohexamlde I s presented i n Figure 2. The interplanar distance d(A) and re la t i ve in tens i t ies 1/10 are shown i n Table 1.

2.6 Spectral ProDerties

2.6.1 U l t rav io le t Spectrum

The u l t r a v i o l e t a b s o r p t i o n s p e c t r u m o f acetohexamide i n methanol was obtained on a Cary 219 spectrophotometer. The spectrum, shown i n Figure 3, i s characterized by two maxima. The one wi th a Xmax a t 247 nm i s t y p i c a l o f s u b s t i t u t e d acetophenones. The absorption a t Xmax 283 nm represents a conjugated aromatic r i ng system.

2.6.2 Infrared SDectrum

The infrared absorption spectrum o f acetohexamide, obtained from a potassium bromide dispersion, was recorded on a Pye Unicam SP 1025 spectrometer and i s shown i n Figure 4. The assignment o f the character ist lc bands are shown i n Table 2.

t I 1 1 i

220 XO nm 300 3 50 400 450

Figure 3 . U l t r a v i o l e t spectrum o f acetohexamide in methanol.

v,. m

.

N

C.

c

c

I--

6,.

al.

Q.

t-.

v),

U *I

V

cn U L

Y

W

E

N

Y

W c

0

c, W

V

tcl

'*- 0

-I-

m "

z! f L c, V

W 0

cn U

fu I

E

ACETOHEXAMIDE 13

Table 1: X-ray d i f f r a c t i o n pattern o f acetohexamfde

d(A) 1/10 d(A) 1/10

15.74 9.47 7.85 7.21 5.30 4.99 4.93 4.55 4.30 4.19 4.08 3.92 3.78 3.60 3.50 3.28 3.26 3.15 3.07 3.01 2.91 2.88 2.74 2.65 2.61 2.58

31.25 30.04

6.89 2.25

100.00 8.28

10.95 4.71 5.30

15.19 23.07

5.44 2.82

24.35 4.52

23.29 9.83 5.72 9.36 1.26 7.99 2.79 4.08 1.51 2.15 1.80

2.55 2.49 2.40 2.36 2.31 2.29 2.27 2.24 2.18 2.15 2.13 2.09 2.04 1.99 1.95 1.94 1.89 1.81 1.77 1.72 1.66 1.64 1.61 1.57 1.47 1.35

1.82 1.75 6.19 4.44 5.26 2.20 1.81 2.54 2.04 2.38 1.20 2.56 4.51 1.69 2.91 4.16 1.50 1.48 1.29 1.77 1 .oo 1.18 1.16 1.32 0.85 0.80

d = Interplanner distance 1/10 = re la t i ve in tens i ty (based on highest in tens i ty of

100).


Table 2: Infrared character ist ic bands and t h e i r assignments.

Frequency (cm- Assignment

3340, 3270

2980, 2940

1710, 1680

1602 , 1600

1455

1345

Amide N-H s t re tch

Aromatic C-H s t re tch 0

Conjugated - E - Aromatic C s t re tch

C - CH3 bending 0

780, 760 Aromatic C-H out o f plane bend i ng .

2.6.3 Proton Nuclear Magnetic Resonance (WRl

Acetohexamide so lu t i on i n DMSO-de was used t o obtain the PMR spectrum on a Varian XL 200 MHr FT NWR spectrometer using TMS as i n te rna l reference. The spectrum i s shown i n Figure 5. The number o f protons i s established by both integrat ion o f the area under the curve and the m u l t i p l i c i t i e s o f the peaks. Table 3 assigns the chemical s h i f t s t o t h e i r respec t i ve protons. Further evidence f o r proton assignment i s obtained from the HETCOR pulse sequence (Figure 9).

Spectrum

Figure 5. PMR spectrum of acetohexamide i n DMSO-dG using TMS as internal reference.

Figure 6. 13C NMR spectrum o f acetohexzmide i n DMSO-ds using TMS as internal reference.

Figure 7 . 1 3 C NMR spectrum o f acetohexarnide using DEPT ex Per i ment .

F i g u r e 8. 13C I M R sDectrum of acetohexanide using APT expe r i rnent .

Figure 9. 13C N M R spectrum of acetohexamide using HETCOR experiment.


Table 3: Assignment o f the NMR chemical s h i f t s t o the d i f fe ren t protons

Chemical s h i f t M u l t i p l i c i t y Proton No. o f ( 6 ) assignment protons

1.09 - 1.71 mu1 ti p l e t Cyclohexyl 11 r i n g 3

0

2.66 s ing let - CH3-0 3

6.45 doublet - CH-NH 1

8.06 - 8.19 mu1 t i p l e t Aromat i c d 4

2.6.4 13C-Nuclear Magnetic Resonance ( 1 3 C NHR) SDect rum

The 1 3 C NMR spectra o f acetohexamide i n DMSO-ds using TMS as internal reference are obtained using a Var ian XL 200 MHz pu lse FT spectrometer and are presented i n F igures 6-9. The assignment o f t h e chemical s h i f t s and the degree o f carbon protonation, presented i n Table 4, are achieved using the DEPT (Figure 7) and APT (Figure 8) experiments as well as t h e HETCOR p u l s e sequence ( F i g u r e 9 ) and t h e approximate addi t ive ef fects o f substituents.

2.6.5 Mass SDectra

The 70 eV e l e c t r o n impact mass spectrum o f acetohexamide, presented i n Figure 10, was obtained on Varian MAT 311 mass spectrometer using i o n source pressure o f 10-0 Torr, ion source temperature o f 180'C and an emission current o f 300 M. The molecular ion i s detectable a t m/e 324 and the base peak a t m/e 56. A proposed fragmentation pa t te rn and the mass/charge ra t ios o f the major fragments are shown I n Scheme 1.

ACETOHEXAMIDE 21

Table 4: Assignment o f the carbon chemical sh i f t s .

Chemical s h i f t Carbon assignment Number o f Protons ( P H I attached

24.26 d 2

25.07 C 2

26.99 e 3

32.33 b 2

48.30 a 1

127.73 i 1

128.73 j 1

140.00 k 0

143.93 h 0

150.45 9 0

197.30 f 0

The chemical ionizat ion spectrum, shown i n Figure 11, was obtained on Finnigan 4000 mass spectrometer using methane gas as a reagent with ion electron energy o f 100 eV, ion source pressure o f 0 . 3 Torr, ion source temperature o f 150’C and emission current o f 300 pA. The spectrum i s dominated by a quasimolecular ion (M + 1 ) . Two peaks appearing a t m/e 353 and m/e 365 are at t r ibutable t o the t ransfer o f carbocations from the c a r r i e r gas. The mass s p e c t r a l assignment o f the

N N

F i g u r e 10. Electron impact mass spectrum o f acetohexamide.

Figure 11. Chemlcal ionization mass spectrum of acetohexarnide.


prominent ions under the chemical ionizat ion conditions are presented i n Table 5.

Table 5: Mass spectral assignment o f acetohexamide using chemical ionization.

M/e Species

365 [M t C3H5]+

353 [M t CzHs]+

325 [M t H (MH)1+

324 [MI+

3. SYNTHESIS

Marshall e t a7 (4) reported a method o f synthesis o f acetohexamide which involves the react ion o f the diazonium s a l t from paminoacetophenone w i t h s u l f u r dioxide t o af ford the sulfonyl chloride which i s then converted t o the sulfonamide by reaction wi th a m n i a . Elaborat ion v i a the carbamate w i t h cyclohexylamine a f fo rds acetohexamide. Another reported method (16) uses p-chloroacetophenone as the s t a r t i n g mater ia l . Both methods are out l ined i n Scheme 2.

ACETOHEXAMIDE 25

Scheme 1: Proposed mass fragmentation pattern o f acetohexamide

n 0

O H

mle 324

0

W-Q-

I 0-H NH

I 1

0

+

m/e 243


Scheme 1 Continued . . .

mle 324 mle183

I -CH,-CO

m /e l41

mle 324

mle76 m l e 104

mle 200

- YN0,S

0

I C H3I; 0

mle 119

-i

ACETOHEXAMIDE

Scheme 1 Continued ...

21

1 [ O N H I +

m/e 324

0 II

2 68 +

i

28 ABDULLAH A . AL-BADR A N D HUMEIDA A. EL-OBEID

Scheme 3: Synthesis o f acetohexamide

Method 1 (4)

SO,-NH-C-NH

Method 2 (16)

0 0

CH,t@ S03Na p0c13*

c H 3 - ! G so, CI SO,NH,-

ACETOHEXAMIDE

4. METHODS OF ANALYSIS

29

4.1 T i t r i m e t r i c Methods

4.1.1 Nonaaueous

A non-aqueous t i t r a t i o n method f o r t h e drug and other hypoglycemic and d i u r e t i c agents was reported by Agarwal and Walash (17). The drug i n t a b l e t o r pure form was d isso lved i n te t ramethy l urea and t i t r a t e d w i t h 0.1 N l i t h i u m methoxide i n benzene-methanol medium. The end p o i n t was determined us ing 0.2% azo v i o l e t i n toluene as ind icator . Recovery ranged from 98.8% t o 101.6%.

Another non-aqueous t i t r a t i o n procedure, f o r t h e q u a n t i t a t i v e a n a l y s i s o f t h e d r u g and o t h e r hypoglycemic s u l f o n y l u r e a s u s i n g HC104 t i t r a t i o n method, was also reported (18).

4.1.2 Gravimetric

Amer and Walash (19, 20a) repor ted a method f o r t h e grav imet r ic determinat ion o f acetohexamide by t r e a t m e n t w i t h 2 , 4 - d i n i t r o p h e n y l h y d r a z i n e t o p r e c i p i t a t e t h e h y d r a z o n e (19). A m i x t u r e o f acetohexamide, tolbutamide and chlorpropamide was also determined grav imetr ica l ly (20a).

4.1.3 Compleximetric

Guerel l o and Dobrecky (21) have d e s c r i b e d a procedure f o r t h e c o m p l e x i m e t r i c e v a l u a t i o n o f m e d i c a t i o n s w i t h h y o g l y c e m i c a c t i o n i n c l u d i n g acetohexamide. The procedure permits the determination o f the hypoglycemic sulphonylureas. A weighed amount o f drug was hydrolysed by heat ing f o r 30 minutes wi th d i l u t e aqueous sodium hydrox ide and t h e s o l u t i o n neutral ized w i th 0.1 N HC1, t reated wi th 0.1 M CuSO4, then wi th bu f fe r so lu t ion t o pH 6, and f i l t e r e d . The excess C U + ~ i n t h e f i l t r a t e was determined by complexometric t i t r a t i o n w i th 0.02 M EDTA disodium s a l t us ing 1-(2-pyridylazo)-2-naphthol as i n d i c a t o r . The method i s appl icable t o evaluate drugs i n tab le t .

30 ARDUI.1.AH A. AL-BADR AND HUMEIDA A. EL-OBEID

4.2 SDect romet r i c

4.2.1 Color imetr ic

Reaction o f acetohexamide w i t h 2,4-dinitropheny’l- hydrazine t o produce the colored hydrazone was used by Amer and Walash (19) t o determine the drug co lor imetr i - c a l l y . The co lored product was d i sso l ved i n KOH and determined a t 480 nm. The accuracy o f the method was claimed t o be 100%. A n inhydr in co lor imetr ic method f o r some o r a l hypoglycemic agents was a lso reported (20b).

Meier e t a7 ( 2 2 ) analysed acetohexamide and o t h e r h y p o g l y c e m i c a g e n t s by d i s s o l v i n g t h e d r u g i n chloroform, adding calcium acetate (1% i n methanol), propylamine (5% i n methanol), d i l u t i n g w i th chloroform and reading the absorbance a t 565 nm a f t e r 15 minutes. Pharmaceutical preparations may be estimated s im i la r l y .

4.2.2 U l t r a v i o l e t (UV)

Solomonova and D v o r n i t s k a y a ( 2 3 ) determined acetohexamide by measuring the absorbance a t 229 nm i n ethanol or 0.1 M sodium hydroxide. Other UV t e s t s f o r the drug are also reported (24, 25).

4.2.3 In f ra red ( I R )

Acetohexamide and o t h e r s u l p h o n y l u r e a s were analysed by IR (22). A t e s t have a lso been described (24). Lazaryan (26) determined t h e drug and o t h e r hypoglycemic agen ts by i n f r a - r e d a b s o r p t i o m e t r i c determination. A sample i s t reated with chloroform and t h e s o l u t i o n from t h e t a b l e t sample i s f i l t e r e d . A po r t i on o f so lu t i on i s d i l u t e d w i t h chloroform and the absorbance i s measured a t 1722 t o 1715 cm-1 i n 0.25 m NaCl c e l l against chloroform.

4.2.4 F1 uoromet r i c

G i r g i s - T a k l a and Chroneos ( 2 7 ) d e s c r i b e d a sensi t ive method f o r the f luorometr ic determination o f t h e d r u g i n plasma o r i n t a b l e t s by means of i t s r e a c t i o n w i t h 1 -me thy ln l co t l namide . The l i m i t o f detect ion was approximately 0.2 Mg o f the drug/mL and the r e l a t i v e standard dev iat ion was 31% f o r 2 Ng/ml i n

ACETOHEXAMIDE 31

plasma. The method i s s u i t a b l e f o r plasma samples containing 0.5-2.5 Mg o f the drug/ml.

4.2.5 Proton Magnetic Resonance

Al-Badr and Ibrahim (28) described a simple, rap id and accurate method f o r the assay o f the drug and other hypoglycemic agents us ing proton magnetic resonance spectrometry. The pure drug o r i n t a b l e t form, can be determined using DMSO-ds as solvent and maleic ac id as in te rna l standard.

The reported recovery i s 100 f 1.5% for pure drug and 98 t o 99.6 f 1.4% f o r tab lets .

4.3 ChromatonraDhic Methods

4.3.1 Thin-Layer ChromatonraDhY (TLC]

Gergis-Takla and Josh1 (29) reported a TLC method f o r the i d e n t i f i c a t i o n , assay and p u r i t y determination o f the drug and other hypoglycemic agents i n powder and i n t a b l e t f o r m u l a t i o n . The d r u g was d e t e c t e d by d i s s o l v i n g p o w d e r e d t a b l e t s o r p o w d e r i n dichloromethane-acetone mix tu re (2: 1) and chromato- graphing the so lut ion on s i l i c a gel F 2 5 4 plates with cyclohexane-chloroform-acetic a c i d and e t h a n o l (10:7:2:1). For quant i ta t i ve determination, the spots were s e p a r a t e d , e l u t e d w i t h m e t h a n o l i c sodium hydrox ide , d i l u t e d w i t h m e t h a n o l i c HC1 and t h e absorbance was measured.

Surborg and Roeder (30) have recommended constant- b o i l i n g s o l v e n t m i x t u r e s f o r t h e development o f chromatograms on s i l i c a gel f o r acetohexamide and other a n t i d i a b e t i c drugs: propanol-cyclohexane (37:163), propanol-benzene-cyclohexene (9:14:27), and cyclohexane - isopropanol (177:23). The R f values o f the drugs are tabulated, spots were located by v iewing i n 254 nm radiat ion.

4.3.2 Gas Liauid ChromatonraDhY (GLC)

Kleber e t a l . (31) determined acetohexamide and hydroxyhexamide i n b i o l o g i c a l f l u i d s u s i n g GLC. Tolbutamide was used as an in te rna l standard and M-HC1 was added t o the sample o f plasma or urine, the mixture


was shaken w i t h to luene and was cen t r i f uged . The separated organic phase was shaken w i th 7.5% KzC03 solut ion and centrifuged again. The aqueous phase was heated a t 6 0 " f o r 10 minutes w i t h methanol and dimethylsulphate, cooled and M-acetate buffer solut ion was added t o a d j u s t t o pH 5.2. The methy lated sulphonylureas were ext racted w i t h hexane and the extract was evaporated t o dryness a t 50' i n a stream of nitrogen. The residue was dissolved i n CS2-CHC13 (l:l), 25 u l and 2 p l was submitted t o GLC on a glass column (61 cm X 3 mn) containing 0.5% o f PEG 20 M on Gas-Chrm Q (80 t o 100 mesh) and the temperature was programed f o r 190 t o 240' a t 5 min-1, wi th helium as car r ie r gas (90 m l min-1) and flame ion i sa t i on detect ion. Peak heights were compared. A t concentrations o f 10 t o 40 ug m l - 1 i n plasma. The mean recoveries (8 determination) were : f o r acetohexamide 9.9 and 39.4 c(g m l - 1 ; f o r the metabolite hydroxyhexamide 14.1 and 40 c(g m l - 1 .

Fricke (32) presented a GLC method f o r the analysis o f the drug and other drugs i n pharmaceuticals, using s imple e x t r a c t i o n s and semiautomated gas - l i qu id chromatography, using Ddxil 300 as the l i q u i d phase and an automatic sample in jector. Results by t h i s method and the o f f i c i a l and other appl icable methods are compared. Content uniformity analysis can be made by using t h i s procedure. The ex t rac t i on and chromatographic conditions were standardized t o make possible a successful interlaboratory study.

4.3.3 Hinh-Performance Liauid ChrmatoqraDhy ( HPLCl

A simple HPLC assay o f the drug i n plasma was developed by Takagish i et a7 (33) . A sample was extracted with a mixture o f benzene and ethyl acetate a t pH 5 and the organic phase was evaporated. A 50% solut ion i n CH3CN o f the residue was chromatographed using a Lichrosorb RP-8 reversed-phase column and a mobile phase composed o f 0.2% acet ic ac id - methyl c y a n i d e ( 1 : l ) . The method can be used f o r b ioava i lab i l i t y and c l i n i c a l pharmacokinetic studies o f acetohexamide.

Beyer (34) used high speed l i q u i d chromatography f o r analysis o f the drug and other ant id iabet ic agents. The reocvery o f the drug from iner t tab le t ingredients by

ACETOHEX AM I DE 33

t h i s method was near 100%. A column (100 cm X 2.1 mm) packed w i t h 1% o f ethylene-propene copolymer on Zipax was used w i t h mobile phase o f 0.01 M disodium hydrogen c i t r a t e containing 15% o f methanol (pH 4.4) . Detection was c a r r i e d o u t a t 254 nm and pack areas were integrated.

Testosterone, chlorpropamide i n 95% ethanol were used i n t e r n a l s tandards. The procedure was a p p l i e d t o compressed tab lets , the powdered sample being extracted with t h e i n t e r n a l standard so lu t ion . Recoveries o f added sulphonylurea were 98.9% t o 100.2%.

5. PHARMACOKINETICS

5.1 In t roduct ion

Acetohexamide i s used as an o r a l a n t i d i a b e t i c agent f o r t h e t reatment o f ke toac idos is - res is tan t diabetes. It i s an in termediate a c t i n g su l fony lu rea der ivat ive. The c l i n i c a l e f f e c t s o f lowering elevated b l o o d g lucose l e v e l s i s s i m i l a r f o r a l l o f t h e sulfonylurea der ivat ives. Acetohexamide, however, i s the only one t o also possess ur icosur ic a c t i v i t y and t h e r e f o r e i s a p r e f e r a b l e agent t o t r e a t d i a b e t i c pat ients w i t h gout.

The durat ion o f act ion o f acetohexamide (12-24 hours) permits once or twice d a i l y dosage. The crossover study o f Fox e t a7. (35) conducted i n 36 p a t i e n t s with matur i ty onset diabetes m e l l i t u s indicated t h a t both chlorpropamide and acetohexamide gave s i m i l a r responses based on fas t ing blood sugar. Acetohexamide was used i n a dose range o f 500-3,000 mg/day and i t i s indicated t h a t primary f a i l u r e on acetohexamide i s more l i k e l y t o respond t o chlorpropamide and v ice versa. Appropriate dosing require ind iv idua l i za t ion o f therapy t i t r a t e d t o the des i red therapeut ic e f f e c t . The usual PO dosage range i s 250-1500 mg/day i n s i n g l e o r d i v i d e d doses (36,37), w i t h a maximum recommended dose o f 1500 mg/day. The 250 mg dose o f acetohexamide i s equivalent t o 500 mg tolbutamlde, 100 mg tolazamide, o r 100 mg chlorpropamide (36). The o r a l an t id iabet ic agents prove more u s e f u l when d i e t a r y r e s t r i c t i o n and we igh t reduction accompany t h e i r use.

34 ABDULLAH A. AL-EADK AND HUMEIDA A. EL-OBEID

Acetohexamide i s l a r g e l y metabolized t o an a c t i v e metaboli te which is excreted i n the ur ine (see below). Therefore, dosage adjustment o r t o t a l avoidance i s necessary i n cer ta in cases. One such case i s the renal f a i l u r e . Azotenic p a t i e n t s may experience prolonged hypoglycemia. A t w i c e d a i l y dose i s recommended f o r p a t i e n t s w i t h m i l d renal f a i l u r e and p a t i e n t s wi th moderate t o severe renal f a i l u r e should not receive the drug (38 , 39).

Dosage adjustment may a lso be required i n pat ients with 1 i v e r insuf f ic iency since acetohexamide i s extensively metabolized i n the l i v e r . Prolonged hypoglycemia may r e s u l t i n pat ients w i th severe l i v e r impairment (36). Dosage r e d u c t i o n may be r e q u i r e d i n e l d e r l y o r d e b i l i t a t e d pat ients, due t o renal o r l i v e r impairment o r hyperresponsiveness (36).

It i s recommended by Bennett e t a7. (39) t h a t no dosage supplementatlon i s requi red i n p a t i e n t s f o l l o w i n g per i toneal d i a1 ys i s . Like other o ra l an t id iabet ic agents, acetohexamide may be used i n combination w i t h i n s u l i n t o reduce i n s u l i n requi rements i n i n s u l i n dependent m a t u r i t y onset d i a b e t i c s and t o reduce t h e p o t e n t i a l f o r a hypoglycemic reaction.

5.2 Mechanism o f Action

Acetohexamide i s a sulfonylurea der ivat ive, t h a t produces i t s hypoglycemic e f f e c t by s t i m u l a t i n g t h e i s l e t t i s s u e t o synthesize and re lease endogenous i n s u l i n (40) . The hypoglycemic e f f e c t s a r e a l s o a t t r i b u t e d t o an increased s e n s i t i v i t y o f i n s u l i n receptors as wel l as improved peripheral u t i l i z a t i o n o f i n s u l i n (37).

A report by Lebowitz and Feinglos (41) indicates tha t , dur ing chronic administrat ion, par t o f the hypoglycemic a c t i o n o f t h e s u l f o n y l u r e a s i s e x t r a p a n c r e a t i c . Peripheral t issues may become more sensi t ive t o a f i x e d dose o f an administered hormone poss ib ly due t o an increase i n the number o f i n s u l i n receptors.

A study on the mode o f act ion o f the sulfonylureas (42) has shown t h a t acetohexamide increased glucose uptake

ACETOHEXAMIDE 35

by r a t diaphragm, inh ib i ted the a c t i v i t y o f glucose-6- phosphatase, triosephosphate isomerase and l ipoprote in 1 i pase . 5.3 Onset and Duration o f Action

B r e i d a h l e t a 7 . ( 4 3 , 4 4 1 r e p o r t e d a peak hypoglycemic e f fec t t o occur between 8 t o 10 hour post ingestion o f acetohexamide.

A duration o f action o f 12 t o 24 hours i s reported by Breidahl et a7. (43,44) and Galloway et a7. (45) which i s s imi lar t o tha t o f tolazamide (up t o 24 hours), less than that o f chlorpropamide (60 hour) and greater than tha t o f tolbutamide (6 t o 12 hours) (37).

The serum c o n c e n t r a t i o n s i n d i a b e t i c p a t i e n t s responding wel l t o the drug had mean acetohexamide levels o f 3.7 mg/dL wi th a ragne f o 2.5 t o 4.9 mg/dL fol lowing dosage regimens o f 0.5 t o 3 g/day (46). No good c o r r e l a t i o n between b lood concent ra t ions o f acetohexamide and therapeutic e f fec t i s established. However, f as t i ng blood glucose concentrations are decreased i n a dose-dependent fashion i n the dosage range between 250 mg t o 1,000 mg (47).

5.4 AbsorDtion

O r a l l y admin is tered acetohexamide i s almost completely absorbed (47). It i s reported t o appear i n the blood wi th in 30 minutes a f te r PO administration and peak levels occur a f t e r 3 t o 5 hours (43,44). Galloway et a7. (45) reported that, fo l lowing single PO doses o f 1 g o f acetohexamide, mean peak blood leve ls o f the drug t o be 47 mcg/ml and f o r hydroxyhexamide mean levels o f 60.3 mcg/ml were achieved. These peak leve ls occurred wi th in 1.5 t o 2 hours f o r the parent compound versus 2 t o 6 hours f o r t he a c t i v e metabo l i te , hydroxyhexami de.

5.5 Dis t r ibut ion

J u d i s ( 4 8 , 4 9 ) r e p o r t e d t h a t acetohexamide extensively binds t o plasma proteins t o the extent o f 65 t o 90%.


5.6 Hetabol ism

Acetohexamide is mainly metabolized by hydroxylation reactions in the liver to inactive and active metabolites. The primary metabolite (47 to 60%) is hydroxyhexamide (47,501. It is an active metabolite and is reported (45,50) to be excreted unchanged in the urine, as well as metabolized to the inactive dihydroxyhexamide (38).

Hydroxyhexamide, like acetohexamide, possesses both hypoglycemic and uricosuric properties (51,52), but it is 2.5 times as potent as its parent drug (36). Impairment of hydroxyhexamide’s el imination has been reported (51) to result in severe hypoglycemia.

Kojima et a7. (53) investigated the effect of various drugs on the i n v i v o metabolic reduction of acetohexamide. Most of the nonsteroidal anti- inflammatory drugs inhibited the acetohexamide reduction in liver, kidney and heart cytosol from rabbits. Ketone-containing drugs including warfarin also inhibited the reduction reaction in both the liver and the kidney; in the heart, acetohexamide reduction was inhibited only by warfarin.

Species differences in the in v i t r o metabolic reduction of acetohexamide were studied (54) in rabbit, guinea pig, hamster, rat and mouse. The rabbit exhibited the highest acetohexamide reductase activity in the cytosol of the liver and kidney among the species tested. The sensitivity to specific inhibitors of cytosolic acetohexamide reductase in the liver and kidney of the rabbit were different from those of the rat. Only rats and guinea pigs showed significant activity of acetohexamide reductase activity in the microsomes of the liver and kidney.

Nagamine et a7 (55) estimated the rates of available fraction for 4-acetamidoacetophenone, 4-acetylbenzene- sulfonamide, and acetohexamide and their respective reduced compounds, 4-substituted a-hydroxyethylphenyl derivatives, in rats. The study indicated that the compounds are in a reversible drug-metabolite relationship. The pharmacokinetic profiles o f the agents were studied after an intraportal administration

ACETOHEXAMIDE 37

i n comparison w i th those a f t e r I . V . admin is t ra t ion using an interconversion model.

5.7 Excretion

Acetohexamide and i t s me tabo l i t es a re main ly excre ted b y the k idneys. The u r i n a r y recovery o f r a d i o a c t i v i t y a f t e r t h e a d m i n i s t r a t i o n o f o r a l 14C-labeled acetohexamide averaged 71.6% i n 24 hours (45). Approximatley one-half t o two-third o f the drug was reported t o be excreted i n u r i ne as the ac t i ve metabolite, hydroxyhexamide (45,501. Fecal excretion o f rad ioact iv i ty fo l lowing ora l administration o f the drug i n one p a t i e n t was 15%. Even a f t e r 1 g I . V . dose ur inary recovery was on ly 85% (451 , suggesting t h a t b i l i a r y excret ion represents a secondary route o f e l iminat ion o f acetohexamide and/or i t s metabolites. However, more data are needed t o confirm the occurrence o f b l l i a r y excretion.

5.8 Hal f -L i fe

Fo l l ow ing o r a l a d m i n i s t r a t i o n o f 14C-labeled acetohexamide t o human subjects, a mean blood h a l f - l i f e o f t he drug o f 1.6 hours was determined, using isotope d i l u t i o n ana lys i s , w i t h a range o f 0 . 8 - 2 . 4 hours (45,56).

Fie ld et a l . (51) , however, reported a range o f 21-70 minutes averaging t o a value o f 55.8 minutes. The combined h a l f - l l f e o f the parent compound and i t s act ive metabolite, hydroxyhexamide, i s reported t o be 5.3 hours (43-45). The h a l f - l i f e o f acetohexamide i s reported be prolonged i n renal f a i l u r e (38) .

The ac t i ve metabol i te, hydroxyhexamide i s reported (45,561 t o have a mean h a l f - l i f e o f 5.3 hours with a range o f 3.7-6.4 hours. The average value of 5.3 hours agrees w i t h the f i n d i n g o f F i e l d e t a l . ( 5 1 ) who reported a range o f 3.2-7.6 hours.

The blood and ur ine data reported by Galloway et a l . (45) agree wi th those reported by Scheldon et a l . (46) and confirm the report by Smith et a l . (56) , tha t the combined half-1 i f e o f acetohexamide and hydroxyhexamide i s comparable wi th that o f tolbutamide.


ACKNOWLEGEMENT

The authors would l i k e t o thank M r . Tanvir A. Butt f o r typing t h i s manuscript.

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ACETOHEXAMIDE 39

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38. B.D. Cohen, J.A. Galloway, R.E. McMahon et a l , Am. J. Med. Sci., 254, 608 (1967).

39. W.M. Bennett, G.R. Aronoff, G. Morrison et a l , Am. J. Kidney Dis., 3, 155 (1983).

40. A.G. Gillman, L.S. Goodman and A. Gillman; Goodman and Gillman’s The Pharmacological Basis o f TheraDeutics, MacMillan & Co. New York. 1980.

41. H.E. Lebowitz and M.N. Feinglos, Diabetes Care, 1, 189

42. K.T. Augusti and P.A. Kurup, Indian J. Biochem., 6(1) ,

(1978).

36 (1969).

43. H.D. Breidahl, G.C. Ennis, F. I . Martin et a l , Drugs, 3,

44. H.D. Breldahl, G.C. Ennis, F.I. Martin et a l , M, 3,

79 (1972).

204 (1972).

45. J.A. Galloway, R.E. McMahon, H.W. Culp, F.J. Marshall and E.C. Young , Diabetes, 16, 118 (1967).

ACETOHEXAMIDE 41

46.

47.

48.

49.

50.

51.

52.

53.

54.

55.

56.

J. Scheldon, J. Anderson and L. Stoner, m, l4, 362 (1965).

R.E. Ferner and S. Chaplin, Clin. Pharmacokinet., 2, 379 (1987). J. Judis, J. Pharm. Sci., 6 l , 89 (1972).

J. Judis, w, 62, 232 (1973).

R . E . McMahon, F.J . Marshal l and H.W. Culp, J. Pharmacol. EXD. Ther., 149, 272 (1965).

J.B. Field, M. Ohta, C. Boyle e t a l , N. Ensl. J. Med., 277, 889 (1967).

T.F. Yu, L. Berger and A.B. Gutman, Metabolism, 17, 309 (1968).

Y. Kojima, Y. Imamura and M. Otagir i , Yakusaku Zasshi, 108(1), 66 (1988).

Y. Imamura, Y. Kojima and M. Otag i r i , Chem. Pharm. Bull., 36(1), 4199 (1988).

S. Nagamine, T . Otawa, H. Nakae and S. Asada, M, 36(11), 4612 (1988).

D.L. Smith, T.J. Vecchio and A.A. Forist, Metabolism, - 14, 229 (1965).

AMODIAQUINE HYDROCHLORIDE

1. 2. 2.1 2.2 2.3 3. 4. 4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11 4.12 5. 5.1 5.2 5.3

Iqbal Ahmad and Tauqir Ahmad

Department of Pharmaceutical Chemistry, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan.

K. Usmanghani

Department of Pharmacognosy, Faculty of Pharmacy, University of Karachi, Karachi-75270, Pakistan.

INTRODUCTION DESCRIPTION Name, Formula, Molecular Weight Appearance, Color, Odor and Taste Proprietary Names SYNTHESIS PHYSICAL PROPERTIES Melting Point Solubility Completeness of Solution Acidity Water Content Residue on Ignition Chromatographic Purity Ultraviolet Spectrum Infrared Spectrum Nuclear Magnetic Resonance Spectrum Mass Spectrum Complex Formation QUALITATIVE TESTS Identification Color Tests Field Test

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS - VOLUME 21

Copyright G 1992 by Academic Press, Inc All rights of reproduction reserved in any form 43

44

5.4 6. 6.1 6.2 6.3 6.4 7. 7.1 7.2 8.

1.

IQBAL AHMAD, TAUQIR AHMAD. AND K . USMANGHANI

Impurity Test for 4-(7-Chloro-4-quinolylamino) phenol Hydrochloride METHODS OF ANALYSIS Titrimetric Analysis Spectrophotometric Analysis Fluorometric Analysis Chromatographic Analysis METABOLISM AND PHARMACOKINETICS Metabolism Pharmacokinetics TOXICITY ACKNOWLEDGEMENT REFERENCES

INTRODUCTION

Amodiaquine is a congener of chloroquine and is employed for the treatment of overt malarial attacks and for suppression. Although it is more active than chloroquine both in vitro and in vivo against certain strains of Plasmodium f’cipamm with decreased sensitivity to chloroquine, amodiaquine is not recommended for routine use in the treatment of such infections (1). It appears that phenolic hydroxyl is essential to the activity of amodiaquine since the removal of this group depresses, and its methylation completely destroys antibacterial activity (2). Amodiaquine has been synthesized and patented under the name of Camoquin by Parke, Davis and Company in 1949 (3). It is used medicinally in the form of its dihydrochloride.

2. DESCRIPTION

2.1 Name, Formula, Molecular Weight

Amodiaquine hydrochloride is 4-(7-chloro-4-quinolylamino)-2- (diethylaminomethyl) phenol dihydrochloride dihydrate (4).

AMODIAQUINE HYDROCHLORIDE 45

CaH2zCIN30,2HC1, 2H20 = 464.8

The CAS registry No. is 6398-98-7.

Official monographs for amodiaquine hydrochloride are given in Argentinian (1966), British (1988), Brazilian (1977), Egyptian (1984), French (1982), Indian (1985), International (1981) and United States (1990) Pharmacopeias.

2.2 Appearance, Color, Odor and Taste

A yellow, odorless or almost odorless, crystalline powder with a bitter taste (5).

2.3 Proprietary Names

CAMAQI, Camoquin, Flavoquine, Miaquin (6,7).

3. SYNTHESIS

Burckhaiter et al. (8) synthesized amodiaquine (111) in 1948 by condensing 4,7-dichloroquinoline (I) with 4-amino-2-diethylamino- methylphenol (11) in dilute hydrochloric acid (Figure 1). In a later method (9), the alkylamino group was added as a last step.

46 IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI

C1 ,q + H2N a c H 2 N ( q H d 2

OH

c1

I

II

100". dilute 2 HCl hours ~ ,@2N(c2H5)2

OH 111

Figure 1. Synthesis of Amodiaquine

The free base was recrystallized from absolute ethanol and converted into the dihydrochloride by treating with hot concentrated hydrochloric acid.

4. PHYSICAL PROPERTIES

4.1 Melting Point

It melts at about 158OC (7).

4.2 Solubility

It is soluble in 22 parts of water and in 70 arts of ethanol (96%), practically insoluble in chloroform and ether &).


4.3 Completeness of Solution

A solution of 200 mg in 10 ml of water is clear (10).

4.4 Acidity

The pH of a 2.0% w/v solution is 3.6 to 4.6 (4).

4.5 Water Content

Not less than 7.0% and not more than 9.0% (10)

4.6 Residue on Ignition

Not more than 0.2% (10)

4.7 Chromatographic Purity

Chromatographic purity of amodiaquine hydrochloride can be examined on thin-layer plate coated with a 0.25 mm layer of silica gel G using solvent system chloroform (saturated with ammonium hydroxide): dehydrated alcohol (9: 1). Under short-wavelength ultraviolet light, the chromatogram shows principal spot at about the same Rf value, and no secondary spot, as obtained with the USP Amodiaquine Hydrochloride RS (10).

4.8 Ultraviolet (UV) Spectrum

The ultraviolet spectra of amodiaquine and amodiaquine hydrochloride have been reported by Sunshine (12) and Clarke (7) respectively. The ultraviolet absorption characteristics are used for the identification of these drugs (4,10,13). The absorption spectrum of amodiaquine hydrochloride as a function of pH in the range 1-11.8 shows a hypsochromic effect at 343 nm, a hyperchromic effect at 305 nm and the isosbestic point at 323 rim (14). The effect of solvents and substitution on the ultraviolet spectra of amodiaquine has been studied and the changes of absorption bands E,K, and B discussed in detail (15).

The ultraviolet spectrum of amodiaquine hydrochloride in 0.1 M hydrochloric acid was recorded on a Shimadzu 240 UV-Visible spectrophotometer and is shown in Figure 2. The uv spectral data reported for amodiaquine and amodiaquine hydrochloride are listed in Table 1.

48 IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANGHANI

1.50 ! r 1 1

A

WAVELENGTH Cnm’l 3 . 0

Figure 2. Ultravlofet Spectrum of Amodluquinc Hydrochloride InO.l M HCI

AMODIAQUINE HYDKOCHLORIDE 49

Table 1

UV Spectral Data for Amodiaquine and Amodiaquine Hydrochloride

Compound Solvent Amax, nm A (l%, Icm) Molar Ref. Absorptivity

Amodiaquine 0.1 M HCl

Amodiaquine Water hydrochloride

0.1 M HCI 0.1 M HCl

Aq. acid

Aq. alkali

0.1 M HCl

283 237 247 224 342

343 223 237 343 237 343 273 287 223 237 342.5

890 530 470

394- 410 366

366 600

836 489 369

41370 24630 21850

18310- 19060 17010

17010 27890

38850 22710 17160

12

6

4 11

7

7

*Values determined by the authors.

4.9 Infrared (IR) Spectrum

The infrared spectrum of amodiaquine has been determined in KBr disc (4). The principal peaks in the infrared spectrum of amodiaquine hydrochloride (KBr disc) are reported at 1565, 815, 1535, 1255, 869, 847 cm" (7). Attenuated total reflectance infrared spectrum is used to detect amodiaquine hydrochloride in the solid state as a layer of crystals on adhesive tape. The method has been applied to the identification of the drug in tablet formulations. Common excipients such as starch, and lactose (absorption in the 1000 to 1200 cm-' region) do not interfere with the method (16).

The infrared spectrum of amodiaquine hydrochloride as KBr disc was obtained with a Shimadzu IR 460 Infrared spectrophotometer and is shown in Figure 3. The assignments for characteristic bands are given in Table 11.

0

0

0

0

0

0

0

m

0

0

0

0

*

I 1

I I

I 0

0

0

0

ID

a

0

0

0

L

, 5.a 4.0 5 o 6 0 7.0 8 . g 5.0 10 0 15.0 z o o . . I I 1 I I I I 1 I

2000 I

3000 I

000 ' I 00" I ' 1 5 0 0

Wave number (em-')

: o o . o

8 0 . 0

6 0 - 0

40 0

20.0

0 . 0 I 500

Figure 3. Infrared Spectrum of Amodiaquine Hydrochloride (KBr disc).


Table I1

IR Spectral Assignments for Amodiaquine Hydrochloride

Frequency, cm- 1 Assignment

3420 3 170 1615 1585,1540,1505

1448 1265,1207 1095 852,840

- NH stretching - OH stretching

C = C stretching (aromatic) C = C, C = N stretching in disubstituted quinoline

C-OH stretching (aromatic) C-Cl stretching (aromatic) isolated CH deformation in disubstituted quinoline

- CH2-N- deformation

4.10 Nuclear Magnetic Resonance (NMR) Spectrum

The 'H-NMR and 13C-NMR spectra of amodiaquine hydrochloride in DMSO-d6 were determined at 300 MHz and 75.4 MHz respectively on a Bruker AM-300 NMR spectrometer using tetramethylsilane as reference standard. The 'H-NMR determinations included spin decoupling experiments, 2D J-resolved and COSY-45 measurements (Figure 4-6). The 13C-NMR spectra comprised DEFT and hetero-nuclear (C-H correlation) measurements (Figure 7-9). The spectral assignments are listed in Table 111.

7 11-1'

1 1 0 I 0

L

..,. ,... 9 0 d , .. 8 0 ,

h

.I'

I , . . ::. , . . 1 0 P O

.. .. : . . .. -* . .

. ... -,r PPM 4 PTL

F l p r c I. Homonuclear Chemlul Shin Cormlaled tCoS)-Ul ~ I I - N S I R Spectrum or Amodisqulnc 1i)dmchloridc

w’ e

I

1 I ,

L

I

‘ I

I .

1 1 1 .. a L - - - “LPr2

1 0

* ‘Iv, h? , , , , , , , , , , , ( 1 ” ’ , ’ , , , , , , , , r* ~ , , , , , , , , ( , , , , , , , , , ,

3 . r- B I O 7 . 0 6 , O 5 0 4 .0 I 0 2 0 P A I

. . , . . . . - , ._.. I ! 1

b u r r 6. Homonuclcar 211 J-Rcsolvcd N M R Spcclrum of Amodiaquinc It)drorhloridr..

CH: CHdCH2

14 1 C.ll..ll

C.5

I I C I . cd‘ l,,c.3tii C.Y

C.2’

I I

-I------

1 A L

C 4

111 1

Figure R. 75 M l l r "c-NMR Off Resonance Decoupled Spectrum ofAmodiaquine Hydrofhloride

-==i I

7 '1 -2

I

L . ' 4

i

t '

t t

58 IQBAL AHMAD. TAUQIR AHMAD. AND K. USMANGHANI

Table I11

‘H- and 13C-NMR Chemical shifts and Coupling Constants for Amodiaquine Hydrochloride

‘H-NMR coupling ”C-NMR Chemical shift Proton constant Chemicalshift Carbon

(PP4 (J in Hz) ( P P d

7.67 (1H,d)

7.35 (lH,dd) 4.22 (lH,d) 4.22 (lH,s) 3.09 (4H,q) 1.27 (6H,t)

8.44 (lH,d) 6.82 (1H’d)

8.% (lH,d) 7.78 (lH,dd)

8.17 (lH,d)

c“ 10.89

3

5 6 7 9’10 11’12

2’ 3’

5’ 6‘

8’

NH/OH

2 5

8.6’2.5 8.6

6.7 1.2

7.0 7.0

9.1 9.1’2.1

2.1

156.04 138.87 l30.00 115.60 128.30 116.85 49.11 46.19 8.41

142.91 100.41 117.54 126.19 127.04 138.15 118.97 154.85 127.92

1 2 3 4 5 6 7 9’10 11’12

2’ 3’ 4’ 5’ 6‘ T 8’ 9’

10’

4.11 Mass Spectrum

The electron impact ionization spectrum of amodiaquine hydrochloride obtained at 70 eV using a solid probe insertion is shown in Figure 10. The spectrum was run on a Finnigan Mat 112s double focusing mass spectrometer connected to a PDP 11/34 (DEC) computer system. It shows a molecular ion peak M+ at m/z 355. Since the molecule contains one chlorine atom, M+2 peak appears at m/z 357. The proposed fragmentation pattern and prominent ions are given in Table IV.

Figure 10. Electron Impact-Mass Spectrum of Amodiaquine Hydrochloride

60 IQBAL AHMAD. TAUQIR AHMAD. AND K. USMANGHANI

Table 4

Proposed Fragmentation Pattern of Amodiaquine Hydrochloride

d z Relative intensity % Ion

355, 357 55.77, 16.83

283

282

253

179

177

43.05

99.00

43.56

8.18

5.81

NH

@CH2 OH

@CH OH c'w -

I-

AMODIAQUINE HYDROCHLORIDE

4.12 Complex Formation

61

Amodiaquine hydrochloride forms 1:l and 1:2 complexes with ferrous sulphate. The infrared spectra indicate that amodiaquine hydrochloride is bonded to iron via N and 0 and that water molecules are coordinated to iron (17). It forms a 1:2 complex with silver nitrate in alcoholic solutions. The average stability constant, log K, for the complex is 7.7 and A E is about 10.8 kcal/mol. (18).

and Fast Green FCF or Orange I1 dye has been reported (19). The formation of 1:l ion association complex between amodiaquine

5. QUALITATIVE TESTS

5.1 Identification (4)

5.1.1 Dissolve 0.1 g of amodiaquine hydrochloride in 10 ml of water and add 2 ml of 2 M sodium hydroxide. Extract with two 20 ml quantities of chloroform, wash the combined chloroform extracts with 5 ml of water, dry with anhydrous sodium sulphate and evaporate to dryness. Dissolve the residue in 2 ml of chloroform. The infrared absorption spectrum of the resulting solution is concordant with the reference spectrum of amodiaquine.

5.1.2 The light absorption in the range 240 to 360 nm of a 0.003% w/v solution of amodiaquine hydrochloride in 0.1 M hydrochloric acid exhibits a maximum only at 343 nm. The absorbance at 343 nm is about 1.1.

5.1.3 To 1 ml of a 2% w/v solution of amodiaquine hydrochloride add 0.5 ml of cobalt thiocyanate reagent. A green precipitate is produced.

5.1.4 Amodiaquine hydrochloride yields the reactions characteristic of chlorides.

The identification tests of amodiaquine hydrochloride based on comparison of infrared and ultraviolet absorption spectra, and reactions of chloride are reported in USP (10).

5.2 Color Tests (7)

Amodiaquine hydrochloride gives a blue color with Folin-Ciocalteu reagent. The Liebermann’s test yields a black color. An orange color is

62 IQBAL AHMAD. TAUQIR AHMAD, AND K. USMANGHANI

produced when amodiaquine hydrochloride is treated with Millon's reagent.

5.3 Field Test (20)

Amodiaquine base is extracted from urine into amyl acetate immediately after alkalinization. The addition of bromophenol blue in 5% boric acid to the organic phase causes a green to blue coloring, depending on the concentration of the drug. The sensitivity of the test is 0.8 mg%.

5.4 Impurity Test for 4-(7-Chloro-4-quinolylamino) phenol Hydrochloride

Carry out thin-layer chromatography, using silica gel G as the coating substance, spread in a layer about 0.5 mm thick, and a solvent system chloroform: butan-2-one: diethylamine (50:40: 10). Apply separately to the chromatoplate 5 pl of each of two solutions in methanol containing (1) 10.0% w/v of the substance being examined and (2) 10.0% w/v of amodiaquine hydrochloride BPCRS and 0.020% w/v of 4-(7-chloro- 4-quinolylamino) phenol hydrochloride BPCRS. After development remove the plate, heat it at 105' for 10 minutes, spray with a freshly prepared mixture of equal volumes of a 10% w/v solution of iron (111) chloride and a 1% w/v solution of potassium hexacyanoferrate (111) and examine immediately. Any spot corresponding to 4-(7-chloro-4- quinolylamino) phenol in the chromatogram obtained with solution (1) is not more intense than the spot with lower Rfvalue in the chromatogram as obtained with solution (2).

(4)-


6.1 Titrimetric Analysis

6.1.1 Nonaqueous titration

The BP method (4) for the assay of amodiaquine hydrochloride as pure drug and in dosage forms is based on nonaqueous titration. A 0.2 g quantity of amodiaquine hydrochloride is dissolved in a suitable volume of anhydrous glacial acetic acid, 7 ml of mercury (11) acetate solution is added and the solution titrated with 0.1 M perchloric acid to a green end point using 1-naphtholbenzoin solution as indicator. In dosage forms, a


quantity of the powdered material equivalent to about 0.2 g of amodiaquine hydrochloride is dissolved in 30 ml of water and 5 ml of 2 M sodium hydroxide is added. Amodiaquine base is extracted with three 30 ml quantities of chloroform, the combined chloroform extracts are washed with 10 ml of water and evaporated to a volume of about 10 ml. To the chloroform extracts, 40 ml of anhydrous glacial acetic acid is added and the solution titrated with 0.1 M perchloric acid using 1-naphtholbenzoin solution as indicator. Each ml of 0.1 M perchloric acid is equivalent to 0.02144 g of CaHzCIN30,2HCl.

Wu et d. (21) have described a simple, rapid and accurate method for the nonaqueous titration of amodiaquine in dosage forms. A powdered sample of 5 milliequivalent weight is dissolved in 7 ml of N hydrochloric acid, made alkaline with 3 ml of 6 N sodium hydroxide, shaken with 30 ml of chloroform for 10 minutes and with 1 g of tragacanth for another 2 minutes, filtered through adsorbent cotton, and titrated (20 ml) with 0.1 N acetic perchloric acid to blue or green end point using crystal violet solution as indicator. For pure chemicals, the digestion with acid and alkali could be omitted. The results agree with those obtained by the official method.

6.1.2 Titration with brominating agents

Amodiaquine can be determined in bulk and in dosage forms by a titrimetric method based on reaction with 1,3-dibromo-5,5- dimethylhydantoin or N-bromosuccinimide as the titrant. The mixture is later treated with potassium iodide solution and the liberated iodine titrated with sodium thiosulphate solution. The recovery is about 100% (22).

A method for the determination of amodiaquine hydrochloride in tablets by titration with N-bromosuccinimide has been developed (23). The sample is dissolved in water, treated with an acetic acid solution of the reagent and mixed with potassium iodide. The iodine released is titrated with sodium thiosulphate solution. The relative standard deviation for the titration is 2.12% and the recovery is 99.4 - 101.0%.

6.1.3 Titration with vanadium (V)

The determination of amodiaquine hydrochloride by oxidation with ammonium metavanadate solution and back titration of the unconsumed reagent with acidic iron (11) ammonium sulphate solution, using

64 IQBAL AHMAD, TAUQlR AHMAD. AVD K. USMANGHANI

N-phenylanthranilic acid as indicator has been reported (24). The recovery of amodiaquine hydrochloride in the pure form and in pharmaceutical preparations is 99.83% (standard deviation 0.49%) and 99.69% (standard deviation 0.78%) respectively. The method is of general applicability and is quick and simple compared with the official methods.

6.2 Spectrophotometric Analysis

6.2.1 Ultraviolet spectrophotometry

The USP assay (10) of amodiaquine hydrochloride in pure form and in tablets involves ultraviolet spectrophotometric determination. A quantity of the drug equivalent to about 300 mg is dissolved in dilute hydrochloric acid (1:lOO) to obtain a concentration of about 15 pg/ml. The absorbance of this solution, along with a solution of undried USP Amodiaquine Hydrochloride RS in the same medium having a known concentration of about 15 pg/ml, is determined at 342 nm using dilute hydrochloric acid (1:lOO) as the blank. The quantity, in mg, of CmH22CIN30, 2HC1 in the portion of amodiaquine hydrochloride taken is calculated by the formula 20C (Ad&), in which C is the concentration, in pg/ml, calculated on the anhydrous basis, of USP Amodiaquine Hydrochloride RS in the standard solution and AU and As are the absorbances of the solution of amodiaquine hydrochloride and the standard solution respectively. The same method is applied to the assay of amodiaquine hydrochloride in tablets after extraction of the base into chloroform and then re-extraction with dilute hydrochloric acid (1:lOO).

Amodiaquine and primaquine can be quantitatively separated by selective precipitation with 4 N ammonium hydroxide, followed by determination of the two compounds at 342 and 282 nm respectively. The method is valid upto primaquine - amodiaquine ratio of 1:40. Recoveries of 98.30 - 100.11% have been reported (25). The presence of higher amounts of amodiaquine yields low results in respect of primaquine as on precipitation with ammonium hydroxide, the primaquine is trapped into the precipitate of amodiaquine (26).

Hassan et al. (27) have developed a method for the simultaneous determination of amodiaquine - primaquine mixtures in dosage forms. The drugs are extracted with 0.1 N hydrochloric acid and absorbance of the mixture is measured at 342 and 282 nm. The concentration of each compound is calculated by solving two simultaneous equations. Excellent

AMODlAQUlNE HYDROCHLORIDE 65

recoveries from authentic samples are obtained and the method is suitable for routine analysis.

6.2.2 Colorimetry

Amodiaquine hydrochloride is determined colorimetrically by complex formation, in aqueous solution, with bromophenol blue, bromocresol green, bromocresol purple, and methyl orange, respectively. The complex with bromophenol blue has the highest molar absorptivity. Recoveries are more than 98.6% for all complexes, and the absorbance is linear with concentration in the range 1-11 pglml. The absorption maxima for the complexes occur at 420 nm except for the bromocresol purple complex which exhibits maximum at 415 nm. The various complexes are extracted with chloroform and absorbance is measured at the respective maxima for quantitative determination (28).

A simple, sensitive, and selective method for the determination of amodiaquine hydrochloride in tablets has been developed. It is based on a color reaction with chloramine-T in the pH range 7.4- 8.0. The chromogen is extracted with chloroform and the absorbance is measured at 442 nm. Beer’s law is obeyed in the concentration range 1-200 p g / l . The coefficient of variation has been found to be 0.64% and the recovery ranges between 100.3 and 102.5%. Chloroquine phosphate or primaquine phosphate do not interfere with the method (29).

Amodiaquine reacts with cobalt and thiocyanate to yield stable ternary complexes. These complexes are readily extractable in nitrobenzene to give a greenish-blue color with maximum absorption at 625 nm that can be used for quantitative determination. The mean recoveries for authentic samples of amodiaquine hydrochloride are 100.81 & 1.77% (p = 0.05). Alternatively, determination of the cobalt content of nitrobenzene extract by atomic absorption spectroscopy provides an indirect method for the determination of the drug with a mean recovery of 99.99 2 2.16%. Both the methods have been successfully applied to the assay of the drug in pharmaceutical preparations (30).

A colorimetric method for the determination of amodiaquine in tablets or powders has been reported (31). The drug is dissolved in 0.1 N hydrochloric acid, treated with acidic ammonium reineckate, the precipitate dissolved in acetone, and the absorbance measured at 525 nm. The results compare favourably with those obtained by the official methods.

66 IQBAL AHMAD. TAUQlK AHMAD. AND K. USMANGHANI

Amodiaquine hydrochloride has been determined in tablets by dissolving it in water and treating with an acetic acid solution of N-bromosuccinimide. An orange-yellow color is produced, whose absorbance is measured at 450 nm. Beer’s law is obeyed in the concentration range 15-160 pglml . The relative standard deviation for the method is 1.44%, and the recovery is 99.7-100.9% (23).

Amodiaquine hydrochloride tablets have been assayed by a method based on the reaction of the drug with 2,3-dichloro-S, 6-dicyano- p-benzoquinone and measurement of the absorbance at 460 nm. The color attains its maximum intensity after five minutes and remains stable for at least one hour. Beer’s law is valid in the concentration range 1-4 mg/100 ml, and the recovery is 99.9-102.6% (32). Another colorimetric method for the determination of amodiaquine in tablets depends on its reaction with chloranilic acid in aqueous solution and measurement of the absorbance at 522 nm. The absorbance is linear over the concentration range 0.04 -0.20 mdml, and the recovery is 99.9-101.3% (33).

A simple, rapid and sensitive method for the colorimetric determination of amodiaquine in bulk and in pharmaceutical preparations has been reported by Sastry et al. (34). It is based on the reaction of amodiaquine with potassium dichromate at pH 1.1 in the presence of sulphanilamide, and measurement of the absorbance of resulting solution at 510 nm. The color is stable for twenty-four hours. Beer’s law is obeyed in the concentration range 20-120pg/ml. The relative standard deviation of the method is 0.94%, and the recovery is 99.0-101.0%. Chloroquine present even in ten-fold excess does not interfere with the determination.

A highly sensitive method is based on the complexation of amodiaquine with ammonium molybdate. The bound molybdenum is converted into its thiocyanate, reduced, and the absorbance of the colored solution measured at 465 nm. The Beer’s law limits, molar absorptivity and Sandell’s sensitivity for the amodiaquine complex are 50-300 pg/25 ml, 1.75 x lo4 M1 cm-l and 0.026 &cm2 / 0.001 absorbance unit, respectively. Recovery ranges from 98-101%. The color obtained is stable for twenty-four hours and common excipients do not interfere with the method (35).

Amodiaquine forms a colored ion association complex with Fast Green FCF or Orange I1 dye. The stoichiometric ratio of the drug-dye complex has been shown to be 1:l. The method can be applied to the assay of amodiaquine in bulk and in pharmaceutical preparations. Sulphur


containing drugs do not interfere with the determination (19).

6.3 Fluorometric Analysis

A fluorometric method for the determination of amodiaquine in serum, plasma, or red cells has been reported (36). Amodiaquine is extracted from alkalinized biological fluids, buffered, and heated to produce a species with marked increase in fluorescence, which could be measured. Standard curves prepared in serum and red cells are linear between 50 and 3000 pgll. Reproducibility of the assay and recovery of amodiaquine from serum and red cells is satisfactory. The specificity of the assay and the nature of the induced fluorophor are not known.

6.4 Chromatographic Analysis

6.4.1 Thin-layer chromatography (TLC)

Amodiaquine can be separated and identified on silica gel G plates using a number of solvent systems. The spots are visualized under short-wavelength ultraviolet light or by spraying with acidified iodoplatinate solution. The following Rr values (Table V) have been reported (37).

Table V

Solvent Systems for TLC of Amodiaquine

Adsorbent Solvent system Rr

Silica gel GFw dipped Methanol: ammonia 0.62 in 0.1 M KOH and dried

Cyclohexane : toluene : 0.08 diethylamine (751510) Cbloroform:methanol 0.40 ( 9 1) Acetone 0.37

(1rn1.5)

The application of principal components analysis to the TLC behaviour of a large number of basic drugs including amodiaquine has been studied (38). A two-component model explains 77% of the total variance in four

68 IQBAL AHMAD, TAUQIR AHMAD. AND K. USMANCHANI

eluting mixtures. For the identification of unknowns, the method provides a drastic reduction of the range of possibilities to a few drugs.

6.4.2 High-performance liquid chromatography (HPLC)

A variety of HPLC packing materials have been prepared and their chromatographic properties evaluated for separating amodiaquine and other basic drugs using a single mobile phase. The three most promising packing materials are silica, a mercapto Pr modified silica and a Pr sulfonic acid modification (39).

A simple and precise HPLC assay for quantitating amodiaquine in tablets and biological fluids involves acid extraction of the drug from tablets and chloroform extraction of its base from the biological fluids after treatment with ammonia. A p-Bondapak Ph column is employed for separation with a mobile phase comprising methanol : water: acetic acid (25:25:1) (pH 2.3), using quinidine as the internal standard. The mean recovery of the drug from tablets is 102.03%, while in the biological fluids, it ranges from 85.2 to 104.6%. Interference from tablet excipients or biological fluids is negligible (40).

A column liquid chromatographic method for the simultaneous determination of chloroquine, amodiaquine and their monodesethyl metabolites in human plasma, red blood cells, whole blood and urine has been developed (41). The drugs and internal standards are extracted as bases with dichloromethane and then re-extracted into an acidic aqueous phase. Separation is achieved using a reversed-phase column and a mobile phase of phosphate buffer (pH 3.0) : methyl cyanide (88:12). The absorbance of the drugs is monitored at 340 nm with a sensitivity limit of 10 pmoVml. The mean overall recovery from each biological fluid is more than 75%. This method can be applied to therapeutic, pharmacokinetic, and epidemological studies.

7. METABOLISM AND PHARMACOKINETICS

7.1 Metabolism

Churchill et al. (42) have isolated four metabolites of amodiaquine in humans using a reversed-phase HPLC method. The two major metabolites have been identified as desethylamodiaquine and 2- hydroxy-


desethylamodiaquine. The importance of these metabolites in the antimalarial effect of amodiquine in humans and on the in vitro sensitivity of persons dosed with amodiaquine is discussed. 2-Hydroxy- desethylamodiaquine has been isolated from urine and characterised by HPLC and NMR spectroscopy. The presence of three additional metabolites of this drug in humans has been suggested and chromatographic confirmation for one of these obtained. The in vitro activity of 2-hydroxydesethylamodiaquine is shown to be 1% that of amodiaquine for two chloroquine sensitive Plasmodium fdcipamm strains (43). The metabolism of 2-amino-4- quinoline derivatives of chloroquine and amodiaquine in humans has been compared by Pussard et al. (41).

7.2 Pharmacokinetics

Amodiaquine hydrochloride is readily absorbed from gastro-intestinal tract after oral administration, and higher concentrations occur in erythrocytes, kidney, liver, lungs and spleen than in the plasma. After absorption it is slowly released into the blood and excreted in the urine for at least seven days after a single dose. The rate of excretion is increased in acid urine (5,7). Amodiaquine is altered rapidly in vivo to yield products which appear to be excreted slowly, and thus have a prolonged suppressive activity (44).

Following a single oral dose of 10 mgkg of amodiaquine to five human subjects, serum concentrations of 0.30 to 0.68 pg/ml (mean 0.5) have been reported after four hours; the ratio of erythrocyte to serum concentration varies with time and between individuals, but erythrocyte concentrations are generally higher than the serum concentrations after forty-eight hours (36).

The metabolic transformation of amodiaquine to monodesethyl- amodiaquine, and its pharmacokinetics in humans have been reported (41).

8. TOXICITY

Amodiaquine hydrochloride is an antimalarial of low toxicity and is three to four times as active as quinine as a suppressive drug against Plasmodium vivav and Plasmodium fakiparum infections (44,45,46). Jn therapeutic doses amodiaquine hydrochloride is generally well tolerated but may occasionally give rise to side-effects, including nausea, vomiting,

70 IQBAL AHMAD. TAUQIR AHMAD. AND K . USMANCHANI

diarrhoea, insomnia, vertigo, and lethargy (5).

The prolonged use of amodiaquine hydrochloride in the dosages necessary to treat lupus erythematosus and rheumatoid arthritis is not recommended, for corneal opacities and retinopathy, peripheral neuropathy, fatal blood dyscrasias, and fatal hepatitis have been reported after these large dosages (47). Patients have experienced involuntary movements, usually with speech difficulty, after large but not excessive doses of amodiaquine (48). It may cause birth defects if taken during pregnancy (49).

A method is described for evaluating the relative toxicity of amodiaquine in rats on the basis of effect on growth, lethal effects, production of pathological changes, and the concentration of drug in blood or plasma. The test can be completed in fourteen days (50).

ACKNOWLEDGEMENT

The authors wish to thank the United States Pharmacopeial Convention, Inc., for donating a sample of amodiaquine hydrochloride.

REFERENCES

1.

2.

3. 4.

5.

6.

7.

8.

Webster, L.T., Jr. (1985). In "Goodman and Gilman's The Pharmacological Basis of Therapeutics", 7th Edition (A.G. Gilman, L.S. Goodman, T.W. Rall and F. Murad, eds.), p. 1032, MacMillan Publishing Co., New York. Dyson, G.M. (1959). "May's Chemistry of Synthetic Drugs", 5th Edition, p. 538, Longmans, Green and Co., London. U.S. Patents (1949). 2,474,819; 2,474,821. "British Pharmacopoeia" (1988). pp. 37, 900, Her Majesty's Stationary Office, London. "Martindale, The Extra Pharmacopoeia" (1989). 29th Edition (J. E.F. Reynolds, ed.), p. 507, The Pharmaceutical Press, London. 'The Merck Index" (1983). 10th Edition (M. Windholz, ed.), p. 82, Merck and Co., Inc., Rahway, New Jersey. "Clarke's Isolation and Identification of Drugs" (1986). 2nd Edition (A.C. Moffat, ed.), p. 347, The Pharmaceutical Press, London. Burckhalter, J.H., Tendick, F.H., Jones, E.M., Jones, P.A., Holcomb, W.F. and Rawlins, A.L. (1948). J. Am. Chem. SOC. 70,1363.


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Burckhalter, J.H., DeWald, H.A., and Tendick, F.H. (1950). J. Am. Chem. SOC. 72, 1024. 'The United States Pharmacopeia" (1990). 22nd Revision 'The National Formulary" 17th Edition, p. 79, United States Pharmacopeial Convention, Inc., Rockville, Md. 'The Pharmaceutical Codex" (1979). 11th Edition, p. 36, The Pharmaceutical Press, London. Sunshine, I. (1981). "Handbook of Spectrophotometric Data of Drugs", p. 24, CRC Press, Inc., Boca Raton, Florida. Brandstaetter-Kuhnert, M., Kofler, A, Hoffmann, R. and Rhi, H.C. (1965). Sci. Pharm. 33,205. Cesaire, G., Fauran, F., Pellisier, C., Goudote, J. and Mondain, J. (1969). Bull. Mem. Fac. Mixte Med. Pharm. Dakar 17, 240; C.A. (1973) 79,97038k. Kracmar, J. and Kracmarova, J. (1974). Pharmazie 29,510. Kang, I.P.S., Kendall, C.E. and Lee, R.W. (1974). J. Pharm. Pharmacol. 26,201. Gupta, S.S., Siddique, S. and Kaushal, R. (1980). J. Indian Chem. SOC. 57,97. Gupta, S.S., Gupta, KK. and Kaushal, R. (1979). J.Sci. Res. (Bhopal, India) 1, 125. Sastry, B.S., Rao, E.V., Tumuru , M.K. and Sastry, C.S.P. (1986). Indian Drugs 24,105. Fuhrmann, G. and Werrbach, K. (1966). Tropenmed. Parasitol. 16, 269; C.A. (1966) 65,12725a. Wu, T.S., Sun, C.C. and Tang, T.H. (1958). Yao Hsueh Hsueh Pao 6, 253; C.A. (1959) 53,20691d. Walash, M.I., Rizk, M., Abou-Ouf, A.A. and Belal, F. (1983). Anal. Lett. 16, 129. Sastry, B.S., Rao, E.V. and Sastry, C.S.P. (1985). Indian Drugs 22, 550. Ahmad, S.J. and Shukla, LC. (1984). Analyst (London) 109,1103. Chatterjee, P.K., Jain, C.L. and Sethi, P.D. (1986). Indian Drugs 23, 563. Sethi, P.D. (1985). "Quantitative Analysis of Drugs in Pharmaceutical Formulations", p. 193, Unique Publishers, Delhi. Hassan, S.M., Metwally, M.E.S. and Abou-Ouf, A.A. (1983). J. Assoc. Off. Anal. Chem. 66,1433. Sane, R.T., Thombare, C.H., Anaokar, P.G. and Pandit, A.D. (1981). Indian J. Pharm. Sci. 43,22.

IQBAL AHMAD. TAUQIR A H M A D . A N D K. USMANGHANI 12

29.

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Rao, G.R., Rao, Y.P. and Raju, I.R.K. (1982). Analyst (London) 107, 776. Hassan, S.M., Metwally, M.E.S. and Abou-Ouf, A.A. (1982). Analyst (London) 107,1235. Dalal, R.R., Bulbule, M.V., Wadodkar, S.G. and Kasture, A.V. (1982). Indian Drugs 19,361. Issa, A.S., Mahrous, M.S., Salam, M.A. and Hamid, M.A. (1985). J. Pharm. Belg. 40,339. Mahrous, M.S., Salam, M A , Issa, A.S. and Hamid, M.A. (1986). Talanta 33, 185. Sastry, B.S., Rao, E.V. and Sastry, C.S.P. (1984). Indian J. Pharm. Sci. 46,186. Sastry, B.S., Rao, E.V. and Sastry, C.S.P. (1986). Indian J. Pharm. Sci. 48,71. Trenholme, G.M., Williams, R.L., Patterson, E.C., Frischer, H., Carson, P.E. and Rieckmann, K.H. (1974). Bull. Wld. Hlth. Org. 51, 431. Stead, A.H., Gill, R., Wright, T., Gibbs, J.P. and Moffat, A.C. (1982). Analyst (London) 107,1106. Musumarra, G., Scarlata, G., Romano, G., Clemente, S. and Wold, S. (1984). J. Chromatogr. Sci. 22,538. Wheals, B.B. (1980). J. Chromatogr. 187,65. Molokhia, A.M., El-Hoofy, S. and Dardiri, M. (1987). J. Liq. Chromatogr. 10,1203. Pussard, E., Verdier, F. and Blayo, M.C. (1986). J. Chromatogr. 374, 111. Churchill, F.C., Patchen, L.C., Campbell, C.C., Schwertz, I.K., Dinh, P.N. and Dickinson, C.M. (1985). Life Sci. 36,53. Churchill, F.C., Mount, D.L., Patchen, L.C. and Bjoerkman, A. (1986). J. Chromatogr. 377,307. White, A.I. (1977). In 'Textbook of Organic Medicinal and Pharmaceutical Chemistry", 7th Edition (C.O. Wilson, 0. Gisvold and R.F. Doerge, eds.), p. 258, J.B. Lippincott Co., Philadelphia. Jenkins, G.L., Hartung, W.H., Hamlin, ICE., Jr. and Data, J.B. (1957). 'The Chemistry of Organic Medicinal Products", p. 361, John Wiley and Sons, Inc., New York. Atherden, L.M. (1969). "Bentley and Driver's Textbook of Pharmaceutical Chemistry", 8th Edition, p. 638, Oxford University Press, London. "New Drugs" (1966). p. 81, American Medical Association, Chicago.

AMODlAQUlNE HYDROCHLORIDE 73

48. 49.

Akindele, M.O. and Odejide, A.O. (1976). Br. Med. J. 2,214. 'The Physicians' and Pharmacists' Guide to Your Medicines", p. 18, United States Pharmacopeial Convention, Ballantine Books, New York. Smith, C.C. (1950). J. Pharmacol. Exptl. Therap. 100,408. 50.

CLOFAZIMINE

Caitriona M . O’Driscoll and Owen I . Corrigan

University of Dublin

Department of Pharmaceutics

School of Pharmacy

Trinity College, Dublin, Ireland

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS - VOLUME 21 75

Copyright 0 1992 by Academic Press, Inc All rights of reproduction reserved in any form

76 CAITRIONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

CLOFAZIMINE

Caitriona M. ODriscoll and Owen I. Corrigan

University of Dublin, Department of Pharmaceutics, School of Pharmacy, Trinity College Dublin, Ireland.

1. Introduction 2. Description

2.1 Structural and Molecular Formulas and Molecular Weight

2.2 Nomenclature 2.3 Official Compendia 2.4 Other Compendia

3. Synthesis 4, Physical Properties

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.10 4.11

Ultraiiolet Absorbance Spectrum Infrared Absorbance Spectrum Mass Spectrum Proton Nuclear Magnetic Resonance Spectrum Carbon-13 Nuclear Magnetic Resonance Spectrum X-Ray Diffraction Melting Point Differential Scanning Calorimetry Dissociation Constants Solubilities Par ti tion Coefficients

5. Methods of Analysis 5.1 Elemental Analysis 5.2 Identification 5.3 Ultraviolet and Visible Spectrophotometry 5.4 Spectrofluorometric Analysis

CLOFAZIMINE

5.5 Thin Layer Chromatography 5.6 High Pressure Liquid Chromatography.

6.1 Bioavailability Considerations 6.2 Distribution, Metabolism and Elimination

7.1 Mechanisms of Action 7.2 Structure - Activity Relationships 7.3 Toxicity 7.4 Dose Schedules Acknowledgements References

6. Pharmacokinetics

7. Pharmacology

1. INTRODUCTION

Clofazimine is active against Mycobacterium leprae and is used clinically to treat leprosy (Hansen's disease). It was synthesised in 1957 by Barry et al., Laboratories of the Medical Research Council of Ireland, Trinity College Dublin. The precise mechanism of the antileprotic action of clofazimine has not been established. The World Health Organisation classify clofazimine as an "essential drug" and recommend its use, in combination, with other agents to treat all cases of leprosy (WHO, 1982).

Clofazimine is also used to treat Mycobacteriurn avium infections which frequently occur in patients with AIDS (acquired immunodeficiency syndrome), (Masur et al., 1987; Woods and Washington, 1987; Gangadharam et al., 1988; Lindholm - Levy and Heifets, 1988; Young, 1988).

clinically useful in controlling erythema nodosum leprosum (ENL) reactions which occur in multibacillary forms of leprosy (Gidoh and Tsutsumi, 1979; Yawalkar and Vischer, 1979; Browne et al., 1981). A study using animal models of rheumatoid arthritis has indicated that clofazimine may be potentially useful to treat this disease (Currey and Fowler, 1972). Although the exact mechanism of clofazimine mediated anti-inflammatory activity is unknown, it may be related to the ability of the drug to increase

Clofazimine also displays anti inflammatory activity which is

78 CAITRIONA M. O’DRISCOU AND OWEN 1. CORRlGAN

the synthesis of anti-inflammatory immunosuppressive prostaglandin E2 (PGE2) by human polymorphonuclear leucocytes (Anderson, 1985; Zeis et al., 1987; Yawalkar, 1988).

2. DESCRIPTION

Clofazimine is a dark red or orange - red fine powder, odourless or almost odourless.

2.1 Structural and Molecular Formulas and Molecular Weight

Q CI

Molecular Formula: C27H22C12N4

Molecular Weight: 473.4

CLOFAZIMINE 19

2.2 Nomenclature

2.21 Generic Name

Clofazimine (BAN, USAN, rI")

2.22 Chemical Names

3-(4-chloroanilino)-10-(4-ehlorophenyl)-2,1 O-dihydrophenazin-2- ylideneisoprop ylamine. N,5-Bis(4-chlorophenyl)-3,5-dihydro-3-[(l-methylethyl) iminol-2- phenazinamine, or 3-(p-chloroanilino)-1O-(p-chlorophenyl)-2, 10-dihydro- 2(isopropylimino) phenazine, or 2-(4-chloroanilino)-3-isopropylimino-5-(4-chlorophenyl)-3,5- dihy drop hen azine, or 2-p-chloroanilino-5-p-chlorophenyl-3,5-dihydro-3- isopropy liminophenazine.

2.23 Trade name

Clofazimine is marketed by Ciba Geigy under the proprietary name "Lamprene".

2.24 Other Names, Abbreviations and Drug Codes

Riminophenazine, 8663, G30320, NSC 141046, chemical abstracts service registry number (CAS no.) 2030 - 63-9.

2.3 Official Compendia

A monograph on clofazimine is included in the British Pharmacoepia and the Indian Pharmacoepia.

2.4 Other Compendia

Clofazimine is included in the Merck Index (19891, the Pharmaceutical Codex (1979), and in Martindale (1989). Clarke (1986) gives a useful summary of physical and chemical data.

80 CAITRIONA M. O'DRISCOU AND OWEN 1. CORRIGAN

iii - + i, ii ~

NHR NHR NHR NHz

( '1 R = aryl

1 i v

I V

(3)

R = Ph, 4-CI-C6H4-

Reagents: i, FeCl3, H+; ii, NH3; iii, R*NH2, alkyamines; iv, benzoquinone/carbonyl compound RkOR3; v, Pt @/H or Pt/C (lO%)/H2; vi, air; vii, Pd/C (lO%)/Hz.

Figure 1. Principal synthetic routes to riminophenazines (Hooper, 1987)

3. SYNTHESIS

The original synthetic routes to riminophenazines (Barry et al., 1956a; 1956b; 1957 and 1958 ) have been modified (O'Sullivan, 1984) to give reproducible high yields. The modifications have been summarised by Hooper (1987) (Figure I) as follows; N-aryl ortho -phenylenediamines (1) undergo regiospecific oxidative dimerization to yield the parent iminophenazines (2) which react further with alkylamines to give substituted iminophenazines (3). Alternatively, oxidation with benzoquinone in the presence of a

CLOFAZIMINE 81

carbonyl compound gives an imidazolophenazine (4) which may be reduced with cleavage of the imino substituent (5) followed by subsequent aerial oxidation to the parent iminophenazine (2). A more selective reduction results in an alternative cleavage of the imidazoline ring (6) which after oxidation gives a substituted iminophenazine (7). The type of catalyst used in the reduction of these compounds is crucial and allows full control of the reactions.


4.1 Ultraviolet Absorbance Spectrum

The ultraviolet spectrum of clofazimine (0.001 % w/v) is shown in Figure 2. The spectrum was obtained using a Hewlet Packard 845 2A diode array UV visible spectrophotometer and 1 em quartz cells. The spectrum, in the range 230 to 600nm, in 0.01m methanolic hydrochloric acid, exhibits two maxima, at 284nm and 486nm. The absorbance at 284nm is about 1.30 and at 486nm is about 0.64.

I I I 1

220 300 400 500 600

WAVELENGTH

Figure 2. Ultraviolet spectrum of clofazimine.

82 CAlTRlONA M. O’DRISCOLL AND OWEN I. CORRIGAN

4.2 Infrared Absorbance Spectrum

The infrared absorbance spectrum of clofazimine is shown in Figure 3. The spectrum was recorded with a Nicolet 5ZDX FT-IR spectrophotometer, from a compressed potassium bromide disc. Structural assignments for some of the characteristic absorption bands in the spectrum are listed in Table I.

Table I. Infrared assignments for clofazimine

W avenumber (cm- l) Assignment

1587,1560,1510,1460,1300 aromatic CH stretching 1389,1360,1130 CH(CH3)2 stretching

4.3 Mass Spectrum

The mass spectrum of clofazimine, shown in Figure 4, was obtained using a Finnigan Quadrupole mass spectrometer, by electron - impact at 70 electron volts. The molecular ion (M-H) at m/z 473 was observed. Major peaks were detected at m/z (%) 474 (66.17),473 (36.22), 472 (1001,457 (93.04), 455 (70.87), 456 (24.13),431 (19.57), 414 (30.43), 380 (22.17), 345 (17.83), 331 (30.43).

4.4 Proton Nuclear Magnetic Resonance Spectrum (IH-NMR)

The IH-NMR spectrum of clofazimine, shown in Figure 5, was obtained in deuterated chloroform containing tetramethylsilane (TMS) as internal standard, using a Joel GX- 270 MHz instrument. A 2D COSY spectrum was also obtained (Figure 6a and 6b). Figure 6b is an expansion showing coupling in the aromatic regions.

4.5 Carbon - 13 Nuclear Magnetic Resonance Spectrum

The carbon-13 NMR spectrum of clofazimine was obtained in deuterated chloroform containing TMS as internal standard using a Joel GX-270 MHz instrument at a frequency of 67 MHz. The carbon-13 NMR spectrum, with DEPT, is shown in Figure 7.

CLOFAZIMINE 83

Figure 3 . Infrared spectrum of clofazimine.

Y

0 . . . : . . . : . . . : . . . : . . . : . . . : . . - : . . . I 2000 I BOO 1 so0 1400 1200 1000 800 SO0 400

W ~ v m u n b r r Cpm-1)

84 CAlTRlONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

Figure 4. Electron-impact mass spectrum of clofazimine.

CLOFAZIMlNE 85

Figure 5 . Proton nuclear magnetic resonance spectrum of clofazimine.

86 CAITRIONA M. O'DRISCOLL AND OWEN I. CORRICAN

Figure 6a. 2-D proton nuclear magnetic resonance spectrum of clofazimine.

CLOFAZIMINE

Figure 6b. 2-D proton nuclear magnetic resonance spectrum of clofazimine.

87

88 CAITRIONA M. O'DRISCOLL AND OWEN I. CORRIGAN

Figure 7. l3c1 nuclear magnetic resonance spectrum of clofazimine, with DEPT.

CLOFAZIMINE 89

4.6 X-Ray Diffraction

The powder X-ray diffraction pattern of clofazimine was obtained on a Siemens D-500 X-ray diffractometer, using a Cu X-ray tube, at 40 kV and 40 mA. The diffraction pattern is shown in Figure 8, indicating the crystalline nature of clofazimine.

of clofazimine, a monoclinic form with a density of 1.3 g cm-3, and a triclinic modification with a density of 1.29 g cm-3. The former was prepared by recrystallization from acetone, and the latter by recrystallization from 12 N-methylformamide/acetone. Cell constants were also calculated. The values obtained for the monoclinic form were a = 7.788 A, b = 22.960 A, c = 13.362 A, p = 98.580. The values for the triclinic form were a = 10.507 A, b =

12.852 A, c = 9.601 A, a = 95.960, p = 97.220, y = 69.730.

Rychlewska et al. (1985) reported two different crystalline forms

4.7 Melting Point

Melting points reported in the literature are in the temperature range of 210 - 215OC, with degradation (Barry et al. 1956a; Clarke 1986; Merck Index 1989; Pharmaceutical Codex 1979).

4.8 Differential Scanning Calorimetry (DSC)

The DSC thermogram of clofazimine obtained using a Mettler DSC 20, scan speed lOOC min-1, is shown in Figure 9. A single sharp melting endotherm was obtained with onset temperature at 214OC. This value is in good agreement with the melting points previously published (Section 4.7). The estimate of the heat of fusion (AH) was 740 joules/gram. However, with some samples there was evidence of degradation on melting.

4.9 Dissociation Constant

The values for the dissociation constant reported for clofazimine are summarised in Table 11.

YO CAlTRlONA M. O'DRISCOLL AND OWEN 1. CORRIGAN

Figure 8. X-ray powder diffraction pattern of clofazimine.

TWO - THETA IOEGREESI

CLOFAZIMINE

200.0- - - -

2 l O . O - - - -

220.0- - - - - 230.0- - - - - 240.0- -

- - -

91

I 20 .000 nU I

Figure 9. Differential scanning calorimetry thermogram of clofazimine.


Table 11. Dissociation constant of clofazimine (pKa)

PKa Method of determination Reference

8.35 Not stated Morrison and Marley

8.37 Potentiometric Canavan et al. (1986) 8.51 Spectropho tome tric Fahelelbom et al. (1989)

(1976a)

4.10 Solubilities

Clofazimine is practically insoluble in water, estimates in the range of 1.03 - 0.49 pg ml-1, at 37OC, have been reported (Fahelelbom, 1989; OReilly, 1991). It is soluble 1 in 700 of ethanol, 1 in 15 of chloroform, and 1 in 1000 of ether. It is also soluble in dilute acetic acid and dimethylformamide (Clarke, 1986).

U I I I I

5 6 7 8

PH

Figure 10. pH solubility profile of clofazimine.

CLOFAZIMINE 93

The effect of pH (range 5.15 - 7.8) on the solubility of clofazimine, shown in Figure 10 (OReilly, 19911, is consistent with the basic nature of the compound. The solubility of the drug is 5.68 and 0.278 mg ml-1 x 10 -3 at pH 5.15 and 7.8 respectively. Values for intrinsic solubility in the range of 2.0 - 2.3 x 10-5 mg ml-1 (Fahelelbom, 1989; OReilly, 1991) have been reported.

systems, containing both naturally occuring surfactants e.g bile salts, and synthetic surfactants, e.g the non ionic Cremophor EL and Triton X100, and the anionic sodium dodecyl sulphate. The incorporation of fatty acids to form mixed micelles brought about a further enhancement in drug solubility in the case of naturally occuring surfactants (approximately 300 fold with sodium cholate: linoleic acid relative to buffer). In contrast, with synthetic surfactants this enhancement decreased (Fahelelbom et al., 1991; ODriscoll et al., 1991).

The solubility of clofazimine was enhanced in aqueous micellar

4.11 Partition Coefficients (Log P)

Partition coefficients for clofazimine have been determined using different solvents and temperatures. The data is summarised in Table 111.

Table 111. Partition coefficients of clofazimine

Solvents Temp (OC) LogP Reference

Octanol: water - +7.48* Morrison and Marley (1 976a,b)

(1 986)

et al. (1990)

Isooctane: buffer pH 5.15 20 5.01 Canavan et al.

N-octanol: buffer pH 5.15 20 4.30 Quigley

N-octanol: buffer pH 5.15 37 4.40 Ibid N-octanol: buffer pH 5.15 45 4.48 Ibid N-octanol: buffer pH 5.15 55 4.54 Ibid

* Estimated



5.1 Elemental Analysis

Carbon Hydrogen Nitrogen Chlorine

% Calculated 68.50 4.68

11.83 14.98

% Found 68.68 4.52

11.48 15.32

5.2 Identification

The B.P. (1988) outlines three methods of identification: (A) By the infrared absorption spectrum, outlined in section 4.2. (B) The light absorption, the UV spectrophotometry is described

in section 4.1. (C) A colour test, dissolve 2mg clofazimine in 3ml of acetone

and add O.lml of hydrochloric acid, an intense violet colour is produced. Add 0.5ml of 5M sodium hydroxide, the colour changes to orange - red.

5.3 Ultraviolet and Visible Spectrophotometry

Quantitative ultraviolet analysis of clofazimine has been performed, in a range of aqueous and nonaqueous media, at 280nm (Canavan et al., 1986; O'DriscolI et al., 1990a,b), and colorimetrically at 482nm (Quigley et al., 1990).

modified by Mansfield (1974) to analyse plasma and tissue levels of clofazimine. The drug was extracted using benzene and concentrated hydrochloric acid, and the absorption read at 540nm. The limit of detection reported was 0.2pg/ml in plasma and O.lmg/gram in tissue.

A colormetric assay was developed by Barry et al. (1960) and

5.4 Spectrofluorometric Analysis

A fluorescent derivative of clofazimine was formed following reduction with titanous chloride (Dill et al., 1970). The fluorescence was measured at 366mp emission. The limits of

CLOFAZIMINE 95

detection reported for this method were in the range of 0.1 - 0.2 pg/ml in plasma (Banerjee et al., 1974; Levy, 1974).

5.5 Thin Layer Chromatography

A thin layer chromatographic (TLC) system suitable for determination of clofazimine in plasma has been developed (Lanyi and Dubois, 1982). The plasma samples were acidified using acetate buffer pH 5 and extracted with toluene, evaporated to dryness under nitrogen, reconstituted in toluene and applied to the TLC plate. The adsorbent used was HPTLC silica gel 60. The plates were developed in toluene - acetic acid - water (50 : 50 : 4), allowed to stand for 30 min at room temperature, the Rf value of clofazimine was 0.36. Detection and quantitation is carried out using a densitometric method. The limit of detection reported for this method was 5ng/g.

5.6 High Performance Liquid Chromatography

Gidoh et al. (1981) developed a high performance liquid chromatographic (HPLC) method with ultraviolet detection to separate and quantify clofazimine (287nm) from other antileprosy drugs, dapsone and rifampicin, in serum on a pBondapak c18 column. This method involved a complicated extraction procedure with the switching of 2 different mobile phase (i.e acetonitrile - water, 20 : 80; and tetrahydrofluran - water containing PIC B-5,50 : 50, the latter reagent contains 1 - pentanesulfonic acid and glacial acetic acid) in order to allow complete resolution of clofazimine from related components. The limit of detection for this method was long ml-1. Recently a modification of this technique was used to study clofazimine and its derivatives (O'Sullivan et al., 1990).

Another HPLC method, was described by Peters et al. (1982), for measuring clofazimine in plasma, with a limit sensitivity of 10 ng ml-1. This method involved extraction of clofazimine into organic solvents and quantifation on a reversed-phase Ultrasphere - octyl column, using a mobile phase of 0.0425M phosphoric acid in 81% methanol and UV detection at 285nm.

The gastrintestinal absorption of clofazimine, using a rat gut perfusion technique, was determined by HPLC (O'Driscoll et al., 1990a,b). The column used was Partisil lOPAC, the mobile phase


was ethanol : N-heptane (50 : 50) and detection was by UV at 283nm. The limit of sensitivity was 0.1pg ml-I.

6. PHARMACOKINETICS

6.1 Bioavailability Considerations

Clofazimine absorption following oral administration is incomplete and varies significantly from patient to patient. Following administration as coarse crystals only about 20% is absorbed, if however, the drug is given as a microcrystalline suspension in an oil wax base an absorption rate of 70% can be achieved (Yawalkar and Vischer, 1979).

anaestheised rat, using an in situ rat gut perfusion model (Komiya et al., 19801, was enhanced by co-administration of simple and mixed micellar systems (O'Reilly et al., 1988; O'Driscoll et al., 1990a,b). The simple micellar systems included various bile salts, and the synthetic emulgents, Cremophor EL (non ionic) and sodium dodecyl sulphate (anionic). The mixed micelles were formulated by the incorporation of various fatty acids. A mixed micellar system containing sodium cholate: linoeleic acid enhanced the rate of absorption of clofazimine by a factor of 840 compared to a buffered solution of the drug. The enhancements were due to a combination of increased solubility and increased membrane permeability. There is also evidence that clofazimine is transported in part via the lymphatic system (Barry et al., 1960; Atkinson et al., 1967).

Clofazimine has a reported pKa of 8.35 and consequently it is highly ionised under physiological conditions. This high degree of ionization, together with its high molecular weight, may be significant factors in the poor oral bioavailability.

Schaad - Lanyi et al. (1987) studied the pharmacokinetics of single oral doses of clofazimine over 11 days following administration. They examined the effect of food on the bioavailability. Following administration with food the area under the plasma concentration versus time curve (AUC) and the peak plasma concentration C,,, were 62 and 30% higher respectively compared to results obtained in the fasted state. The

The gastrointestinal absorption of clofazimine in the

CLOFAZIMINE 97

median time (tmx) to reach Cmax was 8 hours with food and 12 hours without food.

6.2 Distribution, Metabolism and Elimination

Plasma levels of the drug are approximately 0.5mg 1-1 but increase with the dose and at 300mg daily levels of 1.0 - 1.5 mg 1-1 have been achieved (Banerjee et al., 1974; Levy, 1974).

Administration of 50mg of clofazimine daily for 8 days did not achieve steady state (Schaad - Lanyi et al., 1987). The time to reach steady state has been theoretically estimated to be in the range of 30 - 70 days (Schaad - Lanyi et al., 1987; Holdiness, 1989). There is no data available on loading doses. Likewise, there is no information currently available on the pharmacokinetics of clofazimine following intravenous administration.

The appearance of clofazimine in the plasma following absorption is short lived (Banerjee et al., 19741, it rapidly passed out of the circulation and is deposited in various tissues and organs, particularly the fatty tissue, the spleen, lymph nodes, and the cells of the reticulo - endothelial system. Concentrations of 2.1- 5.3 mg g-1 have been reported in the subcutaneous fat (Mansfield, 1974), and 0.6-1.0 mg g1 in the spleen (Desikan and Balakrishnan, 1976; Mansfield, 1974). It is taken up by the macrophages throughout the body (Conalty et al., 1971; Yawalkar and Vischer, 1979). Electrophoretic studies of serum from orally treated mice have shown almost complete binding of clofazimine to the lipoproteins of the a and then phagocytosed by the macrophages (Conalty et al., 1971). Clofazimine crystals have been found at autopsy in the small intestine and in the macrophages of mesenteric lymph nodes (Conalty et al., 1971; Aplin and McDougall, 1975; Desikan and Balakrishnan, 1976; Jopling, 1976). Clofazimine does not appear to cross the intact blood-brain barrier (Mansfield, 1974; Desikan and Balakrishnan, 1976). It does, however, appear to cross the placenta causing pigmentation of the foetus (Holdiness, 1989). There is no data available on the volume of distribution of clofazimine.

Feng et al. (1981; 1982) have used mass, ultraviolet and visible spectrometry to identify three metabolites in the urine of leprosy patients (Figure 11). Metabolite I is the unconjugated compound 3 (p-hydroxyanilino)-lO-(p-chlorophenyl)-2, lO-dihydro-2-

globulin fractions, these lipoprotein are

98 CAITRIONA M. O’DRISCOLL AND OWEN 1. CORRIGAN

isopropyliminophenazine, the other two metabolites are conjugated, metabolite 11 is 3-( P-D-glucopyransiduronic acid)-lO-(p- chloropheny1)-2, 10-dihydro-2- isoproyliminophenazine), and metabolite 111 is 3 - (p-chlorani1ino)-10- (p-chlorophenyl) - 4, 10- dihydro - 4 (PD-glucopyranosiduronic acid) -2- isopropyliminophenazine. Metabolite I is reported to be formed by a hydrolytic dehalogenation reaction, metabolite 11 by hydrolytic deamination followed by glucuronidation, and metabolite III by hydration followed by glucuronidation.

Following administration of 300mg/day of clofazimine, 0.2% of metabolite I, 0.25% of metabolite 11, and 0.2% of metabolite I11 were recovered in the urine over 24 hours (Feng et al., 1981; 1982). No information is available on the pharmacological activity of these metabolites, or whether they are found in faeces or bile. The authors have shown that metabolite I11 may be produced in the laboratory through metabolism by liver enzymes. However, they were unable to demonstrate the same hepatic conversion of clofazamine to metabolites I and 11. In contrast, they suggest that these metabolites are produced by bacterial degradation in the intestine prior to absorption and urinary excretion.

(fatty tissue, skin, lymph nodes, macrophages etc.) and is eliminated very slowly. The kinetics of the drug has been described by both one and two compartment models. Data obtained with relatively low dose, short term administration indicated a one compartment model, with a a plasma tipof approximately 7 days (Levy, 1974; Hastings et al., 1976; Holdiness, 1989). A second compartment is evident with long term, high dose administration and appears to have a ttp of at least 70 days (Banerjee et al., 1974; Levy, 1974).

Following oral administration of 50mg/day of clofazimine to health volunteers Schaad - Lanyi et al. (1987) predicted that steady state (SS) plasma concentrations would occur after approximately 30 days. They calculated an accumulation factor for the drug from the ratio of AUCss: AUC. A value of 4.85 was obtained suggesting a slow accumulation towards steady state. The authors suggest that this may be avoided by administering higher loading doses, followed by daily maintenance doses.

Clofazimine accumulates in certain tissues throughout the body

CLOFAZIMINE

CI

99

N.CH(CH,),

Metabolite I

1. Hydrolytic deamination - - - - - - - - - -

2. Glucuronation N.CH(CH,),

Metabolite II

Clofazimine

Metabolite 111

Figure 11. Metabolic pathways of clofazimine in humans (Feng et al., 1981; 19821)

Up to 50% of a dose of clofazimine is excreted unchanged in the faeces, indicative of poor oral absorption (Banerjee et al., 1974). However, high concentrations of the drug have been found in bile and in the gall bladder. This suggests that part of the ingested drug recovered from the faeces may represent excretion by means of the bile rather than simply the failure of absorption from the gastrointestinal tract (Mansfield, 1974). Urinary excretion in leprosy patients is negligable accounting for an average of 0.1%

I 0 0 CAITRIONA M. O'DRISCOLL AND OWEN I . CORRIGAN

(range 0.01 - 0.43%) of the dose in 24 hours (Levy, 1974). A small amount of the drug is excreted in the sebum and sweat (Vischer, 1969).

7. PHARMACOLOGY

7.1 Mechanisms of Action

Although the precise mechanism of the antileprotic activity of clofazimine has yet to be determined several explanations have been proposed (Hooper 1987).

(a) The drug has been shown to bind to cytosine - guanine DNA base pairs in vitro (Morrison and Marley, 1976a,b). The binding is specific for guanine residues only. The DNA of M. Zeprue has a high guanine - cytosine content, consequently this binding may disrupt the template function of the DNA, causing inhibition of protein synthesis.

(b) The redox properties of clofazimine can divert up to 20% of cellular oxygen (Barry et al., 1957) and thus disrupt normal mitochondria1 oxidation processes (Rhodes and Wilkie, 1973). In addition, it has been suggested that cytotoxic oxygen species, hydrogen peroxide and superoxide, are generated as a result of the presence of the drug (Hooper and Purohit, 1983; Savage et al., 1989). If such a reaction occurred within the macrophages it will enhance the killing of the bacilli which are also found inside the macrophages.

(c) In addition, it has been suggested that the antileprotic effect of clofazimine may be due to its action on the macrophage lysosomal apparatus (Sarracent and Finlay, 1984).

7.2 Structure Activity Relationships (SAR)

The earlier SAR studies, reviewed by Hooper and Purohit (1983), concentrated on three main areas of molecular modification (Figure 12). Firstly, the structure of clofazimine was varied by introducing additional chlorine atoms at positions 4, 7,8 and 9. This resulted in loss of activity, except for the 7- chloro derivative, with was equipotent with clofazimine. The second series was

CLOFAZIMINE 101

based on triaryl derivative. A variety of derivatives with a chloro- or methoxy substituent in various positions showed only modest activity. The third series involved variations at R2 coupled with changes at R1 and R3, and the introduction of various substituents at positions 7 and 9. In general for optimum activity R2 had to be alkyl or cycloalkyl, and R1/ R3 aryl or substituted aryl. When hydrophilic salt forming groups were introduced at R2 activity was greatly reduced.

R’ 9 1 1

Figure 12. Basic structure of iminophenazines

An X-ray crystallographic study (Rychlewska et al., 1985) described the crystal and molecular structures of two crystal forms of clofazimine and of its inactive isomer, isoclofazimine (B3857). The geometric differences between clofazimine and isoclofazimine were probed by CND0/2 molecular orbital calculations. The geometry at the exocyclic amino nitrogen atom N(3) is significantly different in isoclofazimine from that in both forms of clofazimine and in other active analogues (Figure 13). The authors suggest that the value for the intramolecular angle a at N(3) (defined by C(3) - N(3) - C(21) in clofazimine) may play a significant role in the activity. Molecules with values of a in the range 125.5 & 10 were inactive, while those with expanded a angles (i.e 131f 10) were active in vitro. The larger angle in the active compounds is thought to favour intramolecular hydrogen bonding between N(3)-H ... N(2). The capacity to form an intramolecular hydrogen bond was interpreted as evidence of a capacity for intermolecular hydrogen bonding in solution e.g between guanine in DNA and clofazimine.

I02 CAlTRlONA M. O’DRISCOLL AND OWEN 1. CORRICAN

Figure 13. Crystal structure of clofazimine (Rychlewska et al., 1985)

A wide range of clofazimine analogues have been designed as follows; (a) to be active against resistant organisms, (b) not to accumulate in adipose or other tissues, (c) to be rapidly and adequately absorbed from the gastrointestinal tract, and (d) not to crystallize within cells (Barry et al., 1959; Franzblau and OSullivan, 1988; OSullivan et al., 1988; Byrne et al., 1989). These structural modifications generally involve substitution at the imino nitrogen atom by an unbranched alkyl or branched alkyl chain containing a primary, secondary, tertiary, or alicyclic amino group. Frequently the pKa values of these amine containing side chains are approximately 9.5 - 10.5 thus ensuring that these molecules will be substantially ionized under physiological conditions. To counter act this increased hydrophilicity the aliphatic part of the substituents usually contain 6 - 8 hydrophobic methylene groups (Hooper, 1987).

A study, (Canavan et al., 19861, on the influence of lipophilic and stearic properties on the distribution of a range of clofazimine analogues to the spleen of mice following oral administration,

CLOFAZIMINE I03

indicated that lipophilicity of the molecule is a significant factor whereas the stearic properties of the N2 - substituents are not.

The structural features of phenazine derivatives which contribute to stimulation of PGE2 production by polymorphonuclear leucocytes (Zeis et al., 1987) and pro-oxidative interactions with neutophils (Savage et al., 1989), have also been investigated.

7.3 Toxicity

Clofazimine is a relatively non-toxic drug W.S. Leprosy panel, 1976). The acute LD50 was found to be >5 g/kg in mice rats and guinea pigs. It was 3.3 g/kg in the rabbit. Daily oral doses of 30 and 50 mg/kg given for six months were generally well tolerated by monkeys and rats. Reddish discolouration of the skin, faeces and urine was observed. Temporary diarrhoea was occassionally reported in rats (Stenger et al., 1970). Experimental studies in animals did not show any evidence that clofazimine possesses a primary embryotoxic or teratogenic action (Stenger et al., 1970). The drug does not exhibit mutagenic activity (Morrison and Marley, 1976a). A long term study on 51 patients receiving clofazimine for

periods up to 8 years showed that, despite the deposition of the drug in various tissues, it appears to be remarkably free from serious side effects in clinical use (Hastings et al., 1976). Although clofazimine crosses the placenta, no evidence of teratogenicity has been found (Schulz, 1972). The most frequently reported side effects of clofazimine therapy are red-brown hyperpigmentation of the skin and conjunctiva, and abdominal pain (Hastings et al., 1976; Jopling, 1976; Yawalkar and Vischer, 1979; Granstein and Sober, 1981; Moore, 1983; Negrel et al., 1984; Venencie et al., 1986). Cutaneous pigmentation normally fades within 6 to 12 months- Generalised dryness of the skin (xeroderma) ichthyosis, puritis, phototoxicity, acneiform eruptions, exfoliative dermatitis and non specific skin rashes have been reported (Yalwalkar and Vischer, 1979; Pavithran, 1985). Discolouration of sweat, hair, sputum, urine and faeces have also been observed (Yalwakar and Vischer, 1979). Apart from subepithelial pigmentation in the cornea no other side effects on the eye were recorded. Clofazimine crystals were found in the tears of 82% of patients studied (Negrel et al., 1984).

I 04 CAITRIONA M. O'DRISCOLL AND OWEN I . CORRIGAN

Gastrointestinal side effects, nausea, diarrhoea, anorexia, constipation and weight loss have also been reported (Hastings et al., 1976; Moore, 1983). These symptoms have been associated with the deposition of clofazimine crystals in the submucosa of the small intestine and in the mesenteric lymph nodes (Jopling, 1976; Harvey et al., 1977).

The occurence of drug interactions involving clofazimine have also been investigated. Most of the studies show that clofazimine does not exert any effect on dapsone excretion in leprosy patients (Balakrishnan and Seshadri, 1981; Zuidema et al., 1986). Clofazimine has been shown to significantly reduce the absorption of simultaneously administered rifampicin, resulting in delayed time to reach peak serum concentration and increased t; . No significant changes were seen in C,,, or AUC (Mehta et al., 1986).

7.4 Dose Schedules

A dose of 300mg once montly plus 50mg daily or lOOmg on alternative days has been recommended to treat multibacillary forms of leprosy (Martindale, 1989).

The World Health Organisation (1982) has published guidelines for the treatment of leprosy. Dosage schedules are generally not based on serum/plasma concentrations, or pharmacokinetic data. Clofazimine is usually used in combination with other antileprotic agents e.g dapsone and rifampicin, to prevent the emergence of resistance. It is usually given with food in doses adjusted according to body weight and the activity of the disease.

concentration of drug in the immediate environment of M.leprae in the tissues and not on the serum level. Since the drug in not evenly distributed through out the tissues it is impossible to calculate the minimal inhibitory concentration (MIC) in animals (Yawalkar and Vischer, 1979).

The therapeutic activity of clofazimine depends on the

CLOFAZIMINE 10.5

ACKNOWLEDGEMENTS

The authors wish to thank Dr. J. F. OSullivan, formerly of the Health Research Board, Trinity College, Dublin, Dr. Helen Sheridan, Department of Pharmacognosy and Dr. Mary Meegan, Department of Pharmaceutical Chemistry, Trinity College, Dublin for their advice and assistance, Ciba Geigy, England, for the supply of clofazimine, Ms. Mary Lally and Ms. Mary Reilly for technical assistance.

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CLONIDINE HYDROCHLORIDE

Mohamrnad A. Abounassif,' Mohammad Saleem Mian,'

and Neelofur Abdul Aziz Mian'

( 1 ) Pharmaceutical Chemistry Department College of Pharmacy King Saud University Riyadh, Saudi Arabia

(2) Clinical Laboratory Sciences Department College of Applied Medical Sciences

King Saud University Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXCIPIENTS - VOLUME 21

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form. 109

110 M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

Contents

1 , Introduction

2 . Description 2 . 1 Nomenclature

2 . 1 . 1 Chemical Names 2 . 1 . 2 Generic Names 2 . 1 . 3 Trade Names

2 . 2 . 1 Empirical 2 . 2 . 2 Structural 2 . 2 . 3 CAS (Chemical Abstract Service Registry

2 . 3 Molecular Weight 2 . 4 Elemental Composition 2 . 5 Appearance, Color, Odour and Taste

2 . 2 Formulae

Number)

3. Physical Properties 3 . 1 3 . 2 3 . 3 3 . 4 3 . 5 3 . 6 3 . 7 3 . 8 3 . 9 3 . 1 0 3 . 1 1 3 .12 3 . 1 3 3 .14 3 . 1 5 3 .16 3 . 1 7

Melting Range Solubility PH Loss on drying Sulphated Ash Clarity and Color of Solution Stabi 1 ity PK LD5 o Action Half Life Plasma Volume of Distribution Protein Binding Storage X-ray Powder Diffraction Crystal Structure Spectral Properties 3 .17 .1 Ultraviolet Spectrum 3 . 1 7 . 2 Infrared Spectrum 3 . 1 7 . 3 Nuclear Magnetic Resonance Spectra 3 . 1 7 . 4 Mass Spectrum

4 . Synthesis

5 . Phnrmacokirietics 5 . 1 Absorption and Distribution 5 . 2 Uses and Administration 5 . 3 Adverse Effects


5.4 Precautions

6. Methods of Analysis 6.1 Identification 6.2 Colorimetric 6.3 Fluorimetric 6.4 Spectrophotometric Analysis 6.5 Radio-Immunoassay 6.6 Chromatographic Methods

6.6.1 Gas-Liquid Chromatography (GLC) 6.6.2 High-Performance Liquid Chromatography

(HPLC) . 7. Acknowledgements

8. References


Clonidine Hydrochloride

1. Introduction

Clonidine hydrochloride is an imidazoline derivative hypotensive agent (1) which is thought to act through the central nervous system to elicit a hypotensive response ( 2 ) . Although the locus of action in the central nervous system is unlcear, clonidine has been shown to be a potent a-adrenergic agonist in both central and peripheral systems ( 3 ) .

The commercially available transdermal system of clonidine consists of an outer layer of pigmented polyester; a drug reservoir of clonidine, mineral oil, polyisobutylene, and colloidal silicon dioxide; a microporous polypropylene membrane that controls the rate of diffusion of the drug; and a final adhesive layer that provides an initial release of drug and contains those ingredients found in the reservoir. The adhesive layer is covered by a protective strip which is removed prior to application (1) .

2 . Description

2 . 1 Nomenclature


[2-(2,6-Dichlorophenylimino)imidazolidine

2 - ( 2 , 6 - D i c h l o r o a n i l i n o ) - 2 - i m i d a z o l i n e

2,6-Dichloro-N-(imidazolidine-2-ylidene)aniline

2-(2,6-Dichlorophenylamino)-2-imidazoline

hydrochloride (2,4);

hydrochloride ( 4 ) ;

hydrochloride (4);

hydrochloride (5).

2.1.2 Generic Names

Clonidine hydrochloride.


2.1.3 Trade Names

I13

Catapres, Catapresan, Clonistada; Dixarit, D r y l o n , H y p o s y n , I p o t e n s i u m , I s o g l a u c o n , Tenso-Timelets.

2.2 Formulae

2.2.1 Emoirical

C g H g C l z N 3 (Clonidine). CsHsCIzN3 .HC1 (Clonidine hydrochloride).

2.2.2 Structural

2.2.3 CAS (Chemical Abstract Service Registry Number 1

4205.90.7 (clonidine) (4). 4 2 0 5 . 9 1 . 8 (Clonidine hydrochloride) ( 4 ) .

2.3 Molecular Weipht

230.10 (Clonidine) ( 6 , 7 ) . 266.6 (Clonidine hydrochloride) ( 4 ) .

2.4 Elemental Composition

Clonidjne (7): C 46.98%; H 3.94%;

Clondine hydrochloride: C 40.51%; H 3.75% C1 39.98%; Nz 15.75%.

c i 30.82%; N 18.26%.

I14 M.A. ABOUNASSIF, M.S. MIAN. AND N.A.A. MIAN

2 . 5 ADpearance, Color, Odour and Taste

A white o r almost white crystalline powder (8) which has a bitter taste ( 1 ) .

3 . Physical Properties

3.1. Melting Range

Clonidine 130'C ( 7 ) . Clonidine hydrochloride 305'C ( 7 ) . Clonidine hydrochloride 300'C with decomposition (9).

3 . 2 Solubility

Soluble in 13 parts of water, soluble in absolute ethanol, slightly soluble in chloroform ( 8 ) . 1 g soluble in 6 m l H 2 O ( 6 0 ' C ) , about 13 m l H 2 0

( 2 0 ' C ) , about 5 . 8 ml C:H3OH, about 2 5 ml C z H 5 0 H and about 5000 ml of CHCln (9). Practically insoluble in ether (6).

5% solution in H 2 O has a pH of 4.0 to 5 . 0 ( 8 ) .

3 . 4 Loss on Drying

When dried to constant weight at 1 O O ' C to 105-C, loses not more than 0.5% of its weight use 1 g (8).

3 . 5 Sulphated Ash

Hot more than 1% (8).

3 . 6 Clarity and Color of Solution

A 5% w/v solution in carbon dioxide free water is clear (8).

3 .7 Stability

Stable in light air and room temperature (9).

3.8 pk

The drug has a Pk of 8.2 ( 6 , 9 ) .

CLONIDLNE HYDROCHLORIDE I I5

The acute toxicity for clonidine in related species is as follows: ( 7 )

Species LD5o (mg/kg)

Oral I.V.

Mouse 328 18

Rat 270 29

Rabbit 80 45

Dog 30-100 6

Monkey 150-267

3 .10 Action

Clonidine hydrochloride is an antihypertensive agent, whose mechanism of action appears to be central a-adrenergic stimulation. This result in the inhibition of bulbar sympathetic cardioaccelarator and sympathetic vasoconstrictor centers, therapy causing a decrease in sympathetic outflow from the brain. Initially drug stimulates pheripheral a-adrenergic receptors producing transient vasoconstriction ( 5 ) .

3 .11 Half-Life Plasma ( 6 )

Pl.asma half-life, 10 to 25 hours.

3 . 1 2 Volume of Distribution ( 6 )

2 to 4 litres/kg.

3 . 1 3 Protein Binding ( 6 )

About 20 to 40%.

3 . 1 4 Storage

The drug should be kept in a well-closed containers ( 8 ) and protect from sun light ( 4 ) .


3 .15 X-ray Powder Diffraction

The X-ray diffraction pattern of clonidine hydrochloride was determined using philips full automated generator. Radiation was provided by a copper target (Cu annode 2000W, Y = 1.5480 A ) . High intensity x-ray tube operated at 40 kv and 35 Mv was used. The monochromator was a curved single crystal one (Pw 1752/00). Divergence slit and the receiving slit were 0 and 0 . 1 " , respectively. The scanning speed of the goniometer (Pw 1050/81) used was 0 . 0 2 - 20 per second. The instrument is combined with philips PM 8210 printing recorder with both analogue recorder and digital printer. The goniometer was aligned using silicon sample before use. The x-ray pattern of clonidine hydrochloride is presented in Fig. ( 1 ) . The values of scattering angle 2 0 interplanner distance dA and relative intensity 1/10 are shown in the table ( 1 ) .

3 .16 Crystal Structure

Gudmund et a1 ( 1 0 ) have determined the crystal structure of clonidine hydrochloride by x-ray diffraction methods using 3209 observed reflections collected on a counter diffractometer. The crystals are monoclinic, space group ( 2 / c with unit cell dimensions a = 1 7 . 9 5 7 ( 2 ) A b = 1 1 . 9 5 0 ( 1 ) A , c = 13.664 ( 1 ) A and I3 = 128.64 ( 1 ) O ; (t = 18 k 1 . C ) ; V =

1 .546 g cm-3; p = 7 .67 cm-1, 2290.2 A , M = 266 .56 , Z 8 ; F(OOO) = 1088; Dcalc =

Selected interatomic distances and bond angles are listed in Table ( 2 ) . Perspective view of the molecule showing bond lengths is presented in Fig. ( 2 a ) . The stereoscopic view of the crystal structure of clonidine is shown in Fig, ( 2 b ) .

Cody et al. (11) also determined the crystal structure of clonidine hydrochloride in order to determine the conformation of protonated clonidine and to el ucjdate the relationships between its structure and that required for binding to the a-adrenergic re1,epl U I " ,


Table (1) Characteristic lines of x-ray diffraction of clonidine hydrochloride

29 d(A) I / I o %

9.281 9.779

12.463 13.032 14.625 16.801 17.676 19.723 22.232 22.684 23.093 24.634 25.308 25.872 26.334 27.032 28.182 29.088 29.828 30.660 30.889 33.515 33.813 34.788 35.335 36.073 36.754 37.214 38.105 38.104 39.260 39.861 40.252 40.851 41.889 42.175 42.471 43.419 43.747 44.235 44.586 45.623

9.5283 9.0446 7.1020 6.7932 6.0569 5.2768 5.0176 4.5012 3.9986 3.9199 3.8514 3.6138 3.5191 3.4436 3.3842 3.2985 3.1664 3.0698 2.9953 2.9159 2.8948

2,6509 2.5788 2.5401 2,4898 2.4452 2.4161 2.3616 2.3439 2.2947 2.2615 2.2394 2.2089 2.1566 2.1426 2.1284 2,0841 2.0692 2.0475 2,0322 1.9884

2.6738

20.533 58 ,521 22.587 72.142 8.213

16,974 4.859

16.563 33,812 11.362 10.814 46.406 41.204 12.662 100 48.665

6.639 31.211 21.697

6.981 9.582 4.859 8.213 4.996 2.943 5 ,612 6.433 5,270 8.008

10.746 2.943 4.585 6.228 8.418 4.175 5.133 9.924 4.517 4.106 3.764 5.544 3.285

46.963 1.9347 47.821 1.9020 50.711 1.8002 51.129 1.7865 52.072 1.7563 52.667 1.7379

54.372 1.6873 54.903 1.6722 56.132 1.6385 56.476 1.6293 60.422 1.5320 61.337 1.5114 65.051 1.4338 65.578 1.4235 66.518 1.4057 67.342 1.3905 70.397 1.3341 72.236 1.3078 72.918 1.2973

53.648 1.7084

2.943 5.133 7.734 5.886 8 , 0 0 8

12.388 3.901 6.365 5.817 2.395 7.665 3.080 3.011 2.806 3.080 6,707 2.258 2.327 3.832 2.121

20 = scattering angle

dA = Interplanner distance.

I/Io = relative intensity d i s t a n c e ( b a s e d o n highest intensity as 100.

( 2 0 v a l u e )

Fig. (1) The X-ray diffraction pat tern of Clonidine HC1.

CLONIDINE HYDROCHLORIDE 1 I9

Table (2). Distances ( A ) and angles ( " ) in the crystals of clonidine hydrochloride

Bond Length Bond angles

Cl-C2 C2-C3 c3-c4 c4-c5 C5-C6 C6-C1 C1-N1 C2-Cl2 C6-Cl3 N1-C7 N2-C7 N3-C7 N2-C8 N3-C9 C8-C9

1.391 1.382 1.377 1.371 1.385 1.392 1,418 1.733 1.724 1 328 1.322 1.321 1.450 1.447 1.533

C6-Cl-C2 Cl-C2-C3 c2-c3-c4 C3-C4-C5 C4-C5-C6 C5-C6-C1 Cl-C2-C12 c3-c2-c12 Cl-CG-Cl3 C5-C6-C13 C2-C1-N1 C6-Cl -N 1 Cl-Nl-C'I Nl-C'?-NB Nl-C7-N2 C7-N2-C8 N2-C8-C9 C8-C9-N3 C9-N3-C7 N2-C7-N3

Torsional angles (positive for a clockwise rotation) C2-Cl-Nl-C7 - 76.5 Cl-Nl-C7-N2 0.0 Cl-Nl-C7-N3 178.1 C6-Cl-bil-C7 105.2

Hydrogen bonds

Cll-Nl(i - X, - t t y, g - Z) ( A ) C11-HN1 ( A 1 C11-HN1-N1 ( " ) Cll-NX(x,y,~) ( A ) C11-HN2 ( A ) Cll-HN2-N2 ( " )

117.3 121.5 119.8 120.2 119.8 121.4 120.0 118.5 118.9 119.7 121.4 121.3 123.0 123.1 125.2 110.6 103.5 102.6 111.5 111.8

3.094 2.25

3.193 2.38

161.2

163.4

I20 M.A. ABOUNASSIF. M.S. MIAN. AND N.A.A. MIAN

F i g (2b) Stereoscope view o f the crystal structure of Clonidine.

n

F i g ( 2 a ) Perspective view o f Clonidine molecule s h o w i n g bond lengths.

CLONIDINE HYDROCHLORIDE 121

Crystals of clonidine hydrochloride [2,6-dichlorophenylamino)-2-imidazoline HCl] , CgHioN3C13 , were grown by slow evaporation from aqueous solution. The crystals are of exceptional quality. A crystal of columnar shape, 0.2 x 0.2 x 0.6 mm, was screened o p t j cally and by X-ray Weissenberg photography for quality and assignment of space group. The refined cell constants, obtained by a least-squares fit of the I values of 73 high-angle reflections measured ( = 0.707 A ) automatically on a kappageometry diffractometer, are listed in table 3 along with other crystal data. Intensity data were measured in theQ-28 scan mode using Mo h'a radiation and a dispersion-corrected scan sidth of ( 0 . 8 t 0.2tane ) " to a. SinQ,/A maximum of 0.70 8- l . Of the 3335 unique reflections measured, 2112 are greater than or equal to twice their estimated standard deviations.

Table 3. Crystal data for clonidine HC1.

Molecular formula Molecular weight Crystal system Space group 2 Cell dimensions

Cell volume Density (calc. )

Crystal size Final R index

(obs. )

CgHgN3Clz .CH1 266.56 Monoclinic C2/c 8 a = 17.962(3) A b = 11.976(2) W c = 13.672(2) A I3 = 128.62(1)' 2298.2 R 1.541 g ~ m - ~ 1.543 g cm-3 0.2 x 0.2 x 0.6 mm 0.05 (1223 data)

3.17 Spectral Properties

3.17.1 Ultraviolet SDectrun ( U V Y

The UV spectrum (12) of clonidine hydrochloride in H2O (8 mg%) was scanned from 200-600 nm (Fig. 3) using LKB 4054 UV/Vis spectrometer. Clonidine

- I

I I

I I

0

0

0

0

0

0

0

0

0

0

0

In 0

m

0

In

I

0 0

0 0

(D

0

In m

0 0

In

0

m

U

0

0

U

0

m 0

0 0

m

0

Ln C

J

0

0

cv

hl

(v

F

c

0

0


hydrochloride exhibited the following U V data (Table 4 ) .

Table 4: UV data of clonidine hydrochloride

\ax nm Absorbance Molar absorptivity A(1%, 1 cm) (,E) cm-1 gm mol./L

213 2 .488 8290,327

271 0 .214 713 .074

302 0 . 1 0 2 339.876

311

26 .75

1 2 . 7 5

3 . 1 7 . 2 Infrared SDectrum

The I R spectrum ( 1 2 ) of clonidine hydrochloride as KBr disc was recorded on a Perkin Elmer 1310 Infrared spectrometer. Fig. (4) shows the infrared absorption spectrum of clonidine hydrochloride. The structural assignments of clonidine hydrochloride have been correlated with the following frequencies (Table 5).

3 . 1 7 . 3 Nuclear Magnetic Resonance Spectra

4 . 1 7 . 3 . 1 . 'H-NMR Spectrum

The 1 H - N M R spectrum ( 1 2 ) of clonidine hydrochloride in DMSO-ds (Fig. 5-6) was recorded on a Varian X L 200 M H z NMR spectrometer using TMS as an internal reference, The following structural assignments have been made (Table 6 ) .

5

1 JJ

I A

I 0

3

0

0

3

aD (0

U

hl

0 0

to

0

0

.o

7

0

0

m

c

0 0

0

hl

8

d

m

0 0

0

*

I24

Fig. (5) PMR spectrum of Clonidine HC1 in DMS0.D6.

Fig. (6) PMR spectrum of Clonidine HCl in DMSO.D6 (DiO Each.)

CLONIDINE HYDROCHLORIDE I21

Table 5: IR characteristics of clonidine HC1.

Frequencies Approximate description of vibrational modes

3320 NH stretch

3000-3080 Chlorophenyl CH stretch

1650, 1600, 1565 Iaidazolidine ring stretch

1440, 1400 Phenyl ring stretch

1330, 1280

1190, 1100

Chlorophenyl C-H planar bend

Chlorophenyl C-C1 stretch bends.

790, 780 -

Table 6: FMR characteristics of clonidine HC1 structure

Protons 6 (PPM) Multiplicity

g, d (two protons) 8.618 singlet

a,b,c (three protons) 7.439-7.661 multiplet

e, f ( four protons) 3.672 singlet

I28 M.A. ABOUNASSIF. M.S. MIAN, ANDN.A.A. MIAN

3.17.3.2 13C-NMEI S p e c t m

13C-NMR spectrum (12) of clonidine hydrochloride Fig. (7-9) was recorded in DMSO-d6 by Varian XL-200 MHz NMR spectrometer. The multiplicity of the resonances was obtained from DEPT (Distortionless enhancement by polarization transfer) and APT (attached proton test), The carbon chemical shifts are presented in Table ( 7 ) .

Table 7: C-13 chemical shifts of clonidine HC1.

Carbon assignment Chemical shift 6 (ppm)

cs, c9

c1, c 3

CS, c 4

cz

c5

CI

42.647

129.121

133,987

130.772

130.262

157.919

3.17.4 Mass SDectrum

The mass spectrum ( 1 2 ) of clonidine hydrochloride obtained by electron impact ionization (Fig. 10) was recorded on a Finnigan MAT 90 mass spectrometer. The spectrum was scanned from 50 to 500 8.m.a. The electron energy WAS 70 ev. Emission current 1 mA and ion source pressure 10-6 t o r r . The spectrum shows a

Fig. (7) 13C.NMR spectrum of Clonidine HC1 in DMSO-D6.

Fig. (8) 13C,NMR spectrum of Clonidine HC1 i n DMS0.D6 (APT)

Fig. (9) 13C.NMR spectrum of Clonidine HC1 in DMS0.D6 (DEPT).

100.0

50.

- I ' I I

50 100 150 100.01

I 50.

Fig. (10) M a s s spectrum of Clonidine HCJ,


molecular ion M+ at m/z 229 with a relative intensity 100%. The most prominent fragments and their relative intensities are listed in Table 8 .

4 . Synthesis

Clonidine is synthesized (13) by the condensation of 2,6-dichloroaniline and imidazoline.

2,6-Dichloro- Imidazoline Clonidine ani 1 ine

5 . Pharmacokinetics

5.1 AbsorDtion and Dietribution

Clonidine hydrochloride is readily absorbed by ora l route with an absorption time of 2 to 4 hours (9). Drug is well absorbed from the gastro-intestinal tract. I t may also be absorbed when applied topically to the eye, clonidine is well absorbed percutaneously following topical application of a transdermal system t o t h e arm or chest. Plasma clonidine concentrations of 2 ng/mL have been detected one hour after administration of a single 0.39 mg oral dose of radiolabeled drug. Peak plasma concentrations following oral administration occur in approximately 3-5 hours (1).

Reduction in blood pressure is maximal at plasma clonidine concentrations less than 2 ng/mL. Blood pressure begins to decrease within 30-60 minutes after an oral dose of clonidine hydrochloride, the maximum decrease occurs in approximately 2-4 hours. The hypotensive effect lasts up t o 8 hours. Following administration of clonidine by slow intravenous injection in patients with acute hypertensive crises, a hypotensive effect occurred within minutes, peaked in 30-60 minutes and lasted more than 4 hours (1).

I34 M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MlAN

Table 8: The mass fragments of clonidine HC1

m/z Relative intensity % Fragment

230

229

221

207

200

196

194

193

186

174

172

165

147

124

109

73

65

100

10

12

20

22

52

17

11

45

54

20

18

18

20

17


Animal studies indicate that clonidine is widely distributed into body tissues, tissue concentration of the drug are higher than the plasma concentration. After oral administration highest concentrations of the drug are found in the kidneys, liver, spleen, and GI tract. High concentrations of the drug also appear in the lacrimal and parotid glands. Clonidine is concentrated in the choroid of the eye and is also distributed into the heart, lungs, testes, adrenal glands, fat and muscle. The lowest conc. occurs in the brain. Clonidine is distributed in CSF. It is not known whether the drug crosses the placenta. Clonidine is distributed into milk (1).

The plasma half life of clondine is 6-20 hours in patients with normal renal function. The half life in patients with impaired renal function has been reported t o range from 8-41 hours. The elimination half life of the drug may be dose dependent, increasing with increasing dose ( 1).

The drug is metabolized in the liver. In humans, 4-metabolites have been detected but only one, the inactive p-hydroxylated derivative, has been identified ( 1 ) .

In humans 65 % of administered dose of clonidine hydrochloride is excreted by the kidneys, 3 2 X as unchanged drug and the remainder as inactive metabolites. Approximatly 20 % of dose is excreted in feces, probably via entrohepatic circulation. Approximately 85 % of a single dose is excreted with 72 hours and excretion is complete after 5 days (1).

5 . 2 Uses and Administration

Clonidine is an antihypertensive agent which appears to act centrally by stimulating az-adrenergic receptors and producing a reduction in sympathetic tone, resulting in a fall in diastolic and systolic blood pressure and a reduction in heart rate. It also acts peripherally, and this peripheral activity may be responsible for the transient increase in blood pressure seen during rapid intravenous administration as well as contributing to the hypotensive effect during chronic administration. Peripheral resistance is reduced during continuous treatment. Cardiovascular


reflexes remain intact so postural hypotension occurs infrequently. When given by mouth its effects appear in about 30-60 minutes reaching a maximum after 2-4 hours as lasting up to 8 hours ( 4 ) .

Clonidine hydrochloride is used in the treatment of grades of hypertension. The usual initial dose of clonidine hydrochloride is 50 to 100 pg orally thrice daily increased every second or third day according to the response of the patient. The usual maintenance dose is 0.3 to 1 . 2 mg daily but doses of up to 1.8 mg o r more daily may be required. To reduce side effects a similar dose of clonidine may be given in conjunction with a thiazide diuretic but combination w i t h a Ij-blocking agent should be avoided where possible clonidine may also be given in a sustained- release formulation which enables twice-daily dosage, or by a transdermal delivery system which is applied once a week and delivers 100-300 pg daily at a constant rate ( 4 ) .

Drug may be given by slow intravenous injection in hypertensive crises usually in doses of 150 to 300 l.lg (419

It is also used in lower doses for the prophylaxis of migrane or recurrent vascular headaches and in the treatment of menopausal flushing ( 4 ) .

Clonidine hydrochloride has been used topically to reduce intraocular pressure in the treatment of open angle (chronic simple) and secondry glaucoma and hemorrhagic glaucoma associated with hypertension (1).

Because of its GI effects clonidine hydrochloride has been used with some success in a limited number of patients for the management of diarrhea of various etiologies (e.g. narcotic bowel syndrom, idiopathic diarrhea associated with diabetes) ( 1 ) .

5 . 3 Adverse Effects

Serious toxic effects have been reported after ingestion of doses of 0.4 to 4 mg by children and 4 to 11 mg by adults. However, recovery is usually rapid ( 6 ) .

CLONIDLNE HYDROCHLORIDE 137

Drowsiness, dry mouth, dizziness and headache commonly occur during the initial stages of therapy with clonidine. Fluid retention is often transient but may be responsible for a reduction in the hypotensive effect during continued treatment. Constipation is also common and other adverse effects which have been reported include depression, anxiety, fatigue, nausea, anorexia, parotid pain, sleep disturbances, vivid dreams, impotence, urinary retention or incontinence, slight orthostatic hypotension, and dry itching or burning sensations in the eye. Rashes and pruritus may occur and are more common with the use of transdermal delibery systems. Less frequently, bradycardia, including sinus bradjcardia with atrioventricular block, hallucinations, and transient abnormalities in liver function tests have been reported large doses have been associat.ed with initial increases in blood pressure and persist during continued therapy ( 4 ) .

Symptoms of overdosage include transient hypertension or profound hypotension, bradycardia, sedatjon, miosis, respiratory depression, and coma. Treatment consists of general supportive measures. An (1-adrenoceptor blocking agent may be given if necessary ( 4 ) ,

Clonidine withdrawal may result in an excess of circulating catecholamines. Therefore, caution should be exercised in concomitant use of drugs which effect the metabolism or tissue uptake of these amines (monoamine oxidase inhibitors or tricyclic antidepressants, respectively) (1).

5 . 4 Precautions

Clonidine should be used with caution in patients with cerebral, or coronary insufficiency, Raynaud’s disease or thromboangitis obliterans, or with a history of depression. The hypotensive effect may be antagonised by tricyclic antidepressants, and enhanced by thiazide diuretics. Clonidine cause drowsiness and patients should not drive or operate machinery where loss of attention could be dangerous. The effect of other cent.ra1 nervous system depressants mag be enhanced, withdrawal of clonidine therapy should be gradual as sudden discontinuation may cause rebound hypertension which may be severe. Agitation,

I38 M.A. ABOUNASSIF. M.S. MIAN, AND N.A.A. MIAN

sweating, tachycardia, headache, and nausea may also occur. 0-blockers can exacerbate the rebound hypertension and if clonidine is being given concurrently with a D-blocking agent, clonidine should not be discontinued until several days after the withdrawal of the B-blocker. It has been suggested that patients should be warned of the risk of missing a dose or stopping the drug without consulting their doctor and should carry a reverse supply of tablets ( 4 ) .

Although hypotension may occur during anaesthesia in clonidine-treated patients clonidine should not be given intravenously during the operation to avoid the risk of rebound hypertension. Intravenous injections of clonidine should be given slowly to avoid a possible transient pressor effect especially in patients already receiving other antihypertensive agents such as guanethidine or reserpine ( 4 ) .

Abrupt withdrawal of oral clonidine therapy may result in a rapid increase of systolic and diastolic blood pressure, with associated symptoms as nervousness, agitation, restlessness, anxiety, insomnia, headache, sweating, palpitation increased heart rate, tremor, hiccups, stomach pains, nausea, musc1.e pains, and increased salivation (1).

6 . Methods of Analysis

6 . 1 Identification

1) Dilute a volume containing 0.3 mg of clonidine hydrochloride to 5 m l with 0.01 M hydrochloric acid. The light absorption of the resulting solution in the range of 245 to 350 nm exhibits maxima at 272 nm and 279 nm and inflection at 265 nm ( 8 ) .

2 ) To a volume containing 0.15 mg of clonidine hydrochloride add 1 ml of a 10% w/v solution of ammonium reineckate and allow to stand for 5 minutes. A pink precipitate is produced (8).

3 ) The drug gives Libermann’s color test yellow to orange at 100°C ( 6 ) .

4 ) Gives characteristics reaction of chlorides (8).

CLONlDlNE HYDROCHLORIDE I39

5) The infrared absorption spectrum is concordant with the spectrum of clonidine hydrochloride (8).

6 ) Dissolve 0 . 2 g in 70 ml of ethanol (96%) and titrate with 0 . 1 M ethanolic sodium hydroxide vs determining the end point potentiometrically. Each ml of 0 . 1 M ethanolic sodium hydroxide vs is equivalent to 0.02666 g of C6HgClzN3.HCl ( 8 ) .

6.2 Colorimetric

Tawakkol et al, (14 ) developed a method for the colorimetric determination of colonidine in which clonidine reacts with sodium nitroprusside in presence of sodium hydroxide, and on treatment with saturated boric acid it gives a violet color which was measured a t 570 nm.

1.0 mg of powdered tablets were shaked with 10 ml of H20 and centrifuged, decant the clear solution into a 100 ml separating funnel. Repeat two times each Kith 15 ml of distilled H2O collecting in the same separating funnel, then add 1 ml of sodium carbonate and extract three times with chloroform, extract on a water bath and few drops of HC1 were added, evaporate and extract with few ml of distilled H2O. Then standard or test solution was treated with 0.8 ml of 1 N NaOH solution followed by 1 m l of sod. nitroprusside, mixed and leave for 10 min, then 2 ml of 4% boric acid solution was added leave in an ice bath for 10-15 minutes. Complete up to the mark and measure the violet color of both the standard and the test at 570 nm.

Sane (15) developed a method for the estimation of clonidine hydrochloride in pharmaceutical preparations by ion pair extraction and colorimetric method. An acid-dye complexing method with bromophenol blue, broaocresol purple and methyl-orange was used for the ion-pa.ir extraction and colorimetric determination of clonidine hydrochloride in pharmaceuticals containing 100 mg of clonidine hydrochloride was 98.9% and relative standard deviation 0.89%.

I40 M.A. ABOUNASSIF, M.S. MIAN, AND N.A.A. MIAN

6.3 Pluorimetric

A very sensitive fluorimetric method ( 1 6 ) based on the reaction of clonidine hydrochloride with 1-dimethylaminonaphthalene-5-sulphonyl chloride (dansyl chloride) to give a highly fluorescent derivative.

Dissolve 50 mg of clonidine hydrochloride in a mixture of 10 ml of acetone and 40 ml of 0.5 M sod. carbonate solution. Transfer to a 50 ml flask make up to the mark with dansyl chloride and acetone was added and then 4-methyl pentan-2-one was added. Fluorescence intensity was measured after 10 minutes at 455 nm using an excitation wavelength of 345 nm.

6.4 Spectrophotometric Analysis

A simple and rapid spectrophotometric method ( 1 6 ) based on the reaction of clonidine hydrochloride with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone to form a colored product with maximum absorption at 455 nm.

Accurately weighed amount of clonidine hydrochloride equivalent to 50 mg of the base was dissolved in about 20 ml distilled HzO, made alkaline with few drops of 10% w/v NaOH solution and extract with five successive 10 ml portions of chloroform. Pass the chloroform extracts sequentially over anhydrous sod. sulphate and collect the combined chloroform extracts in 50 ml flask make up to volume with chloroform. Heat and dissolve in acetonitrile and 2,3-dichloro-5,6-dicyano-1,4-benzoquinone and measured at 455 nm against. blank.

An other simple and sensitive spectrophotometric method based 011 color reaction with bromocresol green WAS studied by Zivanov-Stakic et a l . ( 1 7 ) for the determination of colonidine hydrochloride in tablet form.

Another method (18) reported the spectroscopic determination of clonidine hydrochloride.

6 . 5 Radio-Immunoassag

Radio-immunoassay for clonidine in human plasma a.nd urine using a s o l i d phase second-antibody separation was studied by Farina et al. ( 1 9 ) . In which


plasma was incubated for 1 8 hour at 4" with 0 . 1 M sodium phosphate buffer (pH 7.4). 1251-labeled 4-carboxyclonidine-tyrosine and antibodies raised in rabbit against 4-carboxyclonidine N-hydrosuccinimide ester conjugated with bovin serum albumin. After a second incubation with immuno beads of goat anti-rabbit immunoglobulin for 2 hours at room temperature, the mixture was centrifuged and radioactivity in the pallets was counted. The detection limit was 10 pg per ml of clonidine, the within and between assay coefficient of variation were 2.8 to 9 and 10 to 13% respectively.

A newly developed and precise and sensitive radio-immunoassay f o r clonidine was done by Arndts et al. ( 2 0 ) . The antiserum is raised in rabbits injected with the pcarboxy derivative of clonidine and is used in the radioimmunoassay for clonidine in the residues of ethyl ether extracts (pH 9.5) of 0.2 ml of plasma [3H] clonidine being used as a tracer in a phosphate buffer medium (pH 7 . 4 ) . After incubation for 18 hours at 4'C unbound antigen is adsorbed on the dextran-coated charcoal, and the bound 3H is determined. A calibration graph is constructed for 0 . 1 to 10 ng per ml of clonidine in plasma.

Another method ( 2 1 ) of clonidine in rats as determined by radio-immunoassay. In which an antigen prepared by reacting 4-hydroxyclonidine with 4-carboxybenzene diazonium chloride and coupling the product t o bovin serum albumin was used to raise an antiserum in rabbits. [3Hl Clonidine or [14C!1 clonidine was used as tracer and separation of the free and bound forms of the antigen was carried out. The sensitivity was 1 0 pg with f3H1 clonidine and 600 pg with [14C] clonidine.

I t has been possible to measure plasma and tissue levels after the administration of rather high doses of radiolabelled clonidine to humans and animals ( 2 2 - 2 5 ) .

6.6 Chromatographic Methods

6.6.1 Gas-Liquid Chromatography (GLC)

1 ) Determination of submicrogram quantities of clonidine in biological fluids by Chu (26). Clonidine


is extracted from plasma, extract is purified by solvent extraction and column chromatography and clonidine is converted to heptaf luorobutyryl derivative for g.1.c at 175' on a partially inactivated column cntaining 3% OV-17 on Chromosorb W AW DMCS with electron capture detection. The 4-methyl analogue of clonidine is used as internal standard. The limit of determination was 2 5 pg ml-1, and the coefficient of variation at the level of 60 pg ml-1 was approximately 8%.

2) The method (27) describes the measurement of clonidine in human plasma and urine by combined gas chromatography-mass spectrometry with ammonia chemical ionization. Addition of [2H4] clonidine to plasma or urine is followed by ethylacetate extraction of clonidine from alkaline medium, back extraction into acid extraction into ethyl ether from alkaline medium and evaporation of the extract to dryness. Trirnethylanilinium hydroxide is added to the residue, and dimethyl derivatives of clonidine are formed by on column methylation with an injection-port temperature of 250' for g.c. -70-eV m , s , , the glass column (1.8 m x 2 mm) packed with 3% of OV-17 on Gas-Chrom Q (100 to 120 mesh) is operated at 245". With He as carrier gas (15 m l min-l); NH3 is admitted to an ion-source pressure of 0.2 Torr, and ions are monitored at m/e 258 and 264. Graphs of peak height ratios (n/e 258 to 264) vs amounts of clonidine in urine (up to 40 ng ml-l) and in plasma (up to 5 ng ml-1) are rectilinear. The precision for assay of clonidine in plasma is 11% at 0.25 ng ml-1 and 5% at 0 . 5 ng ml-l and the lower limit of determination is 0.1 ng ml-1.

3 ) A simple and sensitive gas-liquid chromatographic method ( 2 8 ) has been developed for the quantitative determination of clonidine and some structurally related imidazolidines in rat brain tissues. The aqueous brain homogenates are first purified and t,hen extracted into benzene. Samples are injected directly to GLC column ( 2 m x 2 mm I.D.) pa.cked with 3% OV-17 on chromosorb 750 (80-100 mesh) was used at an oven temperature of 200-270' and an injector temperature of 280" the carrier gas was helium; flow rate 30 ml/min,


4 ) A gas chromatography assay for clonidine in human plasma has been developed by per Olof Edlund (29). The buffered serum is extracted on silica columns, alkylated with pentrafluorobenzyl bromide, clertned up by extractions and analysed by glass-needle injection and electron-capture detection. The packed column (2 m x 3 mm I.D.) was silanized glass and was packed with 3% of OV-17 on 80-100 mesh. Gas-Chrom Q.

5 ) Another method (30) for the determination of clonidine in plasma by G.L .C . 2-(2,4-dichloroaniline)-2-imidazoline is used as internal standard. The column (WCOT: 30 m x 0 . 3 5 mm) was operated at 250'C with H2 as carrier gas.

Other methods used for the GLC determination of clonidine hydrochloride in biological materials (31,321. Recently GC was applied to the measurement of picogram levels of clonidine hydrochloride after derivatization ( 3 3 ) also by (34 -36) .

Advantages of fused silica capillary gas chromatography (FSCC) for conventional GC method (37).

6 . 6 . 2 . High-Performance Liauid ChmmatograDhp ( HPLC )

1 ) A rapid, reversed-phase high-performance liquid chromatography (HPLC) method (38) is described for the determination of clonidine in tablets. Individual tablets or composite samples were sonicated in water, diluted with methanol and filtered. Clonidine formulated at 0.1 or 0.2 mg/tablet was chromatographed on trimethylsilyl-bonded, 5 to 6-ym spherical silica with 65% methanol in pH 7.9 phosphate buffer as mobile phase detection at 254 nm. Mean recovery from 6 synthetic tablet samples was 99.7% (at 0.1 mg/tablet level) with relative standard deviation of 1.55%.

2) A sensitive, selective and reproducible assay for clonidine hydrochloride in tablets and eye drops were described (39). A Nucleosil 5 Cis colum (125 mm x 4 . 6 mm - I.D.) with methanol-water 80:20 containing 0.005% of TEA as the mobile phase at a flow rate of 1 ml per minute at 240 nm UV detection and attenuation, 0.02 a . u . f . s . in tablets and 0.16 a.u.f.s. in eye drops and recorder chart speed, 0.5 crn min-l.

144

7.

8.

1.

2 .

3.

4.

5 .

6.

7 .

a.

9.

10

M.A. ABOUNASSIF, M.S. MIAN. A N D N.A.A. MIAN

Acknowledgements

The authors are highly thankful to Mr. Babkir Awad Mustafa, College of Applied Medical Sciences for his efforts in drawing the spectras and figures, The authors also would like to thank Mr. Tanvir A. Butt, College of Pharmacy, King Saud University, for his valuable and professional help in typing the manuscript.

Beferences

"Drug Information 90" American Society of Hospital Pharmacists Inc. 4630 Montgomery Avenue, Bethesda M.D. 20814, p. 910-915.

Vivian Cody, presented in part at the American Crystallographic Association Meeting, Clemenson, South Carolina, January 1976,

Goodman, L . S. and Gi lman, A . "The Pharmacological Basis of the Therapeutics", 4th ed., 1970, p. 735.

"Martindale", The Extra Pharmacopoeia, 29th Ed. Editor James E.F. Reynolds, The Pharmaceutical Press, London, p. 472 (1989).

"Physician's Desk Reference", 42nd Ed. , Medical Economics Company, USA, p. 718 (1988).

"Clarke's Isolation and Identification of Drugs" 2nd ed., A.C. Moffat Edit., p. 481-482. 'The Pharmaceutical Press' London (1986).

"The Merck Index", 11th Ed., Merck and Co. , Inc., Rahway, N.J., p. 374 (1989).

"The British Pharmacopoeia", Her Majestys Stationary Office, London, p. 147 (1988).

"Remlngton's Pharmceutical Sciences", 16th ed., Mack Publishing Company Easton, Pennsylvania, 18042, p. 785 (1985).

Gudmud Byre, Arvid Mostad and Christian Romming Acts Chemica Scandinavica, m, p. 843-846 (1976).


11.

12 .

13.

1 4 .

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1 7 .

18.

19.

20.

21.

2 2 .

2 3 .

2 4 .

Vivan Cody and Corae J., Detitta, J. of Crystal and Molecular Structure, 9 ( 1 ) , p . 33-43 ( 1 9 7 9 ) .

Mohammad Saleem Mian and Neelofur Abdul Aziz Mian, unpublished data (1992) .

"The Organic Chemistry of Drug Synthesis" Vol. 1, p. 241 , by Daniel Lednicer, Lester A. Mitscher, John Wiley dr Sons, New York.

M.S. Tawakkol, A.I. Jado and H.Y. Aboul-Enein, Arzniem.-Forsch 31, 1064-66 (1981) .

Sane, R.T. , Thombare, C.H. Indian Drum, 1 8 ( 9 ) , 335-7 (1981) .

Fawzy, A,, El-Yazbi, Mona Badair, and Mohamed A. Analyst, Vol. III (1986) .

Zivanov-Stakic, D., Panic, L . J . and Agbaba, G . Farmaco 45, 381-383 (1990) .

Kovar, K.A. and Abdel-Hamed, M., Arch. Pharm., 317, 246 ( 1 9 8 4 ) .

Farina, P.R., Homon, C . A . , Chow, C.T., Keirns, J . J . , Zavorskas, P.A., Esber, H.J. Ther. Drug. Mointo., 7 ( 3 ) , 344-350 (1985) .

Arndts, D., Staehle, H. and Struck, C.J., Arznei,-Forsch., 29(1) , 532-538 ( 1 9 7 9 ) .

Jarrot, Revyn, and Spectro, Sydney. J. Pharmacol. EXP. Ther. 2 0 7 ( 1 ) , 195-202 ( 1 9 7 9 ) .

D. Rehbinder and W. Deckers. Arzneim.Forsch. (Drug Rea), l9, 169 ( 1 9 6 9 ) .

D. Rehbinder, in A. Zahchett and M. Envico (Editors) Ipertension Arteriosa, Boehringer Ingelheim, p . 3 ( 1 9 7 3 ) .

J . P . Fillastre, D , Dubois and P. Brunelle, in A. Zanchett, and Enrico (Editors) IDertension Arteriosa Boehringer Ingelheim, p. 81 (1973) .


25.

26.

27.

28.

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32,

33.

34.

35.

36.

37.

S. Darda, in P. Millies and M. Safar (Editors). Recent Advances in Hypertension, Boehringer, Ingelheim, p. 375 (1975) .

Chu, L.C. , Bayne, W.F. , Tao, F.T., Schmitt, L.G. and Shaw, J.E. J. Pharm. Sci. 6 8 ( 1 ) , 72-74 (1979) .

Murray, S., Waddeil, K . A . and Davies, D.S. Biomed. Mass SDectrom, 8 ( 1 0 ) , 500-502 (1981) .

P.B.M.W.M. Timmermans, A. Brands and P.A. Van Zwieten. J. Chromatogr., 144, 215-222 (1977) .

Per Olof Edlund. J. Chromatogr., 187, 161-169 (1980) .

Hiltunen, R . , Maryola, M., Hirsjarvi, P. and Raisanen, S. Acta Pharm. Fenn., 8 8 ( 4 ) , 161-167 1979) .

A.K. Cho and S.H. Curry, Biochem. Pharmacol., l8, 511 (1969) .

C.T. Dollery, D.S. Davies, G.H. Draffan, H.J. Dargie, C . R . Dean, J.L. Reid, R . A . Clare and '3. Murray, Clin. Pharmacol. Ther., l8, 11-17 (1976) .

S. Murray and D.S. Davies. Biomed. Mass Spectrom, 11, 435 (1984) .

P . O . Edlund and L . K . Paalzow, Acta Pharmacol. Toxicol., 40, 145-152 (1977) .

A . K . Cho and S.H. Curry. Biochem. Pharmacol. 18, p. 511 ( 1 9 6 6 ) .

A. Frydman, Y. Weiss, M. Safar and J.M. Alexandre, in P. Millies and M. Safar (Editors), Recent Advances in Hypertension, Boehringer, Ingelheim, p. 369 (1975) .

J . Settlage and H. Jaeger. J. Chromatogr. Sci. 22, 192-197 (1984) .

CLONlDINE HYDROCHLORIDE I47

38. Walters, Stephen, M., Stonys, Dalia, B. J. Chromatom. &, 2 l ( l ) , 43-5 (1983) .

39. I. Wilezynska-Wojtulewicz and N. Sadlej-Sosnowska. JI Chromatogr., 367, 434-437 (1986).

CYCLANDELATE

Charles M. Shearer

Wyeth-Ayerst Research

Rouses Point, NY 12979


Copyright Q 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

I50 CHARLES M. SHEARER

1. Description

2. Synthesis 3. Physical Properties

I . 1 Name Formul a Mol ecul ar Weight 1.2 Appearance, Color and Odor

3.1 Nuclear Magnetic Resonance Spectra 3.2 Infrared Spectrum 3.3 Ultraviolet Spectrum 3.4 Mass Spectrum 3.5 Melting Point 3.6 Di fferential Scanning Calorimetry 3.7 Solubility 3.8 Crystal Properties

4. Stability and Degradation 5. Metabolism 6. Analysis

6.1 Elemental Analysis 6.2 U1 traviolet Spectrophotometry 6.3 Titrimetry 6.4 Gas Chromatography 6.5 High-Performance Liquid Chromatography 6.6 Thin Layer Chromatography

7. Identity 8. References

CY CLAN DELATE 151

1. D e s c r i p t i o n 1.1 Name. Formula. M o l e c u l a r Weiqht

The name used by Chemical A b s t r a c t s f o r c y c l a n d e l a t e i s a-hydroxybenzeneacet ic a c i d , 3,3,5- t r i m e t h y l c y c l o h e x y l e s t e r . 3 ,3 ,5 - t r ime thy l c y c l ohexy l e s t e r ; 3 ,3 ,5 - t r ime thy l c y c l ohexy l mandel a te; 3 ,3,5- t r imethy l c y c l ohexy l amygdal a te ; and 3,3,5- t r i m e t h y l c y c l ohexanol a-phenyl -a-hydroxyacetate. i n c l ude, Cyc l ospasmol , Nat i 1, Novodi 1 , Pereb ra l , and Spasmocyclon (1).

It i s a l s o c a l l e d mande l i c ac id ,

Trade names

The Chemical A b s t r a c t s number i s 456-59-7.

1.2 Appearance, C o l o r and Odor

powder w i t h a s l i g h t m e n t h o l - l i k e odor. Cyc lande la te i s a w h i t e t o o f f - w h i t e amorphous

'17"24'3 M. W . 276.36

2 . Syn thes is T r i m e t h y l c y c l o h e x y l mandelate was f i r s t syn thes i zed by

r e a c t i n g a - m a n d e l i c - a c i d wi th 3 ,3 ,5 - t r ime thy l c y c l ohexanol ( c o n s i s t i n g o f c i s and t r a n s isomers) (2,3,4). C y c l a n d e l a t e i s now syn thes i zed u s i n g o n l y t h e l o w m e l t i n g ( c i s ) isomer o f 3,3,5-trimethylcyclohexanol (5,6). Es te rs o f mande l i c a c i d wi th t h e h i g h e r m e l t i n g 3,3,5-trimethylcyclohexanol a r e t w i c e as t o x i c as those made w i t h t h e l o w m e l t i n g isomer (7 ) . ma jo r s i d e r e a c t i on p roduc t , tri met h y l c y c l ohexyl phenyl g l y o x a l a t e , can be removed d u r i n g t h e s y n t h e s i s by t r e a t i n g t h e c rude c y c l a n d e l a t e w i t h aqueous sodium bo rohydr ide (8) o r z i n c and h y d r o c h l o r i c a c i d ( 9 ) .

T h i s s y n t h e s i s , u s i n g o n l y t h e c i s isomer, r e s u l t s i n f o u r isomers as d e s c r i b e d i n t h e n e x t s e c t i o n .

The

152 CHARLES M. SHEARER

Figure 1 - Proton NMR Spectrum of Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361) in deuterated chloroform

CYCLANDELATE 153

I

F igure 2 - Carbon -13 NMR Spectrum o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361) in deuterated chloroform


3. Phvsical ProDert ies

i n the synthes is from t h e reac t i on o f a - m a n d e l i c a c i d w i t h - cis-3,3,5-trimethylcyclohexanol and are descr ibed i n Table I (taken from Nakamichi (10)).

3.1 Nuclear Maanetic Resonance SDectra The fou r isomers which make up cyc lande la te a r i s e

Table 1 Isomers o f Cvcl andel a te

Absolute con f igu ra t i on a Absol Ute c o n f i g u r a t i o n Isomer o f mandelic ac id moiety o f c y c l ohexanol moiety

P o s i t i o n 1 P o s i t i o n 5

A B C D

R R S S R R S S

a) The cyclohexanol mo ie t ies o f A,C and B,D are l e v o r o t a t o r y and dex t ro ro ta to ry , respec t i ve l y (11). The absolute con f igu ra t i on o f ( - ) -c is -3 ,3 ,5 - t r imethy l c y c l ohexanol i s assigned as R on the bas is o f i t s chemical c o r r e l a t i o n w i t h pu l egone (12).

The pro ton NMR sample (Wyeth-Ayerst Reference Standard No. 1361) was d isso lved i n deuterated ch lo ro fo rm con ta in ing te t ramethy l s i l a n e as an i n t e r n a l standard. The spectrum was obta ined (13) on a 400 MHz Bruker spectrometer and i s presented as F igure 1. The spec t ra l assignments are l i s t e d i n Table 11. The C-13 NMR sample was a l so prepared i n deuterated ch loroform and i t s spectrum obta ined (13) on a 100 MHz Varian spectrometer. F igure 2 and t h e spec t ra l assignments are l i s t e d i n Table 111. The spect ra are i n agreement w i t h those o f Nakamachi (10).

The spectrum i s presented as

3.2 I n f r a r e d Soectrum The i n f r a r e d spectrum o f a K B r p e l l e t o f

cyc l andel a te (Wyeth-Ayerst Reference Standard No. 1361) was obtained (14) on a N i c o l e t 20 DX inst rument and i s presented as F igure 3. The spec t ra l band assignments are g iven i n Table I V .

CYCLANDELATE

4000 3000 2000 1500 1000 500 Wavenumber (crn-1)

Figure 3 - Infrared Spectrum o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361) KBr pellet


Table I 1 P ro ton NMR Spec t ra l Assisnments o f Cvcl ande la te

Chemical S h i f t Number o f Assignment (ppm f r o m TMS) Protons 7.4 5 Aromat ic CH 5.10 d 1 - H-C-OH 4.95 m 1 - H-C-OC 3.47 exchangeabl e 1 - H-0 2.1 - 0.6 17 A l i p h a t i c CH, CH CH3

0.94 s gem C I - J ~ (AB p a i g j 0.88 s gem CH3 (CD p a i r ) 0.84 d ( J = 6) HC-CI-J3 (AB p a i r ) 0.91 d (J = 6) HC-Ct i3 (CD p a i r )

Table I11 Carbon-13 NMR S p e c t r a l Assisnments f o r Cvc l andel a t e

Carbon PPm

1 73.3 2 43.7 (AB) 43.2 ( C D ) 3 32.2 (AB) 32.1 (CD) 4 47.3 5 27.0 (AB) 26.9 ( C D ) 6 39.7 (AB) 40.1 ( C D ) 7 32.9 (AB) 32.8 (CD) 8 25.4 (AB) 25.3 (CD) 9 22.0 (AB) 22.1 (CD) 1 173.1 2 72.8 1 138.6 2 , 6 126.3 3 , 5 128.4 4 128.1

Table I V I n f r a r e d Spec t ra l Assisnments f o r Cvcl andel a t e

Wavenumber ( C m - l ) V i b r a t i o n Mode

3460 3100 - 2800 1730 1212, 1192 730, 695

OH s t r e t c h CH s t r e t c h CEO s t r e t c h C-0-C s t r e t c h o u t - o f - p l a n e bending of monosubst i tu ted aromat ic

CYCLANDELATE 157

3.3 U l t r a v i o l e t SDectrum The u l t r a v i o l e t spectrum o f c y c l a n d e l a t e (Wyeth-

A y e r s t Reference Standard No. 1361 r e c r y s t a l l i z e d t o remove 0.1% 3 , 3 , 5 - t r i m e t h y l c y c l ohexyl phenyl g l y o x a l a t e ) i n USP e thano l i s presented as F i g u r e 4. The a b s o r p t i v i t i e s a r e as f o l l ows :

X max(nm) 269 258 251

a € 0.57 1575 0.73 2020 0.59 1630

3.4 Mass SDectrum The mass spectrum o f c y c l a n d e l a t e was o b t a i n e d (15)

by e l e c t r o n impact i o n i z a t i o n u s i n g a Finnegan MAT 8230 spect rometer and i s g i v e n as F i g u r e 5. I d e n t i f i c a t i o n o f t h e p e r t i n e n t masses i s presented i n Table V .

Table V Mass Spectrum Fraqmentat ion P a t t e r n o f Cvcl andel a t e

m/e Species

276 Mt

125 '9"17'

107 C6H5CHOHt

83 CH2CHCH2C ( CH3) *t

79 '6"5'

69 CH2CHCH2CHCH3t

55 ( CH3) C C H 2 t

3.5 M e l t i n g Ranqe Observed (16) m e l t i n g range (USP I a ) f o r

c y c l a n d e l a t e (Wyeth-Ayerst Reference Standard No. 1361) i s 55.0" - 56.5"C.


Figure 4 - Ultraviolet Spectrum o f Cyclandelate (Wyet h -Ayers t Reference Standard No. 1361) i n USP alcohol

CYCLANDELATE

20

111

0 50 100 150 200 250

mie

Figure 5 - Mass Spectrum o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361)


3.6 Di fferenti a1 Scanninq Calorimetry The DSC thermogram (14) for cycl andel ate (Wyeth-

Ayerst Reference Standard No. 1361) is presented as Figure 6. The thermogram was obtained at a heating rate of lO'C/minute in a nitrogen atmosphere utilizing a Perkin-Elmer DSC-2. The thermogram exhibits no endotherms or exotherms other than that associated with the melt.

3.7 Solubility

been observed (16). The following s

USP Classificat Sol vent Water Methanol Acetonitrile Ethyl acetate Di met hyl formami de To1 uene Chloroform

lubi ties at room temperature have

ons : Solubil itv i nsoubl e very soluble freely soluble freely soluble freely soluble freely soluble very soluble

3.8 Crystal ProDerties The X-ray powder diffraction pattern of

cycl andel ate (Wyeth-Ayerst Reference Standard No. 1361) obtained (14) with a Phillips diffractometer using copper Ka radiation is presented as Figure 7. The calculated "d" spacings are given in Table VI.

d m; 19.04 11.72 9.55 7.80 7.34 6.77 6.11 5.59 5.27 4.97

Table V I X-Ray Diffraction Pattern

mo

100 4 5 40 34 15 21 13 9 21

- d

4.72 4.56 4.42 3.99 3.90 3.85 3.77 3.71 3.57 2.65

69 11 14 32 15 13 17 15 8 8

CYCLANDELATE 161

I I I I

20 40 60 80 100 120

Temperature (C)

Figure 6 - Differential Scanning Calorimetric Thermogram of Cycl andelate (Wyeth-Ayerst Reference Standard No. 1361)


4 13 22 31 40 DEGREES 2 THETA

Figure 7 - X-Ray D i f f r a c t i o n Pattern o f Cyclandelate (Wyeth-Ayerst Reference Standard No. 1361)

CYCLANDELATE 163

4. Stability and Desradation Cycl andel ate can decompose by hydrolysis to mandel ic

acid and 3,3,5-trimethylcyclohexanol (17). It is oxidized to 3,3,5-trimethyl cyclohexyl phenylglyoxal ate (18).

A study of the formation of 3,3,5-trimethylcyclohexanol in cyclandelate capsules concluded that less than 5% of the cycl andel ate degraded in 66 months at ambient temperatures (17) *

5. Metabol i sm

phenylglyoxyl ic acid and 3,3,5-trimethylcyclohexanol. These are detectable in the urine of rabbits and humans in less than two hours after oral administration (19,20). The ratio o f mandelic acid to phenylglyoxylic acid increases with increased dosage (21). Another metabolic study in humans showed that the maximum blood levels of mandelic acid were reached in 0.5 to 1.5 hours after oral dosing (22).

A pharmacokinetic study using tritiated cyclandelate shows that most organ specimens took up the radioactivity rapidly; usually reaching a maximum within one hour. The brain, diaphragm, stomach and vein specimem showed a maximum level at 24 hours. The levels gradually declined in a non- linear manner over 28 days (23).

6. Analysis

The metabolites of cyclandelate are mandelic acid,


Element Theory Found (24)

C 73.88% 73.95% H 8.75% 8.55%

6.2 Ultraviolet SDeCtrODhOtOmetrY Di rect determination of cycl andel ate by UV

spectrophotometry is not practical since the oxidative degradation product, 3,3,5-trimethyl cycl ohexyl phenylglyoxalate has about 55 times the absorptivity (25). Spectrophotometri c determinations of cycl andel ate after hydrolysis to mandel ic acid and oxidation to benzaldehyde have been reported (26,27).


6.3 Titrimetrv Cycl andel ate can be determined by hydrolyzing the

ester in 0.5 N NaOH under reflux for 0.5 hours, then backtitrating the excess base with 0.1 N HC1 (28,29).

Gas chromatography has been used to analyze cycl andel ate and to separate it from its degradation products and impurities as well as from other pharmaceuticals. Table VI gives column conditions and other necessary data for the various methods.

6.4 Gas ChromatoqraDhv

Column

Table V I Gas ChromatoqraDhv of Cvcl andel ate

Oven Reference Temoera t ure

2 rn x 4 mm i.d.; 5% QF-1 on 160" (30) Chromosorb W(HP) 100/200 mesh

6 ft x 1/8 in; 3% QF 1 t 0.5% (31) HiEFF 8BP on GasChrom Q

25 m x 0.3 mm i.d.; deactivated, 125" for (32) coated w/S E - 3 0 13 min; 3'/min

200 O

to 180", hold 1 min.

30 m x 0.28 mm i.d.; FFAP 170" (10)

6 ft x 1/4 in i.d.; 15% Dexsil 220' (33) 300 on HP Chromosorb W 80/100 mesh

1 m x 3.2 mm; Tenax GC 60/80 mesh 140" for (34) 5 min., 20'/min to 240", lO'/min to 280"

6 ft x 4 mm i.d.; 2.5% SE30 on 200 O (35) 80/100 mesh Chromosorb G

CYCLANDELATE I65

6.5 Hiqh-Performance L i a u i d ChromatoqraDhv An HPLC system c o n s i s t i n g o f a Microbondapak CN (30

x 0.39 cm.) column, 65% methanol , 35% sodium a c e t a t e b u f f e r , a d j u s t e d t o pH 3.7 as t h e e l u a n t : and 254 nm UV l i g h t f o r d e t e c t i o n has been used (36).

6.6 T h i n Laver ChromatosraDhy The f o l l owing TLC systems have been r e p o r t e d :

P1 a tes So lven t Svstem R f Value Reference

S i l i c a Gel 254 Benzene (37)

S i l i c a Gel 254 Hexane 55 0.09 (38) Ch lo ro fo rm 45

S i l i c a G Ch lo ro fo rm 4 0.74 (39) Acetone 1

S i l i c a G E t h y l A c e t a t e 0.71 (39)

7. I d e n t i t y Cyc lande la te can be i d e n t i f i e d amongst many o t h e r drugs,

po i sons and b i o g e n i c compounds by gas chromatography (33 ) . D e t a i l s f o r t h i s procedure a re g i v e n i n S e c t i o n 6.4. odor and c o l o r i d e n t i f i c a t i o n t e s t s a r e g i v e n by Doorenboos and coworkers (28).

Severa l

8. References

1. The Merck Index, 1 1 t h ed., Merck and Co., Rahway NJ,

2 . N. V . K o n i n k l i j k e Pharmaceutische Fabr ieken voor .

(1989) page 421.

Brocades-Stheeman & Pharmacia, Dutch Pa ten t 68,704.

3. K. J. H. van S l u i s , Chemical Products , 11, 374(1954).

4. N. V . K o n i n k l i j k e Pharmaceutische Fabr ieken voorheen Brocades-Stheeman & Pharmacia, B r i t i s h P a t e n t 707,227.

5. A. B. H. Funcke, M. J . E. E r n s t i n g , R. F. Rekker, and W . Th. Natua, A r s n e i m i t t e l - F o r s c h . , 3, 503(1953).


6. N. V . K o n i n k l i j k e Pharmaceutische Fabr ieken voorheen Brocades-Stheeman & Pharmaci a, B r i t i s h Pa ten t 810,888.

Brocades-Stheeman & Pharmacia, Dutch Pa ten t 88,249. 7. N. V . K o n i n k l i j k e Pharmaceutische Fabr ieken voorheen

8. D. F l i t t e r , U n i t e d S t a t e s Patent 3,663,597.

9. H. Takahashi, U n i t e d S ta tes Pa ten t 3,673,239.

10. T. Amano, T. Kasahara and H. Nakamachi, Chem. Pharm.

11. M. J . E. E r n e s t i n g and W . Th. Nauta, Rec. Trav. Chim.

12. N. L. A l l i n g e r and C . K. Riew, J . Org. Chern.,

13. B. Hofmann, Wyeth-Ayerst L a b o r a t o r i e s , Personal

B u l l . , 3, 1106(1981),

Pay-Bas., 8 l , 751 (1962).

- 40, 1316 (1975).

Communication.

14. C . Long fe l l o w , Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication,

15. J . Cantone, Wyeth-Ayerst Labora to r ies , Personal Communication.

16. D. Berg, Wyeth-Ayerst Labora to r ies , Personal Commun i c a t i on,

17. 3. Richard and G. Andermann, Pharm. Ac ta Helv.,

18. M. J . E. E r n s t i n g , R. F . Rekker, J . H. Bos and

- 57, 116(1982).

W . Th. Nauta, Pharm. Weekblad, a, 605(1953).

19. M. J . E. E r n s t i n g , R . F . Rekker, A . 6. H. Funcke, H. M. Tersteege, and W . Th. Nauta, A r s n e i m i t t e l - Forsch, 6 , 245(1956).

20. M. J . E. E r n s t i n g , R. F. Rekker, H. M. Tersteege, and W. Th. Nauta, A r s n e i m i t t e l - F o r s c h , l2, 632(1962).

CYCLANDELATE I67

21. M . J . E. E r n s t i n g , R . F . Rekker, H. M . Tersteege and W . Th. Nauta, A r s n e i m i t t e l - F o r s c h , l2, 853(1962).

22. K. Koj ima, Y , Uezono, T. Takahashi and Y. Nakanish i , J . Chromatogr., 425, 203(1988).

23. A. O r r and J. R. W h i t t i e r , I n t . J . Nucl . Med. B i o l . , - 4, 205( 1974).

24. C. Kraml, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication.

25. R.F. Rekker, H . J . Doorenbos and W. Th. Nauta, Pharm.

26. L . Chafetz , J . Pharm. S c i . , 53, 1192(1964).

27. 6. Andermann, M. D i e t z , and D. Mergel , J. Pharm.

Weekbl ad, lO2, 946( 1967).

Be lg . , 3 4 , 233( 1979).

28. H. J . Doorenbos, H. J . van d e r Pol , R. F. Rekker and W. Th. Nauta, Pharm. Weekblad, 100, 633(1965).

29. J . Zhou, and C . Zhou, Yiyao Gongye, l7, 369(1986) f rom CA( 26) : 232528t.

30. R.T. Sane, V.B. Malkar , and V . G . Nayak, I n d i a n Drugs, - 22, 321(1985) f rom CA103(16):129151z.

31. D. Rodgers, Wyeth-Ayerst L a b o r a t o r i e s , Personal Communication.

32. G. Andermann and M. D i e t z , J

33. B. Kaempe, Arch. Pharm. Chem

365 (1981) .

145(1974).

Chromatogr., 223,

, S c i . Ed., 2,

34. M. D i e t z and G. Andermann, J . High. Resol .

35. B. S . F i n k l e , E . J . Cherry and D. M. T a y l o r ,

Chromatogr. & Chromatogr. Comm., 2, 635(1979).

J . Chromatogr. Sc i . , 9, 393(1971).


36. R. T . Sane, S . V . Desai and R. S. Samant, Ind ian

37. M. He and X . L i , Yaowu Fenxi Zazhi , 4, 40(1984) from

38. B. Kennedy, Wyeth-Ayerst L a b o r a t o r i e s , Personal

Drugs, a, 42(1986).

CAlOO(18): 1 4 5 0 7 5 ~ .

Communication.

39. A. H. Stead , R . Gi l l , T. Wright, J . P. Gibbs and A . C . Moffat , Analys t , 107, 1106(1982) .

FLEC AINIDE

Silvia Alessi-Severini, Ronald T. Coutts,

Fakhreddin Jamali, and Franco M. Pasutto

Faculty of Pharmacy & Pharmaceutical Sciences

University of Alberta

Edmonton, Alberta, Canada, T6G 2N8


Copyright D 1992 by Academic Press, Inc. All rights of reproduction resewed in any form. 169

SILVIA ALESSI-SEVERINI ET AL.

TABLE OF CONTENTS

1. Description 1.1 1.2 Appearance, Color, and Odor 1.3 History

Nomenclature, Formula and Molecular Weight

2. Synthesis 2.1 Synthesis of Flecainide Acetate 2.2 Preparative Separation of Flecainide Enantiomers

3. Physical Properties 3.1 Infrared Spectrum 3.2 NMR Spectra 3.3 Mass Spectrum 3.4 Ultraviolet Spectrum 3.5 Optical Rotation and Absolute Configuration 3.6 Melting Point 3.7 Ionization Constant 3.8 Distribution Coefficient 3.9 Solubility 3.10 Stability

4. Methods of Analysis 4.1 Elemental 4.2 Spectrophotofluorometry 4.3 Fluorescence Polarization lmmunoassay 4.4 Chromatographic Assays

4.4.1 Stereospecific 4.4.2 Non-stereospecific

5. Pharmacodynamics and Pharmacokinetics

6. References

FLECAlNlDE

1. DescriDtion

171

1.1 Nomenclature, Formula and Molecular Weiaht

(f)-Flecainide acetate, USAN, INN; described as R-818 in the early literature; ( f )-N-(2-piperidinylrnethyl)-2,5-bis(2,2,2- trif1uoroethoxy)benzamide acetate. The terms flecainide and flecainide acetate refer to the respective racemates unless otherwise specified.

Registry No.: ( f 1-flecainide acetate 99495-88-2; ( f 1- flecainide free base 99495-87-1 ; ( + )-flecainide acetate 99495- 93-9; ( + )-flecainide free base 99495-92-8; (-1-flecainide acetate 99495-94-0; (-)-flecainide free base 99495-90-6.

Free base c1 7H20F6N203 M.W. 414.36 Acetate c1 7H20F6N203 C2H402 M.W. 474.40

1.2 Amearance. Color. and Odor

Free base: white granular solid from isopropanol/isopropyI ether; odorless. Acetate: white crystalline solid.

1.3 History

Flecainide acetate is a class Ic antiarrhythmic agent which was developed in the Riker Laboratories as part of a broad-based project investigating the effect of fluorine substitution on local anaesthetic or antiarrhythmic activity. The details concerning the development of this drug have been reported l .

I72 SILVIA ALESSI-SEVERINI ET AL.

2. Svnthesig

2.1 Svnthesis of Flecainide Acetate (Fiaure 11

Trifluoroethylation of 2,5-dihydroxybenzoic acid affords 2,2,2-trifluoroethyl 2,5-bis(2,2,2-trifluoroethoxy)benzoate. This is slowly added to a solution of 2-aminomethylpyridine in glyme, under N2, at 25OC. After stirring for 20 hr the reaction mixture is refluxed (3 hr), cooled, and the solvent evaporated in vacuo. Recrystallization of the residue from benzene-hexane gave N42- pyridylmethyl)-2,5-bis(2,2,2-trifluoroethoxy)benzamide as an off- white solid (map 103-105°C) in 91 % yield. This was dissolved in acetic acid and reduced over Pt02 in a Parr hydrogenator. Filtration of the catalyst and evaporation of the filtrate gave a viscous syrup which solidified on trituration with isopropyl ether. Crystallization from isopropanol-isopropyl ether gave flecainide acetate as a white granular solid (m.p. 145-147OC) in 75% yield2.

FIGURE 1. Synthesis of Flecainide Acetate

2.2 PreDarative SeDaration of Flecainide Enantiomers

( + )-Flecainide has been obtained as a salt of ammonium ( + )-ar-bromocamphor-7-sulfonate while (-1-flecainide was isolated

FLECAINIDE I73

with ammonium (-)-a-bromocamphor-?r-sulfonate. In both cases salt formation was accomplished in methanol. The respective enantiopure flecainide free bases are readily recovered by treatment of the salts with dilute alkali. Subsequent reaction with acetic acid affords the enantiopure flecainide acetate$. Flecainide has also been similarly resolved by preparation of diastereomeric salts (in ethyl acetate) of enantiopure mandelic acids followed by fractional crystallization4,

3. Phvsical ProDertieg

3.1 Infrared Soectrum

The infrared spectrum of flecainide free base (KBr disc) was recorded on a Nicolet 71 99 Fourier Transform spectrometer and is presented in Figure 2. The principal absorption bands include (cm-l):

3427 1291 3358 1221 2927 1169 1637 1154 1606 1083 1549 978 1500 863 1458 657

Some suggested assignments include: broad peak centered at approximately 3400 cm-1 (N-H), 2927 (aliphatic C- HI, 1637 (amide I band), 1606 (C=C), 1549 (amide II band), 1500 (C=C str). Aryl ether and CF3 absorptions appear in the same general region; CF3 groups typically show absorptions between 1 120-1 350 and 690-770. Ar-0-CH2 is usually evident as absorptions between 1200-1 275 and 1020-1 075 cm-1.

3.2 NMR SDectra

Proton, carbon-1 3, and fluorine-1 9 spectra of flecainide free base, in CDC13, were obtained on a Bruker AM-300 FT NMR spectrometer. The respective spectra are illustrated in Figures 3,

FIGURE 2. IR Spectrum of Flecainide Base. KBr Pellet.

FLECAINIDE I75

4, and 5 and assignments presented in Tables 1, 2, and 3.

TABLE 1

300 MHz Proton NMR Spectrum of Flecainide Base in CDC13

Chemical Shift Number of Assignment (ppm from TMS) Protons

7.76 broad 7.76 d (J= 3) 7.09 d,d (J= 9, 3) 6.9 d (J= 9) 4.44 q (JH,F = 8) 4.36 q (J,,,= 8) 3.48 m 3.30 m 3.06 m 2.75 m 2.62 m 2.1-1.08 m

1 1 1 1 2 2 1 1 1 1 1 7

TABLE 2

CONH aromatic C6 H aromatic C4 jj aromatic C3 li c2 o c y 2 c 5 o c y 2 CONHCH2 CONHCH2 piperidine CH piperidine NCH2 piperidine NC& NH, piperidine (CH2)3

75 MHz Carbon-13 NMR Spectrum of Flecainide Base in CDC13 a

14 16 3

O C H ~ C F ~ 16 17

Chemical Shift b Assignment C

24.34 26.49 30.57

3 2 or 4 2 or 4

PPM FIGURE 3. Proton NMR Spectrum of Flecainide Base in CDCJ3.

FLECAINIDE 177

46.04 46.68 55.83

65.72 66.19 66.52 66.67 67.00 67.14 67.47 67.95

77

114.88 1 1 7.22 120.49

1 17.47 11 7.67 121.15 121 -36 124.84 125.04 128.52 128.73

124.31 150.23 153.00 163.99

1 o r 6 1 o r 6

5

14and 16 (C-C-F coupling)

CDC13

12 9 1 1

15 and 17 (C-F coupling)

8 10 13 7

a In Figure 4 the CH3 and CH groups are shown as signals possessing an anti-phase with respect to the CDC13 signal, while quaternary carbons, CH2 and carbonyls are in phase.

b ppmfromTMS

C carbon numbering as shown in structure

FIGURE 4. Carbon-13 NMR Spectrum of Flecainide Base in CDC13.

E 9 . S 8 9 . 0 8 8 . 5 8 B . m 8 7 . 5 E 7 . m 8 6 . 5 8 6 . 8 8 5 . 5 PPM

FIGURE 5. Fluorine 19 NMR Spectrum of Flecainide Base in CDC13.


TABLE 3

282 MHz Fluorine-19 NMR Spectrum of Flecainide Base in CDC13

Chemical Shift a Assignment

87.1 1 87.14 87.17

87.44 87.46 87.49

CF3

CF3

a external standard CgFg

3.3 Mass SDectrum

The positive ion electron impact mass spectrum was recorded on a Kratos MS 50 double focusing magnetic sector mass spectrometer. Operating conditions: mass range 31.01 84- 415.1471, sampling rate 25, signal level threshold 1, minimum peak width 7, scan rate (sec/dec) 10.0, # of scans averaged 9. High resolution MS: M + calculated, 41 4.1378; found, 41 4.1 351, Mass spectral data and suggested structures for fragment ions are shown in Figure 6.

Significant Ions Measured Mass %Relative Abundance

c 1 7H1 9N203F6 413.1290 0.17

c1 1 H7°3F6 301.0295 3.64

CgH1 1 97.0889 10.17

FLECAlNlDE

96.081 2

91.0546

84.081 4

5 6.05 20

C E O +

I

mh 301

0 N

1 H

m h 84

2.08

2.51

100.00

6.26

H

mlz 97

N

H mlz 5 6

I

FIGURE 6. Mass Spectral Data

3.4 Ultraviolet Saectrum

The ultraviolet spectrum of flecainide base in ethanol (0.001 6 gm/ l00 ml) is shown in Figure 7. The absorptivities are:

hmax E (1%: 1 cml

205 521 230 (shoulder) 21 9 300 59

3.5 Ootical Rotation and Absolute Confiauration

Optical rotations (sodium D line, 1 dm cells, methanol as solvent) were obtained with a Perkin Elmer Model 241

182

3 5 0 .

4 68

SILVIA ALESSI-SEVERINI ET AL.

+ +

+ +

+ +

f +

FIGURE 7. Ultraviolet Spectrum of Flecainide Base.

FLECAINIDE I83

polarimeter. Optical purity of flecainide free base enantiomers was >99% as determined by 100 MHz NMR using the chiral shift reagent tris[3-heptafluorobutyryl-d-camphoratoleuropium 1113.

( + 1-flecainide free base [a]26, + 3 . 4 O (-1-flecainide free base -3.3O

( + )-flecainide acetate [aI2'D + 4 . 6 O (-1-flecainide acetate [aI27, -4.50

The absolute configurations of flecainide enantiomers have been determined on the basis of CD spectra of the N-chloro derivatives. Thus, ( + )-flecainide has the S-configuration while the antipode is R-(-)-flecainide, The optical rotations of hydrochloride salts were also reported4.

( + 1-flecainide HCI [a12036s (- 1 - f I eca i n id e H C I [a] 203 65

+ 20.0° -2O.OO

3.6 Meltina Points

( f 1-flecainide free base ( + 1-flecainide free base (-1-flecainide free base

105-107°C 104-105°C 3 102-1 04OC 3

( f )-flecainide acetate ( + )-flecainide acetate (-)-flecainide acetate 152.5-1 54OC 3

145-1 47OC 153-1 55OC 3

( + 1-flecainide HCI 222-225OC 4 (-1-flecainide HCI 223-225OC

3.7 Ionization Constanf

The pKa of flecainide acetate has been determined5 as 9.3.

184 SILVIA ALESSI-SEVERINI ET AL.

3.8 Distribution Coefficient

The octanoVwater partition coefficient was determined6 to be 11.4 at pH 7.4 and logP calculated7 as 4.50.

3.9 Solubility

The solubility of flecainide acetate at 37°C is 48.4 mg/ml in water and 300 mg/ml in alcohol5.

3.10 Stabilitv

A solution of flecainide acetate in water has been reported to be very stable at room temperatures. The stability in biological fluids seems to be significantly decreased over a period of 3 months even under storage at -2O"Cg. The tablet formulation must be stored in light resistant containers at 15-30' c5.

4. f Anal is

4.1 Elemental

The calculated elemental analysis for flecainide [Cl7H2oF$J2031:

C 49.28% H 4.87% N 6.76% 0 11.58% F 27.51 %

The calculated elemental analysis for flecainide acetate [C17H20FgN203. C2H402I:

C 48.10% H 5.10% N 5.91% 0 16.86% F 24.03%

FLECAINIDE 185

A method for the determination of (*I-flecainide in plasma utilizes the natural fluorescence of the molecule. Flecainide is extracted from plasma with heptane after addition of 0.5 mol/L Na3P04 and triethylamine. The organic phase is re- extracted with 0.25 mol/L NaH2P04 and the aqueous phase is read in the spectrophotofluorometer (300 nm excitation wavelength, 370 nm emission). The sensitivity is reported to be 25 ng/ml per 2 ml of plasmalo.

4.3 Fluorescence Polarization lmmunoassay

Direct determination of ( f )-flecainide in plasma is possible through the utilization of a commercially available fluorescence polarization immunoassay (Abbott). The reaction is based on the competitive binding of free and fluorescein-labeled flecainide to specific antibodies. Fluorescence polarization measurements are dependent upon the concentration of the free drug in the sample. The method can be performed on 50 pL of plasma, with a sensitivity of 0.1 pg/ml, and is very convenient for therapeutic drug monitoring’ l .

4.4 Chromatoarmhic Assavs

4.4.1 StereosDec ific

Sample preparation and chromatographic conditions are summarized in Table 4.

4.4.2 Non-stereomecific

Sample preparation and chromatographic conditions are summarized in Table 5.

5. Pharmacodvnamics and Pharmacokinetics

The pharmacodynamics and pharmacokinetics of ( f )- flecainide acetate have been studied extensively in animal models and in humans. This drug exhibits potent antiarrhythmic effects

TABLE 4: STEREOSPECIFIC ANALYTICAL METHODS

EXTRACTIONlDERlVATlZATION

( + 1-1 -phenylethyl isocyanate

1 . tetra-O-acetyl-P-D-glucopyranosyl isothio-

2. S-l-(l -naphthyl)ethyl isothiocyanate 3. R-l-(2-naphthyl)ethyl isothiocyanate 4. R-or-methylbenzyl isothiocyanate

cyanate

serum mixed with MeCN, supernatant evaporated; R( + 1-1 -phenylethyl isocyanate

plasma with butyl chloride:2-propanol (95:5); (-)-menthy1 chloroformate

plasma and urine with diethyl ether; 1-1(4- nitrophenyl)sulfonyll-L-prolyl chloride

plasma with 1% 2-propanol in n-hexane; 1. RWphenylbutyric anhydride 2. R( + 1-1 -MeO-1 (CF31phenylacetyl choride 3. N-trifluoroacetyl-L-prolyl chloride 4. f-butyloxycarbonyl-L-alanine

COLUMNlMOBlLE PHASE DETECTION APPROXIMATE RETN TIME SENSITIVITY

silica (250 x 4.6 mm) hexane:EtOAc (55:45)

C18 MeOH:H20

C18 (1 00 x 3mm) MeOH:H20:HOAc (60:40:1) 370em); 0.05 mg/L 20 min

fluorescence (300ex.

silica (250 x 4.6 mm) fluorescence (305ex, hexane:EtOAc:Et3N(84: 16:O.l) 340em); 2.5 ng/ml 22 min UV (298); 40 ng/ml

C18 (300 x 3.9 mm) MeCN:H,O:Et,N (45:55:0.2) 30 min

UV (280); 50 ng/ml

SE 30 fused silica1 GC capillary negative ion chem- column (25 m) ical ionization mass

spectrometer; 0.41 ng/ml

a R REF

1.19 2.37 4

1. 1.16 12 2. 1.05

4. 1.04 3. 1.00

baseline 13 resolved

1.08 14

1.07 1.25 15

1. 1.39 16 2. 1.43 3. 3.38 4. 1.14

plasma with 1 % 2-propanol in n-hexane; Chirasil-L-Val fused silica GC negative ion chem- R = 1 . l -1 .6 16 pentafluoropropionic anhydride capillary column (25 m); XE- ical ionization mass on either col-

60-(R)-phenylethylamide glass spectrometer; < 0.4 umn capillary column (29 m) ng/ml

urine with EtOAc; (-)-menthy1 chloroformate silica (250 x 4.6 mm); hexane: fluorescence (290ex, baseline 17 2-butanol:MeCN(98.75:1:0.25) 340em); 25 ng/ml resolved 20 min

TABLE 5: NON-STEREOSPECIFIC ANALYTICAL METHODS

EXTRACTlONlDERlVATlZATlON

plasma deproteinized, pH adjusted, supernatant injected

plasma or urine washed and extracted with hexane

plasma or serum extracted with methyl r- butyl ether

plasma extracted with hexane

solid phase extraction of plasma with C8 adsorbent

diethyl ether extraction of plasma then back extracted into dilute phosphoric acid

solid phase extraction of plasma with C8 adsorbent

COLUMNlMOBlLE PHASE APPROXIMATE RETN TIME

C18 pBondapak (1 50 x 4 mm) ammonium phosphate buffer:MeOH (60:40); 6 rnin

Zorbax TMS (1 50 x 4.6 mm) MeCN:l% HOAc in 0.01M pentane- sulfonate (45:55); 5 min

Spherisorb S5W silica (1 25 x 5 mm) MeOH:2,2,4-trimethylpentane (80:20) containing d-1 0-camphorsulfonic acid; 4 rnin

pBondapak phenyl (300 x 3.9 mm) MeCN:0.06% H,P04 (40:60); 5.5 rnin

pBondapak phenyl (300 x 3.9 mm) MeCN:0.06% H,P04 (40:60); 5.5 rnin

pBondapak C18 (300 mm) MeCN:H20 (30:70) containing dibutyl- amine phosphate; 7 min

Radial-Pak C18 (1 00 x 8 mrn) MeOH:25% ammonia (99.9:O.l); 7 min

DETECTION SENSITIVITY

fluorescence (300ex, 370em); 50 ng/ml UV (280)

UV (308); 22 ng/ml

fluorescence (200ex. no emission filter); 20 pg/L

fluorescence (300ex. 370em); 3 ng/ml

fluorescence (300ex. 370eml; 3 ng/ml UV (298); 50 ng/ml

UV (214); <30 ng/ 0.5 ml

fluorescence (293ex, 340em); 10 ng/ml

REF

18

19

20

21

22

23

24

serum or plasma extracted with methyl r- octyl microbore column (250 x 2 mm) UV (298); 80 pg/L 25 butyl ether 0.05% triethylamine in MeCN:O. 1 M (250 pL plasma)

sodium acetate (45:55); 11 min

plasma extracted with 1-chlorobutane then phenyl reverse phase (300 x 3.9 mm) UV (297); 0.033 26 back extracted into dilute phosphoric acid MeCN:20 mM sodium acetate mg/L

(42:58); 10 min

serum or plasma extracted with methyl t- octyl microbore column (250 x 2 mm) butyl ether 0.05% triethylamine in MeCN:O.lM 370em); 20 pg/L

fluorescence (300ex, 27

(1 00 pL plasma) sodium acetate (40:60); 8 rnin

microscale protein precipitation of serum Nucleosil 5 C18 (1 50 x 4.2 mm) fluorescence (285ex. 28 with Zn sulfate/MeCN, supernatant injected 300 ml MeCN, 1 ml H,PO,, 0.5 ml 370em); 30 ng/ml

diethylamine in 1L H,O; 5 min

plasma, urine or saliva extracted with diethyl 3% SP-2250 GC column (180 cm x 2 electron capture ether, back extracted into 0.5M HCI, mm); 16 min detection; 12.5 basified, derivatized with pentafluorobenzoyl ng/ml chloride, extracted into hexane

- m

29 rD

190 SILVIA ALESSI-SEVERINI ET AL.

by blocking the cardiac sodium channels and by stabilizing the myocyte membrane without affecting the repolarization30-34, It is used in the suppression and control of ventricular and supraventricwlar a r r h y t h m i a ~ 3 0 - ~ ~ and it has shown some effectiveness in the treatment of atrial arrhythmia~3~-39. The drug is not recommended in non-symptomatic post-infarction patients because of potentially life threatening t o ~ i c i t y . ~ 0 # ~ 1

Flecainide acetate is rapidly and almost completely absorbed from the GI tract following oral administration. Absolute bioavailability of the commercailly available tablets averages 85- 90%. First pass metabolism is negligible. Plasma concentrations must be kept within the 200-1000 ng/ml range in order to maximize the therapeutic effect and minimize the risk of serious side e f f e c t ~ 3 0 - 3 ~ . Plasma levels and dose show a linear correlation42, however considerable inter- and intra-individual variations have been observed and, most recently, evidence on non-linear kinetics has been reported43. After i.v. administration to humans, flecainide is rapidly and apparently widely distributed (Vd ranges from 5-13.4 L/kg; average 5.5-8.7 L/kg). After an oral dose the Vd has been determined to be 10 L/kg44. After i.v. administration to rats, flecainide is distributed extensively to many tissues, including the heart, but only minimally into the CNS, and dose-dependent tissue uptake has been shown in rabbits after chronic admini~trat ion~5,~6. The in vitro protein binding (mainly a1 -acid glycoprotein) is approximately 40-50% and is independent of the drug plasma concentration30-34, The pharmacokinetic profile after i.v. administration follows a two- compartment open model with tr/,ar = 3-6 min and a tr/,B = 11- 14 hr (range 7-19 hr)44. Elimination half-life is prolonged in arrhythmias (1 9 hr), in renal and hepatic impairment (26-49 hr), and in congestive heart failure (up to 50 hrl44f47-52. Urinary pH affects elimination half-life, prolonging it when alkaline (pH 7.2- 8.3) and reducing it when acidic (4.4-5,8)53-55.

Flecainide is extensively metabolized by the liver. The two major metabolites are m-0-dealkylated flecainide and the m-0- dealkylated lactam derivative. These are formed by preferential 0-dealkylation at the meta-position of the benzamide ring and by subsequent oxidation of the piperidine ring of m-0-dealkylated flecainide, respectively. Both metabolites undergo extensive sulfate and glucuronide conjugation at the m-0-dealkylated

FLECAINIDE 191

position. About 80-90940 of a dose is recovered in the urine (30% as unchanged drug, the rest as metabolites and their conjugates). Only 5% is excreted in the faeces and 3% of the total excretion is attributed to at least three unidentified meta bolites44.

Hemodialysis removes about 1% of a dose as unchanged drug. Total apparent plasma clearance of flecainide, by healthy subjects, following oral administration, has been reported to be in the range of 4-20 ml/kg. Renal clearance of the drug is 25- 40% of total plasma clearance. Total apparent plasma clearance is decreased in arrhythmias, congestive heart failure and renal

There are comparatively few reports concerned with the stereoselective pharmacodynamics and pharmacokinetics of flecainide enantiomers. ln vivo studies have revealed equipotency and equiactivity for the two enantiomers in mouse and dog models of arrhythmia3. ln vitro tests on canine Purkinje fibres have also shown that the enantiorners have comparable electrophysiologic a~tivit ies5685~. The two enantiomers have also shown similar affinities to a receptor site associated with cardiac sodium channels in isolated rat cardiac m y o c y t e ~ ~ ~ .

The pharmacokinetic patterns of the enantiomers following oral administration in humans appear to be essentially parallel. The plasma R/S ratio seems to range from 0.67-1.44 in different patient populationsl3#56,59, Stereoselective elimination has been suggested in healthy subjects, which have been classified as poor metabolizers of the sparteine debrisoquine type; R-flecainide is predominant in plasma after oral administration of the racemate. The oral AUCs of the enantiomers as well as the elimination half-lives were slightly, but significantly, different. This has been interpreted as being the result of stereoselective hepatic metabolism1 7.

impairment30-34,47-50,52,

6. References

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SlLVlA ALESSI-SEVERINI ET AL. 192

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13. L. Lie-A-Huen, R.M. Stuurman, F.N. Ijdenberg, J.H. Kingma, D.K.F. Meijer, Ther. Drug Monitor.,

14. J. Turgeon, H.K. Kroemer, C. Prakash, I.A. Blair, D.M. Roden, J.Pharm.Sci., 79 91-95 (1990).

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20. K.K. Bhamra, R.J. Flanagan, D.W. Holt, J. Chromatogr.,

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708-71 1 (1989).

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FLECAINIDE 193

22. S.F. Chang, A.M. Miller, J.M. Fox, T.M. Welscher, Ther.

23. J. Boutagy, F.M. Rumble, G.M. Shenfield,, J. Li9.

24. T.A. Plomp, H.T. Boom, R.A.A. Maes, J. Anal. Toxicol., 10

25. T. Annesley, K. Matz, J. fig. Chromatogr., 11 891-899

26. N. Grgurinovich, J, Anal. Toxicol., 12 38-41 (1988). 27. T. Annesley, K. Matz, J. fig. Chromatogr., 11 1041-1049

28. G. Malikin, M. Murphy, S. Lam, Ther. Drug Monitor., 11

29. J.D. Johnson, G.L. Carlson, J.M. Fox, A.M. Miller, S.F. Chang, G.J. Conard, J. Pharm.Sci., Z3 1469-1471 (1984).

30. J.L. Anderson, J.R. Stewart, B.A. Perry, D.D. Van Hamersveld, T.A. Johnson, G.J. Conard, S.F. Chang, D.C. Kvam, B. Pitt, New Engl. J. Med.,

31. B. Holmes, R.C. Heel, Drugs, 29 1-33 (1 985). 32. S.L. Chase, G.E. Sloskey, Clim Pharm., 33. R.W. Kreeger, S.C. Hammill, Mayo Clin. Proc., 62 1033-

1050 (1987). 34. F. Furlanello, G. Vergara, R. Bettini, G. Mosna, L.

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36. V. Zeigler, P.C. Gillette, B.A. Ross, L. Ewing, Am. J. Cardiol., 818-820 (1988).

37. I.C. Van Gelder, H.J.G.M. Crijns, W.H. Van Gilst, C.D.J. De Langen, L.M. Van Wijk, K.I. Lie, Am. J. Cardiol., 63 112- 114 (1989).

38. M.J. Suttorp, J.H. Kingma, L. Lie-A-Huen, E.G. Mast, Am. J. Cardiol., B 693-696 (1989).

39. S.S. Wafa, D.E. Ward, D.J. Parker, A.J. Camm, Am. J. Cardiol., a 1058-1064 (1989).

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41. D.S. Echt, P.R. Liebson, L.B. Mitchell, R.W. Peters, D. Obias-Manno, A.H. Barker, D. Arensberg, A. Baker, L. Fiedman, H.L. Greene, M.L. Huther, D.W. Richardson, and

Drug Monitor., 6 105-1 11 (1984).

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102-106 (1986).

(1 988).

(1 988).

210-213 (1989).

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839-850 (1 987).

559-562 (1 988).

807-8 14 (1 987).


the CAST Investigators, New Engl. J. Med., 324 781-787 (1991).

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43. G. Boriani, E. Strocchi, A. Capucci, R. Calliva, L. Frabetti, E. Ambrosioni, B. Magnani, Eur. J. Clin. Pharmacol., 41 57-59 (1 991 1.

44. G.J. Conard, R.E. Ober, Am. J. Cardiol., 53 41B-51B (1 984).

45. D. Piovan, R. Padrini, M. Furlanut, R. Moretto, M. Ferrari, Pharmacol. Res. Commun., 18 739-745 (1 986).

46. R. Kannan, A. Matin-Asgari, Drug Metab. Disposit., l§ 228- 231 (1988).

47. J. Braun, J.R. Kollert, J.U. Becker, Eur. J. Clin. Pharmacol.,

48. A.J. Williams, R.L. McQuinn, J. Walls, Clin. Pharmacol. Ther., 43 449-455 (1988).

49. S.C. Forland, R.E. Cutler, R.L. McQuinn, D.C. Kvam, A.M. Miller, G.J. Conard, S. Parish, J. Clin. Pharmacol., 28 727- 735 (1988).

50. S.C. Forland, E. Burgess, A.D. Blair, R.E. Cutler, D.C. Kvam, C.E. Weeks, J.M. Fox, G.J. Conard, J. Clin. Pharmacol., 28

51. R.L. McQuinn, P.J. Pentikainen, S.F. Chang, G.J. Conard, Clin. Pharmacol. Ther., 44 566-572 (1988).

52. A. Cavalli, A.P. Maggioni, S. Marchi, A. Volpi, R. Latini, Clin. Pharmacokinet., 14 1 87-1 88 (1 984).

53. K.A. Muhiddin, A. Johnston, P. Turner, Br. J. Clin. Pharmacol., 17 447-45 1 (1 984).

54. A. Johnston, S. Warrington, P. Turner, Br. J. Clin. Pharmacol., 20 333-338 (1985).

55. R. Hertrampf, U. Gundert-Remy, J. Beckmann, U. Hoppe, W. ElsaBer, H. Stein, Eur. J. Clin. Pharmacol., 41 61-63 (1 991 1.

56. H.K. Kroemer, J. Turgeon, R.A. Parker, D.M, Roden, Clin. Pharmacol. Ther., a 584-590 (1 989).

57. J.K. Smallwood, D.W. Robertson, M.I. Steinberg, Naunyn- Schmiedeberg ‘s Arch. Pharmacol. , 339 625-629 (1 989).

- 31 71 1-714 (1987).

259-267 (1 988).

FLECAINIDE 195

58. R.J. Hill, H.J. Duff, R.S. Sheldon, Molec. Pharmacol., 34

59. S. Alessi-Severini, D.F. LeGatt, F.M. Pasutto, F. Jamali, R.T. Coutts, Clin. Chern., 1 11 -1 12 (1 991 ), Correction, C/in. Chern., aZ 886 (1 991 1.

659-663 (1 988).

GLAFENINE

Adnan A. Badwan,' Muhammad B. Zughul,2

and Mahmoud A l Omari'

( I ) The Jordanian Pharmaceutical Manufacturing Co. Naor, Jordan

(2) Department of Chemistry Faculty of Science

University of Jordan Amman, Jordan


Copyright 0 1992 by Academic Press, Inc. All rights of reproducilon reserved In any form. 197

198 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

CONTENTS 1 - DESCRIPTION

1.1. Nomenclature. 1.1 .l. Chemical Names. 1.1.2. Generic Name. 1.1.3. Registry Number. 1.1.4. Wiswesser Line Notation. 1.2. Formulae. 1.2.1. Emperical Formula. 1.2.2. Molecular Weight. 1.2.3. Structural Formula. 1.3. Colour, Appearance and Odour. 1.4. Therapeutic Use.

2 - SYNTHESIS

2.1. Synthetic Route (I). 2.2. Synthetic Route (11). 2.3. Synthetic Route (111). 2.4. Synthetic Route (IV).

3 - PHYSIC0 - CHEMICAL PROPERTIES

3.1. 3.2. 3.3. 3.4. 3.5. 3.6. 3.6.1. 3.6.2. 3.6.3. 3.6.4. 3.6.5. 3.6.6. 3.6.7.

Melting Range. Differential Scanning Calorimetry. Solubility. Dissociation Constant. Partition Coefficients. Spectral Properties. Ultraviolet Spectra. Fluorescence Spectrum. Single Photon Counting Spectrofluorometry. lnfra - Red Spectrum. Nuclear Magnetic Resonance Spectrum. Mass Spectrum. X - Ray Powder Diffraction.

GLAFENINE

4 - METHODS OF ANALYSIS

199

4.1. 4.1.1. 4.1.2, 4.1 2 . 1 4.1.2.2 4.1.2.3.

4.1 -3. 4.1.3.1 I 4.1.3.2. 4.1.4. 4.1.5. 4.1 5.1. 4.1.5.2. 4.1.6. 4.1.6.1. 4.1 -6.2. 4.2.

Starting Material and Pharmaceutical Dosage Forms. Elemental Composition. Related Materials. 4,7 - dichloroquinoline. Anthranilic acid esters. N - (7 - chloro - 4 - quinolyl) anthranilic acid (glafenic acid). Titrations. Non - Aqueous Titration. Alkalimetric Titration. Gravimetric Analysis. Spectrophotometric Methods. Ultraviolet Absorption. Spectrofluorometric Analysis. Chromatographic Methods. Thin Layer Chromatography. High Performance Liquid Chromatography. Body Tissues and Fluids.

5 - STABILITY

5.1. Stability of The Solid. 5.2. Stability in The Solution.

6 - PHARMACOKINETICS

6.1. Absorption. 6.2. Bioavailability. 6.3. Distribution. 6.4. Metabolism. 6.5. Excretion. 6.6. Half - Life.

200 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARl

1. DESCRIPTION

1 .l. Nomenclature

1.1.1, Chemical names

1.1.1.1 a - glyceryl or 2', 3' - dihydroxypropyl N - (7 - chloro - 4 - quinolyl) anthranilate.

1.1.1.2 2', 3' dihydroxypropyl2"- (7 - chloro - 4 - aminoquinolyl) benzoate. 1.1.1.3 4 - (2" - (2', 3' - dihydroxypropyl carboxyphenyl) amine) - 7 -

chloroquinoline.

1.1.2. Generic name

Glafenine (Listed in The French Pharmacopoeia)

1.1.3. Registry number

CAS - 3820 - 67 - 5

1.1.4. Wiswesser line notation

T66 BNJ EMR 8 V1 YQ1 Q and JG

1.2. Formulae

1.2.1. Emperical formula

1.2.2. Molecular weight

372.8

GLAFENINE 20 I

1.2.3. Structural Formula

1.3. Colour, Appearance and Odour.

Pale yellow, crystalline powder, odourless.

1.4. Therapeutic Use

Glafenine is an analgesic.

2. SYNTHESIS

Different routes of synthesis were proposed. A brief of these is described and a schematic presentation is illustrated.

2.1. Synthetic Route 1.

Reacting of isoatoic anhydride with glycerol to produce glyceryl anthranilate, followed by reacting with 4, 7 - dichloroquinoline to produce glafenine (1).

202 ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL. AND MAHMOUD AL OMARl

Scheme ( I ) Glafenine Synthesis, Route ( I )

2.2. Synthetic Route II.

Condensation of 2 - chlorobenzoate, glyceryl ester with 7 - chloro - 4 - aminoquinoline (1).

I--\

Scheme ( / I ) Glafenine Synthesis, Route (11)

GLAFENINE 203

2.3. Synthetic Route 111.

Condensation of 4, 7 - dichloroquinoline with methylanthranilate. The resulting methyl ester is transesterified with glyceryl acetonide which is further hydrolysed to glafenine (1).

A

HN - glvceryl acetonide I

Scheme (111) Glafenine Synthesis, Route (111).

204 ADNAN A. EADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI

2.4. Synthetic Route IV

Esterification of 2 - nitrobenzoic chloride with glyceryl acetonide, followed by a reduction of the nitro group to the amino group. The resulted ester is condensed with 4, 7 - dichloroquinoline, followed by hydrolysis of acetonide (2).

HO OH

Scheme (IV) Glafenine Synthesis, Route (IV).

GLAFENINE

3. PHYSIC0 - CHEMICAL PROPERTIES

205

3.1. Melting Range

The French Parmacopoeia specifies that the melting range of glafenine is between 170°C - 174 "C, (3).

3.2. Differential Scanning Calorimetry

Glafenine was recrystallized from ethanol, butanol, hexanol and acetonitrile. Thermograms of these crystals were obtained using Mettler TA 3000 - DSC - 20 unit. The heating rate was 10 "C. min-' and the sample size was ranging from 3 - 10mg. The recrystallized glafenine showed a single sharp peak without any decomposition at melting. Melting range of obtained crystals from different solvents was between 170 - 174°C. Recrystallization from hexanol yielded sharper thermogram peak, figure (1). The heat of fusion of these crystals was in the vicinity of 43.8 KJ. mole-' (4).

Heat Flow Exothermal c

E l-

180

Figure ( 1 ) The DSC Profile of Glafenine Recrystallized from Hexanol


3.3. Solubility

Equillibrium solubility of glafenine was determined by shaking an excess of glafenine with the solvent required in a water bath at 30 “C for 48 hours. Table ( I) presents glafenine solubility in commonly used solvents (4).

Table ( I )

Glafenine Equillibrium Solubility in Commonly Used Solvents

Solvent gm/lOOml at 30°C

Hexane Water Chloroform Acetone Ethanol 0.1N HCI

<0.001 0.001 0.260 0.297 0.700 1.295

3.4. Dissociation Constant

The pKa was determined spectrophotometrically at 20 “C in accordance with an earlier reported method (5). Stock solution of glafenine in 10” N HCI was prepared, and diluted with suitable buffer solutions ranging from pH 6 - 10 to obtain afinal glafenine concentration of 1 Oug. mL-’. The absorbance of these solutions were measured at the maximum at 342.5nm. This method yielded 7.2 as pKa of glafenine at 20 “C (4).

GLAFENINE 207

3.5. Partition Coefficients

The partition coefficients of glafenine between n - decanol and aqueous buffers of different pH values were determined at room temperature. Different pH values were obtained by using 0.1 N HCI for pH 1 .O, acetate buffers for pH 3.0,4.0,4.5 and 5.0 and phosphate buffers for pH 6.0,7.0 and 8.0. Figure (2) shows the plot of pH against the partition coefficients (5).

20

15

10

5

2 4 6 8 PH

Figure (2) The Plot of Partition Coefficient of Glafenine Against the pH

208 ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL. AND MAHMOUD AL OMARI

3.6. Spectral Properties

3.6.1. Ultraviolet spectra

The absorption spectra of glafenine dissolved in methanol and 0.1 N HCI are shown in figure (3). It exhibits maximum at 356nm, 225nm and 255nm for methanol and maxima at 342nm, 223nm and a shoulder at 252 nm for 0.1 N HCI. These were recorded using Beckman DU - 7 spectrophotometer.

Wavelength (nm)

The UV absorption Spectra of Glafenine (70 ug.mL-’) in Methanol (-) and 0.7N HCl (...).

Figure (3)

GLAFENINE 209

Table (11) repesents the ultraviolet absorption spectral bands of glafenine in various commonly used solvents (4 , 7).

Table ( I1 )

The UV Absorption Spectral Bands of Glafenine in Different Solvents

Solvent W8Velength (Extinction Coefficient) nm (M-' cm-')

Chloroform 360 (22,900), 255 (18,100) Benzene 360 (14,900), 255 ( 4,100) Methanol 356 (21 ,OOO), 255 (1 8,800) Ethanol 355 (19,600), 255 (1 7,000) Acetone 355 (18,700), 255( 5,600) Ether 351 (19,300), 255 (1 9,000) O.1NHCI 342 (18,300), 252 (Shoulder)

3.6.2. Fluorescence spectra

The excitation and emission spectra of glafenine in ether is shown in figure (4). The excitation and emission maxima of glafenine in different solvents are presented in Table (Ill). Aqueous solutions having pH values lower than 4 do not fluoress while solutions having higher pH values exhibit fluorescence which intensifies with the pH increase. This intensity is maximized in solutions having pH 9 - 10. (7).

210 ADNAN A. BADWAN. MUHAMMAD 8 . ZUGHUL. AND MAHMOUD AL OMARI

60 .- L. u) C Q, C

4-

- 40 .- 9 c Q 0 U -

20

Wavelength (nrn)

Figure (4) The Fluorescence Excitation and Emission Spectrum of Glafenine in Ether (10 ug. mL-'; Aexc = 250,327 and 7 em = 400 nm).

0 2 4 6 8 1 0 l 2 1 4 1 6

Figure (5) The Fluorescence Decay Curve of Glafenine in Ethanol at hexc=340nm and-hem=475nm.

GLAFENINE 21 I

Table ( 111 )

The Fluorescence Characteristics of Glafenine in Various Solvents (5 ug. mL-’)

Solvent exc. (nm) em. (nm) Intensitya

Benzene Ether Chloroform Ethanol 2 - Propanol - 10% (v/v) buffer pH9 (0.1 N glycine-sodium hydroxide). Methanol 2 - Propanol - 10% (v/v) buffer, pH4 (0.1 N citrate-hydrochloric acid). Acetone Methanol - 10% (v/v) ammonia (28% w/w). 2 - Propanol - 10% (v/v) buffer, pH1 (0.2N potassium chloride - hydrochloric acid). Sulfuric acid (10 - 0.005N).

330 392 250b,327 400 250b,340 436 245b,336 439 340 41 0

245b,336 425 26!jb,355 450

330 430 273b,365 455

300 407

20 17 10 6 6

2 2

0.5 0.5

0.5

No signal

a: Referred to a solution of quinine sulfate in a concentration of 1 ug. mL-’ in 0.5N sulfuric acid, of which the relative fluorescence intensity is 100, measured simultaneously.

b: Secondary excitation value with much less intensity.

3.6.3. Single photon counting spectrofluorometry.

A single photon counting spectrofluorometry was used to measure the decay life time of glafenine in absolute ethanol (20ug. mL-’). A hydrogen lamp was used as monochromatic radiation source while the excitation and emission wavelengths were 340nm and 475nm, respectively figure (5). The fluorscence decay curve was obtained using Edinibrugh Instruments single photon counting model 199 - spectrofluorometer. This figure shows that the decay life time of glafenine in ethanol is 0.88 n - sec.(8).

212 ADNAN A. BADWAN. MUHAMMAD 8. ZUGHUL. AND MAHMOUD AL OMARI

3.6.4. Infrared spectrum

Glafenine infrared spectrum is shown in figure (6). This is obtained by screening glafenine - KBr dispersion disc using Perkin Elmer 598 spectrophotometer. Table (IV) shows the band assignments of infrared spectrum of glafenine (4).

Table ( IV ) The I.R. Spectral Assignments of Glafenine

Wavenumber, cm-' Vibration Mode

3500 - 3220 31 00 2900 1680 1620,1580,1530 1260,1050 1 100,980 1450 870,830,750,600

0-H (stretching), N-H (stretching) C-H (stretching), aromatic C-H (stretching),aliphatic C = 0 (stretching) C=C (stretching), aromatic C-0 (stretching), ester C-0 (stretching), alcohol C-H (bending), aliphatic C-H (out of plane bending), aromatic

3.6.5. Nuclear magnetic resonance spectrum

NMR spectrum of glafenine is presented in figure (7). This spectrum was recorded on Varian T60 NMR spectrometer. Table (V) lists the spectral assignments of glafenine in DMSO (1,8).

Table ( V ) The NMR Spectral Assignments of Glafenine

Chemical Shlft Number of Proton Assignment (Multiplicity)

10.1 8.7 - 7.0 4.4 3.8 3.5

Wavenumber (Cm-')

Figure (6) The 1. R. Spectrum of Glafenine.

figure (7) The N. M. R. Spectrum of Glafenine in DMSO.

GLAFENlNE 215

3.6.6. Mass spectrum.

The mass spectrum of glafenine was obtained using mass spectrometer MAT - 112. Some characteristic peaks are listed in Table (VI). Figure (8) represents the mass spectrum of glafenine, (4).

Table ( VI )

The Mass Spectrum of Glafenine

m/z Species I / I,%

372 298 280 253 21 7 121 104 76 74

M+ 40.0

298 - (HZO) 62.9 280 - (CO) 71.4 253 - (CI) 34.3 (C&COOH)' 40.0 (C6H4CO)+ 100.0 (c6H4)+ 70.0 (CH2=CHOHCH20H) 15.7

372 - (CHZ = CHOHCHZOH) 27.1

I / lo = relative intensity (Eased on the highest intensityof 100.0).

The molecular ion peak appeared at m/z ratio of 372. Glafenine mass spectrum showed a distinct peak at m/z 298. This peak corresponds to Maclafferty rearrangement. The cleavage of oxygen - carbonyl bond is evident at m/z 280. Further, the removal of C = 0 group and CI atom is shown at m/z 253 and 217, respectively. The pattern of glafenine mass fragmentation is presented in scheme (V).

Figure (8) The Mass Spectrum of Glafenine

GLAFENINE 217

- CHFCHOHC H,OH - mlz =74

C L U d m/z = 37 2

QtJ m/z=298

CL cl$ c,QI$ m/z = 253 m/z = 280

m/z = 177 m/z =121 QQ

m/z =29 8

m/z =I21 m b =I 04

Scheme (V} The Mass Fragmentation Pattern of Glafenine.

218 ADNAN A. BADWAN. MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARl

3.6.7. X - ray powder diffraction

The x - ray powder diffraction of glafenine powder was determined using Phillips PW 1050 - 81 Goniometer with a PW 1729 Generator with Nickle filtered copper radiation ( = 1.5418nm) as a source of radiation. The scanning rate was 28. (2cm)-’. min-’. The interplanner distance and relative intensity of the major peaks are listed in Table (VII). Figure (9) illustrates the x - ray powder diffraction pattern of glafenine recrystallized from 1 - hexanol (4).

Table ( VII )

The X - Ray Powder Diffraction of Glafenine

5.6 7.6 9.9 11.4 14.9 15.5 16.6 17.6 19.2 20.4 21.2 22.2 22.8

15.781 11.632 8.9341 7.761 7 5.9454 5.71 66 5.3402 5.0390 4.6226 4.3532 4.1907 4.0042 3.9001

2.8 17.3 5.6 70.9 16.8 30.7 51.4 4.5 5.6 79.9 14.0 50.3 22.3

23.4 24.1 25.3 26.0 27.6 29.8 30.9 34.1 35.8 37.9 40.6 41.7 44.1

3.801 5 3.6926 3.5201 3.4269 3.231 8 2.9980 2.8938 2.6292 2.5081 2.3738 2.2220 2.1659 2.0534

32.4 100.0 13.4 16.8 30.7 11.1 6.7 7.8 10.6 22.3 5.0 8.4 21.2

d = Interplanner Distance, l / l o = Relative Intensity (based on the highest intensity of 100).

44 38 32 26 20 14 8 2 Diffraction Angle (28)

Figure (9) The Powder X - Ray Diffraction Pattern of Gla fenine Recrystallized from Hexanol.

220 ADNAN A. BADWAN, MUHAMMAD B. ZUGHUL, AND MAHMOUD AL OMARI


4.1. Starting Material and Pharmaceutical Dosage Forms

4.1 .l. Elemental Composition

C H CI N 0

Calculated Percentage

61.21 4.60 9.51 7.51

17.17

4.1.2. Related Materials

4.1.2.1. 4,7 - dichloro - quinoline : 10 mg glafenine are dissolved in 0.5rnLof hydrochloric acid solution (1 % v/v hydrochloric acid in ethanol). 0.5 rnLof paranitrophenyl hydrazine solution (0.1 % millimole in ethanol) is added. The mixture is cooled in air and then warmed in a water bath at 80°C for one hour in darkness. The mixture is further cooled to 20 "C f 1 "C. 0.25rnbftriethylamine is added, shaken and followed by the addition of 4 ml of dimethyformamide and shaken until a homogenous liquid is obtained. This solution is compared in colour intensity with a standard solution of 0.5rnLof 4,7 - dichlocoquinoline (1 0 ug permL)in 1 % v/v hydrochloric acid in ethanol. The percent concentration of 4,7 - dichloroquinoline in glafenine should not exceed 0.05% (3).

4.1.2.2. Anthranilic acid esters: 50mg of glafenine are dissolved in 5mL of ethanol. 1mL of diluted sulphuric acid is added. The mixture is cooled for 2 minutes in an ice bath. 1mL of 1% millimole per volume of sodium nitrite solution is added and the mixture is left for 10 minutes in the ice bath. 1 mL of freshly prepared and filtered B - naphthol solution of 10% millimole per volume in concentrated ammonia, is added. The obtained solution is compared in colour intensity with a simultaneously prepared standard solution of a mixture of 4.8mL of ethanol and 0.2 mL of methyl anthranilate solution (0.05% millimole per volume in alcohol). The percent concentration of anthranilic acid esters in glafenine should not exceed 0.2% (3).

GLAFENINE 22 I

4.1.2.3. N - (7 - chloro - 4 - quinolyl) anthranilic acid (glafenic acid): Thin layer chromatography can be used to detect glafenine and glafenic acid in the bulk material. The system consists of a plate of silica gel HF 254 impregnated with sodium acetate. The mobile phase is a mixture of chloroform - methanol - glacial acetic acid and water mixed in volumetric proportions of 85:12:2:1, respectively. The developed spots are identified by exposure to U.V. lamp (254nm). Glafenine and glafenic acid solutions having a known concentration are prepared in a mixture of chloroform - methanol and water mixed in volumetric proportions of 3.0:3.0:0.5, respectively. These solutions include 0.5% w/v glafenine sample (solution a), 0.5%, 0.1 %, 0.0025% w/v glafenine standard solutions (b, c and d), 0.0025% w/v glafenic acid standard (solution e), a mixture of 0.5 w/v glafenine standard and 0.0025% w/v glafenic acid standard (solution f). If a secondary spot appears in the chromatogram of solution a with an Rf value slightly inferior to the principal spot of the same chromatogram, it should not be more intense than the principal spot in the chromatogram obtained from solution c. If a secondary spot appears corresponding to glafenic acid, it should not be more intense than the spot obtained from solution e. If secondary spots other than the previous two appear in the chromatogram of solution a, it should not be more intense than the principal spot of solution d. This test could be adopted only if glafenine and glafenic acid were separated in two spots in the developed chromatogram of solution f (3).

4.1.3. Titrations.

4.1.3.1. Non - aqueous titration: Glafenine (300 mg) is dissolved in 30 mL of acetic acid. The end point is detected potentiometrically. When glafenine hydrochloride is used, mercuric acetate is to be added to the titration medium. Each mL of 0.1 M perchloric acid volumetric solution is equivalent to 37.28mg of glafenine. This method is applied for the drugs determination in tablets and suppositories (9).


4.1.3.2. Alkalimetric titration: Powdered tablets equivalent to 50 - 200 mg of glafenine are placed in 150 mL conical flask. 25 mL of 0.1 N HCI are added and shaken for 2 - 3 minutes. The bromocresol green indicator is added to the mixture (2 - 3 drops) and titration is carried out with 0.1 N NaOH. The indicator colour changes from yellow to bluish green at the end point. This method could be adopted for different dosage forms determination. Each mL of 0.1 N HCI volumetric solution is equivalent to 37.28 mg of glafenine (1 0).

4.1.4. Gravimetric analysis

Glafenine is determined in tablets and suppositories by adding 1 mL of 1 N HCI, 10 mL of diluted acetic acid and 6 mL of 0.25M KBi14 with stirring, to 10 mL portions of sample solution prepared from tablets or suppositories described, containing 50mg glafenine. After 30 minutes, the precipitate is filtered off on a sintered glass filter and washed with diluted acetic acid and water. The filter is then dried at 105°C for 60 to 90 minutes, cooled and weighed. Each gram of the precipitate is equivalent to 0.3728 gm of glafenine(l1).

4.1.5. Spectrophotometric methods

4.1 5 1 . Ultraviolet absorption: Glafenine (50 mg) is dissolved in 0.1 N HCI and the volume is made up to 250mL. 5 mL of this solution is further diluted to 100 mL with the same solvent. The solution exhibits a maximum absorption at about 343 nm. The specific absorbance at this maximum is about 490 (4).

4.1 5.2. Spectrofluorometric analysis: Glafenine is determined in tablets by transfering a quantity of fine powder equivalent to 25 mg of glafenine into 500 mL conical flask. About 450 mL of ether are added and the mixture is stirred with a magnetic stirrer for 2 hours. After filtration on a paper filter, the filtrate is diluted to 500 mL with ether. Further 10 mL of this solution is diluted to 100 mL with ether. An analogous standard solution having the same concentration of the sample solution is prepared. It is advisable to extract the tablets simultaneously with the preparation of the standard solution or during at least the same period to avoid incomplete extraction. Pure ether is used as the blank solution. Fluorometric measurement is performed at 327 nm excitation and 400 nm emission (7).

GLAFENINE 223

4.1.6. Chromatographic Methods

4.1.6.1. Thin - layer chromatography (TLC): Glafenine (I), glafenic acid ( 1 1 ) (major metabolite and major photoproduct in the solid state) and methyl N - (7 - chloro - 4 - quinolyl) anthranilate (111) (minor photoproduct in the solid state) can be identified by TLC method using silica gel 60 F254 (2Ox20cm) with thickness of 0.2 mm as stationary phase. 1OuL of 0.20% and 0.01 % of glafenine and glafenic acid in chloroform are spotted. Methyl N - (7 - chloro - 4 - quinolyl) anthranilate is detected after storing a solution of 0.20% of glafenine in methanol for 24 hours under ambient conditions. The system is equillibrated for 15 minutes before the development. The development distance is 10 cm and the plate is air dried. The detection method is UV lamp (254 nm) or by naked eye (yellow colour spots). Table (VIII) lists the Rf values of glafenine and its photodegraded products in different mobile solvents (4).

Table ( Vlll )

The Thin layer Chromatography of Glafenine and its Photodegraded Products in the Solid State (4).

~ ~~~~

Mobile Phase Rf Value Rf Value Rf Value of (1) of (11) of (111)

Ethylacetate - Chloroform (70/30 by Volume) 0.06 0.00 0.37 Chloroform - Methanol - Acetic Acid 0.30 0.22 0.80 (85/12/3 by Volume) Ethylacetate - Methanol - 33% Ammonia 0.45 0.15 0.75 (85/10/5 by Volume) Chloroform - Methanol (80/20 by Volume) 0.57 0.24 0.77


I Rt (min)

4.1.6.2. High performance liquid chromatography: HPLC profile of glafenine is shown in figure (10). This profile was obtained using Beckman HPLC system. An R - Sil C18 column (150 mm x4.6 mm I.D.) with a particle size of 5 um was used. The mobile phase consisted of a mixture of methanol, water and acetic acid (64:27:9 by volume). The chromatographic system was operated at room temperature with an eluent flow rate of 1 .O mL.min-’. It has a sensitivity of 0.01 absorbance unit, attenuation of 64 and chart speed of 0.5 cm.min-’. The wavelength of the detector was set at 344 nm. This method is stability indicating and may be used for tablets, capsules and suppositories (8).

r, 4

Figure (1 0) The HPLC Profiles of Glafenine Dissolved in Ethanol (10 ug. mL-’).

GLAFENINE 225

4.2. Body Tissues and Fluids

Spectrophotometric methods for the determination of glafenine and its metabolites in the body fluids were insensitive and unspecific. More recent and specific methods using HPLC were reported:

- Determination of glafenine and its metabolites involved a separation and extraction procedure in human plasma. Floctafenine was used as internal standard for both the drug and its metabolites. Chromatographic conditions were 5 um R - Sil Cle column, solvent mixture consisted of methanol - water - acetic acid (67:23:10),respectively.pH was adjusted to 4.3 by the addition of ammonia. The flow rate was 0.5 mL.min-’ and the detector was set at 360 nm. For 1 mL plasma, the detection limit was 0.5mg. L-’ for glafenine and hydroxyglafenic acid, and 0.2mg. L” for glafenic acid. This method allowed the deduction of some primary pharmacokinetic parameters (1 2).

- For determination of glafenine (I): Plasma (1mL) containing 50uL of floctafenine (11) as internal standard solution, was made alkaline with glycine buffer of pH 11 (1 mL) and extracted with CHCl3 (2x5 mL).After centrifugation, the combined organic layer was evaporated to dryness at 37OC and the residue was dissolved in the mobile phase (1 00 uL). For determination of glafenic acid (111) plasma (1 mL) containing (11) solution (50 uL) was acidified with 0.1 N HCI (200 uL) and extracted as above. Sample solutions (20 uL) were analysed by HPLC on acolumn (1 5 cm x 4.6 mm) of Spherisorb Ce (5 um) with acetonitrile - water - diethylamine (550:400:3) adjusted to pH 4.5 with anhydrous acetic acid as mobile phase (1 mL.min-’), detection was at 362,358 and 364 nm for I, II and 111, respectively. Calibration graphs were rectilinear from 0.05 (detection limit) to 2.5 mg.L-’, and 0.25 (detection limit) to 2.0 mg. L” for I and Ill, respectively. The coefficients of variation (n = 10) were 8.1 to 13.7 and 7.7 to 10.8% for I and Ill, respectively (13).


5. STABILITY

5.1. Stabillty of The Solid

Glafenine powder is stable against heat and moisture. The powdered drug was stable when stored at 40 "C in the dark for 180 days (14). Glafenine in the solid form readily undergoes photodegradation when exposed to UV - visible or solar radiation. The photodecomposed products are similar regardless of the method of radiation used. The two photodecomposition products were identified as N - (7 - chloro - 4 - quinolyl) anthranilic acid and methyl N - (7 - chloro - 4 - quinolyl) anthranilate. The first was separated and identified as solid while the second was identified in solution of isopropanol and was found to be present in trace amounts. These were cross checked with similar prepared photoproducts. It seems that intramolecular H - abstraction leads to the formation of these photoproducts. Glafenine formulated into solid dosage forms has to be guarded against sources initiating photochemical degradation (8).

5.2. Stability in The Solution

Glafenine is insoluble in water. Heating the drug's suspension at 50 "C for several hours produced no degradation. However, boiling the same solution yielded hydroysed forms of glafenine. In neutral alcoholic solution, galfenine is unstable towards UV/visible radiation where it photodecomposes into two main products, namely glyceryl anthranilate and 7 - chloroquinoline. Glafenine photodegradation is suggested to occur via intermolecular H - abstraction in the presence of proton donor solvents. The rate of photodecomposition in neutral alcoholic solution was found to decrease with the polarity of the solvent, while the increase in viscosity of the solvent was found to be impeded probably due to the cage effect. In nonpolar solvents such as benzene, photodecomposition is very low. In acidic alcoholic and aqueous solutions, glafenine proved to be quite stable (8).

GLAFENINE 227

6. PHARMACOKINETICS

6.1. Absorption

Glafenine is well absorbed from the intestinal wall in gastrointestinal tract. Following oral administration of glafenine, peak concentration of the main metabolite, glafenic acid, is reached after about one hour, The decline in plasma concentration is multiphasic and incompatible with one compartment model. In view of the lack of free glafenine in the central compartment, a substantial first - phase elimination by liver or gut wall can be assumed. Rectal absorption of glafenine or glafenine hydrochloride is extremely slow and incomplete due to the slight water solubility of glafenine at the prevailing pH in the rectum lumen (15).

6.2. Bioavailability

Glafenine suspension was administered and compared with glafenine suppositories and enemas. It is clear from Table (IX) that rectal administration of micro - enemas or suppositories containing this drug is not bioequivalent with oral dosage form (1 5).

6.3. Distribution

It seems that the glafenic acid is deposited in the kidney. Such deposition is manifested by yellow colouration which disappears with biochemical disturbances (16).

6.4. Metabolism

Comparative studies suggest that analgesic activity of glafenine is due to one of its metabolites. The glycerol liberated in vivo following administration of glafenine does not appear to be responsible for the effect of the drug since


Table ( IX )

Absorption Characteristics, Relative Bioavailability and Urine Excretion Pattern of Glafenic Acid (Mean _+ S.D.), After Oral and Rectal Administration of 400mg Glafenine (439mg Glafenine HCI).

Plasma concentation (ug. mL-') at t:

30 (min) 9.2f3.4 60 12.8k2.7 120 4.6k0.7 1 80 1.6k0.5 240 1.1k0.2 300 0.6k0.1

Number 7 Clll, (ug. mL-') 12.6k3.1 tmaw (min) 50+27 AUCo.5 (ug. rnin". mL-') 1308k68 Frei 1 .oo Urine concentation (mg) at t:

60 (min) 31.126.7 120 44.2f8.2 I80 41.0f6.7 240 12.1 k3.1 300 9.20k2.7

0.20*0.02 0.36k0.05 0.40&0.07 0.37+0.05 0.34k0.06 0.30i~0.02 7 7 0.44k0.09 125&28 98+9 0.075

3.8k0.4 0.8k0.07 5.1i-0.3 2.1 20.2 4.8k0.4 1.7k0.1 4.7f0.5 1 -5k0.2 3.5f0.2 0.2k0.04

GLAFENINE 229

equimolar doses of glycerol did not induce any of glafenine characteristic effects. It was reported (17) that glafenine occurs in human urine mainly as corresponding free acid N - (7 - chloro - 4 - quinolyl) anthranilic acid; the process of hydrotysis (enzymatic or not) being still unknown. Glafenine does not seem to be metabolized into simpler molecules such as 4 - amino - chloroquinoline and anthranilic acid. The structure of glafenine and its metabolites are shown in metabolic pathway established in the rat scheme (VI). The excretion patterns in rat and human urine are very similar indicating that the metabolic pathway should be similar in the two species (1 7).

0

Scheme (vl) The Metabolism Pathways of Glafenine in Rat.


6.5. Excretion

In normal subjects 70% of the product is eliminated through the biliary tracts. The remaining 30% are eliminated in the urine. The urine elimination is early, the maximum is between 2 - 4 hours and it is also rapid as about 70% of the quantity eliminated in the urine is achieved in the first 6 hours. It is eliminated almost totally in the form of metabolites and the major metabolite is free glafenic acid. Glafenine is rapidly eliminated from the body even in cases of high doses of renal insufficiency, plasma half - life of glafenine though longer than in normal subjects stays sufficiently short. After the absorption of a 400 mg dose, the serum level after 24 hours is zero or extremely low (1 6).

6.6. Half - Life

The distribution half - life was reported as 75 minutes (15) and the elimination half life was 3.03 hours (18).

GLAFENINE 23 1

REFERENCES

1 . Gilbert Mouzin, Henri Cousse and Jean Marie Autin; Synthesis, 1 ;54 - 55

2 . Netherlands Appl. Patent 296, 793 (CI. C07d), Roussel- Uclaf (1 965), C.

3 . French Pharmacopeia. Glafenine Monograph.

4 . A.A. Badwan and M.M. AL - Omari; Unpublished Data. The Jordanian

5 . Pamela Girgis Takla and Christos J. Dakas; Int. J. of Pharm., 43,225 - 232

6 . Nadia Ghazal; Unpublished Data, The Jordanian Pharmaceutical

7 . W. Baeyens and P. De Moerloose; J. of pharm. Sci., 66 (1 2), 1771 - 1773

8 . M.M. Omari (1987); M.S. Thesis. Universityof Jordan, Jordan.

9 . Mostafa S. Tawakkol and Mohamed E. Mohamedi Analytical Letters14

10. Mostafa S.Tawakkol, Mohamed E. Mohamed and Mahmoud A. Ibrahim; Pharmazie, 36 (H.2), 163 (1981).

11. S. A. Ismaiel, Abdel - Moety, E.M.; Zentralbl. Pharm., Pharmakother Laboratoriumsdiagn, 127 (2), 57 - 59 (1988).

12. Marie Christine Tournet, Catherine Girre and Pierre Etienne Fournier; J. of Chromatography, 224,348 - 352 (1 981).

13. Ennachachibi, A,, Nicolas P., Fauvelle F., Perret G., Petitjean 0;J. Chromatogr. Biomed. Appl., 3 June, 71 (2), (J. Chromatog, (427), 307 - 314(1988).

14. A.A.Badwan; Stability Data on Glafenine, Unpublished Data, The

(1980).

A., 64,3504 e (1966).

Pharmaceutical Manufacturing Company, Jordan.

(1988).

Manufacturing Company, Jordan.

(1 977).

(BlO), 763 - 770 (1981).

Jordanian Pharmaceutical Manufacturing Company, Jordan.


15. F. Moolenaar, J. Visser and T. Huizinga; Int. J. Pharm. 4,195 - 203 (1980).

16. Pharmacology File on Glafenine - JPM.

17. J. Pottier, M. Busigny and J.P. Raynaudi Eur. J. Drug Metab. 4 (2) 109 -

18. M. C. Tournet, S. Giudicelli, C. Girre, J. Crouzetle and P. E. Fournier; C. - R. - Congr. Biopharm. Pharmaco Kinet. lst, 2,288 - 301, (1981). Edited by J. M. Aiache and J. M. Hirtz.

1 1 5 (1 979).

LISINOPRIL

Dominic P. Ip, Joseph D. DeMarco

and Marvin A. Brooks

Merck Sharp & Dohme Research Laboratories

West Point, PA 19486


Copyright Q 1992 by Academic Press, Inc. All rights of reproduction WeNed in any form.

234 DOMINIC P. IP, JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

LlSlNOPRlL

Dominic P. Ip, Joseph D. DeMarco and Marvin A. Brooks

1. History and Therapeutic Properties

2. Description

2.1 Nomenclature 2.1.1 Chemical Name 2.1.2 Generic Name 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 CAS Registry Number

2.2 Formula and Molecular Weight 2.3 Appearance, Color, Odor

3. Synthesis

4. Physical Properties

4.1 4.2 4.3 4.4 4.5 4.6 4.7 4.8 4.9 4.1 0 4.1 1 4.12

Infrared Spectrum 'H - Nuclear Magnetic Resonance Spectrum I3C - Nuclear Magnetic Resonance Spectrum Ultraviolet Spectrum Mass Spectrum Specific Rotation Thermal Behavior Solubility Dissociation Constants Crystal Properties Hygroscopicity Partition Coefficient

LISINOPRIL 235


5.1 Elemental Analysis 5.2 Chromatographic

5.2.1 Thin-Layer Chromatography 5.2.2 High Performance Liquid Chromatography

5.2.2.1 Bulk Drug Analysis 5.2.2.2 Lisinopril in Formulation

5.3 Titration 5.4 Other Methods 5.5 Identification Tests

6. Stability

6.1 Solid State Stability 6.2 Solution Stability

7. Determination in Body Fluids and Tissues

7.1 Radioimmunoassay 7.2 Competitive Inhibitor Binding Assay 7.3 Fluoroentymatic Assay

8. Drug Metabolic Products, Pharrnacokinetics and Bioavailability

9. References


1. History and TheraDeutic ProDerties

Lisinopril, a lysine analogue of enalaprilat, is a long-acting angiotensin converting enzyme inhibitor which differs from captopril by lacking the sulfhydryl group. Lisinopril, discovered and developed by the Merck Sharp & Dohme Research Laboratories (l) , is indicated for the treatment of hypertension and congestive heart failure. Several review articles give a detailed account of the history, design, chemistry and pharmacology of the drug (2-6).

2. Description

2.1 Nomenclature

2.1.1 Chemical Name

(a) L-Proline, 1 -[N2-( 1 -carboxy-3-phenylpropyl)-L- lysyl -dihydrate, (S)-

proline dihydrate

(b) 1 -[N h -[(S)-1 -carboxyl-3-phenylpropyl]-L-lysyl]-L-

2.1.2 Generic Name

Lisinopril

2.1.3 Laboratory Codes

L-l54,826-000T, MK-0521

2.1.4 Trade Names

Prinivil, Zestril, Carace, Novatec

2.1.5 CAS Reqistry Number

9301 5-83-7

2.2 Formula and Molecular Weight

'21 H31 N3°5 ' 2H20 Molecular Weight 441.52

LISINOPRIL 231

H H H O T I T I I

COOH A (CH2)4 NH2 A I H ?OOH

2H20 CH2CH2 .. -C--N--C- -C-N

2.3 Appearance, Color, Odor

Lisinopril is a white to off-white crystalline, odorless powder.

3. Synthesis

Lisinopril has been prepared by the scheme outlined in Figure 1 (7,8). The dipeptide, N,-trifluoroacetyl-L-lysyl-L-proline c1) is subjected to reductive alkylation with ethyl 2-oxo-4-phenylbutanoate (2J over Raney Nickel via a Schiff base (3J to yield a diastereomeric mixture 4 (SSS and RSS). Hydrolysis of the N,-trifluoroacetyl moiety and saponification of the ethyl ester followed by crystallization in ethanol/water and final recrystallization in water yield lisinopril (SSS, 5J of greater than 98% purity in about 65% yield (based on 3. In addition to this synthetic route, others have also been described in the literature (9-12).


4.1 Infrared Spectrum (13)

The infrared spectrum of lisinopril as shown in Figure 2 was obtained in a potassium bromide pellet using a Perkin-Elmer Model 281 -B spectrophotometer. Assignments for the characteristic bands in the spectrum are listed in Table 1.

’ H-Nuclear Maanetic Resonance Spectrum (14) 4.2

The proton magnetic resonance spectrum of lisinopril is shown in Figure 3. The spectrum was obtained using a Bruker Instruments Model WM250 spectrometer and a 10% W N solution of lisinopril in ,lJ solution of deuterium chloride in deuterium oxide. The reference compound (internal) was p-

N W m

- 1 R=CF 3CONHCH 2(CH 2)3

- 2

Ph

(2) Crystallization E~O,C

(1) OH-

*2H20 *

H 0 CO,H

- 5, Lisinopril 4 R'=H 2NCH ,(CH 2)3

Figure 1 Synthesis of Lisinopril

239

Wsvelenglh (rnlcrons)

N w 10

Frequency (CM.'I

Figure 2. Infrared Absorption Spectrum of Lisinopril

240 DOMINIC P. IP. JOSEPH D. DEMARCO, AND MARVIN A. BROOKS

dioxane. An expansion of the spectrum in the 0-6 ppm region is shown in Figure 4. Chemical shifts and assignments for the numbered structure shown below are tabulated in Table II.

COOH 1

Table I

Lisinopril Infrared Band Assianmentsa

Wavenumber (cm-') Assiqnment

3545

Near 3370 81 3290 (broad) 3090 - 2860 -2800 - -2100 1655 1609 1570 1541 1450, -1 443 (strong) 1388 1340, 1299 741, 732,692

OH stretching vibrationb (dihydrate

06 stretching vibration C-H stretching region

Asymmetric -C02: stretch Asymmetric -C02 stretch 'NH or 'NH, bending CH2%ending Symmetric -C02- stretch Not assigned Phenyl out-of-plane bending (2 bands plus -(CH2)4-r~~k)

H 0)

stretching region

a These bands are subject to a reading error range of 25 cm-' above 2000 cm-' and +3 cm-' below. These bands disappeared in a dehydration experiment monitored by infrared spectrometry.

Figure 3. The Proton Magnetic Resonance Spectrum of Lisinopril

Figure 4. The Proton Magnetic Resonance Expanded Spectrum of Lisinopril

LISINOPRIL

Table II

Lisinopril, Proton Maqnetic Resonance Assiqnments

Chemical Shift, SH (ppml Assiqnmenta

a b

C

4.3

7.35 5.06 4.44 4.35 3.84 3.75 3.62 3.04 2.85 2.33

2.03

1.73 1.60

Phenyl ring protons H CI/H D 0 C, protonb C,, proton b

C,,: proton p-dioxane (reference) C, protons C6, protons C, protons C, protons; C, proton (one of

C3, protons; C, protons; C

C5, protons C,, protons

the two)'

proton (other of the twoyc

Assignments refer to number structure above. As in the case of aqueous solutions of captopril (15) and enalapril (1 6), signals attributable to rotamers (rotation about the lysyl-proline tertiary amide bond) are observed in the spectrum of lisinopril. The triplets at 4.62 ppm and 4.13 ppm represent, respectively, the C, and C2, protons in the minor rotamer. Private communication with Dr. B. J. Woodhall, Pharmaceutical Division, Imperial Chemical Industry.

I3C Nuclear Maqnetic Resonance Spectrum (12)

The carbon-13 magnetic resonance spectrum of lisinopril shown in Figure 5 was obtained using a Varian Associates Model XL-1 OOA spectrometer and a 10% (WN) solution of lisinopril in 1 N deuterium chloride in deuterium oxide. The reference compound (internal) was p-dioxane. An expansion of the spectrum in the 12.5-75 ppm region is shown in Figure 6. Chemical shifts and assignments for the numbered structure shown below are tabulated in Table Ill.

243

3

D

rl)

0 : 5 Q

3

3 3 3

N

244

Figure 5. The Carbon-13 Magnetic Resonance Spectrum of Lisinopril

N P VI

I

75 62.5 50 37.5 25 12.5 PPM

Figure 6. The Carbon-13 Magnetic Resonance Expanded Spectrum of Lisinopril

246 DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

NH

COOH 1

Table Ill

Lisinomil, Carbon-1 3 Maanetic Resonance Assianments

Chemical Shift, 6, (ppmla 21.57 (22.04) " 25.39 (22.51) 27.20 29.49 (31.38) 30.02 (30.58) 31.21 31.92 (32.24) 39.89 48.70 (48.1 6) 59.73 (60.29) 60.1 1 60.53 (59.64) 67.40

127.58 129.510~ 1 29.6Eid 140.53 (140.44) 167.32 (1 67.80) 171.44 (1 71 5 5 ) 175.71 (175.14)

Assianmentb-

c43

:;: :;: C, C,.

c5 c21 C,. c2

cP crn

CIS c, I.

C l

'6'

p-dioxane (reference)

:;

Values in parentheses are due to a minor conformational isomer, and many of the assignments for this component are tentative. Assignments refer to numbered structure above. Private communication with Dr. 8. J. Woodhall, Pharmaceutical Division, Imperial Chemical Industry. These assignments could be reversed.

LISINOPRIL

4.4 Ultraviolet Spectrum

241

The ultraviolet absorbance spectra of lisinopril shown in Figure 7 (1 7) were obtained using a Perkin-Elmer Lambda 5 UV-VIS scanning spectrophotometer. The spectrum in 0.1N sodium hydroxide solution is characterized by low intensity maxima at -246 nm, -254 nm, -258 nm, -261 nm and -267 nm with respective Al% 1 cm values of -4.0, -4.5, -5.1, -5.1 and -3.7. The spectrum in 0.1N hydrochloric acid is characterized by maxima at -246 nm, -253 nm, -258 rim, -264 nm and -267 nm with respective Al% 1 cm values at -3.2, -3.9, -4.5, -3.0 and -2.8.

The ultraviolet absorbance arises from the unconjugated phenyl ring in lisinopril molecule.

4.5 Mass Spectrum (18)

The mass spectrum of lisinopril shown in Figure 8 was obtained by direct probe-electron impact (70 eV) method using an LKB Model 9000 mass spectrophometer. The spectrum shows no molecular ion peak. A pseudo-molecular ion peak at m/e 387.2160 is attributed to the diketopiperazine formed during vaporization. Mass fragment assignments are given in Table IV.

4.6 Specific Rotation (19)

Lisinopril contains three chiral centers and is The specific rotation values [a]25

0.25M pH 6.4 zinc acetate) are respectively --120" and --96". and [a]

405 nm 436 nrn

4.7 Thermal Behavior (20)

Differential thermal analysis under vacuum (DTA heating rate = 2OoC/rnin.) as shown in Figure 9 indicated three endotherms with peak temperatures at -98"C, -1 22°C and -1 82°C. The first two endotherms correspond to loss of water of hydration and the one at 182°C to melting.

0.80

0.64

8 0.48 m e !2

C

0.32

0.16

0 220 240 260 280 300 320 340

Wavelength (nrn)

Figure 7. The Ultraviolet Absorption Spectrum of Lisinopril in (a) 0.1N Sodium Hydroxide; Concentration: 1.374 mg/ml and (b) 0.1N Hydrochioric Acid; Concentration: 1.374 mg/ml

Figure 8. The Direct Probe-Electron Impact (70 eV) Mass Spectrum of Lisinopril


Table IV

Lisinopril, Mass Spectrum Assianments

- M/e Assiqnment

387 C,, H2,N304 (M' minus H,O)

369

358

342/343

329

315/316

313

296

283

265

252

245

m/e 387 minus H,O

m/e 387 minus CH3N

m/e 387 minus CO,(H)

m/e 387 minus C3H,N

m/e 387 minus C,H9,,0N

m/e 358 minus C0,H

m/e 387 minus benzyl radical

C,3H,1 N304 (m/e 387 minus styrene)

m/e 283 minus H20

m/e 296 minus CO,

0 CH&H?-N=CH(CH,),NH,

c=o €B

+

1.

LISINOPRIL 25 I

Table IV (Continued)

Lisinopril, Mass Spectrum Assiqnrnents

Mle Assiqnment - 224 '1 1 Hl aN3'2

CH&HzCHzC&NH, CHCH$&CHzNH2

d e 224 rnle 224

207

179

84

CYCH,CH,CH,NH,

.,,dly" tj we224

C, H,,N,O, (rn/e 224 minus NH,)

H Jaco-g e'

H

QH H


Table IV (Continued)

Lisinopril, Mass SDectrum Assianments

- M/e Assianment

70 W" n

4.7 Thermal Behavior (continued)

The thermogravimetric analysis (TGA) curve of lisinopril shown in Figure 10 depicts three inflections corresponding to the loss of free water and the first and second moles of water of hydration. The excess unbound water was 0.6% over the theory of 8.2% for the dihydrate indicating lisinopril is somewhat hygroscopic.

4.8 Solubility (20)

The following approximate solubility data were obtained at ambient temperature.

Table V

Solvent Solubility (mq/ml)

Water 97 Methanol 1 4a Ethanol <0.1 Acetone <o. 1 Acetonitrile <0.1 Chloroform <o. 1 N,N-Dimethylformamide <0.1

a Upon dissolution of lisinopril in methanol, changes in X- ray diffraction patterns indicative of loss of water of hydration were observed. The solubility value obtained becomes dependent upon the water content of the solution.

I ~ I ~ I ~ I ~ I ~ I ~ l ~ ~ l I I I I I

0 4 - -

0 0 - I

c

-0.4 - - Q

2 r g -0.8 - - 0 0 5 e -1.2 -

L

c.

0 0.

-

E - - 1 6 - -

2 0 - c

-

-2 4 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 l 1 1 l l ~

Figure 9. Differential Thermal Analysis (DTA) Curve of Usinopril

Temperelure ( 'C)

Figure 10. Thermogravimetric Analysis Curve of Llsinopril

LISINOPRIL 255

4.9

4.1 0

4.1 1

4.1 2

Dissociation Constants (20)

Aqueous acidichasic potentiometric titration at 25°C yielded four pKa values of 2.5, 4.0, 6.7 and 10.1 for lisinopril.

Crvstal ProDerties (20)

Lisinopril is crystalline as determined by X-ray powder diffraction. A typical X-ray powder diffraction pattern obtained using a Philips APD 3720 X-ray powder diffractometer is shown in Figure 11. A monohydrate which is crystalline, also exists. The monohydrate is readily distinguishable from the dihydrate by its X-ray powder diffraction pattern (Figure 12).

Hvcrroscopicity (21)

Lisinopril is slightly hygroscopic. A sample stored for three months at 98% relative humidity and room temperature showed an increase of 1.1 Yo in the total volatiles content by TGA.

Partition Coefficient (17)

The partition coefficient of lisinopril in the phosphate buffer (O.lM, pH 7)h-octanol system was determined to be 10.2 0.5 at room temperature.

5. Methods of Analvsis

5.1 Elemental Analysis (22)

The elemental analysis found for a reference lot of lisinopril L- 154,826-00T031 was

/Elemental) Analvsis

'21 H31 N3°5 ' 2H20

% Theow*

Carbon 57.13

Nitrogen 9.52 *Anhydrous basis

Hydrogen 7.99

% Found*

56.89 7.69 9.49

256

Figure 11. Powder X-Ray Diffraction Pattern of Lisinopril

L U

Figure 12. Powder X-Ray Diffraction Pattern of Lisinopril Monohydrate

2% DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

5.2 C h ro matoq raph ic

Chromatographic procedures have been developed to separate lisinopril from its principal decomposition product, the diketopiperazine (DKP), a product of intramolecular dehydration. Since there are three optical centers in the molecule, isomers of lisinopril and its DKP degradate are possible. The sequence of thermal decomposition for lisinopril to form isomers of DKP is shown in Figure 13.

The conversion of SSS DKP to SSR DKP has been demonstrated (23) by heating SSS DKP for 15 minutes at 160°C.

Chromatographic procedures also separate in some cases a process impurity, 4-phenyl-2-aminobutyric acid (APBA) and the RSS isomer of lisinopril (Figure 14).

5.2.1 Thin Laver Chromatoqraphy (TLCl(19)

Four TLC systems have been developed and are listed below. Systems (I) and (11) separate lisinopril from its RSS isomer, and APBA. Systems (Ill) and (IV) are of somewhat limited value since they do not separate lisinopril from the RSS isomer. Systems (I) and ( 1 1 ) utilize E. Merck Silica Gel G-60 while systems ( I l l ) and (IV) use Analtech Silica Gel G-60. Visualization of spots is accomplished by reaction with ninhydrin.

Lisinopril (SSS) Lisinopril SSS DKP Lisinopril SSR DKP

Figure 13. Thermal Decomposition of Lisinopril to Its Diketopiperazine Isomers

H H O V H V I I

A A HOOC- -C--N--C--C-N

CH2 (CH2)4 I I H

6 NH2

RSS Isomer

CHzCHpCHNH2

8 Ao2H

2-amino-4-phenyl butyric acid (AP BA)

Figure 14

LISlNOPRlL 26 I

Table VI TLC Systems

Lisinopril Solvent Systems Rf (Approximate)

(1) n - B u tan ol/tol ue ne/g lacial acetic acid/water/acetone

0.34

(1:l:l:l:l)

(11 ) n-Butanol/water/glacial acetic acid (3:l:l)

(111) n-Butanol/water/glacial acetic acid/ethyl acetate (1:l:l:l)

(1V) Chloroforrn/rnethanol/conc. ammonium hydroxide (4:4:1)

0.22

0.43

0.14 to 0.39

262 DOMINIC P. IP. JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

5.2.2 Hiqh Performance Liquid Chromatoqraphv (HPLC)

5.2.2.1 Bulk Druq Analysis (24)

Methods of analysis for lisinopril in bulk drug are summarized in Table VII. Method ( I ) which utilizes a linear gradient is capable of separating lisinopril from two potential process impurities, the RSS isomer of lisinopril and the 2-amino-4-phenylbutyric acid (APBA), and the diketopiperazine SSS and SSR isomer degradation products (see Figures 13 and 14). Under isocratic conditions, method (I) also separates lisinopril from its RSS isomer and APBA. Both gradient and isocratic procedures use a Zorbax (DuPont) RP-8 column at a pH of 5.0 and a column temperature of 50°C. The isocratic procedure has been published in the USP (25). Lisinopril exhibits typical chromatographic behavior (peak broadening) attributed to rotational isomers (26) of proline-containing dipeptides. Higher column temperatures of 950°C appears to be necessary to minimize this effect for acceptable chromatography.

Method (11 ) which utilizes a PRP-1 column (Hamilton Co.) has also been used for the evaluation of lisinopril bulk drug. This method is, however, more cumbersome to use than method (I) which was found to offer better resolution for compounds of interest. Detection for all methods is by UV at 210 to 215 nm.

5.2.2.2 Lisinopril in Formulation (27)

Methods for the analysis of lisinopril in dosage forms are summarized in Table VIII. Methods (I), (11) and (111 ) have been developed to separate lisinopril from its principle degradation product, the SSS diketopiperazine (DKP) and a process impurity APBA (see Section 5.2.1). Method

LlSlNOPRlL 263

(IV) was developed to include the separation of hydrochlorothiazide as well. These methods (I-IV) all utilize a Lichrosorb RP-8 column, 10 pm at 40°C or 50°C. Method (IV) uses a somewhat longer column (300 x 4.6 mm) and it was found that as a result of the increased length, column temperature could be reduced to 40°C while maintaining good peak efficiency (see Section 5.2.2.1). This method is also capable of separating lisinopril diketopiperazine SSS from the SSR isomer as well.

The retention times of both lisinopril and SSS DKP have been found to be a function of the phosphate concentration in methanol (see Figures 15 and 16). Similar data have been obtained in acetonitrile.

The retention of lisinopril and SSS DKP also decreased as the % organic modifier increased. Thus by varying both the molarity of phosphates and % organic modifier in the mobile phase, the resolution of these compounds can be optimized. Methods (V) and (Vl) were developed to quantitate lisinopril for content uniformity and dissolution.

Detection in all of these methods as in the methods designed for the bulk drug is by UV at 210 nrn to 215 nm.

A stability-indicating HPLC method for lisinopril tablets has been published in the USP (28). This compendia1 method employs the same mobile phase composition as method ( 1 1 1 ) but contains an ion-pair reagent.


Table VII

HPCC Systems for Lisinqxil Bulk DruQ

- Merhcd Column Chromatographic Conditions

(1) Zorbax (DuPont) RP-8 A:Acetonitrile 250 x 4.6 mm, 5 p n B:0.02M NaH2P04 pH 5.0

Gradient: T = 50%

- 0% A to 30% A linear over 35 minutes

Legend

a = Lisinopril b = RSS Isomer C = SSS DKP d = SSR DKP

= APBA

PRP-1 (Hamilton Co.) 250 x 4.6 mm. 10 km, T = 50°C

Isocratic: 96% Solvent B - 4% Solvent A

A:O.O2M NaH2P04 at pH 6.8 B:O.O15M NaH2P04 at pH 3.0 C:Acetonitrile

Isocratic: 96% Solvent A - 4% Solvent C

Gradient (11 Solvent NSolvent C (97 .525) for 10 minutes, then linear gradient to Solvent NSolvent C (70:30) in 30 minutes

Gradient [ZJ Solvent &Solvent C (95:s) for 10 minules then linear gradient to Solvent BlSolvent C (70:30) in 30 minutes.

Separation

a.b,c,d,e

a,b,e

LISINOPRIL 265

Table Vll l

HPLC Systems for Lisinopril in Solid Dosage Formulations

Purpose

Stability Single entity



Stability HCTZ Combination

Content Uniformity Dissolution Single entity

Content Uniformity Dissolution HCTZ Combination

Legend

a = Lisinopril b = RSS Isomer C = SSS DKP d - SSR DKP E = APf3A f = Hydrochlorothiazide (HCTZ)

Column

Lichrosorb RP-8 (Hewlen Packard) 200 x 4.6 mm, 10 pm T = 50%

Lichrosorb RP8 (Hewlen Packard) 200 x 4.6 mm. 10 pn T = 50°C

Lichrosorb RP-8 (Hewlea Packard) 200 x 4.6 mm. 10 pm T = 50°C

Lichrosorb RP-8 (E.S. Industries) 300 x 4.6 mm. 10pm T = 40%

Hypersil ODs (Shandon) 50 x 4.6 mm, 5 pn T = 60°C

Lichrosorb RP-8 Hewlen Packard 200 x 4.6 mm, 10 pm T = 50°C

Chromatographic Conditions Mobile Phase (Conditions) Separation

Acetonim’le/0.004M phosphate, a,c,e

45:55 pH 2.0

MethanoVO.WM phosphate, pH 2.0 a.c.e 45:55

Acetonitrile/O.O3M phosphate, a.c,e pH 2.0

2o:ao

AcetonitriIe/O.O05M phosphate, a.c,d,e.f

35:65 pH 2.0

MethanoVO.02M phosphate, pH 2.0 a 12:88

Acetonitrile10.04M phosphate, a

15:85 pH 2.0

Phosphate Molarity

Figure 15. Effect of Phosphate Concentration on Retention of Lisinopril

Figure 16. Effect of Phosphate Concentration on Retention of SSS DKP


5.3 Titration (17)

Lisinopril can be determined by potentiometric titration with aqueous sodium hydroxide and non-aqueous perchloric acid. The sodium hydroxide titration is carried out by titrating the lisinopril potentiometrically with carbonate free 0.1 N NaOH to one endpoint using a combination electrode. Lisinopril can also be determined by titration potentiometrically with 0.1 N perchloric acid in acetic acid to one endpoint. The electrode system consists of a glass electrode (such as a Metrohm Model EA 107 vs. a silver/silver chloride reference electrode such as a Metrohm Model EA 432 filled with 0.1 N lithium perchlorate in glacial acetic acid.

5.4 Other Methods

Lisinopril has also been determined by radioimmunoassay (RIA), fluoroenzymatic assay (FEA) and competitive inhibitor binding assay (CIBA). These procedures are described in Section 7.

5.5 Identification Tests

Identification of lisinopril can be carried out by infrared absorption (see Section 4.1), TLC (see Section 5.2.1) and by HPLC (see Section 5.2.2).

Supportive evidence for identification can be obtained by differential thermal analysis (DTA) (see Section 4.7).

6. Stability

6.1 Solid State - Thermal (29)

Lisinopril is stable as a solid at ambient temperatures (20- 25°C). Degradation can be induced when the solid is stressed at the severe thermal stress conditions of 105°C. HPLC studies have demonstrated that intramolecular dehydration to DKP is the primary degradate. The yield to the diketopiperazine as % of the total chemical loss of lisinopril is higher in nitrogen flushed closed container (80%) than in non- flushed closed container (50-60%) or in open container (6%). Lisinopril, when stressed under these severe conditions, yields

LISINOPRIL 269

additional degradates, the identity of which has not been determined.

Studies have demonstrated that the lisinopril diketopiperazine isomer initially formed is the SSS isomer which can degrade further to the SSR isomer (see Section 5.2).

6.2 Solid State - Photochemical (30)

Very slight surface discolorations have been observed when lisinopril is exposed to intense UV radiation for 24 hours.

6.3 Solution Stability (29)

The solution stability of lisinopril at a concentration of 0.2 mg/ml was studied at pHs ranging from 2.7 to 10.0 and at a constant ionic strength (p = 0.3). Figure 17 depicts a plot of the zero order rate constants from 60" and 80°C data vs. pHs. Lisinopril decomposition proceeds rapidly in acidic media with the major decomposition product being the diketopiperazine. The rate of formation of the diketopiperazine was found to be linear with time. In neutral and basic >pH 7.0, the decomposition rate is minimal.

Figure 17. Plot of Rate Constants at 60" and 80°C as a Function of pH

LISlNOPRlL 27 I

7. Identification and Determination in Body Fluids and Tissues

The quantitation of lisinopril in biological fluids has been reported by standard radioimmunoassay (RIA), competitive inhibitor binding assay (CIBA) and fluoroenzymatic assay (FEA).

7.1 Radioimmunoassay

Radioimmunoassay procedures for the drug in plasma and urine have been developed utilizing a polyclonal antiserum produced to a conjugate in which lisinopril is linked to albumin via a dinitrophenylene bridge (2,31) or succinoylated keyhole limpit haemocyanin (32). In the former procedure, the radiolabel was introduced via radio-iodination of a p- hydroxybenzamidine derived from lisinopril, whereas in the latter procedure, the radiotracer was prepared by acylation of the epsilon amino group of the lysyl side chain of lisinopril with N-~uccinimidyl-(2,3-~H)-proprionate. The limit of sensitivity for the RIA procedures is approximately 0.2-0.4 ng/ml (0.5-1 nM).

7.2 Competitive Inhibitor Bindinq Assay

Binding assays were first described as a technique for the measurement of angiotensin converting enzyme inhibitor (ACE inhibitor) activity (33). This procedure requires incubation (37°C for 2 hours) of serum samples with 1251-labeled ACE in hibitor (p-hydroxybenzamidine derivative of lisinopril) followed by a charcoal precipitation and gamma counting of the precipitate. The ACE value is then determined from a standard curve of inhibitor binding vs. ACE activity determined by an enzymatic kinetic assay (34). The principle of this assay was then extended to a competitive inhibitor binding assay in which the 1251-labeled inhibitor (see above) is displaced from isolated ACE by lisinopril in the biological sample (35-36). The free label is separated by adsorption onto charcoal and related to lisinopril drug concentration. The sensitivity of the assay is reported to be 2-4 ng/ml (35) and correlates well with specific radioimmuno-assays and ACE enzymatic activity (37).

7.3 Fluoroenzymatic Assay

The determination of ACE enzymatic activity via the measurement of the rate of cleavage of the substrates hippuryl-L-histidyl-L-leucine or hippuryl-L-glycyl-L-glycine to


hippuric acid with quantitation by fluorometric, radioassay, liquid chromatographic, radioimmunoassay and enzyme linked immunoassay methods has been reviewed (33). Recent assay procedures have incorporated ACE inhibitors into these enzymatic assays with radioassay (38) or fluorometric (39) measurement. The percent of ACE activity inhibited is correlated with drug (inhibitor) concentration. These procedures (38-39) require extraction of the drug from plasma or urine with methanol to separate the drug (inhibitor) from endogeneous ACE. The methanolic supernatant is evaporated, and then reconstituted in a solution of exogenous ACE containing the substrate. The fluorometric assay (39) utilizes the substrate N-benzoyloxycarbonyl-L-phenylalanyCL- histidyl-L-leucine which has been previously utilized to measure enalaprilat in biological fluids (40). The enzymatic reaction is quenched with ice, o-phthaldialdehyde added, and spectrofluorometric measurement of the derivative is performed at excitation/emission wavelengths of 365490 nm. A logit-log relationship between drug (inhibitor) concentration and percent enzyme inhibitor is used to calculate drug (inhibitor) concentration. The assay has demonstrated a sensitivity limit of 0.7 ng/ml with an RSD of 3-10% over the standard concentration range of 0.4-35 ng/ml for enalaprilat. The assay clearly has the advantage over RIA and ClBA as described of not requiring radiolabel technique to perform the assay. However, utilizing the lengthy sample preparation, assay capacity is limited.

8. Druq Metabolic Products, Pharmacokinetics and Bioavailability

Utilizing the RIA procedure for serum and urine and in vitro isotope dilution procedure for feces, the absorption and elimination profile of lisinopril was determined in 12 healthy male volunteers following oral administration of a 10 mg capsule (2). The observed peak serum concentration was 95 +. 55 nM with a time to peak of 7 +. 1 hours and an AUC (0-72 hours) of 1694 +. 808 nmol liter-’ hr. The serum concentration vs. time profile was polyphasic and the terminal half-life was approximately 30 hours. The renal clearance was 106 2 13 ml/min wit4 urinary and fecal recovery of 29% 2 15% and 69% 23%, respectively, indicating the drug was excreted unchanged.

The half-life for the terminal phase (approximately 40 hours) was not predictive of steady state parameters when 10 daily doses of lisinopril were administered orally to healthy subjects. The mean effective

LISINOPRIL 213

half-life of accumulation was 12.6 hours. The mean accumulation ratio was 1.38 with steady state attained after the 2nd dose (41). The drug is not metabolized but is eliminated via the kidneys. Lisinopril probably undergoes glomerula filtration, tubular secretion and tubular reabsorption (42).

The close correlation between serum concentration of drug and the degree of inhibition of ACE has been demonstrated. Furthermore, there was a close inverse relationship between plasma levels and the ratio of angiotensin II:i, the latter parameter being a measure of the conversion of angiotensin I to II (43).

Age and cardiac failure are reported to be associated with reduced renal clearance of lisinoprii (44). The plasma concentration and drug half-life in patients with chronic renal failure (creatinine clearance 5 30 ml/min) are generally higher than those seen in patients with normally functioning kidneys (45). Food intake had no effect on the pharmacokinetics of lisinopril (42,46).

Acknowledcrements

The authors wish to thank Mrs. Laurie Rittle for typing the manuscript and Mrs. Florence Berg for conducting the literature search.

9. References

1. A.A. Patchett, E. Harris, E.W. Tristram, M.J. Wyvratt, M.T. Wu, D. Taub, E.R. Peterson, T.J. Ikeler, J. tenBroeke, L.G. Payne, D.L. Ondeyka, E.D. Thorsett, W.J. Greenlee, N.S. Lohr, R.D. Hoffsommer, H. Joshua, W.V. Ruyle, J.W. Rothrock, S.D. Aster, A.L. Maycock, F.M. Robinson, R. Hirschmann, C.S. Sweet, E.H. Ulm, D.M. Gross, T.C. Vassal and C.A. Stone, Nature 288, 280 (1980).

2. E.H. Ulm, M. Hichens, H.J. Gomez, A.E. Till, E. Hand, T.C. Vassil, J. Biollaz, H.R. Brunner and J.L. Schelling, Br. J. Clin. Pharmacol. 14, 357 (1982).

3. A.A. Patchett in "Hypertension and the Angiotensin System: Therapeutic Approaches", A.E. Doyle and A.G. Bearn, Editors, Raven Press, New York, NY 1984.

4. T.A. Noble and K.M. Murray, Clin. Pharm. 7, 659 (1988).

274 DOMINIC P. IP, JOSEPH D. DEMARCO. AND MARVIN A. BROOKS

5. C.S. Sweet and E.H. Ulm, Cardiovasc. Drua Rev. 6, 181 (1988).

6. H.J. Gomez, V.J. Cirillo and F. Moncloa, J. Cardiovas. Pharmacol. 9(SuDDI. 31, S27 (1987).

7. T.J. Blacklock, R.F. Shuman, J.W. Butcher, W.E. Shearin, Jr., J. Budavari and V.J. Grenda, J. Ora. Chem. 53, 836 (1988).

8. E.E. Harris, A.A. Patchett, E.W. Tristram and M.J. Wyvratt (Merck

9. M.J. Wyvratt, E.W. Tristram, T.J. Ikeler, N. Lohr, H. Joshua, J.P.

& Co., Inc.), U.S. Patent 4,374,829.

Springer, B. Arison and A.A. Patchett, 2816 (1984).

10. J.S. Kaltenbronn, D. DeJohn and U. Krolls, Ora. Prep. Proceed. Int. 15, 35 (1983).

11. H. Urback and R. Henning, Tetrahedron Lett. 25, 1143 (1984).

12. M.T. Wu, A.W. Douglas, D.L. Ondeyka, L.G. Payne, T.J. Ikeler, H. Joshua and A.A. Patchett, J. Pharm. Sci. 74, 352 (1985).

13. G. Bicker, Merck Sharp & Dohme Research Laboratories, Rahway, NJ.

14. A.W. Douglas, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ.

15. D.L. Rabenstein and A.A. Isab, Anal. Chern. 54, 526 (1982).

16. D.P. Ip and G.S. Brenner, Analvtical Profiles of Druq Substance

17. Merck Sharp & Dohme Research Laboratories, unpublished data.

16, 207 (1987).

18. G.A. Schonberg, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ.

19. R.B. Waters, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ.

20. J.A. McCauley, Merck Sharp & Dohme Research Laboratories, Rahway, NJ.

LISINOPRIL 215

21. R.J. Magliette, Merck Sharp & Dohme Research Laboratories, Rahway, NJ.

22. J. Perkins, Merck Sharp & Dohrne Research Laboratories, Rahway, NJ.

23. C. Bell, Merck Sharp & Dohme Research Laboratories, West Point, PA.

24. T. Novak, Merck Sharp & Dohme Research Laboratories, Rahway, NJ.

25. The United States Pharrnacopeia XXII, 2355 (1990).

26. W.R. Melander, J. Jacobsen and C. Horvath, J. Chrornatoqraphv - 234, 269 (1982).

27. J. DeMarco and P. Kusrna, Merck Sharp & Dohrne Research Laboratories, West Point, PA.

28. The United States Pharmacopeia XXII, 2475 (1991).

29. D.P. Ip, Merck Sharp & Dohrne Research Laboratories, West Point, PA.

30. J. DeMarco, Merck Sharp & Dohrne Research Laboratories, West Point, PA.

31. M. Hichens, E.L. Hand and W.S. Mulcahy, Liqand Quarterv 4, 43 (1981).

32. P.J. Worland and 6. Jarrott, J. Pharm. Sci 75, 512 (1986).

33. F. Fyhrquist, I. Tikkanen, C. Gronhagen-Riska, L. Hortling and M. Hichens, Clin. Chern. 30, 696 (1984).

34. J. Lieberrnan, Am. J. Med. 59, 365 (1975).

35. C. Gronhagen-Riska, I. Tikkanen and F. Fyhrquist, Clin. Chim. Acta 162, 53 (1 987).

36. B. Jackson, R. Cubela, and C.I. Johnston, Biochem. Pharmacoloqy 36, 1357 (1987).

216 DOMINIC P. IP. JOSEPH D. DEMARCO, AND MARVIN A . BROOKS

37. B. Jackson, R. Cubela and C.I. Johnston, J. Cardiovasc. Pharmacol. 9, 699 (1987).

38. B.N. Swanson, K.L. Stauber, W.C. Alpaugh and S.H. Weinstein, Anal. Biochem. 148, 401 (1985).

39. K. Sheplay, M.L. Rocci, H. Patrick and P. Mojaverian, J. Pharm. and Biomed. Awl. 6, 241 (1988).

40. D.J. Tocco, F.A. deLuna, A.E.W. Duncan, T.C. Vassil and E.H. Ulm, Drua Met. Dispos. 10, 15 (1982).

41. B. Beerman, A. Till, H.J. Gomez, M. Hichens, J.A. Bolognese, and I.L. Junggren, Biopharm. Druq Dispos. 10, 397 (1989).

42. 8. Beerman, Am. J. Med. 85, 25 (1988).

43. J. Biollaz, J.L. Schelling, J.L. descombes, D.B. Bruner, G. Desponds, H.R. Brunner, E.H. Ulm and H.J. Gomez, Brit. J. Clin. Pharmacoloav 14, 363 (1 982).

44. P.C. Gautam, E. Vargas and M. Lye, J. Pharm. Pharmacol. 39, 929 (1987).

45. J.G. Kelly, G.D. Doyle, M. Carmody, D.R. Glover and W.D. Cooper, Br. J. Pharm. Pharmacol. 25, 634p (1988).

46. P. Mojaverian, M.L. Rocci, P.H. Vlasses, C. Hoholick, R.A. Clementi and R.K. Ferguson, J. Pharm. Sci 75, 395 (1986).

LOVASTATIN

Gerald S. Brenner, Dean K. Ellison,

and Michael J . Kaufman

Merck Sharp & Dohme Research Laboratories

West Point, PA 19486


Copyright @ 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

278 GERALD S. BRENNER, DEAN K . ELLISON, AND MICHAEL J. KAUFMAN

LOVAST AT IN

Gerald S. Brenner Dean K. Ellison

Michael J. Kaufman

1. History and Therapeutic Properties

2. Description

2.1 Nomenclature 2.1 .l Chemical Name 2.1.2 Generic Name (USAN) 2.1.3 Laboratory Codes 2.1.4 Trade Names 2.1.5 Trivial Names 2.1.6 Chemical Abstract Services (CAS)

2.2 Structure, Formula and Molecular Weight 2.3 Appearance

3. Synthesis


4.1 Infrared Spectrum 4.2 Proton Nuclear Magnetic Resonance Spectrum 4.3 Carbon-13 Nuclear Magnetic Resonance Spectrum 4.4 Ultraviolet Spectrum 4.5 Mass Spectrum 4.6 Optical Rotation 4.7 Thermal Behavior 4.8 Solubility 4.9 Crystal Properties 4.1 0 Dissociation Constants 4.1 1 Partition Behavior

LOVASTATIN 219

5. Methods of Analysis

5.1 Elemental Analysis 5.2 C h ro matog raph y

5.2.1 Thin Layer Chromatography 5.2.2 High Performance Liquid Chromatography

5.3 Flow Injection Analysis 5.4 Identification Tests

6. Stability and Degradation

6.1 Solid State Stability 6.2 Solution Stability

7. Pharmacokinetics and Metabolism

7.1 Absorption and Distribution 7.2 Metabolism 7.3 Excretion

8. Determination in Biological Fluids

9. References

280 GERALD S . BRENNER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN

1. Historv and TheraDeutic ProDerties

It was discovered by the Merck Sharp & Dohme Research Laboratories that a strain of Aspergillus terreus obtained from a soil sample produced the cholesterol lowering fungal metabolite lovastatin (initially named mevinolin). Details of the isolation, structural characterization and biochemical properties of lovastatin have been summarized by Alberts et al. (1). Lovastatin is identical to monacolin K isolated independently from Monascus ruber by Endo (2).

Lovastatin is a prodrug. After oral administration, the inactive parent lactone is hydrolyzed to the corresponding hydroxyacid form. The hydroxyacid is the principle metabolite and a potent inhibitor of 3-

Lactone Hydroxyacid

hydroxy-3-methylglutaryl-coenzyme A (HMG CoA) reductase. This enzyme catalyzes the conversion of hydroxymethylglutarate to mevalonate, which is an early and rate limiting step in the biosynthesis of cholesterol. The effectiveness of lovastatin in lowering cholesterol has been confirmed clinically and it is approved for the treatment of primary hypercholesterolemia.

Several review articles give a detailed account of the discovery, preclinical evaluation, mechanism of action, biological profile, and clinical evaluation of the drug (3-7).

2. DescriDtion

2.1 Nomenclature

2.1.1 Chemical Name [l S-[ 1 a( R*),3a,7P,8P(2S*,4S*),8a~]]-2-Methylbutanoic acid 1,2,3,7,8,8a-hexahydro-3,7-dirnethyl-8-[2-

LOVASTATIN 28 I

(tetrahydro-4-hydroxy-6-oxo-2H-pyran-2-yl)ethyl]-1- naphthalenyl ester; (1 S,3R,7S,8SI8aR)-1 ,2,3,7,8,8a- hexahydro-3,7-dimethyl-8-[2-[(2R,4R)-tetrahydro-4- hydroxy-6-0~0-2H-pyran-2-ylJethyl]- 1 -naphthaten yl (S)- 2-methylbutyrate; 1,2,6,7,8,8a-hexahydro-P,6- dihydroxy-2,6-dimethyl-8-(2-methyl-l -oxobutoxy)-1 - naphthaleneheptanoic acid Glactone; PP,Ga-dimethyl- 8a-(2-methyl-l -oxobutoxy)-mevinic acid lactone.

2.1.2 Generic Name (USAN1

Lovastatin

2.1.3 Laboratow Codes

L-l54,803-000G MK-0803

2.1.4 Trade Names

Mevacor; Mevinacor; Mevlor

2.1.5 Trivial Names

Mevinolin Monacolin K 3-Methyl Compactin

2.1.6 Chemical Abstracts Services GAS1

Registry Number: 75330-75-5

2.2 Structure, Formula, and Molecular Weiaht

Structure:

282 GERALD S . BREWER, DEAN K. ELLISON, AND MICHAEL I. KAUFMAN

Molecular Formula: C H,,O, Molecular Weiaht: 40&5

Lovastatin is a white, crystalline powder.

3. Synthesis

There have been numerous approaches to the total synthesis of lovastatin (8-1 0); however, lovastatin is produced commercially via a multi-stage fermentation process which originates from cultures of a strain of Aspergilks ferreus. The complete details of the isolation and identification of lovastatin from the fermentation media have been described (1). Synthetic approaches have been reviewed (1 1).

4. Phvsical Properties

4.1 Infrared Spectrum

The infrared spectrum of lovastatin is shown in Figure 1 (12). The spectrum was obtained as a potassium bromide pellet using a Nicolet Model 71 99 FT-IR spectrophotometer. Assignments for the characteristic absorption bands are shown below.

Wavenumber (cm-’ 1 3542 301 6 296 7 2929 2866 1725 171 1 1700 1460 1384 1359 1260 1222 1072 1056

969 87 1

Assignment Alcohol 0-H stretch Olefinic C-H stretch Methyl C-H asymmetric stretch Methylene C-H asymmetric stretch Methyl and methylene C-H asymmetric stretch Lactone and ester carbonyl stretch

(hydrogen bonded for 171 1 and 1700 cm-’ )

Methyl asymmetric bend Methyl symmetric bend Methylene symmetric bend Lactone C-0-C asymmetric bend Ester C-0-C asymmetric bend Lactone C-0-C symmetric stretch Ester C-0-C symmetric stretch Alcohol C-OH stretch Trisubstituted olefinic C-H wag

LOVASTATIN 283

.49

a 37

'31

.2 5

. l a

.12

.O 6

0 1 I 1 I 1 1 I d 4000 3600 3200 2800 2400 2000 1600 1200 800

Wavenumbers

Figure 1. Infrared Absorption Spectrum of Lovastatin

2R4 GERALD S. BRENNER. DEAN K. ELLISON. AND MICHAEL J . KAUFMAN

4.2 Proton Nuclear Mametic Resonance Spectrum

The proton magnetic spectrum is shown in Figure 2 (13). This spectrum was obtained on a Bruker Instruments Model AM-300 NMR spectrometer using a 4% w h solution of lovastatin in deuterated chloroform. Chemical shifts (6) are expressed as ppm downfield from tetramethylsilane (internal standard). The tabulated signal assignments refer to the numbered structure of lovastatin shown below.

6 (mml

0.88

1.08 1.11

1.20-2.05

0.89

2.20-2.50

2.55-2.77 4.37 4.64

5.53 5.78

5.38

6.00 7.27

Multiplicit y ' /J

t/J = 7.6 HZ d/J = 7.3 HZ d/J = 7.4 HZ d/J = 7.0 HZ Overlapping Multiplets Overlapping Multiplets Overlapping Multiplets m m m Broad t

6.1, 9.6 Hz

S

d Of d/J =

dN = 9.6 HZ

Assianment

1 Multiplicity: s, singlet; d, doublet; t, triplet; m, multiplet

PPM

Figure 2. Proton Nuclear Magnetic Resonance Spectrum of Lovastatin


4.3 Carbon-1 3 Nuclear Maanetic Resonance Spectrum

The carbon-1 3 nuclear magnetic resonance spectrum of lovastatin shown in Figure 3 was obtained using a Bruker Instruments Model AM-300 NMR spectrometer and an approximately 4% w/v solution of the compound in deuterochloroform. Signal assignments are tabulated below and refer to the numbered structure shown in Section 4.2

Chemical Shift (61, Dpm Assianment

11.69 13.83 16.21 22.79 24.23 26.78 27.39 30.63 32.62 32.90 36.06 36.55 37.24 38.55 41.46 62.52 67.86 76.37 77.00

128.26 129.58 131 5 3 133.03 170.50 176.88

In recent publications, the 1 H and 13C NMR spectra of lovastatin were fully assigned by the use of selective homonuclear and heteronuclear decoupling and two dimensional techniques (1 4,15).

Figure 3. Carbon-13 Nuclear Magnetic Resonance Spectrum of Lovastatin

288 GERALD S. RRENNER. DEAN K. ELLISON. AND MICHAEL J . KAUFMAN


The ultraviolet (UV) absorption spectrum of lovastatin is characterized by absorption maxima at 231,238, and 247 nm with A l% values of 538, 629, and 424, respectively. The absorption maxima at 238 nm is typical for a trisubstituted heteroannular diene chromophore (1 6). A UV spectrum of lovastatin (c = 0.015 mg/mL in acetonitrile) is shown in Figure 4.

4.5 Mass Spectrum

The mass spectrum of lovastatin is shown in Figure 5. This spectrum was obtained by the direct probe electron impact (90 eV) method using a Finnigan MAT 212 mass spectrometer (17). The spectrum exhibits a weak molecular ion signal at m/z = 404 (C H 0 , exact mass calculated = 404.2563; observed = 461.2%6lf. Other pertinent fragment ions are at m/z = 302, 284, and 159; these ions can be rationalized by the fragmentation pattern shown in Figure 6.

4.6 ODtical Rotation

Lovastatin has eight chiral centers and is optically active. The specific rotation [a],,25 is +330" for a 5.0 mg/mL solution in acetonitrile.

4.7 Thermal Behavior

The differential scanning calorimetry (DSC) curve for lovastatin at a heating rate of 2"/min under a nitrogen atmosphere is shown in Figure 7. The thermogram is characterized by a single melting endotherm with an extrapolated onset temperature for melting of 175°C which is independent of heating rate from 2-2O0C/min. In contrast, the DSC thermogram for lovastatin obtained at a heating rate of 2"/min in air (Figure 8) exhibits an exotherm at 154°C which is attributed to oxidative reactions occurring in the non-inerted atmosphere.

The thermal properties of lovastatin, in particular those derived from DSC experiments, have been used to assess the oxidative stability of the compound (18,19).

Wavelength (nm)

Figure 4. Ultraviolet Absorption Spectrum of Lovastatin

198

172 I

159 loo]

15; I/. 60

143 ''{ 105 I 20

0

2 84

302

200 224

!85

404 hi 1

100 150 200 250 300 350 400

Figure 5. Direct Probe Electron Impact Mass Spectrum of Lovastatin

LOVASTATIN 29 I

m/z 302

mlz 159 m/z 284

Figure 6. Proposed Fragmentation Pattern to Explain the Mass Spectrum of tovastastin

292

Figure 7. DSC Therrnogram for Lovastatin under Nitrogen

L

a C

t

m

> 0

J

0

.- .- c

c

3 L

c

E 2

m

0

a r

l- 0

m

n

cd ?? 3 0)

LL .-

293

294 GERALD S . BRENNER. DEAN K. ELLISON, AND MICHAEL J . KAUFMAN

4.8 Solubility

Lovastatin is insoluble in water, and is sparingly soluble in the lower alcohols (methanol, ethanol, and i-propanol). Solubility data obtained at room temperature are tabulated below (20).

Solvent Solubility ImalmL)

Acetone Acetonitrile n-Butanol i-Bu tanol Chloroform N,N-dimethylformamide Ethanol Methanol n-Octanol n-Propanol i-Propanol Water

47 28 7

14 350 90 16 28 2

11 20 0.4

4.9 Crvstal Properties

Lovastatin is a white, crystalline, non-hygroscopic solid. Single crystal X-ray diffraction experiments on a sample crystallized from ethanol indicate that the space group is P2,2 2, with a = 5.974A, b = 17.337A, and c = 22.148A. The calculated density is 1.17 g/cm3. (1)

The X-ray powder diffraction pattern for lovastatin is shown in Figure 9. This spectrum was obtained on a Phillips APD 3720 X-ray diff ractometer using CuKa irradiation. No crystal forms (polymorphs) other than that represented by the X-ray pattern in Figure 9 have been observed.

4.1 0 Dissociation Constants

Consistent with the structure, lovastatin exhibits no acidhase dissociation constants. Potentiometric titration of a sample in 50% aqueous methanol revealed no observable buffering action in the pH range of 2-1 1.

5

I I

I I

I I

I I

I I

00

00

00

00

00

o

mc

o~

ro

-

do

o~

~o

oo

*

r?

cr

?z

0

d

* 4

rc) M

c! 0

M

9

m

N

0

d

N

9

In c

8 c

9

In

0

d


4.1 1 Partition Behavior

In the n-octanol/water test system, lovastatin partitions quantitatively into the organic phase. At room temperature, the partition coefficient is approximately K = 1.2 x 1 04. The partition coefficient for the hydroxyaci82erivative (opened lactone form of lovastatin) between n-octanol and a pH 7.4 phosphate buffer is KO,,,, = 14.1 (21).



Analysis of Merck Sharp & Dohme reference lot L-154,803- 000G102 for carbon and hydrogen gives values compared to calculated values as given below:

Calculated Found

Carbon 71.25 71.26 Hydrogen 8.97 9.18

5.2 Chromatoqraphy

5.2.1 Thin-Layer Chromatoaraphv

Table I lists the thin layer chromatographic systems which have been used for the analysis of lovastatin.

Table 1

Thin-Layer Chromatographic Systems for Lovastatin (221

Solvent System Plate Type - Rf System

Toluenehnethanol 70/30

Toluenelacetone 70BO

Analtech@ Silica 0.77 1 Gel GF

Analtech@ Silica 0.48 2 Gel GF

Cyclohexaneh-butanoI/ethyl acetate Analtech@ Silica 0.43 3 4:l:l Gel GF

Cyclohexane/chloroformlisopropanol E. Merck Silica 0.60 4 5:2:1 Gel 60

F254 High Performance

LOVASTATIN 291

Visualization is either by viewing the developed plate under ultraviolet light or by spraying the developed plate with a dilute methanolic sulfuric acid solution and application of heat. System 4 with sulfuric acid spray detection is the most useful system because non-UV absorbing impurities are detectable.

5.3.2 Hiqh Performance Liquid Chromatoaraphv (HPLC)

A variety of gradient and isocratic reverse phase HPLC systems have been used to chromatograph lovastatin (see Table 2).

Table 2

Hiqh Performance Liquid Chromatoqraphic Systems

Application System No. Column Mobile Phase nm Detection Ref

Drug substance 1 Whatrnan A = Acetonitrile 238 (23) purity Partisil C-8 B = 0.1% (v/v%)

H3P04 aqueous A:B 70:30

Measurement of 2 Whatman Gradient 238 and (24)

in drug substances low level impurities Partisil C-8 A = Acetonitrile 200 nrn

B = 0.1% (vW/O) H3P04 aqueous

Measurement in 3 Sepralyte C-18 lsocratic and 238 nm (25) plasma and bile Gradient

A = 0.05 M ( NH4)3P04 and 0.01 An H3P04 Buffer B = acetonltrile A:B 5050 (isocratic)

Measurement of 4 DuPont A = acetonitrile 260 nrn (26) low levels in Zorbax C-8 B = methanol fermentation broth' C = water

A:B:C 62229

Measurement in 5 Waters A = acetonitrile 238 nm (27) tablets B = water (0.04M

KH2P04 pH s 4) 60:40 A:B

298 GERALD S. BRENNER. DEAN K. ELLISON, AND MICHAEL J . KAUFMAN

Table 2 (Cont'd)

High Performance Liquid Chromatographic Systems

Application System No. Column Mobile Phase nm Detection Ref

Measurement 6 Hypersil 5 230 nm (28) in tablets micron ODS A = 0.025M NaH2P04

pH = 4 B = CH CN C = MebH 33:55:12 A:B:C

Derivatization of lovastatin described.

5.3 Flow lniection Analysis

A flow injection analysis system has been described by Mazzo -- et al. to simultaneously monitor lovastatin and antioxidants in tablets (29).

5.4 Identification Tests

Three methods are routinely used to identify lovastatin: 1. the infrared spectrum; 2. the ultraviolet spectrum; and 3. the chromatographic retention time.

6. Stability and Degradation

6.1 Solid State Stability

Crystalline lovastatin stored at room temperature yields with time trace amounts of oxidation products. The oxidative pathway for degradation has been supported with data generated by chromatography, degradate isolation, and identification, differential scanning calorimetry and heat conduction calorimetry. No products of nonoxidative degradation have been detected. HPLC and TLC studies have demonstrated that samples stored in air generate a complex mixture of largely unidentified trace polar products (30). These products are essentially absent and drug loss prevented in samples stored under nitrogen. For samples stored in air, all isolated and identified degradates result from oxidation and include the 4'-oxolactone which is the major

LOVASTATIN 299

degradate retaining the diene of the parent. The ultraviolet absorption spectra of air degradates indicate

Oxolactone

that more than half of the mass has lost diene, suggesting that oxidation takes place primarily at this site (e.g., epoxidation and subsequent reactions of the resultant epoxides). Heat conduction calorimetry (1 9) and differential scanning calorimetry (1 8) also demonstrate the enhanced reactivity of the compound in an air vs. a nitrogen atmosphere.

6.2 Solution Stability

The hydrolysis of the lactone ring of lovastatin occurs readily in aqueous solution especially under acidic or alkaline conditions (31). The acid catalyzed hydrolysis is reversible leading to a mixture of lactone and hydroxyacid, the equilibrium ratio of the two species being pH dependent. The rate to equilibrium is also pH dependent, being more rapid at acidic pH than near neutrality. In alkaline solution, the lactone ring is irreversibly converted to the hydroxyacid. Solutions of the hydroxyacid demonstrate good stability.

Kaufman (32) has studied and determined the rate and equilibrium constants for the acid catalyzed hydrolysis of mevalonolactone, lovastatin and other structurally related HMG CoA reductase inhibitors in pH 2.0 buffer at 37°C. Under these conditions lactone concentrations decrease with time but do not approach zero indicating that the hydrolysis is reversible. The equilibrium nature of the reaction was further confirmed by repeating the experiment with hydroxyacid as starting material in the same system and demonstrating that an equilibrium composition is achieved that is identical to that achieved starting with lactone. Kinetic points in all studies were carried out to 15 hours and data obtained indicate that

300 GERALD S. BRENNER, DEAN K . ELLISON. AND MICHAEL J. KAUFMAN

7.

there are no side reactions (e.g., oxidation) competing with hydrolysis/lactonization during this time frame.

The solution phase oxidation of a number of HMG CoA reductase inhibitors, including lovastatin, was studied in aqueous surfactant solutions at 40°C (33). Reaction rate constants were determined by monitoring oxygen consumption using an oxygen electrode. In the absence of a free radical initiator, there was no oxygen uptake indicating that the spontaneous rate of oxidation at 40°C was too slow to be detected. With an initiator present, all analogs consumed oxygen with the exception of the one in which the diene is saturated, demonstrating the diene functionality to be most labile to oxidation.

Oxidation of lovastatin in aerated ethylene dichloride solution at 35", containing a free radical initiator, has been monitored kinetically using HPLC (34). The degradates formed in this complex solution system, different from those in the solid state, are primarily oligomers, with peroxide groups within the backbone chain and hydroperoxide end groups. Also, some monomeric epoxides are formed.

Pharmacokinetics and Metabolism

Lovastatin is an inactive prodrug which undergoes in vivo lactone hydrolysis to give the hydroxyacid derivative which is an inhibitor of HMG-CoA reductase. The pharmacokinetic and metabolic profile of lovastatin has been described in detail (33,3537). In the sections below, the absorption, distribution, metabolism, and excretion of lovastatin are briefly reviewed. For this discussion it is helpful to distinguish between active inhibitors (defined as the sum concentration of the hydroxyacid derivative of lovastatin plus other active hydroxyacid metabolites) and total inhibitors (the total concentration of active inhibitors plus lactones and conjugates). Active and total inhibitors can be separately quantitated by assaying samples before and after ex vivo hydrolysis of plasma samples.

7.1 Absorption and Distribution

In studies in laboratory animals, the absorption of lovastatin following oral administration is approximately 30% complete as estimated relative to an intravenous dose of the hydroxyacid. An intravenous formulation of lovastatin for human studies is

LOVASTATIN 30 I

not feasible due to its low aqueous solubility. In all species studied, lovastatin is converted to the hydroxyacid form in viva This conversion is apparently reversible since lovastatin is found in the biological fluids of rats and dogs following administration of the hydroxyacid.

In animals, lovastatin is more efficiently extracted by the liver where it is converted to the active enzyme inhibitor. Accordingly, the systemic bioavailability of active inhibitors is less than 5% of an oral dose of lovastatin. The high hepatic extraction and low systemic availability are desirable features since the liver is the primary site of cholesterol biosynthesis.

Peak plasma concentrations of both active and total inhibitors occur between 2-4 hours post dose, and the area under the curve (AUC) increases proportionally with dose. The hydroxyacid is rapidly cleared; plasma clearance and half-life range from 300-1 248 mUmin and 1.1 -1.7 hrs, respectively. When lovastatin is administered with food, a 50% increase in AUC for inhibitory activity is attained relative to administration in the fasted state.

The plasma protein binding of lovastatin and the hydroxyacid form has been determined by equilibrium dialysis. Both forms are greater than 95% protein bound.

7.2 Metabolism

Lovastatin is extensively metabolized to give both active and inactive compounds. The major active metabolites present in human plasma are the hydroxyacid of lovastatin and its 3- hydroxy-, 3-hydroxymethyl, and 3-exomethylene derivatives. The 3-hydroxylated metabolite is approximately 70% as active as the non-hydroxylated metabolite. In human bile, the 3- hydroxylated metabolite undergoes an allylic rearrangement to give the 6-hydroxy isomer which is inactive (38):

"3C

GERALD S. BRENNER. DEAN K . ELLISON. AND MICHAEL J . KAUFMAN 302

7.3

All of the hydroxyacid metabolites also exist in their corresponding inactive lactone forms. After base hydrolysis to convert lactones to active inhibitors, about 80% of the total enzyme inhibitory activity in human plasma is accounted for by these four lactonelhydroxyacid pairs.

Excretion

The excretion of lovastatin has been assessed following an oral dose of 14C-labeled compound in man. Total recovery of drug equivalents in urine and feces averaged 10% and 83%, respectively. A substantial amount of radioactivity is also recovered in the feces following intravenous dosing of 14C- labeled hydroxyacid, indicating that biliary excretion is an important elimination for orally administered lovastatin.

8. Determination in Bioloaical Fluids

An enzyme inhibition assay capable of measuring total HMG-CoA reductase inhibitors in biological fluids has been described in the literature (1). The basis of this assay is the in vitro inhibition of the HMG-CoA reductase catalyzed conversion of 14C-HMG-CoA to 14C- mevalonic acid. The concentration of inhibitors can be measured before and after base hydrolysis of plasma samples. The measurement before hydrolysis gives the concentration of inherently active species (active inhibitors). Base hydrolysis irreversibly converts inactive but potentially active species (lactones and conjugates) to their corresponding active forms; the inhibition assay of hydrolyzed samples thus provides the concentration of total inhibitors. The enzyme inhibition assay is sensitive (detection limit of ca. 5 ng/mL), but is not specific for lovastatin.

The determination of lovastatin and its hydroxyacid metabolite in plasma and bile can be accomplished by high performance liquid chromatography (25). Plasma samples are prepared for analysis by solid phase extraction and are analyzed using isocratic elution on a C18 column. Bile samples do not require any sample clean-up prior to HPLC analysis, but do require the use of a gradient elution method to separate the compounds of interest. The HPLC assay has a limit of detection of 25 ng/mL.

An analytical method for the determination of lovastatin in serum based on gas chromatography/mass spectrometry has recently been reported (39).

LOVASTATIN

Acknowledclements

303

The authors wish to thank Mrs. Laurie Rittle for typing the manuscript and Ms. Agnes Hendrick for performing the literature search.

9. References

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

A.W. Alberts, J. Chen, G. Kuron, V. Hunt, J. Huff, C. Hoffman, J. Rothrock, M. Lopez, H. Joshua, E. Harris, A. Patchett, R. Monaghan, S. Currie, E. Stapley, G. Albers-Schonberg, 0. Hensens, J. Hirshfield, K. Hoogsteen, J. Liesch and J. Springer, Roc. Natl. Acad. Sci. USA 77, 3957 (1980).

A. Endo, J. Antibiot. 32, 852 (1979).

J.S. MacDonald, R.J. Gerson, D.J. Kornburst, M.W. Kloss, S. Prahalada, P.H. Berry, A.W. Alberts and D.L. Bokelman, Am. J. Cardiol. 62, 16J (1988).

E.E. Stater and J.S. MacDonald, Drugs 36 (Suppl. 3), 72 (1988).

J.M. McKenney, Clin. Pbarm. 7, 21 (1988).

A.W. Alberts, Am. J. Cardiol. 62, 1OJ (1988).

J.A. Tobert, Circulation 76, 534 (1987).

S.J. Hecker and C.H. Heathcock, J. Org. Chem. 50,5159 (1985).

M. Hirama and M. Iwashita, Tetrahedron Letters 24, 181 1 (1983).

D.L.J. Clive, K.S.K. Murthy, A.G. Wee, J.S. Prasad, M. Majewski, P.C. Anderson, C.F. Evans, R.D. Hauger, L.D. Heerz and J.R. Barrie, J. Am. Cbern. SOC. 112, 3018 (1990).

T. Rosen and C.H. Heathcock, Tetrahedron 42, 4909 (1986).

R. Cervino, Merck Sharp & Dohme Research Laboratories, personal communication.

304 GERALD S. BRENNER, DEAN K. ELLISON, AND MICHAEL J. KAUFMAK

13.

14.

15.

16.

17.

18.

19.

20.

21.

22.

23.

24.

25.

26.

27.

R. Reamer, Merck Sharp & Dohme Research Laboratories, personal communication.

J.K. Chan, R.N. Moore, T.T. Nakashima, J.C. Vederas, J. Am. Chem. SOC. 105, 3334 (1 983).

R.N. Moore, G. Bigam, J.K. Chan, A.M.Hogg, T.T. Nakashima, J.C. Vederas, J. Am. Chem. SOC. 107,3694 (1985).

A.I. Scott, Interpretation of the Ultraviolet Spectra of Natural Products, Pergamon Press, Oxford, 1964.

D. Zink, Merck Sharp & Dohme Research Laboratories, personal communication.

J.P. Elder, Thermochim. Acta 134, 41 (1988).

L.D. Hansen, E.A. Lewis, D.J. Eatough, R.G. Bergstrom, D. Degraft-Johnson, Pharm. Res. 6, 20 (1989).

A.Y.S. Yang, Merck Sharp & Dohme Research Laboratories, personal communication.

M.J. Kaufman, Merck Sharp & Dohme Research Laboratories, personal communication.

A.Y.S. Yang, L. Pierson and J. Baiano, Merck Sharp & Dohme Research Laboratories, personal communication.

A.H. Houck, Merck Sharp & Dohme Research Laboratories, personal communication.

A.H. Houck, S. Thomas and D.K. Ellison, Pittsburgh Conference (1 990), manuscript in preparation.

R.J. Stubbs, M. Schwartz and W.F. Bayne, J. Chromatog. 383, 438 (1 986).

V.P. Gullo, R.T. Goegelrnan, I. Putter and Y. Lam, J. Chromatog. 21 2, 234 (1 98 1 ).

L.L. Ng, Anal. Chem. 53, 1142 (1981).

LOVASTATIN 305

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

39.

C.V. Bell and J.C. Wahlich, Merck Sharp & Dohme Research Laboratories, personal communication.

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M. Baum, G. Dezeny, L. DiMichele, R. Reamer and G.B. Smith, Merck Sharp & Dohme Research Laboratories, personal communication.

A.Y.S. Yang, Merck Sharp & Dohme Research Laboratories, personal communication.

M.J. Kaufman, Int. J. Pharm. 66, 97 (1990).

M.J. Kaufman, Pharm. Res. 7, 289 (1990).

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D.E. Duggan and S. Vickers, Drug Metab. Rev. 22, 333 (1990).

R.A. Halpin, K.P. Vyas, P. Kari, B.H. Arison, E.H. Ulrn and D.E. Duggan, Pharmacologist 29, 238 (1987).

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NAPHAZOLINE HYDROCHLORIDE

G . Michael Wall

Alcon Laboratories, Inc.

6201 South Freeway

Fort Worth, Texas 76134


Copyright Q 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

G. MICHAEL WALL 308

NAPHAZOLINE HYDROCHLORIDE

1. DESCRIPTION 1.1 1.2 Appearance, Color, Odor 1.3 History 1.4 Pharmacology

2. SYNTHESIS


Name, Formula and Molecular Weight

3.1 Spectroscopy 3.1.1 Infrared Spectrum 3.1.2 Ultraviolet Spectra 3.1.3 Nuclear Magnetic Resonance Spectra 3.1.4 Mass Spectra

3.2.1 Melting Range 3.2.2 Differential Thermal Analysis 3.2.3 Thermogravimetric Analysis X-Ray Crystallography and Powder Diffractometry

3.2 Thermal Properties

3.3 3.4 Partition Coefficients 3.5 Ionization Constant, pKa 3.6 Solubility 3.7

4.1 Identity

Solution Color, Clarity and pH

4. TYPICAL METHODS OF ANALYSIS

4.1.1 Infrared Spectrophotometry 4.1.2 Ultraviolet Spectrophotometry 4.1.3 Chloride Identity Test 4.1.4 Reaction with Bromine

4.2 Colorimetry 4.3 Elemental Analysis 4.4 Titrimetry 4.5 Chromatography

4.5.1 Thin-Layer Chromatography 4.5.2 High-pressure Liquid Chromatography 4.5.3 Gas Chromatography

5 . STABILITY-DEGRADATION 5.1 Potential Routes of Degradation

5.1.1 Characterization of 1-Naphthylacetylethylenediamine 5.1.1.1 Thin-Layer Chromatography 5.1.1.2 Liquid Chromatography 5.1.1.3 Synthesis of 1-Naphthylacetylethylene-

diamine

NAPHAZOLINE HYDROCHLORIDE 309

5.1.1.4 Physical/Chemical Properties of 1-

5.1.2 Synthesis and Analysis of l-Naphthylacetic Acid Naphth ylacetyleth ylenediamine

5.2 Solid-state Stability 5.3 Solution Stability

6 . DISPOSITION AND TOXICITY

7. ACKNOWLEDGEMENTS

8 . REFERENCES

1. DESCRIPTION

1.1 Name, Formula and Molecular Weight

Naphazoline hydrochloride is an u-adrenergic sympathomimetic agent used in topical nasal or ophthalmic pharmaceutical formulations. Naphazoline has been established as the International Nonproprietary Name (INN) by the World Health Organization for the chemical compound, (2-(l-naphthyl- methyl)-2-imidazoline1$2, which is typically used as either the hydrochloride or nitrate salt. The hydrochloride salt has been given the USAN, naphazoline hydrochloride1. Other chemical names include: (a) 1H-imidazole, 4,s- dihydro-2-(l-naphthalenylmethyl)-, monohydrochloridel, (b) 2-( l-naph- thylmethyl)-2-imidazoline monohydrochloridel, and (c) 4,5-dihydro-2-(1- naphthalenylmethy1)-1H-imidazole, monohydrochloride3. The CAS registry number for naphazoline hydrochloride is 550-99-21; the CAS number for the free base is 835-31-4l.

Empirical Formulal: C14H14N2 * HC1

Molecular Weightl: 246.74

Structure:

310 G. MICHAEL WALL

1.2 Appearance, Color and Odor

Naphazoline hydrochloride is a white to almost white, odorless, crystalline powdefl with a bitter taste5y6.

1.3 History

An investigation of the vasoconstrictor activity of substituted imidazolines by Fritz Uhlmann at Ciba in Basle, Switzerland during the early 1940’s resulted in the introduction of the sympathomimetic drug, naphazoline; its analogs, xylometazoline and oxymetazoline, used as decongestants; and also the a-adrenoceptor antagonist, tolazoline798. Patents include: U.S Patent 2,161,938 (1939) and Danish Patent 62,889 (1944)6. Naphazoline has been marketed under a variety of trade names around the world29399,

1.4 Pharmacology

Naphazoline is a potent a-adrenergic sympathomimetic agent. It is a vasoconstrictor with a rapid and prolonged action in reducing swelling and congestion when applied to mucous membranes, hence, its use for the symptomatic relief of rhinitis and sinusitis. Rebound congestion and rhinorrhea are common after prolonged use. Nasal drops or spray are used as a 0.05% aqueous solution of the hydrochloride or nitrate, with a usual recommended dosage of 2 drops in each nostril every 3 hours. Aqueous solutions have also been used as ophthalmic conjunctival decongestants4.

2. SYNTHESIS

Naphazoline hydrochloride has been prepared through a series of synthetic chemical steps beginning with (1-naphthy1)-acetonitrile, I (Figure l)3910. The starting material, I, is treated with ethanol and hydrochloric acid to obtain the naphthyl-(1)-acetiminoethylether hydrochloride, II1o. A solution is made of 2.7 parts II and 12 parts absolute alcohol3. One part of ethylenediamine is then added and the mixture is heated to gentle boiling with stirring under nitrogen until the evolution of ammonia ceases. The alcohol is then distilled and the residue is dissolved in 40 parts of benzene and 1.8 parts of caustic potash. The benzene is removed and the residue is recrystallized several times from toluene. Reaction with hydrochloric acid gives naphazoline hydrochloride, III3. The preparation of radiolabelled naphazoline with 14C in the 2-position of the imidazoline ring has also been reported11 using a-chloromethylnaphazoline, potassium cyanide-14C, and ethylenediamine.

I I11

Figure 1. Synthesis of naphzolinc hpirochbr&ie.

G. MICHAEL WALL 312


3.1 Spectroscopy

3.1.1 Infrared Spectrum

The infrared spectrum of naphazoline hydrochloride was obtained. A mixture of the drug substance and potassium bromide was pressed into a pellet and analyzed using a Perkin-Elmer Model 1750 FTIR. The spectrum is shown in Figure 2. The major absorption bands for the infrared frequencies and the corresponding assignments are listed in Table I.

3.1.2 Ultraviolet Spectra

The ultraviolet absorption spectra of naphazoline hydrochloride in absolute ethanol, pH 3 buffer (0.05M phosphate), pH 7 buffer (0.05M phosphate) and pH buffer (0.05M borate) were obtained using a Perkin-Elmer 559A W/VIS spectrophotometer and 1 cm cells. A representative W spectrum in ethanol is shown in Figure 3. Samples of naphazoline hydrochloride in these solvents were scanned from 200 to 400 nm and the absorption coefficients at wavelengths of maximum absorption were calculated (Table II).

3.1.3 Nuclear Magnetic Resonance Spectra

The lH-NMR spectrum (100 MHz) of naphazoline hydrochloride has been reported and the chemical shifts have been assigned for the methylene groups12. The IH-NMR spectrum (300 MHz) of naphazoline hydrochloride (143 mgmL DMSO-d6 at 1OOOC) was obained using a Vanan VXR 300 spectrometer (Figure 4). In order to assign all of the aromatic proton signals, a series of 2-D experiments were carried out: these spectra were not shown but the assignments are listed in Table III.

The l3C-NMR spectrum (22.5 MHz) of naphazoline hydrochloride has been reported and the chemical shifts have been assigned for the methylene and imidazoline carbons12. The %NMR spectrum (75 MHz) of naphazoline hydrochloride (143 mg/mL DMSO-& at 1OOOC) was obtained using a Varian VXR 300 spectrometer (Figure 5 ) and the assignments are listed in Table III. The Attached Proton Test (APT) (Figure 5 ) and extensive 2-D studies were performed in order to assign aromatic carbons.

The resonances for the methylene protons were shifted downfield for the HC1 salt compared to the base: h 6 (ppm) (+ indicates downfield shift compared to base); C&C&, +0.44 and aryl-C& +0.53)12. The reso-

Figure 2. In@zrd spctnm (KBr) Of Mphotolinc hyhxhbnkk.

3 14 G. MICHAEL WALL

I .E

0.c I I I I 1

200 250 300 350 6 0 0

Wavelength (nm)

Figure 3. UVspctrum of naphamline hydnxhloride (0.019mghL in ethanol).


Table I. Infrared spectral assignments for naphazoline hydrochloride.

Wavelength (cm-I) Assignment

3150-2500 C-H and N-H stretch 1618 Amine salt N-H

801, 765 Imidazoline C-H 602, 561, 525, 481

1302, 1198 20 N-H

Out of plane ring bend

Table II. Ultraviolet absorption of naphazoline hydrochloride.

E (l%, 1 cm) Solvent 223nm 270nm 280nm 287nm 291nm

Ethanol 3622 239 286 196 198 pH 3 Buffer 3214 246 287 198 193

pH 9 Buffer 3294 236 274 191 187 pH 7 Buffer 3246 238 279 193 188

I - - - . . - - - - - - - 1 - - - . . - - - I - . - . ..-.,.... ....,.... . . . . I . . . , ...,,..., ....,.,.. 1)

rrr 0

F i R m 4. H-NMR Siuctn#n (300 MHz) of Mphatolne hyhxhlofi (143 m g h L in DMso-d6 at I W C ) .

I

Figun 5. JJC-NMR Spanun (75 MHz) of napAazoline hydrrnrhloride (143 m g h d inDMSOd6 a! IcK)DC).

318 G. MICHAEL WALL

Table III. lH- (300 M H Z ) and 1% (75 MHz) NMR Data for Naphazoline HCl(l43 mg/mL in DMSO-& at 1OOOC).

1 2 3 4 5 6 7 8 9

10 11 12

14 & 15

7.64 (lH,d) 7.49 (lH,m) 7.91 (lH,d) 7.96 (lH,d) 7.54 (lH,m) 7.58 (lH,m) 8.15 (1H,d) -

4.45 (2H,s)

3.82 (4H,s)

128.71 128.09 125.61 128.47' 128.66* 126.06 126.83 123.34 131.37 133.57 29.41

169.78 44.40

* Interchangeable assignments


nances for carbons attached to the imidazoline ring were shifted for the HC1 salt compared to the base: A 6 (ppm) (- and + indicate upfield and downfield shifts respectively compared to base); zIHgH2, -5.09; aryl- CH2, -3.63; and N-IZ-N, +3.9611.

3.1.4 Mass Spectra

Mass spectra were obtained for naphazoline hydrochloride using a Finnegan MAT TSQ46 GC/MS/MS unit. A small amount of naphazoline hydrochloride was volatilized by heating at a linear rate of 5 mA/sec from 0 mA to about 500 mA and ionized by either chemical ionization (CI, 0.3 Torr pressure of isobutane) or by electron impact (EI) at 70 eV. The CI and EI mass spectra were presented in Figures 6 and 7 and the interpretation presented in Table IV. The fragmentation pattern was consistent with the chemical structure of naphazoline hydrochloride (Figure 8).

3.2 Thermal Properties

3.2.1 Melting Range

The melting point of naphazoline hydrochloride has been reported as 257OC13 with a range of 255-60 (decomposition)5*6; the melting point for the base has been reported as 115-120OC13.

3.2.2 Differential Thermal Analysis

A 2-mg sample of naphazoline hydrochloride drug substance was heated from 40OC to 3000C at a linear rate of 200C/min using a Perkin-Elmer DSC- 4. One single, sharp endotherm was observed with an onset of 259OC and a maximum of 261OC, corresponding to the melting range, after which decomposition occurred (Figure 9).

3.2.3 Thermogravimetric Analysis

A 7-mg sample of naphazoline hydrochloride was heated using a Perkin- Elmer System 4 Thermogravimetric Analyzer from 4OOC to 298OC at a linear rate of 200C/min. The drug substance exhibited a gradual weight loss near the melting range (Figure 10).

3.3

Naphazoline hydrochloride exists as a crystalline powde8. Podder et a1.15 described the crystal structure: Mr = 246.73, monoclinic, P21/c, a = 11.895 (3), b = 9.228 (2), c = 12.820 (3) A, J3 = 117.18 (2)0, V = 1252 813, 2 = 4, Dm - 1.30, Dn = 1.29 Mg m-3, h (Cuka) = 1.5418 A, p = 2.48 mm-1, F(OO0) = 524, T = 277 (1) K. Final R = 0.040 for 1291 observed reflec-

X-Ray Crystallography and Powder Diffractometry

I P x ii d

E p

320

Figure Z EJ Mass spectrum of napfuwlitu h@mhlon&.

322 C. MICHAEL WALL

Tablc IV . t I and CI Mass splrital Assignnrcnls for naphamline hydrochloridc

~~~

El Rel. Assignment Rel. CI

21 I 4 209 100 I95 7 181 6 I53 9 141 9 1 I5 12

11s I --,-I

153 I ----

NAPHAZOLINE HCL VT. 228 g

SCAN RATE. 20.00 w a i n

Figurc 9. DSC of naphazolinc hyhchloride.

W N P

I T , 7.290. ag RATE. 20.00 d.g/rln

m a

I#c .Q¶J T Q -84

TEMPERATURE (C) TC

Figwv 10. Z A of naphawline hydrochlode.

W N Ln

x U .r(

u)

c Y

C U

Y

5 10 15 28 2s 30 35 45

20

Figun 11. X-Ray pmvdPr difiaction pancrn of naphamline hyirochloride.

326 G. MICHAEL WALL

Table V. X-Ray powder diffraction data for naphazoline hydrochloride obtained using CuKa radiation and indexed on the basis of a monoclinic cell: P2ljc, a = 11.895 (3), b - 9.228 (2), c - 12.820 (3) A, fl- 117.18 (2)O.

V h k 1 d Intensity

1 2 1 3.760 18

1 0 0 10.7 47

0 1 1 7.21 29

1 1 0 1 - 2 1 3 1 6.97 13

-1 1 1 ) 1 2 2 J -3 1 2 3.598 100 -3 'I -1 0 2 6.38 41

0 0 2 5.71 3

2 0 0 ) 0 2 2 J

} 3.529 23 0 1 3

3 1 0 3.300 4

3.135 18

1 1 1 5.28 8

-2 0 -2 I 0 1 2 4.87 29 -1 0 4 j

-2 1 1 5.01 25

0 2 0 1 4.60 27

1 2 2 1

2 1 0 ) 2 2 1 3.091 6

0 2 1 2 1 2 3.042 8 I 4.29 30 1 0 2 1 -2 1 4 3.016 14

- 3 0 2 i . 3.881 6

1 1 2 1


tions. The bond lengths of the N-C-N group of the imidazoline ring were short and indicative of double bond character. One nitrogen atom was protonated and both nitrogen atoms participated in hydrogen bonding. Each chlorine atom was involved in two intermolecular hydrogen bonds of the form Nl-H-Cl-H-N2, that linked the molecules into continuous parallel chainsls.

To obtain an x-ray powder diffraction pattern, a sample of the drug substance was irradiated using a Philips powder diffractometer equipped with a diffracted beam graphite monochronometer. CuKa (1 - 1.5405 A) radiation was used for obtaining the powder pattern (Figure 11). All of the diffraction lines could be assigned hkl indicies on the basis of the unit cell parameters proving that the material was single-phase (Table V).

3.4 Partition Coefficients

Partition coefficients were determined for naphazoline hydrochloride between pH 3 buffer (0.05 M phosphate), pH 7.0 buffer (0.05 M phosphate) and pH 9 buffer (0.05 M borate) versus l-octanol. All solutions were prepared using octanol-saturated buffers and buffer-saturated octanol. Tubes containing 100 mg of naphazoline hydrochloride, 10 ml of buffer and 10 ml of octanol were agitated for 2 hours at 23OC and allowed to partition overnight. Analysis (HPLC) of the aqueous phases of each mixture revealed the following partition coefficients: pH 3.0 = 0; pH 7.0 = 0; pH 9.0 = 7.4.

3.5 Ionization Constant, pKa

The pKa of naphazoline HC1 has been reported as 10.9 at 200C4, 10.35 k 0.02 at 25OC16, 10.13 ? 0.02 at 35*C16, and 9.92 f 0.03 at 450C16.

3.6 Solubility

The solubility of naphazoline hydrochloride in various solvents at room temperature is presented in Table VI.

Table VI. Solubility of Naphazoline Hydrochloride

Solvent Solubility Reference

Water 1 in6 13 Ethanol 1 in 15 13 Chloroform Very slightly soluble 4 Diethyl Ether Practically insoluble 4

328 G . MICHAEL WALL

3.7

An aqueous solution (1 in 100) of naphazoline hydrochloride in carbon dioxide-free water is clear, colorless and exhibits a pH value between 5.0 and 6.617.

Solution Color, Clarity and pH

4. TYPICAL METHODS OF ANALYSIS

4.1 Identity

4.1.1 Infrared Spectrophotometry

The identity of naphazoline hydrochloride may be determined by comparison of its infrared spectrum (KBr) (see Figure 2) to an authentic reference standardl7.

4.1.2 Ultraviolet Spectrophotometry

The identity of naphazoline hydrochloride may be confirmed by comparison of its ultraviolet spectrum (1 in 50,000) to that of an authentic standard and the observation of a maximum at 280 nm17.

4.1.3 Chloride Identity Test

An aqueous solution of naphazoline hydrochloride (1 in 100) is treated with 6N ammonium hydroxide to precipitate naphazoline base. The filtrate then yields a white, curdy precipitate upon the addition of 0.1N silver nitrate. The precipitate is insoluble in nitric acid but is soluble in a slight excess of 6N ammonium hydroxide5~~~.

4.1.4 Reaction with Bromine

A 10-mL aliquot of an aqueous solution of naphazoline hydrochloride (1 in 100) when mixed with 5 mL of bromine-saturated water yields a yellow precipitate. Upon boiling, a deep purple color is produced5.

4.2 Colorimetry

Naphazoline has been analyzed by colorimetry using reagents such as sodium nitroprussidel8, ceric sulfatelg, chloranillg, bromocresol green20, bromophenol blue20, bromothymol blue20, methyl orange20, cobaltous acetate in chloroform-methanol21, iodine in chloroform22-25, and 2,6- dichlorophenol-indophenol in CHC1326.



Elemental analysis of a sample of naphazoline hydrochloride was performed: Anal. (C14H1&HCl) C (calcd 68.14; found, 68.52), H (calcd, 6.14; found, 6.28), N (calcd, 11.36; found, 11.48), C1 (calcd, 14.37; found, 14.54).

4.4 Titrimetry

Naphazoline hydrochloride drug substance may be titrimetrically assayed as described in the USP monographl7. The hydrochloride salt is dissolved in glacial acetic acid with mercuric acetate and titrated with dilute perchloric acid using crystal violet as the indicator. Also, nonaqueous titration of n a p hazoline hydrochloride in acetic anhydride/glacial acetic acid with dilute perchloric acid and potentiometric detection has been described as an official method in the Japanese Pharmacopoeias. In addition, nonaqueous titration with dioctylsulfosuccinate sodium salt using 3'3"5'5"-tetrabromophenolph- thalein as the indicator has also been reported25.

4.5 Chromatography

4.5.1 Thin-Layer Chromatography

The USP describes a TLC method for ordinary impurities in naphazoline hydrochloride drug substancel7. A sample of the drug substance dissolved in methanol (10 mg/mL) is spotted on a silica gel TLC plate, eluted with a mobile phase of methanol-glacial acetic acid-water (8:1:1, v/v/v), and visualized with iodoplatinate spray.

4.5.2 High-pressure Liquid Chromatography

Naphazoline has been analyzed in ophthalmic preparations by HPLC using a 10 pm octadecylsilane column (3.9 X 300 mm), a mobile phase of 0.08 M HClO4 (PH 2.2)-methanol(7/3, v/v), a flow rate of 2 mL/min and UV detection at 265 nm28; in ear and eye drops using a 10 pm octadecylsilane column (4 X 250 mm), a mobile phase of methanol-water (40/60, vh), a flow rate of 2 mL/min and UV detection at 279 nm29; in ophthalmic formulations or raw material using a 5 pm cyano column (4.6 X 150 mm), a mobile phase of dilute phosphate solution (PH 3)/-acetonitrile (60:40, v/v), a flow rate of 2.0 mL/min and W detection at 225 nm30 or a 5pm octylsi- lane column (4.6 X 250 mm), a mobile phase of 0.05 M phosphate solution (pH 5,6)-acetonitrile (4:1, v/v) containing 0.07 M triethylamine, a flow rate of 1.5 mL,/min and W detection at 270 nm3O; in tablets and capsules using a 10 pm phenyl column (4 X 300 mm), a mobile phase of water-methanol- glacial acetic acid (55:44:1, v/v/v) containing 0.005 M heptane sulfouic acid sodium salt, at a flow rate of 2.0 d / m h and W detection at 254 nrn3l.

330 G. MICHAEL WALL

4.5.3 Gas Chromatography

Naphazoline has been analyzed by gas chromatography using various stationary phases including OV-l3%, OV-3 3%, OV-7 3%, OV-17 256, and QF-1 5%32*

5. STABILITY-DEGRADATION

5.1 Potential Routes of Degradation

Naphazoline has been shown to be relatively stable in acidic or neutral solutions but readily prone to hydrolysis in basic solution. The first step in the hydrolytic reaction33 results in the formation of l-naphthylacetylethylenediamine which upon vigorous treatment34, undergoes further cleavage to form 1-naphthylacetic acid and ethylenediamine (Figure 12). The kinetics of this reaction have been describedl6. The major degradation products of naphazoline, 1-naphthylacetylethylenediamine and 1-naphthylacetic acid, have been prepared33 and investigated30*33*34.

5.1.1 Characterization of 1-Naphthylacetylethylenediamine Hydrochloride

5.1.1.1 Thin-Layer Chromatography of 1-Naphthylacetylethylenediamine

An adaptation of the USP TLC procedure17 for the determination of ordinary impurities in naphazoline hydrochloride allowed for the detection of 1- naphthylacetylethylenediamine in the presence of naphazoline. Using silica gel 60 high-performance TLC plates (20 X 20 cm) and a mobile phase of methanol-glacial acetic acid-purified water (8:1:1, v/v/v), spots were visible after spraying with ninhydrin: naphazoline Rf = 0.54; l-naphthylacetylethylenediamine Rf - 0,6330. A similar method has been described in the European Pharmacopoeia for 1-naphthylacetylethylenediamine in naphazoline nitrate35.

5.1.1.2 Liquid Chromatography of 1-Naphthylacetylethylenediamine

1-Naphthylacetylethylenediamine has been quantitated in the presence of naphazoline and 1-naphthylacetic acid using column chromatography followed by W assay33934. A modem HPLC procedure has been developed for the analysis of 1-naphthylacetylethylenediamine in the presence of naphazoline by HPLC using a 5 pm cyano column (4.6 X 150 mm), a mobile phase of 0.025 M Na2HPO4 buffer (pH 7,4)-acetonitrile (35:65, v/v), a flow rate of 2.0 mL,/min and W detection at 270 nm30. Retention times were: naphazoline, 6.3 min; 1-naphthylacetylethylenediamine, 3.1 min (Figure 13).

NH2 N H

OH-

/ /

Naphazolinc HCI 1 -Naphthylacetyluhylenadiamine

d? HC'

1 -Naphthylacdc Acid M y lmdiarnine

Figure 12. Lkgradaion producrs of nuphazolinc hydrochloridc undcr alkaline conditions.

332 G. MICHAEL WALL

c L i

Figure 13. HPLC of I-nriphrhylacerylerh~l~~diurnine HCI (5.2 pg), L and naphamtine HCl (1.2 ps), 2 IS pm qano column, 4.6 X 150 nim, 5.025 M phosphate bufler (pH 7.4)-acetonirrile (35:6S. vh) . 2.0 ml l in in , UV 2701.


5.1.1.3. Synthesis of 1-Naphthylacetylethylenediamine Hydrochloride

The synthesis of 1-naphthylacetylethylenediamine was described by Schwartz et ~1.33 using a modification of previous work by Miescher et uP6. Five grams of naphazoline hydrochloride were refluxed with 100 ml of 0.5N NaOH for 30 minutes. The mixture was then cooled, made alkaline, and extracted with CHC13. The CHCl3 extract was evaporated, leaving a yellowish oil which solidified upon chilling to give the base as an off-white solid recrystallized from CHC13-petroleum ether (1:l) mp 93-95OC. The base in chloroform was treated with HC1 gas to obtain the HCl salt as an off-white solid mp 142-8%

5.1.1.4. Physical/Chemical Properties of 1-Naphthylacetylethylenediamine Hydrochloride

A sample of 1-naphthylacetylethylenediamine hydrochloride was prepared and evaluated30. The substance appeared as off-white powder with a melting point of 153.8-154.2OC. The material was not hygroscopic. The IR spectrum (Table W, Figure 14), UV spectrum (Figure 15), lH-NMR spectrum (Table VIII, Figure 16), 13C-NMR and APT spectra (Table WI, Figure 17), and mass spectrum (Table IX, Figure 18) were consistent with the proposed chemical structure. The DSC (Figure 19) of l-naphthylacetylethylenediamine hydrochloride was consistent with the metling range.

Table W. Infrared spectral assignments for l-naphthylacetylethylenediamine HC1.

Wavelength (cm-1) Assignment

3392

1641 1599,1482,1438 1520 802,795,779

3220-2400 10 amide N-H stretch C-H and N-H stretch amide C-0 aryl C-C stretch 20 amide N-H substituted aromatic

5.1.2. Synthesis and Analysis of 1-Naphthylacetic acid

The synthesis of another naphazoline degradation product, 1-naphthylacetic acid, was described b Schwartz et aP3. Five grams of naphazoline HC1 were refluxed with 5 (r ml of 1N NaOH for 2 hours. The mixture was cooled

7-

m a I U 1 2 1 CI.

Figun 14. Inrfrand spanun (KBr) of I-~phrhylaccrykthyktudicdiamirv HCI.


1.0

0

0 e 8 4

0.0

r

Figum 15. U V Spctrum o f l - M ~ h y l ~ ~ l ~ h y ~ M m i ~ HCI (0.02 m g M in ethanol).

W W 01

Figure 16. IH-NMR (200 MHz) of l - ~ p h r h y l o c t r y l c t h y l e ~ i ~ i ~ HCI (20 mg in DMSOdd.

41 W

338 G. MICHAEL WALL

7.44-8.60 7.44-8.60 7.44-8.60 7.44-8.60 7.44-8.60

Table WI. NMR assignments for 1-naphthylacetylethylenediamne HC1 (20 mg in DMSOQ6).

124.34J25.47, P 125.59,125.96,

127.08,127.88, 128.31

1

2 3 4 5 6 7 8 9 10 11

12 13 14 15 16


and acidified with HCI, producing a flocculent white precipitate. The precipitate was filtered, washed with cold H20 and recrystallized from hot H20 mp 133-134OC; W spectrum, A max 283 pm, E (l%, 1 cm in CHC1-j) =

360. 1-Naphthylacetic acid has been quantitated in the presence of naphazoline and 1-naphthylacetylethylenediamine using column chromatography followed by UV a~say3373~.

Table IX. EI Mass spectrum of 1-naphthylacetylethylenediamine HC1.

EI Relative (We) Abundance (%) Assignment

228 4 [MI+ 199 14 [M-NHCH$ 185 73 [ M-NHCH2CH2]+ 141 100 [ M-C3H7N20]+ 128 14 [ M-C4H8N2O]+

5.2 Solid-state Stability

Naphazoline hydrochloride drug substance has been shown to be stable for at least 6 months under the conditions of room temperature, 4OC, 350C, 40OC at 75% relative humidity, 50OC and exposure to light (1000 foot-can- dles) at room temperature. The drug substance was found not to be hygroscopic.

An investigation was performed to determine if naphazoline hydrochloride would exhibit polymorphism. Samples of the drug substance were treated with (a) heat at lOOOC for 4 hours or (b) vigorously ground with a mortar and pestle. Analytical data from IR, DSC, and x-ray powder diffractometry showed no changes compared to a control sample, suggesting no evidence of polymorphism for naphazoline hydrochloride30.

5.3 Solution Stability

The stability of naphazoline hydrochloride in aqueous buffers (PH 4.5,7.0, 9.0) and oxygen-saturated water was evaluated under the conditions room temperature, 350C, 55OC, and light exp0sure3~. The drug was relatively stable under all conditions at acidic and neutral pH and in oxygen-saturated water for at least 26 weeks. The alkaline solutions, however, turned a range of dark colors and showed severe degradation after only 4 weeks.

0

1m.a 7

n.0 -

I

1 s

T F i p n 18. El Mars s p c m m of 1 -naphrhylacctyltthyknedim~ne Ha.

u f

342 G . MICHAEL WALL

6 . DISPOSITION AND TOXICITY

Naphazoline hydrochloride is commercially available in concentrations of 0.01 to 0.1% as a nasal or ocular decongestant. Local effects are obtained after topical administration to the eye and nasal passages. Metabolic studies could not be found in the literature, despite the report of the synthesis of 14C-labelled drug substance intended for that purpose1'. Dittgen et a1.37 studied the elimination of naphazoline from the isolated pig eye after topical application. Reports of systemic effects of poisoning with naphazoline have been scarce38. The LDSO S.C. in rats has been reported as 385 mgAcg6.

ACKNOWLEDGEMENTS

The author expresses his sincere thanks to the following persons who have provided data and/or information for this chapter: R.E. Hall for solid-state data; G. Havner for confirming the bromine ID test; D.D. Taylor for partition coefficients, UV, MS, and NMR data; R. Conroe and S. Spruill for the synthesis and B. Scott and P. Ritter for analytical data on 1- naphthylacetylethylenediamine, all at Alcon Laboratories; Professor John Baker, Department of Medicinal Chemistry, University of Mississippi, Oxford, Mississippi for NMR data and interpretation; and Professor Hugo Steinfink, Department of Chemical Engineering, University of Texas, Austin, Texas for x-ray powder diffraction and crystallographic information.

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3.

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5 . The Pharmacopeia of Japan, Eleventh Edition (English Version), The Society of Japanese Pharmacopeia: Tokyo, Japan; 1986, p.761.


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Reference 14, Naphazoline Hydrochloride Monograph, p. 916.

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Sane, R.T.; Sane, S. Indian Drugs, 1979,16, 239.

Bult, A.; Klasen, H.B. Pharm. Weekbld., 1974, 109, 513.

Kovar, V.K.A.; Abdel-Hamid, M. Arch. Pharm., 1984,317, 246.

Lajosne, S. Acta Pharm. Hung., 1980,50, 130.

G . MICHAEL WALL 344

24.

25.

26.

27.

28.

29.

30.

31.

32.

33.

34.

35.

36.

37.

38.

Lajosne, S . Acta Phurm. Hung., 1980,50, 270.

Lajosne, S . Acta Pharm. Hung., 1982, 52, 61.

Salam, M.A.; Issa, A.S.; Mahrous, M.S. Anal. Lett., 1986, 19, 2207.

Solimar, S.A.; Abdine, H.; Mocos, M. Can. J . Pharrn. Sci., 1976, 11, 36.

Bauer, J.; Krogh, S . J . Pharm. Sci., 1983, 72, 1347.

Al-Kaysi, H.N.; Salem, M.S.; Al-Khalili, N . Dlrasat, 1985, 12, 101.

Alcon Laboratories, Inc., Unpublished data on fire.

Koziol, T.R.; Jacob, J.T.; Achari, N. 1. t'harm. Sci., 1979, 68, 1135.

Quaglio, M.P.; Cavicchi, G.S.; Cavicchioni, G. Boll. Chim. Farm., 1973, 112, 760.

Schwartz, M.; Kuramoto, R.; Malspeis, L. J . Am. ktiarm. Assoc., 1956,15, 814.

Stern, M.J. Drug Standards, 1958,26, 158.

European Pharmacopoeia, Second Edition, Maisontleuve S . A.: Sainte-Ruffine, France; 1982, p. 147.

Miescher, K.; Marxer, A.; Urech, E. Helv. C h b . Acta, 1951,34, 1 .

Dittgen, M.; Oe.stereich, S.; Eckhardt, D. F'harmazie, 1991,46, 716.

Montfrans, G.A.; van Steenwijk, R.P; Vyth, A.; Borst, C. Acta Med. Scand., 1981, 209, 429.

NAYKOXEN

Fahad I . Al-Shamnlary .' Neelofur Abdul Aziz Mian.'

and Mcrhamrnad Saleem Mian'

( I ) Clinical Laboratory Sciences Department College of Applied Medical Sciences

King Saud University Riyadh, Saudi Arabia

(2) Pharmaceutical Chemistry Department College of Pharmacy King Saud University Riyadh, Saudi Arabia

ANALYTICAL PROFILES OF DRUG SUBSTANCES Copyright c 1992 by Academic Press, Inc AND EXCIPIENTS - VOLUME 21 345 All rights of reproduction reserved in any form

346 F. I. AL-SHAMMAKY. N. A. A. MIAN. A N D M . S. MIAN

CONTENTS

1 Introduction

2 Description

2.1 Nomenclature 2.1 .l Chemical Names 2.1.2 Generic Names 2.1 .3 Trade Names

2.2.1 Empirical 2.2.2 Structural 2.2.3 CAS (Chemical Abstract Service Registry Number) 2.2.4 Optical Rotation

2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Colour, Odour and Taste

2.2 Formulae

3 Physical Properties

3.1 Melting Range 3.2 Solubility 3.3 Dissociation Constant 3.4 Loss on drying 3.5 Hal f - l i fe 3.6 Volume of Distribution 3.7 LD50 3.8 Action 3.9 Sulphated Ash 3.1 0 Stability 3.1 1 X-Ray Powder Differaction 3.1 2 Spectral Properties

3.1 2.1 Ultraviolet Spectra (UV) 3.12.2 Infrared Spectrum 3.1 2.3 Nuclear Magnetic Resonance Spectra

3.12.3.1 PMR Spectrum 3.1 2.3.2 13C-NMR Spectrum

3.1 2.4 Mass Spectrum

4 Synthesis

NAPROXEN 347

5 Pharmacokinetics

5.1 Absorption and Distribution 5.2 Adverse Effects and Precautions 5.3 uses

6 Methods of Analysis

6.1 Identification Methods 6.2 Spectrophotometric 6.3 Nuclear Magnetic Resonance Method 6.4 Titrimetric 6.5 Polarometric 6.6 Fluorometric 6.7 Chromatographic Methods

6.7.1 Thin Layer Chromatography (TLC) 6.7.2 High Performance Liquid Chromatography(HPLC)

7 Acknowledgements

8 References

348 F. J . AL-SHAMMARY. N. A. A. MIAN. AND M. S. MIAN

NAPROXEN

1 INTRODUCTION

Naproxen, a propionic acid derivative, is a nonsteroidal anti- inflammatory agent (NSAIA). The drug is structurally and pharmacologically related to fenoprofen and ibuprofen (1).

Naproxen is a nonsteroidal compound that has anti-inflammatory, analgesic and antipyretic activities. The mode of action is unknown, except that inhibition of prostaglandian synthesis may have an action role. While various manifestations of anti-inflammatory and analgesic actions are evident in patients with rheumatoid arthritis under treatment with naproxen or its congeners, there is no evidence that the progressive source of the underlying disease is altered (2) I

2 DESCRIPTION

2 . 1

1 ) (S)-6-Methoxy- CY -methyl-2-naph?halene acetic acid (3)

( 2 ) d-2-(6-methoxy-2-napht hy1)propionic acid(3)

( 3 ) 2-naphthalenacetic acid (2,4)

( 4 ) 6-methoxy- CY -methyl-,(+)(2,4)

( 5 ) (+)-2-(6-Methoxy-2-naphthyl)propionic acid (5,6,7)

( 6 ) (+)-6-Methoxy- CY -methyl-2-naphthaleneacetic acid ( 2 , 8 )

2.1.2 Generic Names

Naproxen

NAPROXEN 349

2.1.3 Trade Names

Axer; Bonyl; calasen; Diocadal, Dysrnenalgit N; Equiproxen; Floginex; Laraflex; Laser; Naixan, Napren, E; Naprux; Naxen; Prexan; Prirneral; Proxen; Reuxen; Veradol; Xenar.

2 . 2 I

. . 2.2.1 Dir ica l (3)

c14 H14 03

2.2.2 S t r u c t u r a l

2.2.3 GAS (Chemical Abstracf Service Reaistrv Numbe r)

(22204-53-11

2.2.4 Qpt ica l Rotat i o n

[ a ] ~ + 65.5O (C = 1 in chloroforrn)(9) In 4% w/v solution in chloroform +63.0° - 68.5' (6)

2 . 3 Jvlo lecular Weiaht (3)

230.26

2 . 4 Elemental Cornmsi t ion (3)

C = 73.03% H = 6.13% 0 = 20.84%

350 F. J. AL-SHAMMARY. N. A . A. MIAN. AND M. S. MIAN

2 . 5 m e a r a n c e . Colour Odour and Taste

White to off white crystalline powder with bitter taste (2) odourless or almost odourless (6).

3 PHYSICAL PROPERTIES

3 . 1

152 - 1540 (3) 155O (2) 155.30 (9) about 156O (6,7)

. . 3 . 2 So lub i l i ty

Practically insoluble in water. Soluble in 25 parts of ethanoI(96%), in 20 parts of methanol, in 15 parts of chloroform and in 40 parts of ether (6).

Practically insoluble in water at pH 2; freely soluble in water at pH 8 or above, sparingly soluble in alcohol (2).

3 . 3 Dissociation ComtaqJ . .

Pka = 4.2 (25O) (7)

3 . 4 Loss 0 n drvina (6.8)

Dry it at 105O for 3 hours; it loses not more than 0.5% of its weight.

3 . 5 H a l f - l i f e

Plasma half fife, 10-20 hours (mean 14) (7)

. . . 3 . 6 Volume of Distribution (7)

About 0.1 litre/kg

3.7 LDgo(3)

In mice (mg/kg): 435 intraveinous;

In rats (mg/kg): 575 ip;

1235 orally

534 orally

NAPROXEN

3 . 8 A C t i Q n (2,6)

35 I

Anti-inflammatory: analgesic and antipyretic activities.

3 . 9 Sulphated A s h (1 0)

Not more than 0.1%. Use 1.5 g and ignite at a temperature of about 6000.

3 . 1 0 Stabi l i tv

Commercially available preparations of naproxen should be stored at room temperature, exposure of suspension to temperature exceeding 4OoC should be avoided (1). It should be kept in well-closed containers, protected from light (10).

3 . 1 1 X-Rav Powder Differaction

X-ray powder differaction data for Naproxen is determined by De Camp, W.H. (11). X-ray powder differaction pattern was obtained by the use of Cu K a radiation on a Philips goniometer. The X-ray tube was typically operated at 40 KV and 20 mA. Detection was effected with a Nal (TI) scintillation counter coupled to a pulse-height analyser.

3 . 1 2 Sr>ect ral PrsDertieS

3.12.1 Ultraviolet SDectra CUV)

UV spectra of Naproxen (12) in Ethanol (6 mg%) was scanned from 200-400 nm (Fig. 1) using LKB 4054 UV/vis spectrophotometer. Naproxen exhibited the following UV data (Table 1).

3.12.2 In f ra red SDec t r u m

The IR spectrum of Naproxen as KBr disc (12) was recorded on a Perkin Elmer 1210 infrared spectrometer and is presented in Fig. (2). The structural assignments of Naproxen have been correlated with the following frequencies (Table 2).

3.12.3 Nuclear Maanetic Resonance Spectra

3.12.3.1 PMR Spec t rum

The PMR spectra of Naproxen (12) in BMSO-d6 (Fig. 3, 4) was recorded on a varian XL 200 MHZ NMR spectrometer using TNS as an internal reference. The following structural assignments have been made (Table 3).

352 F. J. AL-SHAMMARY. N. A. A. MIAN. AND M. S. MIAN

Frequency cm-1

31 80

3000

2940, 2930

Table 1: UV Data of Naproxen in Ethanol

Absorbance Molar Absorptivity A: nrn (E ) c i ' am moVL

Assignment

(Carboxylic) -OH.

Aromatic C=C stretch.

Alipathic C-H stretch.

21 2 2.81 3

21 5 2.812

10795.355 468.833

10791.51 7 468.666

263 1.345 51 61.66 224.1 66

27 1 1.325 5084.907 220.833

31 7 0.467

33 1 0.518

1792.1 89 77.833

1987.91 86.333

Table 2: 1.R. Characteristics of Naproxen

1730

1600

0 (Carboxylic) I I

6-

Aromatic stretch.

-- -- -cz

%_

-. I

I i

I 0

0

0

0

0

0

Ln

0

0 0

mo

N

cv c

-

0

0

U

0

a3 m

0

'W

0

0

a

m

0

(v

m

0

'0

m

0

.%

0

.w

hl

0

-Y

hl

-0

(v

hl

0

0

0

0

Ln

0

0

TR

AN

SM

ITT

AN

CE

0

0

u3

0

0

03

0

0

0

- 0

0

2

0

0

-4

- 0

0

lo - 0

0

03

- 0

0

0

cy

0

0

m

N

0

0 0

#

0

0

Ln

cr,

0 0 0

.t

.c.r 0

aJ Q

v,

w

Y

CI

N

Y

Cj,

.- LL

354

00 4 0

I

Fig. ( 3 ) PMR SPECTRUM OF NAPROXEN IN C D C l 3

.-

Fig. ( 4 ) PMR SPECTRUM OF NAPROXEN ( D20 Exchange 1

NAPROXEN

6 H aromatics (at d, e f, g, h, i)

3 H Cj)

5 H (at a, b, c )

3.51

7.087 - 7.136 (t)

7.656, 7699 (d)

3.869, 3.878 (d )

1.552 - 1.590 (d)

C C H,

I h I

CH-COOH 9 b a

i C H,O

f e

Table 3: 'H-NMR Characteristics of Naproxen

Proton Assignment I Chemical Shift 6 (ppm)

I

d = douolet, t = triplet.

3.12.3.2 1 3C-NMR Spect rum

I3C-NMR spectrum of Naproxen (12) in DMSO-d6 (Fig. 5, 6) was recorded on varian XL-200 NMR-spectrometer. The multiplicity of the resonance was obtained from APT (Attached Proton Test) program. The BC-NMR spectrum displayed all the fourteen carbon resonances. The narrow resonance range of some of the carbons makes the spectrum rather complex. The carbon chemical shifts assignments are presented in (Table 4).

3.1 2.4 Mass SDectrurn

The mass spectrum of Naproxen (12) obtained by electron impact ionication (Fig. 7) was recorded on a Finnigen MAT 90 spectrometer. The spectrum wc?s scanned from 50 to 500 a m a . Electron energy was 70 ev. Emission current 1 mA and ion source pressure torr. The most prominant fragnents and their relative intensities are presented in (Table 5).

358 F. J. AL-SHAMMARY, N. A. A. MIAN, AND M. S. MIAN

3 7 5

14

10 12

Table 4: Carbon-13 Chemical Shifts of Naproxen ~~~ ~~~ ~~

Carbon Assianment :hemica1 Shift ( w m l

18.1 15

45.306

55.267

128.867

133.81 8

134.825

157.696

181.052

105.559

11 9.027

129.292

126.137, 126.174 Linterchangable A

127.21 7

180 160 140 1

I A

3 100 80 60 40 2 o P P M 0 I 13

Fig. ( 5 ) C -NMR SPECTRUM OF NAPROXEN IN C D C l 3

--A- -

] ) I I I I ’ I I I I I I I I I l q I I I I l I I I I I I I I I I 1 I I I l l 1 1 ) I I I1 I I I I I l i l l l mlr 1-40 120 100 80 60 40 20 PPM

I;;; I I i I I I ’ I t ) ’

13 Fig. ( 6 1 C -NMR OF NAPROXEN I N C O C l ( A P T )

3

100.0 2 3

Fig. ( 7 ) MASS SPECTRUM OF NAPROXEN

362 F. 1. AL-SUMMARY. N. A. A. MIAN. AND M. S. MIAN

Table 5: Mass Spectrum of Naproxen

r n l z

230.1

21 5

185

169.9

153.2

141.1

Relative intensitv %

100

2

5 8

10.2

4

7

ions

NAPROXEN 363

4 SYNTHESIS

SCHEME

Naproxen is prepared (13) by the acylation of 6 substituted naphthalenes by AcCl forming the 2-acetyl derivative, which is further converted to 2-naphthyl acetic acid. Esterification and alkylation of 2-naphthyl acetic acid in the prescence of H2S04, MeOH, NaH, Me1 and with NaOH gave after hydrolysis the naphthyl propionic acid. Resolution of 2-(6-methoxy-2-naphthyl) propionic (Naproxen) was readily achieved by crystallization of the cinchonidine salt.

SCHEME

6 - s u b s t i t u t e d Naphthalene

2-acetyl d e r i v a t i v e

(1) Morpholine, S

( i ) H2SO4, CH30H CH, ( i i ) NaH, C H s I

I

( i i i ) NaOH

&bOH- D C O O H

C H30 CH,O

Naproxene. (6 subs t i tu tcd)2-Naphthyl - a c e t i c ac id .

364 F. 1. AL-SHAMMARY. N. A. A. MIAN, AND M. S. MIAN

5 PHARMACOKINETICS

i a 5 . 1 Absorption and Distribut . .

When administered as the acid or the sodium salt, naproxen is completely absorbed from the gastrointestinal tract; the sodium salt is absorbed more rapidly than the acid (1). Peak plasma levels (about 55 pg/ml) are reached in 2 to 4 hours after a 500 mg dose, and steady state levels are attained after 4-5 doses at 12 hours intervals. More than 99% is bound to serum albumin. The mean plasma half life is about 13 hours (2). Approximately 95% of a dose is excreted in the urine, principally as conjugates of naproxen and it's inactive metabolite 6-desmethyl naproxen (2).

The apparent volume of distribution of naproxen averaged abut 8.3 L in healthy adults and about 11.9 L in patients with severe renal failure (serum creatinine 5.4-1 2.5 mg/dl)(2). In healthy adults, plasma half-life of naproxen reportly ranges from 10-20 hours.

After entering the stomach naproxen sodium readily dissolves in gastric juice and about 30% of dose of naproxen is metabolised in the liver to 6-desmethyl naproxen, which is inactive. Most of the drug is excreted in urine as unchanged naproxen (10%) and 6- desmethyl-naproxen (5%) and their glucuronide or other conjugates (82%). Some data, however suggest that renal excretion of unchanged naproxen may be negligible or absent. In patients of with severe renal failure, total body clearance of naproxen may increase apparently because of decreased binding of the drug serum proteins. A small amount (less than 5%) of the drug excreted in feces (2), precipitates is out as fine particles of naproxen. These particles provide a greater surface area for dissolution than the larger particles that result from naproxen tablet disintegration (1 4). When using conventional tablet formulation of naproxen sodium and naproxen, the former consequently produces earlier and higher plasma conc. of naproxen (15). The same holds true during administration of suppositories of naproxen sodium and naproxen (16). Mean time to peak plasma conc. (tmax) is about 1 hour for naproxen sodium and 2 hours for naproxen when administered to fasting subjects (14).

With respect to concomitant antacid administration past studies have shown that sodium bicarbonate enhances the rate of naproxen absorption, magnesium carbonate caused a slight reduction, and a mixture of magnesium oxide and aluminium hydroxide gave a clear reduction in the rate of naproxen absorption (17).

NAPROXEN 365

5 . 2 Adve rse E ffects a nd Precaut ions

Adverse reactions to naproxen mainly involve the GI tract, constipation, heartburn, abdominal pain, and nausea occur in about 3-9% of patients recieving the drug, less frequently, dyspepsia, diarrhea, stomatitis, vomiting, anorexia, flatulence occur (1).

Adverse effects of naproxen is similar to that of ibuprofen. The most frequent adverse effects occuring are gastrointestinal disturbances. Peptric ulceration and gastro-intestinal bleeding have been reported, other side effects include headache, dizziness, nervousness, skin rash, pruritus, tinnitus, oedema, depression, drowsiness, insomnia, and blurred vision and other occular reactions. Hypersensitivity reactions, abnormalities of liver function tests, impairment of renal function including interstitial nephritis or the nephrotic-syndrome, agranulocytosis, and thrombocytopenia have occasionally been observed (5).

Naprosyn (Naproxen) should not be used conmitantly with the related rug anaprox (naproxen sodium) since they both circulate as the naproxen anion (4). All aspirin-sensitive asthmatic patients developed reactions such as rhinorrhoea, tightness of chest, wheezing, dyspnoea, after taking naproxen in doses of 40-80 mg ( 1 8 ) .

Gastrointestinal reactions tended to be more frequent and severe when naproxen dosage was increased from 750 mg/day to 1500 mglday in patients with rheumatoid arthritis. In studies involving almost 500 children with juvenile arthritis the incidence of rash and prolonged bleeding time was increased, gastrointestinal and CNS reactions were reported at approximately the same rate, and other adverse effects were observed less frequently in children than in adults. However naproxen was not associated with a higher frequency of adverse effects in elderly (> 65 years) patients with rhematoid arthritis or osteoarthritis compared with younger patients (19, 20).

Naproxen should be given with care to patients with asthma or bronchospasm, bleeding disorders, cardiovascular disease, peptic ulceration or a history of such ulceration, renal failure, and in those who are recieving coumarin anticoagulants. Patients who are sensitive to aspirin should generally not be given naproxen (5).

Naproxen may interfere with some tests for 17-ketogenic steroids (5).

F. 1. AL-SHAMMARY, N. A. A. MIAN, AND M. S. MIAN

Naproxen has analgesic, anti-inflammatory, and antipyretic properties; it is an inhibition of prostaglandin synthetase. The drug is used in rheumatic disorders such as ankylosing spondylititis, osteo arthritis, and rhematoid arthritis, in mild to moderate pain such as dysmenorrhoea, migrane and some musclokeletal disorders, and in acute gout (43).

Naproxen is used to relieve mild to moderately severe pain. The drugs are also used for anti-inflammatory and analgesic effects in the symptomatic treatment of mild to moderately severe, acute and chronic muscleskeletal and soft tissue inflammation (1).

The usual dose of naproxen or naproxen sodium is the equivalent of 500 mg to 1 g of naproxen daily in 2 divided doses. A dose of 10 mg per kg body-weight daily of naproxens in 2 divided doses has been used in children over 5 years of age with juvenile rheumatoid arthritis (5 ) . In painful conditions such as dysmenorrhoea the usual initial dose is the equivalent of 500 mg of naproxen followed by 250 mg every 6 or 8 hours. In accute gout an initial dose equivalent to 750 mg of naproxen followed by 250 rng every 8 hours has been suggested (5). Rectal administration of naproxen is sometimes employed naproxen has also been used orally as the piperazine salt (5).

Naproxen is comparable to aspirin in controlling disease symptoms, but with lesser frequency and severity of nervous system and milder gastrointestinal adverse effects (2).

Naproxen has been used effectively to relieve pain, fever, redness, swelling and tenderness in patients with accute gouty arthritis (1).

One study indicates that single oral dose of naproxen (2.5 or 7.5 mg/kg) was at least as effective as a single oral dose of aspirin (15 mg/kg) in the reduction of fever in children. The result of one study suggested that the combination of naproxen sodium and ampicillin was more effective than ampiciline alone in elleviating fever, dyspnea, and coughing associated with accute respiratory infections in children (1). A favourable antipyretic effect with naproxen 5 mg/kg twice daily in children with fever caused by infection (21).

Naproxen 7.5 mg/kg twice daily in children (22) and 250-375 mg twice daily adults (23) has been shown to be extremely effective

NAPROXEN 367

for controlling fever associated with malagnancy. Naproxen has been proposed as a test to distinguish neoplastic fever from infectious fever in cancer patients with fever of unknown origin ( 2 3 ) .

6 METHODS OF ANALYSIS

6 . 1 JDENTlFlC ATION METHO DS

The infrared absorption spectrum of a potassium bromide dispersion of its exhibits maxima only at the same wavelengths as that of a similar preparation of USP Naproxen R.S. (8).

It gives liebermann's test black-green; Marquistes-brown. Sulphuric acid-orange (7).

The light absorption in the range of 230 to 350 nm of a 0.004% w/v solution in methanol, exhibits four maxima, at 262, 271, 316 and 331 nm. The absorbance at 262 nm is about 0.91, at 271 nm, about 0.92 at 316 nm, about 0.26 and at 331 nm, about 0.30 (6).

It melts at 156OC (6).

Dissolve about 500 mg of Naproxen, accurately weighed in a mixture of 75 ml of methanol and 25 ml of water that has been previously neutralized to the phenolphthalein end point with 0.1 N NaOH. Dissolve by gentle warming, add phenolphthalein and titrate with 0.1N NaOH VS. Each ml of 0.1N NaOH is equivalent to 23.03 mg of C14H1403 (8).

6 . 2 SPECTROPHOTOMETRIC

Spectrophotometric determination of naproxen in tablets form was done by Tosunoglu, S. (24). A portion of the crushed tablets containing about 250 mgs of naproxen was shaken with 50 mi of 96% ethanol for 30 minutes and the mixture was diluted to 100 mi with 96% ethanol and filtered. A 1 ml portion of diluted solution was mixed with 4 mM-rosaniline in 20% ethanol (3 ml) and extracted with 5 mi of CHC13 and the absorbance was measured at 545 nm against blank. The calibration range was 1 to 15 pg per ml. Recovery was 99.9%.

Quantitation of naproxen with other drugs in pharmaceutical dosage forms by first and second derivative uv spectrometry by

368 F. J. AL-SHAMMARY, N. A. A. MIAN. AND M. S. MIAN

Mahrous, M.S. et al (25). Derivative uv spectroscopy was used to determine the cited drugs in capsules and tablets. First derivative spectroscopy was used to determine indomethacin in 0.1 N-HzS04 solution, and second derivative spectroscopy was used to determine naproxen and iboprofen in O.1N-NaOH solution. The method is rapid and accurate.

3 Powdered tablets (26) equivalent to 50 mg of naproxen were dissolved in 10 ml of methanol and boiled under reflux with 20 ml of 5M-HCI for 45 mins. The solution was cooled and the excess of HCI was removed under vacuum. The residue was dissolved in 10 ml of methanol, adjusted to pH 7.0 with NaOH solution and diluted to 100 ml with H20. To a portion of this solution, were added 1 ml of 0.05% P-NN-dimethyl phenylenediammonium chloride and 1 ml of aq. 0.2% K2Cr207 and H20 to 25 ml. The absorbance was measured after 10 minutes (but with in 1 h) at 600 nm vs, a reagent blank. Beer's law was obeyed from 5-40 pg ml-1 ( E = 2760).

6 . 3

Tosunoglu, S . ; et al (27) determined naproxen by NMR spectrometry. Powdered tablets equivalent to 85 mg of naproxen were mixed with acetanilide (40-45 mg; internal standard) and extracted with CHC13 (25 ml). After ultrasonic agitation for 30 minutes, the mixture was filtered and a 10 ml portion of the filtrate was evaporated to dryness in vacuo. The residue was dissolved in 0.8 ml of CHC13 and the spectrum was recorded on a Bruker NMR spectrometer operated at 250 and 300 MHz with the probe of 370. Chemical shifts were measured relative to tetramethylsilane at 1.58 and 2.09 ppm for naproxen and the internal standard respectively.

6 . 4 TlTRlMETRlC DETERMINATION

The determination of naproxen involved oscillometric titration (28) of its solution in aq. 20% acetone (10 ml, 10 mM in naproxen and containing 2 ml of aq. 0.1 M-NH3) with O.1M-KOH, a Radelk is type OK-302 apparaturs being used for determining the end point. Results obtained for naproxen indicated the good precision of the oscillometric technique.

6 . 5

Polarometric determination (29) of naproxen by dissolving in various heterocyclic, aromatic and aliphatic basic solvents and the

NAPROXEN 369

optical rotation was determined at Na and Hg iines. The total specific rotation was calculated, the results obtained showed variations of up to 22O at the Na line and of 30° at the Hfg line with heterocyclic solvents with aromatic amines the values obtained were lower than those of published in U.S.P. and B.P; Negative values of optical rotation were determined when naproxen was dissolved in aliphatic amines.

6 . 6 FLUOAIMETR IC DETERMINA T l W

Naproxen contents in sugar coated tablets was determined by fluorescence (30) spectrophotometry. Naproxen tablets with their sugar-coating removed, were powdered and a sample equivalent to about 25 mg of naproxen was dissolved in 5 ml of 1% NaOH and diluted with Y20. A 0.5 ml portion of the supernatant solution was mixed with 1 ml of 1M-HCI and dilute to 100 ml with H20 and the fluorescence of the solution was measured at 356 nrn (excitation at 274 nm) vs. 0.01 M-HCI. Recovery was 39.5% with a coeff. of variation of 0.8%

6 . 7 CHROMATOGRAPHIC METHODS

6.7.1 Thin laver chromcrtowhv (TLQ

Naproxen and its metabolites were measured (31) in urine. 1- 2 ml ot sample was acidified with 0.1 rnl of O.1M-HCI and extracted with 5 rnl of CHCI3. The extract was evaporated, and the residue was dissolved in 100 jd of ethanol. 10 pI portions were spotted on to silica gel 60 F254 plates and TLC was carried out with CHCl3:methanol (1 7:3) as mobile phase. Spots corresponding to naproxen and its 6-demethylmetabolite (Rf 0.54 and 0.41, respectively) were visualized under 254 nrn radiation, scraped off and extracted with aq. 95% ethanol (4 x 5 ml). The extracts were diluted to 25 ml, and the absorbance was measured at 232 nm. The calibration covered the range 0.2-0.3 pg m1-l in the final solution.

graph

6.7.2 Hiah Performa nce Liauid Chromatoa raDhy w

* A summary of the sum of the HPLC methods for the analysis of Naproxen are given in the Table (6).

ACKNOWLEDGEMENTS

The authors are highly thankful to Mr. Babkir Awad Mustafa, College of

TABLE (6 ) Summary of HPLC conditions of Naproxen.

(10 cm x 4.6 mm) of Brownlee RP 18 (5um)

(25 cm x 4 rnm) of Hypersil ODS (5 urn)

(22 cm x 4.6 mm) of underivetized 5-urn Brownlee silica with 7-um silica pre-column (1.5 cm x 4.6)

Octadecylsilane column (25 cm x 4.5 mm i.d)

Dctadecy Isi I ane acetic acid (1 125:1375:8)

25 um.amm.phosphate buffer (pH 3.0) in 75% methanol

acetonitrile-acetate buffc (pH 4.8 or 4.2)

5mM-aq. sod. phosphate- H3P04 buffer of pH 2.6 (19:l) containing 0.9% acetonitrile

methanol.sod. acetate buffer

Flow rate rnl/rnin.

t t f

. * *

1 ml/min.

1.5 ml/rnin.

Ietectior

240 nm

254 nrn

240 nm

uv

235 nm

Sample

'lasma or blood

'lasma

'lasrna or serur

>apsules or ablets

Plasma

- 3ef. - 3 2

33

3 4

35

3 6

NAPROXEN 37 1

Applied Medical Sciences for his efforts in drawing the spectrums and figures. The authors also would like to thank Liberty S. Matibag, College of Applied Medical Sciences for her valuable and professional help in typing the manuscript.

REFERENCES

1

2

3

4

5

6

7

8

9

10

11

12

13

14

"Drug Information 91" p. 1124. American Society of Hospital Pharmacists.

"Pharmaceutical Sciences" p. 1059. Mack publishing Company Easton, Pennsylvania, U.S.A. (1 980).

"The Merck Index" 11 th ed. p. 1014 (1 989).

Physician's Desk Reference 42nd ed. p. 2101 (988).

"Martindale" "The extra pharmacopeia" 29th ed. p. 28. The Pharmaceutical Press, London (1 989).

"British Pharmacopoeia" Her Majesty's Stationary Office London p. 384 (1988).

"Clark's Isolation and Identification of Drugs" 2nd ed. The Pharmaceutical Press London (1 986).

"T h e U n it ed States P h a r ma cop o e i a" United States Pharmacopoeia1 Convention, Inc., 12601. Twinbrook Parkway, Rockville, M.D. 20882 p. 917 (1990).

"The Merck Index" 10th ed. p. 920 (1983).

'* B r i ti s h P ha r ma co p oe ia " Her Majesty's Stationary Office London p. 300 (1980).

De Camp, W.H. J. Assoc. Off. Anal. Chem. 67(5) 9 2 7 -

Mohammad Saleem Mian, Neelofur Abdul Aziz Mian unpublished data (1992).

Jan T. Harrison; Brian Lewis; Peter Nelson; Wendell Rooks; Adolph Rostkowski; Albert Tomolonis and John H. Fried. J. Med. Chem. 13 203-205 (1970).

A, Moyer S. Cephalgia 6 (Suppl. 4) 77-88 (1986).

933 (1984).

372 F. J. AL-SHAMMARY. N. A. A. MIAN, AND M. S. MIAN

1 5

1 6

1 7

1 8

1 9

2 0

2 1

2 2

23

2 4

2 5

2 6

2 7

2 8

2 9

Sevelius H., Runkel R., Segre E., Bloomfield, S.S. Br i t i sh Journal of Clinical Pharmacology 10 259-263 (1980).

Gamst, O.N., Vesje, A.K., Arabakke, J., International Journal of Clinical Pharmacology, Therepy and Toxicoloty

Weber, S.S., Bankhurst, A.D., Mroszczak, E., Ding, T.L. Therapeutic Drug Monitoring 3 75-83 (1981).

A. Szczeklik, et. al. Br. Med. J. 2 231 (1977).

22 99-103 (1984).

Geczy, M., Peltier, L., Wolbach, R. J. Rheumatology 14

Husby, G. American J. Medicine 81(Suppl. S.B.) 6-10 (1 986) .

Szmyd, L., Perry, H.D. American Journal of Bpthamology 99 598 (1985).

Azeemuddin, S.K.; Vega, R.A.; Kim, T.H.; Ragab, A.H.; The Effect of Naproxen on Fever in Children with Malignancies Cancers 59

348-354 (1 987).

1966-1968 (1987).

Peter A. Todd and Stephen P. Clissold Drugs 40(1} p. 91- 137 (1990).

Tosunoglu, S. Acta. Pharrn. Turc. 31(3) p. 119-122 (1 989).

Mahrous, M.S.; Abdel-Khalek, M.; Abdel-Hamid, M.E.; J . Assoc. Off. Anal. Chem. 68(3) 535-539 (1985).

Sastry, C.S.P.; Prasad, Tipirneni, A.S.R.; Suryanavayana, M.V.; Aruna, M. India Drugs 26(11) 643-644 (1989).

Tosunaglu, S.; Buyuktimkin, N. Acta. Pharm. Turc. 31(4)

Kolodziejska, T. Acta. Pol. Pharm. 40(3) 357-360 (1 983)(Pol.).

Ceccarin, G.; Maione, A.M. J. Pharm. Sci. 78(12) 1053- 1054 (1989).

149-152 (1989).

NAPROXEN 313

30 Tang, H.; Wang, K. Yaown Fenxi Zazhi lO(6) 358-359

31 Abdel.-Moety, E.M.; Al-Obaid, A.M.; Jado, A.I.; Lotfi, E.A. Eur.

32 Levine, B.; Caplan, Y.H. Clin. Chem. 31(2) 346-347

33 Kazernifard, A.G.; Moore, D.E. J. Chromatogr. 533 125-

34 Streete, P.J. J. Chromatogr. 495 179-193 (1989).

3 5 Larnpert, B.M.; Stewart, J.T.; J. Chromatogr. 504(2)

36 J. Phar. Sci.

(1990) (Chinese)

J. Drug Metab. Pharmokinet. 13(4) 267-271 (1988).

(1 985).

132 (1990).

381 -389 (1 990).

Shirnek, J.L.; Rao, N.G.S.; and Wahba Khalil, S.K. 71(4) 436-439 (1982).

PERGOLIDE MESYLATE

Delores J . Sprankle and Eric C. Jensen

Lilly Research Laboratories

Eli Lilly and Company

Indianapolis, IN 46285

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS -VOLUME 21

Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form, 375

316 DELORES J. SPRANKLE AND ERIC C. JENSEN

TABLE OF CONTENTS

1. Description 1.1 Name, Formula, and Molecular Weight 1.2 Appekance and Color 1.3 History

2. Synthesis

3. Physical Properties 3.1 Confirmation of Structure

3.1 1 Spectroscopic Data 3.12 Potential Isomerism

3.2 Infrared Spectrum 3.3 Nuclear Magnetic Resonance Spectrum 3.4 Mass Spectrum 3.5 Fluorescence Identification 3.6 Fluorescence Spectrum 3.7 Ultraviolet Spectrum 3.8 Melting Range 3.9 Differential Thermal Analysis 3.10 Thermogravimemc Analysis 3.11 Optical Rotation 3.12 Crystal Properties 3.13 Solubility 3.14 Partition Coefficient 3.15 Ionization Constant, pKa 3.16 Color Identification Test

4. Methods 4.1 Identity 4.2 Elemental Analysis 4.3 Ultraviolet Spectrum 4.4 Chromatography

4.41 Thin Layer 4.42 High Performance Liquid

5 . Stability - Degradation 5.1 Degradation Profile 5.2 Stability in Dosage Form

6. Drug Metabolism and Pharmacokinetics

7. References

8 . Acknowledgments

PERGOLIDE MESYLATE 377

1. DESCRIPTION

1.1 Name, Formula, and Molecular Weight

name Permax@. It is also commonly referred to as LY 127809.

monomethane sulfonate. The CAS Registry Number is 66 104-23-2.

Empirical Formula: C19H26N2SCQ03S

Molecular Weight: 410.6

Structure:

Pergolide mesylate is marketed by Eli Lilly and Company under the trade

Chemically, it is known as 8~-[(methylthio)methyl]-6-propylergoline,

1.2 Appearance and Color

Pergolide mesylate occurs as white to off-white crystals or a crystalline powder .

1.3 History

Permax (pergolide mesylate) is a dopamine receptor agonist at both DI and D2 receptor sites.' Permax is indicated as adjunctive treatment to levodopalcarbidopa in the management of signs and symptoms of Parkinson's disease. Administration of Permax should be initiated with a 0.05 mg/day dosage for the first 2 days. The dosage should then be gradually increased by 0.1 or 0.15 muday every third day over the next 12 days of therapy. The dosage may then be increased by 0.25 mg/day every third day until an optimum therapeutic dosage is achieved. Permax is usually administered in divided doses three times per day. During dosage titration, the dosage of concurrent I-dopalcarbidopa may be cautiously decreased. 2

DEWRES J. SPRANKLE AND ERIC C. JENSEN

2. SYNTHESIS

Pergolide mesylate can be synthesized from dihydroelymoclavine.3 In the first step of this method, dihydroelymoclavine is demethylated by von Braun cleavage. The resulting cyanamide intermediate I is then cleaved to intermediate I1 via sodium hydroxide. The Wallach reaction utilizing propionaldehyde and formic acid then propylates N-6 to give intermediate n1. A mesylate ester functional group is added to intermediate 111 using methanesulfonyl chloride in pyridine to give intermediate IV. Displacement of the mesylate ester with methanethiol and sodium methoxide results in sodium thiomethoxide to produce crude pergolide base, intermediate V, which is isolated, and using methanesulfonic acid, is converted into its final form as the crystalline mesylate salt from methyl alcohol. The isolated pergolide mesylate is finally recrystallized from methanol.

Dlhydrodymoclavlna

- CH,SH, NaOCH.

DMF cn2cn2cn3

( I V )

Figure I .

Pergollde Mesllste

Chemical synthesis for pergolide mesylate

PERGOLIDE MESYLATE


379

3.1 Confirmation of Structure

3.11 Spectroscopic Data The chemical structure of pergolide mesylate was determined from the

data of synthetic method, elemental analysis, ultraviolet (UV) spectra, infrared (IR) absorption spectra, hydrogen (1H) and carbon (13C) nuclear magnetic resonance (NMR) spectra, and mass spectra. The following is a summary discussion of the spectroscopic data and potential isomerism of pergolide mesylate to support the confirmation of structure of this compound?

Pergolide mesylate (I), is an ergoline alkaloid that is structurally related to lysergic acid and its derivatives.5-6 The structures of several lysergic acid derivatives have been determined by X-ray crystallography 6, and the proton NMR spectra of lysergic acid and iso-lysergic acid diakylamides have been reported.5 The proof of structure for pergolide mesylate is provided by spectroscopic data and supported by correlation with lysergic acid, its derivatives, and other ergoline alkaloids.

SCH 3 I

CH - SO 3 3

The proton-decoupled 13C NMR spectrum of pergolide mesylate indicates the presence of twenty carbons, which are further identified as four non- protonated carbons, six methine carbons, and ten methyl and methylene carbons via a 1% Distortionless Enhancement by Polarization Transfer (DEFT) spectrum.7 The eight aromatic and olefinic carbon resonances in the 134 ppm to 106 ppm region have chemical shifts consistent with an indole structure (2).8 The 1H NMR spectrum contains four aromatic proton resonances, from 6.88 to 7.22 ppm, whose chemical shifts are also indicative of an indole-type structure. The assignment of these carbon and proton resonances were confirmed with IH correlation spectroscopy (COSY) and W-1H heteronuclear correlation spectroscopy (HETCORR). The UV spectrum also indicates the presence of an indole chromophore.

3 80 DELORES J. SPRANKLE AND ERIC C. JENSEN

2

The NMR data shows the molecule contains a methyl with carbon and proton shifts at 10.78 and 0.93 ppm, respectively. The COSY experiment identifies the coupling of these methyl protons to a methylene site, with 13C and 1H chemical shifts at 15.53 and 1.68 ppm, which in turn is coupled to another methylene site. The 13C and 1H shifts of this last site (53.83 and 3.36 ppm) indicate this methylene is attached to a heteroatom. Correlation experiments c o n f m the coupling to an NH+ site, and the IR absorption band at 2556 cm-1 is indicative of MI+, allowing us to propose substructure 3.

H 3

The COSY experiment shows the protonated amine of 3 residing in a ring system as shown in 4. The 13C, 'H, COSY, and HETCORR NMR data are consistent with the structure proposed in 3. The l3C data indicate a methylene carbon, located at 55.53 ppm, attached to a heteronuclear site. The corresponding protons, located at 2.95 and 3.59 ppm, were assigned from the HETCORR experiment. The COSY experiment shows that these two protons are coupled to a methine site, whose proton is coupled to protons located at 1.44 ppm, 1.55 ppm, and 2.8 ppm. These protons were assigned to the corresponding carbons using the HETCORR data, and these l3C resonances were identified as methylenes from the DEPT spectrum. The COSY and HETCORR experiments also show a methylene carbon whose protons are coupled to a methine carbon bonded to a heteroatom, based upon IH and 13C chemical shifts, 2.55 and 36.44 ppm, respectively.

PERGOLIDE MESYLATE 38 I

4

The MS spectrum of pergolide mesylate contains a fragment with m/z 267, which is consistent with the combination of substructures 2 and 4 to yield 5.

CH

5

MS spectrometry also indicates the presence of an -S-CH3 group from the m/z 285 fragment. The IH and l3C spectra also shows resonances, located at 2.08 and 15.19 ppm, which are consistent with the -S-CH3 group. Attachment of the -S-CH3 group to a methylene follows from the 13C data of the methylene. Presence of the mesylate moiety was confirmed by the carbon resonance located at 39.64 ppm and its correlation to the protons at 2.36 ppm.8 This is supported by IR absorption bands for an S-0-C stretch at 1038 cm-1 and 0-S-0 stretches at 1157 cm -1 and 1331 cm-1.

The structure for pergolide mesylate (1) is supported by other spectroscopic evidence. For example, aromaticity is observable in the IR spectrum and in fragments ( d z 144,267,285) of the mass spectrum. The mass spectrum fragmentation pattern also supports the connectivity of the ring systems of the molecule.


3.12 Potential Isomerism Protonation of the tertiary nitrogen to produce the quaternary amine salt

gives rise to the possibility of both an a and p isomer at the nitrogen position. The 1H NMR spectrum indicates the presence of two NH+ protons, confirmed by the results of a deuterium exchange experiment, which are consistent with 6-a (6) and -p (7) isomers. These assignments are further confirmed by the coupling of the 6-a-NH+ and 6-p-NH+ protons to lH (the protons attach to the carbon at the 1' position). In anhydrous solvents, such as dimethylsulfoxide-ds, it is possible to observe both isomers.

In aqueous solvents, the rate of exchange is such that these isomers rapidly interconvert, resulting in only one NH+ resonance.

SCH 3

I -

3 3 r2 C H - S o

N

SCH 3

I - CH - SO

3 3 f"2

6 7


3.2 Infrared Spectrum

The infrared spectrum for pergolide mesylate as a potassium bromide pellet is illustrated in Figure 2. The spectrum was recorded on a Nicolet Model 60SXB Fourier Transform infrared spectrophotometer. The infrared spectrum of pergolide mesylate is positively identified with maxima at the following approximate wave numbers: 3183 cm-l,2556 cm-1, 1456 cm-1, 1157 cm-l, 1038 cm-l, and 775 cm-l. The major absorption bands for the infrared frequencies and the corresponding assignments are listed in Table I.

Table I. Infrared Band Assignments for Pergolide Mesylate

Wavenumber (cm-1)

3183

3040

2556

1619, 1606

1456, 1443

1331, 1157

1038

794.775

Assignment

N-H pyrole with hydrogen bonding: N-H stretch

Aromatic: C-H stretch

NH+: N-H stretch

Aromatic: C-C stretch

Aliphatic: C-H deformation

RS03-: 03-0 stretch

RS03-: S-0-C stretch

N-H pyrole with hydrogen bonding: N-H deformation

m

00 1

08

09

06

02

33NW

LlIW

SN

tJklL %

0


3.3 Nuclear Magnetic Resonance Spectrum

The 300 MHz 1H spectrum of pergolide mesylate (10 mglmL) in dimethylsulfoxide-dg (2.5 ppm) is shown in Figure 3. The spectrum was obtained on a Varian Unity spectrometer using the following instrumental parameters: 5 mm 1H/13C dual probe; spectral width, 4416 Hz; 90" pulse; 64K time-domain data points; acquisition time, 7.421 seconds; 100 scans and probe temperature, 35OC. The spectrum was provided with 0.1 Hz Lorentzian line broadening.

The proton-decoupled 13C spectrum of pergolide mesylate (50 mg/mL) in dimethylsulfoxide-& (39.5 ppm) is shown in Figure 4. These data were obtained using a 5 mm lH/13C dual probe; spectral width, 11 658.5 Hz; 90" pulse width; 64K time-domain data points; acquisition time, 5.49 seconds; relaxation delay, 2.4 seconds; WALTZ- 16 proton decoupling; 4000 scans and probe temperature, 35°C. The spectrum was processed with 1.0 Hz Lorentzian line broadening followed by the addition of 64K zero-fill data points.

in Table 11. Assignments are based on 'H, l3C, lH-lH COSY, DEPT, and lH-13C HETCORR experiments.

Structural assignments for both proton and carbon NMR spectra are listed

8' CHZ-S-CH, I

13 1' 3'

N- 1 2

1 .Dm

Figure 3. IH NMR spectrum of pergolide msylate in dimethylsulfoxide-dg (35 "C)

W m 4

i w( IY IU I 1 I" 11, ," s " n " Y * 1 e# I . .F

Figure 4 . l3C NMR spectrum of pergolide mesylate in dimethylsdfoxide-dg (35 "C)

388 DELORES 1. SPRANKLE AND ERIC C . JENSEN

Table 11. NMR Chemical Shift Assignments of Pergolide Mesylate

d: doublet m: multiplet q: quartet t: triplet s: singlet bd: broad doublet bs: broad singlet

3.4 Mass Spectrum

The electron impact mass spectrum of pergolide mesylate is shown in Figure 5. The spectrum was obtained using a VG Model 7070E magnetic sector instrument operating at 70 eV ionizing potential. A molecular ion for the free base at m/z 314 was observed. Accurate mass measurements on these fragments support these assignments. Several of these fragment assignments are illustrated in Table 111.

IUk

9s.

98.

65.

88.

E .

78.

65.

68.

55.

5%.

15.

49.

35.

3% 4ie

285 154

Figure 5. Electron impact mass spectrum of pergolide mesylate

390 DELORES J . SPRANKLE AND ERIC C. JENSEN

Table 111. Assignment of Ion Fragments of the EI-MS Spectrum -

m/z

-

314

299

-

285

Elemental composition

observec mass -

314.181

299.36C

285.144

- mmua

-

1.1

-

-2.1

-

-1.7

-

- D B E ~

-

8.0

-

8.5

-

8.5

-

Parent ion relative

intensities

100

4

34

Structure

SCH3

I

I

CHZ

$- H’ N

S+ I & 0

N H’

SCH3 I

Q$ “CH:

H*


Table 111. Assignment of Ion Fragments of the EI-MS Spectrum (continued)

25 1

-

223

-

Elemental composition

C17H19N2

C15H15N2

3bserved mass

267.183

251.151

223.123

- mmu

3 .O

3.3

-

0.7

-

8.5

9.5

9.5

Parent ion relative intensities

11

6

5

Structure

dN- N

H*

@ N

H'


Table 111. Assignment of Ion Fragments of the ELMS Spectrum (continued)

Elemental Zompositior

c 14H13N2

c 12H 1oN

observed mass

209.107

194.095

168.079

~~

167.071

- mmua

-

0.4

-

2.2

-

2.1

-

2.1

- D B E ~

-

9.5

-

9.5

8.5

-

9.0

-

Parent ion relative

intensities

4

7

10

12

structure

[@] H'

[&] CH2

@ , H'

+



miz -

154

-

154

-

144

-

144

-

Elemental :ompositior

observec mass

154.056

154.056

144.069

144.078

- mmua

-

-3.5

-

-0.1

-

0.2

-

2.8

-

- D B E ~

-

9 .O

-

4.0

-

7.0

-

6.5

-

Parent ion rclative

intensities

33

11

Structure

H-

iJH2

394 DELORES J. SPRANKLE AND ERIC C . JENSEN


structure

a: milli mass unit (difference between observed mass and elemental composition mass expressed in thousandths of mass units)

b double bond equivalents

3.5 Fluorescence Identification

When pergolide mesylate is viewed under long wavelength UV light (366 nm), the product is observed to fluoresce. Pergolide mesylate has a characteristic, pale bluish-white fluorescence.

3.6 Fluorescence Spectrum

The fluorescence spectrum of pergolide mesylate in methanol is shown in Figure 6. The fluorescence excitation and emission analyses of the compound were obtained using a Perkin-Elmer MPF-66 fluorometer.

M

Wavelength (nm)

Figure 6 . Fluorescence spectrum of pergolide mesylate



absorption pattern for compounds with an indole chromophore. The electronic transition of the indole chromophore of the pergolide mesylate molecule is observed as follows: the absorption bands at 280 and 274 nm are A to A* electronic transitions separated by lLb and 'La vibrational transitions. Additional vibrational transitions for the indole chromophore in pergolide mesylate are observed by the 0-0 'Lb transition at 291 nm and the 1B transition at 224 nm.

The maximum absorptions (h m a ) and the molar absorption coefficients (E m a ) of pergolide mesylate agree with those observed in 3-methylindole? a model compound with an indole chromophore, and support the structure of pergolide mesylate. A maximum absorption at or about 280 nm, a shoulder at or about 290 nm, and a minimum at or about 244 nrn are useful for identification of an indole structure. The UV absorption data of pergolide mesylate and 3-methylindole are shown in Table IV.

The UV absorption spectrum of pergolide mesylate is consistent with the

Table IV. Maximum Absorption and Molar Absorption Coefficients

Compounds

pergolide mesylate

3-methylindole

Maximum absorption

(nm)

29 1

280

274

224

290

282

223

Molar absorption coefficient W-lcm-1)

474 1

5667

5290

26930

4700

5640

35500


Solvents

Water

Methanol

Dehydrated ethanol

Spectral acquisition was performed on a Perkin Elmer Model Lambda 6 spectrophotometer using 1-cm quartz cells. The ultraviolet spectra of pergolide mesylate in water, methanol, and dehydrated ethanol are shown in Figure 7. UV absorption spectra of pergolide mesylate in water, methanol, and dehydrated ethanol exhibit different absorbances but similar spectral patterns.

The ultraviolet spectra of pergolide mesylate in buffers of various pH values are shown in Figure 8. Nearly consistent spectra are observed from pH 2-6. No absorption pattern is observed for pH values above 6. This is due to the insolubility of pergolide mesylate in alkaline media.

1%

hmax (nm) Elcm E max

219 155 6385

280 170 6980

28 1 170 6993

3.8 Melting Range

Pergolide mesylate melts between 258-260°C (decomposition).

3.9 Differential Thermal Analysis

The DTA thermogram for pergolide mesylate shows a large, sharp endotherm at 263OC, indicating a melt (decomposition).

3.10 Thermogravimetric Analysis

at 255°C followed by a continuous weight loss, indicating decomposition. The TGA thermogram for pergolide mesylate shows a weight loss starting

1.500

ABS

I .ooo

0 .500

0.000 240 265 290 315

WAVELENGTH (nml 340

Figure 7. Ultraviolet absorption spectra of pergolide mesylate, in water, methanol, and ethanol

2.950

ABS

1.900

0.850

.200 240 265 290 315

WAVELENGTH Inn1 340

Figure 8. Ultraviolet absorption spectra of pergolide mesylate, obtained at pH 2-8


3.1 1 Optical Rotation

are chiral in the drug. The absolute configurations at the three asymmetric centers of the molecule, C5, C8, and C10, have been confirmed by anomalous x-ray dispersion techniques.3>10 The centers at positions C5 and C10 are fixed in the starting material (dihydroelymoclavine) and are not changed during the course of the synthesis. The third chiral site occurs at position C8, where the methylthiomethyl side chain may occur in either an alpha or beta position. The stereochemistry at this position is known based on the known stereochemistry of dihydroelymoclavine, which contains a beta hydroxymethyl group in the C8 position. The synthetic chemistry involved in synthesis of the bulk drug is not expected to alter this configuration. The results of the structural determination by x-ray confirm these results.

Pergolide mesylate contains three asymmetric carbon centers, all of which

H

CH3S03-

Because the stereochemistry is fixed at all three chiral centers of the molecule, one would expect to observe optical rotation by the molecule. The optical rotation of pergolide mesylate was measured with the sodium d line (589 nm) as the light source at a concentration of 10 mg/rnl in dimethylformamide @MF) in a 100 mm cell using a Perkin-Elmer Polarimeter Model 241MC. The specific rotation measured at 2OoC has been observed to be between - 18.0 and -23.0'.

3.12 Crystal Properties

I, is shown in Figure 9. The pattern was obtained using a Nicolet powder diffractometer using copper Ka irradiation (1.5418 A) with a graphite monochrome ter .

Pergolide mesylate has been observed to demonstrate two different crystalline forms. Only one of these is observed routinely in the manufacture of the bulk drug substance and has been designated Form I. A mixture of

The X-ray powder diffraction pattern of pergolide mesylate, crystal Form

2 0 0 . 7 4

180-

160-

z 140- CI -d (0

C

c, C H

a, 120-

u 100- Ill (0 \ (0

C 3 0 U

4J BO-

60-

2 0 1 1 1

5.0 10.0

. 1

15.. 0 20.. 0 25.0 Two-Theta

30.0 35.0

Figure 9. Powder X-ray diffraction pattern of pergolide mesylate (crystal form I )


Form I and Form I1 has been observed experimentally in laboratory lots where the recrystallized solutions were cooled very rapidly. On larger scale equipment where the cooling takes place more slowly, only Form I is observed.

Form I and a mixture of Form I and I1 have been studied by x-ray diffraction and by IR spectroscopy. Figure 10 shows the IR spectrum for Form I. Figure 11 shows the IR spectrum for a mixture of Forms I and 11. An additional peak at angle 5.788 degrees is noted in a mixture of Forms I and I1 material, while this peak is absent in the pattern for the Form I material. Bands appear to be sharper in Form I material, and subtle differences are observed in the groups of peaks centered at about 775 cm-1,607 cm-l, and 544 cm-*. In the IR spectrum of Form I, these groups appear as a triplet, a doublet, and a triplet, respectively. In the IR spectrum from the mixture of Form I and 11, the groups of bands appear as a doublet, a singlet, and a doublet, respectively.

3.13 Solubility

The solubility properties of pergolide mesylate are listed in Table VI. The measurement was performed by adding an excess amount of sample to a solvent, shaking for thirty seconds at five-minute intervals for a total of thirty minutes, filtering the saturated solution, and determining the concentration of the drug in the filtered solution with a UVNIS spectrophotometer. (For dehydrated ethanol, ether, dimethylformamide, acetonitrile, dichloromethane, acetone, and chloroform, the filtered solutions were evaporated to dryness and reconstituted in methanol).

Table VI. Solubility of Pergolide Mesylate

i i

I 1

4000 3600 3iOO 2800 2400 2 b O O 1600 l i 0 0 800 4 00 WAVENUMBER

Figure 10. Infrared spectrum of pergolide mesylate (crystal form I)

I I I I I I0 3600 3200 2800 2400 2600 1600 1500 800 400 WAVENUMBER

Figure I I . Infrared spectrum of pergolide mesylute (cryml forms I & II)

PERGOLIDE MESYLATE

System

pH 2.19 Buffer

pH 4.02 Buffer

pH 6.10 Buffer

pH 7.95 Buffer

405

Concentration in Concentration in

(rndml) (mdml) organic layer aqueous layer Partition coefficient

1.42 0.23 6.14

1.93 0.02 119.6

1.322 0.00 -

0.02 0.00 -

3.14 Partition Coefficient

The partition coefficient was determined as follows. Saturated aqueous solutions of the product in various pH buffer solutions were prepared and an equal amount of chloroform was added and the solutions were shaken at 25°C for thirty minutes. The aqueous and organic layers were separated and filtered. A portion of the organic layer was evaporated to dryness and reconstituted using a mixture of equal parts of methanol and a solution of methionine (0.01 mg/ml) in 0.01 N hydrochloric acid. A portion of the aqueous layer was prepared in a mixture of equal parts of methanol and a solution of methionine (0.01 mg/d) in 0.01 N hydrochloric acid. The test solutions were assayed by subjecting them to the isocratic conditions described under high performance liquid chromatography (see Section 4.42). The chlorofodwater partition coefficients were calculated as the ratio of the concentration of pergoIide mesylate in the organic phase to the concentration of pergolide mesylate in the aqueous phase.

The results are shown in Table VII. A partition coefficient was observed in the acidic buffers. No partition coefficient was observed in alkaline buffers due to the insolubility of the pergolide mesylate in alkaline media.

3.15 Ionization Constant, pKa

The pKa of pergolide mesylate in a 66% dimethylformamide solution was measured by potentiometric titration with 2 N potassium hydroxide. The pKa of the secondary amine is 7.8.


3.16 Color Identification Test

Pergolide mesylate contains an indole ring system. Indole derivatives with free 2- or 3- positions in the pyrrole ring condense with p-dimethylaminobenzaldehyde in a sulfuric acid solution to produce a deep purplish blue color (Neubauer Rhode Reaction). Since the more reactive 3- position in pergolide mesylate is blocked, the condensation reaction of the less reactive 2-position results in a slow color development. The addition of femc chloride catalyzes this color reaction.

A 0.1 mg/ml solution of pergolide mesylate is prepared in 2.5 N sulfuric acid. The addition of 1 ml of a p-dimethylaminobenzaldehyde TS and 3 drops of ferric chloride TS to 2 ml of the pergolide mesylate solution results in the immediate formation of a dark purplish blue colored solution (the control solution remains a pale yellow solution).

4. METHODS

4.1 Identity

The identity of pergolide mesylate is determined using the specificity of infrared spectroscopy, which differentiates it from any synthetic intermediates, process related substances or degradation products. Pergolide mesylate is triturated with potassium bromide and pressed into a transparent pellet for spectroscopic analysis. The identity is confirmed by comparison to a reference standard spectrum obtained under similar conditions.


Equipment Corporation Elemental Analyzer Model 240XA. The following elemental composition was obtained using a Controlled

Table VIII. Elemental Analysis

Element % Calculated 96 Found

C 58.51 58.26 H 7.37 7.46 N 6.82 6.89 S 15.62 15.49



The identity of pergolide mesylate can be determined by ultraviolet spectroscopic assay at 280 nm. Using a 1 cm cell, the procedure is done at a concentration of 0.026 mg/mL pergolide mesylate in a mixture of equal parts of methanol and a solution of methionine (0.01 mg/ml) in 0.01 N hydrochloric acid.

4.4 Chromatography

4.41 Thin Layer A TLC method can be used for the identity and purity of raw material for

pergolide mesylate. Precoated silica gel 60 F254 TLC plates are used as the stationary phase and a tertiary solvent system consisting of chlorofomdrnethanolfethyl acetate (90: 10: 10) is used as the developing solvent. Visualization is performed by viewing the plate under long wavelength UV light (366 nm) and also by exposing the plate to iodine vapors prior to viewing under short wavelength UV light (254 nm). This developing solvent system will resolve known process related substances and degradation products from pergolide mesylate.

4.42 High Performance Liquid Conditions for quantifying pergolide mesylate have been optimized using

isocratic reversed-phase HPLC. The mobile phase consists of a 1/1 mixture of methanol and acetonimle added to an equal part of a 2 mg/ml octanesulfonic acid, sodium salt buffer (0.1% glacial acetic acid v/v). A DuPont Zorbax RX column (25 cm x 4.6 mm; 5 micron particle size) is used in conjunction with a flow rate of 1.5 mL/min. Detection is obtained with an UV detector set at 280 nm. The sample is analyzed at a concentration of approximately 0.065 mg/mL,. The method is stability-indicating as indicated by its ability to separate pergolide mesylate and known degradation products. Figure 12 shows the separation of pergolide mesylate and its primary known degradation product, the sulfoxide.

mesylate and its potential related substances (synthetic impurities and degradation products). A Supelco LC-18-DB column (25 cm x 4.6 mm; 5 micron particle size) is used in conjunction with a flow rate of 1 mL/min. Detection is obtained with a UV detector set at 280 nm. The mobile phase components are a 0.5% morpholine buffer (v/v) in water (pH 7.0 with phosphoric acid) (A) and HPLC-grade methanol/acetonitrile/tetrahydrofuran (1 : 1 : 1) (B). A linear gradient is initiated at 30% (B) and increased 2%/minute for 35 minutes to a final concentration of 100% (B); then returned to 30% (B) and re-equilibrated for 20 minutes. The sample is analyzed at a concentration of approximately 3 rngJmL.

Gradient reversed-phase HPLC methodology is used to quantify pergolide

Figure 12. HPLC chromatogram of stabiliry-indicating assay for pergolide rnesylate and degradation products . Peak identification: ( 1 ) pergolide mesylate sulfoxide, (2) pergolide mesylate


5. STABILITY - DEGRADATION

5.1 Degradation Profile

Pergolide mesylate is stable as the bulk drug substance stored at 25°C for up to 2 years and is also stable under stress conditions (100°C for 4 weeksklosed container, 3 x 106 lux-hr. light exposure, 65'C for 3 months, 40°C/75% Wclosed container for 3 months, 25"C/75% Wclosed container for 3 months, and 4@C/75% RWopen container for 6 months).I4 A trace level of (8~)-8-[(methylsulfinyl)methyl]-6-propyl-D-ergoline (the sulfoxide, I) is observed to remain relatively constant under both long term and stress storage conditions.

Pergolide mesylate was also subjected to degradation experiments involving water, acid, base, heat, and light to generate degradation profiles. In all of these studies, the compound was evaluated by specific HPLC or GC, and TLC procedures. The studies revealed the following resu1ts:ll

1. Pergolide mesylate is stable in base (a slurry in 0.1 N sodium

2. Pergolide mesylate is stable in acid (a slurry in 0.1 N hydrochloric

3. Pergolide mesylate is considered unstable in water when exposed

hydroxide at 40'C for 7 days).

acid at 40'C for 7 days).

to severe light and heat. When exposed to 3 x 106 lux-hr., pergolide mesylate undergoes degradation to yield two degradation products, the sulfoxide (I) and (8~)-8-[(methylsulfonyl)methyl]- 6-propylergoline (the sulfone, 11). The sulfone probably results from further oxidation of the sulfoxide. When exposed to 40°C for 7 days in a slurry of water, a slight increase in the sulfoxide is observed.

The following structures represent potential degradation products of pergolide mesylate:

Sulfoxide (I) Sulfone (11)


The conclusions of these studies demonstrate that, while some decomposition was observed under extreme conditions, pergolide mesylate bulk drug substance is very stable.

5.2 Stability in Dosage Form

Pergolide mesylate is marketed as 0.05,0.25, and 1 mg Permax tablets. The active ingredient in the formulated tablet for all three dosage strengths is stable for up to two years when stored in a cold-formed aluminum blister package. 12

6. DRUG METABOLISM AND PHARMACOKINETICS

Pergolide mesylate is a potent dopamine receptor agonist. Pergolide is 10 to 1,OOO times more potent than bromocriptine on a milligram per milligram basis in various in vitro and in vivo test systems. Pergolide mesylate inhibits the secretion of prolactin in humans; it causes a transient rise in serum concentrations of growth hormone and a decrease in serum concentrations of iuteinizing hormone. In Parkinson’s disease, pergolide mesylate is believed to exert its therapeutic effect by directly stimulating postsynaptic dopamine receptors in the nigrostriatal system.

Information on oral systemic bioavailability of pergolide mesylate is unavailable because of the lack of a sufficiently sensitive assay to detect the drug after the administration of single doses. However, following oral administration of 14C radiolabeled pergolide mesylate, approximately 55% of the administered radioactivity can be recovered from the urine and 5% from expired C&, suggesting that a significant fraction is absorbed. Nothing can be concluded about the extent of presystemic clearance, if any.

N-despropylpergolide, pergolide sulfoxide, and pergolide sulfone. Pergolide sulfoxide and pergolide sulfone are dopamine agonists in animals. The other detected metabolites have not been identified and it is not known whether any other metabolites are active pharmacologically.

The major route of excretion is via the kidneys. Pergolide is approximately 90% bound to plasma proteins. This extent of

protein binding may be important to consider when pergolide mesylate is co- administered with other drugs known to affect protein binding.

Permax is indicated as adjunctive treatment to levodopa/carbidopa in the management of the signs and symptoms of Parkinson’s disease.

Data on post absorption dismbution of pergolide are unavailable. At least 10 metabolites have been detected, including


Evidence to support the efficacy of pergolide mesylate as an antiparkinsonian adjunct was obtained in a multicenter study enrolling 376 patients with mild to moderate Parkinson’s disease who were intolerant to I-dopalcarbidopa as manifested by moderate to severe dyskinesia and/or on- off phenomena. On average, the sample of patients evaluated had been on I-dopdcarbidopa for 3.9 years (range, 2 days to 16.8 years). The administration of pergolide mesylate permitted a 5 to 30% reduction in daily dose of I-dopa. On average, these patients treated with pergolide mesylate maintained an equivalent or better clinical status than they exhibited at baseline.

412 DELORES 1. SPRANKLE AND ERIC C. JENSEN

7. REFERENCES

1.

2.

3.

4.

5 .

6.

7 .

8.

9.

10.

11.

12.

13.

14.

Package Insert, PermaxB, Eli Lilly and Company.

Langtry, H.D. and Clissold, S.P. (1990). Drugs 39,493.

Kornfeld, E.C., Bach, N.J. (1979). U.S. patent 4,166,182.

McCune, K.A., Maple, S.R., Cooke, G.G., and Underbrink, C.D. (Eli Lilly and Company, Lilly Research Laboratories) “Confirmation of Structure of Pergolide Mesylate” internal report, 29 July 1991.

Bailey, K., Grey, A.A. (1972). Can J. Chem. 2, 3876.

Baker, R.W., Chothia, C., Pauling, P., Weber, H.P. (1973). Mol. Pharm. 9, 23.

Derome, A.D. (1987). in Modern Techniques for Chemistry Research; Chapter 6. Pergamaon Press, New York.

Pretsch, E., Seibl, J., Simon, W., Clerc, T. (1989). in Tables of Spectral Data for Structure Determination of Organic Compounds; p. C160. Springer-Verlag, New York.

The Stadtler Handbook of Ultraviolet Spectra (1970). Stadtler Research Laboratories, Philadelphia.

Ma, L., Camerman, N., Swartzendruber, J. Jones, N., and Camerman, A. (1987). Can. J. Chem. 65, 256.

Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories) “Physiochemistry of Pergolide Mesylate”, internal report, 1990.

Jensen, E.C. er af . (Eli Lilly and Company, Lilly Research Laboratories) “Permax Stability Reports”, Japanese New Drug Application.

Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories) U.S. New Drug Application for Pergolide Mesylate, 1985.

Jensen, E.C. et al. (Eli Lilly and Company, Lilly Research Laboratories) “Pergolide Mesylate Stability Reports”, Japanese New Drug Application.


8. ACKNOWLEDGMENTS

The authors wish to express their sincere thanks to the following individuals who have provided information for portions of this chapter: K.A. McCune, S.R. Maple, G.G. Cooke, C.D. Underbrink, and G. Stephenson for the spectroscopic data ; A.G. Wich and T. Wozniak for chapter review; and B.T. Farrell for method development.

PREDNISOLONE

Syed Laik Ali

Zentrallaboratorium Deutscher Apotheker

6236 Eschborn

Germany

ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS -VOLUME 21 415

Copyright D 1992 by Academic Press, Inc All rights of reproduction reserved In any form

416 SYED LAIK ALI

Prednisolone

Syed Laik A l i

1. H i story

2. Nomen c 1 a tu re

3. Description

3.1 Name, Formula, Molecular weight

3.2 Appearance I Colour Odour I Taste

4. Svnthes is

5.

5.1 5.2

5.3

5.4

5.5

5.6

5.7

5.8

5.9

5.10

5.11 5.12

5.13

5.14

5.15

5.16

Phvs ical properties

Solubi 1 ity Loss on drying

Melting point

Specifical optical rotation Residue on ignition Selenium Light absorption

Related impurities Colour reactions

Ultraviolet spectrum Infrared spectrum Nuclear magnetic resonance spectrum

Mass spectrum

Crystal structure Po 1 ymor p h i sm Circu 1 ar dichroi sm

6. Stability and deqradation

PREDNISOLONE 417

7.

7 . 1

7.2

7.3

7.4

7.5

7.5.1

7.5.2

7.5.3

7.5.4

Methods of analysis

Colorimetric and spectrophotometr ic determination

Polarography

Radiochemistry and radioimmunoassay

NMR determination Chromatographic methods

Thin layer chromatography

High performance liquid chromatography

Gas chromatography-mass spectrometry Supercritical fluid chromatography

a. I n vitro dissolution

9. Pharmacokinetics and druq metabolism

10. Acknowledqements

11. References

418 SYED LAlK ALI

Prednisolone

1. History Hershberg and co-workers (1) observed at first that

the dihydroderivative of hydrocortison, prednisolon, possesses a 4 to 5 times stronger antirheumatic and

anti a 1 1 erg i c activity , showing s imu 1 taneous 1 y 1 esser undesired side effects.

2. Nomenclature 1 , 2-Dehydrohydrocortisone; Pregna-1 , 4-diene-3 , 20-dione , 11 , 17 21-tri - hydroxy-l1R-1

7a,21-Trihydro~y-l,4-pregnadien-3~2O-dion. The

formula is illustrated at the next page (Fig. 1).

3. Descriution

3.1 Name, Formula, Molecular weiqht

Prednisolone; C21H2805 360,45 (anhydrous)

3.2 Appearance, Colour, Odour, Taste

A white or almost white, crystalline hygroscopic, odourless powder with a bitter taste.

4. Svnthesis Prednisolone can be obtained with a chemical

dehydration of hydrocortisone with selendioxide in tertiary butanol (2) or microbiological ly through

the dehydration action of corynebacterium simplex in Al,Z-position ( 3 , 4). A methanolic solution of the substrate was mixed with a 24 hours old bacteria

PREDMSOLONE 419

PREDNI SOLON

ch20h C=O i

&-QH

0-

Fig. 1

Structural Formula

420 SYED LAIK ALI

culture (0.1 % yeast extract, buffer, pH 7.0) and

was shaken for 3 to 34 hours at 28 "C. The contents were then extracted with chloroform and prednisolon could be crystalised from aceton in very good yield.

Wettstein and Co-Workers have further found that an enzymatic introduction of a 1,Z-double bond in hydrocortisone could best be obtained with mushrooms

of Genus Didy mella type (5 , 6).

The 1,Z-double bond could also be introduced in the

molecule of hydrocortisone chemically through 2,4-dibromination of 3-ketone and then the

subsequent dehydrobromination ( 7 ) . The yield is only

10 - 15 %- The squibb company used this classical

method for the production of 9 -Fluorprednisolon (8).

5. Physical properties

5.1 Solubility (9, 10)

Prednisolone is very slightly soluble in water,

soluble in 27 parts of absolute ethanol, in 30 parts of ethanol, in 50 parts o f acetone and in 180 parts o f chloroform. It is soluble in dioxane and methanol.

5.2 Loss on dryinq (10. 111

Anhydrous prednisolone loses not more than 1.0 % of its weight when dried at 100 - 105 "C. Hydrous prednisolone loses not more than 7.0 % of its weight.

5.3 Meltina point (10) About 230 "C with decomposition.

PREDNISOLONE 42 1

5.4 Specific optical rotation (10)

In a 1 % m/v Solution in 1,4-dioxanet +96 to +120 '.

5.5 Residue on ignition (11)

Negligible, from 100 mg.

5.6 Selenium (11)

0.003 %, a 200 mg test specimen being used.

5.7 Liqht Absorption (10)

The A ( 1 % , 1 cm) in 96 % ethanol at the maximum of

240 nm i s between 400 to 430.

5.8 Related impurities (10)

Between 1 to 2 % using silica gel TLC plates

containing a fluorescent indicator and a mixture of

dichlormethane + ether + methanol + water,

77:15:8:1.2 as mobile phase and detection under UV

254 nm.

5.9 Colour reactions

The simplest reagent, used for more than 40 years in steroid analysis is concentrated sulfuric acid. They

exhibit intense spectra in the range 220 - 600 nm.

Prednisolone shows 2 hours after dissolution in concentrated sulphuric acid an absorption maximum at

470 nm with a specific extinction o f 89.

Prednisolone gives after dissolving in conc.

sulphuric acid ( 1 mg/ml) instantaneously a red colour which after dilution with water changes to

violet-brown ( I ) . Prednisolone (1 mg in 5 ml

nitromethane or nitrobenzene) reacts with aluminium

422 SYED LAIK 4LI

chloride ( 4 g anhydrous aluminium chloride in 10 ml nitromethane or nitrobenzene, 2 ml reagent) to give a weak orange colouration (12).

5.10 Ultraviolet spectrum Prednisolone shows absorption maximum in methanol at 242 nm. The E 1 %, 1 cm in this solvent is reported

to be 416 and the molecular extinction coefficient 15000 (13). The UV spectrum is shown in Fig. 2.

5.11 Infrared spectrum The infrared spectrum i s given in Fig. 3. The

spectrum was obtained with a Perkin-Elmer 1420 Ratio Recording Infrared Spectrophotometer from a KBr pel let.

5.12 Nuclear maanetic resonance spectrum The nuclear magnetic resonance spectrum o f

prednisolone was taken with a Varian 60 MHZ

spectrometer i n deuterated dimethyl sulfoxide. The spectrum is reproduced in Fig. 4.

5.13 Mass spectrum

The mass spectrum was recorded with a Varian Mat 311

mass spectrometer using direct inlet in EI-mode at 80 ev and source temperature of 300°C. The spectrum is illustrated in Fig. 5.

PREDNISOLONE 423

F i g . 2 (13)

UV Spect rum o f P r e d n i s o l o n e i n Methanol

Fig . 3 IR Spectrum o f Prednisolone, KBr Pel le t Perkin-Elmer 1420 Spectrophotometer

k

a, c,

a, E

0

k

c, 0

a,

v)

N I

z

0

\o

C

0

*rl

k

0

w W

C 0

4

0

u)

-4

C 0

W

k

L

+ 0 E

2

k

c, 0

a,

v)

mtx

-rl z

LL

Z

n

.

-tn

.

425

426 SYED LAlK ALI

F i g . 5 Mass Spectrum o f P r e d n i s o l o n e

PREDNISOLONE 427

5.14 Crystal structure

Inclusion complexation o f prednisolone with three Cyclodextrins, a p and -phomologues, in aqueous solution and in solid phase was examined using UV

absorption, CD, C 13 NMR, C 13-CP/MAS-NMRI X-ray

d iff ractometry and thermal analysis. The spectroscopic data suggested the different inclusion mode of prednisolone within the three cyclodextrin cavities. X-ray diffraction patterns of the

complexes differed significantly from those of the physical mixtures (14).

5.15 Polvmorphism

Prednisolone shows the phenomenon of polymorphism

(15, 16). The results o f investigation on po 1 ymorph i sm and Pseudopo 1 ymorph i sm (formation of

solvates) of about 100 steroids including prednisolone is described. Prednisolone forms

solvates with water and chloroform. These

polymorphic forms have melting points between 218 - 234 "C and 210 - 225 "C. The hydrate always

represents the most stable form. The analytical methods of thermomicroscopy, differential scanning

calorimetry (DSC) and infrared spectrophotometry were applied for investigation (17, 18, 19, 20, 21).

The therapeutic activity varies for the different polymorphs o f the same chemical substance. This behaviour is attributed to various factors but especially to the differences in the crystalline structure (22, 23, 24).

428 SYED LAIK ALI

Veiga and co-workers have isolated three forms I ,

11, 111, of prednisolone which were identified by IR

spectroscopy and characterized by scanning electron

microscopy, X-ray powder diffraction and hot stage

microscopy (25) . The study by optical microscopy as well as scanning electron microscopy revealed that

the crystals have a very different appearance. Form I has small, tridimensional crystals with irregular shape and granular surface. Crystals of form I1 have

a lamellar structure, well defined edges with

smaller and very differently shaped crystals grown on their surfaces. Crystals of form I11 are reported

to be similar in size to those of form 11; they have regular shape, but are formed by layers, so their

edges are not well defined and are rather opaque (25). Diffractometer patterns are shown in fig. 6, interplanar spacings are presented in table 1 and unit cell parameters in table 2. The study was

carried out by a Phillips PW 1010 powder diffactometer using the incident radiation Cu K (Y

(X=1.5418A0) filterd with Nickel. The unit-cell

parameters and the interplanar spacings were

determined using a counter diffractometer and powdered samples containing si 1 icon powder as

internal standard.

Unit cell parameters have been assigned to forms I

and 111, automatic indexation tests have failed to

yield a solution for from I 1 (25) . Table 3 shows the results of hot stage study. During the heating process forms I and I11 remained unchanged until the melting point was reached. Form I1 showed in the

PREDNISOIBNE 429

x L)

a c Y 3 c H

FORM I

FORM I t

FORM 111

Fig. 6 (25) X-ray D i f f r a c t i o n P a t t e r n s o f P redn i so lone

430 SYED LAIK ALI

- 10.9743 85840 6.3657 5.7120 5 m 3 5.4335 50924 5.0349 4.5484 4.1874 41)188 3.9139 35729 3.5173 5.4635 3.3985 52407 31)868 3M)55 2 m 5 2-7945 2.7526 2.7121 2.5686 2.4693 23599 233018 22739 22176 2-1 392 2.1 157 2M52 2 f i 5 I 2.0128 I .9%0 I .979s I8827

16.9SO3 10.9743 6.8044 6.1672 5.9806 56577 5.5546 50349 49239 4&670 4.5600 45709 4.3080 4 m 7 0 3.9224 38471 3.7124 36897 3.4635 3.4113 33483 331 I 6 3246s 3m73 2 3 7 I2 2 m 2.8290 2.7363 2.6685 26766 261% 25129 2.4662 2.4275 2.-*2 22s22 2.~65 1 2.0107 I .%33 1.9240 1 S226 1.7730

- 10.9743 8.3S40 6:4116 5.7304 516043 5.4335 5.0349 4.5484 43392 4.1726 4.0188 3.9054 3.7825 3.7048 3.587 I 3.524 I 3.4615 3.3965 32465 3.1951 3.1079 2.9956 2.9144 250m 2.5810 2.4959 25x29 23103 zm22 I S772

PREDNISOLONE 43 I

TABLE I1 (25)

Unit cell parameters.

Momclinic Form I

Crystal S>1ICm &lonoclinic Form II

1 ID48 (A) 16.904 (A)

13.3% 13.167

6.3 I5 7.909

9(r 90-

9 1-76 s3m

90 90

TABLE 111 (25)

ram II Form I Form Ill

Partial melting and recrystallization ienip. ("1 115-130

Melting point (") 2 10224 205-2 18 2 10-220

432 SYED LAlK ALI

range of 115 - 130 "C a partial, intracrystalline melting with further recrystallization which then

remains unchanged up to their melting point. Veiga

and coworkers deduced from these facts that I and

I 1 1 are anhydrous polymorphic forms of prednisolone while the form I 1 is a 1.5 hydrate and probably a twin (26).

5.16 Circular dichroism Circular dichroism (CD) is much more useful than any other spectroscopic technique in control 1 ing the sterospecificity of the reactions in the total

synthesis of steroids. Prednisolone can be determined simultaneously along hydrocortisone.

Hydrocortisone can be measured selectively at 326 nm where prednisolone does not show dichroism, while at

314 nm prednisolone can be measured on the basis of

its selective dichroism (27, 28).

6. Stabilitv and deqradation

One of the identified decomposition products formed during the anaerobic decomposition of prednisolone

at pH 8 is 17-deoxyprednisolone. This decomposition product differs from prednisolone only in the side chain. The hydroxyl group at C 17 disappears thus giving 17-deoxyprednisolone (29). Gutman and Meister

(30) found that with the dihydroxyacetone side chain of prednisolone two reactions predominate which

yield the 17-ketosteroid and the hydroxy acid ( 3 0 ) . The isolation through TLC and HPLC methods and structural e 1 uci dat ion through po 1 arograph i c and

PREDNISOLONE 433

masspectrometric techniques is described. A decomposition mechanism of prednisolone leading to 17-deoxyprednisolone is postulated.

Studies on the stability of corticosteroids and

degradation patterns in aqueous solution are

described ( 3 1 ) . Formation and degradation kinetics of 21-dehydrocor t icostero ids, key intermediates in the oxidative decomposition of 21-hydroxycorticoids (32) , kinetics and mechanism of the acid-catalyzed degradation of corticosteroids are reported ( 3 3 ) .

Another decomposition product formed during the

anerobic decomposition of prednisolone i s

17-deoxy-21-dehydroprednisolone. This can only be

detected when the decomposition of prednisolone

takes place at about pH 6 or lower pH-values. This product has been isotaled through chromatographic

techniques and structure elucidated through mass spectrometry ( 3 4 ) . This product differs only from

prednisolone in the side chain where the hydroxyl

group at C 17 has disappeared and at C 21 the hydroxyl group has been changed to an aldehyde group

(341. Another decomposition product, the 17-Ketosteroid where at C 17 the hydroxyl group has been converted to a keto group, is described. The isolation and the structural elucidation of 17-Ketosteroid is reported as well as decomposition mechanisms of prednisolone leading to the 17-ketosteroid are discussed ( 3 5 ) . The major

decomposition product of prednisolone phosphate formed under anerobic decomposition conditions in

434 SYED LAIK ALI

aqueous solution at pH 8.3 i s identified as 17 a- h ydroxy - 1 7a - hydroxyme t hy 1 - 1 7 - ke to- D- homosteroi d phosphate. Isolation, structural elucidation and a

mechanism leading to this compound are postulated

(36).

In another publication the stability and

interactions between prednisolone and urea in ointments and the influence of one component on the other are discussed (37). Factors influencing

stabi 1 ity, the rate of disappearance of prednisolone from aqueous solution have been investigated (38).

The rate exhibited a marked dependancy on buffer

concentration. Prednisolone is susceptible to degradative reactions which involve the

17-di hydroxyacetone side-chain. Transformation and elimination of the side-chain have been shown to

occur both in the presence and absence of oxygen.

Autoxidation, however, appears to be the mode of destruction which is most likely to be responsible

for stability problems in drug products. The involvement of trace metals in catalyzing the autoxidation i s an obvious possiblity. Trace-metal impurities which were present in the buffer reagents were catalytically involved in the degradation. Rate constants were determined over a wide range of pH in borate and phosphate buffers and in the presence and

absence o f ethylenediamine tetraacetic acid. The rate of the apparent metal-catalysed reaction was pH

dependant above pH 7 and below pH 5 and exhibited a first-order dependency on the hydroxide-ion in the

PREDNISOLONE 435

7.

7 . 1

intermediate range. In the presence of EDTA, the rate of reaction was strongly dependant on

OH-concentration above pH 8 but exhibited only

slight dependency at lower pH values. Addition of EDTA provided a method to isolate and quantitate the

rate o f the apparent metal-catalyzed reaction (38).

The stability of prednisolone in an aqueous-organic solvent system (40 % lI2-propandiol + 38 %

tetraglycol + 30 % water) under accelerated conditions has been studied. It was possible to demonstrate six different decomposition products. The four major products were identified by TLC. The

accelerated stability tests were evaluated using a stability-indicating assay procedure. Although

decomposition of prednisolone in solution is complex, stability prediction via Arrhenius plotting

is possible. The degradation of prednisolone in this

system is a first order kinetic reaction. The

temperature dependence of degradation process in this organic-aqueous system is illustrated in fig. 7

and the Arrhenius plot of this diagramm is shown in fig. 8 (39).

Methods of analvsis

Colorimetric and spectrophotometric determination

Blue tetrazolium (BT) and Phenylhydrazine H2SO4 (PH)

reagents react with the intact side chain at C 1 7 o f

prednisolone while isonicotinic acid hydrazide (INH) and UV-absorption depend upon conjugation in Ring A

at the other end of the prednisolone molecule. Since

e w m

I

100-

90

eo

70.

7.

f\sr Y -4

F ig . 7 ( 3 9 ) Temperature-Dependence of Degradation o f Prednisolone

a, K

0

4 0

v)

-4

5

73

a, k

k

0

Lc

a, E

E

0

k

m

0

n

2 +

J 0

431

438 SYED LAIK ALI

the reactions in these four methods occur with different portions of the molecule, they can be used to detect and distinguish between decomposition products (40). The PH method is described by Silber and Porter (41) and the I N H method is given by Umberger (42). The Porter-Silber reaction for the colorimetric determination of corticosteroids is

applicable only to steroids with a side chain at position 17 or their derivatives that are readily

hydrolysed in the strongly acid medium (43) . Prior

to the determination by BT, PH, I N H and UV method a

column chromatographic clean-up and removal of decomposition products and interfering substances

has been performed (44). The method of tetrazolium assay of prednisolone consists of reaction of the

substance (34 - 36 ug/ml) with a 0.5 % solution of triphenyl-tetrazol ium chlorid (TTC) solution in aldehyde-free ethanol (96 %) and measuring the

extinction at 485 nm (45) . A colorimetric method of determination of prednisolone in powder and tablet

forms using ammonium molybdate has been described. Prednisolone gives a blue colour with an absorption

maximum at 655 nm. The range of sensitivity for prednisolone is given between 5 - 30 ug (46 ) . The prednisolone contents in low concentrated ointments

and creams were measured with blue tetrazolium reaction after several steps of extraction and

clean-up (44 ) . A large number of prednisolone dosage forms have been examined using tetrazolium blue

method ( 4 8 ) . A review of methods of practical

importance for the determination of steroids in

PREDNISOLONE 439

pharmaceutical formulations i s given (49) . The most

generally used method for the assay o f formulations containing unsaturated 3-Ketosteroids is their

condensation with isonicotinoyl hydrazide ( I N H ) and

measurement of the formed hydrazones in strongly

acid medium at 410 nm (E 17000). For the

determination of prednisolone in dosage forms on the basis of their side chain at C-17 tetrazolium

methods based on the reducing properties of the side chain are stability indicating. Both the triphenyl tetrazol ium chloride and tetrazolium blue methods

are fairly sensitive having molar absorptivities of

16200 and 24000 respectively (49, 50). Condensation of the glyoxal, obtained by cupric acetate oxidation of prednisolone, with aqueous phenylhydrazine

reagent affords a near UV chromophore at 366 nm with

a molar extinction coefficient of 17000 (51) . The TTC and BT methods have a relative standard deviation o f less than 1 % in the assay o f bulk

corticosteroids and not more than 2 % for dosage

forms (52) . The TTC method has been used for the determindtion of prednisolone in tablets (53, 54) , ointments (55, 56) and for the kinetic investigation of its decomposition in alkaline media (57). The sodium borohydride method has been appl ied by Gorog

for the analysis of prednisolone in ointments (58).

An alcoholic solution of ointment containing 10 - 15

mg prednisolone is treated with 1 N Sodium hydroxide followed by the addition of 100 mg sodium

440 SYED LAIK ALI

borohydride. The mixture is refluxed for 1 h, cooled

and treated with 1 N HC1. The absorption of the resulting solution is measured at 243 nm against a

corresponding blind solution.

7.2 Polaroaraphv The electoanal yt ical behaviour of predn i solone along with other corticosteroids has been studied in supporting electrolytes. Dependence of the peak potentials on the structure of the steroids at

concentrations of 10-4 M in 0.03 M tetramethyl ammonium hydroxide (TMAH) in methanol, in Britton-Robinson buffer pH 10 (50 % V/V in methanol) and in 0.02 M TMAH in dimethyl formamide (87 % V/V) has been studied. In prednisolone the reduction of C-3 and C-20 keto groups takes place. Both reduction steps can be used for analytical purposes. The

differential pulse peak height i s linear with the concentration down to 10-6 M. The wave pattern of the differential pulse polarography in methanol shows for prednisolon reduction at -1.60 V and an

additional peak at -1.76 V versus SCE. The reduction in DMF is similar to that in methanol. The peak potential in a methanol-buffer mixture at pH 10 for prednisolone is given as -1.50 V . Prednisolone has

also been analysed by constant potential coulometry (59). The differential pulse polarographic determination of prednisolone in single component

tablets is described. After extraction o f

prednisolone with methanol from tablets it was

PREDNISOLONE 44 1

analysed in a supporting electrolyte of 0.03 M TMAH in methanol with a dropping mercury electrode, a

Ag/AgCl reference electrode and a platinum wire

prednisolone to be determined was in the range of 10-3 - 10-5 M (60) . Prednisolone gives waves in d. C. and normal pulse polarography and peaks in differential pulse polarography which correspond to

a one-electron uptake. Mechanism of polarographic

electroreduction of prednisolone is described. The

effect of pH using different buffers (acetate,

phosphate, borate and ammonia buffers) on half-wave

potential and limiting current for prednisolone is

given. The half-wave potential of the prednisolone wave remains pH-independent up to pH 10.3 but is

shifted towards more negative potentials at higher

pH-values. Dependence of the peak-heights in pulse polarography on pH for the first wave for

prednisolone closely resembles the pH dependence of

these waves obtained by d-c-polarography. In linear sweep vol tammetric curves the dependence of the values of peak potentials of prednisolone on the

logarithm of the scan rate was linear over a wide pH range (61).

auxiliary electrode. The concentration of

A differential pulse polarographic method for the determination of prednisolone in tablets is

described. The method is more sensitive than dc polarography and the measurement of diffusion

current is greatly simp1 if ied. Sorenson phosphate buffer pH 5.6 was used as the supporting

442 SYED LAlK ALI

electrolyte. The peak potential was found to be -1.19 V versus SCE for prednisolone. The position of the peak was independent of concentration and peak heights were linear over the 5 - 20 ug/ml range

(62). Predn i so 1 one and predn i sone can be determined

in tablets using ethanol as an organic solvent, acetate buffer pH 5.6 as a supporting electrolyte and polarographing between -1 and -1.5 V ( 6 3 ) .

7.3 Radiochemistrv and radioimmunoassav Strict limits on allowable residual quantities of ethylene oxide and its major reaction products have been imposed due to their possible mutagenic and cancerogenic properties. Co-60 irradiation is a

major goal o f a sterilization alternative programme. The cobalt 60 radiolytic degradation products have

been identified for many corticoids including prednisolone. Two major types o f degradation processes have been identified: loss of the

corticoid side chain on the D ring to produce the

C-17 ketone and conversion of C 11 alcohol, if present, to C 11 ketone. Minor degradation products

derived drom the other changes affecting the side

chain are also identified. These compounds are

frequently associated in corticoids as process

impurities or degradation compounds. No new radiolytic compounds unique to Co-60 irradiation

have been found. Through cobalt 60 radiolytic

degradation pathway of prednisolone prednisone and llB-hydroxy-l,4-androstadiene 3,17-dione are formed.

PREDNISOLONE 443

Conditions for isolation of cobalt 60 radiolytic degradation products as well as paths and schemes of degradation are described. For prednisolone

methylchloride was used as enrichment solvent, a HPLC Brownlee RP 18 column C 18 bonded phase and a mobile phase o f methanol-water, 55:45 were used. The rate of radiolytic degradation in prednisolone is

given as 0.7 %/Mrad (64).

Radioimmunoassay has been used for the estimation of

prednisolone after prednisone intake. Taking

advantage of the similarity of structure of prednisone to cortisone and of prednisolone to

cortisol , urinary prednisolone was estimated by

radioimmunoassay for cortisol and found to be

linearly correlated with the dose o f prednisone

administered. Free prednisolone in urine was estimated by the "clinical Assay" R1A kit for

cortisol, which gave a very high cross-reactivity with prednisolone. In estimating free prednisolone

by RlA, cortisol cross reactivity was found to be

91,6 % in the range of 2-100 ug prednisolone. When RIA kits with differing specificity of the antibody

to cortisol were used, the cross-reactivity also

differed as expected. The less specific the

antibody, the higher were the results obtained with the same blood sample (65).

The predn i solone rad ioimmunoassay developed by

Colburn and Buller (66) used an antiserum raised

against prednisolone-21-hemisuccinate conjugated to

444 SYED LAIK ALI

bovine serum albumin (BSA) and showed roughly 10 %

cross-reactivity with endogenous cortisol. The

plasma samples were treated at 70 "C for 30 min prior to radioimmunoassay so as to eliminate interference by endogenous corticosteroid binding

globulin with the binding of prednisolone to the

antiserum ( 6 7 ) .

The smallest amount of prednisolone which can be

assayed by radioimmunoassay with confidence was 0.5

ng giving a usable range for the assay of 5 - 400

ng/ml in the plasma. The within-batch precision of

the assay was between 3 - 5 %. The cross-reactivity of the antiserum with various metabolites of

prednisolone and some endogenous steroids was between 6.4 and less than 1 %. The crossreaction

with cortisol and corticosterone was 6.4 % and 3.8 %

respectively (68). A method for measuring prednisolone using an antiserum raised against dexamethasone-21,-hemisuccinat-bovine serum albumin

conjugate in sheep is reported, which reacts poorly

with endogenous steroids. The results o f

radioimmunoassay were compared with those obtained by using competitive protein binding method (69). This method is based on the high affinity of predn i solone for p 1 asma cort i costeroi d binding

globulin (69, 70). The data show that binding of prednisolone to dexamethasone antiserum is

sufficient for this to be used as the basis for a

prednisolone radioimmunoassay as we1 1 as perfectly adequate for the measurement o f prednisolone plasma

PREDNISOLONE 445

levels in routine clinical investigations. Nevertheless, the technique is less sensitive than

that which uses an antiserum raised against a predn i solone-21- hemi succi nat- bov i ne serum a1 bumine

conjugate. The dexamethasone antiserum used did not cross-react significantly with any of the several cortisol metabolites tested (68). Comparison of the

results obtained by the competitive protein binding method and the radioimmunoassay method showed good agreement, although over the whole range of concentrations the latter technique always gave

slightly lower values. This discrepancy was

attributed either to the presence of cortisol in the

pooled plasma used for preparing the standard curve or to some metabolite of prednisolone which cross-reacts in protein binding method. The method permits measurement of prednisolone in the presence

of prednisone, since the latter does not cross-react

with the antiserum (68).

7.4 NMR determination

A highly selective NMR method for the determ nation of prednisolone in tablets is described. After

extraction of prednisolone with 95 % ethano , the solvent is evaporated and the residue is dissolved

in diemthyl sulphoxide containing fumaric acid as internal standard. The NMR spectrum of the resulting solution is recorded with a 60 MHZ instrument. The

prednisolone content is calculated from the integral

of the signal of the C 1 proton at 7.9 ppm w i t h the aid of the integral of the signal of internal standard at 6.9 ppm (71).

446 SYED LAIK ALI

7.5 Chromatographic methods

7.5.1 Thin layer Chromatography (TLC) All important parameters of TLC separation are given in table 4. Prednisolone has been separated from its oxidation product through TLC on fluorescent silica gel plates. In some cases the quantity of the oxidation product could amount upto 2 %. By applying

100 ug of corticosteroid, the oxidation products can be detected at 0.4 % level and their presence is easily demonstrable at the 1 % level (72, 73) . Knopp

(74) applied three different mobile phases and HPTLC

plates for the detection of decomposition products

of prednisolone through UV 254 nm and INH reagent. 5

- 50 ul o f 0.1 % ethanolic solution of prednisolone were applied on TLC plates (74) . Prednisolone could

also be determined through densitometry at 250 nm

(74 ) . A simple TLC screening procedure was developed

for the detection of prednisolone as adulterant in Chinese herbal preparations. Depending on its

complexity the sample may be directly extracted into aqueous ethanol, or stepwise fractionated into acidic, basic and neutral components. Extracts were analysed on silica gel TLC plates with fluorescent

indicator with the aid of four solvent systems and detected under short and long wavelength UV light and iodine vapour (75) . Prednisolone and

chloramphenicol in oily 1 iquids could be separated

through TLC (76) . Quantitative HPTLC coupled with densitometry was developed for the determination of

PREDNISOLONE 441

prednisolone in human plasma, saliva and urine. For HPTLC analysis the residues after extraction were

reconstituted in 10 ul acetone and 5 ul were applied

on TLC plates and developed with two solvent systems. The plates were then sprayed with a mixture

o f sulfuric acid-ethanol and heated at 60°C for 45

min. The fluorescent intensity of the steroid bands were determined by densitometry at a wavelength of

598 nm, with excitation at 254 nm. The method allows

simultaneous measurement of endogenous cort is01 in plasma following administration of prednisolone. The calibration curve was linear over a wide range of concentration in all biological fluids (0.025 - 4 ug/ml). The limit of detection was 10 ng/ml in plasma and saliva and 25 ng/ml in urine. The method was reproducible with an inter- and intra-assay

coefficient of variation of < 10 %. No interference from endogenous steroids was found (77). TLC

behaviour of prednisolone in creams is described. As

the content of corticosteroids like prednisolone in creams is generally low, the detection of these

active components can be a problem. Detection was not sufficiently sensitive for creams containing about 0.1 % or less of a particular corticosteroid

(78). In B.P. 88 and european pharmacopeia a TLC

method for identification of prednisolone and for the examination of related substances is given (79)

K. Macek has tabulated the chromatographic data with number of mobile phases for the identification and

separation of prednisolone from innumerable

corticosteroids and related substances (80).

448 SYED LAIK ALI

Levarato has developed a series of quantitative

methods for the determination of prednisolone along

other corticoids after extraction from preparations and separation by TLC on silica gel plates and

determination by spectrophotometric (absorption at 240 nm) or colorimetric methods (INH-hydrazone formation, tetrazol ium reaction) (81). A simple, fast and quantitative TLC-method for the determination of prednisolone in tablets is described. The method is stability-indicating with respect to accuracy, specificity, sensitivity and

precision. The coefficient of variation was between

1.26 and 1.96 % and the sensitivity was about 25 ng.

The chromatographic separation was performed on a silicagel plate using two step development of the

plate (82 ) . A simple TLC method of separation of

prednisolone from other corticosteroids is reported

(83). Prednisolone and the dephosphorylated

D-homosteroid can be separated on silanised silica gel TLC plates (36).

Fluorimetry is a useful method for the direct

determination of steroids on chromatograph i c p 1 ate. The method is based on the measurement of the fluorescence produced on irradiation of the chromatogram with UV light. The quenching of the

fluorescence of various activated layers by unsaturated ketosteroids 1 ike prednisolone can also be used for quantitative measurements. The basis of the quantitative evaluation is the difference between the fluorescence of the background and that

PREDNISOLONE 449

of the dark spots. A direct densitometric evaluation

o f prednisolone has been performed (84) * The method for detecting prednisolone on TLC plates i s assigned

according to the functional group involved in the reaction. The name and composition of the reagent,

the colour o f fluorescence observable on spraying

and the sensitivity is given. Detection of predn i sol one with sulphur ic ac i d , orthophosphor ic acid and antimony (111) chloride reagents is described (85 ) . The limit of detection of

prednisolone on silica gel fluorescent TLC plates

with a mobile phase acetone-cyclohexane-ethyl

acetate, 1:l:l in U V light 254 nm is given as 0.3 ug

(85). Compernolle and coworkers (86) reported on the purity checking of commercial prednisolone samples

by TLC, where several impurities such as

hydrocortisone, prednisone etc. could be detected.

Excellent separations could be achieved by TLC with

solvent mixtures such as dichlormethane-dioxane- water (2: 1 : l ) or dichlormethane-diethylether-

methanol-water (77:15:8:1). The Rf-values for prednisolone 0.42 and for prednisone 0.62 were found with this system (87 ) .

Table 4 Stationary Phase Mobile Phase Detect ion References

hRf -values

Silica gel 60 with methylene chloride- UV 254 nm

fluorescence indicator dioxane-water 100:50:50

prednisolone: 34

oxidation product: 77

VI P same as above 3

same as above

120: 30: 50

prednisolone: 13

oxidation product: 55

UV 254 nm

HPTLC plates, Merck Methylene chloride-ether- UV 254 nm pre washed with methanol- methanol-water INH reagent chloroform (80+20); 77:15:8:1.2

15 cm prednisolone: 29

decomposition products: 0-71

(73)

(74)

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Table 4

Mobile Phase Detection

hRf-values

References

same as above

same as above

P VI N

Silica gel UV 254

ethyl acetate-toluene-formic same as above (75) acid-dimethyl formamide-water

75:75:2:4:4; prednisolone: 12

acid-dimethyl formamide

75:75:2:4; prednisolone: 15

ethyl acetate- to1 uene-formi c same as above (75)

Ether; prednisolone: 11 UW 254 nm (76)

Table 4 Stationary Phase Mobile Phase Detection References

hRf -values

HPTLC plates without Chlorof orm-ethano 1 -water H2SOq-ethano1, 6.5:3.5 fluorescent indicator BDH 90:10:2; prednisolone: 25 spray reagent; densi-

10 x 20 cm tometric determina-

tion at 598 nm with

excitation at 254 nm e ?n W

same as above Ch lorof orm-ethano 1 -water

45:5:7.5; prednisolone: 29

Silica gel 60 F HPTLC

plates 10 x 10 cm Merck, acetic acid, 20:5:2

distance o f 7 cm under prednisolone: 72

saturated conditions

n-bu tan0 1 -water-g 1 ac i a 1

same as above

UV 254 nm

(77)

(78)

Table 4

Stationary Phase Mobile Phase Detect ion References

hRf-values

Silica gel 60 F 254 TLC

plates

same as above e VI P

Silica gel 60,

20 x 20 cm Merck without fluorescent

indicator

methyl ene c h 1 or i de-ether- UV 254 nm, spraying (79)

methanol-water; 77:15:8:1.2 with ethanolic sulfuric acid (20 % ) ,

heat at 120°C for

ether-toluene-butano1 10 min and UV 365 nm (79)

saturated with water, 80:15:5

a) to1 uene-ether-acetone 250 nm, densitometry; (82)

b) methylene chloride-ethyl

18:19:3 development first with

mobile phase a, drying

acetate-ether-formic acid the plate with air and again 10:10:10:0.3 development with mobile

prednisolone: 25 phase b

Table 4

Stationary Phase Mobile Phase Detect ion References hRf -values

Silica gel 60 F 254 met hy 1 ene c h 1 or i deace tone UV 254 nm, spraying (83) plates Merck 75:25; prednisolone: 20 with 20 % H2S04 in ethanol

and drying at 120°C 10 min

reddish brown spot

% Silanised silica plates methanol-water - 0.4 M sodium U V 254 nm; blue 60 F 254 Merck, 0.25 nm phosphate solution 50:50:1 colouration with

prednisolone: 54 tetrazolium blue

VI

456 W E D LAlK ALI

7.5.2 Hiah performance liquid chromatoaraphy A sensitive, specific, HPLC procedure for the determination of prednisolone in plasma is described. The organic solvent extract from plasma

is chromatographed on a silica gel column using a mobile phase of 0.2 % glacial acetic acid, 6 %

ethanol and 30 % methylene chloride in n-Hexane at 254 nm. Quantitation of plasma samples containing 25

ng/ml prednisolone is reported. Metabolites and endogenous hydrocortisone do not interfere with prednisolone (88). For the simultaneous analysis of prednisone and prednisolone in plasma the internal

standard was dexamethasone and the mobile phase consisted of glacial acetic acid-ethanol-

methylene ch 1 or i de-n-hexane (89, 90). A brief discussion of the merits and limitations of HPLC relative to other

chromatographic methods and special problems in the application to steroids are discussed (91) . A

sensitive, specific and reproducible HPLC assay for

the simultaneous determination of prednisone,

prednisolone and cortisol in biological fluids was

developed with dexamethasone as the internal standard. Samples were extracted with methylene

chloride, washed with sodium hydroxide and then water and chromatographed on a microparticulate

silica gel column with a mobile phase

methanol-methylene chloride, 3:97 at a flow rate of 2 ml/min and detected at 254 nm. Sensitivity was greater than 15 ng for all four steroids. A constant

ratio of peak height of a steroid at the wavelengths

(0.2 : 3.5 : 30 : 66.3)

PREDNISOLONE 451

280 and 254 nm served as an added measure of specificity o f the assay. The 280:254 nm ratio for prednisolone was 0.09 (92) . While monitoring at dual

wavelengths improves assay specificity, the 254 nm wavelength yields nearly optimum absorbance (92).

Determination of prednisolone in plasma by HPLC

using a water saturated mobile phase of equal

volumes o f ethanol and methylene chloride and 1 %

glacial acetic acid was used on a stainless steel 30

cm x 3.9 cm porasil column (10um porous silica) with

a flow rate of 2 ml/min and detection at 254 nm. A 1 inear relationship exists over the concentration

range 25 - 150 ng/ml. The effects of sample storage on reproducibility of results were examined. The samples were stored at -20°C for upto four weeks.

The mean recovery was between 101.6 to 103.9 % for

prednisolone concentrations between 20 and 100 ng/ml respectively (93). A normal phase micro-bore column packed with 10 um microsphere silica (50 cm x 1 mm

i .D.), mobile phase o f water-saturated butyl

chloride- buty 1 acetic

acid (450:450:105:53:44) , and the detection at 254

nm were used for the separation of prednisolone from other corticosteroids (94). HPLC retention values of

prednisolone along various other corticoids on a Bondapak C18/corasil column using methanol or acetonitrile of different compositions in water at a

flow-rate of 1.5 ml/min and detection at 254 nm were evaluated. The values for prednisolone lie between 1.06 t o 3.35 i n relation to acetone with a value

1.00 (95) . An improved HPLC separation of

ch lor ide-THF-met hano 1 -g laci a1

458 SYED LAIK ALI

decomposition products of prednisolone by adding sodium sulphite to the mobile phase is reported. To

a methanol-water, 1:1, solution 1 % of 0.4 M sodium phosphate solution pH 7.0 was added. Sodium sulphite was optionally added t o give a concentration of 0.1

% w/w. The flow-rate was 1.0 ml/min and the

detection was performed at 240 nm. A C18 M Bondapak co 1 umn was used. With this system

21-dehydro-prednisolone, a decomposition product of prednisolone is separated and detected (96).

Prednisolone was separated along other

corticosteroids in topical pharmaceuticals on a

reversed phase microparticulate HPLC column Zorbax

c8, Dupont (25 cm x 4.6 mm) with a mobile phase of THF-methanol-water, 25:12.5:62.5 with a flow-rate of

1 ml/min and at a detection wavelength of 254 nm. Prednisolone had a relative retention value of 0.94 with respect to hydrocortisone (value 1 .OO). Various commercial topical formulations of these corticosteroids were prepared by both simple

dilution and by extraction for analysis by the

proposed HPLC procedure, by the blue tetrazolium

procedure and by the isoniazid procedure and/or by phenylhydrazine method (97). Prednisolone and chlorhexidine have been separated on a Nucelosil C18

10 um column, 30 cm x 4 mm along with a Vydac 201 RP guard column 5 cm x 4 mm with a mobile phase

methanol-water, 120:80 containing the PIC reagent

87, Waters with a flow-rate of about 1.35 ml/min at 50 "C and detected at 240 nm. Prednisolone and chlorhexidine dichloride were thus well separated in hydrophilic emulsions and lipophilic ointments (98).

PREDNISOLONE 459

A normal phase HPLC method for the determination of

prednisolone in tablets and bulk drugs was studied. The HPLC system consisted of the mobile phase methanol-water washed ethylene dichloride-acetic

acid ( 6 + 94 + O . l ) , 25 cm x 4.6 mm column packed with 5-6 um porous spherical particles (Du Pont), flow-rate 1.5 ml/min, detection at 254 nm and

injection volume 10 - 15 ul. The bulk drugs and tablets containing prednisolone were extracted with

a mixture of methanol and methylene chlorid (4:96)

and an internal standard of 1 mg/ml solution of

f luoxymesteron was used. The coefficient of

variation of the analysis results ranged from 1.34 %

for bulk drugs to 2.14 % for tablets (99) .

Extraction-monitoring and rapid flow fractionation

for determination of serum corticosteroids is

described. The HPLC system applied for the analysis

of serum corticosteroids including prednisolone

consisted of a mobile phase 0.1 % water, 4 %

methanol, 30 % methylene chloride in n-hexane, Lichrosorb Si 60.5 um, 25 cm x 4 mm column

pretreated with 5 % H2SO4, flow-rate 2 ml/min and

240 nm UV detection (100). A HPLC analysis of prednisolone and endogenous cortisol is described in plasma samples of kidney transplantation patients using dexamethasone as an internal standard. A glass column (15 cm x 3.1 rnm filled with Separon Six, 5

urn, Laboratorni Pristroe, Prag, CSSR), a mobile

phase methylene chloride-methanol, 9 7 : 3 , flow-rate 1

ml/min and detection at 254 nm were the HPLC parameters. The calibration curve for prednisolone

460 SYED LAIK ALI

( Y = 0.0046 x) is linear up to 500 ng/ml and the detection limit is given between 2-5 ng/ml. The use of glass column permitted a higher sensitivity and less consumption of mobile phase (101).

Application of HPLC procedure to the determination of binding of prednisolone to high-affinity binding sites in human serum is reported. Prednisolone binds to globulin and albumin in human serum. The binding affinity of the steroid for globulin is high whereas the capacity is low. In contrast, albumin has a low affinity for the drug but the binding capacity is high. The method describes a HPLC gel permeation procedure which a1 lows prednisolone bound to albumin to completly dissociate during chromatography while the binding of the drug to high affinity proteins is unaffected. The column (Bio-Sil TSK-250, 3 0 0 ~ 7 . 5 mm, 10 um, Bio-rad Labs) with a molecular mass range of 1000-300,000 was preceded by a guard column. The mobile phase consisted of 0.1 M Sodium sulfate and 0.02 M Sodium phosphate monobasic adjusted to pH 6.8

with 0.1 M NaOH. The flow-rate was 5.4 ml/h and detection was performed at 280 nm. The data about the effect of prednisolone concentration on the binding in serum and comparison o f binding of prednisolone by HPLC and equilibrium dialysis is given (102).

HPLC determination of prednisolone incorparated in gel ointment i s reported. The gel ointment is composed of carboxy vinyl polymer (1.3 % W / W ) and a

PREDNISOLONE 46 1

large amount of an aqueous organic solvent. A

methanol extraction system offered simultaneous advantages of the removal of the polymer and the recovery of active ingredients from the gel phase.

The recovery of the drug was 100 %. The following

chromatographic conditions were used: mobile phase methanol-water, 6:4, reversed phase, Bondapak C 18,

10 um column, 30 cm x 3.9 mm, flow-rate 1 ml/min, detection at 254 nm. The prednisolone content in gel ointment was well maintained for 3 months or longer

when stored at 5 "C (103).

Retention data of 12 corticosteroids including prednisolone is given on dynamically modified silica

by cetyltrimethylammonium bromide added to the

eluent with various organic modifiers. Separation

factors between hydrocortisone and 11 other corticosteroids including prednisolone measured on 8

different silica columns and six different

ODs-sil ica columns are presented. The variations in selectivity were found to be substantially smaller

than those of chromatographic systems based on chemically bonded ODs-silicas from the same sources

(104).

A HPLC

predn i so

method

one a

for the simultaneous determ

ong other corticosteroids

plasma i s described. Extraction of the

nation of

in swine steroid

mixture from swine plasma with dexamethasone as

internal standard was accompl ished by sol id-phase

(SPE) extraction or by the more traditional

462 SYED LAIK ALI

liquid-liquid extraction (LLE) techniques. A

Lichrosorb Si 60, 5 um silica column, 25 cm x 4.6

mm I mobile phase methylene chloride-water saturated met h y 1 ene chlor ide-tetrahydrofuran-

met hano 1 -g 1 ac i a 1 acetic acid (664.5 : 300 : 10 : 25 : 0.5) , flow-rate 0.8 ml/min and detection at 254 nm were used. Calibration curves were found to be linear

between 10 and 100 ng/ml by the LLE technique. Within-day and inter-day variability for the measurement of the plasma samples spiked with

prednisolone is given (20 ng/ml, 10 % and 100 ng/ml,

8.4 %). The average recovery of prednisolone at 20

ng/ml is between 70 and 90 % (105). Cox et a1 (106) used a weak cation-exchange column and a mobile phase of 0.05 M ammonium formate in 2.5 % aqueous

ethanol, flow rate 0.5 ml/min, detection 240 nm, to separate prednisolone from prednisone. The retention

times were about 18 and 23 min for prednisone and prednisolone respectively (106). Prednisolone and

prednisone could not be resolved with a ODS 10 cm

column, methanol-water (1:l) mobile phase, flow rate

1 ml/min, detection 240 nm (107). Retention values of prednisolone along with a number o f other steroids relative to acetone on a u Bondapak C 18 column using methanol-water and acetonitrile-water

mobile phases o f different compositions at a flow

rate of 1.5 ml/min are reported (108). The extraction efficiency of ethyl acetate, diethyl ether and dichlormethane in extracting prednisolone from pooled plasma is reported (109). Dichlormethane appears to be the best solvent for extraction of

corticosteroids from plasma (109). Trefez et a1

PREDNISOLONE 463

(110) showed that prednisolone could be separated

from other corticosteroids in plasma samples on a 25

cm Zorbax Sil (Dupont) column. Two techniques, HPLC

and HPTLC were developed for the determination o f

prednisolone in human plasma, salvia and urine. Both

methods shared a single and simple step o f an organic extraction procedure and separation using a

normal phase column or HPTLC plates. A normal phase

HPLC column (0.45 x 20 cm) packed with Zobrax SIL 5

um, mobile phase dichlormethane-methanol-acetic

acid, 95:1:3:75, flow-rate 2.5 ml/min and detection

at 254 nm were the chromatographic parameters. The method allows simultaneous measurement o f endogenous cortisol in plasma following administration of prednisolone and methyl prednisolone. The calibration curves of steroids in a1 1 biological

fluids were linear over a range of concentration o f

0.025 - 4 ug/ml. The limit o f detection for

prednisolone was 10 ng/ml in plasma and salvia and 25 ng/ml in urine. The method was reproducible with an inter- and intra-assay coefficient o f variation

of < 10 % over a wide range of concentration in all biological fluids. No interference from endogenous steroids was found (111).

7.5.3 Gas chromatoaraphy - mass spectrometrv Pentaf luorobenzyl hydroxylamine has been used as a derivatization reagent in the analysis of cort i costero ids inc 1 uding predn i sol one by gas chromatography-negative ion chemical ionization mass

spectrometry (NCI). The resulting pentaf luorobenzyloxime (PFBO) trimethylsi lyl (TMS)

464 SYED LAlK ALI

derivatives were generally formed in moderate yield

but, despite this, the use of these derivatives resulted in a 10-fold improvement in the capability of identification of corticosteroids by GC/NCI mass spectrometry in comparison with the methoxime/TMS derivatives. The NCI mass spectra of PFBO/TMS dervatives were simple with most o f the ion current

being carried by the (M-CfjF6CH2)- or (M-PFB)- - ion and by a reagent-specific peak at m/z 196.

The PFBO/TMS derivatives are suitable for the

analysis o f pi cogram quantities o f cort i costero i ds in biological media by GC/NCI mass spectrometry

(112). The negative ion chemical ionization mass

spectra of the methoxime-TMS dervatives of the

cort icosteroids including predni solone have been

chroamtography-mass spectrometry. Fig. 9 shows a NCI spectrum of the methoxim-TMS derivative of

prednisolone. The spectra showed abundant diagnostic ions at m/Z greater than 300 allowing for clear

discrimination between prednisolone and other

steroid dervatives (113). A capi 1 lary GC-MS method using negative ion chemical ionization mass spectrometry has been developed to confirm the presence of the parent steroids in horse urine following the administration of proprietary

preparations o f prednisolone and betamethasone. For

this purpose standard steroids (40 ug) were treated with methoxylamine hydrochloride in dry pyridine (8

% w/v; 100 ul) and heated at 80°C for 30 min. The sol vent was removed under N2 and

obtained using capi 1 lary co 1 umn gas

Fig. 9 (113) NCI Spectrum o f Methoxime - TMS D e r i v a t i v e o f Predniso lone

441

300 400

512

466 SYED LAIK ALI

trimethylsilylimidazole (50 ul) was added and sily- lation was carried out at 80°C for 2 hours. Excess dervatization reagents were removed by filtration

through a 2 cm Sephadex LH-20 column using chloroform-n-hexane (1 : 1) as eluent. The steroid MO-TMS derivatives were eluted in the first 2 ml of eluent. The solvent was removed under nitrogen and the

residue was dissolved in n-hexane (100 ul) for

analysis by GC and GC/MS. The base peak in the spectrum o f prednisolone-Mo-TMS occured at m/Z 457. The abundant diagnostic ions in higher mass regions of the spectra render these derivatives amenable to

analysis by SIM. Capillary GC/MS-NCI analysis of a

mixture of 1 ng derivatives of prednisolone Mo-TMS and dexamethasone Mo-TMS and monitoring ions 441,

457, 473 and 489 demonstrates the applicability of this technique. The sensitivity that can be achieved

by this technique is 250 pg of each steroid derivative (113). The use of capillary GC-MS/NCI for the confirmatory analysis of corticosteroids in horse

urine is more sensitive than the liquid chromatography-mass spectrometry method (114). The combination of HPLC and mass spectrometry for the analysis

of prednisolone has been reported (115). Mass spectra for prednisolone has been obtained in the thermospray discharge mode. Thermospray is a reli-

able HPLC/MS interface. The spectra obtained are

reproducible but fragmentation is not predictable. The sensitivity o f the technique i s compound-depen-

dent and variable. Because of the dependence of ion production on solvent composition, it is not easy to

use the interface with gradient elution (115).

PREDNISOLONE 461

7.5.4 Supercritical fluid chromatoqraphy (SFC) The coupling of supercritical fluid chromatography

(SFC) with mass spectrometry (MS) seems to be easier than liquid chromatography (LC) and MS. This follows

from consideration of the facility of the mass spectrometer vacuum system pumping excess carbon

dioxide from SFC eluent rather than aqueous reversed-phase eluents from HPLC. The other important factor in SFC/MS is whether capillary or packed columns are used. A synthetic mixture of five corticosteroids including prednisolone was analysed by packed-column E 1 SFC/MS (Fig. 10). The

separation was accomplished on a 2 mm x 250 mm, 3 um S3CN spherisorb column maintained at 70°, a

flow-rate of 0.8 ml/min 92:8 C02-methanol and an

inlet head pressure of 3000 Psi. The corticosteroids are difficult to analyse even by GC/MS because they

are relatively polar and thermally labile. Although

they can be characterised by capillary GC/MS either

as parent drugs or TMS derivatives, their long

retention time and thermal instability suggest a

need for alternative means of confirmation.

Prednisolone along with other corticosteroids could

be separated within 6 min by EI SFC/MS. The packed column SFC/MS was applied for the analysis o f a TLC

scrape of a urine sample collectd two hours after the intramuscular administration of 100 mg of prednisolone to a horse. The extracted ion current

profile for the abundant fragment ion at m/z 122 and the molecular ion at m/z 360 easily identify the prednisolone in this sample. Packed-column SFC/MS

468

80.

60 - 40.

20 1

0'8

SYED LAIK ALI

P R E OH t 80 LO H E

~.- . . - 1 . . . . . . a 1

- 1 2 3 4 5 6

1- UELEWOESTROL ACEPITE 2. CORT180NE 8. PREONISONE 4- H YDROCORT180N E 6. PREOHl8OLONE 6- BETAUETHASONE

MIN

Fig. 10 (116) Packed Column SFC/MS Separa t ion o f P redn iso lone

Fig. 10 (116) EI Mass Spectrum of Predn iso lone

100 - 80

60 - 40.

20.

0

80 100 720 140 160 180 200 220

PREDNISOLONE

with a two-stage momentum separator is feasible for

obtaining E I mass spectra of compounds amenable to

chromatographic separation by this route. The

sensitivity afforded by this approach is

nevertheless not suitable for trace analysis (116,

117).

469

Prednisolone has been analysed by capi 1 lary

supercritical fluid chromatography in equine urine

extract and was identified by matching retention

time of pure standard. Supercritical fluid carbon

dioxide was used as the mobile phase in conjunction

with a methylpolysiloxane stationary phase capillary

column and a flame ionization detector. SFC can thus

be successfully applied for the estimation of

prednisolone without derivatization (118).

8. In vitro dissolution

USP XXII requires that not less than 70 % of the

labelled amount of prednisolone is dissolved in 30 minutes in dissolution medium water (900 ml) with

paddle stirring element test apparatus (apparatus 2)

at 50 rpm (119). I n vitro dissolution profiles of

sustained release formulations of prednisolone are

given. For each tablet formulation 20 tablets were placed in a 100 ml beaker of 5.5 cm diameter; 35 ml

of distilled water were added and the contents were

stirred for one hour, at 37°C. Sustained-release

formulation gives a more uniform blood level of

prednisolone and avoids high peaks of plasma prednisolone (120).

470 SYED LAIK ALI

Two nonporous and three porous-amorphous silicas were used as dispersion media to convert corticoid

solutions into free flowing powders. Prednisolone was dissolved in N,N-dimethylacetamide- polyethylene

glycol 400, 7:3, and their 10 % (w/v) solutions were mixed with silicas (1:3 V / W ) . Dissolution rate from

such powdered solution was more rapid than those of

their micronised powders in various aqueous media. Dissolution in simulated gastrointestinal media of solution of prednisolone dispersed on various silicas is reported (121).

HPCMP tab

A solid d sorbitol ,

Prednisolone tablets, enteric coated with neutral ised hydroxypropyl methylcel luolsoe phthalate (HPMCP) were compared with Delta cortril tablets (Pfizer) by compendia1 in vitro testing. For this

study tablets were tested using the disintegration and gastroresistance tests o f both the USP and

european pharmacopeia. The dissolution o f prednisolone from coated tablets followed the USP

X X I procedure which involved monitoring drug release

in a pH 6.8 phosphate buffer after two hours in 0.1

M HC1. Percent of drug (prednisolone) released in different pH media for neutral ised HPMCP coated tablets is illustrated in fig. 11. The dissolution performance closely reflected the disintegration

characteristics and was independent o f coating

weights between 5 and 25 mg for the neutralised

ets (122).

spersion technique with PEG, PVP, urea, mannitol and cremophor has been used for

Percent of Drug Released i n Different pH Media for Neutralised HPMCP Coated Tablets

412 SYED LAIK ALI

improving prednisolone dissolution. The optimum di ssolut ion-rate composition was for dispersions containing 10 % w/w prednisolone (123). A marked increase in the dissolution rate of prednisolone in solid dispersion was observed compared with that of drug alone or with that of a physical mixture with a carrier (123). Prednisolone, which is poorly soluble in water, was chosen to prepare solid dispersion systems with water-soluble carriers. I t was further determined whether the quantities of these carriers and their chemical structure influenced the dissolution rate of prednisolone from such systems. The results of the studies showed that the

dissolution rate of prednisolone from all solid dispersions increased markedly from those o f the physical mixture and the drug alone. Nevertheless, there was no observed relationship between the higher dissolution rate and chemical structure of the carriers, Nor was it possible to predict quantitatively to what extent any carrier would improve the dissolution rate o f the drug in solid dispersion. For example sorbitol and mannitol, which are chemically similar, produced different effects on dissolution rate. Results indicated that sorbitol was one of the better carriers and the maximum drug dissolved (100 %) was achieved after 3 h. Yet at the same time only 63.17 % prednisolone was dissolved from the mannitol solid dispersion. When PEG, PVP and urea were used as carriers, they gave similar results. The amount of prednisolone dissolved from the solid dispersions with above carriers was twice as great as that from the drug alone. Similar

PREDNISOLONE 413

investigations with physical mixtures showed that smaller amounts of drug were dissolved than solid

dispersions. Comparison of these results with those of the dissolution rate of prednisolone in carrier solutions showed that a solubilizing effect had taken place and this had evoked a better dissolution

rate. The mechanism of the enhanced dissolution properties of prednisolone in solid dispersion with different carriers could not be explained. The results showed that improved wettability of drug molecules and their solubilization by the carriers

were not basic processes. Molecular dispersion of drug through the matrix o f the carriers was of greater importance. A1 though changes in crystalographical structure of the drug during

preparation of solid dispersion were evident, x-ray diffraction studies nevertheless indicated an amorphous form of prednisolone in solid dispersion with PVP. In contrast the presence of identical prednisolone diffraction peaks in the spectrum of

pure drug and solid dispersion systems with PEG,

urea, mannitol, and sorbitol showed that these solid

dispersions contained prednisolone in crystalline

form (123).

The dissolution behaviour of ground mixtures of prednisolone with chitin and chitosan were prepared

by co-grinding in a ball mill. The x-ray diffraction patterns and results of differential scanning

calorimetry suggested that the size of prednisolone crystals decreased in the ground mixtures. The dissolution rate o f prednisolone from the ground

414 SYED LAIK ALI

mixtures was significantly greater than that from

the physical mixtures or from intact prednisolone powder. These results indicate that chitin and chitosan can improve the dissolution properties of

prednisolone. The ground mixture with chitosan gave slightly greater dissolution than that with chitin

and this difference reflected the reducing effect of chitosan on the relative enthalpy change o f

prednisolone (124). The dissolution profiles of a model formulaion of prednisolone tablets containing different disintegrants have been investigated.

Marked increase was observed in disintegration and dissolution rate with increased concentration of

microcrystalline cellulose, methylcellulose, maize starch, whereby a decrease in dissolution rate was

recorded with increasing concentration of sodium

carboymethylcellulose and pregelatinized starch

(125) .

9. Pharmacokinetics and drua metabolism

Prednisolone is efficiently absorbed through the gastrointestinal tract, with approximately 75 - 98 %

o f the dose given being abosorbed. Inactivation of prednisolone is achieved mainly in the liver through

reduction of the double bonds in ring A and the Keto

groups to form tetrahydroprednisolone which

conjugates with glucoronic acid and sulphate groups to form water-soluble compounds that are excreted in the urine (126, 127) . Prednisolone plasma concentrations are commonly determined by either radioimmunoassay or competitive protein binding

PREDNISOLONE 415

techniques. Prednisolone is absorbed completely and rapidly after oral administration reaching peak plasma conentrations after 1 to 3 hours. The bioavailabiltiy of prednisolone afte oral prednisone administration is approximately 80 % of that after prednisolone. A wide intersubject variation in prednisolone concentration is evident, which may

suggest impaired drug absorption in some

individuals. Prednisolone shows dose-dependent pharmacokinetics, where an increase in dose leads to an increase in volume o f distribution and plasma

clearance. This can be explained in terms of the

non-linear binding of the drug to plasma proteins.

The degree of binding will determine the

distribution and clearance of free drug. Prednisolone pharmacokinetics is also dependent on age, the half-life being shorter in children. Liver

disease prolongs the prednisolone half-life and also increases the percentage of unbound drug. In these cases prednisolone rather than prednisone is the

drug of choice in active liver disease owing to the poor conversion of prednisone to prednisolone.

However, the reduced plasma concentration of prednisolone in such patients is compensated for by delayed clearance. Thus, there is little advantage

of one preparation over the other (126).

Hepatic conversion o f prednisone to prednisolone i s

extensive and the two compounds are generally considered to be therapeutically equivalent when used systemically. It is however suggested that orally dosed prednisone resulted in lower

416 SYED LAIK ALI

circulating prednisolone concentrations compared

with equivalent oral doses of prednisolone. The results provided evidence that oral prednisone products may not be bioequivalent to oral

prednisolone products and suggest that substitution

of one drug from for another can result in marked changes in circulating concentrations o f active steroid (128). The half-life of prednisolone was found to be about 4.2 h , the apparent volume of distribution in the B-phase was about 0.6 l/kg and

the systemic clearance about O.ll/l.h.kg (111). In Fig. 12 concentration-time profile of prednisolone in biological fluids is illustrated (111). Prednisolone is cleared from the body primarily by hepatic metabolism and greater than 90 % of

radioact i vt i y admi n i stered ora 1 1 y or intravenous 1 y as 4-C14-prednisolone is recovered in urine (127,

128). Only approximately 7 - 15 % of an oral dose of prednisolone is excreted as unchanged prednisolone

in the urine, the rest being recovered as a variety of metabol ites (70).

The plasma half-life of prednisolone following the

oral administration of prednisone to normal subjects ranges form 2.5 to 3.5 h (126, 130, 131). Similar

half-1 ife values for prednisolone were observed

after oral prednisolone is administered (126, 132,

133, 134). Nugent et a1 (135) found after an intravenous dose of 1 mg/kg body weight of predn i so 1 one ( sodi um succ i nate sa 1 t ) an average half-life o f 3.5 h. After an intravenous dose of 0.3 mg/kg prednisolone as phosphate the mean plasma

PREDNISOLONE 411

URINE

2 4 6 8 lo 12

TIME (hr) Fig. 12 ( 1 1 1 ) Concentration-Time Profile o f Prednisolone in Biological Fluids Following Intravenous Administration o f 64 mg o f Prednisolone

478 SYED LAIK ALI

half-life was 4.2 h, plasma clearance 97.3 ml/min/1.73 m2, volume of the central compartiment

28.2 1/1.73 m2 and that of peripheral compartiment 36.9 1/1.73 m2 (136). In another study eight

subjects received an intravenous bolus dose of 12 mg

prednisolone phosphate and four received 48 mg dose. Similar pharmacokinetic parameters were found. Neither half-life nor clearance was statistically different between the two dose levels (137). In a later study the mean metabolic clearance rate was

measured as 1.16 ml/min/kg, mean half-life 3 . 2 h and the values for the apparent volume o f distribution

were 0.14 l/kg for V and 0.15 l/kg for V2 (138).

The plasma clearance, half-life and volume of distribution of prednisolone is reported to be

independent in the range o f the doses 10, 20 and 30

mg prednisolone adminstered orally (139). Pickup et a1 (140) studied pharmacokinetics of prednisolone at

different levels in ten subjects, four normal subjects and six patients with osteoarthritis after intravenous administration o f prednisolone. Average

prednisolone half-lives were found to be between 2.6 to 3.8 h, mean volume distribution between 0.22 to 0.64 l/kg, and plasma clearance between 1.02 to 2.0 ml/min/kg following the tracer 0.15 mg/kg and 0.3

mg/kg doses. This data showed sginificant increases in volume of distribution and plasma clearance of prednisolone with inceasing dose. An increase in half-life was also observed. Pickup et al. (140)

thus postulate that the observed dose-dependent kinetics is primarily due to the non-linearity in

PREDNISOLONE 479

plasma protein binding of prednisolone. The percentage of unbound prednisolone increases with increasing dose and results in a larger apparent

volume of distribution and plasma clearance. The net

effect of these changes causes the observed

prolonged prednisolone half-life following larger doses (140).

In another study the area under the plasma concentration-time curve for prednisolone for the 20

mg dose was 77.89 % of that calculated for the 10 mg

dose. This change in area represented an increase in prednisolone clearance from 1,7 ml/min.kg to 2.2

ml/min.kg when the dose was increased (141). Rose et a1 . (142) found dose-dependent pharmacokinetics of prednisolone where the plasma half-life increased from 3 to 5 h as the oral dose of prednisone was

increased from 5 t o 50 mg. T.anner et a1 (143)

reported the pharmacokinetics of prednisolone at

different dose levels in 43 subjects. Each subject

received only a single dose, 5 - 200 rng of oral prednisolone. Kinetic parameters of oral

prednisolone are presented in table 5 and fig. 13

illustrates concentration-time profile of

prednisolone. The mean half-life of prednisolone remained fairly constant between 3.4 to 3.8 h.

Bioavailability of prednisolone was 98.5 2 4 %.

Furthermore as the prednisolone dose increased, the

area under the curve increased but not proportionally to the dose, such that a fivefold increase in dose from 20 to 100 mg resulted in only a two-to threefold increase in area under the curve.

Table 5 (143)

a W 0

Kinetic parameters for oral prednisolone, 6 to 200 rng

I07 198 2 9 250 2 21 320 2 24 299 f 27 625,549 761 -c 177 676,66 1 I409

467 1,079 f 108 1,243 2 85 1,739 f 150 1,664 2 52

4,361 f 1141 4,694,4186 8,692

3 , ~ m s

2-9 45 3,7 2 0 ,2 49 ,c 5 3.8 t 0.2 66 f 6 3.5 2 0.2 58 2 5 3.7 2 3.3 I60 t I 1 3.4,3.4 99,109 3.7 2 0. I 3,6,3.6 134,146 3 , 8 I26

1.72 5 40

10.7 9.3 f 0.8

12.1 2 0.9 11.5 t 0.9 30.0 2 2.6 19,23 22.9 t 5.2 26,28 23.0

* 20 m o of thle preparation waa equlvalent to 18 m o prednleolone and 100 mg woe equlvslent to 90 mg prednleolone; numbere In Parentheeee lndlcate the number of eublects atudled a t that pertloular doaage and from whloh the data are derlved.

48 1

Fig. 13 (143) Concentration-Time Relationship o f Prednisolone and Prednisone Following the Oral Administration of 100 mg Prednisolone PREDN180LON 0- 0

PREDNISON O--o

482 SYED LAIK ALI

The apparent volume of distribution divided by the extent of avai labi 1 ity (Vd/F) and clearance (CL/F)

for prednisolone was found to increase with dose.

For example Vd/F at 10 mg was 49 1 increasing to 132

1 if 100 mg dose was given and CL/F increased from

155 ml/rnin to 382 rnllrnin. The authors (143)

postulated that since they found no change in the plasma protein binding of prednisolone over the dose

range studied, the increase in volume may be due to increasing binding of prednisolone at extravascular

sites. There was a constant amount of prednisolone

bound to cortisol-binding-globulin (CBG; 145 2 16

ng/ml ) .

It appears that prednisolone may exhibit

dose-dependent pharmacokinetics, so that with increasing dose values volume of distribution, plasma clearance and half-life may increase. It is

believed to be related to changes in the plasma

protein binding of prednisolone. Prednisolone appears to bind to plasma proteins in a non linear manner over the range of doses used (131).

It is believed that it is the free non-protein-bound fraction of the circulating steroid wich is biologically active and which is metabolised. Prednisolone has been shown to bind to albumin and to specific a- and P-globuline in plasma. Human cort icostero i d-bind ing g lobu 1 in (CBG , transcort in) binds prednisolon with a high affinity but low capacity due to relatively low concentrations of

PREDNISOLONE 483

about 10-7 in plasma. Thus there is a saturation in protein binding as the steroid concentration

increases above physiological levels. However, albumin, although it has a lower affinity for

prednisolone, has a larger capacity for binding due to its higher plasma concentrations o f about 10-4 M. It is therefore suggested that changes in predn i solone plasma protein binding are responsible

for the varience in volume of distribution,

half-life and metabolic clearance (144) . At low prednisolone concentrations binding to CBG is

important, which becomes then saturated so that a higher concentration of albumin plays the major role

(145) . In another study it is suggested that variations in the level of circulating cortisol

could cause variation in the protein binding o f

prednisolone (146). Uribe et a1 (147) determined the effect of a liquid diet on the serum protein binding

o f prednisolone in normal healthy subjects. It was

observed that the percentage of prednisolone bound to plasma proteins measured at the time of peak

levels was 80 %, whether administered with the meal or with water.

Prednisone and prednisolone tablets are on the list

o f drugs with high risk potential for therapeutic inequivalence because o f differences in

bioavailability. Levy et a1 reported a case of a patient with arthritis who was successfully treated with a proprietary brand o f prednisolone 5 mg

tablets. Subsequently when prednisone 5 mg tablets

484 SYED LAIK ALI

as generic brand was given even at fourfold dose,

but the response failed. It appears that prednisone tablets with low dissolution rates may be clinically

ineffective (148).

Gambertoglio et a1 (131) have given in a detailed review the pharmacokinetics of prednisolone in healthy volunteers, patients with different diseases as well as effect of other drugs such as

barbiturates , phenytoi n , r if ampi n and oral contraceptives.

Much attention has been focussed on comparisons between standard and susstai ned-re lease preparations

(70, 149) and between standard and enteric-coated tablets (150). Enteric coated prednisolone has been

introduced to reduce gastrointestinal distress

(151). The ability of enteric coated and sustained-release preparations to produce prolonged

plasma concentrations were considered to be of no

greater value than conventional tablets (152). The distribution and elimination of prednisolone have

been described in terms of a 2-compartiment open model, with rapid distribution within the first half-hour followed by a slower terminal elimination

phase (126).

The absolute bioavailability of prednisolone from a rectal capsule was tested in 12 healthy volunteers. the bioavailability from this dosage form was

PREDNlSOLONE 485

calculated with about 48 % compared to a short

infusion. The maximum plasma levels occured between

1.2 to 3.4 h (153).

10. Acknowledqement

The author is indebted to Miss Michaela Schiavulli

who has taken great pains in typing this manuscript.

Mrs. Petra Grotsch kindly assisted in drawing the

figures.

486 SYED LAIK ALI

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PREDNISOLONE 481

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PREDNISOLONE 489

( 34

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494 SYED LAIK ALI

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(109

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PREDNISOLONE 491

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PREDNISOLONE 499

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(141) J . C. K. Loo and Coworkers, J. Pharm. Pharmacol. 3 736 (1978)

(142) J . Q. Rose and Coworkers, In seventh International Congress o f Pharmacology, Abstracts, Paris, P. 400

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(147) M. Uribe and Coworkers. Gastroenterology 71 362

(1976)

(148) G. Levy and Coworkers, Am. J. Hosp. Pharm. 21 402

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(1971)

500 SYED LAIK ALI

(150) C. G. Wilson and Coworkers, Bri. J. Clin. Pharmacol.

- 4 703 (1977b)

(151) W. H. Kammerer and Coworkers. Arthritis and

Rheumatism L 122 (1958)

(152) J. Y. , Thiessn, J. Am. Pharm. Assoc. 16 143 (1976)

(153) N. Mehlhaus-Barlet, M. Kummer and K. Kunz,

Therapiewoche 3 209 (1988)

SOTALOL

Robert T. Foster and Robert A. Carr

Faculty of Pharmacy & Pharmaceutical Sciences

University of Alberta

Edmonton, Alberta, Canada, T6G 2N8



502 ROBERT T. FOSTER AND ROBERT A. CARR

1 . Description 1.1 Nomenclature

1 .1 .1 Chemical Names 1.1.2 Nonproprietary Names 1.1.3 Proprietary Names

1.2.1 Empirical 1.2.2 structural

1.3 Molecular Weight 1.4 Appearance, Color and Odor

1.2 Formula

2. Synthesis

3. Physical Properties 3.1 Infrared Spectra 3.2 NMR Spectra

3.2.1 Proton NMR 3.2.2 13C NMR

3.3 Mass Spectra 3.4 Ultraviolet Spectra 3.5 Optical Rotation 3.6 Melting Point 3.7 Ionization Constants 3.8 Partition Coefficient 3.9 Solubility

4. Methods of Analysis 4.1 Elemental 4.2 Chromatographic

4.2.1 Thin-Layer 4.2.2 Gas 4.2.3 High-Performance Liquid

5. Pharmcokinetics 5.1 Absorption 5.2 Distribution 5.3 Metabolism 5.4 Excretion

6. References

SOTALOL 503

1. DESCRIPTION

1.1 Nomenclature


N-[4-[ 1 -Hydroxy-2-[ (1 -meth ylethyl)amino]ethyl]phenyl]- methanesulfonamide; 4 ' -[ 1 -hydroxy-2-(isopropylamino)ethyl]- methanesulfonanilide (1,2); MJ-1999 (2). Chemical abstracts registry no. : 3930-20-9, sotalol; 959-24-0. sotalol hydrochloride (2).

1.1.2 Nonproprietary Name

Sotalol (1)

1.1.3 Proprietary Names

Beta-Cardone, Betacardone, Betades, Sotacor, Sotalex, Sotapor (192).

1.2 Formula

1.2.1 Empirical

C ~ ~ H ~ O N ~ O ~ S , sotalol base; C12H21ClN203S, sotalol hydrochloride

1.2.2 structural

Figure 1 depicts the structure of sotalol.

C Hg SO 2NH <ob C!! HC HZ NHC H ( C Hg ) 2 *

FIGURE 1. Structure of sotalol, where the asterisk denotes the chiral center.



272.36, sotalol base; 308.82, sotalol hydrochloride


An odorless, white, crystalline solid (1).

2. SyNntIESIs

The synthesis of several sulfonamidophenethanolamines, including sotalol, has been described previously (3). The synthesis of the sulfonamidophenethanolamine compounds relied upon firstly obtaining a 2-aminoacylsulfonanilide precursor. The alcohol formed from the ketone precursor is via either palladium-catalyzed low-pressure hydrogenation or sodium borohydride chemical reduction (3).

Two schemes outlining the synthesis of the 2- aminoacylsulfonanilide precursor have been reported (3). The first scheme (Figure 2) introduces the amino moiety last, whilst holding the suIfonamido constant. The second scheme (Figure 3) introduces the sulfonamido last, thus introducing the amine moiety first and holding it constant.

3. PEYSICAL PROPERTIES

3.1 Infrared Spectra

The infrared spectrum of sotalol hydrochloride is depicted in Figure 4. The spectrum was obtained on a KBr disk using a Nicolet 20 SX Fourier Transform infrared spectrometer. Diagnostic peaks were observed at 3570 cm-l (secondary alcohol, free); 3410 cm-1 (secondary alcohol, H-bonded); 2700-2800 cm-l and 2950-3200 cm-1 (hydrochloride); 1325 cm-1 (S =O asymmetric stretch); 1154 cm-1 (S=O symmetric stretch). The peaks are presented in Table I.

SOTALOL

0 I t

505

0 II

pczr2 N H S 0 2 R I Br2

R ,S02CI

0 I I

t NH2

0 I1

N H S 0 2 R

FIGURE 2. Synthetic Pathway for Ketone Precursor to Sotalol (from ref. 3). R1 =CH3; R2=H; R3Rq=CH(CH3)2


0 I I 0

H2N

H N R 3 R 4 - 3 4 Fci:NR

NHSO R 2 1

FIGURE 3. Synthetic Pathway for Ketone Precursor to Sotalol (from ref, 3). R1 =CH3; R2=H; R3Rq=CH(CH3)2

FIGURE 4. Infrared Spectrum of Racemic Sotalol Hydrochloride. Instrument: Nicolet 20 SX Fourier Transform infrared spectrometer


Table I. I.R. Spectrum of (+)-Sotalol HC1. KBr pellet. Instrument: Nicolet 20 SX Fourier Transform I.R.

Wavenumber cm- Relative Intensit I

s broad 2700-3 100 s broad

m 1585 1508 S

1460 m

bl

,1393 m 1325 S

1225 m 1201 W

1071 m 1016 m 985 m 962 W

904 m 864 W

837 m

773 m 689 S

655 W

,793 W

11 635 I w 8, s=strong; m=medium; w=weak.

SOTALOL 509

3.2 NMR Spectra

3.2.1 Proton NMR

The 300 MHz proton NMR spectrum of (+)-sotalol in CD30D is described in Table 11. The spectrum was obtained on a Bruker AM-300 spectrometer. Instrumental settings were: time domain (data points), FT NMR 16K; aquisition time, 1.819 sec.; spectral width, 4504.51; receiver gain, 32; line broadening, 0.200. The spectrum is shown in Figure 5.

and 7, respectively. There is no difference between the spectra for either pure enantiomer of sotalol and that of the racemate (all run in CD30D). It is worth noting that the coupling of protons (e.g., -CHC&NH, -CH(C&)2, and -Cg(OH)CH2-) is altered probably as a function of the chiral center of sotalol.

The D20 exchange NMR spectrum of sotalol is shown in Figure 8. The exchangeable protons (OH and NH) are absent, and are replaced by a single HOD peak at 4.886.

The spectra for S( +)- and R(-)-sotalol are depicted in Figures 6

3.2.2 13C NMR

The 300 MHz 13C NMR spectrum of sotalol in CD30D is described in Table 111. The spectrum was obtained on a Bruker AM-300 spectrometer. Instrumental settings were: time domain (data points), FT NMR 16K; aquisition time, 0.4424 sec.; spectral width, 18518.52; receiver gain, 400; line broadening, 2.00. The spectrum is shown in Figure 9.

3.3 Mass Spectra

Mass spectra were obtained on a AEI MS9 (Manchester, U.K.) instrument equipped with a fast atom bombardment source (Figures 10 and 11). The medium was either glycerol or Cleland and the sample was introduced by means of direct insertion. Instrument settings were: 92- 963; total scans in run, 4; sampling rate, 256; signal level threshold, 30; minimum peak width, 5; scan rate (sec/dec), 10.0.


Table II. 300 MHz Proton NMR of (f)-Sotalol in CD30D.

J-

i i

1, L r

I -

5 9 3 2 1 1 6 P r n

FIGURE 5 . Proton NMR Spectrum of Racemic Sotalol. Instrument: Bruker AM-300 FT NMR spectrometer

r I

JL 1 I I

9 8 7 6 5 3 2 1 PPfl

FIGURE 6. Proton NMR Spectrum of S( +)-Sotalol. Instrument: Bruker AM-300 FT Nh4R spectrometer

-4 -I I I

9 8 7 6 5 9 3 2 1 PPW

FIGURE 7. Proton NMR Spectrum of R(-)-Sotalol. Instrument: Bruker AM-300 FT NMR spectrometer

I

I !I

I 4 7 6 5 3 2 I

P P V

FIGURE 8. Proton NMR Spectrum of Racemic Sotalol. D20 Exchange. Instrument: Bruker AM-300 FT NMR spectrometer

SOTALOL 515

Table III. 300 MHz l3C NMR of (+)-Sotalol in CD30D.

b - 7 Chemical Shift

139.22 1 c-4 140.81 1 c- 1

I wa I t m 1 mm a m 6m rm 21 PP 11

FIGURE 9. l3C NMR Spectrum of Racemic Sotalol. Instrument: Bruker AM-300 FT NMR spectrometer

SOTALOL 517

Regardless of the medium, MH+ peaks were found at m/z 273. Both spectra also exhibited peaks at m/z of 545, corresponding to M2H+; at m/z of 581, corresponding to M2H+ + HC1; and at m/z of 255, corresponding to MH+-H20. The spectrum with glycerol as the medium showed a base peak at m/z of 93, which suggests CH3NSOZ. Table IV summarizes the fast atom bombardment spectral data and suggests the structures for the fragments.

Additionally, a positive ion electron impact mass spectrum was obtained on a Kratos MS 50 double focusing magnetic sector mass spectrometer. The sample was introduced by means of direct insertion. Instrument settings were: mass range, 5 1.0235-279.1606, total scans in run, 1; sampling rate, 25; signal level threshold, 1; minimum peak width, 7; scan rate (seddec), 10.0, number of scans averaged, 11. The calculated M+ is at m/z 272.1195; a M+ was found at m/z 272.1196. Furthermore, diagnostic peak (100% relative abundance) was found at m/z 72.0817 which suggests a C ~ H ~ O N fragment.

Table IV. FAB Mass Spectral Data of Sotalol HCl.

Ton Measured Mass 9% Relative Abundance

C12H21N203S 272.93 100.00 (Cleland) 272.93 97.05 (glycerol)

C12H19N202S 255.01 62.77 (Cleland) 255.02 56.46 (glycerol)

CH3NS02 93.04 100.00 (glycerol)

Previously, spectral data for sotalol were reported using negative ion chemical ionization mass spectrometry (4). The base peak (M-79)- corresponded to the loss of (-S02CH3). Other characteristic ions were found at m/z 163 (C2FsCOO)-; m/z 147 (C2F5CO)-; m/z 144 (C2F4COO)-; and at (M-147)- and (M-166)-.


Figure 12 depicts the ultraviolet spectrum of sotalol free base in chloroform. The spectrum was obtained using a Phillips PU8700 series UV/VIS scanning spectrophotometer (Cambridge, U.K.) . Qualitative results depict maximal wavelengths at 242.2 and 275.2 nm.

FIGURE 10. Positive Ion FAB Mass Spectrum of Racemic Sotalol. Instrument: AEI, MS9. Medium: glycerol

8 0

6 0 - 1 1 9 - 255

238 195

213 3 3

2 0 - 1 5 5 177 w’ 1.. il. I,

FIGURE 11. Positive Ion FAB Mass Spectrum of Racemic Sotalol. Instrument: AEI, MS9. Medium: Cleland

. I,, .#.ul . / < I l -


+ +

+

+ +

f

c c

FIGURE 12. Ultraviolet Spectrum of Racemic Sotalol Base in Chloroform. Instrument: PU8700 series scanning UV/VIS spectrophotometer

SOTALOL 521

3.5 Optical Rotation

Optical rotations of the two pure enantiomers of sotalol HC1 were obtained using a Perkin Elmer Model 241 polarimeter. The rotations were measured in a 10 cm cell (water as solvent) at the sodium D-line (589 nm). The optical rotations (specific rotations) of sotalol HCl were:

(+)-Sotalol HC1 [ c Y ] ~ ~ D +35.80" (-)-SOtalol HC1 -34.75 "

The specific rotations of sotalol HC1 in methanol were reported (5) as:

(+)-sotal~i HCI +39.9" (-)-SOtalol HC1 [ c Y ] ~ ~ D -36.3"

3.6 Melting Points

Utilizing a Uni-Melt capillary melting point apparatus (Arthur H. Thomas Company, Philadelphia, PA), the melting points of racemic sotalol HCl, S- and R-sotalol HCl were 218 to 219, 210 to 211 and 204 to 205 "C, respectively. The melting point of racemic sotalol HCl has previously been reported as being within the range of 206.5 to 207 (1).

3.7 Ionization Constants

The pka values for sotalol are 9.8 and 8.3 for the amine and the sulfonamide, respectively (6).

3.8 Partition Coefficient

The watedn-octanol partition coefficient (log P value) has been reported to be 0.24 (7). Using octan-1-ol/phosphate buffer @H 7.4) at 37" C, sotalol was reported to have a partition coefficient of 0.09 (8).

3.9 Solubility

Sotalol HC1 is freely soluble in water and only slightly soluble in chloroform (1).


4.1 Elemental

The elemental analysis of sotalol(1) is:

C 52.92% H 7.40% N 10.28% 0 17.62% S 11.77%

4.2 Chromatographic Analysis

4.2.1 Thin-layer

A number of methods have been reported for the analysis of sotalol(9- 12). These methods are summarized in Table V.

Table V. Rf Values of Sotalol under Various Thin-Layer c Conditions Solvent -System

methano1:ammonium hydroxide (100: 1.5) cyclohexane: toluene: diethylamine (75: 15: 10) chloroform: methanol (9: 1) acetone ethyl acetate: methanol: 30 % ammonia (85: 105)

Rf Value

where both constants represent principle component scores.

01 962

Ref. - 3 , l O

continued.. .

SOTALOL

Table V continued.. .

523

Silica Gel 60 F254

Polygram Sil

254 N-HR UV

ethyl acetate: methanol: ammonia (85: 105) methanol: ammonia (100: 1.5) methanol: butanol (60:40 and 0.1 M NaBr) methanol: water:HCl (5050: 1) ethyl acetate: methanol: concentrated

22

56

75 (bad spot shape)

71

0.7

11

12

4.2.2 Gas

The use of gas chromatography has been reported by others (4, 13-15). Generally, these methods have only been utilized for the analysis of sotalol in urine. Table VI summarizes most gas chromatographic methods reported to date.

4.2.3 High-Performance Liquid

a. Nonstereospecific. Numerous HPLC methods have been reported for the analysis of sotalol (16-23). Generally, most nonstereospecific HPLC assays utilize reverse-phase chromatography with isocratic flow. Table VII summarizes the more recently reported methods.

b. Stereospecific. The enantiomers of sotalol have been reported utilizing HPLC methods (24-28). These methods employed either chiral stationary phases (24,25), or pre-column derivatization with a homochiral reagent and subsequent separation utilizing either reverse- phase (26) or normal-phase (27) chromatography. For the most part, however, chiral columns have primarily been used for preparative-scale

Table VI. Conditions of Reported Gas Chromatographic Analyses.

Extraction I Derivatization I C o l d Retention Time

diethyl ether; pentafluoro-propionic capillary, fused silica diethyl ether: anhydride: pyridine (1=25 m, 0.32 mm i.d.) dichlorometh- (2: 1) and SE-30 methylsilicone ane (1:l) bonded phaseI8.64 min

two acetic capillary, cross-linked extractions; anhydride: pyridine methyl-silicone (1 = 12 m, dichlorometh- (3:2) 0.2 mm i.d.), 0.33 pm ane:isopro- film thickness/ panol : ethyl acetate (1:1:3) 12)

ret. index, 2675 (see ref.

electroncapture 4 detector (ECD)

flame ionization 13 detector and a nitrogen-sensitive detector

continued.. .

Table VI continued.. .

Ln N Ln

diethyl ether followed by t- butyl alcohol: diethyl ether (1 5) diethyl ether, followed by chloroform- pentanol (3:l) Bond-Elut C2, C18 and CN solid-phase diethyl ether

N-methyl-N- trimethylsilyl- trifluoracet-amide,

methylbistri- fluoracetamide trifluoroacetic anhydride:ethyl acetate (2: 1) dried 1-butaneboronic acid in ethyl acetate

followed by N-

capillary, J & W Durabond 1 (1=30 m, 0.25 mm i.d.), 25 pm film thickness/ 3.3 min.

none capillary, fused silica, SE54 (1=25 m, 0.32 mm i.d.), 0.3 mm film thickness/ret. time not reported

Finnigan-MAT ion trap detector

Ion trap detector gn>

~

14

15

Table VII. Conditions of Reported Non-Stereospecific High-Performance Liquid Chromatographic Analyses.

Extraction

no extraction, qualitative

benzyl alcohol: chloroform (60:40)

1 -butanol: chloroform (20: 60)

Baker- 10 SPE Octyl, elution with ethyl acetate: acetonitrile ( 1 : 2)

Mobile Phase Composition; Retention Time Detector; Ref. Flow Rate Sensitivity

methano1:hexane (85:15) with rel. ret. time, 0.68 UV, 215 nm; 16 0.02 % perchloric acid (1.85 mM); 2mVmin

methanol: water: acetonitrile (55:45:20) with 1 % acetic acid and 0.005 M dodecyl sodium sulphate; 1 mVmin 0.01 M phosphate buffer @H 3.2):acetonitrile (20: 80) with 3 mM n-octylsodium sulphate;

(rel. to prazepam, qualitative use where rel. ret. time of 1.0=9.20 min) approx. 5 min. U V , 227nm; 17

10 ng/ml

10 min W, 226 nm; 18 0.03 pmoV1

1.5 ml/min water:methanol:acetonitrile: 0.1 8.3 min fluorescence, 19 M dibasic ammonium phosphate (45:48:6: 1); 1.5 mVmin (ex., em.);

240/3 10 nm

10 ng/ml (plasma), 0.5 p g / d (urine)

continued.. .

Table VII continued.. .

25 cm Altex, ODs-5-pm

25 cm Hypersil, ODs-5-pm

25 cm LiC hrosorb , CN 10-pm 22 cm Brownlee Labs, ODS 5-pm

Baker-10 SPE Octyl, elution with acetonitrile: ethyl acetate (2: 1) n-pentanol : chloroform (1 : 3)

1 ethyl acetate

1 -pentanol: chloroform ( 1 : 3)

0.01 M potassium phosphate dibasic buffer @H 2.4) containing 0.002 M nonylamine; 2 mYmin

4.5 min

acetoniae: water:acetic acid (20:79: l), adjusted to pH 2.5 by NaOH; 0.005 mol/l heptanesulfonic acid and 0.0005 moYl sodium dodecylsulfate added; 1 mYmin

approx. 5 min

methanol:2-propanol: 1.16 M 4.4 min perchloric acid (75:25:0.5); 2.5 mYmin water:acetonitrile (60:40) with 1 !% heptanesulfonic acid:glacial acetic acid (75); 1 ml/min

9.3 min

diodearray, 20 235nm; 20 ng/ml

fluoreGnce, 235 nm/no emission filter; 50 ng/ml (plasma), 2 pg/ml (urine) fluorescence, 235/310 nm; 2 ng/ml fluorescence, 235 (excitation)/ no emission filter; 25 ng/ml

- 21

22

23

Table VIII. Conditions of Stereospecific High-Performance Liquid Chromatographic Analyses for Sotalol.

Column

25 cm amylose tris (3,5-dimethyl- p hen y lcarbamate)

10 cm alphal-acid glycoprotein (Enantiopac, LKB)

10 cm, Partisil ODS 5-pm

25 cm, Partisil5-pm

Extraction

No extraction; qualitative analysis

Mobile Phase Composition; Flow

Rate hexane: 2- propanol: diethyl- mine (80:20:0.1); 0.5 mYmin

quantitation acid in 0.02 M phosphate buffer @H

acetonitrile (60:40); 1 mYmin

methanol (65: 33: 2); 2 mYmin

Retention Time (d);

Detector; Sensitivity

(+)- and (-)- sotalol at approx. 17 and 23 min,

UV, h not specified; sensitivity not

respectively reported (+)- and (-)- U V , 230 nm; sotalol at approx. 15 and 10 min, respectively.

sotalol at approx. 28 and 30 min, respectively.

(-)- and (+)-

(+)- and (-)- sotalol at 7.5 and 8.7 min,

sensitivity not reported

fluorescence, 232 nm ex/no emission filter; sensitivity not

fluorescence, 220 nm ex/no emission filter;

~ reported

respectively. I 20 ng/ml

Ref.

24

25

26

27

SOTALOL 529

enantiomer separations of sotalol. Table VIII summarizes the stereospecific HPLC assays for sotalol.

5. PaARMAcoKINEllcs

5.1 Absorption

Although the lipid solubility of sotalol is relatively low compared with other P-blocking adrenoceptor drugs (28), oral bioavailability is deemed to be 100%. Sotalol is absorbed somewhat slower than most other @-blockers, with peak concentrations occuring within 2-3 hours (29). Although food may impair the absorption of sotalol (28), administration of either calcium carbonate or aluminum hydroxide antacids has little effect on absorption (30). After administration of a single 160 mg oral dose of sotalol, both enantiomers reached maximal plasma concentrations in approximately 3 hours (31) and, hence, did not exhibit stereoselective absorption.

5.2 Distribution

Sotalol is only negligibly bound (28) to plasma proteins (albumin and alphal-acid glycoprotein). The volume of distribution of sotalol is 1.3 L/kg. As expected, the more lipophilic @-blocking drugs, including metoprolol and propranolol, have greater reported volumes of distribution of 5.5 and 2.8-5.5 Wkg, respectively (28). Interestingly, the volume of distribution appears to be somewhat reduced in elderly hypertensive subjects (32). For example, values of 3.55k0.51 and 2.22k0.28 Wkg were reported for healthy young and elderly hypertensive subjects, respectively. As sotalol has a very low lipid solubility compared with other P-blocking drugs, there is slow entry of drug into brain; the brain:plasma ratio was determined as 0.52 in anesthetized cats (29). At present, there is no evidence for stereoselective distribution of sotalol after administration of the racemate (31).

5.3 Metabolism

Sotalol does not undergo first-pass metabolism after oral administration (29). Following intravenous administration of 3H-sotalol to dogs, over 90% of the drug was excreted renally; less than 1 % of the drug was excreted in bile (33). In a stereospecific study of sotalol(31), nonrenal clearance constituted a mean of approximately 23% of the oral clearance. As sotalol does not a D m u to be metabolized in man. it was


suggested that either biliary excretion and/or direct secretion of drug across gut wall may occur in humans (31).

5.4 Excretion

Sotalol is excreted by glomerular filtration with approximately 75% of the drug being excreted within 72 hours (29). The reported elimination half-life ranges from 7-18 hours (29). As expected, reduced renal function (i.e., reduced creatinine clearance) results in reduced renal clearance values of sotalol. For example, renal clearance has been reported (34) to be reduced from a mean of 4.99 Wh (creatinine clearance > 80 ml/min) to a mean of 0.27 L/h (creatinine clearance C 10 ml/min). In fact, after chronic administration of sotalol, the serum half-life was reported to be 69 hours in an anuric patient (35). Although there is no difference in the enantiomeric clearance of sotalol (31), it has been suggested that the clearance of (+)-sotalol after administration of such may be reduced (36) as compared to its clearance when administered with an equal proportion of (-)-sotalol (i.e., when administered as racemate).

individuals and control subjects (37). In elderly hypertensive subjects, however, renal clearance was reduced from a value of 4.10f0.60 ml/min/kg which was observed in healthy young subjects, to 1.93f0.32 ml/min/kg (32). Presumably, the reduction in sotalol renal clearance in the elderly is a reflection of the changed physiology in the elderly (e.g., reduced glomeruIar filtration).

concentration ratios ranged from 2.43-5.64 (38). Consequently, breast- fed infants may be exposed to relatively large sotalol concentrations.

The disposition of sotalol appear to be comparable between obese

Finally, sotalol is excreted in breast milk, whereby mi1k:serum

6.

1.

2.

3.

4.

REFERENCES

Windholz M., editor. The Merck Index, 10th edition. Rahway, NJ, Merck & Co., Inc. 1983: 1248. Reynolds J.E.F., editor. Martindale: the Extra Pharmacopoeia, 29th edition. London, The Pharmaceutical Press. 1989:807-808. Uloth RH, Kirk JR, Gould WA, Larsen AA: Sulfonanilides. I. Monoalkyl- and arylsulfonamidophenethanolamines. J. Med. Chem.

Cartoni GP, Ciardi M, Giarrusso A, Rosati F: Detection of p- blocking drugs in urine by capillary column gas chromatography- negative ion chemical ionization mass spectrometry. 1. High Resolution Chromatogr. Com. 1988; 11 528-532.

1966; 9: 88-97.

SOTALOL 531

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

15.

16.

Garrec LL, Delee E, Pascal JC, Jullien I: Direct separation of d- and l-sotalol mandelate and hydrochloride salts by high performance liquic chromatography. J. Liquid Chromatogr. 1987; 10:3015-3023. Doerge RF, Editor. Wilson and Gisvold's Textbook of Organic Medicinal and Pharmaceutical Chemistry, 8th edition. Philadelphia, J.B. Lippincott Company. 1982:845. Burgot G, Serrand P, Burgot JL: Thermodynamics of partitioning in the n-octanol/water system of some &blockers. Internat. J . Pharmaceutics 1990;63: 73-76. Jack DB, Hawker JL, Rooney L, Beerahee M, Lobo J, Pate1 P: Measurement of the distribution coefficients of several classes of drug using reversed-phase thin-layer chromatography. J. Chromatogr. 1988;452:257-264. Musumarra G , Scarlata G, Romano G, Clementi S, Wold S: Application of principal components analysis to TLC data for 596 basic and neutral drugs in four eluent systems. J. Chromatogr. Sci.

Musumarra G, Scarlata G, Cirma G, Romano G, Palazzo S, Clementi S, Giulietti G: Qualitative organic analysis: 1. identification of drugs by principal components analysis of standardized thin-layer chromatographic data in four eluent systems. J. Chromatogr. 1985;350: 151-168. Ojanpera I, Vuori E: Thin layer chromatographic analysis of basic and quaternary drugs extracted as bis(2-ethylexy1)phosphate ion- pairs. J. Liq. Chromatogr. 1987; 10:3595-3604. Jack DB, Dean S, Kendall MJ, Laugher S: Detection of some antihypertensive drugs and their metabolites in urine by thin layer chromatography: 11. A further five beta blockers and dihydralazine. J. Chromatogr. 1980;196: 189-192. Maurer H, Pfleger K: Identification and differentiation of beta- blockers and their metabolites in urine by computerized gas chromatography-mass spectrometry. J. Chromatogr. 1986;382: 147- 165. Leloux JS, De Jong EG, Maes M A : Improved screening method for beta-blockers in urine using solid-phase extraction and capillary gas chromatography-mass spectrometry. J. Chromatogr.

Koppel C, Tenczer J, Peixoto-Menezes DM: Formation of formaldehyde adducts from various drugs by use of methanol in a toxicological screening procedure with gas chromatography-mass spectrometry. J. Chromatogr. 1991;563:73-81. Flanagan RJ, Storey GCA, Bhamra RK: High-performance liquid chromatographic analysis of basic drugs on silica columns using non-aaueous ionic eluents. J. Chromatogr. 1982:247: 15-37.

1984;22 1538-547.

1989;488:357-367.


17. Lemmer B, Ohm T, Winkler H: Determination of the beta- adrenoceptor blocking drug sotalol in plasma and tissues of the rat by high-performance liquid chromatography with ultraviolet detection. J. Chromatogr. 1984;309: 187-192.

determination of sotalol in biological fluids. J. Chromatogr.

19. Bartek MJ, Vekshteyn M, Boarman MP, Gallo DG: Liquid

18. Karkkahen S: High-performance liquid chromatographic

1984;336: 3 13-3 19.

chromatographic determination of sotalol in plasma and urine employing solid-phase extraction and fluorescence detection. J. Chromatogr. 1987;421:309-318.

20. Hoyer GL: Improved high-performance liquid chromatographic method for the analysis of serum sotalol. J. Chromutogr. 1988;427: 181-187.

21. Gluth WP, Sorgel F, Gluth B, Braun J, Geldmacher-v. Mallinckrodt M: Determination of sotalol in human body fluids for pharmacokinetic and toxicokinetic studies using high-performance liquid chromatography. Anneim. Forsch. Drug Res. 1988;38: 1:408- 411.

chromatographic determination of sotalol in plasma. J. Chromotagr. 22. Poirier JM, Lebot M, Cheymol G: Rapid and sensitive column

1989;493 :409-4 13. 23. Morris R: Improved liquid chromatographic fluorescence method

for estimation of plasma sotalol concentrations. Ther. Drug Mon. 1989; 11 :63-66.

24. Okamoto Y, Aburatani R, Hatano K, Hatada K: Optical resolution of racemic drugs by chiral HPLC on cellulose and amylose tris(pheny1carbamate) derivatives. J. Liq. Chromatogr. 1988; 11~2147-2163.

25. Le Garrec L, De le E, Pascal J-C: Direct separation of d- and 1- sotalol mandelate and hydrochloride salts by high performance liquid chromatography. J. Liq. Chromutogr. 1987; 10:3015-3023.

racemic adrenergic drugs utilizing precolumn derivatization with (-)- menthyl chloroformate. J. Chromatogr. 1989;492:402-408.

27. Carr RA, Foster RT, Bhanji NH: Stereospecific high-performance liquid chromatographic assay of sotalol in plasma. Phamz. Res.

28. Riddell JG, Harron DWG, Shanks, RG: Clinical pharmacokinetics

26. Mehvar R: Stereospecific liquid chromatographic analysis of

1991;8: 1195-1198.

of 0-adrenoceptor antagonists: An update. Clin. Pharmacokinet. 1987; 12:305-320.

29. Singh BN, Deedwania P, Nademanee K, Ward A, Sorkin EM: Sotalol: A review of its pharmacodynamic and pharmacokinetic properties, and therapeutic use. Drugs 1987;34:311-349.

SOTALOL 533

30. Kahela P, Anttila M, Sundqvist H: Antacids and sotalol absorption. Acta Pharmacol. et Toxicol. 1981;49: 181-183.

31. Carr RA, Foster RT, Lewanczuk RZ, Hamilton PG: Pharmacokinetics of sotalol enantiomers in humans. J. Clin. Pharmacol. 1992; accepted.

32. Ishizaki T, Hirayama H, Tawara K, Nakaya H, Sat0 M, Sat0 K: Pharmacokinetics and pharmacodynamics in young normal and elderly hypertensive subjects: A study using sotalol as a model drug. J. Pharmacol. &p. Ther. 1980;212: 173-181.

33. Bourne GR: The metabolism of P-adrenoceptor blocking drugs. Progress in Drug Metab. London, U.K. , John Wiley & Sons, 198 1 ; 6: 77- 1 10.

34. Dumas M, D'Athis P, Besancenot JF, Chadoint-Noudeau V, Chalopin JM, Rifle G, Escousse A: Variations of sotalol kinetics in renal insufficiency. Znt. J. Clin. Pharmacol. Ther. Tox. 1989;27:486- 489.

35. Berglund G, Descamps R, Thomis JA: Pharmacokinetics of sotalol after chronic administration to patients with renal insufficiency. Eur. J . Clin. Pharmacol. 1980;18:321-326.

Pharm. Res. 1991;8:S265.

FC: Comparison of propranolol and sotalol pharmacokinetics in obese subjects. J. Pharm. Pharmacol. 1990;42: 344-348.

Excretion of sotalol in breast milk. Br. J. Clin. Pharmacol. 1990; 29: 277.

36. Carr RA, Foster RT: Enantiospecific study of sotalol in rats.

37. Poirier JM, Le Jeunne C, Cheymol G, Cohen A, Barre J, Hugues

38. Hackett LP, Wojnar-Horton RE, Dusci LJ, Ilett KF, Roberts MJ:

THIOPENTAL SODIUM

Michael J . McLeish

School of Pharmaceutical Chemistry

Victorian College of Pharmacy (Monash University)

Parkville, Victoria, Australia


Copyright 0 1992 by Academic Press, Inc. All rights of reproduction reserved in any form.

536

1. Description

MICHAEL J. MCLEISH

1.1

1.2

1.3 1.4

1.5

Nomenclature 1.1.1 Chemical Names 1.1.2 Nonproprietary Names


Formulae

1.2.1 Empirical 1.2.2 CAS Registry Numbers

1.2.3 Structural

Molecular Weight

Elemental Composition

Appearance, Color and Odor


2.1 Melting Range 2.2 Solubility Data 2.3 Dissociation Constant

2.4 pHRange

2.5 Spectral Properties 2.51 Ultraviolet Spectrum


2.5.3 Nuclear Magnetic Resonance Spectrum 2.5.4 Mass spectrum

3. Synthesis

4. Stability

THIOPENTAL SODIUM 531


5.1 Extraction

5.2 Identification 5.2.1 USP Analysis

5.2.2 BP Analysis Colorimetric, Spectrophotometric and Fluorimetric Analysis 5.3

5.4 Chromatography 5.4.1


5.4.3 High Performance Liquid Chromatography

Paper, Thin-layer and Column Chromatography

5.5 Radioimmunoassay

6. Metabolism

7. Uses, Administration and Contraindications

8. Pharmacokinetics

9. Acknowledgements

10. References

538

1. DESCRIPTION

MICHAEL J. MCLEISH

1.1 Nomenclature


(a) (rt)-5-Ethyldihydro-5-( 1-methylbutyl)-2-thioxo 4,6( lH,5H)-

pyrimidinedione monosodium salt [ 1,2]

(b) (f)-S-Ethyl-5-( 1 -methylbutyI)-2-thiobarbituric acid sodium salt

[1,2,3,41

1.1.2 Nonproprietaw Names

Thiopental sodium, Thiopentone sodium, thionembutal, thiomembumal

sodium, penthiobarbital sodium [l J, soluble thiopentone [3J.


Pentothal Sodium, Nesdonal Sodium, Intraval Sodium, Trapanal,

Thiothal Sodium, Farmotal, Hypnostan, Sandothal[ 1,3].

1.2 Formulae

1.2.1 Empirical

1 lH 17N2Na02S (Thiopental sodium)

cl 1H18N202S (Thiopental)

1.2.2 CAS Registry Numbers

71-73-8 (Thiopental sodium) [1,2,3]


76-75-5 (Thiopental) [3]

1.2.3 structural

H I

H I

NaS yJ N ,CH,

CHCH,CH,CH, CHCH,CH,CH, 0 I 0 I

CH, CH,

Thiopental Sodium Thiopental


264.3 1 (Thiopental Sodium)

242.33 (Thiopental)


Thiopental Sodium:

C. 49.98%

0. 12.11%

Thiopental:

C. 54.52%

0. 13.21%

H. 6.48%

Na. 8.70%

H. 7.49%

N. 10.60%

S. 12.13%

N. 11.56%

S. 13.23%

S40 MICHAEL J. MCLEISH


Thiopental sodium is a yellowish-white, crystalline powder or pale

greenish hygroscopic powder with an alliaceous, garlic-like odor

[1,3,4]. Thiopental sodium for injection is a sterile mixture of

thiopental sodium and anhydrous sodium carbonate as a buffer [ 1,2,4]

2. PHYSICAL PROPERTLES

2.1 Melting Range

The free acid (thiopental) melts at 158-160 OC [5,6].

2.2 Solubility Data

At 20 "C, thiopental sodium is soluble in 1.5 parts water [3,4,5]. It is

partially soluble in alcohol [1,3,4,5] and is practically insoluble in

ether [I ,3,4,5], benzene [l] and petroleum ether [ 1,3].

The partition coefficent of unionized thiopental between isoamyl

alcohol and water at 37 "C, is 991 [7].

2.3 Dissociation Constant

pK, 7.6 at 20 OC IS].

2.4 pH Range

An 8% solution for injection has a pH of 10.2 to 11.2 [2,5].

THIOPENTAL SODIUM


54 1

2.5.1 Ultraviolet Spectrum

The ultraviolet absorption spectra of thiopental sodium, in both 0.02M

HCl and 0.02M NaOH, were obtained on a Shimadzu UV-160A

recording UV-Vis spectrophotometer. The spectra (shown in Figure

One) exhibited maxima at 295 and 305nm, respectively. The

pH-dependence of the W absorption spectrum has also been

determined 181.


The infrared spectrum of thiopental sodium and/or thiopental has

undergone considerable investigation [S-1 11. The infrared spectrum of

thiopental sodium and thiopental, both as KBr disks, were obtained on

a Hitachi 270-30 infrared spectrophotometer. The spectra, presented

as Figure Two and Three, respectively, display absorption

characteristics in good agreement with those previously reported.

Frequency assignments for some of the characteristic bands are given

in Table 1.

Frequency cm-I

3270 2940 1720 1655 1525 1420 1350 1300 1220

Infrared Assignment

N-H stretch C-H stretch C=O stretch C=O stretch

NCS stretch (asymm) C-N stretch

NCS stretch (symm)

N-C=S

C-N

T a b l e 1. IR characteristics of Thiopental

E

I.

W a

f

- 3

0

\ E

z

m

a

In

I

v

m

m

W

n,

a

m v)

a

m W

6,

4

.. a

W

m

m

+

542

-\

0

0

0

0

0

W

0

0

m

0

0

0

.-I

0

0

cy rl

h

7

01

:g

v

t) z

fl 2

E

3

C

0,

iQ

0

0

0

N

0

0

n

N

0

0

0

F1

0

0

ul m

0

0

0

* 0

543

0

0

-4

0

0

W

0

0

m

0

0

0

d

0

0

Y h

Y

8 'E 2s

%E

04

::z d

1

C

23

0

0

0

N

0

0

(Y

m

0

0

0

m

0

0

m

m

0

0

0

*


2.5.3 Nuclear Magnetic Resonance Spectrum

The 'H nmr spectrum of thiopental in dimethyl SdphOXide-d6 has been

recorded at 60 MHz [ 121. The spectrum showed considerable overlap

which made assignment difficult. A later study, carried out in CDCL3,

showed that lanthanide shift reagents could be used to simplify the

spectrum [13].

The 'H nmr spectrum of thiopental sodium in DMSO-d6 has been

recorded on a Bruker AMX300 nmr spectrometer and is presented in

Figure Four. Spectral assignments for both thiopental sodium and

thiopental are given in Table 2. Initial assignments were based on

integrals and expected splitting patterns, and were later confi ied

using two dimensional proton correlation spectroscopy (COSY).

Figure Five shows the proton decoupled 13C nmr spectrum of

thiopental sodium in DMSO-d6. Spectral assignments for thiopental

sodium and thiopental are provided in Table 3. The assignments are

based on those of Fratiello et al. [14].

2.5.4 Mass Spectrum

The fast atom bombardment (FAB) mass spectrum of thiopental

sodium was recorded using a JEOL JMS-DX 300 mass spectrometer

and is presented in Figure Six. The spectrum shows an (M+H)+ peak

at m/z 265 (relative intensity lo%), a peak at m/z 287 (45%)

corresponding to (M+Na)+ , and a further peak at m/z 309 (28%)

corresponding to (M+2Na-H)+. The base peak is at m/z 115.

The electron impact (EI) mass spectrum of thiopental was also

obtained, at an ionization voltage of 70eV, on the same instrument, and

is presented in Figure Seven. The spectrum corresponds to that

Figure Four. '8 nmr spectiurn of thiopental sodium in D M S O - 4

'1 ppm 12 10 8 6 4 2


Table 2 . 'H NMF4 Assignments

a c d s CHCH,CH2CH,

0 I b

Thiopental Sodium

6 @pm)

0.583

0.785

0.825

0.990

1.310

1.730

10.42

Thiopental

6 (PPW

0.696

0.807

0.906

1.090

1.340

1.900

12.56

Multiplicity 'H

triplet (3 H) H,

triplet (3H) He

(No. H) Assignment

doublet (3H) Hb

multiplet (2H) H,

muliplet (2H) H,

approx quartet Ha, H, on multiplet (3H)

broad singlet 4

Figure Five. Proton &coupled *C nmr spectrum of thiopental sodium

Y

I " " I ' l ' ' ' ' l ~ ' i ' I

ppm 150 100 50


Table 3. 13C NMR Assignments

H I

0 11 12 13

CHCH,CH,CH, I

CH,

H’

O 10

Carbon Assignment

Thiopental Sodium 6 (ppm)

Thiopental 6 @pm)

185.6

177.0

176.5

58.3

41.3

33.6

27.4

20.5

14.3

14.1

9.8

178.8

171.0

170.6

59.9

41.8

33.4

27.5

20.1

14.1

13.8

9.4

* May be interchanged

LA LA 0

Figure Six. FAB mass spectrum of thiopental sodium

loo r 115

+ ( M - N a )

287

(M+2Ya-H)+ 1 309

100 150 200 250 300 350

M/Z

Figure Seven. Electron impact (EI) mass spectrum of thiopental

100

50

0

172

157

50 100 150 200 250 300

M / Z

Figure Eight. Fragmentation pathway for thiopental

H I

CH3 m/z 242

/% ~-c5xlo

H I

OH + m/z 173

H H

- OH OH +' +

m/z 172 m/z 157


reported by Maurer [ 151. The most prominent ions were at m/z 242

(%), 173 (21%), 172 (42%), 157 (26%) and 29 (100%). A possible

fragmentation pathway is shown in Figure Eight.

3. SYNTHESIS

Two methods of preparing thiopental are outlined in Figure Nine.

In method A, ethyl (1-methylbutyl) malonate (I) was condensed with

thiourea (II) in the presence of sodium ethoxide in absolute alcohol.

After heating at reflux for several hours the solvent was removed and

the residue dissolved in cold water. Thiopental (III) was precipitated

by the addition of dilute hydrochloric acid. Further purification could

be accomplished by dissolving III in dilute sodium hydroxide and

precipitating with carbon dioxide [6].

In method B, the appropriately substituted nitrile (IV) was condensed

with thiourea (II), again in the presence of sodium ethoxide in absolute

alcohol. Heating at reflux for several hours resulted in the 4-imino

compound (V) which was hydrolysed to thiopental[16].

Thiopental sodium can be prepared by treating an alcoholic solution of

thiopental with one equivalent of sodium hydroxide and removal of the

solvent [6].

These two methods formed, respectively, the basis of the American

and British patents for the production of thiopental[17]. Upon

comparison of the two methods under laboratory conditions it was

concluded that method A was preferable [17].

Figure N i n e . Synthesis of thiopental

Method A.

c H 3 c H 2 2 H 3

CH,CH, CHCH,CHzCH3 0 CH,

I 11

Method B.

CH3CH20

CN CH, IV + o

i) NaOCH2CH3 - ii) H+

H I

H I

'YJ N ,CH,

H' CHCH,CH,CH, 0 I

III CH,

H I - CH,CH, H20 'YJ ,CH,

N

H' CHCH,CH,CH, H' CHCH,CH,CH,

NaOCH,CH3

I NH I 0 . .. .

CH, V

CH, 111


4. STABLLITY

According to the Codex [5], aqueous solutions of thiopental sodium

decompose upon standing and solutions should not be used if they have

become cloudy or contain precipitates or crystals. A shelf life of 5

days has been reported for thiopental in glycine/NaOH buffer at pH 9.0

[18] while a thiopental sodium for injection solution (2.5% in water) is

stable for at least 10 days at 25 O C [19].

In normal saline (0.9%), when stored in plastic infusion bags,

thiopental sodium loses up to 23% of its activity in one day [20]. This

loss was attributed to sorption onto the polyvinyl chloride (PVC) of the

infusion bags [20]. Furthermore, absorption onto the plastic matrix of

an intravenous delivery system and concomitant loss of activity, has

also been observed [21]. Conversely, a solution of thiopental sodium

stored in disposable plastic syringes showed only negligible loss of

potency after 5 days at 25 OC and 45 days at 5 "C [22]. A recent study

has examined the interaction between thiopental sodium and infusion

containers and found no decrease in potency when stored for 24 hours

at 21 OC in the dark [23]. The initial study [20] was carried out at pH

6.0 while the more recent study was carried out at pH 9.1 [23]. The

higher sorption rate at low pH was attributed to a greater fraction of

thiopental being nonionized [23]. This is consistent with no loss of

thiopental activity being seen in the study using plastic syringes 1221

which was also carried out at high (10.1) pH.

The effect of y-irradiation on thiopental has also been investigated

124). No evidence of decomposition was observed with a 2.5 Mrad

radiation dose. It was concluded that thiopental, in powder form, may

be sterilised by y-irradiation [24].

556 MICHAEL .I. MCLEISH


5.1 Extraction

Thiopentone sodium, although soluble in water, in acid solution is

converted to the free acid (thiopental) which is water insoluble. The

assay of thiopental sodium in both biological fluids and proprietary

preparations takes advantage of these acid-base characteristics.

Extraction is accomplished by acidification followed by shaking with

organic solvents. Solvents commonly employed include chloroform

[25-271, methylene chloride [28-311 and diethyl or petroleum ether

[32-361. Less frequently used are benzene [37], toluene [38,39], ethyl

acetate [40,41], n-hexane [15,28] and n-butyl chloride [42]. Additional

selectivity and sensitivity was obtained by back-extraction into sodium hydroxide [25,28,3 5,36,42,43].

More recently an extraction procedure using a solid phase column

(Bond-Hut C18) has been reported [MI.

5.2 Identification

5.2.1 USP Analysis 121

Dissolve about 500 mg thiopental sodium in 10 mL water in a

separator, add 10 mL of 3 N hydrochloric acid, and extract the

liberated thiopental with two 25 mL portions of chloroform. Evaporate

the combined chloroform extracts to dryness. Add 10 mL of ether,

evaporate again and dry at 105 OC for 2 hours: the infrared absorption

spectrum of a potassium bromide dispersion of the residue so obtained

exhibits maxima only at the same wavelengths as that of a similar

preparation of USP Thiopental reference standard.


5.2.2 B.P. Analysis 141

A.

carbon dioxide free water with 2 M hydrochloric acid. The solution

effervesces. Shake the solution with 20 mL of ether, separate the ether

layer, wash with 10 mL of water and dry over anhydrous sodium

sulphate. Filter, evaporate the filtrate to dryness and dry the residue at

100 to 105 OC. The infrared absorption spectrum of the residue is

concordant with the spectrum of thiopental EPCRS.

Acidify 10 mL of a 10% w/v solution of thiopental sodium in

B. Determine the melting point of the residue obtained in test A

and of a mixture of equal parts of the residue and thiopental EPCRS.

The difference between the melting points, which are about 160 OC, is

not greater than 2 O C .

5.3 Colorimetric, Spectrophotometric and Fluorimetric Analysis

Early estimations of thiopental and other thiobarbiturates depended on

color reactions with either cobalt [27] or copper [34]. These

estimations were neither accurate nor particularly sensitive and were

superseded by ultraviolet spectrophotometric methods [33,35,45].

Thiopental has W absorption maxima at 290 nm and 305 nm in acidic

and alkaline media, respectively. In contrast, the oxobarbiturates have

absorption maxima at 220 nm and 255 nm, respectively 1461. These

differences provided a means of distinguishing thiopental from its

major metabolite, pentobarbital, and as a consequence most

determinations were carried out at around 280 nm [33,35,45]. In one

case greater selectivity was provided by a change in extraction solvent,

which permitted the determination of the carboxylic acid metabolite of

thiopental[35]. Additional sensitivity could be provided by the use of

back-extraction methods (vide supra).

558 MICHAEL J . MCLEISH

The UV methods reported minimum detectable limits of approximately

0.5 pg/mL. The development of a spectrofluorimetric method (hex= 305 nm, A,= 505 nm) lowered this limit to 0.1 pg/mL [36].

5.4 Chromatography

5.4.1 Paper, Column and Thin-Layer Chromatography

Raventh 1461 lists a number of methods using paper chromatography

for the identification of thiopental and other barbiturates. These

methods all show relatively poor sensitivity with a minimum of 50 pg

required for the positive identification of most barbiturates.

Alumina column chromatography using 2% methanol in chloroform as

eluant was employed in the determination of thiopental in tissues, urine

and blood 1341. Thiopentone (>2 mg) showed as a dark band under

ultraviolet light.

Thin-layer chromatography using silica get has been used to isolate

and identify thiopental[36,47]. Elution from silica gel was achieved

with benzene-glacial acetic acid (1:9). The Rf under these conditions

was 0.47, the recovery better than 95% and the sensitivity as low as 0.5 pg [36]. Other solvents have included chloroform-acetone (9:l) and

dioxan, benzene and aqueous ammonia (20:75:5) [47]. Using a

potassium permanganate spray, thiopental could be identified as a

yellow spot on a purple background [47].


Table 4, although by no means exhaustive, provides a summary of the

numerous methods that have been developed for the gas

T a b l e 4. GC Methods for the determination of thiopental ~~

COLUMN / SUPPORT DETECTOR DEIUVAT'IZATION SENSITIVITY REF.

3% Neopental Adipate

3% Poly A-103 on Gas Chrom Q

3% SE-30 on HP Chromsorb WP

3% OV-17 on Gas Chrom Q

5% OV-1 on HP Chromosorb W

3% OV-17 on Gas Chrom Q

2% SP2110 - 1% 2510 DA on Supelcoport

5% OV-101 on HP Chromosorb G

FID

Alkali - FID

FID

ECD

m

NP-FID

F!ID

FID

none

none

TMPAH methylation

Th4AH methylation

none

Iodomethane methylation

none

none

25

37

38

40

28

43

29

15

560 MICHAEL J. MCLEISH

chromatographic analysis of thiopental [ 15,25,28,29,37,38,40,43].

Generally these methods have achieved much greater selectivity and

sensitivity than colorimetric methods, with low nanogram levels being

measured using alkali-flame or nitrogen-phosphorus detectors [37,43].

However, the lowest detection limit (100 pg) was obtained using an

electron capture detector [40].

In most cases the extraction of thiopental was achieved using

procedures described in section 5.1. When required, methylation was

the favored method of derivatization with reagents including

trimethylphenyl ammonium hydroxide F/IpAH, 381,

trimethylanilinium hydroxide [TMAH, 401 and iodomethane [43].

Gas chromatography has also been combined with mass spectrometry

to develop a computerised general screening procedure for

barbiturates, including thiopental [ 151.

5.4.3 High Performance Liquid Chromatography

In recent times HPLC appears to have become the method of choice

for the assay of thiopental. As detailed in Table 5 all the methods have

employed reversed phase columns and ultraviolet detection. The

variation in mobile phases and detector wavelength has been primarily

to enable determination in different body matrices or to permit the

simultaneous determination of thiopental and either its metabolites or

another drug. For example, particular attention has been paid to the

simultaneous measurement of thiopental and its active metabolite,

pentobarbital [26,31,42,44,48 1.

When developing these assays much attention was focussed on sample

preparation. For many of the methods sensitivity and selectivity were

of prime importance and consequently extraction methods were

Table 5. Conditions employed for the HPLC determination of thiopental

COLUMN MOBILE PHASE DETECTOR INTERNALSTANDARD SENSITIVlTY REF.

c18 -5p MeCN / H,O w, 254nm Nucleosil (32 : 68)

c8 - l o p Radial-Pak

-04 ( pH 7.7) / MeoH / THF (13 : 7 : 4)

W, 254nm

c g - l o p MeOH / H20 W, 29Onm (60 : 40)

c18 - 7 p NaP04 (0.05M, pH 4.6)MeCN W, 195nm LiChroCart (1 : 1)

c18 - 5 p MeOH / KPO, (O.OlM, pH 4.4) W, 284nm pBondapak (1 : 1)

c18 5p MeOH / H20 W, 280nm Spheri-5 (60 : 40)

Secobarbital 1 oong 48

none 125ng 44

Phenolphthalein n.s. 30

Hexobarbital n.s. 39

5 -Ethyl-5 -p-tolylbarbituric 30Ong 31 acid

Phenolphthalein 30Ong 49

- - - - - - - - - - - - - - continued

c6 - 5 p NaOAc (O.OlM, pH 3.6) / MeCN U V , 28Onm Flunitrazepam 50Ong 50 Spherisorb (70 : 30)

c18 - 5 p NaP04 (0.16M, pH 6.6) /THF (86 : 14)

c18 - 1op-n @ondapak

KP04(pH 7.8) / MeCN / THF (78 : 22 : 4)

ci8 - 5cUn Spheri-5 (52 : 48)

Do4 (O.OMM, pH 6.5) / MeOH

low MeOH / H,O Spherisorb (1 : 1)

c18 KCL (0.2M, pH 2.0) / MeOH Wondapak (1 : 1)

CIS - l o p Partisil10/25 (55 : 45)

NaCit (0.1%, pH 6.5) / MeOH

KPO4 (0.2M, pH 4.0) / MeCN sil-x-1 (9: 1)

W, 24Onm Barbital l0Ong 42

W, 254nm Pentobarbital l0Ong 26

W, 28Onm Phenolphthalein n.s. 51

W, 2541x11 Methohexitone n.s. 32

W, 254nm none 90% 52

W, 254nm Quinoline 50Ong 53

W, 205nm Bupivicaine 5 M 41

0


favored. However, others have concentrated on the rapidity and ease

of sample preparation. In these cases the preparation was limited to

the precipitation of plasma proteins with either acetonitrile [49-511 or

ethanol [52], with the supernatant being injected directly on the

column. In the most extreme example untreated plasma was also

injected directly onto the column [53]. Not unexpectedly, this method

suffered in that column efficiency was rapidly lost.

The sensitivity of most methods was 300 ng/mL, or better, which is

ample for monitoring plasma levels during thiopental infusion. During

continuous treatment plasma levels of even unbound thiopental are

generally greater than 500 n g h L [541.

5.5 Radioirnmunoassay

Flynn and Spector [55] developed a radioimmunoassay for a number of

barbiturates, including thiopental. The sensitivity for the latter was

100 ng, a figure tenfold higher than for its oxo-analogue, pentobarbital.

This indicated that the urea portion of the ring was critical in

determining antibody specificity [%I.

6. METABOLISM

The metabolism of thiopental and other thiobarbiturates has been

reviewed extensively [46,56-581. In mammals the biotransformation

of thiobarbiturates appears to take place by up to four different

pathways [46,56-581:

i) Side-chain oxidation

ii) Desulphuration

iii) Hydrolysis of the thiobarbiturate ring


iv) N-dealkylation

7.

In man, hydrolysis of the thiobarbiturate ring of thioperital does not

occur [58], and biotransformation by the liver microsomal oxidase

system appears to be the main route of elimination from the body [59]. However, in spite of the many studies of thiobarbiturate metabolism,

the fate of the majority of the administered dose is yet to be identified

[58]. What is clear is that less than 0.5% of the dose is excreted as

unchanged thiopental [35,60].

The major known pathways [58] of thiopental metabolism in humans

are shown in Figure Ten. Of these, conversion to pentobarbital seems

to be of minor significance, with only a small proportion of the

administered dose being excreted as the desulphurated metabolite

[25,60]. Oxidation of the side-chain appears to be the major pathway

for metabolism in humans, 10-25% of the administered dose being

excreted in urine as the carboxylic acid metabolite [35,60]. Carroll et

al. 1601 also demonstrated the formation of the hydroxy metabolite,

albeit in small amounts.

USES, ADMINISTRATION and CONTRAINDICATIONS

Thiopental sodium is a barbiturate which is administered intravenously

for the induction of general anaesthesia or for the production of

complete anaesthesia of short duration [3]. Other uses include the

supplementation of regional anaesthesia or low potency agents such as

nitrous oxide, the control of convulsive states and as a hypnotic [3,61].

In psychiatry it has found some use as an aid in diagnosis, and as a

treatment of some disorders 1611.

Thiopental sodium is administered intravenously as a 2.5% or 5%

Figure Ten. Metabolism of thiopental in humans

H I

/ H I

oyJ N H2CH3

H/ CHCH2CH2CH3 0 I

Pentobarbital

ST-@---. N CH2CH3

0 I H’ CHCH2CH2CH3

I CH3 \ H I

syJ N H2CH3

H/ CHCH2CH2COOH 0 I

CH3

Thiopental Carboxylic Acid

H I

sTJ N CH2CH3

0 I CHCH2CH(OH)CH3 H/

Thiopental Alcohol

566 MICHAEL I. MCLEISH

solution, the dose for induction being 100-250 mg administered over

10-20 seconds [3,61]. For longer operations it may be given as an intravenous drip, or additional injections of 50-100 mg may be given

as required [3].

The physical incompatabilities of thiopental sodium which are

sometimes observed [62] have been attributed to:

a. acidic solutions that precipitate the free acid (thiopental)

b. calcium or magnesium solutions that form insoluble carbonates

c. amine salts that liberate the free base in alkaline solutions

There are few absolute contraindications to the use of thiopental

sodium, but porphyria is generally considered to be completely

restrictive [61]. Extra care with both dosage and rate of administration

is requited in cases of severe haemorrhage, burns dehydration, severe

liver disease, status asthmaticus, severe anaemia, raised intracranial

pressure, and some metabolic diseases such as thyrotoxicosis and

diabetes 1611.

8. PHARMACOKINETICS

Thiopental sodium is rapidly and efficiently absorbed following either

oral or rectal administration [63,64]. However, clinically thiopental is

administered as an intravenous injection whereupon it is extremely

rapidly taken up by the brain. Equilibrium of brain and plasma

thiopental is achieved within about one minute 165,661 and is followed

by a speedy decrease in the brain concentration, approximately half the

maximal concentration remaining after five minutes [65,66]. The rapid

decline in brain and plasma concentration has been attributed to the

redistribution of the drug to other body tissues [58, and references

therein] and is responsible for its extremely short duration of action.


The plasma concentration curve for thiopental shows phases

corresponding to its distribution to lean tissue, to adipose tissue and to

its elimination from the body [67]. Although early studies showed the

elimination half-life to be of the order of 2-5 hours [35,68,69], these

studies were carried out using inadequate sampling times (less than

three half-lives). Recent studies have determined the elimination

half-life to be approximately 12 hours [67,70,71], although the half-life

has been shown to be significantly longer in babies [72,73] and the

elderly [74-761. Conversely, in patients aged between 5 months and 13

years the elimination half-life is considerably shorter than in adults

[77], an observation attributed to children having a relatively higher

hepatic mass [59].

To produce surgical anaesthesia, it has been seen that a plasma

thiopental concentration of 39-42 pg/mL is necessary [78]. The

average dose required for induction is essentially independent of age in

patients between 20 and 60 years [59]. A reduction in dose may be

required for patients over the age of 60; with severely deteriorated

hepatic function, with moderately affected kidney function [59], or

those heavily prernedicated with narcotics and other central

depressants [61].

In some cases thiopental is used as a primary hypnotic and is

administered as an infusion, over several days. If the plasma

concentration does not exceed 15-20 p g / d the pharmacokinetics are

essentially the same as those for bolus administration [54,79].

9. ACKNOWLEDGEMENTS

The author would like to thank Abbott Australia Pty. Ltd. for providing

568 MICHAEL .I. MCLEISH

10.

1.

2.

3.

4.

5.

6.

7.

8.

9.

10.

11.

12.

13.

14.

the sample of thiopental sodium (Lot No: 58355WB) used for spectral

analyses. Thanks are also due to Denis Morgan and Malea Kneen for

critical reading of the manuscript.

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TICLOPIDINE HYDROCHLORIDE

Fahad J . Al-Shammary and Neelofur Abdul Aziz Mian

Clinical Laboratory Sciences Department

College of Applied Medical Sciences




Copyright Q 1992 by Academic Press, Inc. All rights of reproduction reserved in any form. 573

514 F.J. AL-SHAMMARY AND N.A.A. MIAN

CONTENTS

1 Introduction

2 Description 2.1 Nomenclature

2.1 . 1 Chemical Names 2.1 .2 Generic Names 2.1.3 Properietary Names

2.2.1 Empirical 2.2.2 Structural 2.2.3 CAS (Chemical Abstract Service Registry

Nu m ber) 2.3 Molecular Weight 2.4 Elemental Composition 2.5 Appearance, Colour and Odour

3 Physical Properties 3.1 Melting Range 3.2 Solubility 3.3 Action 3.4 Indications 3.5 Partition Coefficient 3.6 LD50 3.7 Compression Properties 3.8 X-Ray Powder Differaction 3.9 Spectral Properties

2.2 Formulae

3.9.1 Ultraviolet Spectrum (UV) 3.9.2 Infrared Spectrum 3.9.3 Nuclear Magnetic Resonance Spectra

3.9.3.1 1H.NMR.Spectrum 3.9.3.2 1 3C .NMR .Spectrum

3.9.4 Mass Spectrum

4 Synthesis

TICLOPIDINE HYDROCHLORIDE 575

5 Pharmacokinetics 5.1 Absorption and Distribution 5 .2 Metabolism 5 . 3 Elimination and Excretion 5.4 Adverse Effects and Precautions 5.5 uses

6 Methods and Analysis 6 .1 Elemental Analysis 6 .2 Spectrophotometric Determination 6 .3 Chromatographic Methods

6.3.1 Gas Liquid Chromatography (GLC) 6.3.2 Thin Layer Chromatography (TLC) 6.3.3 High Performance Liquid Chromatography

(HPLC)

7 Acknowledgements

8 References


TICLOPIDINE HYDROCHLORIDE

1 INTRODUCTION

Ticlopidine ( l ) is an inhibitor of platelet action that has been used in the treatment of a variety of disease states in which platelet play a prominent role. Studies in animal and in man have demonstrated that ticlopidine is a potent inhibitor of platelet aggregation induced by adenosine diphosphate (ADP) and variably inhibits aggregation due to collagen, adrenaline (epinephrine), archidonic acid, thrombin and plate activating factor. Inhibition of platelet aggregation is both dose and time-related, with it's onsset of activity being 24 to 48 hours. It's maximal activity occuring after 3-5 days and its activity still being present 72 hours after a final dose.

Ticlopidin (2) is potent and specific platelet aggregation inibitor and antithrombotic agent, exhibiting a sustained effect and wide spectrum of activity.

Ticlopidine (3) is superior to aspirin and dipyridamole as anti-thrombotic agent towards different kinds of experimental thrombosis .

2 DESCRIPTION

2 . 1 m e nclature


( a ) 5- [ (2-c h I or op h e n y I) met h y I] -4 ,5,6,7- tetra h y d ro - ethieno [3,2-C] pyridine (2.4);

( b ) 5- (0-c hloro bent y I) -4 ,5,6,7- t et r a h y dro t h ie no [3,2-C} pyridine (1, 2, 4)

( c ) 5-(2-chlorobenzyl)-4,5,6,7-tetrahydrothieno [3,2-C] pyridinehydrochloride (5)

2.1.2 Generic Names

Ticlopidine hydrochloride, Ticlopidina.


Anagregal, Aplaquette, Caudaline, Opteron, Panaldine, Ticlid, Ticlodix, Ticlodone, Tiklyd, Ticlosan.

2.2.1 EmDi r icaL (4,5)

C14H14CI N S (Ticlopidine) C14HlqCI N S.HCI (Ticlopidine Hydrochloride)

2.2.2 S t r u c t u r a l

Tic lo pi dine Hydrochloride (1 )

CAS (Chemical Abs- Service Reaistrv Number1 (2, 5)

2.2.3

15 5 1 42 - 85 -3) [ 5 3 8 8 5 - 3 5 - 1 ]

(Ticlopidine) (Ticlopidine Hydrochloride)

2 . 3 Molecu lar Weiaht

263.78 (Ticlopidine) (4) 300.2 (Ticlopidine Hydrochloride) (5)


Ticlopidine: C 63.75% H 5.35% CI 13.44% N 5.31 % S 1 2.1 5 %


Ticlopidine Hydrochloride: C 56.01 % H 5.03% CI 23.65% N 14.006% S 10.68%

2 . 5 &Jl!m3nce.Color.-~

Ticlopidine hydrochloride is a white, odorless crystalline powder.

3 PHYSICAL PROPERTIES

3 .1 Meltina Range (4)

MP = 18900 for Ticlopidine Hydrochloride

3.2 A c t i o n (2)

Potent and specific platelet aggregation inhibitor and antithrombotic agent, exhibiting a sustained effect and wide spectrum of activity.

3 . 3 Jnd ica t lons ( 2 )

Prevention and correction of platelet disorders in surgical patients undergoing extracorporal circulation and in long term hemodialysis patients.

3 . 4 p a r t i w n Coefficient . . . .

The PKa of Ticlopidine is 7.64 (6)

3 . 5 LR50 ( 4 )

55 mg/kg/24 hrs (IV in mice). >300 mg/kg/24 hrs (orally in mice).

. . 3 . 6 S o l u u

Almost soluble in water, soluble in 95% alcohol also soluble in methanol, chloroform and insoluble in ether.

Comparative evaluations of aqueous film coated tablet formulations by high humidity aging was studied by Chowhan, Z. T; et a1.(7) Dissolution rate studies of 3 film coated formulations of ticlopidine. HCI compared by storage under


95% relative humidity at 23 and 37% showed that tablets coated with a formulation containing Eudragit E 30D dissolved more slowly before storage and the dissolution became very slow after storage. Tablets coated with 10% hydroxypropyl Methyl cellulose or Ethyl. Cellulose colloidal dispersion also dissolved slower after storage except that the dissolution rate of tablets coaled with 10% hydroxy propyle-Me-Cellulose increased af'ter 12-15 week storage at 25%. In general the decrease in dissolution rate is related to the nature of the film coating temp. of storage, amount of moisture gain, and tablet core formulation. Thus to maintain good dissolution throughout the shelf life of film coated tablets they should be protected from high relative humidity.

3.7 Comp ression Proberties

Z.T. Chowhan and Y.P. Chow (8) studied the role of the granulation moisture content on compression properties of granules made with selected binders. The results suggested that at lower pressures, higher moisture containing granules were slightly more compressible than lower moisture- containing granules. However at higher pressures, the reverse was true because of the water lubrication effect. At lower moisture levels, the crushing strength of the tablets was dependent on the binder, at higher moisture levels, binder differences became less significant.

3 . 8 X-rav Powder Differaction

The X-ray differaction pattern of ticlopidine hydrochloride was determined using Philips full automated x-ray differaction spectrogoniometer equipped with PW 1730/10 generator. Radiation was provided by a copper target (Cu annode 2000W, y= 1.5480 Ao). High intensity x-ray tube operated at 40 Kv and 35 Mv was used. The monochromator was a curved single crystal one (Pw 1752/00). Divergence slit and the receiving slit were 0 and 0.10 respectively. The scanning speed of the goniometer (Pw 1050/81) used was 0.02-2 0 per second. The instrument is combined with Philips PM 8210 printing recorder with both analogue recorder and digital printer. The goniorneter was aligned using silicon sample before use. The x-ray pattern of Ticlopidine hydrochloride is presented in Fig,(l). The values of scattering angle 2 8 interplanner distance dAO and relative intensity 1/10 are shown in the table ( 1 ) .

( z e- VALUE)

Fig. ( 1 1 X-Ray powder Diffraction of Ticlopidine Hydrochloride

TICLOPIDINE HYDROCHLORIDE 58 I

Table (1): Characteristic Lines of the X-ray Powder Diffraction of Ticlopidine Hydrochloride.

20 dA I / lo% 20 dA I / lo%

9.236 11.817 12.622 14.422 16.233 16.466 17.658 18.825 19.009 19.847 20.073 22.578 23.043 23.876 24.929 25.673 26.425 27.128 27.91 28.566 29.213 29.751 30.037 30.800 31.75 32.974 33.981 34.41 6 25.578 26.01 0 36.436

9.5746 7.4889 7.01 27 6.1416 5.4602 5.3834 5.0227 4.71 39 4.6686 4.4734 4.4234 3.938 3.8596 3.7268 3.571 7 3.4698 3.3728 3.287 3.1 966 3.1247 3.057 3.0028 2.975 2.9029 2.8183 2.7164 2.6381 2.6058 2.5233 2.494 2.4658

7.193 10.973 0.057 100 9.836 5.386 11.408 19.003 14.31 9 12.345 4.884 10.806 14.084 25.46 12.947 24.422 16.627 8.999 3.029 20.809 3.345 2.709 3.41 2 3.479 9.769 3.51 2 9.501 2.408 5.31 9 2.843 4.583

36.859 2.4385 37.725 2.3845 38.192 2.3564 39.461 2.2835 40.261 2.2400 40.710 2.2163 42.240 2.1 395 42.960 2.1 053 43.496 2.0805 44.423 2.0393 44.824 2.022 46.72 1.9442 48.41 8 1.8799 48.71 9 1.8690 50.099 1.8207 50.288 1.8143 52.095 1.7556 52.61 1.7396 53.453 1.71 41 53.773 1.7047 55.35 1.6598 56.273 1.6347 56.99 1.6159 58.879 1.5685 61.61 3 1.5052 64.749 1.4397 70.693 1.3325 77.876 1.2266 78.81 8 1.21 43 83.01 3 1.1 633

2.843 4.884 2.877 3.278 3.445 2.375 2.107 2.944 2.475 3.479 2.107 3.21 1 3.613 3.41 2 3.178 3.579 1.706 2.643 2.810 1.806 1.873 2.576 2.074 1.304 11.572 1.706 1.438 1.271 1.237 1.572

2 0 = scatterina anale. dA = intemlanner distance. - - I/lo% = relative intensity based on highest as’ 100.


3 . 9

3.9.1 Ultraviolet Spectrum tuvl

The UV spectrum (9) of ticlopidine hydrochloride in H20 (7 mg %) was scanned from 200 to 400 nm (Fig. 2) using LKB 4054 UV/Vis spectrophotometer. Ticlopidine hydrochloride exhibited the following UV data (Table 2).

Table (2) UV Data of Ticlopidine

1

x (€1 n.m max Absorbance Molar Absorptivity A'

cm-1 gm mol/L

21 4 2.127 9121.79 303.8

268 0.092 394.5 13.14

295 0.014 60.04 2

3.9.2 Infrared Seect r u q

The 1R spectrum (9) of Ticlopidine hydrochloride as KBr disc was recorded on a Perkin Elmer 1210 Infrared Spectrometer. Fig. (3) shows the infrared spectrum of Ticlopidine hydrochloride. The structural assignments of Ticlopidine hydrochloride have been correlated with the following frequencies (Table 3).

3.9.3 Nuclear M aanetic Reso nnance SD ectra

3 .9 .3 .1 PMR Spectrum

The PMR spectra (9) of Ticlopidine HCI in D M s 0 - d ~ (Fig. 4-6) was recorded on a varian XL 200 MHZ NMR spectrometer using TMS as an internal reference. The following structural assignments have been made (Table 4).

0 0

-#

0

OD

m

0

u)

m

0

-s m

0

(Y

m

0

0

m

0

N

@

0

lo

N

0

Y N

0

(Y

(Y

0

0

cy

aJ E

B -c 0

0

U r I

aJ C

L

.- 2

n

0

0

I- 0

A

.- rc

E

l5 3

L

aJ c1 v,

7

3

c

c\

- 0

.- LL

jb I I I I I

4 000 3000 2 000 1500 1 boo 6r WAVEN UM 6 ER S

Fig. ( 3 1 Infra Red spectrum of Ticlopidine Hydrochloride

0

6.908 7

LD

V

I

0

r/) x 0

c

aJ U

L

0 r

U

0

L

U

%

11 aJ c U a

0

U

I- %- 0

.C

.- d

.- .- - .- 5 L t

U

a, P

v)

a

z z I

I

7 C

I

4

--c

m

.- u,

1 (Expansion Fig. ( 5 1 H-NMR Spectrum of Ticlopidine Hydrochloride of peak

N

m s- w

I

4.J I

1 1 1 I 1 1 I I I I I 1 I I

12 10 a 6 ’ L 2 0 PPM

1 Fig. ( 6 1 H-NMR spectrum of Ticlopidine Hydrochloride in DMSO- d 6

( DzO Exchange )


Table (3) IR Characteristics of Ticlopidine Hydrochloride

Frequency cm-1

3400

3020, 3040

2260

1590, 1560

1430, 1425

1280

1220, 1200

1160

1080, 1060, 1020, 1000

750, 560, 720

Assignment

NH stretch and plannar bend

Chlorophenyl CH Stretch

C-S-C stretch

Chlorophenyl ring stretch

Pyridine methylene wag

Methylene twist

Chlorophenyl C-CI stretch and bends

Pyridine ring stretch

Pyridine-methylene rock

Chlorophenyl spatial bend

3.9.3.2

l3C-NMR spectrum (9) of ticlopidine in DMSO-de (Fig. 7-9) was recorded on varian XL-200 NMR-spectrometer. The multiplicity of the resonances was obtained from APT (Attached Proton Test) and DEPT (Distortionless Enchancement by Polarization Transfer) programs. The 13C- NMR spectrum displayed all the fourteen carbon resonances. The narrow resonance range of some of the carbons makes the spectrum rather complex. The carbon chemical shifts assignments are presented in table (5).

L'J I

I

180 160 140 120 100 80 60 40 20 0

13 F i g . ( 7 ) C- NMR spectrum of Ticlopidine Hydrochloride

P PM

065

13 Fig. ( 8 1 C - NMR Spectrum of Titlopidine Hydrochloride in DM SO -d 6 (APT)

C H 3

c MX

13 Fig, ( 9 ) C-NMR SPECTRUM OF TICLOPIOINE H d IN DMSO-d (DEPTI

6


TABLE (4 ): PMR Characteristics of Ticlopidine HCI

Structure

g C ____------__________--__----__----____

Protons I (PPm) I Multiplicity

a,b 8.086 - 8.131 m

g.h,i,j 7.457 - 7.577 m

f 6.908 - 6.934 d

d 4.609 S

C 4.277 S

e 3.424 S

The mass spectrum (9) of Ticlopidine HCI obtained by electron impact ionization (Fig. 10) was recorded on a Finnigen MAT 90 spectrometer.

The spectrum was scanned from 50 to 500 a.rn.a. Electron energy was 70 ev. Emission current 1 mA and ion source pressure 10-6 torr. The most prominent fragments and their relative intensities are presented in Table (6 ).

4 SYNTHESIS

4 . 1 Scheme I

Ticlopidine Hydrochloride is prepared (1 0) by the treatment of 4, 5, 6,7-tetrahydrothieno [3,2-C] pyridine with

0

0

m

0

0

0

c

Fig. (10 1 Mass spectrum o f Ticlopidine Hydrochloride

594 F.J. AL-SHAMMARY AND N.A.A. MlAN

. . Table (5) Carbon-13 Chamical Shifts of TiclqpLQUle

Chemical Shift

21.529

48.91 1

49.698

54.336

134.61 9

131.401

127.726

127.763

127.581

124.898

133.863

129.793

131.333

125.257

2-chlorobenzyl chloride in the presence of Pot. Flouride. The reaction mixture is stirred at 50% for three hours in THF. (Scheme I).

4.2 Scheme II

Yamanochi et al (11) developed a method for the preparation of 4, 5, 6, 74etrahydrothieno [3,2,-C] pyridine by treating 2-(24hienyl)ethylarnine and HCHO at 9OOC for three hours. The reaction mixture was extracted with CgHg which was recrystallised with CgHe/hexane mixture to give 1, 3, 5-


tris(thieny1 ethyl) triazine. A solution of 1, 3, 5- tris(thieny1 ethy1)triazine in (CH3)zCHOH was added dropwise to (CH3)zCHOH containing HCI at 50oC and reaction mixture was stirred at 50oC for 5 hours to give 81% of 1,3,5-tris(thienyI ethy1)triazine. (Scheme 11).

4 . 3 Scheme Ill

Ticlopidine HCI has also been synthesized (12) by the reaction of 4, 5, 6, 7-tetrahydrotheno[3,2-C] pyridine with O-CIC6H6COCI in CHCl3 - aq. NaOH at room temperature for overnight. Which was further treated with AIH(CH&HMe2)2 in toluene at 90-95OC for 2 hours.

Ticlopidine has also been synthesised by other methods (1 3- 16) .

TABLE (6): The Mass fragments of Ticlopidine HCI

m / z

110.4

125.2

Relative Intensity

100

25%

Ions


Scheme I CI

OS) + &cH2c'

Ticlopidine Scheme I1

r ; S f ' z i W

for 90°C for 3h.,~,

4 , 5, 6, 7, tetrahydrothjeno [3,2-C] pyridine


Scheme 111

0 5- (O-~hlorobenzoy1)-4,5,6,7, tetrahydrothieno

[ 3,2-C] pyridine

/ CH3

A1..H(CH2CH 1 2 ' CH3 1 in toluene

Ticlopidine


5 . 1 Absorption and Distribution

About 80-90% of an oral dose of the drug absorbed after oral administration in rat or man (17, 18). After a single dose in rats or man, peak plasma concentrations occured at 1-3 hours (17, 18, 19, 20).

In human volunteers and patients given single doses of 500 mg, peak plasma ticlopidine concentrations were 0.61 and 0.82 mg/L respectively. Accumulation was not noted in multiple-dose studies (18). In volunteers given a single oral dose of 1000mg, peak plasma concentrations were 2.13 mg/L those given repeated doses of 250 mg twice daily for 21 days. had peak concentrations of 0.90 mg/L (18).

In rats given single or repeated doses, highest ticlopidine concentrations were measured in the liver, kidneys, duodenum and fat tissues. In pregnant rats, conc. in fetal blood were 40- 90% of those in maternal blood, and fetal, placental and amoniotic conc. were appreciable (20). Plasma protein binding has not been studied in vivo, but in rats 60% of circulating radiolabelled ticlopidine was distributed to plasma and 40% blood cells (1).

Ticlopidine HCI (21) is readily absorbed from the gastrointestinal tract after oral dosing.

The oral bioavailability of ticlopidine was increased by 20% when taken after a meal. In contrast, absorption of ticlopidine administered after antacid treatment was approximately 20% lower than under fasting conditons. Administration of drug with food is recommended to maximize gastrointestinal tolerance (22).

5 . 2 Metabol ism

The metabolic disposition of ticlopidine is complex with at least four metabolites isolated in man and thirteen in rats (23). The rate of metabolism in man is rapid as even shortly after dosing, when ticlopidine concentrations are at their peak, only 22% of the total radioactivity in plasma represents unchanged ticlopidine (23), and by 15 hours past dose, unchanged ticlopidine represents 6% or less of the total dose in man (17).


The main quantitative metabolic route in man is N.dealkylation, followed by oxidation with opening of the thiophene ring (17) but another metabolic pathway is responsible for the 2-keto derivative of ticlopidine called PCR-3787. This metabolite, which has been found in small concentrations in rat bile, has been found to be 5 to 10 times more potent than ticlopidine itself as an antiplatelet agent, although its potential contribution to ticlopidine's effect is as yet uncertain (24).

Anne Tuang et al (21) has been studied the metabolism of Ticlopidine on rats, the compound was quickly absorbed as evaluated by the time of the peak plasma concentration. The metabolism of drug involved N-oxidation, cleavage of the N-C bond oxidation of aliphatic carbon followed by glycine conjugation. Urinary excretion took place essentially in the first 24 hours and biliary excretion was most pronounced in the hour following dosing. Small amounts of drug were excreted unchanged. The major urinary metabolites were 2- chlorohippuric acid (16% of the dose) and tetrahydrothienopyridine (8%) while Tic1opidine.M predominated in the bile (2% in 0-5 h). The peak plasma concentration of Ticlopidine occured at 0-5 hour. The plasma concentrationAime curve displayed a biphasive profile and the terminal half life of Ticlopidine was tentatively estimated to be 6-10 h in the rat.

Metabolic Path of Ticlopidine

Unchanged Ticlopidine (25) and three metabolites Ticlopidine N-oxide, (T-NO), Tetrahydrothieno pyridine (THTP), and 2,chloro-hyppuric acid (CI-HPA) were isolated from rat urine by differencial solvent extraction and characterized by their behaviour on TLC and GLC. Their identities were confirmed by comparison with authentic standards. A fourth metabolite (T-M) gave rise upon acid hydrolysis to a compound, which co-chromatographed with authentic (T), both on TLC and GLC. The original structure of this metabolites is not yet elucidated. (T) and (T-M) were also found in bile wxtracts, whereas (T-NO), (THTP), and (CL- HPA) were not detected in the bile, under the conditions used.

Urine and bile samples were assayed for the supposed intermediates of (CI-HPA), i.e. 2-chlorobenzyl alcohol (CI- BzOH), 2-chloro-benzaldehyde (CI-BzAld), and 2- chlorobenzoic acid (CI-BzA), however, under the conditions used, only trace amounts of (CI-BzA) were detected by GLC.

F.J. AL-SHAMMARY AND N.A.A. MIAN

Glucuronides or sulphate conjugated metabolites may account for only insignificant amounts, as the TLC patterns of enzyme hydrolyzed and untreated samples were quite similar. Following acid hydrolysis, however, the TLC patterns showed the presence of several though minor compounds. Attempts to characterize these (except T-M) or to make derivatives suitable for GLC have so for failed.

The possibility of hydroxylated metabolites was investigated using various TLC spray-techniques and spot-test reactions upon silicagel eluted materials, but no net reactions resulted.

A scheme for the known metabolic pathway of Ticlopidine is shown in Fig. (11).

5.3 Elimination and Excretion

In man, approximately 60% a radiolabelled dose is recoverable in urine, and 25% infaeces following oral administration (1 7). Ticlopidine concentrations measured as detectable nitrogen by gas chromatography (thus, probably not specific for the parent compound) dropped rapidly from 0.70 mg/L at 2 hours post-dose to 0.15 mg/L at 6 hours post-dose following a single 500 mg oral dose (18). Plasma concentration of unchanged ticlopidine fell rapidly after oral administration of a single 750 mg dose in volunteers (18).

After repeated doses of 250 mg twice daily for 21 days, peak concentrations of 0.90 L 0.18 mg/L fell to trough concentrations of 0.20 L 0.07 mg/L. Elimination half-lives of 24 2 7.5 hours and 33.2 f 3.8 hours have been reported ( 1 8 ) .

Ticlopidine plasma or blood concentrations do not correlate with ex vivo activity as an antiaggregant of platelets (17, 18).

Anne Tuong et al (21) have studied that also high concentrations of unchanged ticlopidine were found in various organs (liver, kidney, and adipose tissues mainly) although only minute amounts of drug were excreted in urine (0.1% of the dose) and in bile (0.02% of the dose).

5.4 Adverse Effects and Precautions

Approximately 10-1 5% of patients receiving ticlopidine have experienced side effects, the most common of which have been

TICLOPIDINE HYDROCHLORIDE 60 I

Ticlopidine (T)

(C1 - B ZOH) (THTP) -----,

(T-NO)

+ acid I 1 T

C1 -BzAld.

C l

C1-BzA

+ Glycine 1 HOOC H2C H N O C

ti

C1-HPA

Fig. (11) Metabolic pathway of Ticlopidine.

602 F.J. AL-SHAMMARY AND N.A.A. MlAN

gastrointestinal complains and skin rash. About 10% experience gastrointestinal discomfort, dyspepsia, abdominal pain, nausea and diarrhoea (1).

Gastro-intestinal disturbances and skin rashes are the most commonly reported side-effects associated with Ticlopidine therapy. Blood dsycrasias, particularly serious in elderly patients have been reports of vertigo and occasional reports of cholestatic jaundice (5)- Gastrointestinal distress may necessitate discontinuation of the medication but may be markedly reduced if the drug is given after meals (26).

Bleeding during ticlopidine therapy is an unusual side effect, but is dangerous in patients who must undergo surgery or another invasive procedure (1). In patients undergoing AV access insertion, there has been no increase in bleeding (27). But in patients undergoing open heart surgery the risk of bleeding may be increased with ticlopidine (28).

Agranulocytosis. neutropenia, thrombocytopenia, and erythroleu-kaemia have been reported during therapy with ticlopidine. Elevation of liver function tests are unusual with ticlopidine therapy, but occasionally cholestatic jaundice or hepatitis have been reported. Drug may increase total serum cholesterol, as well as LDL- and VLDL-cholesterol and other lipoproteins, without effecting HDL-cholesterol (1).

Ticlopidine should not be administered to patients with haemorrhagic diathesis, gastrointestinal ulcers or severe liver dystfunction. It should not be given to patients receiving aspirin, anticoagulants or corticosteroids (5).

5 . 5 Uses

Ticlopidine is an inhibitor of platelet aggregation. It has been given in the treatment of atherosclerotic disease and intermittent claudication in doses of 250 mg once of twice daily by mouth, with meals. Regular haernotological monitoring has been recommended (5).

Ticlopidine (29) is equally effective in both men and woman and also improves symptoms of claudication in patients with peripheral arterial disease and appears to reduce anginal pain. Patients with subarchnoid haemorrhage and sickle cell disease have shown some improvement with ticlopidine ad m i n is t rat ion.


Giuffetti, G; et al (30) studied the treatment with ticlopidine improved the neurologic outcome and the hemorheologic pattern in the postacute phase of ischemic stroke.

The drug (31) is to be effective in influencing the rheological measures of red cell filteribility and membrane microviscosity filteribility was increased and microviscosity was decreased.

Davi G., et al (32) concluded that in schemic hear disease patients the association of ticlopidine and low dose aspirin seems superior toe each drug alone in inhibiting platelet activity and according to Uchiyama S; et al (33) combination of aspirin plus ticlopidine is a potent antiplatelet strategy, in ischemic attach or cerebral infarction. Balsano F; et al (34) concluded that long term treatment with ticlopidine improves walking ability and ankle systolic blood pressure in patients with claudication.

6 METHO DS OF ANA LYSlS

6 . 1 Elemental Analvsis

The elemental analysis of ticlopidine is as reported (4).

Element Composition C 63.75% H 5.35% CI 13.44% N 5.31% S 12.15%

For Ticlopidine Hydrochloride:

Element Composition C 56.01 % H 5.03% CI 23.65% N 4.01 yo S 10.68%

6.2 SDectroDhotornetric Determination

A spectrophotometric study of ticlopidine was carried out by Sanchez Perez (35). Ticlopidine reacts slowly with iodine in CHC13 forming a mol. complex with two change transfer bands


(hmax = 295 nm and Amax = 360 nm) which was also observed after the extraction, into CHC13 of the ticlopidine- iodine complex formed in aqueous solution. Two spectrophotometric methods for the determination of drug based on the formation of the ticlopidine-iodine complex were studied. The first involves the extraction of ticlopidine base from aqueous samples into CHC13 and addition of a solution of ticlopidine. The second involves the formation of the mol. complex in aqueous solution (pH = 7.0) over 90 minutes and its later extraction into CHC13. In both procedures Beer's Law was followed with the ticlopidine concentration rage of 1.6 x l o m 6 - 1.6 x 1 0-5 M for the two max.

6 . 3

6.3.1 Gas Liauid Chromatoa raphv (G LCL

1. Ticlopidine and its metabolites in biological fluids are being analysed by GLC.

Ticlopidine (T), and Ticlopidine N-oxyde (T-NO), were simultaneously solvent extracted and separated by column chromatography (T-NO) was converted to (T) by reduction with SO2 (36) before analysis due to degradation of (T-NO) within the injection port of gaschromatograph. Ticlopidine-M (T-M) was processed as (T) after acid hydrolysis of the aqueous phase. GLC analysis was performed on Hewlett Packard model 5710 gaschromatograph equipped with a nitrogen-phosphorous detector and a HP 3352 data system using a 6 ft x 2 mm ID glass column, packed with 3% OV 17 on Chromosorb WHP 100/120. Injection port temp. 2500, detector temp. 3000, oven temp. 2000. He flow 25 ml/min. The retention times for Ticlopidine was 6.1 min and for internal standard was 3.2 min (25).

2. Another method used to analyse the drug and its metabolites ie. 2-chloro-hippuric acid (CI-HPA) after extraction and methylation of the dry residue with diazomethane, GLC analysis was performed on a Hewlett-Packard model 5830 gaschromatograph equipped with a flame-ionisation detector and an automatic sampler, using a 4 ft. x 2 mrn ID glass column, packed with 1% OV 25 on Chromosorb WHP 100/120, injection port temp. 2400, detector 2500, oven temp. 210°,for 5 minutes then raised to 230° at 10°/rnin.


The methylated derivatives of CI-HPA and the internal standard had the reaction times of 4.0 min. and 8.4 min., respectively (25).

6.3.2 Jhin Laver Chromatoaraphv fTLC)

Folowing extraction and coupling in aqueous medium with sodium naphtoquinone-sulfonate (37) the tetrahydrothienopyridine (THTP) derivative extracted into methylene chloride. The residue after solvent evaporation was quantitatively applied on a silica gel plate (Merck 60F 254, 0.25). and the plate was developed in chloroform- methanol (9O:lO v/v). The orange coloured derivative migrated as a single spot (Rf 0.69) well resolved from co- extracted, absorption measured at 480 nm (25).

2. Giuseppe Musumarra et al (38) analysed Ticlopidine HCI by T.L.C. The drug dissolved in methanol (5 ml) or extracted from an alkaline aqueous solution with ethyl acetate and prepared as a solution containing about 2 rng/ml of drug. The freshly made drug solution were applied approximately 1 cm apart to 20 x 10 cm silica gel 60 F254 HPTLC plates (Merck).

. . 6.3.3 ah Performance L i a U ChrornatoaraDhvl [HPLC)

An HPLC (39) method was developed for determination of the drug and its metabolites in human and rat bile. A stainless- steel column (15 cm x 4.6 mrn I.D.) packed with LiChrosorb RP-8 (Pore size 5pm) or Nucleasil C i 8 (pore size 5pm) was used. The columns were packed by means of a balanced density slurry method specially developed for the ammonia elution system. Gradient elution was performed with water (0.005 M ammonia) to which methanol was added, according to the desired programme. The final elution was usually effected with 100% methanol. Flow rate was lml/min. A wavelength of 235 nm was found suitable for the detection of drug and its metabolites.

7 ACKNOWLEDGEMENTS

The authors are highly thankful to Liberty S. Matibag and Mr. Babikir Awad Mustafa, College of Applied Medical Sciences, King Saud University for their Secretarial and technical assistance respectively in preparing the manuscript.


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1

2

3

4

5

6

7

8

9

1 0

1 1

1 2

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Bruno JJ, Molony BA. Ticlopidine, In Scriabine (Ed) New drugs anual, cardiovascular drugs p. 295-31 6. Raven Press New York (1983).

Panak E. Maffrand JP, Picard-Fraire C, Vallee E, Blanchard J et. al Haernostasis 13 (Suppl. 1): 1-54, (1983).

Knudsen JB, Gormsen J. The effect of ticlopidine on platelet function in normal volunteers and in patients with platelet hyperaggregability in virto. Thrombosis Research 16:

Takegoshi T, Ono K, Mutsubayashi K, Hasirnoto F, Sano M, Metabolic disposition of ticlopidine hydrochloride, a new anti-thrombotic agent, in rats, Pharrnacornetrics 19:

663-671, (1 979).

349-361, (1980).

Anne Tuong; Anne Bouyssou; Josiane Paret; and Tuon Ghi Cuong. European Journal of drug metabolism and pharrnacokinetics 6(2) p. 91-98 (1981).

Sha J; Fratis A; Ellis D; Murakami S; Teitelbaum P; J . Clin. Pharrnacol. 30(8), p 733-6 (1990).

Picard-Fraire C. Pharrnakokinetics and metabolic characteristics of ticlopidine in relation to its inhibitory properties on platelet function. Agents and Actions supplements. Ticlopidine: Quo Vadis, 15(Suppl): 68- 75, (1984).

Aubert D, Bernat A, Ferrand JC, Maffrand JP, Szygenda E. et al. Pharmacological profile PCR 3787; a metabolite of


ticlopidine. From the Seventh International Congress I of Thrombosis, October, Valencia, Spain (1 982).

25 Anne Tong, Anne Bouyssou, Josiane Paret and Tuaong Ghi Cuong, Eurp. J. of Drug Metab. and Pharmakokinetics, 6 (20) p 91-98 (1981).

Goyan JE. Adverse reactions in man, Agents and Actions supplements, Ticlopidine; Quo vadis? 15. 11 6-1 25, (1989) .

2 6

2 7 Ell S, Mihindukulasuriya JCL, OBrien JR, Polak A, Vernham G, Ticlopidine in the prevention of blockage of fistuale and shunts. Abstract 332 from the 7th international congress on Thromasis, p. 180, Valencia, Spain, October 13-1 6, (1982) .

lnstalle E, Gonzalez M, Schoevaerdts JC, Tremouroux J. J. Cardiovascular Pharmacology 3: 1174-1 183 (1 9 8 1 ) .

2 9 McTavish D; Faulds D; Goa KL, "Drugs" United States

2 8

40(2) p 238-59 (1990).

3 0 Giuffetti, G; Aisa G; Meercuri M; Lombardini R; Paltriccia R; Neri C; Senin U; Angiology 41(7) p. 505-11 (1990).

31 Caimi G; Lo Presti R; Serra A; Francavilla G; Catania A; Sarno A; J. Int. Med. Res. 18(2) p.161-3 (1990).

3 2 Davi G; Catalano I ; Spatola A; Alaimo P; Notarbartolo A; Cerbone AM; Strano A. Cardiologia. 344 p. 69-71 (1 989 ) .

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3 4 Balsano F; Coccheri S; Libretti A; Nanci GG; Catalano M, Fortunato G; Grasseli S; Violi F; Helemans H; Vanhove P. J. Lab. Clin. Med. 114(1), p. 84-91 (1989).

3 5 Sanchez Perez A; Montero Garcia, J. Quim, Anal. (Barcelona) 6(2) 204-14 (1987).

3 6 Breyer U. Urinary metabolites of 10-[3'-(4"-methyl- piperaziny1)-propyll-pheno thiazine (perazine) in


psychiatric patients. 1. Biochem. Pharmacol., 18, p. 777-788 (1969).

3 7 Pesez M and Bartos J Colorimetric and fluorimetric Analysis of Organic Compounds and Drugs. Chapter 4, Aliphatic Amines, p. 132, Marcel Dekker Inc. New York ( 1 974) .

3 8 Guiseppe Mausumarra; Giuseppe Scarlata and Gurseppe Cirma. J. of Chromtgr. 350 p. 151-168 (1985).

3 9 F. Overzet, A. Rurak, H. Vander Voet, B.F.H. Drenth, R.T. Ghijsen and R.A. De Zeeuw. J. of Chrom. 267 329-345 (1 9 8 3 ) .

VINBLASTINE SULFATE

(SUPPLEMENT)

Farid J . Muhtadi and Abdul Fattah A. A . Afify

Department of Pharmacognosy

College of Pharmacy



ANALYTICAL PROFILES OF DRUG SUBSTANCES AND EXClPlENTS -VOLUME 21 61 1

Copyright 0 1992 by Academic Press. Inc. All rights of reproduction reserved in any form.

612 FARID J. MUHTADI AND ABDUL FATTAH A. A. AFIFY

V I N B LAST I N E SU LF AT E

Contents

Foreword

1. Description

1.1 Nomenclature 1.2 Empirical Formulae 1.3 Molecular Weight 1.4 Structure 1.5 Elemental Composition 1.6 CAS Registry Number 1.7 Appearance, Color and Odor

2 Physical Properties

2.1 Melting Range 2.2 Solubility 2.3 Specific Optical Rotation 2.4 pH Range 2.5 Loss on Drying 2.6 Dissociation Constant 2.7 Spectral Properties

2.7.1 Ultraviolet Spectrum 2.7.2 Infrared Spectrum 2.7.3 'H-NMR Spectrum 2.7.4 Carbon-I3 Spectrum 2.7.5 Mass Spectrum

3. Isolation of Vinblastine

4. Total Synthesis of Vinblastine

4.1 Total Synthesis of ( 2 ) - Vindoline 4.2 Total Synthesis of ( 2 ) - Catharanthine 4.3 Total Synthesis of Vinblastine

5. Biosynthesis of Vinblastine

6. Pharmacokinetics

6.1 Drug Absorption 6.2 Drug Distribution 6.3 Metabolism

VINBLASTINE SULFATE 613

6.4 Drug Excretion 6.5 Half-Life

7. Preparation and Preservation

8. Uses of Vinblastine Sulfate

8.1 Precautions 8.2 Contra-indications


9.1 Identification Tests 9.2 Titrimetric Determinations 9.3 Voltametric Determination 9.4 Spectrophotometric Determinations

9.4.1 UV Spectrophotometry 9.4.2 Colorimetric Determinations


9.5.1 Paper Chromatography 9.5.2 Thin Layer Chromatography 9.5.3 Gas Liquid Chromatography 9.5.4 High Performance Liquid Chromatography

9.6 Radioimmunoassay Methods

Acknowledgement

References

614 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFlFY

Foreword

Vinblastine or vincaleukoblastine is an indole alkaloid obtained from Madagascan periwinkle, Catharanthus roseus G. Don., (FamiZy Apocynaceae) which has been formerly designated Vinca rosea L. Vinblastine is one of the antineoplastic agents and is mainly used for the treatment of Hodgkin's disease and other lymphomas as well as choriocarcinoma (1). It is used as vinblastine sulfate which is formulated as IV injections . 1. Description

1.1 Nomenclature

Vinblastine; vincaleukoblastine; VBL; 29060 - LE. (The Base).

Vinblastine sulfate; vincaleukoblastine sulfate; vincaleukoblastine sulfate ( 1 : 1) (salt) ; Exal; Velban; Velbe (The Salt).

1 . 2 Empirical Formulae

(Vinblastine) . C46H58N409 C46H58N409 .H2S04 (Vinblastine sulfate) .

1 . 3 Molecular Weight

810.98 909.06

(Vinblastine) . (Vinhlastine sulfate).

1 . 4 Structure

The following is the absolute configuration of vinblastine ( 2 ) .

The structure of vinblastine was deduced by a combination of chemical degradation and spectral data which indicated that the molecule is a dimeric indole- indoline (bisindole) and thus composed of two parts, vindoline which i? connected through a carbon to carbon bond to 16 B-carbomethoxyvelbanamine.


The X-ray c r y s t a l - s t ruc tu re determinat ion of v inc r i s - t i n e methiodide d ihydra te (3) defined t h e abso lu te stereochemistry of v i n c r i s t i n e ; v i n b l a s t i n e should the re fo re has t h e above absolu te s t r u c t u r e i n view of t he known r e l a t i o n - sh ip

1.5

1.6

1 . 7

between these two a lka lo ids .

Elemental Composition

C, 68.13%; H, 7.21%; N, 6.91%; 0, 17.75% (Vinblas t ine) .

C, 60.78%; H, 6.65%; N , 6.16%; S, 3.53%; 0, 22.88% (Vinblas t ine s u l f a t e ) ,

CAS Registry Number

[ 865 -21 -4 ] [143-67-91 Vinblas t ine s u l f a t e .

Vinb 1 ast i n e .

Appearance, Color and Odor

The base occurs as so lva ted needles from methanol (4) .

Small c o l o r l e s s needles from e thanol (5) o r a white c r y s t a l l i n e powder o r white t o s l i g h t l y yellow amorphous powder; odor less ; very hygroscopic (1, 6) (The s u l f a t e s a l t ) .

2. Physical P rope r t i e s

2 . 1 Melting Range

Vinblas t ine melts a t 211-216' (4). Vinblas t ine s u l f a t e mel t s a t 284-285' (4 ,7) .

616 FARID 1. MUHTADI AND ABDUL FATTAH A. A. A F I R

2.2 So lub i l i t y

Vinblastine p r a c t i c a l l y insoluble i n water, soluble i n alcohols, acetone, e thylacetate and chloroform (4) .

One p a r t of v inb la s t ine s u l f a t e i s soluble i n 10 p a r t s of water; i n 50 p a r t s of chloroform; very s l i g h t l y soluble i n ethanol (96%); p r a c t i c a l l y insoluble i n e the r (6).

2.3 Specif ic Optical Rotation

[a]DZ6 + 42" ( i n CHC13) f o r vinblast ine (4,7).

The following data have been reported f o r v inb la s t ine su l fate :

[a]D26 - 28' (c = 1.01 in methanol) (4,7).

[a]D - 28" t o - 35' i n a 2% w/v solut ion i n methanol (6)

[u]D between - 28' and - 35', calculated on t h e d r i ed basis , determined i n a solution of methanol containing 200 mg i n each 10 m l (8).

PH Range (The s u l f a t e s a l t )

Between 3.5 and 5.0 i n a solut ion prepared by dissolving 3 mg i n 2 m l of water (8). 3 . 5 t o 5 . 0 i n a solut ion of 0.15% w/v (6).

2.4

2 .5 Loss on Drying

When v inb la s t ine s u l f a t e i s dr ied a t 60' a t a pressure not exceeding 0.7 kPa f o r 16 hours, loses not more than 17.0% of i t s weight (6). The USP (8) requires t he determination t o be performed by thermogravimetric analysis ,

2.6 Dissociation Constants

pKa 5.4, 7.4 ( 1 , 7 ) .



2.7.1 Ultraviolet Spectrum (UV)

The UV absorbance spectrum of vinblastine sulfate in methanol was scanned from 200 to 400 nm using a Pye- Unicum SP 8-100 Spectrophotometer. shown in Figure 1. Vinblastine sulfate exhibited the following absorptivity values (Table 1).

The spectrum is

Table 1 : UV Absorptivity Values

X max. nm log E A (l%, lcm)

212 262 284 292

4 . 7 5 4.28 4.22 4.18

627.50 209.25 185.0 167.50

Other reported UV data for vinblastine

Solvent X max. nm (Ref. )

Ethanol 214 (log E 4.74) 259 (log E 4.22)

296 (log E 4.12)

(7) 288 (log 4*151 shoulder

Aqueous acid 268 (A = 176) (9)

2.7.2 Infrared Spectrum (IR)

The IR absorption spectrum of vinblastine sulfate as a KBr-pellet (1%) was recorded on a Pye-Unicum SP 3- 300 Infrared Spectrophotometer. sented in Figure 2 . Assignment of the functional groups have been correlated with the following frequencies (Table 2 ) .

Table 2 : IR Characteristics of Vinblastine

The spectrum is pre-

-1 Frequency cm Functional Group

3420 (very broad) Free OH 3035 2950 C-H stretch 1725 Ester C=O (acetoxy)

N-H stretch of indole ring

618 FARID J. MUHTADI AND ABDUL FAITAH A. A. AFIFY

2

FIGURE 1 : UV SPECTRUM OF VINBLASTINE.

6 0

40.

20-

0*4000 3500 3000 2500 2000 1800 1600 ti00 1200 1000 800 600 400 20'0

FIGURE 2 : IR SPECTRUM O F VINBLASTINE.

620 FARID J. MUHTADI AND ABDUL FATTAH A. A. AFlFY

Frequency ern-' Funct ional Group

1610 Lactam C=O 1580, 1500, 1455 Aromatic C=C 1225 c-0-C

The fol lowing p r i n c i p a l peaks a t wave numbers 1227, 1136, 1111, 1724, 1176, 1613 cm-1 were repor ted f o r v i n b l a s t i n e sulfate a s K B r d i s c (9) . Other I . R . da t a have been a l s o repor ted (10-12).

2 .7 .3 'H-NMR Spectrum

The proton magnetic resonance spectrum of v i n b l a s t i n e s u l f a t e i s shown i n Figure 3. I t was obtained on a Varian XL 200 NMR spectrophotometer f o r a s o l u t i o n i n D20. Table 3.

The proton chemical s h i f t s a r e presented i n

1 Table 3 : H-NMR Assignment t o Vinblas t ine

Chemical S h i f t 6 (ppm) Assignment

7.18-7.48 (m) 4 , H aromatic protons of ca tharanth ine ( a t C9'

ll', 1 2 3 . 9

6.68(s) H , aromatic proton of vind-

6.42 (s) H, aromatic proton of vind-

3.878(s) 3H, e s t e r protons of

3.867 ( s ) 3H, methoxy protons of

3.692 (s) 3H, ester: protons of

2.755 (s) 3 H , N-methyl protons of

2.123 (s) 3H, ester protons of vind-

o l i n e ( a t Cg).

o l i n e ( a t C12).

ca tharanth ine ( a t Cl6/).

v indol ine ( a t C ) . vindol ine (a t C16).

v indol ine .

o l i n e ( a t C ) .

11

1 7

s = s i n g l e t , m=multiplet

Other repor ted (13-16).

1 H-NMR s p e c t r a f o r v i n b l a s t i n e have been

FIGURE 3 : 'H - NMR SPECTRUM OF VINBLASTINE.

622 FARID J . MUHTADI AND ABDUL FA'ITAH A. A. AFlFY

2 . 7 . 4 13C-NMR

The carbon-13 NMR spectra of vinblastine and some derivatives have been exhaustively studied and complete assignments for all the 46 carbon atoms in the structure have been made (17-19).

These are presented in Table 4. This table includes the reported 13C-chemical shifts of vinblastine, its sulfate salt and two of its derivatives i.e. the desacetylvinblastine and vinblastine N-oxide. Figure 4 represents the reported proton decoupled l3C-NMR spectrum of vinblastine which was measured on a Jeol PFT-100 Spectrometer (19).

a r - T r i t i a t e d v i n b l a s t i n e (C9,12,91,101,11 ',121-3H ) v i n b l a s - t ine has been prepared and ana lyzed b y means of t r i t i u m NMR s p e c t r o s c o p y , this t e c h n i q u e p r o v i d e s a r a p i d , nondes t ruc - t ive and d i r e c t method for the a n a l y s i s of t r i t i u m on a v e r y small s c a l e and can be a p p l i c a b l e t o the a n a l y s i s of vinblas- t ine recovered from animal t i s s u e s i n b i o l o g i c a l e x p e r i m e n t s (20).

tents with the conformation shown in structure below f o r the piperidine ring in the velbanamine residue (18).

6

The NMR data f o r vinblastine are considered to be consis-

' HO COOCH,

FIGURE 4 : 13C - NMR SPECTRUM OF VINBLASTINE.

624 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFlFY

Table 4 : Carbon-13 Chemical Shifts of Vinblastine and Derivatives 6 (ppm) .

Vinblastine V L B Desacetyl V B L VLB N-Oxide W B I H2S04

Carbon

Vindol ine Moiety

c2

c3

c5

c7

c9

clo

c1 1

‘6

2

‘13

‘14

‘15

‘16

‘1 7

c18

c19

c20

c21 - COOCH3

COOCH3 - ArOCH3 - NCH3 -

OCOCH3 -

OCOCH3 -

1(18)

83.1

50.0

50.0

44.3

52.8

122.6

123.1

120.4

157.8

93.8

152.5

124.3

129.7

79.3

76.1

8.1

30.5

42.3

65.2

170.6

51.8

55.3

38.0

171.4

20.7

2 (19)

83. 3a

50.2

50.2

44.6

53.2

122.6

123.5

121.1

158.0

94.2

152.5

124-4

129.9

79.7

76.4

8.3

30.8

42.7

65.5‘

b

170.8

52. la

55. 8a

38.3‘

171.6

21.1

(19)

80.7

50.4

50.4

44.4

53.9

124.1

122.5

120.7

159.4

95.5

153.7

124,l

131.0

80.7

75.6

7.9

31.7

43.2

66.6

172.9

52.9

56.8

38.6

173.4

20.9

82.8

50.4

49.8

44.7

53.2

122.8

123.9

120.9

158.0

93.9

152.5

124.2

130.0

80.7

74.1

8.6

32.9

42.4

66.4

173.1

52.8

55.8

38.6

- -

83.0

50.4

50.4

44.5

53.2

123.6

123.1

120.5

157.7

93.8

153.0

124.6

130.0

79.7

76.4

8.1

30.7

42.7

65.5

170.9

52.2

55.8

38.0

171.6

21.1

contd ... ,.


Table 4 contd.. . .

Vinblastine VLB Desacetyl VBL VLB N-Oxide W B ) H2S04

Carbon

Velbanamine Moiety

ci

c; cs' c; cs' cs' clo'

cli

cli cl<

cis' clB

cli c19'

c2d

c21'

'14'

'1 7'

- COOCH3

130.9

47.5

55.5

28.7

115.9

129.0

118.1

122.2

118.8

110.2

134.7

29.2

40.0

55.3

34.1

6.7

34.1

68.6

63.1

174.6

52.0

131.4

48.0

55.8

28.2

117.0

129.5

118.4

122.1

118.7

110.4

135.0

30.1

41.4

55.8

34.4

6.9

34.4

69.4

64.2

174.9

52.3

131.5

61.1

54.5

26.9

114.5

128.7

121.1

121.1

119.0

112.3

135.9

35.8

45.8

56.1

34.8

6.9

36.1

68.9

61.1

175.2

52.8

131.3

48.1

55.8

28.7

117.0

129.4

118.4

122.2

118.7

110.4

134.9

30.2

41.4

55.8

34.3

6.9

34.3

69.5

64.3

175.1

52.3

123.6

64.0

67.9

21.4

113.9

129.7

119.1

123.9

119.2

110.1

134.4

30.5

39.1

56.1

35.5

7.1

35.5

71.9

77.8

175.1

52.7

Specific decoupling frequency (sdf) a = 3.76; h = 5.46 6;

c = 2.7 6.

626 FARlD J . MUHTADI AND ABDUL FATTAH A. A. AFIFY

2.7.5 Mass Spectroscopy

Conventional mass spectra (21) as well as high resolution mass spectra of vinblastine and vinblastine hydrazide have been reported (22). High resolution mass spectrometry has established the correct elemental composition of vinblastine, provided completely independent additional information regarding the point of attachment of the two parts (vindoline - velbanamine) and showed that this alkaloid is thermally labile. Some characteristic ion peaks, their corresponding element composition and element lost have been reported (22).

3. Isolation of Vinblastine

Initial methods for the isolation of vinblastine from the periwinkle plants ( v i n c a r o s e a ) had been described (5,7,23-25) and well documented in several texts including the previous profile of vinblastine sulfate (12). Isolation of vinblastine and vincristine from C a t h a r a n t h u s r o s e u s continues to receive attention, and several procedures have been reported (mainly in the patent literature) for the isolation and separation of these alkaloids (24-29). Extracts of Catharan thus r o s e u s have been found to contain N-demethylvinblastine and this can be used to prepare vincristine by formylating the alkaloid mixture before separation and purification (30). In summary, vinblastine is extracted from Catharan thus r o s e u s plants with aqueous acid or with aqueous alcoholic- acid, isolating the alkaloids from the extracts by the usual precipitation and solvent techniques, followed by purifying by chromatography (usually on alumina oxide columns), vinblastine is then obtained (31).

Vinblastine sulfate Conversion to the (1:l) sulfate is effected by dissolving the alkaloid in an equimolar quantity of dilute sulfuric acid and either evaporating to dryness o r precipitating with a suitable organic solvent (31).


4 . Tota l Synthes is of Vinblas t ine

Since v i n b l a s t i n e i s a dimeric a lka lo id , c o n s i s t s of v indol ine moiety and carbomethoxyvelbanamine p a r t , schemes f o r t h e t o t a l syn thes i s of both a r e requi red followed by jo in ing t h e two monomeric u n i t s t o produce t h e dimeric a l k a l o i d . The t o t a l syntheses of v indol ine and d ihydroca tharanth ine (a d e r i v a t i v e of carbo m ethoxyvelbanamine) have been r e - por ted (32-34).

4 . 1 Tota l Synthes is of (?) -Vindoline (32)

6-Benzyloxyindole [ 11 underwent Mannich condensation with dimethylamine [ 2 ] and formaldehyde [3] i n aqueous a c e t i c a c i d t o g ive t h e condensate [4] . This a f t e r qua te rn iza t ion with dimethyl s u l f a t e , was t r e a t e d with aqueous sodium cyanide t o g ive t h e n i t r i l e [ 5 ] . Methylation of [51 with methyl iodide-sodium hydride i n dimethylformamide, followed by hydrogenation over Pd/C i n methanolethyl a c e t a t e a t 50 p s i , gave t h e phenol [ 6 ] . This was t r e a t e d with t o s y l ch lo r ide - sodium hydride i n te t rahydrofuran followed by hydro- genat ion over platinum i n aqueous e thanol -e thyl a c e t a t e conta in ing hydrochlor ic ac id t o produce t h e t r y p t a - mine [7 ] . The hydrochlor ide of [7] was condensed with 1-chloro-3-ketobutene-1 i n e thanol - t r ie thylamine provided t h e l i q u i d Z-enamino ketone [8] ( i n 83% y i e l d ) . [8] was converted t o i t s E-acetamide [9 ] by t reatment with a c e t y l chloride-sodium hydride i n te t rahydrofuran ( i n 89% y i e l d ) . [9] was sub jec t ed t o c y c l i z a t i o n by hea t ing a t 90' i n boron t r i f l u o r i d e e t h e r a t e f o r 16 minutes t o a f f o r d t h e amine [ l o ] i n 89% y i e l d . The l a t t e r was t r e a t e d with 20% potassium hydroxide i n methanol-water a t r e f l u x , t o g ive t h e phenol which was hea ted with dimethyl s u l f a t e i n acetone over suspended potassium carbonate t o a f f o r d t h e methyl e t h e r [ll] i n q u a n t i t a t i v e y i e l d . Removal of t h e ace ty l group i n [ l l ] was accomplished with t r i e t h y - loxonium f luo robora t e i n methylene c h l o r i d e a t room temperature over suspended sodium bicarbonate t o pro- v ide t h e amine [ 1 2 ] i n 82% y i e l d . Condensation of [12 ] with a c r o l e i n i n methanol conta in ing sodium methoxide followed by dehydrat ion with methanesulfonyl ch lo r ide i n pyr id ine gave t h e unsa tura ted ketone [13] i n 60% y i e l d . E thyla t ion of [13] with e thy l i od ide i n t e r t - b u t y l alcohol-dimethylformamide conta in ing potassium t e r t -bu tox ide y i e lded t h e e thy l unsa tura ted ketone [14] i n 53% y i e l d . Condensation of t h e sodium

628 FARlD .I. MUHTADI AND ABDUL FATTAH A. A. AFIFY

Scheme I : Total Synthesis of (*)-Vindoline.


'15' I

630 FARID J. MUHTADI AND ABDUL FAITAH A. A. AFlFY

hydr ide generated eno la t e of ketone [14] wi th dime- thy lcarbonate gave t h e k e t o e s t e r [15]. Hydroxylation o f t h i s alcohol-dimethoxyethane conta in ing potassium t e r t - butoxide a f forded t h e 8-hydroxy ketone [16] i n 76% y i e l d . t e t rahydrofuran) followed by reduct ion with sodium b i s (2-methoxyethoxy) aluminum hydr ide (-20°) t o g ive s i n g l e epimer a lcohol . wi th a c e t i c anhydride-sodium acetate af forded (?) - vindol ine [ 171. T h i s synthes is i s presented in scheme I .

with 38% hydrogen peroxide i n t e r t - b u t y l

[16] was t r e a t e d with aluminum ch lo r ide (-2S0,

Acety la t ion of t h i s a lcohol

4.2 Tota l Synthes is of (+)-Dihydrocatharanthine (33,34)

Ethyl 2-carbethoxy-4, 4-diethoxy butanoate [l] (prepared from dimethylmalonate ( 35 ) underwent condensation with 0.5 molar excess of methyl-a- e t h y l a c r y l a t e [ 21 (prepared from methyl -2-carboxybuta- noate ( 36,37) i n t h e presence of f r e s h l y prepared sodium ethoxide as t h e c a t a l y s t t o g ive t h e condensate, methyl-2-ethyl-4, 4-dicarbethoxy-6, 6-diethoxy hexan- o a t e [3] i n 86% y i e l d . This was ref luxed wi th 1.5 equiva len ts of dry sodium cyanide i n dry dimethyl su l fox ide t o a f f o r d [4] i n 70% y i e l d . Substance [4 ] was d i r e c t l y condensed with t ryptamine [S] by r e f l u x - ing i n aqueous acetic ac id under Nz f o r 6 hours t o produce the lactam ester [63. Product [6] was reduced by r e f lux ing a so lu t ion of i t i n te t rahydrofuran (THF) with LAH t o g ive t h e amine a lcohol [7 ] . Mesyla- t i o n of [ 7 ] with anhydrous methane su l fonyl ch lo r ide and trimethylamine in anhydrous e t h e r , followed by r e f lux ing t h e mesylate i n anhydrous a c e t o n i t r i l e f o r several hours t o y i e l d t h e qua ternary salt [ 8 ] . This s a l t was heated a t 200° KCN i n d igo l , conversion t o 16-cyanodihydro cleavamine [9] was e f f ec t ed . Methan- o l y s i s of [9] under mild cond i t ions by us ing anhydrous methanol and bubbling d ry HC1 gas a t 25' a f forded (+)-16-methoxycarbonyldihydrocleavamine [ l o ] . Subst- ance [ l o ] was subjec ted t o oxida t ion with mercuric a c e t a t e t o g ive (+)-dihydrocatharanthine [ll]. T h i s t o t a l s yn thes i s is presented i n scheme 11.

Dihydrocatharanthine [ l l] can be converted i n t o ca th- a ran th ine [ 121 by an e s t a b l i s h e d method (38).

Other syntheses o f v indol ine (39,40) and o f ca tharan- t h i n e (41,42) have been repor ted .

VINBLASTINE SULFATE 63 I

S c h e m e I1 T o t a l Syn thes i s of (+)-Dihydrocatharanthine C 0 2 E t C 0 2 E t

( E t O ) 2CH /\i + H2C=C-C02CH3 I ( E t O ) 2 C H q ^ ( c 0 2 c H 3 E t -

C 0 2 E t C 0 2 E t

NaCN DMSO I [31

[I1 [21

P I [41

LAH

THF -

1. C H 3 S 0 2 C 1

2 . CH3CN

CH2OH (-1

O S 0 2 C H 3 [71 q \ T H - KCN D i g o i

[81

MeOH/HCl

25'

632 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY

J

4.3 Tota l Synthes is of Vinblas t ine (43-47)

Catharanthine [l] underwent ox ida t ion with m-chloro- perbenzoic ac id t o g ive t h e N-oxide. Catharanthine N-oxide [2] was t r e a t e d with v indo l ine i n methylene chloride-trifluoroacetic anhydride a t - 5 0 ° , coupl ing occurred, t o give t h e immonium ion [3] which wa r,edu,ced with sodium borohydride t o provide t h e A 1 5 ' q 2 0 1 2 0 -deoxyvinblast ine (anhydrovinblast ine) [ 4 ] . followed by borohydride reduct ion a f forded v inb la s - t i n e [ S ] . T h i s s y n t h e s i s i s presented i n scheme III.

This upon t reatment with tha l l i um t r i a c e t a t e

A highly e f f i c i e n t and commercially important syn thes i s of v i n b l a s t i n e from ca tha ran th ine and v in - do l ine has r e c e n t l y been descr ibed ( 4 8 ) . Other s y n t h e t i c methods have also been repor ted ( 49,SO).


Scheme 111 : Total Synthesis of Vinblastine

-0

Vindol ine coup 1 ing

/ Vindoline [41

3 I i) Tl (0Ac)

3. ii) NaBH4

H3COOC

/

H : OCOCH3 H3C

CH3 ~ O ~ C H ~

/

/ [51 Vindoline

634 FARID I. MUHTADI AND ABDUL FATTAH A. A. AFIFY

5. Biosynthesis of V inb la s t ine

I t has long been proposed t h a t t h e i n d o l i c moiety of t h e indole a l k a l o i d s i s der ived from t h e aminoacid "tryptophan" ( 51-53). This has been j u s t i f i e d when r a d i o a c t i v e t r y p t o - phan o r t ryptamine (decarboxytryptophan) were incorpora ted i n t o seve ra l i ndo le a l k a l o i d s ( 5 4 - 5 7 ) . I t has a l s o been p red ic t ed t h a t t he non-tryptophan p o r t i o n s of t h e s e a lka l - o ids a r e formed from two mevalonate u n i t s t o a f f o r d a cyc lo- pentane monoterpenoid p recu r so r (58,59). This was proved upon feeding d l - [2-I4C] -mevalonic a c i d lac tone , and sodium (?)-[2-14C] mevalonate i n t o V i n c a r o s e a p l a n t s and r e s u l - t ed i n t h e i s o l a t i o n of r a d i o a c t i v e v indol ine , ca tharan- t h i n e and a jma l i c ine ( 60-64). I t was f u r t h e r p red ic t ed t h a t t h e monoterpenoid p recu r so r could wel l be "the g lucos ide loganin" ( 6 5 ) . I t i s now known t h a t loganin a r i s e s i n t h e p l a n t s from two mevalonate u n i t s . i n t o i sopentenyl diphosphate (66) and t h e o t h e r i n t o dime- thyal lylpyrophosphate (67 ) . Combination of t h e s e two u n i t s leads t o geranio l (68-73), then t o loganin (74-76) and f i n a l l y i n t o secologanin (77, 7 8 ) . Evidence sugges ts t h a t t ryptamine ( o r L-tryptophan) [ 11 r e a c t s wi th secologanin [2] t o form s t r i c t o s i d i n e ( i s o - vincoside) [3] ( 66,79-84). I t has been observed t h a t labe led s t r i c t o s i d i n e [3] ; g e i s - soschiz ine [4] (80,85,86); stemmadenine [7] ( 8 4 , 8 7 ) and tabersonine [7b,9] (86-88) were a l l incorpora ted i n t o both ca tharanth ine [8] and v indo l ine [ 101 i n Catharan thus r o s e u s p l a n t s , i n d i c a t i n g t h a t t h e s e a r e t h e main p recu r so r s i n the b iosyn the t i c pathway t o t h e Aspidosperma-Iboga alkaloids.

Other in t e rmed ia t e s such as ge i s sosch iz ine oxindole [ 5 ] , preakuammicine [6] have been de tec t ed 28-40 hours a f te r germination of C. r o s e u s seeds (85,87,89) provided s t rong evidence f o r t he formation of ca tha ran th ine [8] and vindo- l i n e [ l o ] as presented i n schemes I and 11. Feeding r ad ioac t ive [8] as [3H-C02CH3] and [ l o ] as [14C- OCOCH31 i n t o a p i c a l c u t t i n g of 3-4 month-old C. r o s e u s p l a n t s a f forded low but d e f i n i t e i nco rpora t ions of bo th a l k a l o i d s i n t o v i n b l a s t i n e [12] demonstrat ing t h a t t h e s e monomeric a l k a l o i d s a r e t h e p recu r so r s of [ 1 2 1 (90) . Feed- ing both [acetyl-14C] v indol ine and [OC3H3] ca tha ran th ine t o 6 week-old d i f f e r e n t i a t e d c. r o s e u s p l a n t s f o r 6 days, l a b e l l e d anhydrovinblas t ine [ 111 was i s o l a t e d ( 91) . This was incorpora ted i n t o v i n b l a s t i n e by c e l l - f r e e p repa ra t ions Of C a t h a r a n t h u s r o s e u s (92 ,93 J . Later it was found t h a t anhydrovinblas t ine [ l l ] can be con- v e r t e d i n t o v i n b l a s t i n e [12] by c e l l - f r e e homogenates of

One of which i s transformed by a s e r i e s of s t e p s

VINBLASTINE SULFATE 63.5

Scheme I : B i o s y n t h e s i s of C a t h a r a n t h i n e

+ -

[51

I -

C02CH3 CH20H

636 FARID 1. MUHTADI AND ABDUL FATTAH A. A. AFIFY

Scheme 11: Biosynthesis of Catharanthine and Vindoline

0-B 0 N [91

H I (-)-form COZCHS C02CH3

(+)-form

[7b1 1

VINBLASTINE SULFATE

Vindoline

Cell-free extracts leaves C. roseus

Scheme 111: Biosynthesis of Vinblastine

Catharanthine [S] + Vindoline [ l o ]

- or Cell-free homoyenates of C. roseus cell suspen-

Cell-free extracts from Catharanthus roseus plants

sion cultures 1

638 FARlD J . MUHTADI AND ABDUL FATTAH A. A. AFlFY

C . roseus cell suspension cultures ( 3 4 ) , thus administration of [21’a-3H] anhydro-VLB to a cell-free homogenate of suspension culture cells C. roseus afforded radioactive [3H] -vinblastine ( 94 ) . The biosynthesis of vinblastine is presented i n scheme III.

6. Pharmacokinetics

6.1 Drug Absorption

Vinblastine is poorly absorbed after oral administration (9,951. It is readily absorbed after intravenous administration (IV) or intraperitoneal injection (IP).

6.2 Drug Distribution

Vinblastine is rapidly distributed with high tissue binding, readily binds to platelets, red blood cells and white blood cells; subject to enterohepatic circulation; volume of distribution, 86 to 111 liters ( 1,961. After IV radioactively labeled, vinblastine is detected mostly in the liver in less than an hour (97). Protein binding: It is highly protein bound ranging from 98 to 99.7% ( 9 8 ) . Binds in plasma to a- and 8 - globulins (1,96). The drug does not penetrate the CNS o r other fatty tissues ( 99). Drug concentration levels: vinblastine (2 mg/square meter) will produce cytotoxic concentrations of approximately 2 ng/mL ( 100). After an IV dose of 15 mg, a plasma concentration of about 16 ng/mL is obtained in 24 hours; an additional dose of 15 mg at this time produces a plasma concentration of about 55 ng/mL 4 hours later ( 1,96).

continuous infusions of

6.3 Metabolism

Vinblastine is metabolized in the liver. Metabolic reactions in rats is deacetylation to give desacetylvinblastine which is the major metabolite of vinblastine ( 1,9). A significant amount of vinblastine is metabolized in the liver to the active metabolite desacetylvinblastine ( 95).

6.4 Drug Excretion

In 72 hours, 25 to 40% of an intravenous dose is excreted in the feces and 19 to 23% is excreted in the urine, most of the urinary excreted material is


unchanged, wh i l s t t h a t i n t h e f aeces i s i n t h e form of metabol i tes (1,961. About 14% of a r a d i o a c t i v e l y labe led dose i s excre ted i n t h e u r i n e i n 72 hours and 10% is e l imina ted i n t h e f aeces i n t h e same pe r iod ( 9 ) . Following I V v i n b l a s t i n e dosing depending upon t h e r a d i o a c t i v e l a b e l technique used only 13.6 t o 23% of t h e t o t a l dose was excre ted i n t h e u r i n e and t h a t excre ted i n t h e f eces ranged from 9.9 t o 41% wi th 72 hours ( 101) .

6.5 Half-Life

Plasma h a l f - l i f e ( t o t a l a c t i v i t y ) , about 20 hours

In whole blood, a-phase, about 4 minutes and 8-phase, about 190 minutes, f o r drug p l u s metabol i tes (1). Vinblas t ine f i t s a 3-compartment pharmacokinetic model wi th a lpha , b e t a and gamma ( te rmina l phase) , h a l f - l i v e s of 0.062, 0.164 and 25 hours r e s p e c t i v e l y were obtained ( 9 9 ) .

( 9 ) *

7 . Prepara t ion and Preserva t ion

Vinblas t ine s u l f a t e should be s t o r e d i n a i r t i g h t con ta i - ne r s , a t a temperature between 2" and 10" pro tec t ed from l i g h t . Vinblas t ine s u l f a t e i s adminis tered by t h e IV i n j e c t i o n of a so lu t ion of 1 mg pe r mL i n water f o r i n j e c t i o n o r i n sodium ch lo r ide i n j e c t i o n (preserved with phenol o r benzyl- a l coho l ) . Usually t h e ampoule conta ins 10 mg s te r i le v in- b l a s t i n e s u l f a t e . The drug d i s so lves i n s t a n t l y t o g ive a c l e a r s o l u t i o n having a pH i n t h e range of 3.5-5.0. Via ls of v i n b l a s t i n e s u l f a t e should be s t o r e d i n a r e f r i g e r a t o r between 2 O and 8°C t o a s su re extended s t a b i l i t y . After r e c o n s t i t u t i o n with 10 mL b a c t e r i o s t a t i c Sodium Chloride I n j e c t i o n USP (preserved with benzyla lcohol ) , s o l u t i o n may kept i n a r e f r i g e r a t o r a t 2" t o 8OC f o r 30 days without s i g n i f i c a n t l o s s of potency (102) . If t h e i n j e c t i o n conta ins no bac te r ioc ide , i t should be used a s soon a s poss ib l e a f t e r p repa ra t ion , and i n any case wi th in 4 days. r i o c i d e such a s 0.5% phenol, i t may be used f o r up t o a one month when s to red at 2 O t o 10°C ( 1 ) .

In t h e presence of a s u i t a b l e bac te-

640 FARID J . MUHTADI AND ABDUL FATTAH A. A. AFIFY

8 . Uses of Vinblastine Sulfate

Vinblastine is an antineoplastic drug which apparentlyacts by binding to the microtubular proteins of the spindle and arresting mitosis at the metaphase. It also interferes with aminoacid metabolism and nucleic acid synthesis. It has some immunosuppressant activity, as it suppresses the immune response and in high doses it is neurotoxic. Like other cytotoxic drugs it is teratogenic (95). Vinblastine sulfate is mainly used in association with other antineoplastic agents, in the treatment of Hodgkin's disease and other lymphomas including mycosis fungoides. It is also of use in the treatment of some inoperable malignant neoplasms including those of the breast, female genital tract, testis, lung, gastrointestinal tract and in neuroblastoma, choriocarcinoma, Kaposi's sarcoma and histiocytosis X ( 9 5 ) . In the treatment of Hodgkin's disease, it is often given with cyclophosphamide or mustine, procarbazine and prednisone or with doxorubicin, bleomycin and dacarbazine. In carcinoma of testis, vinblastine is given with bleomycin and cisplatin (95).

I n clinical dosage it depresses bone-marrow activity, affecting mainly the white cells, with relative sparing of the erythroid elements. reversible on stopping the drug (1).

The bone-marrow depression is

8.1 Precautions

Vinblastine sulfate should be used with care in cach- ectic patients. Its use in pregnancy is not advised as it is teratogenic. Care should be applied when it is injected intravenously as perivenous infiltration may cause cellulitis, phlebitis and venous thrombosis ( 1).

8.2 Contra-indications

Vinblastine sulfate should not be given if the white- cell count is below 4000 per cubic millimeter, if bacterial infection is present, or if the bone-marrow is infiltrated with neoplastic cells ( 1).

VlNBLASTlNE SULFATE 64 I


9 .1 I d e n t i f i c a t i o n Tests

The following identification tests are mentioned in the B P (6).

To lmg v i n b l a s t i n e s u l f a t e add 0.2 m l of a f r e s h l y prepared 1% w/v s o l u t i o n of v a n i l l i n i n hydrochlor ic ac id . co lo r i s produced i n about 1 minute ( d i s t i n c t i o n from v in - c r i s t i n e s u l f a t e ) . Mix 0.5 mg v i n b l a s t i n e s u l f a t e wi th 5 mg of 4-dimethylaminobenzaldehyde and 0.2 ml of g l a c i a l acetic ac id and 0.2 ml of s u l f u r i c ac id ; a reddish-brown c o l o r i s produced. Add 1 m l of g l a c i a l acetic ac id ; t h e c o l o r changes t o p ink .

the USP (8).

The i n f r a r e d absorp t ion spectrum of a pot.assium d i spe r s ion of v i n b l a s t i n e s u l f a t e , previous ly d r i ed i n vacuum a t 60' f o r 16 hours , e x h i b i t s maxima only at t h e same wavelengths a s t h a t of a s i m i l a r p repa ra t ion of USP v i n b l a s t i n e s u l f a t e RS . Other i d e n t i f i c a t i o n t e s t : Marquis T e s t ( s u l f u r i c ac id - formaldehyde) g ives wine-red co lo r with v i n b l a s t i n e (103).

A pink

The following identification tests are mentioned in

9.2 T i t r i m e t r i c Determinations

Non-Aqueous T i t r a t i o n s

Quan t i t a t ive determinat ion of t h e a l k a l o i d s inc luding v i n b l a s t i n e i n drugs and e x t r a c t s a r e repor ted as fo l lows ( 104). Aer i a l p a r t s of Vinca rosea were ground, t r e a t e d with 25% NH40H f o r 30 minutes, and ex t r ac t ed with MeOH. The e x t r a c t was evaporated and t h e r e s idue was d isso lved i n 2% H2SO4 on a water ba th . The H2S04 e x t r a c t was a l k a l i n i z e d with NH40H, r eex t r ac t ed with CHC13, t h e e x t r a c t was d r i e d , and evapora- t ed . The r e s idue was d i s so lved i n HOAc and t i t r a t e d wi th

Aer ia l p a r t s of vinca minor were t r e a t e d with 25% NH40H f o r 30 minutes, ex t r ac t ed with CHC13, t h e concent ra ted e x t r a c t was reex t r ac t ed with 2% t a r t a r i c a c i d ad jus ted t o pH 9.0 with 25% NH40H, and a l k a l o i d s were ex t r ac t ed with CHC13, d i sso lved i n HOAc and t i t r a t e d with H C l O 4 by us ing c r y s t a l v i o l e t i n d i c a t o r .

- A mixture of v i n b l a s t i n e s u l f a t e and t e s t o l a c t o n e i s determined by p r e c i p i t a t i o n a t pH 3.7 with a measured volume of Na t e t r apheny lbora t e so lu t ion , and t i t r a t i o n of unconsumed r e a - gent with hexadecylpyridinium ch lo r ide us ing bromophenol b l u e a s an i n d i c a t o r (105) .

0 . 1 ~ ~ ~ 1 0 4 .


9.3 Voltametric Determination

Ultrasensitive voltammetric measurements based on coupling hydrogen catalytic systems with controlled interfacial accumulation of the catalyst has been reported (106). By combining adsorptive stripping voltammetry with a catalytic hydrogen process, trace Pt can be measured with a detection limit of -1X10-12M. The supporting electrolyte (1.08 M H2SO4) contains 0.04% (wt./vol.) formaldehyde and 0.001% (wt./vol.) hydrazine. The adsorption of the Pt- formazone complex (the catalyst) results in a well-defined catalytic H peak at - 0.92 V, with a peak half-width of 60 mV. The peak height increases rapidly with increasing preconcentration time, indicating a large enhancement of the complex on the surface of the hanging Hg drop electrode (SDS) and gelatin cause serious interference by competing with the catalyst on adsorption sites. ammoniacal buffer (pH 9 . 3 ) , the vinca alkaloids vinblastine and vincristine can be determined at subnanomol levels (106)

By using 0.25 M

9.4 SDectroDhotometric Determinations

9.4.1 UV Spectrophotometry

The official methods for determination of vinblastine sulfate are UV spectrophotometric techniques(6,8). The BP ( 6 ) recommends the following procedure: Dissolve 10 mg of vinblastine sulfate in sufficient methanol to produce 500 ml and measure the absorbance of the resulting solution at the maximum at 267 nm. Calculate the content of C46H58N409, H2SO4 taking 185 as the value of A (1%, 1 cm) at the maximum at 267 nm. The USP (8) recommends the following procedure: Dissolve about 5 mg of vinblastine sulfate, accurately weighed in methanol and dilute quantitatively and stepwise with methanol to obtain a solution containing about 20 pg per ml on the dried basis. Dissolve an accurately weighed quantity of USP Vinblas- tine Sulfate RS in methanol and dilute quantitatively and stepwise with methanol to obtain a standard solution having a known concentration of about 20 pg per ml on the dried basis. Concomitantly determine the absorbances of both solutions in 1-cm cells at the wavelength of maximum absorbance at about 267 nrn, with a suitable spectrophotometer, using methanol as the blank. Calculate the quantity in mg of C46H58N40g. H2S04 in the portion of vinblastine sulfate taken by the formula


0.25 C (Au/As) , i n which C i s t h e concent ra t ion , i n pg pe r m l of USP Vinblas t ine S u l f a t e RS i n t h e Standard so lu t ion and Au and As a r e t h e absorbances of t h e so lu - t i o n of v i n b l a s t i n e s u l f a t e and t h e Standard so lu t ion r e spec t ive ly . The USP r e q u i r e s t h a t t h e weighings t o be performed r a p i - d ly and wi th a minimum of exposure of t h e subs tance t o a i r .

9.4.2 Color imet r ic Determinations

A co lo r ime t r i c method was f i r s t deviced f o r t h e a s say of pure v i n b l a s t i n e s u l f a t e ( 107). The method depends on t h e formation of a deep rose c o l o r upon hea t ing v inb la s - t i n e s u l f a t e a t 80' with a reagent c o n s i s t i n g of p y r i - d ine (35 ml) , concent ra ted s u l f u r i c a c i d (1 m l ) and a c e t i c anhydride ( 3 5 ml) conta in ing 0.05% a c e t y l c h l o r i d e . The c o l o r so produced i s measured a t 574 nm. The s e n s i t i v i t y i s 5 t o 70 pg of v i n b l a s t i n e s u l f a t e p e r ml . To check p u r i t y of t h e sample, t h e absorbances of t h e c o l o r produced a r e measured i n 1 cm c e l l s a t 574 and 538 nm a g a i n s t water a s a r e fe rence . The r a t i o of A574 nm/A538 nm should be i n t h e range of 1.20 - 1.25 (107) . Another co lo r ime t r i c method has been r epor t ed f o r t h e q u a n t i t a t i v e de te rmina t ion of v i n b l a s t i n e and l eu ros ine i n Vinca rosea ( 1 0 8 ) . The sequen t i a l s t a g e s used were - ( i ) s e l e c t i v e ex t r ac - t i o n of t h e a l k a l o i d s i n t o benzene o r to luene (wet t ing t h e raw ma te r i a l with aq. 5% Na a c e t a t e improved t h e e x t r a c t i o n ) , ( i i ) back-ext rac t ion of t h e a l k a l o i d s i n t o 2% c i t r i c o r t a r t a r i c a c i d , ( i i i ) adjustment of pH t o 6 .0 with aq. 5% NH3 and r e - e x t r a c t i o n of t h e a l k a l o i d s i n t o to luene , ( iv ) s epa ra t ion of t h e a l k a l o i d s on LH-20 with methanol - CHC13 (7 :3 ) , s epa ra t ion of t h e dimeric a lka lo id f r a c t i o n on s i l i c a g e l , with CHC13 - benzene - acetone - e t h y l a c e t a t e - methanol (20:20:15:5:3) ( v i ) e l u t i o n of t h e a l k a l o i d s with 1% H C 1 , and ( v i i ) add i t ion of 0.2 m l of 1% t ropaeo l in 000-1 (C.I. Acid Orange 20) and then CHC13. The c o l o r so produced i s measured a t 490 nm.

644 FARlD J . MUHTADl AND ABDUL FATTAH A. A. AFlFY


9.5.1 Paper Chromatography

Clarke ( 1 0 3 ) described the following system for the identification of vinblastine. System : Reversed phase, Whatman no. 1 or no. 3 paper chromatography, impregnated by dipping in 10% solution of tributyrin in acetone and drying in air. Sample : A 5 1-11 of 1 to 5% solution in ethanol o r chloroform. Solvent : Acetate buffer (pH 4.58). Location : Under UV light gives purple fluorescence. Location spray : Iodoplatinate. Rf value : 0.10.

9.5.2 Thin Layer Chromatography (TLC)

The following TLC systems were recommended for the identification and separation of vinblastine.

Chromatograni Solvent System Rf value Ref. ~~

1. Silica gel G, 250 methanol-strong

diethylamine (75:15:10) 0.10

(90 : 10)

diethylamine (80:40:6) - (6)

alcohol (3 : 1) 0.21 (109 1

alcohol (1 : 1) 0.33 (110)

Oe60 0.60 i (9 1-1m thick, dipped ammonia (100:l.S) in or sprayed with Cyclohexane-toluene- 0.1 M KOH in methanol and dried chloroform-methanol

2. Silica gel Gf 254 toluene-chloroform-

3 . Silica gel G ethylacetate-absolute

4. Silica gel G ethylacetate-absolute

1 5. Alumina ethylacetate-absolute

alcohol (3: 1) 0.66 (110)

6. Silica Gel G n-butanol-acetic acid- water (4 : 1 : 1) 0.19 (109)

7 . Silica Gel G met han o 1 0.46 (109)

(95 : 5) 0.24 (111)

9. Alumina chloroform 0.17 (110)

acetate (1:l) 0.25 (110)

8. Silica gel G chloroform-methanol

10. Alumina chloroform-ethyl-


Detection : The spots can be detected by: 1- Under short UV light (254 nm) 2- Spraying with:

a) Dragendorff's reagent ( 103) b) Acidified iodoplatinate solution (103) s) 1% Ceric ammonium sulfate in 85% phosphoric

acid (110)

- A double development technique was recommended f o r the separation of vinblastine from the other dimeric catharanthus alkaloids on alumina layers(ll2). The loaded chromatoplates were first developed in the solvent ethylacetate, then after drying, a second development in ethylacetate-absolute alcohol (3: 1) was carried out. Vinblastine in this technique gave Rf value of 0.73 ( 112).

- Two dimensional TLC technique was reported for the separation of more complex mixture of catharanthus alkaloids (115) . ence of related alkaloids in vinblastine sulfate sample (checking the purity of the sample): TLC chromatoplates are coated with silica gel GF 254. of the following three solutions in methanol are separately applied to one chromatoplate. 1- 1.0% w/v of the substance being examined. 2 - 0.02% w/v of standard vincristine sulfate BPCRS. 3- 1.0% w/v of standard vinblastine sulfate BPCRS. The chromatoplate is then developed in the solvent toluene- chloroform-diethylamine (80:40:6). After development, the plate is allowed to dry in air and examined under UV light (254 nm). Any secondary spot in the chromatogram obtained with solution (1) is not more intense than the spot obtained with solution (2).

described. Two dimensional TLC is performed followed by densitometric scanning of the spots at 289 nm. The CO-

efficient variation of the method was 7.5% ( 114).

.- The BP (6) adopted a TLC technique to test for the pres-

5r.tl

- A TLC - Densitometric determination of vinblastine was


9.5.3 Gas Liquid Chromatography (GLC)

The following GLC system has been reported for the identification and separation of vinca alkaloids including vinblastine ( 115).

Column condition: Glass column (lm x 3.2mm), precoated with hexamethyldisilazane and packed with 3% OV-101 on Gas chrom Q (80-100 mesh), with temperature programmed from 200' to 300' at 5' min.-l

Carrier gas: Nitrogen at a flow rate of 30 ml/min.-l

Detection: F.1.D

Condition: Vinca alkaloids including vinblastine were derivatized before application by heating for 5 minutes at room temperature with t r i f l uo rob i s - (Tr ime thy l s i ly l ) acetamide-pyridine (1 : 1).

9.5.4 High Performance Liquid Chromatography (HPLC)

Several HPLC methods have been employed to determine vinblastine and its metabolites in biological fluids and tissues. Some of these methods are as follows:

System 1: The following system has been recommended for quantitative determination of vinblastine and other vinca alkaloids in plasma and urine ( 116).

Conditions:The drugs are extracted from biological mate-

Mobile phase :

System 2:

Column : Mobile phase :

rials by an ion pair extraction with sodium octylsulfate as counter ion at pH 3.0. The extracts are injected onto a reversed-phase system with a cyano column as stationary phase. MeCN -phosphate buffer pH 3.0 (65:35).

The following system is a reversed-phase with electrochemical detection. It is employed for quantitative determination of vinblastine and its metabolites in plasma and urine. Quantifi- cation of substances in human plasma and urine is possible down t o 1 ng/ml ( 117). Hypersil ODs. Methanol - 10m M phosphate buffer pH 7.0.


System 3: This system is employed for the analysis of Catharanthus alkaloids including vinblastine (118).

Column: A stainless steel (25cm x 4mm), packed with Li-Chrosorb RP-8; operated at ambient temperature.

Mobile 0.01M ammonium carbonate-acetonitrile (53:47). phase :

Flow rate: 1.5ml min.-l

Retention 12 .37 minutes forvinblastine. time :

Detection: UV at 298 nm.

System 4 : This system has been employed f o r the separation, detection and correlation of plate height and molecular weight of vinblastine and other Vinca alkaloids (119).

octadecyl-silica gel. Gradient elution with aqueous 50 to 85%

Column: 25cm x 4.6mm, packed with R SiL C1g HL-D

Mobile phase : methanol containing 0.1% ethanolamine.

Flow rate: 2 ml min.-l

Detection: UV at 290 nrn.

System 5: The following reversed-phase system has been used for the analysis of Catharanthus alkaloids including vinblastine by thermospray liquid chromatography-mass spectrometry (120).

column. Column: p Bondapak C18 (30cm x 3.9mm), reversed-phase

Mobile Isocratic solvent, 0.1M ammonium acetate phase : (pH 7.2) - MeCN (51:49). Flow rate: 1 ml min.-l

Detection: Electrochemical and UV (The limit of detection

System 6 :

being 4 ng/injection €or each alkaloid).

The following reversed-phase system has been reported f o r the determination of vinblastine and other alkaloids of Catharanthus roseus leaves ( 121).

Column: p Bondapak Cis.

Mobile 0.1M diammonium hydrogen orthophosphate - MeCN phase : (25:75), pH 7.0.

Detection: UV at 254 and 280 nm.


System 7: This reversed-phase system is descr ibed f o r t he determinat ion of v i n b l a s t i n e and o t h e r Catharanthus a l k a l o i d s . The r e l a t i v e s t a n - dard dev ia t ion , t h e l i m i t of de t ec t ion and the recovery were 1.63-3.52%, 10 ug/ml and 96.6-102% respec t ive ly (122) .

Column: A c a r t r i d g e column packed with Spheri-5RP.

Mobile 2 g rad ien t systems conta in ing MeOH, MeCN, phase : 0.025M ammonium a c e t a t e and t r i e thy lamine

i n d i f f e r e n t r a t i o s . I n t e r n a l 5-Methoxytryptamine. s tandard :

Detect ion: UV a t 280 and 254 nm.

System 8: This i s a l s o a reversed-phase system which has been appl ied f o r s epa ra t ion and quan t i t a - t i o n of a l k a l o i d s from c e l l suspension c u l t u - res of Catharanthus roseus inc luding v inb la s - t i n e (123) .

Column: 1.1 Bondapak C18.

Mobile A mixture of methanol and (NH4)2HP04 i n water . phase :

Detect ion: UV a t 298 nm.

Other HPLC systems f o r v i n b l a s t i n e have a l s o been repor ted (124-127).

9.6 Radioimmunoassav Methods

Quan t i t a t ive determinat ion of v i n b l a s t i n e i n t i s s u e cu l - t u r e s of Catharanthus roseus by radioimmunoassay was repor ted ( 128). Antibody was obta ined by t h e immunization of r a b b i t s aga i - n s t a conjugate of v i n b l a s t i n e with bovine serum albumin. The ant ibody had a h igh a f f i n i t y (Ka = 1.2 x 109 L/mol) and s p e c i f i c i t y f o r v i n b l a s t i n e . s tandard curve f o r assay was 0.5-10 ng/ml. Crude a l k a l o i d e x t r a c t s of t i s s u e c u l t u r e s could be assayed and many sam- p l e s could be processed i n one time. The v i n b l a s t i n e con- t e n t s of mul t ip le shoot c u l t u r e s were lower than t h a t of i n t a c t p l a n t s b u t much h igher than t h a t of c a l l u s c u l t u r e s .

Another Radioimmunoassay method was repor ted f o r v inb la s - t i n e and v i n c r i s t i n e a s fol lows ( 1 2 9 ) : Radioimmunoassay developed f o r determining t h e neoplasm i n h i b i t o r s v inb la s - t i n e (I) and v i n c r i s t i n e (11) i n blood involves t h e use of antiserum r a i s e d i n a r a b b i t immunized with ( I ) bovine

The usable range of


serum albumin conjugate . Detect ion l imits (ng ml-1) o r 2 . 1 f o r ( I ) and 3.8 f o r (11) with use of t r i t i a t e d ( I ) under non-equi l ibr ium assay condi t ions . The ant iserum showed no c r o s s - r e a c t i v i t y with 25 o t h e r a l k a l o i d s and cy to tox ic drugs used t h e r a p e u t i c a l l y i n combination with ( I ) and (11).

A t h i r d method of Radioimmunoassay for Vinca a l k a l o i d s v i n b l a s t i n e and v i n c r i s t i n e was r epor t ed ( 130) a s fo l lows : An t i se ra were r a i s e d i n r a b b i t s by immunisation a g a i n s t compounds prepared by coupl ing carboxyl ic a c i d d e r i v a t i - ves of v i n b l a s t i n e and v i n c r i s t i n e t o human serum albumin. For assay , ant iserum was incubated with t h e sample, e.g. p l a n t e x t r a c t and t h e appropr i a t e t r i t i a t e d a l k a l o i d f o r 1 h a t 37', and t h e mixture was allowed t o r e a c t wi th a goat a n t i - r a b b i t serum o r with polyoxyethylene g lycol overn ight a t 4', and then cen t r i fuged ; t h e p r e c i p i t a t e was d isso lved i n NaOH so lu t ion f o r s c i n t i l l a t i o n count ing. E i t h e r compound could be determined i n amounts down t o <1 pmol. The s p e c i f i c i t y of t h e a n t i s e r a i s discussed; t h a t r a i s e d a g a i n s t v i n c r i s t i n e bound t h i s a l k a l o i d 200 times more e f f e c t i v e l y than it bound v i n b l a s t i n e . Severa l o t h e r compounds, inc luding a n t i n e o p l a s t i c drugs, showed no c ross - r e a c t i v i t y . The assays were app l i ed t o t h e blood o f rabb- i t s i n j e c t e d wi th t h e drugs , and a l s o t o e x t r a c t s of Vinca rosea a f t e r pre l iminary f r a c t i o n a t i o n by HPLC.

A s e n s i t i v e radioimmunoassay method f o r v i n b l a s t i n e and v i n c r i s t i n e was a l s o repor ted a s fol lows ( 131 ) : Antiserum which was used was r a i s e d i n r a b b i t s a g a i n s t a 4 -deace ty lv inb la s t ine carboxazide-bovine serum albumin conjugate; [3H] - v i n c r i s t i n e o r r3H] -v inb la s t ine was used as rad io- l igand . Rates of b inding of v i n c r i s t i n e and v i n b l a s t i n e t o t h e ant iserum were s i m i l a r . Af t e r incuba- t i o n f r e e and bound l igand were sepa ra t ed with use o f a dext ran - charcoa l suspension; a f t e r c e n t r i f u g a t i o n t h e a c t i v i t y of t h e superna tan t s o l u t i o n was measured by l i q u i d s c i n t i l l a t i o n count ing. S e n s i t i v i t y was improved by a sequen t i a l s a t u r a t i o n procedure ( incubat ion wi th unlabe l - l e d drug, followed by incubat ion with r ad io l igand) . O f t h e drugs t e s t e d , only bleomycin (>0.1 u n i t ) i n t e r f e r e d .

650 FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFlFY

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108. L.A. Sapunova, A.V. Gaevskii, G.A. Maslova and E.I. Grodnitskaya, Khim-Farm. Zh., - 16, 708 (1982); Anal. Abst. , 44, 5E 13 (1983).

109. N.R. Farnsworth, R.N. Blomster, D. Damratoski, W.A. Meer and L.V. Cammarato, Lloydia, 27 , 302 (1964).

110. N.J. Cone, R. Miller and N. Neuss, J. Pharm. Sci., 52, -

688 (1963).


111. N.R. Farnsworth and I.M. H i l i n s k i , J. Chromatog., - 18, 184 (1965).

112. G.H. Svoboda, Llyodia, - 24, 173 (1961).

113. A.N. Masoud, N.R. Farnsworth, L.A. S c i u c h e t t i , R.N. Blomster and W.A. Meer, Lloydia , 31, 202 (1968).

114. P . Horvath and G . Ivanyi , Acta Pharm. Hung., 52, 150 (1982).

115. M. Gazdag, K . Mihalyfi and G . Szepesi , Fresenius Z . Anal. Chem., - 309, 105 (1981).

116. M. De Smet, S.J.P. Van Belle, G.A. Storme and D.L. Massart, J. Chromatog., 345, 309 (1985).

117. D.E.M. Vendrig, J. Teeuwsen and J . J . M . Hol thuis , Ib id . , - 424, 83 (1988).

118. S. Gorog, B. Herenyi and K . Jovanovics, I b i d . , 139, 203 (1977).

119. M. Verzele, L. D e Taeye, J . Van Dyck, G. De Decker and C . De Pauw, Ib id . , - 214, 95 (1981).

120. S. Auriola , V.P. Ranta, T. Naaranlah t i and S.P. Lapinjoki , Ib id . , 474, 181 (1988).

1 2 1 . S. Mandal and M.L. Maheshwari, Indian J. Pharm. S c i . , 49, 205 (1987).

1 2 2 . T . Naaranlah t i , M. Nordstrom, A . Huhtikangas and M . Lounasmaa, J . Chromatog., - 410, 488 (1987).

123. J.P. Renaudin, Ib id . , - 291, 165 (1984).

124. D.E.M. Vendrig and J.J.M. Holthuis , Trace Trends Anal. Chem., g , 141 (1989).

125. Atta-ur-Rahman, M. Bashir , M. Hafeez, N . Perveen, J . Fatima and A . N . Mistry, P lan ta Med., - 47, 246 (1983).

126. S. Tafur , W.E. Jones, D . E . Dorman, E .E . Logsdon and G.H. Svoboda, J. Pharm. S c i . , - 64, 1953 (1975).

127 . D.E.M. Vendrig, B.P.G. Smeetis, J.H. Beijnen, O.A.G. Vander Hauwen and J.J.M. Holthuis , Int. J. Pharm., 43, 131 (1988).

658 FARlD J. MUHTADI AND ABDUL FATTAH A. A. AFIFY

128. K ; Hirata, M. Kobayashi, K . Miyamoto, T. Hoshi, M. Okazaki and Y , Miura, P lan ta Med., 55, 262 (1989).

129. J . D . Teale , M.C. Jacquel ine and V. Marks, B r . J. Cl in . Pharmacol., - 4 , 169 (1977).

Biochem., - 95 , 214 (1979). 130. J.J. Langone, M.R. D'onofrio and H. Van Vunakis, Anal.

131. V.S. S e t h i , S.S. Burton and D.V. Jackson, Cancer Chemo- t h e r . Pharmacol., - 4 , 183 (1980).

ACKNOWLEDGEMENT

The au thor would l i k e t o thank Mr. Uday C . Sharma, Dept. o f Pharmacognosy, College of Pharmacy, Riyadh, Saudi Arabia f o r h i s va luable and s i n c e r e e f f o r t s i n typing t h i s manuscript.

TITANIUM DIOXIDE

Harry C. Brittain, Gary Barbera,

Joseph DeVincentis, and Ann W. Newman

Bristol-Myers Squibb Pharmaceutical Research Institute

Bristol-Myers Squibb Company

New Brunswick, NJ 08903



660 HARRY G. BRlTTAlN ET AL.

CONTENTS

1.

2.

3.

Description 1 . 1 1.2 Appearance 1.3 General Chemical properties 1.4 Uses and Applications

Name, Formula, and Molecular Weight

Method of Preparation

Physical Properties 3.1 Particle morphology 3.2 Crystallographic Properties 3.3 Thermal methods of analysis 3.4 Particle size distribution 3.5 Surface area 3.6 Density 3.7 Powder Flow characteristics 3.8 Hygroscopicity 3.9 Solubility 3.10 Spectroscopy

4. Methods of Analysis 4.1 Compendia1 Tests 4.2 Identification 4.3 Elemental Analysis 4.4 Spectrophotometric Methods of Analysis 4.5 4.6 4.7 High Performance Liquid Chromatographic

Thin Layer Chromatographic Methods of Analysis Gas-Liquid Chromatographic Methods of Analysis

Methods of Analysis

5. Stability 5.1 Stability 5.2 Incompatibilities with functional groups

6. References

TITANIUM DIOXIDE

1. Description

66 1

1.1 Name. Formula. and Molecular Weight

Titanium dioxide is the most stable oxide of titanium, and can be obtained from either natural or synthetic sources. The material exists naturally in three crystal modifications, known as rutile, anatase, and brookite [ I ] .

The Chemical Abstracts identification number is CAS- 13463-67-7. In the United States, it is identified in the Color Index as 77891, and is denoted as C.I. Pigment White No, 6. It is also identified as EEC No. E 171 [2].

The chemical formula is TiO,, which corresponds to a formula weight of 79.90 Daltons. The elemental composition is Ti 59.95% and 0 40.05 % .

1.2 Appearance

Naturally occurring titanium dioxide may appear red, or reddish brown to black. The color is normally due to the presence of iron, chromium, or vanadium contamination, which may amount to 10% of the total titanium content [3].

Synthetically purified titanium dioxide exists as a white, odorless, tasteless powder.

L3 Ge neral Chemical Properties

A very detailed description of the chemistry associated with titanium oxygen systems is available [4]. The material is very thermally stable, and extremely resistant toward chemical degradation. It can be partially reduced when heated in the presence of hydrogen or carbon monoxide, the products being either lower oxides or mixtures of titanium carbide and lower oxides. Reduction by active metals (Na, K, Ca, or Mg) can

662 HARRY G . BRIITAIN ET AL.

only be partially effected, and chlorination of the oxide phase is possible only in the presence of a reducing agent.

The reactivity of titanium dioxide toward acids is very dependent on the temperature of the reaction mixture. It may be slowly dissolved by boiling concentrated sulfuric acid, with the dissolution rate being promoted by the addition of ammonium sulfate. Titanium dioxide may be readily dissolved by hydrofluoric acid. The material is completely insoluble in aqueous alkalies, but is readily dissolved in molten sodium (or potassium) hydroxide, carbonate, or borate. An equimolar molten mixture of sodium carbonate and sodium borate is partially effective as a dissolution medium.

The most important use of titanium dioxide is as a white pigment, owing to its very high reflectance at visible and ultraviolet wavelengths [ 5 ] . The refractive index of this material is so extremely high that fine particles scatter light with almost total efficiency. At the same time, the films are almost totally opaque. The ability of industry to produce titanium dioxide in appropriate particle size ranges has made it the most important white pigment in existence.

In ointments or lotions, titanium dioxide is a very efficient reflector of sunlight [6] . Its ability to act as a sunblock has led to its widespread use as a protective agent toward sunburn.

2. Method of Preparation

Although titanium dioxide can be obtained naturally as one of three crystal polymorphs (anatase, rutile, or brookite), pharmaceutically acceptable material is produced synthetically.

The bulk of pure titanium dioxide is obtained from purification of the abundant ore, illmenite (FeTiO,), using the su&m process [7]. In this

TITANIUM DIOXIDE 663

procedure, the raw ore is first digested with concentrated sulfuric acid. The product of this step is termed the "sulfate cake", which is then leached with water to produce a mixture of FeSO,, Fe;?(SOJ,, and TiOSO,. At this point, scrap iron is used to reduce all Fe(IT1) to Fe(II), whereupon the FeSO, is removed by filtration. The TiOSO, solution is boiled to hydrolyze the solute into a suspension of hydrated TiO,. This material is filtered, and finally calcined at 800-900°C to produce the final product. When calcined at 8OO0C, the hydrous oxide normally converts to the anatase phase.

The other important process for production of titanium dioxide is termed the chloride process [7]. The raw material used in this process is natural rutile, which is first heated at 950°C in the presence of carbon (in the form of coke) and chlorine. This produces crude TiCI,, and this product is heated at 1OOO"C in the presence of oxygen to produce the final titanium dioxide product. Under these conditions, the final product is the rutile phase.

The chloride process accounts for over half of the TiOz production in the United States, and is the most economical when high-grade ores are available. The sulfate process cannot use rutile as its starting material, but is able to make use of low-quality ores (or slags remaining after iron processing is complete) as input materials.

The physical properties of the titanium oxide pigments can be further improved by slurrying the products in water, and then selectively precipitating a surface coating of either S i 9 , A1203, or TiO, itself on the fine particles.


A concise summary of the physical properties of titanium dioxide is available in the Handbook of Excipients [S]. Also summarized in this publication are commercial availability, methods of manufacture, and various pharmacopeial specifications.

664 HARRY G. BRITAIN ET AL

For the present work, three representative samples of U.S.P. grade titanium dioxide were characterized. Two of these were obtained from Warner Jenkinson, and were described as being either oil ("Atlas White" material, lot 402239) or water ("Kowett" material, lot 401 398). The third lot was obtained from Spectrum Chemical Co., and was also certified to be U.S.P. grade (lot HB098). In addition to these U.S.P. materials, a non-U.S.P. grade of titanium dioxide (obtained from Johnson Matthey, lot MTI50W) was also studied as part of the crystallographic characterization.

Photomicrographs of the titanium dioxide samples are shown in Figures 1-4. All materials were found to exist as aggregate species which were built up from the consolidation of exceedingly fine subparticles. When viewed at 2O,OOOx, the subparticles of the U.S.P. materials are uniformly round in nature, and appear to be approximately 100 nm in diameter. No difference in the subparticle size was noted for materials prepared for different applications, or obtained from alternate vendors. This would indicate that the subparticle size was determined by the method of manufacture. The aggregates formed from these subparticles were very compact in nature, indicating efficient close-packing of the subparticles. The particle size of the aggregate species did vary among vendors, and this difference will be discussed in a different section.

The subparticles of the non-U.S.P. grade material were found to be much coarser. The size distribution of these was also quite variable, but averaged approximately 1 pm in diameter. These subparticles were also spherical in their appearance, and the aggregate species formed from these appeared to be looser than those of the U.S.P. materials.

Titanium dioxide is known to crystallize in three polymorphic forms, anatase, brookite, and rutile. While the rutile and anatase polymorphs are commonly encountered, the brookite phase is quite rare. The


Figure 1. Scanning electron photomicrographs of U.S.P. grade titanium dioxide ("Atlas White" material, Warner Jenkinson), obtained at 500x (upper photo) and 3 0 0 0 ~ (lower photo).

666 HARRY G. BRIlTAIN ET AL.

Figure 2. Scanning electron photomicrographs of U.S.P. grade titanium dioxide ("Kowett" material, Warner Jenkinson), obtained at 500x (upper photo) and 3OOOx (lower photo).


Figure 3. Scanning electron photomicrographs of U . S .P. grade titanium dioxide (Spectrum Chemical Company), obtained at 505x (upper photo) and 3OOOx (lower photo).

668 HARRY G. BRI'ITAIN ET AL.

Figure 4. Scanning electron photomicrographs of non-U .S.P. grade titanium dioxide (Johnson Matthey), obtained at SoOx (upper photo) and 3000~ (lower photo).


relative stabilities of rutile and anatase are almost equivalent, but it appears that rutile is the most stable polymorph of the two. The crystal structures of these three polymorphs are well known, and the important properties summarized in Table I 191.

Although anatase and rutile are both tetragonal, they are not isomorphous. Anatase is usually obtained in near-regular octahedra, which has given it the alternate name of octahedrite. Rutile is found as slender prismatic crystals, which often grow as twins.

The rutile structure has been discussed in great detail, since it is commonly taken as one of the prototype crystal structures [9]. The unit cell is depicted in Figure 5. The structure consists of chains of TiO, octahedra, in which each octahedron shares a pair of opposite edges and vertices with neighboring octahedra. Another way to envision the overall structure is to consider it as a slightly distorted hexagonally close packed array of oxygen atoms with half the octahedral interstices being occupied by titanium atoms.

Powder x-ray diffraction can be used to easily differentiate between the polymorphs of titanium dioxide. For the anatase phase, the most intense diagnostic scattering peaks correspond to d-spacings of 3.52 and 1.89 A, with relative intensities of 100:4. For rutile, the most intense scattering peaks correspond to d-spacings of 1.69, 3.26, and 2.49 A, and exhibit relative intensities of 100:97:70. Although brookite is never encountered in synthetically produced samples of titanium dioxide, its three most intense scattering peaks would correspond to d- spacings of 3.47, 2.90, and 1.88 A (relative intensities of 100:85:75).

The U.S.P. grade of titanium dioxide most commonly marketed is the anatase phase. The x-ray powder pattern for this material is shown in Figure 6, while the scattering angles, d-spacings, and relative intensities are found in Table 11. For comparison purposes, a powder pattern obtained for the rutile phase (the non-U.S.P. material described earlier) is shown in Figure 7, and its corresponding crystallographic information is presented in Table 111.

670 HARRY C . BRITAIN ET AL.

Table I

Crystallographic Data for the Three Polymorphs of Titanium Dioxide

Property Anatase Brookite Rutile

Crystal system orthorhombic

14, Space group

tetragonal

Pcab

tetragonal

P4/mnm

Number Ti02 in unit cell 4

Cell dimensions (nm) a 0.3785 b C 0.9514

Cell volume (mL x i d 4 ) 136.3

anisotropic refractive indices "1 2.554 n2 n3 2.493

density (glmL) 3.893

8

0.5456 0.9182 0.5143

257.6

2.583 2.586 2.741

4.1 19

2

0.4594

0.2962

62.5

2.616

2.903

4.245

TITANIUM DIOXIDE 67 I

Figure 5 . Structure of the unit cell of titanium dioxide, rutile phase.

I I I I I I I

e /

0

- l r\ @ - I I I

Oo @ Ti

612 HARRY G. BRITTAIN ET AL.

Figure 6. Powder x-ray diffraction pattern of titanium dioxide, anatase phase. The intensity scale is presented in arbitrary units.

0 . 0 10 .a 20.0 30.0 40.0 50.0 4 1

60.0 70.0

Degrees 2-8


Table II

Crystallographic Data for the 10 Most Intense Scattering Peaks of Titanium Dioxide, Anatase Phase

Angle D-Spacing Relative Intensity (degrees 2-0) (Angstroms) w n a x )

25.2625 36.9150 37.7600 38.5500 48.0 100 53.8500 55.0200 62.6150 62.82OO 68.6800

3.5226 2.4330 2.3805 2.3335 1.8935 I .7011 I .6677 1.4824 1.4817 I .3655

100.00 7.12

27.59 8.41

42.40 28.61 28.32 23.58 11.98 11.14

674 HARRY G . BRI'ITAIN ET AL.

Figure 7. Powder x-ray diffraction pattern of titanium dioxide, rutile phase. The intensity scale is presented in arbitrary units.

0.0 10.0 20.0 30 .O 40 .O 50 .O 60 .O 70.0

Degrees 2-8

TITANIUM DIOXIDE

Table 111

Crystallographic Data for the 1 1 Most Intense Scattering Peaks of Titanium Dioxide, Rutile Phase

Angle D-Spacing Relative Intensity (degrees 2-8) (Angstroms) (1 4naJ

675

27.3025 36.0200 39.0275 41.1400 43.8625 54. I700 56.3075 62.7175 63.8 100 68.7850 69.7850

3.2638 2.4914 2.3061 2.1924 2.0624 1.6918 1.6325 1.4802 1.4575 1.3637 1.3466

97.07 69.69 4.64

37.65 10.34

100.00 23.69 23.69 13.60 37.29 30.43

616 HARRY G. BRIITAIN ET AL

The rutile polymorph of titanium dioxide is refractory, and melts around 1850°C. Melting points for the anatase and brookite polymorphs have not been established, since these materials convert to the rutile phase at elevated temperatures.

3.4 Part icle size distribution

The particle size distributions of the three U.S.P. titanium dioxide samples were obtained using optical microscopy and image analysis. This method provides information on the size of the aggregate species only, and does not contain any data pertinent to the subparticles from which the aggregates are composed. Full particle size distributions for these three Ti@ lots are provided in Table IV. The particle diameters were obtained from analysis of the particle cross-sectional areas (provided by the image analysis system). All materials were found to be composed of very small particles, the basic units of which were smaller than 10 pm. The average particle size (shown in Table V) of the two Warner Jenkinson lots were equivalent, while the material obtained from Spectrum Chemical Co. was slightly coarser in nature.

32 su rface a r a

The surface area of the three titanium dioxide lots was determined using a five-point B.E.T. analysis procedure, and the results of this study are found in Table V. The three materials exhibit fairly high surface areas (approximately 10 m2/g), and were found to be mutually equivalent.

The true density (measured by helium pycnometry) of titanium dioxide differs with the polymorphic state of the material. Rutile is the most dense (4.25 g/mL), followed by brookite (4.12 g/mL) and anatase (3.89 g/mL).

611 TITANIUM DIOXIDE

Table IV

Particle Size Distributions for Various Titanium Dioxides, Obtained Using Optical Microscopy (aggregate species having diameters larger

than 10 pm have been excluded)

Band Size (Pm) 401398 402239 HB098

0.0 - 0.5 0.5 - 1.0 l . 0 - 1.5 1.5 - 2.0 2.0 - 2.5 2.5 - 3.0 3.0 - 3.5 3.5 - 4.0 4.0 - 4.5 4.5 - 5.0 5.0 - 5.5 5.5 - 6.0 6.0 - 6.5 6.5 - 7.0 7.0 - 7.5 7.5 - 8.0

12.7 45.4 26.5 8.2 4.0 l .3 1.1 0.0 0.5 0.0 0.0 0.3 0.0 0.0 0.0 0.0

9.6 52.2 26.7 5.4 3.6 1.1 0.0 0.5 0.0 0.4 0.2 0.0 0.0 0.2 0.0 0.0

8.0 15.4 14.1 14.6 14.9 7.5 8.8 5.3 4.8 1.6 0.5 1.9 1.1 0.8 0.8 0.0

678 HARRY 0. BRITTAIN ET AL.

Table V

Micromeritic Properties Obtained for Various Titanium Dioxides

property 401398 402239 HB098

Average Particle Size (pm) 1.05 1.02 2.19

Surface Area (m2/s) 10.5 8.2 9.0

Bulk Density (g/mL) 0.4 0.4 0.5

Tap Density 0.7 0.6 0.8

Compressibility 39 37 35


The bulk densities of the commercially supplied U.S.P. grade materials were found to average around 0.4 g/mL (Table V). The tap densities of these lots were found to increase to approximately 0.7 g/mL, corresponding to a compressibility factor of approximately 37%. No significant difference among the three lots studies was evident when comparing the micromeritic properties.

3.7 HVgroscoDicin!

Titanium dioxide is not hygroscopic, and does not form true hydrate phases. It is possible to prepare hydrated titanium oxide materials through the addition of alkali-metal hydroxides to a solution of a Ti(I1) or Ti(II1) salt. The resulting titanium hydroxide precipitate is extremely unstable, is a powerful reducing agent, and rapidly converts to a hydrated oxide material [4].

If the precipitation is performed at room temperature, one obtains a compound known as orthotitanic acid, and which has the approximate formula of Ti0;2H20’Ti(0H),. If the suspension is boiled, or if the precipitation is effected from a hot solution, a compound known as metatitanic acid is obtained. This less hydrated oxidic compound has the approximate formula of TiOz-H20TiO(OH)2, Metatitanic oxide is commonly obtained in the colloidal state, and is the preferred intermediate in the manufacture of titanium dioxide pigments.

U Solubility

Titanium dioxide is completely insoluble in water, dilute acids, or common organic solvents. It can be dissolved in concentrated sulfuric or hydrofluoric acids at elevated temperatures, with the accompanying production of salt species.

322 Spe ctroscopy

Titanium dioxide transmits through the visible and near infrared regions

680 HARRY G. BRlTTAlN ET AL.

of the spectrum, having no absorption bands in these regions. It becomes completely opa ue at wavelengths below 400 nm, and at energies below 2000 cm- . The reflectivity of TiO, is approximately 90% of that of a MgO standard [4].

9


4.1 Compendia1 Tests

The U.S.P. compendia1 requirements [ 101 for titanium dioxide are that it cannot contain less than 99.0% and not more than 100.5% TiO,, when calculated on a dried basis. In addition, the material may not contain more than 0.001 % of lead, not more than 2 ppm of antimony, and not more than 1 ppm of mercury.

The compound is tested as to its identification, loss on drying, loss on ignition, water-soluble substances, acid-soluble substances, and arsenic content. A full method is provided for the potency assay. The details of these tests are as follows:

Identification: The compound is suspended in hot concentrated sulfuric acid, and diluted with water. Undissolved solid is filtered off, and a few drops of hydrogen peroxide test solution are added to the clear filtrate. The positive identification consists of an orange-red color which develops immediately.

Loss on drying: The general test method <731> is followed. After being dried at 105°C for 3 hours, the material cannot lose more than 0.5% of its weight.

Loss on ignition: Following general test method < 733 > , the material is ignited at 800 k 25°C to constant weight. The material cannot lose more than 0.5% of its weight.

Water-soluble substances: The sample is suspended in water, mixed, and allowed to stand overnight. Ammonium chloride

TITANIUM DIOXIDE 68 1

test solution is used to clarify the suspension, which is then filtered. A 100 mL portion of the filtrate is collected, dried, and ignited to constant weight. The residue cannot amount to more than 0.25% of the original sample weight.

Acid-soluble substances: The solid is suspended in 0.5 N HCI, and heated on a steam bath. The suspension is filtered, with the filtered solids being washed with 0.5 N HCI. These washings are combined with the original filtrate, dried, and ignited to constant weight. The residue cannot weigh more than 0.5% of the original sample weight.

Arsenic: The arsenic content is determined according to general test < 21 1 > . The solid is suspended in water, to which is added appropriate amounts of hydrazine sulfate, potassium bromide, sodium chloride, and sulfuric acid. Any evolved arsine is collected, and determined. The limit is 1 ppm.

Assay: The initial sample is dissolved in a mixture of hot sulfuric acid and ammonium sulfate. After the dissolution is complete, the mixture is allowed to cool, and diluted with water. The suspension is then filtered, and neutralized with ammonium hydroxide. This filtrate is reduced in a Jones reductor (making use of a zinc amalgam), and then titrated with 0.1 N potassium permanganate volumetric reagent. IJnder these conditions, each mL of 0.1 N potassium permanganate reagent is equivalent to 7.988 mg of TiO,.

All identification tests for titanium dioxide first require solubilization of the oxide, typically by concentrated acid solutions. After production of aqueous solutions of titanium ions, a number of colorimetric reactions may be used for identification purposes.

Hydrogen peroxide causes a yellow color to develop in acidic solutions containing dissolved titanium [ 1 11. In solutions containing sulfuric


acid, the color is due to a complex peroxidic anion formed from free peroxodisulfatotitanic acid. This reaction is the one used for the cornpendial identification test since it is not interfered with by common metallic ions,

Several other color reactions may be used for the identification of titanium dioxide, after solubilization of the material. Pyrocatechol yields a yellowish-red color with weakly acidified solutions of titanium salts [12]. When chromotropic acid is reacted with solubilized titanium between pH 2.5 and 5.0, a wine red colored solution is obtained 1131. An intense brown color is given by the addition of acidic solutions of 3,5,7,2’,4’-pentahydroxyflavone (morin) to titanium salts, although the composition of the product is unknown [ 141,

Disodium- 1,2-dihydroxybenzene-3,5-disulfonate (tiron) yields a strong yellow color when reacted with titanium salts in the pH range of 4.3- 9.6 [15]. Titanium salts also react with 5-sulfosalicylic acid at pH 3-5 to yield yellow solutions [16]. Other reagents which have found limited use in identification testing are thymol [17], gallic acid [18], and salicylic acid [ 191.

4J Gravimetric Met hods of Analysis

As one of the transition elements, titanium can be readily precipitated with ammonium, sodium, or potassium hydroxide. The hydrated oxide is ignited to constant weight at any temperature exceeding 350°C. The method is not useful for complicated samples containing other cations which could be precipitated by hydroxide, but is very applicable to the analysis of U.S.P. grade titanium dioxide.

Titanium salts can also be determined using cupferron (the ammonium salt of nitrosophenylhydroxylamine) [20] or tannin [21]. In addition, 2’-hydroxy-4’-methylpropiophenone oxime has been shown to form an insoluble 1:l complex with Ti salts [22].


- 4.4 Titrimetric Methods of Analysis

Most titrimetric methods for titanium depend on the reduction of Ti(1V) to Ti(III), followed by subsequent titration with a standard oxidizing solution [23]. The methods vary with the choice of reductant, the titrant, and with the method of detecting the endpoint.

The standard dissolution procedure for titanium oxides is the ammonium sulfate-sulfuric acid mixture developed by Rahm [24]. The most commonly used method in industry is based on the use of metallic aluminum as the reductant, and ferric ammonium sulfate as the titrant. The use of ferric ion as the reagent is preferred, since relatively few species will interfere with its reaction with reduced titanium solutions.

Other reductor systems can be used, which will yield equally satisfactory results [25]. These can be the Jones, lead, cadmium, iron, nickel, or bismuth reactors, with the Jones reactor being chosen for use in the compendia1 assay method. Liquid mercury amalgams can also be used as reductors, being prepared with zinc, cadmium, bismuth, lead, or tin. While the liquid amalgams are easier to handle, and are more rapid than are column reactors, none of these is as simple as the aluminum foil reductor.

Besides ferric ammonium sulfate, permanganate solutions can be used to titrate the reduced titanium. Although many cations interfere with the permanganate titration, this reagent is still useful in the assay of highly purified titanium dioxide materials. Potassium dichromate can be used to titrate Ti(II1) solutions (with the aid of 0.2% indigo as the indicator), and Ce(1V) reagents can also be used as titrants.

Ethylenediaminetetraacetic acid can be used as a reagent for the titration of Ti(1V). However, the reaction proceeds slowly, and the Ti(1V) species tend to hydrolyze during the titration, so that a back-titration method is necessary to make the complexometric method work properly [261.

684 HARRY G. BRI7TAIN ET AL.

U Polarographic Methods of AM lysis

It has been demonstrated that the half-wave potential for the reduction of Ti(1V) to Ti(I1I) is -0.81 V (against the standard calomel electrode) in O.1M HCI [27]. The further reduction of Ti(II1) to Ti(l1) can be observed in alkaline media, but this reaction has no useful analytical significance. In these methods, oxalate, tartrate, or citrate buffer systems are used as supporting electrolytes to prevent the hydrolytic precipitation of hydrated titanium oxides. In the presence of tartrate buffer, well defined waves are obtained only at pH values less than 2, or between 6 and 7. The Ti(1V)-Ti(II1) couple is reversible only in tartrate buffer at pH values less than 1.

When 0.4M citrate ion is used as the medium, the reduction is well defined at all pH values, but the reduction potential was found to vary with the solution pH [27]:

PH E,,, V.S. S.C.E.

0.0 3.0 7.0

11.5

-0.28 -0.80 -0.95 -1.49

Ethylenediaminetetraacetic acid has been found to be satisfactory as a supporting electrolyte [28]. It was demonstrated that the half-wave potential for a 0.4M solution of Ti(1V) in 0.25N EDTA varied from - 0.22 V (vs. S.C.E.) at pH 3.0 to -0.82 V at pH 8.7. The half-wave potential was found to be independent of the solution acidity below pH 2.

AC voltammetry using the fundamental and second harmonic wave of Ti at a semistationary mercury drop electrode has been used for the direct determination of Ti [29]. This method has the distinct advantage of being able to tolerate large quantities of metal ion impurities. In another method, Ti salts were chelated with dihydroxyazo dyes, adsorbed onto a hanging mercury drop electrode, and then determined

TITANIUM DIOXIDE

by cyclic voltammetry [30]

685

- 4.6 Atomic Spectroscopic Methods of Analysis

Historically, emission spectrography has been very important in the quantitative assay of titanium-containing materials. It should be pointed out that where Ti is a major constituent of the material (as would be the case for titanium dioxide), the spectrophotometric and titrimetric methods of analysis are more appropriate. Nevertheless, a variety of quantitative methods have been developed for the determination of Ti in oxidic phases.

After spark-source excitation, the Ti lines at 3239.04 or 3349.41 A are determined against an internal standard. The 3239.04 A Ti line is normally quantitated against the 3232.61 A line of Li, while the 3349.41 A Ti line is quantitated against the 3126.11 A line of Cu.

After dissolution of the oxide, Ti may be directly determined by atomic absorption using either the nitrous oxide-acetylene [3 I] or oxygen- nitrogen-acetylene [32] flame systems. The method is straight-forward, and no interference has been noted from Cr, Co, Mn, Mo, Nb, W, Ta, or Cu [33]. An improvement in the determination of Ti using the nitrous oxide-acetylene flame system was noted when the analysis was performed in a buffered HF-boric acid mixture [34].

X-ray fluorescence has been found to be useful in the quantitation of titanium oxides, with internal standards also being used [35]. The methods all make use of the Ti Ka emission at 2.750 A, and differ in their choice of internal standards.

Atomic absorption spectroscopy has been used to determine the trace quantities of other metal contaminants in titanium dioxide pigments [36]. Auger electron spectroscopy has been used to directly determine the levels of Ti in oxide layers [37].


4.7 Spectrophotometric Methods of Analysis

Theoretically, any colorimetric method useful for identification purposes can be developed into a quantitative spectrophotometric assay. The number of reagents proposed for use in photometric assay methods is extensive, and interested readers should consult appropriate review articles. Although many of these reagents have been developed for use in areas relating to nonferrous metallurgy [38], they may be applied to the spectrophotometric assay of Ti in TiO,. Assays involving spectrophotometric reagents probably represent the most extensive range of methods developed for determination of Ti in any sample matrix.

The peroxide method has proven to be the most useful for this purpose, owing to the high acidity of the medium in which the reaction is conducted. Interferences are observed only in the presence of V, Mo, or F, but these species are not normally present in U.S.P. grade titanium dioxide. In the spectrophotometric assay method, the absorption maximum at 410 nm is used to determine the titanium concentration after the oxide is dissolved [39]. The spectrophotometric endpoint of the peroxide method has been combined with flow injection analysis techniques to yield an automated procedure “1.

Reagents containing oxygen-donor atoms (phenolic or alcoholic hydroxy groups) are most suitable as spectrophotometric reagents, but nitrogen- donor functional groups can also be used. A detailed review of photometric reagents for Ti has been written by Sommer [41].

The yellow colors developed by titanium salts with tiron [15] and sulfosalicylic acid [16,42] have also been used to develop quantitative spectrophotometric assay methods. Other useful reagents include tichromin and dibromotichromin [43], chromotropic acid [MI, chlorophosphonazo I [45], and diantipyrylmethane [46]. The diantipyrylmethane reagent has also been used to measure Ti salts after these have been stripped off a silica gel column [47].


4J3 Chromatographic Methods of Analvsis

Chromatographic methods for titanium dioxide have not been investigated vigorously, owing to the wealth of alternate methods available. Spectrophotometric or atomic spectroscopic measurements can be conducted more rapidly, are more sensitive, and are not affected by the oxidation state of the metal. A chromatographic method would be appropriate only if the Ti species was to be separated from a complicated sample matrix, but this is not anticipated in the analysis of U.S.P. titanium dioxide.

In addition, Ti salts are prone to hydrolysis, and ultimately form insoluble hydrated oxides. For a chromatographic procedure to be developed, the eluent would have to contain strongly coordinating agents capable of preventing hydrolysis. Although such agents have been proposed for the ion chromatographic assay of other transition elements [48], they do not appear to have been applied to the analysis of titanium salts.

5. Stability

Stability

Titanium dioxide reacts only with hot, concentrated mineral acid solutions. It is completely stable with respect to light, oxidation, changes in pH of suspensions, and microbiological attack. It has been found to be stable at all temperature values up to its melting point (1850°C). If heated strongly under vacuum, there is a slight loss of oxygen corresponding to a change in composition to TiO,.,. This product is dark blue, but reverts to the original white color when heated in air.

Incompatibilities with functional srouDS

Titanium dioxide is chemically unreactive, and its only chemistry takes place after the oxide has been dissolved in strong acids. It is a known

688 HARRY G . BRlTTAlN ET AL

catalyst, however, and is capable of inducing solid state chemical reactions under certain conditions. However, these reactions require the application of temperatures exceeding lOO"C, which should not be encountered in the normal range of pharmaceutical formulations.

6.

I .

2.

3.

4.

5 .

6.

7.

8.

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CUMULATIVE INDEX

Bold numerals refer to volume numbers.

Acehutolol, 19. 1 Acetaminophen, 3, I ; 14.551 Acetohexamide, 1.1; 2,573; 21, I Allopurinol, 7, 1 Alpha-tocopheryl acetate, 3.111 Amantadine, 12.1 Amikacin sulfate, l2.37 Amiloride hydrochloride, IS, I Aminoglutethimide, 15, 35 Aminophylline, 11, 1 Aminosalicylic acid, 10, 1 Amiodarone, 20.1 Amitriptyline hydrochloride, 3, 127 Amoharbital, 19, 27 Amodiaquine hydrochloride, 21,43 Amoxicillin, 7, 19 Amphotericin B, 6, I; 7,502 Ampicillin, 2, I; 4. 518 Apomorphine hydrochloride, 20, 121 Ascorbic acid, ll, 45 Aspirin, 8, 1 Astemizole, 20, 173 Atenolol, 13, 1 Atropine, 14, 32 Azathioprine, 10, 29 Azintamide, 18, 1 Aztreonam, 17,l Bacitracin. 9, 1 Baclofen, 14, 527 Bendroflumethiazide, 5, I; 6,597 Benperidol, 14,245 Benzocaine, 12.73

Benzyl benzoate, 10.55 Betamethasone dipropionate, 6,43 Bretylium tosylate, 9.71 Bromazepam, 16.1 Bromocriptine methanesulfonate, 8,47 Bupivacaine, 19.59 Busulphan, 16.53 Caffeine, 15,71 Calcitriol, 8, 83 Camphor, 13, 27 Captopril, ll, 79 Carbamazepine, 9,87 Cefaclor, 9, 107 Cefamandole nafate, 9. 125; 10, 729 Cefazolin, 4, 1 Cefotaxime, 11. 139 Cefoxitin, sodium, ll, 169 Ceftazidime, 19.95 Cefuroxime sodium, 20,209 Celiprolol hydrochloride, 20, 237 Cephalexin, 4.21 Cephalothin sodium, 1,319 Cephradine, 5,21 Chloral hydrate, 2, 85 Chlorambucil, 16, 85 Chloramphenicol, 4.47,518; 15,701 Chlordiazepoxide, 1, 15 Chlordiazepoxide hydrochloride, 1,39; 4,518 Chloroquine, 13, 95 Chloroquine phosphate, 5, 61 Chlorothiazide, 18.33 Chloropheniramine maleate, 7.43

693

Chlorprothixene, 2 , 63 Chlortetracycline hydrochloride, 8 , 101 Chlorthalidone, 14.1 Chlorzoxazone, 16,119 Cholecalciferol, see Vitamin D, Cimetidine, U, 127; 17, 797 Cisplatin. 14.77; L5,7% Clidinium bromide, 2,145 Clindamycin hydrochloride, 10.75 Clioquinol, 18.57 Clofazamine, 18,91 Clofazimine, 21,75 Clofibrate, 11, 197 Clonazepam, 6,61 Clonidine hydrochloride. 21, 109 Clorazepate dipotassium, 4,91 Clotrirnazole, ll, 225 Cloxacillin sodium, 4, 113 Cocaine hydrochloride, 15,151 Codeine phosphate, 10,93 Colchicine, 10, 139 Cyanocobalamin, 10,183 Cyclandelate, 21,149 Cyclizine, 6, 83; 7 , 502 Cyclobenzaprine hydrochloride, 17,41 Cycloserine, 1.53; 18,567 Cyclosporine, 16,145 Cyclothiazide, 1 ,66 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 and dibucaine hydrochloride, l2.105 Diclofenac sodium, 19, 123 Diethylstilbestrol, 19, 145 Diflunisal, 14,491 Digitoxin, 3.149 Digoxin, 9 ,207 Dihydroergotoxine methanesulfonate, 7 , 81 Diwtyl sodium sulfosuccinate, 2.199; 12,713 Diperodon, 6 ,99 Diphenhydramine hydrochloride, 3,173 Diphenoxylate hydrochloride, 7,149 Disopyramide phosphate, W , 183 Disulfiram, 4,168 Dobutamine hydrochloride, 8,139 Dopamine hydrochloride, 11,257 Doxorubicine, 9,245 Droperidol, 7 , 171 Echothiophate iodide, 3, 233 Emetine hydrochloride, 10,289

Enalapril maleate, 16, 207 Ephedrine hydrochloride, 15, 233 Epinephrine, 7, 193 Ergonovine maleate, 11,273 Ergotamine tartrate, 6 , 113 Erythromycin, 8 , 159 Erythromycin estolate, 1,101; 2,573 Estradiol, 15,283 Estradiol valerate, 4, 192 Estrone, 12, 135 Ethambutol hydrochloride, 7,231 Ethynodiol diacetate, 3,253 Etomidate, 12, 191 Etoposide, 18, 121 Fenoprofen calcium, 6, 161 Flecainide, 21, 169 Flucytosine, 5, 115 Fludrocortisone acetate, 3,281 Flufenamic acid, ll .313 Fluorouracil, 2,221: 18.599 Fluoxetine, 19, 193 Fluoxymesterone, 7,251 Fluphenazine decanote, 9,275; 10,730 Fluphenazine enanthate, 2,245; 4,524 Fluphenazine hydrochloride, 2,263; 4, 519 Flurazepam hydrochloride, 3,307 Folic acid, 19,221 Furosemide, 18, 153 Gentamicin sulfate, 9,295; 10,731 Glafenine, 21, 197 Glibenclamide, 10,337 Gluthethimide, 5, 139 Gramicidin, 8 , 179 Griseofulvin, 8,219; 9 ,583 Guanabenz acetate, 15,319 Halcinonide, 8 , 251 Haloperidol, 9 , 341 Halothane, 1,119; 2,573: 14,597 Heparin sodium, 12,215 Heroin, 10,357 Hexestrol, ll, 347 Hexetidine, 7.277 Homatropine hydrobromide, 16,245 Hydralazine hydrochloride, 8 ,283 Hydrochlorothiazide, 10,405 Hydrocortisone, 12,277 Hydroflumethiazide. 7,297 Hydroxyprogesterone caproate, 4,209 Hydroxyzine dihydrochloride, 7,319 Impenem, 17.73 Imipramine hydrochloride, 14,37 Indomethacin, 13,211 Iodamide, 15,337

694

Iodipamide, 2,333 lodoxamic acid, 20,303 Ioparnidol. 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 Lactose, anhydrous, 20,369 Leucovorin calcium, 8,315 Levallorphan tartrate, 2,339 Levartereno1 bitartrate, 1 ,49; 2,573; 11,555 Levodopa, 5 , 189 kvothyroxine sodium, 5,225 Lidocaine base and hydrochloride, 14,207; 15,

Lisinopril, 21, 233 Lithium carbonate, 15, 367 Lobeline hydrochloride, 19,261 Lomustine, 19.315 Loperamide hydrochloride, 19,341 Lorazepam, 9, 397 Lovastatin, 21, 277 Maprotiline hydrochloride, 15,393 Mebendazole, 16,291 Mefloquine hydrochloride, 14, 157 Melphalan, l3, 265 Meperidine hydrochloride, 1, 175 Meprobamate, 1,209; 4,520; 11,587 6-Mercaptopurine, 7,343 Mestranol. ll, 375 Methadone hydrochloride. 3,365; 4,520; 9,601 Methaqualone, 4,245,520 Methimazole, 8,351 Methotrexate. 5 ,283 Methoxamine hydrochloride, 20, 399 Methoxsalen, 9,427 Methyclothiazide, 5, 307 Methylphenidate hydrochloride, 10,473 Methyprylon, 2, 363 Metipranolol, 19, 367 Metoclopramide hydrochloride, 16.327 Metoprolol tartrate, 12, 325 Metronidazole. 5.327 Mexiletine hydrochloride, 20,433

76 I

Minocycline, 6,323 Minoxidil, 17, 185 Mitomycin C, 16,361 Mitoxantrone hydrochloride, 17,221 Morphine, 17,259 Moxalactarn disodium, W , 305 Nabilone, 10,499 Nadolof, 9,455; 10,732 Nalidixic acid, 8,371 Naloxone hydrochloride, 14,453 Nalorphine hydrobromide, 18, 195 Naphazoline hydrochloride, 21,307 Naproxen. 21,345 Natamycin, 10,513 Neomycin, 8 ,399 Neostigmine, 16,403 Nicotinamide, 20,475 Nifedipine, 18,221 Nitrazepan], 9 ,487 Nitrofurantoin, 5, 345 Nitroglycerin, 9, 519 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, 20,601 Oxazepam, 3,441 Oxyphenbutazone, 13, 333 Oxytocin, 10,563 Papaverine hydrochloride, 17,367 Penicillarnine, 10,601 Penicillin-G, benzothine, 11,463 Penicillin-G, potassium, 15,427 Penicillin-V, 1,249; 17,677 Pentazocine, W, 361 Pergolide mesylate, 21, 375 Phenazopyridine hydrochloride, 3 ,465 Phenelzine sulfate, 2,383 Phenformin hydrochloride, 4,319; 5,429 Phenobarbital, 7,359 Phenolphthalein, 20, 627 Phenoxymethyl penicillin potassium, 1, 249 Phenylbutazone, l l .483 Phenylephrine hydrochloride, 3 ,483 Phenylpropanolarnine hydrochloride, 12,357; 13,

Phenytoin, 13,417 Physostigmine salicylate, 18,289 Phytonadione, 17,449 Pilocarpine, 12,385

77 1

695

Piperazine estrone sulfate, 5, 375 Pirenzepine dihydrochloride, 16,445 Piroxicam, 15, 509 Polythiazide, 20, 665 Pralidoxine chloride, 17,533 Prazosin hydrochloride, 18,361 Prednisolone, 21.415 Primidone, 2,409; 17,749 Probenecid, 10,639 h-ocainamide 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 Pyrimetharnine, 12,463 Quinidine sulfate, 12,483 Quinine hydrochloride, 12,547 Ranitidine, 15,533 Reserpine, 4, 384; 5,557; W,737 Riboflavin, 19,429 Rifampin, 5,467 Rutin, 12,623 Saccharin, 13,487 Salbutamol, 10,665 Salicylamide, 13,521 Scopolamine hydrobromide, 19,477 Secobarbital sodium, 1,343 Silver sulfadiazine, 13,553 Sodium nitroprusside, 6,487; 15,781 Sotalol, 21,501 Spironolactone. 4,431; 18,641 Streptomycin, 16,507 Strychnine, 15,563 Succinycholine chloride, 10,691 Sulfadiazine, ll.523 Sulfadoxine, 17,571 Sulfamethazine, 7,401 Sulfamethoxazole, 2,467; 4,521 Sulfasalazine, 5, 515 Sulfisoxazole, 2,487 Sulfoxone sodium, 19,553 Sulindac, 13,573 Sulphamerazine, 6,515 Sulpiride. 17,607 Teniposide, 19,575

598

Terazosin, 20. 693 Terbutaline sulfate, 19,601 Terfenadine, 19,627 Terpin hydrate, 14, 273 Testolactone. 5,533 Testosterone enanthate, 4,452 Tetracaine hydrochloride, 18,379 Tetracycline hydrochloride, W, 597 Theophylline, 4,466 Thiabendazole, 16,611 Thiamine hydrochloride. 18,413 Thiopental sodium, 21,535 Thioridazine and Thiondazine hydrochloride, 18, 459

6, Thiostrepton, 7,423 Thiothixene, 18.527 Ticlopidine hydrochloride, 21,573 Timolol maleate, 16,641 Titanium dioxide, 21.659 Tolbutamide, 3,513;s. 557; 13,719 Trazodone hydrochloride, 16, 693 Triamcinolone, 1,367; 2,571; 4, 521,524; ll,593 Triamcinolone acetonide, 1,397.416; 2,571; 4,

Triamcinolone diacetate, 1,423; 11, 651 Triamcinolone hexacetonide, 6,579 Triclobisonium chloride, 2.507 Trifluoperazine hydrochloride, 9,543 Triflupromazine hydrochloride, 2,523; 4,521; 5,

Trimethaphan camsylate, 3,545 Trimethobenzamide hydrochloride, 2,551 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 nbamate, 4,494 Valproate sodium and valproic acid, 8,529 Verapamil, 17,643 Vidarabine, 15,647 Viblastine sulfate, 1,443; 21,611 Vincristine sulfate, 1,463 Vitamin D,, 13,655 Warfarin, 14,243 Xylometazoline hydrochloride, 14, 135 Yohimbine, 16,731 Zidovudine, 24,729 Zomepirac sodium, 15.673

521; 7,501 ; U, 615

557

696

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35107468 Analytical Profiles of Drug Substances and Excipients Vol 21 1992 ISBN 0122608216 9780122608216