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Studies of carbamazepine metabolism Webster, Donald Shaw 1989

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STUDIES OF CARBAMAZEPINE METABOLISM By DONALD SHAW WEBSTER B.Sc.  (Honours), The U n i v e r s i t y  of B r i t i s h Columbia,  1987  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE  in THE FACULTY OF GRADUATE STUDIES Department of Pharmacology Faculty  3 Therapeutics  of Medicine  We accept t h i s t h e s i s as conforming to the r e q u i r e d  standard  THE UNIVERSITY OF BRITISH COLUMBIA AUGUST 1989 ©DONALD SHAW WEBSTER,  1989  lo;  In  presenting this  degree at the  thesis  in  University of  partial  fulfilment  of  of  department  this or  thesis for by  his  or  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  representatives.  an advanced  Library shall make  it  agree that permission for extensive  scholarly purposes may be her  for  It  is  granted  by the  understood  that  head of copying  my or  publication of this thesis for financial gain shall not be allowed without my written permission.  Department  of Pharmacology & T h e r a p e u t i c s  The University of British Columbia Vancouver, Canada  Date  DE-6 (2/88)  August 2 1 .  1989  _  ii  ABSTRACT  The  objective  metabolism,  in  examination  of  of  this  order  to  how the  study  was  to  contribute  metabolism  examine  to  of  a  aspects  long  term  carbamazepine  of  goal  is  carbamazepine of  a  thorough  influenced  by  other  drugs. The f i r s t values  for  s e t of experiments were designed w i t h the i n t e n t of  the  pharmacokinetic  parameters  male New Zealand white r a b b i t s . t  l/2  (90-122  constant cases,  min),  unexpected  these parameters. in  reaching  maintained  five  peak level  concentration for  differences  between  rates  of  emptying,  rabbits  period is  also  place.  of  were  fasted  required possible  The  rabbits  have  may  a factor  that  relative  responsible  of  to  some  positions  hepatic  of  did  allow  of  remainder  of  calculation  of  been may  an  apparent  peak,  due  differences  in  the  influenced  by  the  have  been  thought  experiments. been  of  Alternatively,  underestimated.  enterohepatic curves  for  that  there  may  monooxygenases  and  f o r the m e t a b o l i c f a t e s of  but  It  to  is  delayed  time.  the  suggest  the  elimination  i n excess of the 12 hours t h a t  the  degree  In  reached  period  sampling may have that  it  the  cases.  in  (60-90 m i n ) ,  m a x  and  not  metabolism  c o n c e n t r a t i o n was e i t h e r  an extended  prior  carbamazepine-10,ll-epoxide activities  which  or  food eaten by the animals i n a p e r i o d the  test  were observed  these  gastric  in  1  ml/min/kg),  The plasma carbamazepine  its that  (46.2-142.4  min" )  results  carbamazepine  Values were o b t a i n e d f o r t  clearance  (0.0057-0.0077  of  determining  is  differences  glucuronysyl  carbamazepine.  the  carbamazepine be  some o f  In a d d i t i o n ,  circulation  that  time it  taking and  for  in  the  transferases  i The some  of  second its  set  of  experiments  principal  This  study  rabbits.  is  a  acetylisoniazid, containing co-factors. inhibition isoni'cotinic range  prelude  Three  The of  in  to  hydrazine,  on  the  S9  fraction  and  of  each  indicated  carbamazepine These  of carbamazepine  types  of  that  studies  of  of  of  rat of  acid  were  isoniazid  liver the  so t h a t  to  homogenate.  interaction  in  acetyl hydrazine,  tested and  in  a  of  isoniazid,  i n h i b i t i o n can be d e t e r m i n e d , as can be done M i c h a e l i s - M e n t o n  dependent  hydrazine,  should expanded to an e v a l u a t i o n  system  essential  t h e r e was a c o n c e n t r a t i o n by  and  carbamazepine  isoniazid,  experiments  concentrations  of  carbamazepine  metabolism of  influence  conversion  l'sonicotinic  concentrations  results  acid.  the  the  planned j_n v i v o  concentrations  constant  examined  metabolites  carbamazepine-10,ll-epoxide  ii  of  and  include  the  type  kinetics.  a of  iv  TABLE OF CONTENTS  CHAPTER  Page  ABSTRACT  rr  LIST OF TABLES LIST OF FIGURES ACKNOWLEDGEMENT 1 PHARMACOKINETICS OF CARBAMAZEPINE IN RABBITS  v vi viii 1  1.1  INTRODUCTION: 1.1.1 PHARMACOKINETICS: 1.1.2 CARBAMAZEPINE: 1.1.3 PHARMACOKINETICS OF CARBAMAZEPINE: 1.1.4 INDUCTION OF CARBAMAZEPINE METABOLISM: 1.1.5 STATEMENT OF PROBLEM:  1 1 3 17 18 19  1.2  EXPERIMENTAL: 1.2.1 MATERIALS: 1.2.2 ADMINISTRATION AND SAMPLING: 1.2.3 EXTRACTION: 1.2.4 ANALYSIS OF S A B L E S :  20 20 20 21 23  1.3 RESULTS: 23 1.4 DISCUSSION: 2 THE INFLUENCE OF ISONIAZID ON THE IN VITRO METABO 2.1  2.2  INTRODUCTION:  34 43 43  2 . 1 . 1 CARBAMAZEPINE: 2 . 1 . 2 ISONIAZID: 2 . 1 . 3 STATEMENT OF PROBLEM:  45 45 54  EXPERIMENTAL: 2 . 2 . 1 MATERIALS: 2 . 2 . 2 MICROSOME PREPARATION: 2 . 2 . 3 INCUBATION: 2 . 2 . 4 ANALYSIS OF SAMPLES:  54 54 55 56 57  2.3 RESULTS:  57  2.4 DISCUSSION:  67  3 REFERENCES:  73  V  LIST OF TABLES  TABLE  Page  I  Pharmacokinetic parameters f o r those t r i a l s parameters c o u l d be d e t e r m i n e d .  II  E s t i m a t e s of a and b f o r i s o n i a z i d t e s t s .  65  III  E s t i m a t e s of a and b f o r hydrazine t e s t s .  66  IV  E s t i m a t e s of a and b f o r i s o n i c o t i n i c  66  V  P-values  from run a n a l y s i s  f o r which the  acid t e s t s .  of i n v i t r o i n t e r a c t i o n  studies.  34  67  vi  LIST OF FIGURES  FIGURE  Page  1  Imipramine  4  2  Imi nodi benzyl  4  3  Iminostilbene  4  4  Carbamazepine  4  5  Three-dimensional r e v e a l e d by X-ray  6  S t r u c t u r e s of carbamazepine m e t a b o l i t e s i s o l a t e d from human u r i n e and major pathways of b i o t r a n s f o r m a t i o n .  11  7  Serum carbamazepine and carbamazepine-10,11-epoxide i n r a b b i t s a d m i n i s t e r e d carbamazepine (25 mg/kg) and fed ad l i b .  24  8  Serum carbamazepine and c a r b a m a z e p i n e - 1 0 , l l - e p o x i d e i n r a b b i t s a d m i n i s t e r e d carbamazepine (25 mg/kg) and fasted.  27  9  Serum carbamazepine and carbamazepine-10,11-epoxide in r a b b i t s a d m i n i s t e r e d carbamazepine (12.5 mg/kg) and f a s t e d .  30  10  Sample chromatogram from carbamazepine p h a r m a c o k i n e t i c s study i n r a b b i t s .  32  11  Isoniazid  48  12  Relevant a s p e c t s of i s o n i a z i d metabolism.  48  13  Sample chromatogram from i n v i t r o  14  I n f l u e n c e of i s o n i a z i d on the c o n v e r s i o n of carbamazepine to carbamazepine-10,11-epoxide i n the S9 f r a c t i o n of r a t l i v e r homogenate.  59  15  I n f l u e n c e o f a c e t y l i s o n i a z i d on the c o n v e r s i o n of carbamazepine to carbamazepine-10,11-epoxide i n the S9 f r a c t i o n of r a t l i v e r homogenate.  60  s t r u c t u r e of carbamazepine as diffraction.  interaction  study.  6  58  VI1  LIST OF FIGURES  (cont'd)  FIGURE  Page  16  I n f l u e n c e of a c e t y l hydrazine on the c o n v e r s i o n of carbamazepine to carbamazepine-10,11-epoxide i n the S9 f r a c t i o n of r a t l i v e r homogenate.  61  17  I n f l u e n c e o f h y d r a z i n e on the c o n v e r s i o n of carbamazepine to carbamazepine-10,11-epoxide i n the S9 f r a c t i o n of r a t l i v e r homogenate.  62  18  I n f l u e n c e of i s o n i c o t i n i c a c i d on the c o n v e r s i o n of carbamazepine to carbamazepine-10,11-epoxide i n the S9 f r a c t i o n o f r a t l i v e r homogenate.  63  viii ACKNOWLEDGEMENTS I for  would  like  giving  continuing  me  express the  advice  my s i n c e r e s t  opportunity  and guidance.  to His  c o m p l e t i o n of t h i s degree a p l e a s a n t Thanks  must  Adams f o r t h e i r many y e a r s o f  teaching,  be extended  technical  to  assistance  work  in  A.  Wall  for  his  knowledge and d e d i c a t i o n  made  his  extensive  to  Dr.  Richard  laboratory  and  experience. Ms.  Maureen  Murphy  and  to  Mr.  Stephen  and a l l o w i n g me the chance to tap  their  experience.  Thank-you to  also  appreciation  to  the  faculty  which enabled  the s e c r e t a r i a l  staff,  whose a s s i s t a n c e  members of to  learn  the department  what  I did  for  about  their  commitment  pharmacology,  Margaret Wong, E l a i n e J a n , and J a n e l l e  Harris,  I would never have been a b l e to complete t h i s  and  to  without  thesis.  I must a l s o express my g r a t i t u d e to the members of my examining committee f o r working under the time c o n s t r a i n t s t h a t were r e q u i r e d . Finally, tant  they  thank-you  are  f a r as I have.  to me.  to my f a m i l y . Without  their  Mere words cannot support,  I could  describe  never  how impor-  have made  it  as  ix  LIST OF ABBREVIATIONS  AcHz  A c e t y l hydrazine  AcINH  Acetylisoniazid  CBZ  Carbamazepine  CE  Carbamazepine-10,11-epoxide  Cl  Clearance  Hz  Hydrazine  INA  Isonicotinic  INH  Isoniazid  H/2  Apparent  V  d  Volume of  acid  half-life distribution  WEBSTER, D. S.  1  1 PHARMACOKINETICS OF CARBAMAZEPINE IN RABBITS;  1.1  INTRODUCTION:  The f i r s t mazepine  in  p o r t i o n of t h i s rabbits.  hepatotoxicity study of  is  In our  isoniazid  to examine the  the  Since  in  of  there  rabbits. of  One  is of  an  in  future To do  the  pharmacokinetic  and s i n c e an a p p r o p r i a t e  ongoing  the  carbamazepine.  carbamazepine,  the a p p r o p r i a t e  i n the l i t e r a t u r e  examines the p h a r m a c o k i n e t i c s  laboratory,  influence  pharmacokinetics  required. able  of  thesis  same  liquid  drug  absorption,  excretion). for  the  of  a  animal  the that  knowledge model,  were  not  is  avail-  chromatographic  assay  project.  PHARMACOKINETICS:  The study of p h a r m a c o k i n e t i c s of  of  goals  procedure had been d e v e l o p e d , the study was done as p a r t of t h i s  1.1.1  carba-  study  so,  parameters  of  There  direct  and u r i n e .  involves  distribution, are  and  sensitive,  measurement  of  These measurements  the i n v e s t i g a t i o n  elimination  accurate  drugs of  in  and  (i.e.,  precise  biological  of the  metabolism  analytical  samples,  drug c o n c e n t r a t i o n s  kinetics  such  methods  as  plasma  can be used to  deter-  mine p h a r m a c o k i n e t i c parameters such as b i o a v a i l a b i l i t y ,  the  elimination  rate  and  the  constant,  the  apparent  volume  of  and  distribution,  elimination  half-life. Bioavailability the  systemic  1987). one.  Thus,  defined  circulation for  F o r an o r a l  several  is  reasons.  as  the  following  an i n t r a v e n o u s  fraction  of  administration  dose,  the  unchanged by  bioavailability  d o s e , the b i o a v a i l a b i l i t y  may be l e s s  The  is  most  obvious  reason  any  the  drug  reaching  route  (Benet,  will  be equal  than one f o r  incomplete  to  any  of  absorption  of  WEBSTER, D. S.  the  drug.  As  intestinal  well,  the  drug  mucosa c o n t a i n s  of  residing  b o l i s m i n the p o r t a l the  systemic  metabolized  absorption.  gastrointestinal  b o l i s m by b a c t e r i a  be  sulphate-conjugating  c e r t a i n drugs d u r i n g t h e i r the e f f e c t s  may  circulation.  absorption.  enzymes which may  The drugs may a l s o  secretions  i n the l i v e r  As a r e s u l t ,  be s u s c e p t i b l e  to  to meta-  Drugs may a l s o undergo meta-  without  the  The  inactivate  and they may be s u b j e c t  i n the i n t e s t i n e s .  blood or  during  2  ever  being  bioavailability  able  to  can be  reach  signifi-  c a n t l y decreased from u n i t y . The fluid the  apparent  i t would occupy  same  vary  volume if  concentration  widely  of  distribution  the t o t a l as  in  depending on the  the pK  (V^)  of  a  drug  is  the  volume  of  a solution  at  amount i n the body were  in  plasma.  distribution  of  The  the d r u g ,  volume the  of  degree  of  may  plasma  protein  tissues,  and the  a binding, degree  the p a r t i t i o n c o e f f i c i e n t  of  binding  that i s t i g h t l y 0.06  L/kg,  weight.  to  other  tissues  which  corresponds  to  some drugs  elimination  between  volume  of  plasma  that  are  per  example,  selectively  by c e l l s  can  bound to  it  indicates  the  stopping) it  in has  of  body  constituents volume  hundred times body volume.  that  from  drug  of  have an apparent  (tjy =0.693  in  a  distribution  kilogram  clearance  parameter  However,  volume  and  s t a t e or to decay  or  For  distribution  steady  regimen).  body.  (t^)>  kinetic  or  the  half-life  useful  starting  the  o r are taken up s e l e c t i v e l y  of d i s t r i b u t i o n t h a t i s s e v e r a l The  within  bound t o plasma p r o t e i n may have a volume of  In c o n t r a s t ,  of t i s s u e s  of the drug i n the f a t t y  steady  n  state  a particular little  a  value  rate  expression  the  2  time  conditions of  of  drug  relationship V /Cl),  x  required  after  of  a  to  attain  a change  (i.e.,  administration  as an i n d i c a t o r  is  d  drug  (dosing  elimination  distribution. Clearance  i s the measure of  the b o d y ' s a b i l i t y  to e l i m i n a t e  a drug.  The  WEBSTER, D. S.  organs  of e l i m i n a t i o n  can only  clear  i n d i r e c t c o n t a c t w i t h the organ. will  drug from the  Thus,  blood  the time course  depend on both the volume of d i s t r i b u t i o n and the  1.1.2  [5-carbamyldibenz-(b,f)-azepin],  t i v e r e l a t e d to imipramine  (Figure  1 ) , was f i r s t  i n t r o d u c e d f o r the t r e a t m e n t of t r i g e m i n a l  to  was approved  its  that  is  drug i n the body  clearance.  an  iminostilbene  for  Carbamazepine metabolism  mechanisms of  neuralgia  use as an a n t i c o n v u l s a n t  agent  has been s t u d i e d e x t e n s i v e l y ,  and  pharmacokinetics,  its  deriva-  s y n t h e s i z e d by S c h i n d l e r  the l a t e 1950's and i t was patented i n 1961 ( S c h i n d l e r ,  1974.  of  plasma  CARBAMAZEPINE:  Carbamazepine  it  or  3  1961a).  (Blom, in  I t was  1962,  the  particularly  toxicities,  and  first  1963),  United  in  and  States  with its  in  respect  potential  action.  1 . 1 . 2 . 1 DEVELOPMENT AND CHEMISTRY:  Iminodibenzyl first cally  described as  the a  anaesthetic  and  of  by T h e i l  precursor  synthesized  activity.  (10,ll-dihydro-5H-dibenzo[b,f]azepine),  number  and of  of  1899,  carbamazepine.  antihistamine  was combined w i t h i m i n o s t i l b e n e  Schindler  and  group  anticonvulsant  properties  may be c o n s i d e r e d  derivatives  properties  (carboxamide)  considerable  strongest anticonvulsant  in  iminodibenzyl  When a carbamyl  iminodibenzyl,  Holzinger  shown i n F i g u r e  some  and that  activity  (1954)  possessed  local  at  was  histori-  Hafliger  modest  was added  2,  anticonvulsant the  5-position  observed.  were observed when a carbamyl  side  The chain  ( F i g u r e 3 ) , a s t r u c t u r e analagous to imino-  WEBSTER,  Figure 3:  Iminostilbene  Figure 4 :  D. S.  Carbamazepine  4  WEBSTER, D. S.  dibenzyl,  but  having  structure  has become known as carbamazepine,  i n 1961 ( S c h i n d l e r , Carbamazepine 5-carboxamide) formula  crystalline  (Kutt  and P a r i s - K u t t ,  that  a  in  and o t h e r  structure  is  distance  30°,  between  (Gagneux,  1976).  parameters,  a  It  is  11  positions.  s y n t h e s i s was  This  described  236.26  point  with  g/mol,  between  lipophilic  dissolves  an  in  empirical  appears  190°C  as  and  193°C  substance.  ethanol,  a  It  is  chloroform,  solvents.  of  of  carbamazepine  tricyclic  the angle of the  of  of  the  similarity  produced drugs.  a,  is  53°,  torsion,  y,  is  benzene  to  have  psychoactive  flexure,  angle  centres  The  of  a neutral it  5H-dibenzo[b,f]azepine-  derivative  melting  but  studies  and the  weight  with  water,  characteristic  6,  and i t s  iminostilbene  molecular  1982).  diffraction  dimensional  an  compound  insoluble  are  tion,  4),  and  white  X-ray  10 and  1961b).  (Figure  dichloromethane,  bond between the  (5-carbamyl-5H-dibenzo[b,f]azepine;  C^H^^O  virtually  a double  5  rings  imipramine  In  the  the angle 3"  in  three annela-  5).  4.85  obvious  except f o r the t o r s i o n angle which i s 20° f o r  of  (Figure  measured  is  measurements  The  Angstroms the  steric  imipramine.  1 . 1 . 2 . 2 THERAPEUTIC USES:  Carbamazepine administered 1963), most  to  was  patients  a disorder  dramatic,  afflict  the  Fothergill fourteen  first  if  human  suffering  that  is  not  the  face,  introduced  also most  from  in  the  trigeminal  referred  to  as  common,  of  all  trigeminal  early  neuralgia  tic  (1773), who c o m p i l e d , with thoroughness, patients  Blom's r e p o r t s ,  with  trigeminal  a number of c l i n i c a l  neuralgia.  In  investigators  when  it  neuralgia  (Blom,  douloureux.  By  the was  1960's,  painful  in  the c l i n i c a l short  1962,  far  disorders  described  a  was  the that  detail features  time  published similar  by of  following obser-  WEBSTER,  < \n  X  i  D. S.  n = 53* '5 = 4.85 A  0 = 30*  y = 3-  Figure  5:  T h r e e - d i m e n s i o n a l structure of c a r b a m a z e p i n e r e v e a l e d by Above:  X—ray  diffraction.  frontal  view  Middle:  top v i e w  Below:  side  Note  that  slightly  view  the c a r b a m a z e p i n e  twisted,  as  molecule is bent  (from Gagneux.  1976)  and  6  WEBSTER, D. S.  vations  (Taylor,  Amols,  1966).  1963;  Carbamazepine i s  of  trigeminal  is  also e f f e c t i v e  Westberg,  neuralgia.  disorder in  has  1963;  Dalessid  now the drug of c h o i c e  Later  also  investigations  was  the  and A b b o t t ,  i n the  indicated  anticonvulsant.  It  both  complex  t h a t of p h e n y t o i n and e s p e c i a l l y  1966;  treatment  that  carbamazepine  neuralgia  (Ekborn and  et a l . ,  broadened. depression  Carbamazepine (Post,  et  al.,  and a l c o h o l withdrawal  for  attacks  lightning  paroxysmal,  use  in  pains brief,  use and  and  the  United  Its  efficacy  the t r e a t m e n t  Carbamazepine  of  1972;  is  very the  cord.  States  as  tonic-clonic is  in the  of d e m y e l i n a t i o n of  with g e n e r a l i z e d  (Meinardi,  an and  comparable  to  grand mal  epilepsy  Cereghino,  et  now a f i r s t - l i n e  al.,  drug  in  epilepsy.  therapeutic has  for  seizures.  1974).  the  are  associated  r o o t columns of the s p i n a l  in p a t i e n t s  psychomotor  years,  pains  pains  of carbamazepine  tabetic  and d o r s a l  partial  the treatment of most forms of recent  The b a s i s  was approved  useful  lightning  symptoms and s i g n s  and p h e n o b a r b i t a l  for  1974; L i v i n g s t o n ,  is  treat  between The  root,  to  1972).  presents  carbamazepine  and  1966,  neuralgia.  p o s t e r i o r columns, d o r s a l  simple  used  similarity  Tabes d o r s a l i s  In 1974,  been  (Ekborn,  trigeminal  intense.  In  al.,  1966).  with tabes d o r s a l i s  pains  et  i n the treatment of glossopharyngeal  Carbamazepine  this  Bonduelle,  7  been  1984),  indications  suggested  excited  for  psychosis  syndrome ( R i t o l a and M a l i n e n ,  for use  carbamazepine in  cases  (Klein,  et  have  of  bipolar  al.,  1984),  1981).  1 . 1 . 2 . 3 MECHANISMS OF ACTION:  The easy  elucidation  task.  of  the  Carbamazepine  of onset of a c t i o n and the  mechanism has  several  dose  of  action  clinical  required  for  of  carbamazepine  effects, these  but the  effects  is time  varies  not  an  course  greatly.  WEBSTER, D. S.  This  suggests  different  there  clinical  biochemical  and  of  may  In  carbamazepine's effects  different fact,  on a v a r i e t y  neuropeptide  antidepressant  be  effects.  effects  messenger, course  that  carbamazepine  systems  (Post,  provide  of  action  exerts  of n e u r o t r a n s m i t t e r ,  anticonvulsant,  may  mechanisms  a  The  the  the  plethora  of  second  differential  antinociceptive, into  for  neuromodulator,  1988).  insight  8  time  antimanic,  mechanisms  of  and  action  r e l e v a n t to each syndrome. The  anticonvulsant  readily  apparent  i n the f i r s t  and  within  week,  antinociceptive  hours  to  and maximal  days,  ity  of  suitable  partial  animal  elucidation  anticonvulsant  effects  "peripheral-type" of  sodium  glutamate 1985). with  (Steardo,  ill  et a l . ,  exerted  al.,  1983).  minergic  via  et  et  and  treatment  and,  al.,  1985)  et  and  and  allowed  in  The  involving inhibition 1985),  decreased  Zeiglgansberger,  has  fluid  to  availabil-  al.,  possibly,  carbamazepine  cerebrospinal et  1986),  MacDonald,  Volger  tend  has  mechanisms  et a l . ,  1985),  1985;  with  in  (Rubinow,  1984;  occurs  carbamazepine.  to  (Weiss,  The  disorders of  tied  sleep  effects  1988).  pain  effects  al.,  al.,  in  are  been  associated  somatostatin epileptic  in  patients  1986).  has been suggested  are  al.,  carbamazepine  improvement  (Post,  closely  receptors  (Post,  reduction  patients  be  et  (Olpe,  chronic  significant  affectively  It  well,  to  (Willow,  potentiation  seizure  anticonvulsant  appear  responsivity  As a  the  of  and a n t i d e p r e s s a n t  respectively  for  benzodiazepine  currents  noradrenergic  models  of  whereas  antimanic  occur w i t h i n two and t h r e e weeks,  effects  a  that  mechanism  Foong and Satoh  mechanisms.  the a n t i n o c i c e p t i v e that  (1985)  involves also  GABAg  implicated  effects  of  receptors  carbamezepine (Terrence,  noradrenergic  et  and dopa-  WEBSTER, D. S.  The makes  relative  the  specific every and  task  lack of  of  suitable  linking  biochemical  the  depression  ranging  from  (Post,  therapeutic  performed by Post added to the l a r g e depressant  1988),  (1988)  a  high  to  that  candidates  mania of  and  for  dose  The  adenosine its  affects  to be i n v o l v e d  significant toxic.  depression  carbamazepine  Carbamazepine  system hypothesized  suggests  range of  for  effects  difficult.  though  to  models  psychotropic  mechanisms  neurotransmitter-modulator  animal  range  time  and  to  almost in  is  mania  required,  course  substance  putative  9  analysis  P should  antimanic  and  be  anti-  effects.  1 . 1 . 2 . 4 BIOTRANSFORMATION:  The  term  substances  "biotransformation"  undergo  ably catalysed occur  are  concerned al.,  in  biological  by enzymes,  dependent  on  applied  systems.  to  the  and the enzymatic  physico-chemical complement  of  chemical  These changes  so the form they take  the  and the  are  almost  rate  properties  the  changes  of  biological  which invari-  at which the  system  they  substance (Faigle,  et  1976). As s t a t e d p r e v i o u s l y ,  substance organic  various  based  and  property  on  lipid  exogenous  characteristics  media. to  its  sites  lipophilic  of  carbamazepine i s c l a s s i f i e d  dose  of  action.  substances,  of  of  of  its  lipophilicity  the  active it  also  as a n e u t r a l  solubility  the  and b a r r i e r s ,  excreted in a  Since  diffusion  membranes  can be r e a d i l y percent  the  aqueous  conducive  the drug to  2  is  of  substance  those  unchanged form (Weiner,  carbamazepine  can  be  partition  carbamazepine through  facilitates  The body possesses particularly  and  lipophilic  the  the  transport  a neutral  1967).  recovered  of  which  character,  In f a c t , unchanged  a  body's  no mechanism by  of  is  in  at most in  human  WEBSTER, D. S.  urine  or  bile  substances  (Levy,  must  hydrophilic  al.,  be  transformed  first  metabolites  kidney.  Thus,  duration  of  from  the  organism  than  by  renal  Feldmann,  et  1975;  which  can  biotransformation  pharmacologic is  or  biliary  of  a metabolite  excretion  et  al.,  identified  was  species,  as  determined  in  1972).  result,  to  excreted  elimination  primary the  unchanged  highly  through  of  metabolic  such  more  f o r both the i n t e n s i t y  the  of  a  organism  readily  i n the e a r l y 1972,  Since  when  then,  (Lertratanangkoon  r a t e of b i o t r a n s f o r m a t i o n  the  the  the  As  the  and the  carbamazepine  reactions drug  rather  (Faigle  and  1982).  (Frigerio, been  since  by  Carbamazepine was i n t r o d u c e d cation  more  important  effects,  1975).  within  be  is  controlled  Pitlick,  10  and  by  plasma,  would appear  same b a s i c  it  mechanisms,  c e r t a i n biochemical  the  10,11-epoxide  more  than  Horning,  i n man i s d r a s t i c a l l y  reflected  in  1960's but the f i r s t  the  making  findings  it  1982).  identified  metabolites the  have  overall  from t h a t i n animal  half-lives  of  they m e t a b o l i z e  apparently  was  While  different  elimination that  thirty  identifi-  carbamazepine  carbamazepine  permissible  to  from animal models to man ( F a i g l e  by  extrapolate  and Feldmann,  1982). Radiotracer Richter,  et  structures proceeds  studies  al., that  by  attributable epoxidation hydroxylation  1978) suggest  four  radioactivity  as to of  (Faigle  100  the  N-glucuronidation  carbamazepine  that  major  the  of  of  the  different 10,11  bond  six-membered  at the carbamoyl  6).  following  pathways  Faigle,  administration  (Figure  the  double  1975;  biotransformation  pathways  percent,  the  and Feldmann,  or of  the  side chain,  the  total  ring, 25  15 p e r c e n t ;  in  are  metabolites: 40  percent; and  man  urinary  percentages  corresponding azepine  1976;  metabolite  carbamazepine  Taking  rings,  al.,  reveal  approximate  the  aromatic  of  et  percent; direct  substitution  WEBSTER, D. S.  Figure  6:  Structures of carbamazepine metabolites urine and major  pathways  (from Faigle and Feldmann.  of  isolated  biotransformation,  1982)  from human  11  WEBSTER, D. S.  of  the  six-membered  remaining  rings  radioactivity  drug and to  products  with  sulfur-containing  excreted  of  by the  kidneys  combined m e t a b o l i c  groups,  5  percent.  can be a t t r i b u t e d  attack.  For  12  to  example,  The intact  there  may  be m e t a b o l i t e s t h a t have been both h y d r o x y l a t e d and N - g l u c u r o n i d a t e d . The  reaction  mediate al.,  of  producing  pathway  1976).  1)  is  carbamazepine-10,ll-epoxide  catalyzed  Most of the epoxide  by  The epoxide accounts  w h i l e the d i o l partly  accounts  present  portion  of  as  the  for  such  f o r o n l y one percent  and  epoxide  partly  as  intermediate  its  elucidated.  proceeds  directly  conjugated (Faigle, The starts  In  with  with  phenols,  the  glucuronic  acid  pathway,  rings.  i.e.,  Single  1-,  2-,  of  additionally,  a methoxy  1978).  trace  this  The  O-glucuronate amounts  not  diol. at  also  hydroxylation  intermediates  al.,  is  the  radioactivity,  In u r i n e ,  converted  to  the d i o l  a  A  The  known  whether  acridan  hydroxymethyl  is  is  smaller  ring-contracted  by a mechanism t h a t  yet  (Oesch,  has y e t  this  almost  group  to  reaction completely  before  excretion  1976).  the  six-membered  or  it  via  et a l . , second  addition,  hydrase  mono-O-glucuronide.  is  et  trans-10,11-  of e x c r e t e d  35 p e r c e n t .  compound, 9-hydroxymethyl-10-carbamoylacridan be  by epoxide  inter-  (Frigerio,  c o n v e r t e d to  i n the l i v e r  approximately  first  monooxygenase  is enzymatically  dihydro-10,11-dihydroxycarbamazepine 1973).  hepatic  (the  bulk  the  of  pathway group of  to  be  and carry  in  these  monooxygenases,  various  positions  in  four  results  a  hydroxy 1 or  metabolites  are  at  by  all  4-hydroxycarbamazepine.  position  conjugates  phenols  catalyzed  carbamazepine  substitution 3-,  and 0 - s u l f a t e of  thought  in  group in  are  a ratio  excreted  in  excreted of  Two  by  about  3  other 2  and,  (Richter,  the 2:1,  unconjugated.  the  possible  postion  position  of  kidney while  et as only  Additional  WEBSTER, D. S.  metabolites  have  hydroxymethoxy 1978), a l l  been  compounds  by  other  and t h r e e  investigators,  dihydroxy  compounds  including  (Lynn,  et  three  al.,  1977,  of which were g l u c u r o n i d a t e d .  The t h i r d  important  carbamazepine  with  to  group  the  found  13  amino  the  conjugation  and  Feldmann,  glucuronide  route  glucuronic of  is  the  biotransformation  acid.  In the  carbamoyl  metabolized  1982).  of  The  by  side  chain.  It  the  has  glucuronyl  e-glucuronidase  from most conjugated s p e c i e s ,  direct  conjugate,  a hepatic  enzyme  is  but i t  is  is  conjugation ligand  is  bound  been assumed  transferase able  unable  to  of  that  (Faigle  cleave  the  to do so i n  this  case. The f o u r t h major pathway i n v o l v e s the i n t r o d u c t i o n of a substituent  sulfur-containing  i n t o one of the six-membered r i n g s of the carbamazepine m o l e c u l e .  Four p r o d u c t s r e s u l t i n g  form t h i s  pathway were found i n human u r i n e :  3-methylsulfinylcarbamazepine  and  (Faigle  The  and Feldmann,  1982).  2-  and  2-  and  3-methylsulfonylcarbamazepine  mechanism  by which  these  conjugates  are  generated i s unknown. Additional by  products  Lertratanangkoon  formed by  and  Horning  unidentified fraction in urine, A  study  hydrolysis zepine  has of  been  done  to  pathways  1,  (1982).  Such  et  al.,  examine  the  in  vitro.  However,  1987).  During  attempts  guinea p i g and with c y t o s o l i c their the  hydrolytic diol  being  activity formed  with  fractions  on the after  products  steric  been  will  the  protracted  of  described  reduce  the  metabolite  study,  the  the  enzymatic  of  carbama-  intention  was  to  epoxide h y d r o l a s e from animal  microsomes  from r a t  epoxide  course  a primary  s u b j e c t the epoxide to the a c t i o n of microsomal liver  and 4 have  but they are r e l a t i v e l y minor components.  carbamazepine-10,11-epoxide,  (Bellucci,  2,  was very  from  and guinea low,  with  incubations.  As  rabbit, pig  rat  and  showed  that  only a  traces  result,  of the  WEBSTER, D. S.  investigators  were  forced  under carbamazepine treatment  with  enantiomeric  Both the  of  80  percent,  (R,R)-diols  (Bellucci,  diol free  substrates,  of  from  the  diol,  urine  and t h a t  were found to  prevalant  This  the  but i s  i n most o t h e r  et a l . ,  the  configuration.  enantioselectivity  meso and racemic of  the  e-glucuronidase/arylsulfatase,  absolute  pronounced  isolate  treatment.  excess  (-)-10S,llS  to  microsomal  of  is  in  having  hydrolase  hydrolysis  an the  indication  i n c o n t r a s t with the p r e v a l e n t  known cases of enzymatic  after  be formed  an  epoxide  patients  obtained  enantiomer  finding  14  of  toward  formation  of  epoxides  1987).  1 . 1 . 2 . 5 ADVERSE EFFECTS:  I t has been e s t i m a t e d  t h a t 33 to 50 p e r c e n t of a d u l t s  t r e a t e d with carbamazepine  experience  effects  common  seem  (Masland, divided  to  be  1982).  In  into  more  general,  two c l a s s e s  -  side  with  the  and/or  polytherapy  adverse  clinical  effects  side  effects effects  and c h i l d r e n toxicities.  than of  with  laboratory  Side  monotherapy  carbamazepine  and  being  can  be  abnormali-  ties.  the  Most of  the c l i n i c a l  dosage  is  reduced  common  side  vision,  diplopia,  origin. therapy  as  or  effects  if  in only side  nausea,  and s l u r r e d  five  percent  effect  that  are m i l d ,  initiation  include  These s i d e e f f e c t s  associated ticals  side effects  All  are s e r i o u s  is  cases  syndrome,  but  gradual.  vertigo,  the  first  The  ataxia,  most  blurred  enough to warrant d i s c o n t i n u a t i o n  of  1987). reported  are  if  in  most f r e q u e n t l y  Lyell's  is  and r e v e r s i b l e  neurotoxic  (Pellock,  and the FDA i n the U n i t e d S t a t e s  Stevens-Johnson  treatment  drowsiness,  speech.  of  of  transient,  The  to Geigy  are s k i n and a l l e r g i c  syndrome,  carbamazepine-  exfoliative  Pharmaceu-  reactions  such  dermatitis,  and  WEBSTER, D. S.  erythema m u l t i f o r m e . been  attributed  C e r t a i n movement d i s o r d e r s  to  carbamazepine  include  chorea,  usually  been observed  pine,  most  seizures There  commonly  and  have  increase  dystonia,  a  patients  significant  and  of  involved  and wave  carbamazepine et  Hurst,  Horn,  pine t h e r a p y , malities. are  fatal  cases  they  of  anemia  are  not  are  is  the  most  in  ceuticals static  hepatic  include  icterus,  bone the  common.  order  a condition  c h i l d r e n and a d u l t s common  1986).  abnormalities.  The  myoclonic,  and  absence-type  seizures.  of  most  frequency,  estimated  to  carbamaze-  hepatitis, liver  hematologic  include  dysfunction,  rare,  and  potentially  side  thrombocytopenia,  (Hart  liver  tests,  to  and  to  but Geigy  aplastic As  well,  10 p e r c e n t  and E a s t o n ,  reported  function  they  effcts,  reported  i n approximately  abnormalities  abnormal  are  and bone marrow d e p r e s s i o n . occur  abnor-  Agranulocytosis  frequently  t r e a t e d w i t h carbamazepine and p a n c r e a t i c  serious  depression.  serious  Those  pancytopenia,  carbamazepine  can produce  marrow most  to  and  with  have been observed with  reactions  carbamazepine  perhaps  agranulocytosis,  leukopenia  most  since  protracted  Pharmaceuticals, anemia,  hematologic  important,  aplastic  et a l . ,  the most important of which are hematologic and h e p a t i c  Although  very  effects  seizure  seizures  but t h e s e can p r o g r e s s to g e n e r a l i z e d t o n i c - c l o n i c side  1982).  Sachedo  nonconvulsive  seizures,  laboratory  hard-to-control  1984;  that  of  atonic,  have  carbamaze-  (Masland,  al.,  1985;  of  which  and  associated  electroencephalograph^  generally  rare  for  dysfunction  Johnson,  1985;  very  polytherapy  neurologic of  are  have  disorders,  seizures  A variety  are  movement  increases  plasma l e v e l s  with g e n e r a l i z e d  generalized  arise  with t o x i c  1983;  patients  seizure  and myoclonus,  reports  Saslow,  The  receiving  1985; Snead and Hosey,  Most of these slow s p i k e  in  number  (Shields  Chokroverty,  asterixis,  in conjunction  having  been  therapy.  and  15  1982).  Geigy  of The  Pharma-  jaundice/chole-  hepatomegaly/hepatosplenomegaly,  and  WEBSTER, D. S.  pancreatitis. enzymes, zepine  The most common o b s e r v a t i o n s  r e p o r t e d to o c c u r  (Pellock,  i n 5 to 10 p e r c e n t of p a t i e n t s  of  some aromatic  z e p i n e and some of factors.  effect  in v i t r o  on the  mutagenic city  polycyclic  metabolites  survival  in mice,  while  other  Wray, e t a l . ,  receiving  rate tester  the  yielded  In humans,  (Oesch,  was  mice)  found  to  be  leukemia L1210 (Frigerio  (Glatt,  et a l . ,  reported p o s i t i v e negative  1975).  no i n c r e a s e  induce  sister-chromatid  chromosomes correlation in vitro an  carbamazepine  monotherapy  of  been  al.,  The s t u d i e s  detectable  in epileptic  concentrations  u g/ml.  et  et  al.,  1980).  studied  by  (SCE)  and  1985).  In  observed between chromosome breaks  pine at therapeutic pine  have  exchanges  (Schaumann,  studies.  absence  Nakane,  chromosome  and SCE i n  effects  the  f o r a minimum of subjects  in  18 months.  the  study  al., 1976;  mutagenic  ability  to  in  the  was  no  e i t h e r J_n v i v o  or  there  results, of  was  pregnancy  aberations  indicating  carbamazepine  human s u b j e c t s who had been a d m i n i s t e r e d  levels of  damaging  al.,  the  its  studies,  produced n e g a t i v e _i_n v i v o  nor  teratogeni-  during  examining  structural  was no  1975)  et  Recently,  such  there  to  malformations  1974;  al.,  these  (Eluma, et  (Fritz,  in congenital  to  cytotoxic  In  results  results  carbama-  respect  and M o r s e l l i ,  (Livingston, of  carbama-  1976),  (i.e.,  of mothers t r e a t e d with carbamazepine  effects  liver  carcinogenic  neither  observed i n o f f s p r i n g et  and  have been examined with  strains  some s t u d i e s  studies  1982).  of  mutagenic,  hydrocarbons  and to mice b e a r i n g  to b a c t e r i a l  studies  1981)  its  teratogenic,  Carbamazepine-10,11-epoxide  human c e l l s  of  1987).  In view of the known c y t o t o x i c , properties  are t r a n s i e n t e l e v a t i o n  16  in  carbamaze-  The serum carbamaze-  ranged  from  4.3  to  9.0  WEBSTER, D. S.  1.1.3  PHARMACOKINETICS OF CARBAMAZEPINE:  The pharmacokinetics sively,  and F r i g e r i o , cokinetics and Levy,  1975;  carbamazepine  1983;  Levy,  doses, hours  1975),  a  immense  has  been  (Faigle of  variation  1984),  has been  this  L/kg  (Sumi,  et  al.,  et a l . ,  1982).  ± 0.143  hours,  distribution the  of  1975), a  hours  et a l . ,  1978) and the into fatty  and i n the  The i n t r a v e n o u s a clearance 1.086  rate  * 0.287 was  2.35  hours  of  (Morselli  the  pharma-  hours  and  L/kg  1975).  This  al.,  et  1973), et  al.,  (Strandjord al., is  (Palmer,  and  1985).  The  obvious.  The  et  al.,  value can be  carbamazepine  hydrophobicity  following  (Morselli,  individuals  as 1.3  et  hours  (Eichelbaum,  of  (Wedlund 1986)  half-life,  (Palmer,  20-65  binding  exten-  1987).  plasma  between  of plasma p r o t e i n  1987)  are a v a i l a b l e  (Chang and Levy,  31-55  range  parameter  In one study  half-life  be 35-37  ± 6.1  There have been two s t u d i e s rabbits.  to  with  (Morselli,  (Bertilsson,  studied  In a d d i t i o n ,  rat  apparent  has been r e p o r t e d  l e a d to i t s p a r t i t i o n i n g  in  i n the  the  Feldmann,  hours  of  1986).  1982; Sumi, et a l . ,  reported  and 26.2  i n p a r t by the l e v e l 80 percent  al.,  and  35  1975),  and as 0 . 8 2 - 1 . 0 4  orally,  humans  have been examined i n the rhesus monkey  subjects,  volume of d i s t r i b u t i o n  pine  in  and Tomson,  et a l . ,  human  mean  Johannessen,  et  (Siegers,  healthy  single  Bertilsson  of carbamazepine  i n the r a b b i t  40-41  of  and very thorough reviews of the i n f o r m a t i o n  In  17  1973)  explained  being  70 to  of the drug which  could  tissues.  i n v o l v i n g the p h a r m a c o k i n e t i c s carbamazepine other  it  was a d m i n i s t e r e d  was  administration of L/kg.  1.219  carbamaze-  intravenously  orally  produced a h a l f - l i f e  ± 0.470  When  * 1 hour.  administered  of  L/hr/kg,  carbamazepine Since  these  (Siegers, of  0.648  and a volume was  studies  of  administered either  used  WEBSTER, D. S .  the  wrong r o u t e  parameters was  for  of  administration  the experiments  or  did  not determine  all  planned f o r our l a b o r a t o r y ,  of  18  the  desired  the c u r r e n t  study  initiated.  1.1.4  It  INDUCTION OF CARBAMAZEPINE METABOLISM:  is  well  established  and t o h e t e r o i n d u c t i o n . own metabolism comitant  auto-  with  is  pathway  that  is  is  time  studied  in  course  three  (Bertilsson, (CBZ-D^) three  was  On  therapy), 4  the  dose.  Thus,  as  6  The  subject  with  of  a  and  of  children  of  after at  is  1985).  Wad,  al.,  of 4  There  induces 1979,  are  developed  before  five least,  than  doubled  months, the  1985). both  trans-diol  kinetics  et  al.,  has  been  psychomotor  maintenance  carbamazepine  was  the  indications  Eichelbaum,  carbamazepine  was g r e a t e r 4  Con-  induced d u r i n g  1984;  carbamazepine  CBZ-D  its  1982).  recently  regular  CBZ-D  clearance  of  dose  dose  1975).  further et  that  induces  al.,  Tetradeuterium-labelled  single the  autoinduction  but a l s o the f o r m a t i o n of the  1980). a  et  Eichelbaum, route  to  carbamazepine  phenytoin  et a l . ,  autoinduction  increased in  or  1973;  (Bourgeois  (second  clearance  was not f u r t h e r given.  of  part  day  is  Eichelbaum,  1981; Wedlund, et a l . ,  al.,  given  occasions,  CBZ-D^.  Dam,  (Eichelbaum,  children  et  1980;  the m e t a b o l i c  induced  1985; T y b r i n g , e t a l . , The  al.,  not o n l y the e p o x i d a t i o n  metabolite  CBZ-D  and  and h e t e r o i n d u c t i o n  that i t  et  phenobarbitone  (Christiansen  The e p o x i d e - d i o l  carbamazepine  During long term t h e r a p y ,  (Bertilsson,  treatment  metabolism  that  it  therapy, was  during was  from  when the  for  21  last  autoinduction  metabolism seems to be complete during the f i r s t  carbamazepine  dose  given  epilepsy  to  on  replaced  by  maintenance the  initial  36  days  and  CBZ-D  dose  was  of  3-5 weeks of  and  4  carbamazepine treatment.  WEBSTER, D. S.  Autoinduction adults of  of carbamazepine metabolism has a l s o  (Eichelbaum,  healthy  al.,  volunteers  carbamazepine. received  et  The  that  subjects,  group  the  plasma  The  attributed  the  of  adults,  a  significant  Unfortunately, clearly  the  a  for  degree  influence  prior  to  the  of  months.  plasma  clearance  six  in  pathway.  month  200  of  who  had with  patients was  Thus,  within time  mg  Compared  higher  occurs  human  consisted  patients  3-fold  induction  induction  of  microsomal  enzymes  also  been examined (Wagner and Schmid,  four  days  with  daily  oral  equimolar  killed  by d e c a p i t a t i o n  24  hours  livers  were  excised,  and  it  liver  weight  increased  the  not the c o n c e n t r a t i o n an i n d u c t i v e e f f e c t  1.1.5  mainly in  six  point  on  human  months.  is  not  as  was  of microsomal  rat  1987).  liver  by  carbamazepine  has  were t r e a t e d  for  The animals  doses  of  the  last  dose  that  carbamazepine  found  the  in  315  nmol/kg.  concentration protein.  of  of  This  The  rats  were  carbamazepine.  The  significantly  cytochrome  indicates  P4gQ»  that,  but  in  rats,  this  study  i s o c c u r r i n g w i t h i n f o u r days.  STATEMENT OF PROBLEM:  determine  the  metabolism  indicates  after  and  As s t a t e d at the beginning  pine  six  study  dose  epileptic  epoxide-diol  of  the  oral  least was  in  in  defined.  The  was to  of  increased the  group  single  at  clearance  monotherapy. induction  first  consisted  monotherapy  carbamazepine to  The  received  second  carbamazepine  healthy  1985).  been s t u d i e d  19  that  in  rabbits.  i n other s p e c i e s .  that  this  pharmacokinetic  knowledge  d e s p i t e the f a c t  of  of  The the  introduction, parameters  information parameters  the goal  associated  of  with  presented  in  for  species  this  the  the metabolism has been s t u d i e d e x t e n s i v e l y  Thus, the b a s i s of t h i s  study i s  justified.  carbamazeintroduction is  sparse,  i n man and  WEBSTER, D. S .  1.2  EXPERIMENTAL:  1.2.1  MATERIALS:  Carbamazepine, Co. (5  20  Acetonitrile percent)  the  heparin,  and methanol  was obtained  supplier  of  and  ethanol were  obtained  from Abbott  nitrazepam.  were  obtained from  was  Sigma  Chemical  BDH C h e m i c a l s .  Laboratories,  Nar^PO^  from  and  obtained  Dextrose  Hoffman-LaRoche  from  Fisher  was  Scientific  Co. Male  New Zealand  white  rabbits  Care U n i t of the U n i v e r s i t y  (2-3  of B r i t i s h  kg)  were  obtained  from  the  Animal  5  percent  Columbia.  1.2.2 ADMINISTRATION AND SAMPLING:  Carbamazepine, dextrose  as  rabbits  (2-3  local  tion  required, kg).  supplier  subclinical of  rabbit.  the  suspended was  This  lmL  of  of  under  rabbit  2  kg  was  by  The doses were chosen based on the  done  dently, the  the  current  et  al.,  1982),  existence  of  study  was  the  to  to  study  essentially  New Zealand  because to  a  animals  lethal  Administra-  12.5 mg/kg or 25 mg/kg,  and  prior  administration  that rats  rats  this  are  the  the  doses of to  of  white  from  frequently  and handled.  back  often  throat  of the  i n which  group  complete.  a comparable  dose was In  was not  40  drug.  administered  have a much more r a p i d  carbamazepine by  male  and  the  reference  the  the  mg/mL)  of  fact  of 80 mg/kg and t h a t  In the one a v a i l a b l e  (Siegers,  orally  susceptible  syringe  they were e i t h e r f a s t e d o r f e d ad l i b .  metabolism.  (0.5  i n f e c t i o n when t r a n s p o r t e d  suspension  range  20  was chosen  are  The r a b b i t s were a d m i n i s t e r e d  dose i n the  Tween  administered  size  weighing  respiratory  in  rate  of  study was  mg/kg.  discovered  studies  a  in  Inciuntil which  WEBSTER, D. S.  carbamazepine  was  administered  10 mg/kg (Rimerman, cation  for  et a l . ,  the doses used,  250 mg/kg was carbamazepine  accidently obtained  concentrations.  least  seven  days  1979; Sumi,  administered  was very  Each  effect,  a l l o w i n g each animal  monitor  the  levels  of  the  1987).  the  f o r the  were  over  constant was  duration  by f l u s h i n g  taken  time.  approximately  samples  were  of  the  the  the  drug  over  catheter  in  Eppendorf C e n t r i f u g e 3200. until  but  study  the  two  so  a dose  of  there  with  no  at  period  carryover  period  and of  Blood  its  to  with the  of  up  primary  samples  to  six  metabolite,  (approximately (Jelco  hours,  22g)  volume  microcentrifuge  of  of  that  heparinized  blood  tubes  for  The plasma was c o l l e c t e d  mL)  remained  in  relatively saline  that  The  blood  removed. one  carbama-  0.7-0.8  Blood volume was kept  a volume  to  minute  using  and was s t o r e d at  an  -20"C  EXTRACTION:  Carbamazepine  and  carbamazepine-10,ll-epoxide  were  extracted  plasma samples u s i n g a s o l i d phase e x t r a c t i o n procedure d e s c r i b e d by al.  a  extracted.  1.2.3  et  as  carbamaze-  This  was  of  plasma  obtained  administrations,  that  about  justifi-  maximum  administrations.  a  experiment.  equivalent  centrifuged  further  concentration  were taken from the ear v e i n through a c a t h e t e r place  As  generally  the c u r r e n t  rabbit,  to  was  to be used as i t s own c o n t r o l .  of carbamazepine  zepine-10,ll-epoxide,  a  underwent  between  elimination  samples  to  similar  rabbit  separation  complete  blood  al.,  dose  as was the r e s t of the time p r o f i l e  allowed  Ten  et  the  i n one experiment of  r e s u l t of a 25 mg/kg dose, pine  intravenously,  21  (1986).  extraction  This  type  because the l a t t e r  of  extraction  was  chosen  over  a  from  the  Hartley,  liquid-liquid  have a tendency to form emulsions t h a t  decrease  WEBSTER, D. S.  the e f f i c i e n c y  of the e x t r a c t i o n s .  a  simple  relatively  procedure  H a r t l e y and co-workers and i t  to use t h i s  were  similar  those  bonded  Bond-Elut  (manufactured  were  types.  The Co.,  prior  use  to  acetonitrile  were  and  with  vacuum  Chemical  sample  silica  applied  and  a  water  minute  and  one  of  was  NJ).  With  all  of  water/acetonitrile  and i t s extract  was  evaporated  until  procedure  respect  to  column  under  since  analysed. it  hydrophobicity the  same  were two  is  Nitrazepam  column  (J.T.  Baker  immediately  of  each  the  ng/mL,  in  The  ethanol)  vacuum  (80:20).  one  The  of  plasma  Upon r e l e a s e , with  and  columns  l g  volumes  minute.  was  there  volume  of  compounds  of  were e l u t e d with 750 nL  dryness  under  a  nitrogen  i n 250 uL of mobile phase and was  chosen  to  carbamazepine  and w i l l ,  as  a result,  and  C  Both  CA)  between  nL  100  octade-  used.  system  250  similar  conditions  study  PA)  column  washing  to  current  conditioned  epoxide m e t a b o l i t e )  stored this  SPE  released,  before  deci-  City,  efficiency  one  to the  were  Horsham,  in  for  the  Harbor  of the plasma i n t o the column. period  efficient.  Reversed-phase  (nitrazepam,  equilibrate  led  capacity,  vacuum,  The r e s i d u e was r e c o n s t i t u t e d  -20"C  during  Baker-10  stream at 55"C. at  ni  columns  vacuum  standard  to  a  under  the  equilibration  this  The  through,  internal  volume  and  used  that  group.  Monitoring,  apparatus  (Carbamazepine  ethanol,  reasonably  International,  difference  allowed  two  interest  Worldwide  2.8  no s i g n i f i c a n t  a p p l i e d to t r a n s p o r t was  obtained  a  by  water. of  with  Analytichem  by drawing  25nL  and  i n the range of 90 t o 93 p e r c e n t ,  by H a r t l e y ' s  by  Phillipsburg,  and  quick  recoveries  columns,  (manufactured  utilized,  The  reported  cylsilane  Clean-Up  was  hand, the column method was  and the ease of the technique  method.  to  that  reported recoveries  was these v a l u e s  sion  On the o t h e r  22  provide  and  elute a  as  the the  from  similar  standard epoxide  the  in  with  extraction  recovery.  In  WEBSTER, D. S.  addition,  the compounds  the  length  wave  used  of  interest  for  have  detection  similar  following  extinction their  23  coefficients  separation  at  by  liquid  performance  liquid  chromatography.  1.2.4  The  ANALYSIS OF S A B L E S :  samples  chromatography  were (HPLC)  analysed using  by  reversed-phase  a Spectra-Physics  high  SP8000B  liquid  A 125 x 4 . 6 mm bore column was packed w i t h S p h e r i s o r b temperature was s e t at 48"C. a  0.01M  NaH P0 2  buffer.  4  a Spectra-Physics tion  of  both  The mobile  The  flow  SP8400 u v / v i s  carbamazepine  m e t a b o l i t e (Rambeck,  et a l . ,  The  oven  phase was 42 p e r c e n t methanol  with  rate  was  detector  set  and  chromatograph.  5 um 0DS2.  lmL/minute. at  Detection  210 nm, a l l o w i n g  carbamazepine-10,ll-epoxide,  1981).  The r e s u l t s  the its  were o b t a i n e d  was  by  detecprimary  as peak  areas  as determined by the data system of the chromatograph.  1.3  RESULTS: The  results  of  the  are shown i n F i g u r e s in Figure  10.  7,  Figure  study  of  carbamazepine  7 shows the  results  access to  8,  given  animals  were  in  8, and 9, with a sample chromatogram being  25 mg/kg and allowed f r e e the  pharmacokinetics  also  food.  a dose  of  i n animals For  those  25 mg/kg,  The animals used to get the data i n F i g u r e 9 were a l s o  administered trials but  rabbits displayed  a dose  shown i n  they  were  fasted,  of  Figure fasted.  but they were  a d m i n i s t e r e d a dose o f o n l y 12.5 mg/kg. Initially, When t h i s  did  the not  period  over  seem adequate  time p e r i o d was extended to s i x  which for  samples  rabbit  Dl  were  taken  (25 mg/kg,  was fed  five  hours.  ad l i b . ) ,  the  hours f o r r a b b i t D2 (25 mg/kg, fed ad l i b . ) ,  Plasma Concentration <uo/ml)  Plasma Concentration (ug/ml)  Plasma Concentration (ug/ml)  WEBSTER,  Figure  7:  Seurm  carbamazepine  rabbits administered The  data  for e a c h  and  carbamazepine-10.11—epoxide  carbamazepine animal  D. S.  (25 mg/kg) and  is presented  in an  26  in  f e d a d lib.  individual  frame.  ro  Plasma Concentration (ug/ml)  Plasma Concentration (ug/ml)  Plasma Concentration (ug/ml)  co —I m  o GO  ro co  RABBIT D 8 25  mg/kg: fasted  WEBSTER, D. S .  - • - CE  csz  e |  a O  30  60  9 0 1 2 0 150 180 2 1 0 2 4 0 2 7 0 3 0 0 3 3 0 Time  360  (minj  RABBIT D9 25  mg/kg: fasted  —*— cez  - • - CE  i  O  30  60  9 0 1 2 0 1 5 0 1 8 0 2 1 0 2-40 2 7 0 3 O 0 3 3 0 Time  360  (minj  RABBIT D1 1 25 — * — C8Z  mg/kg: fasted -•- ce  a O  30  60  9 0 1 2 0 150 180 2 1 0 2 4 0 2 7 0 3 0 0 3 3 0 Time  Figure 8:  Serum  carbamazepine  rabbits administered The  360  <minj  a n d c a r b a m a z e p i n e — 10,11 — e p o x i d e in carbamazepine  ( 2 5 mg/kg) a n d f a s t e d .  d a t a for e a c h animal is presented in a n individual frame.  29  Plasma Concentration (ug/ml)  Plasma Concentration (ug/ml)  Plasma Concentration (ug/ml)  Plasma concentration (ug/ml)  Plasma Concentration (ug/ml)  Plasma Concentration (ug/ml)  WEBSTER,  Figure 9:  Serum  carbamazepine  rabbits administered The  Figure  D. S.  32  and carbamazepine-10,1 1—epoxide in carbamazepine  (12.5 mg/kg) and f a s t e d  d a t a for e a c h animal is p r e s e n t e d in a n individual frame.  10:  Sample chromatogram  from  carbamazepine  pharmacokinetics study in rabbits. sample a of  shown w a s  The  t a k e n a t 1 2 0 min from  rabbit f e d a d lib. a n d administered 2 5 mg/kg.  T h e chromatogram  a  dose  shows  carbamazepine-10.11 - e p o x i d e (293), nitrazepam  (579). a n d carbamazepine (632).  WEBSTER, D. S.  at which time an a p p r o p r i a t e for  rabbit  usable  D3.  But,  the  was o b t a i n e d .  as more animals were  concentration-time  Subsequently,  profile  tests  profile were  could  repeated  administration sampling). of  the  (most  animals  did  were  allowed  The animals  experiment.  The  the  drug  to  the  site  extended p l a t e a u animals  phenomenon ( F i g u r e  accurately tives  of  group t h a t  the  study,  for  all  ad l i b .  values  for  and  w  for  e  r  e  9  0  the  (Figure  8),  were  a n <  * ^  Of  the  rabbits  clearance  a lower  were  of  group  to  in  cases.  all  of  a result the  for  food  delayed  small  of  the  in  the  blood  stomach  presentation  profile  (12.5 mg/kg)  drug  duration  intestine.  pharmacokinetic  a  overnight  period  water  was o b s e r v e d ,  of  But,  for  displayed  parameters,  animals  in  the  profile.  and  90  for  and  minutes,  clearance  constant  f o r the  type of  (k  e l  )  101.0  a dose  - 1  of  a  usable  apparent  were were  administered gave  for  was not  outlined  a usable  D3 and D8  administered  it  a dose  rabbits  142.4  the  c o n s t a n t were 0.0077 and 0.0057 m i n In the  that  fasted  the  presence  dose  obvious  a t l e a s t one hour a f t e r  and a d m i n i s t e r e d  minutes,  were 60 and 90 m i n u t e s , for  60  elimination  respectively.  the  rabbits  during  namely  pharmacokinetic  D2 and D3 produced m a w  the  access  the  of the phenomenon t h a t  rabbits  ^1/2^  at  in  be  became  observed  an  several  a  similar  9).  was f e d  t  seen  obtained  and as  absorption,  Tests  determine this  of  was again  (Figure 8 ) .  As a r e s u l t  emptying,  not  eat  free  i d e a was t h a t  may have d e l a y e d g a s t r i c  it  and f o r  not  was a l s o  tested,  with  p r i o r being a d m i n i s t e r e d carbamazepine,  This  33  study  of  the 91.6  0.0077  dose  of  profiles.  half-life  mL/min/kg,  mg/kg  the  groups.  (Figure  7),  tests,  the  and 46.2  25  half-life mL/min/kg,  0.0071  mg/kg  The  objecthe  apparent  and  to  In  these  min" ,  and  values  1  fasted  for  t  m a v  were 90 and 122 minutes, and  for  f o r r a b b i t s D3 and D8, 12.5  in  25 mg/kg For  possible  (Figure  9),  the  elimination  respectively. only  rabbit  D8  WEBSTER, D. S.  gave  a  standard  profile.  The  t  v  was  60 m i n u t e s ,  110  minutes.  as  in  the  34  previously  max mentioned 121.8  test.  ni/min/kg,  The  half-life  and  the  was  elimination  r e s u l t s are summarized i n T a b l e  Table  I:  Pharmacokinetic  constant  The  clearance  was  0.0063  trials  for  rate  min" .  was These  1  I.  parameters  for  those  t  K  clearance  min  mL/min/kg  which  the  para-  meters c o u l d be determined.  Treatment  Rabbit  max min  elimination constaijit min"  25 mg/kg Ad l i b .  D2 D3  60 90  90 98  91.6 46.2  0.0077 0.0071  25 mg/kg Fasted  D3 D8  60 90  90 122  142.4 101.0  0.0077 0.0057  12.5 mg/kg Fasted  D8  60  110  121.8  0.0063  1.4  DISCUSSION:  The been  pharmacokinetics  examined by  1982)  the  drug  carbamazepine of  in  rabbits  investigators.  orally  The  (40 mg/kg),  had  first  as was  been  previously  (Siegers,  done  in  et  the  al.,  current  B u t , the o n l y parameters t h a t were determined were the time at which  the  maximum  and  the  were  two teams  administered  study.  of  plasma  half-life  similar  compartment  to  concentration ( t ^ ) '  those  which  obtained  pharmacokinetic  was  achieved  was from  profiles  2.35  hours.  animals in  the  (t^  ),  which  The  exhibiting  current  study.  was  values  2  hours,  for  t  conventional This  held  m £ ( X  one true  WEBSTER, D. S.  for  each  current  of  the  tests  co-workers,  dosing/feeding  were  but  they  used to determine closely as  those  still  tablets  the  studies  (Bertilsson,  results  model  and they  (1.086  significantly  less  the v a l u e s o b t a i n e d  at  drug  is  1979),  an o r a l l y  concurrent  and  various  dose  clearance  drug  second  (1.219  (0.684  current studies  study  described studies  hr).  model  available  follows  the  team of  two  investi-  intravenously  L/hr/kg), These  studies.  in  administered  commercially  the carbamazepine  The  (10  volume  values  are  in  of all  differences  may be a t t r i b u t e d ,  administered  giving  an  immediate  occurring orally,  administered  uptake  the  in t h i s  have been  the  The  is  a period  situation,  in  of  and  profiles  which they  single  when  1977).  for  The  by the one compartment  disposition  dose  and i n t r a v e n o u s  current  In  the  Siegers  parameters  group,  in  in  part,  to  of animals used.  attained.  for  whereas  by  range.  suspensions  and h a l f - l i f e  i n these  activity  is  the  values  those  administered  time as uptake  the o r a l  than  once,  the o n l y  reported  be d e s c r i b e d  and B e n e t ,  L/kg),  intravenous  stream  or  measured  reported  model.  1987) a d m i n i s t e r e d  in strains  concentration  the  could  determined  distribution  of  that  (Ronfeld  mg/kg)  an  the  by the S e i g e r s  solutions  Pynnonen,  (Sumi, e t a l . ,  When  half-lives  average  compartment  have been a d m i n i s t e r e d  differences  the  within  obtained  single  gators  the  fell  i n which  1978;  compartment  point,  than  The  the v a l u e s of the pharmacokinetic  to  have d i s p l a y e d  blood  less  resembled those  conforming  man,  all  regimens.  35  well,  of  time.  routes  the of  peak  entire  is  elimination  elimination  elimination.  of  the If  drug this  before  is  the  occurring  when t h e r e will  administration.  uptake  enters  On the other  a delay  Normally,  dose  concentration.  elimination.  there  As  drug,  the  is  the  be  It was  is  is  is  the  From  this  hand,  when  peak at  same  same  uptake  for  possible  occurring,  plasma  the  rapid  slowed,  the  both  that,  in  resulting  in  the  values  WEBSTER, D. S.  obtained  for  clearance,  volume  of  distribution,  i n t r a v e n o u s dose would a l l  appear to be l e s s  dose.  that  This  is  v a l u e s of the  the  was  half-life  than f o r an o r a l l y  actually  observed,  with  for  the  administered  respect  to  the  that  the  parameters.  A recurrent  pattern  single  compartment  cases,  the  minutes,  result  and  36  model  plasma  began  emerged i n was  not  concentration  to  decrease,  p a t t e r n was observed  for  several  satisfactory  of  in  carbamazepine  and e i t h e r  animals  animals which  in  the  cases.  reached  plateaued  each of  all  indicated  or  a  In  peak  increased  several at  60-90  again.  This  dosing/feeding  groups  that  were t e s t e d . The most experiments The  likely  explanation  for  were to  be repeated  employing  reasoning  sequence  of  behind  steps  this  statement  involved  in  the  F o l l o w i n g i n g e s t i o n of the d r u g , case of t a b l e t s .  This i s  by  a  electrical hand, area. is  thick,  It  is  facilitating  hydrophobic since  it  epithelial the c e l l , the  thin,  has the  drug,  is  cells  of  primary  the  small  of  an  if  orally  one  intestine  absorption  lipid  be  of  i n the stomach,  portal  quite  Thus,  intestine.  readily it  surface  will  be  On  its  the  large  the  small  quickly  into  the  portal  is  high other  function being  a  intestine  taken  The drug must then d i f f u s e  and  the  surface  primary  membrane, and then make i t s  vasculature  in  and  Carbamazepine, in  drug.  The stomach  has an extremely and  the  s i n c e the c a r b a -  digestion.  resistance,  the  sampling.  administered  small  nutrients.  absorbed  soluble.  is  of  if  considers  particles.  with  function  low e l e c t r i c a l  of the small  the  time c o u r s e  obvious  membrane  a g a i n pass through the c e l l  membrane  be  may be obvious  i t must be d i s s o l v e d  Its  should  highly  may  as a suspension of f i n e  resistance. of  a longer  uptake  mucus-covered  the e p i t h e l i u m  observation  not a problem i n the c u r r e n t case  mazepine was a d m i n i s t e r e d lined  this  by  the  through  way a c r o s s  circulation.  WEBSTER, D. S.  From t h i s first  point  pass  processes  tion,  described, is  the  absorption,  the  rate the  rest  transported then  only  the  long  period  It  the  food  small  in  for  the  emptying  also  possible  slowing g a s t r i c foods  that  that  this  gastric  emptying  are " d e t e c t o r s " types.  If  carbamazepine. chemical  that  for  surfactant  are  this Since  property  responsible  is  is it  with  delayed Tween  in  and  is  is  Of  the  degree  majority  will  be  of  to  primarily  the  overnight  that  slow  rate  be absorbed  any w o r r i e s about  from  diges-  necessarily  delayed,  the animals  possible  of  responsible  able  contents  carbamazepine some of a  itself  rabbits  the  will  prior  to  require  stomach.  by the  at  differential  As  feeding  may have been  the a n i m a l s .  greater  period  and p r o t e i n s .  of  a a  the  to  This  It  is  be  responsible  well  passed  known  the  is  has been a t t r i b u t e d  for  case,  very  fat.  As  gastric  unknown.  some chemical perhaps  a  hydrophobic, a  It  result,  emptying.  suspension  may  is  possible  feature  parallel  carbamazepine carbamazepine  Alternatively, have  been  that  be  itself  the the to  there  different drawn  shares  the  to  signal  of the can  that  through  but the e x a c t mechanism by which the  specific  the  the  process  is  food  initiated  is  undergoes  fasting.  require  c o n t e n t of the f o o d ,  it  for  may have been i n f l u e n c e d  stomach than do c a r b o h y d r a t e s caloric  it  significant  stomach  fasting  but  of  emptying i n  generally  where  circulation.  any  tract  the  intestine  I t was thought  the observed r e s u l t s  is  fatty  the  displays  responsible  Should  animals p r i o r t o the p e r i o d of  food  systemic  the g a s t r o i n t e s t i n a l of  liver,  emptying.  emptying between a n i m a l s ,  result,  slow  the  the  that  organ  emptying.  enters  to  to  experiment would be adequate to a l l e v i a t e  gastric  for  on  one  presence  gastric  drug  is of  the  a l s o be slowed. the  and  the  stomach  for  of  is  the r a t e o f g a s t r i c  the  while  which  drug  metabolism,  variability Since  the  37  a  to key  may  be  presence  of  resposible,  due  its  WEBSTER, D. S.  structural  similarity  determinant  of  the  to  fatty  acids.  If  rate  of  gastric  caloric  content  emptying,  it  d e x t r o s e used to make the suspension was s u f f i c i e n t One of the m e t a b o l i c onidated  compounds  possibility Due to the  that  can  be  intestine.  bile,  it  is  If  possible  glucuronides  using  uptake.  The  observed  plateau)  amount  or  glucuronide  that  intestinal  i n the  samples of  bile.  for  (Johnson  rats  enterohepatic adapted f o r  be  even  an  increase  in  apparently  and  the  bile,  A technique  for  this  in  can  rabbits.  The e x c r e t i o n  this  information,  In f a c t , man. as  was e x c r e t e d  level  circulation  being of  used  (Terhaag,  be r u l e d  particularly only  and  the  the  duct  the  neglect  The  and  collecting described  excretion could in  and  easily  be  bile  of  administered  dose  of  Based  on  1978).  negligible  other  the to  of  glucuronide  to any measurable  since  (the  the  al.,  in  for  future.  has been  ruled  out  parent  be f r e e  the  biliary  et  might  as o c c u r r i n g  cannot  here,  carbamazepine  bile  as  technique  of  hydrolyze  concentration  carbamazepine  1 percent  circulation  has not been d e s c r i b e d  rabbits  the  the  enterohepatic  Enterohepatic  the  note  it  in  of  the  the  procedure  This  in  elimination  in  bile  wherein  the  can a g a i n  circulating  the  in  case,  slower  the  considered.  occurs  to  the  tested  Glucur-  excreted  able  being  type of  be a s s e s s e d .  is  presumably  cannulating  1978),  are  be  the  fact,  be  uptake  and i t  the  should  man has been e v a l u a t e d and o n l y about carbamazepine  in  by  Rising,  its  much  determined  and  must  carbamazepine  This  that  on t h i s  occurring  intestine  an  can  Based  most of  enzymes.  small  possible  a major  glucuronidation.  bile. is  really  t o slow emptying.  is  microflora  be  circulation use  of  will  carbamazepine can  the  of carbamazepine,  postulation  conjugate,  in  circulation  glucuronidase  result  This  of  excreted  the  drug can be regenerated  drug.  of carbamazepine  enterohepatic  lipophilicity  small  the  fates  is  is  38  man.  extent  species,  bile  refer  in  in  such  measurements to  conjugated  WEBSTER, D. S.  metabolites. different  from  conjugating being  a  in  the  man,  intestinal and  This  glucuronysyl  excreted  it  factor  would  a p l a t e a u with be  reasonable  experiencing  that  estimate  enterohepatic  better  activity,  able the  since  is  to  very  importance if  If  one were  show the  there  that  circulation  15 t o in  to compare  traditional 20  order  of  the there  were  more  circulation the  profiles  type  of  profile,  of  the  percent to  likely  hydrolyze  the degree to which e n t e r o h e p a t i c  those  to  be  of the r a b b i t  would i n c r e a s e  transferase  i n the b i l e  flora  may  o c c u r r i n g would be more pronounced.  displaying it  that  connection.  high  conjugate is  In a d d i t i o n ,  39  produce  the  quite  clear,  dose  is  observed  plateau. The d i f f e r e n c e s some  display  others  usable  obviously  entirely  between r a b b i t s single  do  not.  it  is  clear,  in  this  compartment While  quite  the  study  are  concentration-time  reasons  possible  for  that  this  a  profiles,  observation  genetic  factor  responsible.  For example,  those animals which are suspected of  enterohepatic  circulation  may  greater  of  rate  glucuronysyl  glucuronide  transferase  is  have a more c o n v e n t i o n a l conjugation  (or,  forming  glucuronide  the  possible producing  that a  genetic  greater  the m e t a b o l i t e s  of  carbamazepine  greater).  a faster  of  influence  on  activity  excreted will  in  may have  enzymatic can  glucuronidated experience  hand,  the  display  urine the  not  with  conventional  the it  a greater  single  may  be  that of  reaction  a  of  a of  rate  is  will  degree  experience  not  rabbits  Thus,  metabolites  are  activity  control,  exist.  while  produces  a slower  Since  a greater  a rabbit will  the  that  experiencing  that  those  oxidation).  under  On the o t h e r  and  profile  is  and w i l l  (i.e.,  Alternatively,  rate  of  complement  formation  conjugates  i n the b i l e  of  genetic  conjugate  proportion  hepatic c i r c u l a t i o n .  a  pharmacokinetic  possibly  a  more m e t a b o l i t e  have  in  quite rabbit excrete  entero-  proportion  recirculation compartment  WEBSTER, D. S.  profile. above, tion,  presence  not  often  is  a strain  would, the  The  been  able  study,  to  negate  obtain have  acetylation  apparent  hepatic  a  same  the  decrease  is  Using F i g u r e  indicates  that  a  manner  concentration.  It  is  P  carbamazepine  in  polymorphisms  polymorphism that  may  convert  7 as  of  also  monooxygenases,  that  influences  of  auto-induction the is  two is  time c o u r s e possible  that  Despite  was  used to  have  in  this  rates  polymorphisms  do  of  exist  study and t h a t  increases  the  the  the  animals  (are)  for  glucuronysyl  resultant  genetic occurring  It  over  be too  carbamazepine  the  time  plasma  be  able  for to  the  of  due it the  its  of D2,  does  not  carbamazepine have  a  faster in  the  metabolism  of  likely,  if  these  and  for  hepatic  to  the  combined  is  possible  experiment,  such an o c c u r r e n c e . induce  of  greater  differences  quite  are  is  and  above  transferase  course  that  of  for  relations  For rabbits  time  Alternatively,  short is  would  observations  factors.  the  same  genetic  responsible  animals.  in  noted  activity  carbamazepine-10,  to a l e v e l  decrease  the  to  the  can be s e e n .  parallels that  from  differences  at  is  the  seems to  which  laboratory  respect  carbamazepine  an example,  possible  exist  that  our  arise  carbamazepine  individual  do  defini-  possible.  that  4 5 0  By  uniformity  that  genetic  described  paragraph.  in  with  r a t e o f o x i d a t i v e metabolism which may be due t o cytochromeCs)  species.  this  strain  to the carbamazepine curves  concentration in  in  phenotypes  nature  genetic  investigators  D3, and D4 the c o n c e n t r a t i o n o f epoxide than  a given  presented  the  the  of r a b b i t s t h a t were used i n t h i s  genetic  the epoxide c u r v e s  of  of  high degree o f  strain, from  This  monooxygenases  11-epoxide.  a strain  differential  activity.  e x p l a n a t i o n put f o r t h  the  of  had  polymorphism,  the proposal  rabbits,  amongst the p o p u l a t i o n  The  genetic  found w i t h i n  uniformity  that  a  should have a f a i r l y  in e f f e c t ,  supposed  of  40  That  own metabolism  that though is, in  it the  WEBSTER, D. S.  rabbits  noted.  This  effect  may  the apparent i n d u c t i o n occurs fact of  t h a t they  the  were a l l  initial  more l i k e l y  rates  also  t o be t r u e ,  a  reaction  the  of  indicates  that  likelihood In  fact,  pine  of  it  Siegers  doses,  at  As  this  complete. that  former  taken p r i o r  over  a single (1982)  weeks  dose  in  expected  and  t  dose  examination  explanation  the  to  The  rates  children,  is  slim  examine  the  with  the  at  best.  carbamaze-  differences  problem  of  autoinduction  human  no  is  greater  initial  rabbits  the  in  the  their  study  (10 mg/kg) was a d m i n i s t e r e d and b l o o d  to and 2 hours  effects  in  found  .  complete c l e a r a n c e  inductive  3  attempted  rabbits  oral  had the f a s t e s t  i n the i n t r o d u c t i o n ,  take  in  the  daily  must be examined f u r t h e r , the data base  over  phenomena  essentially  and any  can  and co-workers  was t h a t o n l y a s i n g l e  a result,  as s t a t e d  occurring  maximum c o n c e n t r a t i o n s  samples were o n l y  if,  metabolism  autoinduction  However an  the  since  despite  s i n c e those animals d i s p l a y i n g an a p p a r e n t l y  In a d d i t i o n ,  carbamazepine  predisposition,  same d o s e .  r a t e of o x i d a t i v e metabolism over time a l s o reaction.  genetic  i n some animals and not i n o t h e r s ,  administered  of  have  41  after  drug a d m i n i s t r a t i o n .  of carbamazepine  may have time t o  is  allowed  reverse.  s i n c e any i n s i g h t can o n l y  These  be gained by  As  between  phenomena increasing  significantly. discussion  A great  remain.  indicates,  the  deal  of  research  One might  be  tempted  work  must to  be  in  this  done  explain  the  field  to  is  answer  by the  observations  no  means  questions for  which  F i g u r e 7 was used as an example by c l a i m i n g t h a t ,  s i n c e the animals were  fed  ad l i b . ,  of  food  there  content  examined,  it  may have been d i f f e r e n t i a l  of  the  is  obvious  where the animals  gastrointestinal that  were f a s t e d  f o l l o w i n g drug a d m i n i s t r a t i o n  uptake  tract.  But,  similar  patterns  overnight  prior  are  the if  drug,  Figures seen  to and f o r  for at  based  on  the  8 and  9  are  these  trials  least  one  hour  (most animals d i d not e a t d u r i n g the p e r i o d  of  WEBSTER, D. S.  blood  sampling  previously,  results  carbamazepine  a l o n g e r time c o u r s e ,  administration  under these  the  of  conditions,  carbamazepine. the  can be c o n c l u s i v e l y  circulation  was a d m i n i s t e r e d ) .  the data base must be expanded  current study, the  after  in  the  rabbit  issue  of  settled. should  be  using  Thus,  the The  a series effect  of  of  situation  examined  in  as  the method employed  and a l o n g e r p e r i o d of f a s t i n g If  42  experiments  gastric  detail.  In  time course of carbamazepine a u t o - i n d u c t i o n must be e l u c i d a t e d .  in  the  prior were  contents  regarding  stated  on  to  done the  enterohepatic addition,  the  WEBSTER, D. S.  43  2 THE INFLUENCE OF ISONIAZID ON THE IN VITRO METABOLISM OF CARBAMAZEPINE:  2.1  INTRODUCTION:  The second p o r t i o n of t h i s  thesis  carbamazepine metabolism i n r a t  examines the i n f l u e n c e  l i v e r microsomes,  planned e x t e n s i o n of the pharmacokinetic metabolic  interactions  between  study  isoniazid  and  The change i n p l a n s was a r e s u l t of an i l l n e s s  of  isoniazid  r a t h e r than the  on  originally  to the examination of  in  carbamazepine  rabbit.  in  the  vivo  t h a t prevented me from working  the i n t e n s i v e 12-15 hour days t h a t would have been r e q u i r e d . Instead, series  of  an  experiments  progression, in  vitro  rn  study are  using  rat  closely  related  but they d i d not r e q u i r e  v i t r o model  had been developed  liver  in  route  by undergraduate  laboratory  analysis  the  original  The  our  a  of  the  done.  long working days.  Band and Lawrence S e l b y .  complete  to  was  such extremely  students C h r i s t i a n more  microsomes  This  scenario  study  than  was able  did  to  their  of The  summer provide  preliminary  observations. It  has  been  metabolism, normally  as  indicated  therapeutic  carbamazepine Wright,  suggested  et  that by  clinical  1982;  and/or  Valsalan  blurred  symptoms  of  carbamazepine considered valid  to  vision,  toxicity  concentration  sufficient  postulate  reports  for  that  to  in  and  1982;  included  with  above  effectiveness.  either  produced  inhibits  that  signs  (Block, et  al.,  headaches,  The an  carbamazepine  coadministration  Barbare,  confusion.  well  of  hepatotoxicity  conjunction  therapeutic isoniazid  wherein  intoxication  levels  inhibitor  and i s o n i a z i d  Cooper,  drowsiness,  occurred  an  isoniazid  and  The observed s i g n s of carbamazepine vomiting,  is  doses of carbamazepine  intoxication  al.,  isoniazid  of of  1982; 1986). nausea,  onset  of  these  increase  in  serum  which  Thus,  it  is  generally  would  carbamazepine  appear  metabolism  WEBSTER, D. S.  or  alters  the d i s t r i b u t i o n  much more  likely  Carbamazepine  is  inhibited  half-lives  are  carbamazepine.  postulation  process t h a t i s d i f f i c u l t  readily  of  to  since  (Pippenger,  e l e v a t e d as a r e s u l t  distribution  and  by a h e p a t i c 1987).  Drug  steady  state  of i n h i b i t i o n .  added to reports  in  the  metabolic  patient's  indicated  s t r a t i o n of symptoms  p e r c e n t decrease  of  microsomal  is  a  diffusionary  clearance serum  system t h a t is  drug  as  soon  regimen. patients  as  This  the  decreased,  Wright  and  were undergoing  appear  drug  occurred chronic  as  in  is the  carbama-  and the admini-  l e d to the  (1982)  i n carbamazepine c l e a r a n c e 3-5 days a f t e r  are  concentration.  disorders,  co-workers  drug  concentrations  situation  seizure-causing  can be  inhibiting  i n a d d i t i o n t o the carbamazepine,  intoxication.  be a  above the minimum t o x i c  begin  therapeutic  the c o n t r o l  isoniazid,  of  pattern  above where the  z e p i n e therapy f o r  to  Symptoms of drug i n t o x i c a t i o n  soon as the serum c o n c e n t r a t i o n r i s e s The changes  tends  of metabolism  alter.  metabolized  prolonged,  Inhibition  44  observed  reported  isoniazid  a  45  admini-  stration. In v i t r o (S9  fraction)  this  system,  being  slowed  1989).  This  studies  of  the  indicated is  dependent  by  greater  thesis  will  that  influence the  on the  of  rate  of  the  in  metabolism  concentration  concentrations examine  isoniazid  of  of  of  goal the  isoniazid,  of t e s t i n g inhibition  acetyl  hydrazine,  the m e t a b o l i t e s of  carbamazepine  hydrazine,  liver  the  and  microsomes  carbamazepine,  isoniazid,  and those of experiments with the primary m e t a b o l i t e s acetyl  of  isoniazid  results  rat  with  (Webster,  studies  the  rate  et  al.,  with  isoniazid  of i s o n i a z i d ,  including  isonicotinic  acid.  was to determine which were r e s p o n s i b l e metabolism  and,  i n f o r m a t i o n to the p r o d u c t i o n of h e p a t o t o x i c i t y  by  in  possibly, isoniazid.  to  relate  The for this  WEBSTER, D. S.  2.1.1  The  45  CARBAMAZEPINE:  development  and  biotransformation,  and  chemistry,  therapeutic  toxicities  i n t r o d u c t i o n to the p r e v i o u s  of  uses,  mechanisms  of  action,  carbamazepine  were  section.  As such,  information w i l l  drug f o r  the chemotherapy  this  discussed  in  the  not be  repeated h e r e .  2.1.2  ISONIAZID:  Isoniazid all  patients  tubercle  is  the primary  with  bacillus  and Sande,  tuberculosis should r e c e i v e  1985).  Isoniazid,  duced i n 1952 ( B e r n s t e i n , then,  z i d has been t e s t e d f o r i t s  the  by  drug  1952).  they of  can  Despite a great  and d e s c r i p t i o n  of the most important of which i s  since  Isonia-  opportunity  and adverse  isoniazid-induced  intro-  disorders.  has p r o v i d e d the  of many t o x i c i t i e s  of  (Mandell  of work  elucidated.  v a l u e i n a number of  isoniazid  it  a c i d was  deal  and  strains  tolerate  isonicotinic  has not been c o n c l u s i v e l y  of  tuberculosis,  isoniazid-sensitive if  hydrazide  therapeutic  use and t e s t i n g  the o b s e r v a t i o n  the  et a l . ,  i t s mechanism of a c t i o n  The e x t e n s i v e  caused  of  effects,  for one  hepatotoxicity.  2 . 1 . 2 . 1 DEVELOPMENT AND CHEMISTRY:  The  synthetic  chemical limitless century  tuberculostats,  laboratory,  lend  potential.  For  saw a g r e a t  deal  produce a t h e r a p e u t i c a l l y  i.e.,  themselves  of  this  readily  reason,  synthetic  effective  those  the  which to  creative  first  chemistry  were  half  performed  devised  in  the  manipulation  and  of in  agent a g a i n s t t u b e r c u l o s i s  the  twentieth  an attempt (Fox,  1953).  to  WEBSTER, D. S.  The  first  tubercle  bacillus  effective  in  man  considerable  with  Most  the  of  In  was  to  exhibit  marked  than  in  laboratory  Subsequently,  increased compounds  activity that  were  solubility  synthesized  early  1940's,  certain  and i n h i b i t  organism  tuberculosis.  responsible  p-aminosalicylic variations  for  acid,  commonly  against  proved  addition,  to  the  be  less  it  exhibited  were made to  synthesize  and  were  decreased  more  toxicity.  soluble  and  less  activity.  benzoates  decrease the oxygen uptake  it  In  many attempts  and  activity  but  animals.  but they a l s o l a c k e d the necessary the  vivo  vn  4,4-diaminophenylsulfone,  toxicity.  sulfones  toxic,  compound  46  the  known  and  salicylates  were  growth of  tubercle  The  common  as  most PAS  bacillus, of  (Lehmann,  i n the PAS s t r u c t u r e were i n v e s t i g a t e d ,  found  to the  these  was  1944).  Many  but none were found to be  s u p e r i o r to the parent compound. In  the  late  1940's,  the  tuberculostatic  zones was d i s c o v e r e d as a r e s u l t drugs of  in  tuberculosis.  thiosemicarbazones  of  Behnisch and  found  zone  (TbI),  which was the most  the advent of the h y d r a z i d e s A tested the  series in  the  parent  co-workers compound  of  pyridine  late  compound in  with  1948.  study  pyridine  and h i s  is  active  (Fox,  1940's.  to  The  be  acid  (1950)  of  tested  the a  sulfa series  tuberculostatic.  benzaldehyde  The  thiosemicarba-  tuberculostat  derivatives  tuberculostatic  group,  was  This  was  followed  by  three  resistant acid  thiosemicarba-  in  use  until  1953).  this  carboxylic  the  investigation  strongly  synthetic  of  But,  of  co-workers  p-acetamido  carboxylic  approximately  n i c o t i n a m i d e or PAS.  a systematic  them  b e s t known compound of the s e r i e s  activity  times  activity  discovered  synthesized of  by  discovery  the  tuberculostatic  rapidly closely  of  and  pyrazinamide,  emerged. related  and  nicotinamide,  Kushner  the  strains  derivatives  were  to  his a  activity  of  The d e s i r e  to  nicotinamide  WEBSTER, D. S.  led  to  attempts  1952).  Since  carboxylic studied,  to  prepare  isom'cotinoyl  hydrazide  acid  derivative,  was  investigated  it  be more a c t i v e  than  E x t e n s i v e chemical isonicotinic  acid  any  isonicotinaldehyde  and for  known  (Figure  related  to  tuberculostatic  substance  and chemotherapeutic or  thiosemicarbazone  (isoniazid)  thus  pyrazinamide,  -  11)  the  a  pyridine being  and was found  synthetic  or  showed t h a t ,  isom'cotinoyl hydrazide  i n many ways w i t h o u t a b o l i s h i n g i t s  is  (Fox,  structures  activity  whether  studies  47  antibiotic.  unlike  could  to  be  3-aminomodified  activity.  2 . 1 . 2 . 2 THERAPEUTIC USES:  it  Isoniazid  is  has  since  its  isoniazid  has  been  addition,  the primary  other d i s o r d e r s . ations to  arises  which  structural  introduction been  is  administered.  relationship  Huntington d i s e a s e  which acid  loss  of  probably (GABA)  as  Bernstein's its  for testing  extent,  et a l . ,  to  in  1979, the  a population  inhibitory  its  1982).  caudate of  neurotransmitter.  When l a r g e  a  result  ferase,  that  increases  was thought  the f i r s t  in  in  takes  In  several  in d i f f e r e n t in  and  1952.  value  drug  to  (Perry  doses  the  that  the  situsubject  advantage  of  the  i n the treatment disease,  and  of  of a of  y-aminobutyric in  with the  are g i v e n  1973; P e r r y ,  is  most  decrease  in patients  inhibition  there  putamen,  utilize  A marked  isoniazid  and Hansen,  be due t o  of two s e q u e n t i a l  of  value  In t h i s  cells  (Perry,  b r a i n GABA c o n t e n t  the  nucleus  areas has been observed  1973).  of this  c o n t e n t i n the a f f e c t e d et a l . ,  group  isoniazid  influences  an  tuberculosis,  therapeutic  has been t e s t e d f o r  neurons  belong an  by  of  between i s o n i a z i d and endogenous compounds.  (Perry,  small  To  the chemotherapy  for  physiological  F o r example, i s o n i a z i d  marked  tested  The j u s t i f i c a t i o n  from the  it  drug f o r  et  to  GABA  disease animals,  al.,  1974),  GABA-aminotrans-  enzymes t h a t degrade GABA i n the b r a i n .  WEBSTER, D. S.  ONHNH. Figure  11:  Isoniazid  N  Figure  12:  Relevant aspects of isoniazid metabolism.  48  However,  in  a double-blind  cerebrospinal iazid  fluid  therapy,  disease  there  was a l a c k  associated  et  1985;  al.,  1987).  The e a r l y  in part, observer  et  were markedly  clinical  al.,  the  1982),  increased  improvement  to  bias.  to  metry  demonstrated  found  role,  a  In tested  The  the a  clinical that,  of  this  et  in  to  evaluation,  studies al.,  trial  1986) of  et  while  during  most  ison-  Huntington  usefulness  al.,  al.,  reduction only  appears  warranted  in  such  of  have  multiple  et  al.,  that  were  to  as  patient  polarised  (Bozek,  Polarised  marginal to  Bozek,  subject  tremograms  against  Duquette,  results  techniques  and  1982;  1986;  which was  isoniazid.  showed  isoniazid  is  used  three-fold  assessment  et  its  type gave c o n t r a d i c t o r y  effectiveness two  for  (Sabra,  1985; F r a n c i s ,  later  although  clinical  postural  al.,  (Francis,  evaluate  group  et  sclerosis  the mechanism of  1987)  of  has been examined  with m u l t i p l e  studies  goniometry  methods  isoniazid  Hallett,  light  al.,  of  (Perry,  49  patients.  tremors  and  trial  GABA c o n c e n t r a t i o n s  In r e c e n t y e a r s ,  due,  clinical  WEBSTER, D. S.  tremor  et  light when  goniostandard  improvement. a limited  sclerosis  al.,  Bozek's  therapeutic  patients  with  tremor. addition  for 1988)  its and  to  those  value  in  disorders the  rheumatoid  (McConkey and S i t u n a y a k e ,  already  therapy  of  arthritis  discussed,  Parkinson's  (when  isoniazid  disease  administered  has  been  (Gershanik,  with  et  rifampicin)  1988)  2 . 1 . 2 . 3 MECHANISMS OF ACTION:  The mechanism by which i s o n i a z i d culosis cellular  is  unknown,  mycolate  but  there  synthetase  are  is effective several  activity  of  i n the treatment of  hypotheses.  Mycobacterium  For  tuber-  example,  tuberculosis  the H37Ra  WEBSTER, D. S.  has been shown t o be very a-Mycolic a c i d , one  of  a  aliphatic  homologous  fatty  acids  of  (Minnikin  wall.  synthesis acids  are  thought  amide  the  adenine  greater to  be  than  inhibit  to  inactivate  by c e l l - f r e e  inhibitor  of  NAD  (Gopinthan,  et  of  Bricker,  H37Rv, 1967).  however, Thus,  isoniazid  and  ligase  bacteria  of  DNA l i g a s e  is  1966).  the  i n DNA s y n t h e s i s ,  a direct  lowering depends  essential  not  for  of  the u n a v a i l a b i l i t y  to i n h i b i t i o n of DNA s y n t h e s i s .  fatty  are  acids  al.,  and  2 6  1975).  acids,  and  These  and  thus  cells.  nicotinamide,  where  (NADH)  of  nicotin-  concentrations  of  isoniazid  both  sensitive  isoniazid  H37Rv,  present by  of is  in  the  human  H37Rv,  an i s o n i a z i d  concentrations  vital  enzyme NADase  resistant and  lethality of  1971)  polydeoxynucleotide this  of  able  (Bekierkunst  between the  (Olivera,  is  the  the r e l e a s e  and  virulent  isoniazid  isoniazid  exists  of NAD f o r  C  isoniazid  important  a cofactor of  long  are  The r e s u l t  intracellular  elongation  NAD  is  and  pathways  glycohydrolase  correlation  on NAD as  et  In a d d i t i o n ,  inactivated  a  w a l l s of growing  homonis  The i n h i b i t o r is  of  1969).  f o r the d e g r a d a t i o n of NAD. al.,  high  var.  responsible  strain  1972).  e-position,  than  mycolic  involve  extracts  and Ramakrishnan,  the  the  reduced  relatively  Mycobacterium t u b e r c u l o s i s  (Sriprakash  greater  between i s o n i a z i d  or  +  example,  of  action  (NAD )  the  These  (Takayama,  2 4  containing  has been shown t h a t  d i s r u p t the c e l l  of  at  1967).  acids  precursors  mechanisms  NAD s y n t h e s i s  resistant strain  For  C  acids  group  It  fatty  similarity  dinucleotide  intermediates.  1971).  saturated  structural  proposed  a hydroxyl  (Lederer,  of  fatty  Because of the of  (Wang and Takayama,  fatty  and P o l g a r ,  i n h i b i t i o n of t h e i r f o r m a t i o n w i l l  many  isoniazid  C^-Cg^  a-position,  rings  p r e s e n t i n the c e l l  unsaturated  series  at the  two cyclopropane  the  to  a major mycolate component of Mycobacterium t u b e r c u l o s i s ,  chain  inhibits  sensitive  50  NAD.  DNA  and  chains  of  since formed  r e a c t i o n may  lead  WEBSTER, D. S.  It  has  r e l a t e d to and  been its  Fe.  ability  The  (Middlebrook, system of organic  postulated  1954;  Arora  breast  nitroreductase related  the  to complex  inhibitory  pigeon  presumably  that  to  certain  effects and  gram-negative  sequestering  of  on  1960),  and K r i s h n a bacteria  essential  of  isoniazid  heavy m e t a l s  isoniazid Murti,  (Arora  effect  essential  of  Krishna  muscle of  bacteriostatic  on  the  catalase  succinoxidase  1954),  (Arora,  et  is  such as Cu  hepatic  Murti,  metal  51  or  al.,  on  the  1959)  are  from  the  ion moieties  enzymes.  2.1.2.4  Since  BIOTRANSFORMATION:  the i n t r o d u c t i o n of  the e a r l y  1950's,  metabolic  pathway,  injury.  The  metabolic  there  identification introduction  particularly aspects  of  isoniazid  metabolites  the d r u g ,  when s t u d i e s  the  man  studies  in  isonicotinic  hydrazide, identified  in  to  accepted  Figure  12.  occurred i n dogs  1952). et  nicotinamide,  nicotinoylglycine,  isoniazid-induced  in  The the  scheme first  al.,  1953),  in  isonicotinamide, and  of  in  which  the  published as  isonicotinic  T h i s was f o l l o w e d ,  its liver  same y e a r  identified  in  the acid  1953, by nicotinic  nicotinic  acid  isom'cotinoyl glycine  were  urine.  By the mid 1960's r o u t e of i s o n i a z i d and c h a r a c t e r i z e the c o n v e r s i o n  et a l . ,  in  of t u b e r c u l o s i s  work done to e l u c i d a t e  currently  (Cuthbertson,  acid,  isoniazid,  shown  of  relates  the  are  (Kelly,  acid,  of  it  isoniazid  as a major m e t a b o l i t e first  as  of  of  f o r the treatment  has been a g r e a t deal  relevant  fate  isoniazid  it  had been e s t a b l i s h e d  inactivation  the  (Jenne,  acetylation  is  the major  i n man, with work having been done to  N-acetyltransferase 1965).  that  enzymes  responsible  for  purify  catalyzing  P r o d u c t i o n of a c e t y l i s o n i a z i d was found to be  WEBSTER, D. S.  the step t h a t  is  lism  rapid  between  established genetic  solely  that  and  isoniazid At  the  were  had  same  et  al.,  inactivation  as  a c e t y l hydrazine (Timbrell,  et  the  1965).  capacities  It  are  was  under  1977).  and as  Second,  it  acetylated  urine.  Third,  the  reactive  the  First,  it  to  which i s  at  excreted acid  is  it  by  and  diacetyl hydrazine,  a c e t y l hydrazine  1976; N e l s o n , et a l . ,  body  is  a-oxoglutaric  enzyme system,  intermediate  the  responsible  that  for  production  was  which  and  urine acid  is  by  is  the  of  (Black that routes as  free  hydrazones.  excreted  in  the  by  the  produce  the  metabolism  thought  the  their  three  the  pyruvic  al.,  observed  least  in  eliminated  a pathway  et  and c a r c i n o g e n s  Subsequently,  from  pyruvate  a c e t y l hydra-  Hydrazines  mutagens,  isonia-  (Mitchell,  for  injury.  to  and  particularly  responsible  hepatic  1973).  eliminated  al.,  to  hydrazone  metabolites  metabolites,  be h e p a t o t o x i n s ,  Druckrey,  is  microsomal  led  isoniazid  as  metabolites  which  a c e t y l hydrazine is  identified  hydrazide  were known to 1970;  a-ketoglutarate  been  time,  intermediate"  Thomas,  et a l . ,  in  (Peters,  i n i s o n i a z i d metabo-  when much work was being done w i t h r e s p e c t  identified  derivatives  hepatic  inactivators  differences  hepatotoxicity,  hydrazone  "reactive  and  f o r the d i f f e r e n c e s  control.  zid-induced  zine,  slow  these  In the mid 1 9 7 0 ' s ,  1975).  responsible  52  to  hepatotoxicity  (Mitchell,  1975).  2 . 1 . 2 . 5 ADVERSE EFFECTS:  The adverse e f f e c t s rence of 1 to 2 p e r c e n t  of  isoniazid  at c o n v e n t i o n a l  and 15 p e r c e n t at 10 mg/kg/day reactions  are  rash,  are dose r e l a t e d ,  fever,  low dose therapy  (Goldman and Braman,  jaundice,  w i t h a r a t e of  and  peripheral  occur-  (3 to 5 mg/kg/day)  1972).  The most common  neuritis  (Mandell  and  WEBSTER, D. S.  Sande,  1985).  The  range  ding:  hypersensitivity  of  reactions  reactions,  and m o b i l l i f o r m , maculopapular, cal  reactions  anemia,  and w r i s t s .  terminate  transient  self-control, One of the 5  ataxia,  of  symptoms  memory,  side e f f e c t s of  of  those  common,  treated  toxic  (Girling,  optic  stupor,  separation  is  of  of  the  most  hepatitis, 1982).  bilateral  the  knees,  neuritis,  muscle  encephalopathy as  euphoria,  reality,  loss  of  studied,  of  1985).  extensively  which  It  hematologi-  of  such and  and Sande,  hepatitis,  pain,  toxic  ideas  inclu-  thrombocytopenia,  back  abnormalities  and c e r t a i n l y  isoniazid  rashes;  arthralgia  been r e p o r t s  mental  broad,  eruptions,  such as  and  parasthesias,  and  skin  quite  eosonophilia,  involvement,  have a l s o  is  and u r t i c a r i a l  and f l o r i d psychoses (Mandell  the most  percent  purpuric,  joint  fatally),  impairment  such as f e v e r ,  and a r t h r i t i c  There  dizziness,  isoniazid  agranulocytosis,  interphalangeal  twitching, (may  as  and v a s c u l i t i s ;  proximal elbows,  such  to  53  is  occurs  in  manifested  less as  than  hepatic  n e c r o s i s , with i n c r e a s e d l e v e l s of l i v e r enzymes i n the serum being e v i d e n t . I t was f i r s t necrosis  is  gested t h a t  suggested,  al.,  the  1976;  acetylation Thus,  the  to  t o the key  et  non-toxic  determinant  one would  al.,  et  al.,  due to  the  toxicity  and  to  risk  (Mitchell,  1975).  of  acetylation,  acetylator  1974).  there  et a l . ,  (Wright  i.e.,  liver  later  sug-  activation  1976; is  the  by  may  between  (Lauterburg, to  play  be  of  Mitchell, a  and T i m b r e l l ,  isoniazid  phenotype  is  was  the m e t a b o l i c  with r a p i d a c e t y l a t o r s  But,  for  It  Detoxification  d i a c e t y l hydrazine  expect  responsible  (Nelson,  d i a c e t y l hydrazine  i n the i n c i d e n c e o f t o x i c i t y , al.,  is  species  1980).  role  et  the  intermediate  of  activation  and metabolism  Based on t h i s ,  injury  a reactive  Timbrell,  that  (Snodgrass,  hepatocellular  between m e t a b o l i c production  1974,  acetylisoniazid  a c e t y l hydrazine et  in  the  further 1978). balance  a c e t y l hydrazine et a  al.,  1985).  significant  being at a g r e a t e r  abundant c l i n i c a l  evidence  to  WEBSTER, D. S.  show t h a t no  the  greater  Riska, be at  risk  in  rapid  1976).  studies  risk  by  than  reactions  in  slow  (Lai,  et  al.,  and  during  1972;  Smith,  t h e r e has been a r e c e n t r e p o r t  logically  demonstrated  itself,  rather  interest  since  than  in  rabbits  as  a c e t y l hydrazine  hydrazine  is  In  were  support  of  a  result  (Noda,  of  et  involved the  metabolism of  one  in  the  latter  obser-  n e c r o s i s was  histo-  by  1983).  hydrazine  This  metabolic  is  pathway  i n c r e a s e d i n slow  influence  carbamazepine  of of  in  this  introduction,  isoniazid rat  liver  and  its  the goal  major  of  whose  acetylators.  microsomes.  resultant t o x i c i t i e s  of  this  metabolites  This  will,  p r o v i d e a b e t t e r u n d e r s t a n d i n g of the nature of the c l i n i c a l  study on  the  hopefully,  interaction  and  t h a t were n o t e d .  EXPERIMENTAL: 2.2.1  MATERIALS:  Carbamazepine,  NADP , +  glucose-6-phosphate,  dehydrogenase were o b t a i n e d from Sigma Chemical methanol, the  may  STATEMENT OF PROBLEM:  examine the  2.2  is  1981;  including  induction  al.,  al.,  acetylators  1972),  who  produced by a d i f f e r e n t  As s t a t e d at the beginning was to  al.,  isoniazid  et  slower  i n which h e p a t i c  r e l a t i v e p r o p o r t i o n of i s o n i a z i d metabolism i s  2.1.3  et  (1985)  hypothesis.  with  (Dickinson,  have shown t h a t  co-workers  the o p p o s i t e  treatment  acetylators  some s t u d i e s  Lauterburg  proposing  vations,  hepatic  In f a c t ,  higher  published  of  54  and KC1  supplier  buffer,  and  of  were  MgCl^,  NaH P0 . 2  4  obtained  and  Co.  from BDH C h e m i c a l s .  hydrazine,  tris  A c e t y l hydrazine  glucose-6-phosphate  Isoniazid, Fisher  (hydroxymethyl) was  obtained  acetonitrile, Scientific  aminomethane  from  Aldrich  was  (TRIS)  Chemical  WEBSTER, D. S.  Co.  and i s o n i c o t i n i c  a c i d was o b t a i n e d  a z i d was s y n t h e s i z e d of von S a s s e n , excess  of  et a l .  acetic  continous  stirring,  ether  U n i t of the U n i v e r s i t y  The  anhydride  and  Sprague-Dawley  2.2.2  the  Acetylisoni-  s u p p l i e d by BDH, a c c o r d i n g  to the method  at  room  product  for  was  1.5  four-fold  hours  with  recrystallized  in  (1:4). rats  (265-340g)  of B r i t i s h  were  obtained  from  the  Animal  Care  Columbia.  MICROSOME PREPARATION:  microsome  preparation  and  the  subsequent  incubation  were  based  on  (1984).  Rats were s a c r i f i c e d by a blow t o the head, an i n c i s i o n  through the neck  the  mid-section  spinal  exposed  and  via  portal  the  cava  was  cord,  the  and  liver vein.  occluded.  completely,  attaining  homogenized  using  homogenizing  buffer  was  slowly  exsanguination.  perfused  When the  liver  Further  perfusion  a  a  thorough  tan  colour.  Potter-Elvejhem (20mM  TRIS;  1.15  Model JA21 r o t o r . kept  tions  on  ice  cuvettes  2 mL o f with  to  NaHoPO^ expand,  allowed The  The  the  the  liver  with  KC1)  7.4)  superior  vena  to  to  excised a  until  used.  using  the  the microsome  teflon  stoppers.  The  4:1  liver  P^^Q  described  suspension  expand and  ratio (v:w).  was of The  15 minutes at 4"C using a Beckman  cytochrome  method  was  pH  liver  was  apparatus, percent  buffer,  The S9 microsome f r a c t i o n was o b t a i n e d as the  was determined  method,  (0.1M  ceased  homogenate was c e n t r i f u g e d at 10,000 g f o r  and  temperature  synthesized  the methods of G r a s e l a and Rocci  to  Co.  (1985), wherein i s o n i a z i d was r e a c t e d with a  acid  methanol - d i e t h y l Male  from m a t e r i a l s  from Eastman Kodak  55  by Mazel  was p l a c e d  An LKB U l t r o s p e c  content  in  4050  of  supernatant  the  (1971).  each of  two  preparaIn  this  matched  spectrophotometer  was  WEBSTER, D. S .  used  t o determine  the baseline  monoxide was g e n t l y milligrams was  of  placed  gassed  with  density -1  of  Each  .  Total  incubation  concentration),  I.U./ni  10  and one o f  its  isonicotinic assays four  protein  additional of  t h e cap  t h e sample was seconds.  20  sodium  The  d i t h i o n i t e and  were a g a i n measured a t 4 5 0 and P ^ Q was c a l c u l a t e d  concentration  male  incubation  consisted  uL o f  150  extinction  from  the  coefficient  was determined  mM M g C l  250  uL o f  60  or  17  mM  metabolites acid).  2  (0.490  mM f i n a l  mixtures  incubated  were  or  1 0 0 \il samples  (that  being  (0.981  using  the  NADP  concentration)  there  samples size  water  removed a t 0 , 5 ,  (0.115  mM f i n a l or of  acetyl hydrazine,  metabolite  i n a shaking  final  of i s o n i a z i d ,  hydrazine,  150  concentration),  mM  4  mM  mM f i n a l  I.U./mL  mM f i n a l  t h e sample  (0.721  (4.327  (0.721  mM) on microsome is,  +  concentration),  uL of  60  and each  0.490  rats  lOmM  dehydrogenase  (acetylisoniazid,  mM, and  uL o f  mM f i n a l  (4.327  mM carbamazepine  For isoniazid  0.115  150  mM g l u c o s e - 6 - p h o s p h a t e  60  Sprague-Dawley  with  of  glucose-6-phosphate  34  (control,  minutes,  and then  nm) and t h e molar  nl o f homogenizing b u f f e r ,  60  concentration) of  cytochrome  (450-480  mixture  uL o f  36  concentration),  one  an  added,  (1976).  concentration),  of  was i n v e r t e d , for  were  4  a few  INCUBATION:  2.2.3  uL  2  a few m i l l i g r a m s  of  f o r 2 0 seconds,  (Na S 0 ) 2  Carbon  -1  method o f B r a d f o r d  final  cuvette  o f each c u v e t t e  quantity  difference  mM cm  91  with  The a b s o r p t i o n s The  dithionite  monoxide  only  a t 4 5 0 and a t 4 8 0 nm.  t h e sample  the cuvette  carbon  was t r e a t e d  4 8 0 nm.  optical  into  sodium  on s e c u r e l y ,  mixed w e l l . at  solid  again  reference  bubbled  absorptions  56  were  three  from each o f  was f o u r ) .  bath  or  (37"C)  The  for 4 0  1 0 , 1 5 , 2 0 , 3 0 , and 4 0  WEBSTER, D. S.  minutes  and  being  placed  acetonitrile.  The  3200  minute.  for  vials,  one  tubes  samples were then c e n t r i f u g e d The  supernatants  were  in  containing  samples  temperature sole  as  were  an Eppendorf  removed,  uL  of  Centrifuge  placed  in  storage  analysed.  samples.  analysed  described  difference  production of  was  using  previously that  the  for  standards  same  the  apparatus,  rabbit  were  run  solvents,  pharmacokinetic  frequently  to  and  study.  allow  standard c u r v e s and the d e t e r m i n a t i o n of c o n c e n t r a t i o n s  An example o f the chromatograms o b t a i n e d i s  2.3  shown i n F i g u r e  the  of  the  13.  RESULTS:  The  epoxide  preparations azid,  metabolic  at  three  14 to  18.  carbamazepine  manner. diverge  The with  Significant  ratio-time  different  acetyl hydrazine,  Figures  hydrazine,  differences  isoniazid  of  the time,  the  of  isonicotinic  inhibited  representing  progression  and  with  was a p p a r e n t l y  the  in  concentrations  In the t r i a l  lines  profiles  by  three a very  S9  liver  isoniazid, acid  (Figure  isoniazid  are  were observed between the  the  on  indicator  blank  There with  were  no  points  acetylisoniazid use  of  three  displaying  (Figure  15)  concentrations  concentration related pattern.  or of  significant  of  acetylisoniazid  Instead, there i s  (Figure did  dependent  the  figure  the  trend.  and 0.490 mM i s o n i -  differences  a c e t y l hydrazine  in  metabolism  a dose  concentrations good  presented  14), in  microsome  acetylisoni-  a z i d samples at the 30 and the 40 minute marks ( u s i n g s t u d e n t ' s  the  200  ANALYSIS OF SAMPLES:  The  of  microcentrifuge  and s t o r e d at -20°C u n t i l  2.2.4  The  into  57  not  t-test). in  the  16). even  some degree of  In  trials fact,  produce  variability  a  WEBSTER,  D. S.  D  O  r  •"I fMiS..  .  (Nji ; CO CS" in  13:  Sample chromatogram from the in vitro interaction study. C the left is a standard s a m p l e of c a r b a m a z e p i n e ( 7 6 9 ) and c a r b a m a z e p i n e - 1 0 . 1 1 - e p o x i d e ( 3 3 9 ) and on the right is a sample t a k e n a t 4 0 minutes.  58  WEBSTER, D. S.  CBZ/INH — ' —  Control  INTERACTIONS  -•€>-• 0.115 mM  — 0 . 4 9 0  INH _ N  1.00 r  T  0.80 -  59  mM  INH  T i m e (min)  Figure  14:  Influence  of isoniazid  (INH) on the conversion  of carbamazepine  (CBZ) t o carbamazepine-10,1 1 - e p o x i d e (CE) in the S 9 of rat liver homogenate.  D a t a represent  mean  +/—  fraction  S £ M (n=4)  WEBSTER,  CBZ/AclNH — • —  Control  N CD U  60  INTERACTIONS  --©-• 0.115 mM  — ± — 0.490  AclNH _  D. S.  mM  AclNH  0.14 r 0.12 "  +  0  5  10  15  20  25  30  35  40  T i m e (min)  Figure  15:  Influence of a c e t y l i s o n i a z i d carbamazepine the Data  S9  (AclNH) on the conversion o f  (CBZ) to carbamazepine-10.11-epoxide  fraction o f rat liver  represent  mean  +/-  homogenate.  S.EM  (n=4)  (CE) in  WEBSTER, D. S.  CBZ/AcHz — i —  Control  INTERACTIONS  --©-• 0.115  mM  — 0 . 4 9 0  AcHz _  N CD  0.12  5  10  15  20 Time  16:  mM  AcHz  r  0  Figure  61  Influence  of acetylhydrazine  25  30  35  40  (min)  (AcHz) on  the conversion  of  carbamazepine (CBZ) t o carbamazepine-10,1 1 - e p o x i d e (CE) in the Data  S9  fraction of rat liver homogenate.  represent  mean +/-  S.EM  (n=4)  WEBSTER, D. S.  CBZ/Hz •  INTERACTIONS 0.115  Control  mM  —A—  Hz  _  62  0.490  mM  Hz  0.14 p  N CO  5  10  15  20  25  30  35  40  T i m e (min)  F  ,  g  u  r  e  1  7  :  Influence of hydrazine (CBZ)  (Hz) on the conversion o f carbamazepine  t o carbamazepine-10.1 1-epoxide (CE) in the S9  of rat liver homogenate.  Data  represent mean +/-  fraction  SE.M.  (n=4)  WEBSTER, D. S.  C B Z / I N A  63  I N T E R A C T I O N S  T i m e (min)  Figure  18:  Influence of isonicotinic carbamazepine  ( C B Z ) t o c a r b a m a z e p i n e - 1 0 . 1 1 - e p o x i d e (CE)  in the S 9 fraction Data  represent  a c i d (INA) on the conversion o f  o f rat liver  mean +/-  S E M  rK>mogenate. (n=4)  WEBSTER, D. S.  i n the r e l a t i v e is  not t r u e  has  the  course,  positions  of  the  points  at each time  i n the case of a c e t y l h y d r a z i n e ,  greatest  values  although  there  for are  the no  of measuremment.  where the c o n t r o l  metabolic  ratio  significant  64  over  sample  the  differences  This clearly  complete  between  time  specific  points. In the c a s e s of h y d r a z i n e the  line  ting  incubation  trials, the  representing  the  mixtures  t h e r e are  isonicotinic  is  Thus,  between  effort  doses,  to  differences there  it  achieve  another  type  clearly  distinct  metabolite.  is  In  acid  (Figure  from those  fact,  in  the  hydrazine  at 30 and at 40 m i n u t e s ,  a significant  difference  the metabolism of  a comparison of  of  analysis  the  was  while  in  observed  at  isonico-  carbamazepine.  whole  also  18),  represen-  would appear t h a t both h y d r a z i n e and  a c i d are capable of i n h i b i t i n g  In an  17) and i s o n i c o t i n i c  containing  trials,  the 20 minute p o i n t . tinic  control  significant acid  (Figure  spectrum  attempted.  of  A  points  nonlinear  estimation analysis  was performed u s i n g the S y s t a t  software package with  modelling  equation  being  represents  ratio  t  time.  and  is  the  estimates  were  obtained,  metabolic  ratio  attained  combined r a t e c o n s t a n t of rate  of  first be  exhaustion  or  where  b t  The  variables  with (i.e., several  essential  a  MR  a and b  representing  the  plateau  first  value)  order  components  a  saturfor  ating this  of v a r i a t i o n of  one  reaction.  of  its  functioning  type o f  and  first  experiment.  In c o n t r a s t ,  would  the  for not  one would expect  to  the  the  order  for  theoretical and  b  which  maximum  representing  rate processes in  metabolic  values  that  reaction  infers  mixture.  functions  a,  since  be  the  the  are to  level  limiting  see d i f f e r e n c e s  the see  of  factor in  the  a the The  d i s p l a y e d what appears  One would not expect  i n the value o b t a i n e d metabolites  are the  order p r o c e s s was chosen because the r e s u l t s  observed deal  of  MR=a(l-e ),  the  to  usual  a  great  isoniazid in value  the of  WEBSTER, D. S.  b,  since  if  any  inhibition  n e c e s s a r i l y be a l t e r e d . which the obtained numbers  t-tests using  are  showed s i g n i f i c a n c e  the  software  package  examined c l o s e l y ,  less  than the  That  is,  the  occurring  values  of  it  is  are  shown  obvious  approximations.  the  metabolic  group.  interactions but f o r  were  for  tested.  the a n a l y s i s  of  the a n a l y s i s y i e l d e d 0 . 1 0 . p . 0 . 0 5  Trial  of  reaction  a and b f o r  Tables the  those t e s t s  the  at  the  The  II  -  for  increase  If  the  package  was  a appeared  40 minute  in  estimates  IV.  software  The v a l u e s  will  time  i n the v a l u e  to  be  point. of  the  r a t i o t h a t occurs between 30 and 40 m i n u t e s .  d i f f e r e n c e s were observed o n l y  Table I I :  in  that  ratio  program seems to be i g n o r i n g  v a r i a n c e on each i n t e r a c t i o n  p.0.25,  rate  at one or more p o i n t s .  The v a l u e s of a and b f o r each i n d i v i d u a l  azid  the  Values were o b t a i n e d f o r  unable to make a p p r o p r i a t e  metabolic  is  65  By t h i s method of a n a l y s i s ,  this  group,  p,0.0005.  Control  analysis  the t e s t s  f o r both parameters.  E s t i m a t e s o f a and b f o r t e s t s with  Parameter  For  the  of  with  of  significant  the group i n which carbamazepine  For b,  run were t e s t e d by a n a l y s i s  a  isoniyielded  hydrazine,  F o r the t e s t s  with  isoniazid.  C o n c e n t r a t i o n of i s o n i a z i d 0.115 mM 0.490 mM  1  a b  0.041 -6.897  0.041 -6.903  0.037 -6.892  2  a b  0.052 -6.927  0.053 -6.919  0.046 -6.896  3  a b  0.043 -6.912  0.034 -6.888  0.032 -6.875  4  a b  0.042 -6.911  0.044 -6.911  0.044 -6.907  WEBSTER, D. S.  Table I I I :  E s t i m a t e s of a and b f o r t e s t s with  Trial  Parameter  66  hydrazine.  C o n c e n t r a t i o n of hydrazine 0.490 mM 0.115 mM  Control  1  a b  0.084 -7.027  0.056 -6.940  0.059 -6.955  2  a b  0.059 -6.948  0.049 -6.929  0.042 -6.903  3  a b  0.073 -6.987  0.047 -6.916  0.036 -6.899  4  a b  0.048 -6.924  0.038 -6.895  0.045 -6.927  Table  IV:  E s t i m a t e s of a and b f o r  Parameter  Trial  t e s t s with i s o n i c o t i n i c  acid.  C o n c e n t r a t i o n of i s o n i c o t i n i c a c i d 0.115 mM 0.490 mM  Control  1  a b  0.069 -6.970  0.049 -6.924  0.043 -6.923  2  a b  0.040 -6.915  0.036 -6.908  0.046 -6.926  3  a b  0.060 -6.953  0.043 -6.903  0.045 -6.914  §  a b  0.039 -6.980  0.035 -6.885  0.038 -6.896  isonicotinic and b, tests  acid,  the  respectively.  r e s u l t s of This  done w i t h each of  apparent  that  differences  over  the  developing.  is  the  a n a l y s i s gave  somewhat  unexpected.  these compounds  are  course  experiment  The  r e f l e c t e d i n the s t a t i s t i c a l  of  the  problem  p=0.25 and p.0.25  is  If  examined  that  the f i g u r e s  visually,  there  these  procedures presented thus  are  it dose  differences far.  for  for is  a  the quite  related are  not  WEBSTER, D. S.  If are  the  probability  somewhat  procedure  different.  p o i n t s of a d j a c e n t  In  lines  of  this  run  type  analysis  of  being d i f f e r e n t  done on the  curves  p-value  the  for  from the  comparison  employed,  analysis,  is  m u l t i p l i e d over the course of the l i n e .  is  0.5.  the  the  For example,  control  to  the  results  probability  of  These p r o b a b i l i t i e s if  carbamazepine/isoniazid of  67  the  this  can be  procedure were  interaction 0.490  two  study,  mM curve  would  the be  5 (0.5)  or  0.03,  since  the  points  of  the  control  line  of the 0.490 mM l i n e at 5, 15, 20, 30, and 40 minute T a b l e V:  P-values  from run a n a l y s i s of i n v i t r o  P-value Control vs 0.490 mM  Treatment  are  above  the  points  points.  interaction  studies.  for: Control vs 0.115 mM  0.115 mM vs 0.490 mM  Isoniazid  0.03  0.03  0.02  Acetylizoniazid  0.03  0.03  0.06  A c e t y l hydrazone  0.02  0.02  0.06  Hydrazine  0.02  0.02  0.13  0.02  0.02  0.13  presented i n t h i s  study would appear  Isonicotinic  2.4  acid  DISCUSSION:  The r e s u l t s confirmation  of the c l i n i c a l  carbamazepine  metabolism,  Block  and by  (1982),  suggest t h a t are  Valsalan  hydrazine,  metabolites  l i v e r microsome  of  as  observations reported and  Cooper  isonicotinic  isoniazid  fractions.  that  by  that  isoniazid  Wright (1982).  acid, inhibit  to p r o v i d e  and  is  able  co-workers  In a d d i t i o n ,  and p o s s i b l y  an j_n v i t r o to  inhibit  (1982), the  by  results  even a c e t y l hydrazine  carbamazepine  metabolism  by  S9  WEBSTER, D. S .  It  i s not p o s s i b l e  carbamazepine dependent  metabolism  nature  particularly  t o determine  o f the  since  at  this  and i s o n i a z i d  (Barbare,  1986).  action  i s taking  doing  binding  studies.  An i r r e v e r s i b l e  al., of  in a clinical  that  of certain  cytochrome  P  without  interaction  i s less  likely,  though,  seem t o r e t u r n t o normal  i s ceased  (Block,  s  capable  potent  inhibiting  observed i n the c u r r e n t series  o f experiments  preliminary of  studies  a more potent  capability  study.  was chosen  t i o n o f what i s o c c u r r i n g other  used  in this  studies  concentrations  where  study  were  close effect.  may  because  i n the  to zero-order,  resulted  kinetic  a 70  above  percent  was  not  used i n t h i s  the best  response i n  species.  of isoniazid  situation.  procedure  The  suggests  t h e high  end,  lack  that the  i n t h e systems with  when compared  the concentration  b u t not o u t o f range.  used i n some o t h e r  profile,  In f a c t ,  was u s e d ,  1981) t o 1.0 mM (van B o x t e l ,  have  i s a suicide  may have been t o o high and not a r e f l e c -  a similar towards  effects  in press).  described  i t provided  presence  i n the c l i n i c a l  o f carbamazepine  mM ( T y b r i n g , e t a l . , carbamazepine  isoniazid  done i n the absence o f any i n h i b i t i n g  difference  o f the  The carbamazepine c o n c e n t r a t i o n  chosen carbamazepine c o n c e n t r a t i o n  with  of isoniazid  within  1982; Wright, e t  of causing  decrease i n acetaminophen metabolism ( E p s t e i n , e t a l . , The  inter-  be made  subsequent o b s e r v a t i o n s  45Q'  situation  an i r r e v e r s i b l e  i s o n i a z i d on acetaminophen metabolism suggest t h a t  inhibitor  t o be impaired  cannot  administration  Despite t h i s observation,  1982).  reported  interaction,  conclusion  o f carbamazepine  isoniazid  but t h e c o n c e n t r a t i o n  are c o a d m i n i s t e r e d possible  of the i n h i b i t i o n o f  a competitive  has been  but a d e f i n i t e  s i n c e plasma c o n c e n t r a t i o n s a few days a f t e r  would suggest  I t i s also  place,  nature  i n time,  metabolism  when carbamazepine et a l . ,  point  inhibition  isoniazid  t h e exact  68  studies  et a l . , adopting  the i n h i b i t o r  The  ranged from 0.05  1977).  The  higher  a zero-order, or having  very  little  WEBSTER, D. S.  When  discussing  conversion  of  diol,  is  experiments,  carbamazepine  process that hydrolase,  these  is  able  the  to  to occur  present.  in  As  must  remain  aware  carbamazepine-10,ll-epoxide the  enzyme r e s p o n s i b l e  still  one  system.  for  well,  the  the  is  69  that  the  the  only  not  In the S9 p r e p a r a t i o n  conversion  enzymes  of  the  epoxide  epoxide  responsible  for  the e p o x i d e . a  balance  Thus, of  reaction  is had  In  responsible been  fact, for  allowed  p o s s i b l e t h a t the m e t a b o l i c  might  on  an  expect  factor  at  drugs,  provided  It  is  side  the  possible chain  part,  of  active that  to  activity isoniazid  of  compound,  same  If  namely  site,  the  isoniazid  long  structures  enzyme  the  enough,  from  epoxide  hydrazide  the  the an  If  quite is  drugs, group  from  the  inhibition relative  is  acid.  the  see  carbamoyl  of  are, If  in this  inhibitory  breakdown  of  Such an  a competitive  importance  would l i k e l y  site.  to  the  process.  enzyme.  simple  not  of  isoniazid,  the  one  recognition  metabolism  namely of  on  the  compounds,  important  one would expect  and i t s m e t a b o l i t e s  is  a competitive  and i s o n i c o t i n i c  r a t h e r than f o r the a c t i v e  two  for  is  compounds  result  it  s i n c e the r a t i o  responsible  was o c c u r r i n g ,  hydrazine  be  the two  the  that  to  of  inhibition  of  and the  about  of  to  processes.  structure  the mechanism of  postulations  f e a t u r e s of  the  positioning  influences  activity  for  enzymic  side chains  from the m e t a b o l i t e s  was o b s e r v e d .  latory  of  carbamazepine  r a t i o curve has to a r i s e the  proceed  mechanism of  the  for  of ring  site the  that  responsible  combination  the  aromatic  is  o r d e r with r e s p e c t  r a t i o may have d e c r e a s e d ,  examination  the  it  first  present,  the curve not q u i t e being e x p o n e n t i a l .  consequence of a balance of s e v e r a l Based  is  a p l a t e a u of the m e t a b o l i c  processes.  hydrolase that the  Each of these p r o c e s s e s  the  glucuronid-  a t i o n and f o r the c o n v e r s i o n of the epoxide to the a c r i d a n are a l s o but these are m i n o r .  to  the  action  process,  the  structural  hold true  f o r a regu-  WEBSTER, D. S.  The s l i g h t reasons.  effect  First,  d i s p l a y e d by a c e t y l hydrazine may be due to one of  the  structural  hydrazine may p l a y  a role,  the  of  effectiveness  lism.  is  conversion  being  has  not  reaction  may  apparent  inhibition  similarity  with the  reason  converted yet  occur.  is  to  been  at  p r e v e n t i o n of access  of  the  a c e t y l hydrazine  acetyl  inhibiting  that  a small  hydrazine  described,  Acetylisoniazid, of  between  presence  a c e t y l hydrazine  The second p o s s i b l e  hydrazine  it  is  possible  other  carbamazepine metabolism.  This  required  recognition  the  that  hand, is  sites  tested  evaluation for  their  of  trials  relative  ability  the hydrazine moiety the  the  is  to  hydrazine  compared, i t  is  obvious t h a t  lead  surmise  one t o  inhibit  likely  with  of  carbamazepine  isonicotinic  (Figures  activity.  primarily  responsible  correlate  well  isoniazid  and  ity,  with its  evidence  the  et  enzyme to produce the r e a c t i v e and  human  liver  microsomes  is  to  the  identity  studies  examining  In  studies  of  1975;  by  a c y l a t i n g agents et  al.,  a  et  the  in in vitro  are might  species do  not  effects  of  hepatoxic-  is  1976).  microsomal  If  responsible  toxic  al.,  1976).  that  16)  This  isoniazid-induced  Nelson,  the  compounds  and  is  that  no  group,  reveals  of  the  be the m e t a b o l i t e  oxidized  (Nelson,  the  o f carbamazepine metabolism,  of  shown to  al.,  a c e t y l hydrazine  regard  inhibition  results  has been  (Mitchell,  that  the  metabolites.  acetyl hydrazine  responsible  for  with  a  due to  effect.  f o r a g r e a t e r p r o p o r t i o n of the observed i n h i b i t o r y obtained,  such  acid.  15  isoniazid  results  moiety  a  the a c t i v i t y .  of  The  hydrazide  of  for  hydrazine exerted a greater  the  Such  by the a c e t y l  metabolism  acid  acetyl-  displayed  likely  each  to be most r e s p o n s i b l e  and  that  effectiveness  and  metabo-  preparation.  the  to the  on  the  of  two  decreasing  carbamazepine  proportion  in  but  group  a f a c t o r not i n f l u e n c i n g the a c t i o n s of i s o n i a z i d o r i s o n i c o t i n i c An  70  primarily There  cytochrome s t u d i e s with  Subsequent  is P ^ Q rat  results  WEBSTER, D. S.  showed  that  cysteine,  the  entire  thereby  would  and be  oxidation  Mitchell,  expected  inhibiting  to  be  such  as  of  the  of  ketene  as  the  information,  on  this  effective  metabolism,  due  ^ Q ' S  of  to  that  effect.  Thus,  it  would  its  as  a  result  of  a c e t y l hydrazine  microsomes  ability  to  species  for  mechanism least  the  described  It  structure  of  a  would be  of any t h a t are  acylated.  be  exclusion  The  cannot  the  lack  positioned  cysteines  present  acylation  at  It  be confirmed  is in  reasonable  the  such a s i t e  active would  of  exposed  if  this  in  is  some  or  cysteine  bound  lead  to  bound for  likely by  the at  carbamazepine metabolism  if  is  moeities,  of  or  postulate,  likely  But,  degree  The most  covalently  fact  regulatory  bind  responsible  the  case,  f o r carbamazepine e p o x i d a t i o n to  at  d i d not have a  enzyme  being  so as to a l t e r  of the enzyme r e s p o n s i b l e  elucidated.  from  tested  t h a t can be c o v a l e n t l y  reactivity.  protein  (Nelson,  covalently  carbamazepine metabolism would seem to be one such p r o t e i n . reason  agent  carbamazepine.  there  by  a c e t y l hydrazine  a c e t y l hydrazine that  trapped  metabolites  metabolize  appear  s p e c i f i c i t y w i t h r e s p e c t to the p r o t e i n  liver  the  is  acetylating  Based  i n the experiments being presented c u r r e n t l y , significant  reactive by  most  cytochrome  a c e t y l hydrazine  a c e t y l hydrazine  1976).  carbamazepine  proteins,  group  eliminating  formed d u r i n g the Hinson,  acetyl  71  though sites  the  that  of  since has y e t  there  the  are  enzyme,  inhibition  the  of  to no  since  carbamaze-  pine metabolism. The  results  obtained  s t u d i e s wherein a m e t a b o l i c carbamazepine  hepatic  o x i d a t i v e metabolism,  1984).  The  (Daniel obvious  and  this  other  to  are  consistent  drugs  1988), pursue  with  or competitiveness with  cyclic  such as e r y t h r o m y c i n  Netter,  route  study  interaction  between  imipramine  and  in  and in  (Barzaghi,  future  number  has been  moeities  cimetidine  the  a  observed  that  undergo  et a l . ,  1987),  (Grasela would  of  be  and to  Rocci,  continue  WEBSTER, D. S.  studying above. will of  the It  interactions  is  assist  hopeful  that  clinicians  drugs.  This  may  between  compounds  information  that  derived  i n making wise d e c i s i o n s  result  in  fewer  patients  e f f e c t s t h a t sometimes r e s u l t from drug  fit  from  criteria  these  about that  the  types  the  examine  tions.  tion  more This  that  isoniazid addition,  is  done,  thoroughly  observed,  is  competitive  if  it  over will  by  experience  the  adverse  interactions.  a wide  range  be p o s s i b l e  plotting  studies  of  it  is  carbamazepine  to examine  Michaelis-Menten  the and  necessary concentra-  kinetics  studies designed  the enzyme,  it  t u r e of the a c t i v e  i.e., or  the of  question a  suicide-type  are done with c h e m i c a l l y for  should site.  specific  types  be p o s s i b l e  of  to  of get  whether should  of  the be  inhibi-  inhibition  by  answered.  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