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The effect of carbamazepine on valproic acid metabolism Panesar, Sukhbinder Kaur 1987

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THE  E F F E C T OF  CARBAMAZEPINE  ON VALPROIC  ACID  METABOLISM  By SUKHBINDER  THESIS THE  SUBMITTED  KAUR  PANESAR  IN  PARTIAL  REQUIREMENTS  FOR THE  MASTER  OF  FULFILLMENT D E G R E E OF  SCIENCE  in THE  FACULTY  OF  GRADUATE  STUDIES  Division  of  Pharmaceutical  Chemistry  Faculty  of  Pharmaceutical  Sciences  We  accept to  THE  this  the  thesis  required  UNIVERSITY  OF  Sukhbinder  Kaur  conforming  standard  BRITISH  April ©  as  COLUMBIA  1987 Panesar,  1987  In presenting t h i s thesis i n p a r t i a l fulfilment of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t freely available for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission.  BeWMent of  Pharmaceutical Chemistry Faculty o f Pharmaceutical  The U n i v e r s i t y o f B r i t i s h 1956 Main Mall V a n c o u v e r , Canada V6T 1Y3  Date  April  30, 1987  Columbia  Sciences  ABSTRACT  Modifications to metabolites were and  the GCMS  attempted  derivatizing  with  reagents.  assay for valproic acid and 12 respect to internal standards Four  new  internal  standards,  octanoic acid and 2-methylglutaric acid for analysis of VPA and metabolites and hexanoic acid and di-n-butylacetic acid for the analysis of hexadeuterated VPA and metabolites were used. Two new tested as  d e r i v a t i z i n g reagents, alternatives to  (MSTFA) and sensitivity,  the reagent  tBDMS derivatives stability,  derivatives formed  MSTFA  and  MTBSTFA,  were  previously used.  TMS  were compared  and  with respect  to  time.  The  chromatographic  from MTBSTFA  were extremely stable a major  drawback was the formation of a diderivative of 3-keto VPA upon increased heating time and storage. Preliminary data in one  volunteer.  on the  metabolism of Dg-VPA was obtained  The substitution of six deuterium atoms for  six hydrogen atoms resulted in an isotope effect with decreased serum trough concentrations of 4-ene VPA and 2,4-diene VPA. Valproic  acid  coadministered in  and  microsomal  p o t e n t i a l l y toxic VPA were  are  frequently  e f f o r t s to optimize seizure c o n t r o l .  extensively metabolized hepatic  carbamazepine  while  enzyme  CBZ  i s known  system,  and  interaction.  to  thus,  Pharmacokinetic  VPA i s  induce this  the is a  parameters for  obtained before and after CBZ administration in f i v e ,  healthy male  volunteers.  Increased plasma  i i  clearance of  VPA  accompanied by l i f e , and This was  decreased plasma  AUC values  were observed  consistent with  hepatic microsomal  concentrations, after  the a b i l i t y  enzyme  systems  CBZ  half-  comedication.  of CBZ  in a  serum  to  induce  manner  similar  the to  phenobarbital. Serum trough and steady state concentrations and AUC values for  12  metabolites  administration.  were The  metabolites decreased  determined  AUC  values  after CBZ  before for the  after  potential hepatotoxin, administration  of  were increased  in  administration.  the  was not  CBZ.  diunsaturated metabolites,  The  of  the  CBZ  monounsaturated the AUC  The amount of 4-ene  increased in the serum amounts  2,3'-diene VPA  serum  after  administration while  values of the polar metabolites increased. VPA, a  and  of  the  and 2,4-diene  volunteers  after  two VPA, CBZ  The amount of 2-ene trans VPA in the serum was  s i g n i f i c a n t l y decreased  after CBZ  administration,  while  the  amount of 3-keto VPA did not increase. Urinary metabolic p r o f i l e s were determined i n d i v i d u a l l y and grouped in pathways for the five CBZ administration. VPA, and  volunteers before and after  Increased recoveries of 4-ene VPA, 4-keto  2-PSA after  CBZ administration  were consistent with  enhanced CJ-1 oxidation. Formation  clearance,  metabolized were the individual  metabolic  clearance,  and  fraction  determined for the metabolic pathways and for  metabolites.  CBZ  adminstration  increased formation clearances for a l l pathways.  resulted  in  The results caused a  obtained from  general induction  of  t h i s study VPA  metabolism  s p e c i f i c a l l y affect a particular pathway. the beta-oxidation cause a  metabolic  indicate  that  and  i n h i b i t beta-oxidation  d i d not  The effect of CBZ on  pathway i s not c l e a r l y understood. shift  CBZ  CBZ may  away from beta-oxidation, or actually to some  beta-oxidation may be involved.  iv  extent.  As well,  peroxisomal  TABLE OF CONTENTS  Page ABSTRACT  ii  L I S T OF TABLES  ix  L I S T OF FIGURES  xiii  L I S T OF ABBREVIATIONS I.  xx  INTRODUCTION A. B.  C.  Mechanism o f a c t i o n Pharmacokinetics  2 5  1. 2. 3.  5 6 7  2.  Drug 1. 2.  F.  8  Fatty 1.1. 1.2. 1.3.  a c i d metabolism Beta-oxidation Ketone b o d i e s Role of c a r n i t i n e i n f a t t y metabolism 1.4. Peroxisomal b e t a - o x i d a t i o n 1.5. c j - O x i d a t i o n Metabolism of v a l p r o i c a c i d 2.1. M e t a b o l i s m o f 4-ene VPA 2.2. M e t a b o l i t e s  Adverse e f f e c t s , metabolic and t o x i c i t i e s 1. 2. 3.  E.  A b s o r p t i o n and d i s t r i b u t i o n Plasma p r o t e i n b i n d i n g Elimination  Metabolism 1.  D.  1  8 9 10 acid 1 1 11 12 12 15 16  disturbances, 19  Adverse e f f e c t s Metabolic disturbances Toxicity 3.1. Hepatotoxicity  19 21 25 26  interactions  28  D r u g s w h i c h a l t e r VPA c o n c e n t r a t i o n s E f f e c t s o f VPA on o t h e r d r u g s  I n t e r a c t i o n between v a l p r o i c carbamazepine  v  29 30  a c i d and 32  1.  Enzyme induction 1.1. Metabolism 1.2. Types of reactions 1.3. Factors influencing enzyme induction 1.4. Properties of enzyme inducing agents 1.5. Types of enzyme inducing agents 1.5.1. Phenobarbital and 3-methylcholanthrene 1.6. C l i n i c a l implications 1.7. Markers of enzyme induction  32 33 33 34 34 35 36 36 37  OBJECTIVES  42  II.  44  EXPERIMENTAL A.  B.  C.  Reagents and materials  44  1. 2. 3.  Valproic acid and metabolites Internal standards Reagents  44 44 45  4.  Drugs  46  Drug interaction study  46  1.  46  Volunteer Details  Analysis  49  1.  49 49 49  2.  3. 4.  5. 6.  Valproic acid and metabolites 1.1. Stock solutions of internal standards 1.2. Preparation of urine and serum standards 1.3. Extraction and d e r i v a t i z a t i o n of standards and patient samples 1.4. Preparation of tBDMCS reagent with 5 % catalyst Carbamazepine and carbamazepine-10,11-epoxide in serum 2.1. Preparation of stock solutions 2.2. Extraction of serum samples for CBZ and CBZE Determination of urinary creatinine Instrumentation 4.1. Valproic acid and metabolites 4.2. Carbamazepine and carbamazepine - 10,11-epoxide S t a t i s t i c a l analysis Pharmacokinetic model development and calculations  vi  51 53 54 54 55 55 57 57 58 58 59  III.  RESULTS A.  Assay development  64  1. 2. 3. 4.  64 65 69 71 71  Modifications to the assay Analysis of deuterated samples Preparation of internal standards Derivatizing reagents 4.1. Comparison of TMS and tBDMS derivatives 4.2. Comparison of tBDMCS reagent and MTBSTFA reagent  75  B.  Analysis of serum and urine samples after administration of deuterated VPA  78  C.  Analysis of carbamazepine and carbamazepine10,11-epoxide in serum  81  Interaction between valproic acid and carbamazepine  85  D.  1. 2. 3. 4. 5.  IV.  64  Analysis of serum samples Analysis of urine samples Analysis of serum samples Analysis of urine samples Pharmacokinetic analysis 5.1. Pathway analysis 5.2. Metabolite analysis  for for for for  VPA VPA VPA metabolites VPA metabolites  DISCUSSION A.  B.  85 97 97 141 161 161 164 170  GCMS analysis of valproic acid and metabolites  170  1.  170 170 172  Assay 1.1. Internal standards 1.2. Choice of d e r i v a t i z i n g reagent 1.3. Stable isotopes in pharmacokinetic studies  Effect of carbamazepine on valproic acid metabolism 1. 2.  Inducing properties of carbamazepine Volunteer data 2.1. CBZ and CBZE concentrations in serum 2.2. VPA data 2.2.1. Protocol 2.2.2. Serum VPA data 2.2.3. Serum metabolite data 2.2.3.1. Metabolite serum concentrations 2.-2.3.2. Metabolite AUC values  vii  173 176 176 179 179 181 181 182 186 186 190  2.2.4. Urinary data 2.2.4.1. Recovery expressed as a percent of dose 2.2.4.2. Recovery expressed as a percent of t o t a l excreted 2.2.5. Effect of CBZ on VPA metabolism 2.2.6. Metabolite pathways 2.2.7. Pharmacokinetic analysis 2.2.7.1. Pathway analysis 2.2.7.2. Individual metabolites 2.2.8. C l i n i c a l significance of VPA and CBZ interaction  191 191 194 194 196 197 197 199 201  SUMMARY AND CONCLUSIONS  203  REFERENCES  207  APPENDIX  224  viii  LIST OF TABLES  Serum trough concentrations (mg/L) for VPA and metabolites after administration of VPA and DgVPA following 2 weeks of carbamazepine therapy for FS. Serum AUC values (mg.h/L) for VPA and metabolites obtained after VPA and Dg-VPA administration following 2 weeks of carbamazepine therapy for FS. Amount (jumol) of VPA and metabolites recovered in the urine over 12 h after VPA and Dg-VPA administration following 2 weeks of carbamazepine therapy for FS. Serum CBZ concentrations (iug/mL) in healthy volunteers after 7 and 14 days of CBZ administration. A t o t a l daily dose of 200 mg was taken for the f i r s t week and 300 mg for the second week. Serum samples are C i (prior to morning dose), 3 h, and 5 h post close. m  n  Serum CBZE concentrations (uq/mL) in healthy volunteers after 7 and 14 days of CBZ administration. A t o t a l daily dose of 200 mg was taken for the f i r s t week and 300 mg for the second week. Serum samples are C i (prior to morning dose), 3 h, and 5 h post dose. m  n  Percent r a t i o of serum CBZE to serum CBZ concentrations in healthy volunteers after 7 and 14 days of CBZ administration. A t o t a l d a i l y dose of 200 mg was taken for the f i r s t week and 300 mg for the second week. Serum samples are C ^ (prior to morning dose) 3h, and 5 h post dose. m  n  Valproic acid kinetic parameters for f i v e healthy volunteers before and after administration of carbamazepine. ix  8.  9.  10.  11.  12.  13.  14.  15.  Mean serum valproic acid and metabolites trough concentrations (mg/L) before and after administration of carbamazepine in five volunteers. Numbers in parentheses represent range.  129  Mean serum AUC (mg.h/L) for VPA and metabolites over 12 h before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  130  Sum of serum AUC (mg.h/L) for polar metabolites of valproic acid over 12 h for the five healthy volunteers before and after administration of carbamazepine.  134  Sum of serum AUC (mg.h/L) for unsaturated metabolites of valproic acid over 12 h for the five healthy volunteers before and after administration of carbamazepine.  134  Mean serum AUC (mg.h/L) over 12 h for valproic acid and metabolites expressed as pathways before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  135  Mean 'average serum steady state concentrations' (mg/L) for valproic acid and metabolites before and after administration of carbamazepine. Numbers in parentheses represent range (n=5).  142  Mean 'average steady state serum concentrations' (mg/L) of valproic acid and metabolites expressed as pathways before and after administration of carbamazepine. Numbers in parentheses represent range (n=5).  143  Mean valproic acid and metabolites (nxmol) recovered in urine over 12 h before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  144  x  16.  17.  18.  19.  20.  21.  22.  23.  Mean valproic acid and metabolites (Mmol) recovered in the urine over 12 h expressed as pathways before and after carbamazepine administration. Numbers in parentheses represent range (n=5). Sum of polar metabolites of VPA (/nmol) recovered in the urine over 12 h for the f i v e healthy volunteers before and after administration of carbamazepine.  145  149  Sum of unsaturated metabolites of VPA (Mmol) recovered in the urine over 12 h for the five healthy volunteers before and after administration of carbamazepine.  149  Mean valproic acid and metabolites recovered in the urine over 12 h as percent of VPA dose before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  150  Percent of valproic acid dose recovered in the urine as VPA and metabolites over 12 h for the five healthy volunteers before and after carbamazepine administration.  151  Mean valproic acid and metabolites (umolar basis) recovered over 12 h expressed as percent of t o t a l recovered before and after carbamazepine administration in f i v e volunteers. Numbers in parentheses represent range.  155  Mean valproic acid and metabolites (Mmolar basis) recovered over 12 h in the urine expressed as percent of VPA recovered before and after carbamazepine administration in five volunteers. Numbers in parentheses represent range.  156  Mean formation clearances ( C l f , L/h) for pathways 1 - 6 before and after CBZ administration for f i v e healthy volunteers.  xi  162  24.  25.  26.  27.  28.  29.  30.  31.  32.  Mean metabolite clearances ( C l , L/h) for pathways before and after CBZ administration for five healthy volunteers.  163  Mean fraction metabolized ( f ) by each pathway before and after CBZ administration for five volunteers.  165  m  m  Mean metabolite formation clearances ( C l f , L/h) for the VPA metabolites before and after CBZ administration. Numbers in parentheses represent range (n=5). Mean metabolite metabolite clearances ( C l ^ , L/h) for the VPA metabolites before and after CBZ administration. Numbers in parentheses represent range (n=5). Mean fraction ( f ) of metabolite metabolized before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  166  168  m  Comparison of serum VPA and metabolite concentrations (nq/mL) in volunteers with patient data. Numbers in parentheses represent range.  169  188  Comparison of mean serum steady state VPA and metabolite concentrations (uq/mh) from two volunteer studies. Numbers in parentheses represent range.  189  Comparison of urinary recovery of VPA and metabolites expressed as a percent of dose in two volunteer studies. Numbers in parentheses represent range.  193  Comparison of recovery of VPA and metabolites as a percent of the t o t a l amount recovered. Numbers in parentheses represent range.  xii  195  LIST OF FIGURES Page 1.  Human metabolism of valproic acid.  14  2.  Extraction and d e r i v a t i z a t i o n scheme for valproic acid and metabolites from urine and serum.  52  3.  Extraction scheme for carbamazepine and carbamazepine-10,11-epoxide from serum.  56  4.  Pharmacokinetic model applied carbamazepine study.  60  5.  Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample.  6.  C a l i b r a t i o n curve for 2-PSA in serum using 2methylglutaric acid as the internal standard.  67  7.  C a l i b r a t i o n curve for 2-PGA in serum using 2methylglutaric acid as the internal standard.  68  8.  Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample.  70  9.  Selected ion chromatograms of TMS derivatives of valproic acid and metabolites from a patient urine sample.  73  Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample.  74  10.  xi i i  to valproic acid-  66  11.  Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample.  76  12.  Peak area r a t i o of tBDMS derivatives of 2-ene trans VPA, 5-OH VPA, 4-keto VPA, 2-ene c i s VPA, and 2,4-diene VPA from MTBSTFA reagent versus heating time.  77  13.  Change in peak area r a t i o of 3-keto VPA monoand diderivative to D -2ene c i s VPA with increased heating time.  79  14.  Change in peak area r a t i o of 3-keto VPA monoand diderivative to D3~2-ene c i s VPA with storage.  80  15.  Liquid chromatogram of 1O-methoxycarbamazepine from a spiked serum sample.  3  89  16.  Liquid chromatogram of 10methoxycarbamazepine, carbamazepine and carbamazepine-10,11-epoxide from a spiked serum sample.  90  17.  Liquid chromatogram of a patient serum sample 3 h post dose, after one week of carbamazepine 200 mg d a i l y .  91  18.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for BA before CBZ and after CBZ administration.  92  19.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for FS before CBZ and after CBZ administration.  93  20.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for MS before CBZ and after CBZ administration.  94  xiv  21.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for RM before CBZ and after CBZ administration.  95  22.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for WT before CBZ and after CBZ administration.  96  23.  Plot of VPA h a l f - l i f e ( t , / , h) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  99  24.  Plot of VPA clearance ( C l , L/h) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  100  25.  Plot of VPA elimination rate constant (Ke, h ) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  101  26.  Plot of VPA AUC (mg.h/L) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  102  27.  Plot of VPA volume of d i s t r i b u t i o n (Vd, L/kg) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  103  28.  Representative semilogarithmic plot of 4-OH VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  104  29.  Representative semilogarithmic plot of 4-ene VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  105  30.  Representative semilogarithmic plot of 3-ene VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  106  2  p  - 1  xv  31.  Representative semilogarithmic plot of 2-ene c i s VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  107  32.  Representative semilogarithmic plot of 2-ene trans VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  108  33.  Representative semilogarithmic plot of 3-keto VPA concentration (mg/L) versus time before CBZ and a f t e r CBZ administration.  109  34.  Representative semilogarithmic plot of 4-keto VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  110  35.  Representative semilogarithmic plot of 5-OH VPA concentration (mg/L) versus time before CBZ and a f t e r CBZ administration.  11 1  36.  Representative semilogarithmic plot of 2-PSA concentration (mg/L) versus time before CBZ and after CBZ administration.  112  37.  Representative semilogarithmic plot of 2-PGA concentration (mg/L) versus time before CBZ and after CBZ administration.  113  38.  Representative semilogarithmic plot of 2,3'diene VPA concentration (mg/L) versus time before CBZ and after CBZ administration.  114  39.  Representative semilogarithmic plot of 2,4diene VPA concentration (mg/L) versus time before CBZ and a f t e r CBZ administration.  115  40.  Plot of 4-OH VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  116  xvi  41.  Plot of 4-ene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  117  42.  Plot of 3-ene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  118  43.  Plot of 2-ene c i s VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) administration in volunteers (n=5).  119  44.  Plot of 2-ene trans VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) administration in volunteers (n=5).  120  45.  Plot of 3-keto VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) administration in volunteers (n=5).  121  46.  Plot of 4-keto VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  122  47.  Plot of 5-OH VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  123  48.  Plot of 2-PSA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  124  49.  Plot of 2-PGA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  125  50.  Plot of 2,3'-diene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  126  xvi i  51.  Plot of 2,4-diene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  127  52.  Histograms of AUC (mg.h/L)values of polar metabolites before CBZ and after CBZ administration in volunteers (n=5).  131  53.  Histograms of AUC (mg.h/L)values of unsaturated metabolites before CBZ and after CBZ administration in volunteers (n=5).  132  54.  Pathway 1 (beta-oxidation of valproic a c i d ) .  136  55.  Pathway 2 (dehydrogenation  of valproic a c i d ) .  137  56.  Pathway 3 (dehydrogenation  of valproic a c i d ) .  138  57.  Pathway 4 (CJ-1 oxidation of valproic a c i d ) .  139  58.  Pathway 5 (a>-oxidation of valproic a c i d ) .  140  59.  Histograms of mean recovery (Mmol) of unsaturated metabolites before CBZ and after CBZ administration in volunteers (n=5).  146  60.  Histograms of mean recovery (Mmol) of polar metabolites before CBZ and after CBZ administration in volunteers (n=5).  147  61.  Histograms of mean recovery (Mmol) of unsaturated metabolites expressed as percent of dose before CBZ and after CBZ administration in volunteers (n=5).  152  62.  Histograms of mean recovery (Mmol) of polar metabolites expressed as percent of dose before CBZ and after CBZ administration in volunteers (n=5).  153  xvi i i  63.  Histograms of mean unsaturated metabolites recovered in the urine expressed as a percent of the t o t a l amount recovered before CBZ and after CBZ administration in five volunteers.  157  64.  Histograms of mean polar metabolites recovered in the urine expressed as a percent of the t o t a l amount recovered before CBZ and after CBZ administration in five volunteers.  158  65.  Histograms of mean unsaturated metabolites recovered in the urine expressed as a percent of VPA recovered before CBZ and after CBZ administration in five volunteers.  66.  Histograms of mean polar metabolites recovered in the urine expressed as a percent of VPA recovered before CBZ and after CBZ administration in five volunteers.  xix  159  160  LIST OF ABBREVIATIONS  2,3'-Diene VPA  2-[(E)-1'-propenyl]-(E)-2-pentenoic acid  2,4-Diene VPA  2-Propyl-(E)-2,4-pentadienoic acid  2-Ene VPA  2-Propyl-2-pentenoic acid  2-PGA  2-Propylglutaric acid  2- PSA  2-Propylsuccinic acid  3- Ene VPA  2-Propyl-3-pentenoic acid  3-Keto VPA  2-Propyl-3-oxopentanoic acid  3- OH VPA  2-Propyl-3-hydroxypentanoic  4- Ene VPA  2-Propyl-4-pentenoic acid  4-Keto VPA  2-Propyl-4-oxopentanoic acid  4- OH VPA  2-Propyl-4-hydroxypentanoic  acid  5- OH VPA  2-Propyl-5-hydroxypentanoic  acid  [ H ]-2-Ene VPA  [3,5,5, H]-3-heptene-4-carboxylic ac id  [ H ]-VPA  [ Hg]-Valproic acid  AUC  Area under the serum concentration versus  2  3  2  6  acid  2  2  time curve CBZ  Carbamazepine  CBZE  Carbamazepine-10,11-epoxide  ci  Plasma clearance  p  D -2-Ene VPA  [3,5,5, H]-3~heptene-4-carboxylie  D -VPA  [ Hg]-Valproic acid  Degrees °  Degrees Celsius  DMAP  4-Dimethylaminopyridine  DNBA  Di-/z-butylacetic  3  6  2  2  xx  acid  acid  LIST OF ABBREVIATIONS (CONT'D)  GCMS  Gas chromatography-mass spectrometry  GLC  Gas l i q u i d chromatography  HA  Hexanoic acid  HPLC  High performance l i q u i d chromatography  Ke  Elimination rate constant  MCBZ  1O-Methoxycarbamazepine  MGA  2-Methylglutaric acid  MSTFA  N-methyl-N-trimethylsilyltrifluoroacetamide  MTBSTFA  N-tert-butyldimethylsilyl-N-methyltr i fluoroacetamide  OA  Octanoic acid  SD  Standard deviation  SIM  Selected ion monitoring  ti/  2  tBDMCS tBDMS  Half-life Tertiarybutyldimethylsilylchlorosilyl Tertiarybutyldimethylsilyl  TMS  Trimethylsilyl  Vd  Volume of d i s t r i b u t i o n  VPA  Valproic acid (2-propylpentanoic  xxi  acid)  ACKNOWLEDGEMENT  The  author  would  like  to acknowledge  provided by Dr. F. S. Abbott. Dr. Andrew t h i s study,  Acheampong for Dr. James  The author i s deeply indebted to  the synthesis  Orr for  modelling, Dr. Kevin F a r r e l l volunteer study, computer help. following people P h y l l i s Abbott, Chan, Ms. Ms. Barbara  the supervision  of compounds used in  assistance in pharmacokinetic  for assistance and advice in the  and Mr. Roland Burton The author also  for his technical and  wishes  to acknowledge the  for their contributions to t h i s project: Mr. Bruce  A l l e n , Mr.  Shawn Black,  Ms.  Ms. Grace  Leanne Embree, Ms. Barbara Fraser, Mr. Rajesh Mahey, McErlane, Ms.  Radana Vaughn,  and Mr. Matthew Wright.  XX >  I  Ms. Janice Woodley,  DEDICATION  To my parents for their support.  I.  INTRODUCTION  The anticonvulsant  a c t i v i t y of  serendipitously discovered  by Meunier  almost 100  years after  u t i l i z e as  a solvent (Burton,  clinic trial  on  valproic  acid  (VPA) was  and coworkers  in 1963,  Burton had synthesized the compound to 1881).  The r e s u l t s of the f i r s t  the effectiveness  of VPA's  anticonvulsant  a c t i v i t y were reported in 1964 by Carraz and coworkers. VPA  has  been available  anticonvulsant agent  since 1978.  treatment of a variety myoclonic,  tonic-clonic,  convulsive  (Rimmer  e f f e c t i v e as convulsions  in North  It  of seizure  types  infantile,  and  is effective  Richens,  including  partial,  1985).  VPA  and  in the absence, photo-  i s equally  diazepam in the prophylactic treatment of f e b r i l e  (Lee et a l . ,  prophylaxis of  1986).,  It i s also e f f e c t i v e in the  post-trauma epilepsy  (Rimmer and Richens, 1985). in a  America for use as an  variety of  and in status e p i l e p t i c u s  VPA has been employed successfully  other conditions  including acute  mania and  alcohol withdrawal. Unlike other  a n t i e p i l e p t i c drugs, VPA i s a branched, short  chain fatty acid as i l l u s t r a t e d below.  CH -CH -CH 3  2  2  \ CH-COOH  / CH ~CH2 Valproic acid 3  1  VPA  i s extensively metabolized in the l i v e r and a number of  metabolites have Some of while  been i d e n t i f i e d  the metabolites  others  may  be  (Acheampong et a l . , 1983).  also possess responsible  anticonvulsant a c t i v i t y  for the adverse  effects  associated with VPA usage (Granneman et a l . , 1984a). Since anticonvulsant  therapy in most instances  i s a long  term treatment, the potential for interactions with other drugs i s greatly with  increased.  other  The majority of these interactions are  anticonvulsant  agents  since  polytherapy  is  frequently i n i t i a t e d in an e f f o r t to optimize seizure control. The interaction  between VPA and carbamazepine (CBZ) i s the  subject of t h i s investigation as these two drugs are frequently coadministered Because CBZ  in an  i s capable  attempt  to maximize  of inducing  seizure  the metabolism  compounds in addition to i t s own, t h i s combination  control. of  other  of drugs i s  a very important one to investigate.  A.  MECHANISM OF ACTION  Although the mechanism understood,  i t s anticonvulsant  mediated through VPA may brain  of  effects on  (Johnston D.,  competitive 1984; Wilder  of VPA  activity  increasing GABA  inhibition and Bruni,  2  i s not well  i s thought  gamma-aminobutyric  exert i t s effects by through  action  acid  to be (GABA).  levels in the  of GABA-transaminase 1982; Glaser  et  al.,  1980; Alkadhi and Banks, 1984; Perlman and Goldstein, 1984), by enhancing the neuronal responsiveness membrane effect  (Johnston,  between increases has been  1984).  to GABA, or by a direct A  positive  correlation  in GABA  levels and  anticonvulsant a c t i v i t y  observed (Wilder  and Bruni,  1982).  VPA  also  may  interfere with the axonal and g l i a l reuptake of GABA. VPA may  act on  enhance GABA limits  a chloride  ionophore or a related s i t e to  mediated i n h i b i t i o n  sustained  repetitive  firing  responsiveness (MacDonald et a l . , The  major  locus  of  suprahypophyseal, although  in mice  pentylenetetrazole (Wilder  also  altering  GABA  of  VPA  appears  to be  there i s the p o s s i b i l i t y of direct 1984).  provide protection,  seizures induced  without  VPA  1985).  action  p i t u i t a r y action (Jones et a l . , VPA w i l l  (Meldrum, 1986).  by  at the  picrotoxin,  and Bruni,  same  dose,  against  bicucculline,  1982).  and  Seizure control  has been maintained for up to two weeks after withdrawal of the drug in a chronically infused model (Glaser et a l . , One hypothesis same membrane 1984).  i s that VPA and ethanol may act through the  disordering mechanism  After therapeutic  s t r u c t u r a l changes  1980).  in the  (Perlman  and  Goldstein,  concentrations in rats, VPA causes internal mitochondrial  membrane by  a l t e r i n g the conformation of the membrane proteins (Rumbach et al.,  1986).  At  low doses,  membranes in frog s c i a t i c Decreased sodium  VPA has  a d i r e c t effect on nerve  nerve (Van Dougen et  and potassium  3  a l . , 1986).  conductances accompanied  by a  reduced e x c i t a b i l i t y  have also  been observed with VPA in this  p a r t i c u l a r model. VPA acts  i n d i r e c t l y as  (GABA-T) at  doses as  low as  administration (Loscher, GABA-T i n h i b i t o r s  an i n h i b i t o r 125 mg/kg  1981a).  with respect  of GABA-transaminase in  mice  after  i.p.  VPA i s superior to s p e c i f i c to anticonvulsant  potency  at  doses of 170 mg/kg in mice (Loscher, 1981b).  The metabolite 2-  ene  inhibiting  VPA  i s more  potent  than  VPA  in  aminotransferase in v i t r o (Nau and Loscher, 1984). metabolites, 3-OH the enzyme  nerve endings VPA, and 4-OH  The hydroxy  VPA and 4-OH VPA, also s i g n i f i c a n t l y  ijri v i t r o .  GABA-  inhibit  The enzyme i s also inhibited in vivo in  from mouse  brain by  2-ene VPA, 3-ene VPA,  3-OH  VPA.  Other hypotheses for the mechanism of action of VPA include 1) the  formation of a highly reactive aldehyde intermediate of  biogenic amine  metabolism  a c t i v i t y (Javors  and Erwin,  beta-oxidation pathway metabolic acidosis. the brain  which  may  possess  1980), and  anticonvulsant  2) stimulation of the  by VPA which results in a s h i f t towards The  and result  ketone bodies formed are u t i l i z e d by  in  a  greater  tolerance  to  transient  stimulation (Schreiber, 1981). VPA  decreases  diaphragm and  cat gastrocnemicis  manner (Mansuri due to  indirectly  et a l . ,  a neuromuscular  1984).  evoked muscle  contractions in  dose  related  This effect i s thought to be  blockade caused  by VPA.  have an e f f e c t on d i r e c t l y evoked responses.  4  a  of rat  VPA did not  B.  PHARMACOKINETICS  1.  Absorption and d i s t r i b u t i o n After oral administration in man, VPA i s rapidly and almost  completely absorbed  from the gastrointestinal tract (Bruni and  Wilder, 1979; Delgado et a l . , 1983; Pinder et a l . , 1977). plasma l e v e l s in  are attained  absorption  may  be  concommitantly (Bruni levels of  VPA are  within one to four hours. observed  and Wilder,  when  food  1979).  is  Peak  A delay ingested  Plasma therapeutic  50 - 100 ug/mL (Bruni and  in the range of  Wilder, 1979; Delgado et a l . , 1983; Pinder et a l . , 1977). A sustained yielded a  more prolonged  when compared 1984a).  release preparation  dosage form.  The  b i o a v a i l a b i l i t y of form.  The area  ingestion of be almost dosage  pharmacokinetic an enteric  absorption of  tablet)  absorption rate in dogs  dosage  form  (Bialer  et  al.,  levels were also obtained with this  sustained release 0.84 with  food.  preparation had  respect to  the regular  an oral dosage  Enteric coated tablets have been found to  bioavailable when in man  (Hoffman  compared to et  the  a l . , 1986).  standard Similar  parameters have been reported for a regular and  coated dosage  et a l . , 1984).  (matrix  under the blood curve was not effected by the  100 %  forms  and uniform  to the regular  Sustained plasma  of VPA  form in e p i l e p t i c patients (Albright  The only difference noted was a lag time in the the enteric  coated form.  5  The b i o a v a i l a b i l i t i e s  of a  1 g standard tablet, 1 g enteric coated tablet, and a 0 . 8  g gelatin  capsule were  not  significantly  different  in man  in the body,  and i s  (Bialer et a l . , 1984b). VPA undergoes  rapid  distribution  detectable in the brain et a l . , - 95  1983).  %) and  within a few minutes (Delgado-Escueta  VPA i s extensively bound to plasma albumin (85  thus has  a small apparent volume of d i s t r i b u t i o n  (Vd) of 0.15 - 0.40 L/kg which corresponds to the e x t r a c e l l u l a r space.  2.  Plasma protein binding Albumin i s the only serum protein to s i g n i f i c a n t l y bind VPA  (Kober et  a l . , 1980).  VPA  binds at two different s i t e s , the  diazepam (Ka = 3.1 x 10 M ) 4  s i t e s , on  _1  and warfarin (Ka = 1.7 x 10  the albumin molecule in human serum.  M" )  4  1  In dog, VPA i s  78.5 % plasma protein bound, in rat 63.4 %, and in mouse 11.9 % (Loscher, 1978).  In  dog, VPA  binding  i s independent  of  concentration in the 5 - 7 0 Mg/mL range but decreases at higher concentrations. doses the 1985).  The binding  free fraction  of VPA  may be  i s saturable and at high  as high  as 50  %  (Chadwick,  However, serum VPA concentrations of 500 to 700 nM are  required to saturate the binding s i t e s on the albumin molecules (Holtzman, 1983).  The  degree of  VPA binding  i s affected by  pregnancy, old age, uremia, hepatic disease and hypoalbuminemic states (Levy affected by  and Moreland,  1984).  diurnal variations  6  The free fraction i s also  in plasma  free  fatty  acids  (Perucca, 1984).  VPA  and consequently,  the free fraction increases when drugs which  are displacing of  can be displaced from i t s binding sites  agents are coadministered.  f a t emulsion  (Intralipid ) R  to rhesus  The administration monkeys lead  to an  elevation in free VPA levels (Kutt, 1984).  3.  Elimination VPA  i s rapidly eliminated  h a l f - l i f e of  12 -  plasma h a l f - l i v e s  15 h of 8  polytherapy are observed rats, after  from the body, with  in healthy  volunteers.  a  plasma  In patients,  - 15 h in monotherapy, and 6 - 10 h in (Levy and Morland, 1984).  In dogs and  intravenous administration, VPA has a h a l f - l i f e of  1.7 and 4.6 h, respectively (Loscher, V978). VPA  elimination i s decreased  (Gram and  in neonates and in the elderly  Bentsen, 1985; Nau e t . a l . , 1981).  In the elderly,  free levels of VPA are increased with an accompanying reduction in i n t r i n s i c  clearance  possibly due  to decreased  metabolizing  capacity  clearance of  free drug.  varies from 1982b; Nau al.,  1981).  l i f e and al.,  10 -  70 h  (Perucca  et a l . , 1984).  protein binding  which The  results  or decreased  in decreased  h a l f - l i f e of  (Nau et  This i s  VPA  drug  hepatic  i n neonates  a l . , 1981; Nau et a l . , 1982a,  et a l . , 1984) with a mean h a l f - l i f e of 47 h (Nau et In guinea pigs under 10 days of age, longer half-  higher clearance  values for VPA are observed  1985).  7  (Yu et  In a  24 day  clearance  (Cl)  old neonate, the h a l f - l i f e of VPA was  (Irvine-Meek et of age,  0.18  mL/min/kg,  a l . , 1982).  the h a l f - l i f e  increased to 0.53  and  Vd  was  was  17.2  0.28  h, L/kg  In the same patient at six months  of VPA  decreased to  7.5  h  and the Cl  mL/min/kg without a s i g n i f i c a n t change in Vd.  Hepatic enzyme maturation and/or polytherapy  may  be  responsible  for these changes.  C.  METABOLISM  A brief VPA  may  review of fatty acid metabolism is presented since  share some  common metabolic  pathways with  the fatty  ac ids.  1.  Fatty acid metabolism Fatty acids  a l k y l side  consist of  chain and  (Devlin, 1986). formed into  a terminal  have the  Fatty acids may  carboxyl group  and  an  basic formula, CH3-(CH2) COOH -  n  be stored as t r i a c y l g l y c e r o l s ,  complex l i p i d s which are u t i l i z e d in the  synthesis  of c e l l structures, or used in the t r i c a r b o x y l i c acid cycle. Fatty acids beta-oxidation, from the  are mainly metabolized via beta-oxidation. 2  carbon fragments  carboxyl terminal  are  sequentially  after dehydrogenation,  oxidation, and t h i o l y s i s (Devlin, 1986;  8  In  removed  hydration,  Stryer, 1981).  1.1.  Beta-oxidation The  first  step  activation of coenzyme A  1986).  the fatty  (CoA),  reticulum or  which  so a  across the membrane. the activated  occurs  either  mitochondrial  the  for transportation  across the mitochondrial membrane. the hydroxyl  from the sulphur atom  group on the  of CoA on the outer  by c a r n i t i n e palmitoyl transferase I.  mitochondrial  transferred from  (Devlin,  Carnitine i s responsible for transporting  the membrane  inner  membrane  membrane i s impermeable to CoA and  group i s transferred to  surface of  i s the  in the endoplasmic  c a r r i e r i s necessary  acyl groups  carnitine molecule  cycle  acid by the formation of a fatty acyl  The mitochondrial  The acyl  the beta-oxidation  in the outer  i t s derivatives,  At  in  membrane,  c a r n i t i n e back  the acyl  group i s  to CoA by c a r n i t i n e palmitoyl  transferase I I . Inside oxidized by 1986).  the mitochondrion,  derivatives  may  be  one of a group of acyl CoA dehydrogenases (Devlin,  These enzymes are s p e c i f i c for a c e r t a i n chain length;  palmitoyl CoA  dehydrogenase for medium and  acids, while  the other  three enzymes,  butyryl dehydrogenases, acids.  the CoA  The function  hydrogen atoms between the  are s p e c i f i c of these  to form  second and  atoms are accepted by  an enoyl  octanoyl for shorter  CoA,  fatty and 2  chain fatty  dehydrogenases i s to remove 2 CoA with a trans double bond  t h i r d carbon f l a v i n adenine  9  long chain  atoms.  The 2  hydrogen  dinucleotide (FAD) and  ultimately,  2  electrons  are  channelled  into  the  electron  transport system. The alpha,beta-unsaturated water, a  reaction catalyzed  beta-hydroxyacyl CoA. beta-hydroxyacyl CoA is  then  further  ketothiolase. carbon to  by enoyl  accepts a molecule of CoA hydrase  L-beta-hydroxyacyl dehydrogenase to  oxidized  CoA  acyl CoA  in  CoA  is  to form Loxidized by  beta-ketoacyl CoA which  the  beta-position  by  beta-  i s inserted and cleavage occurs at the beta-  y i e l d acetyl  CoA and  a saturated acyl CoA with two  fewer carbons than the o r i g i n a l substrate. The steps  described above  are repeated  until a  4 carbon  butyryl CoA  remains as the intermediate.  oxidized to  y i e l d acetoacetyl CoA and subsequently 2 molecules  of acetyl  1.2.  Butyryl CoA  i s beta-  CoA.  Ketone bodies Ketone bodies  reduction  are composed  product  Acetoacetate  beta-hydroxybutyric  undergoes  decarboxylation  to  a  slow,  acetone  hydroxybutyrate depending ratio.  of acetoacetic  is  and  (Devlin,  spontaneous,  or  on the  acid  acid  its  1986).  nonenzymatic  reduced  to  beta-  intramitochondrial NADH/NAD+  Under normal conditions the serum concentrations of the  two constituents concentrations may ketoacidosis, the  are less than 0.2 mM. be as  high as  concentrations of  10  3 -  During starvation, the 5  mM.  ketone bodies  In  diabetic may  be  as  high as 20 mM. for  Ketone bodies replace glucose as the major fuel  respiration for the CNS  1.3.  during prolonged starvation.  Role of c a r n i t i n e in fatty acid metabolism Carnitine i s  required for the transport of activated fatty  acids of chain length 12 to 18 carbons across the mitochondrial membrane.  However,  short  diffuse across  the membrane  derivatives in  the matrix  i.e.  fatty  acids  and become  can  directly  activated to  compartment of  the  the  CoA  mitochondrion,  the oxidation of short chain fatty acids i s independent of  carnitine.  Deficiency of  cramps which fat  chain  c a r n i t i n e leads  to  aching  muscle  are precipitated by fasting, exercise, or a high  d i e t . These are a l l instances where fatty acid oxidation i s  the major energy-yielding  1.4.  source.  Peroxisomal beta-oxidation Beta-oxidation of fatty acids was  mitochondrial event, peroxisomes are 1986). present  but recent  capable of  Peroxisomes  are  initially  work  has  performing  thought to be a  demonstrated  that  beta-oxidation (Devlin,  subcellular  organelles  which  are  in the kidney, l i v e r , and other various tissues.  Peroxisomal  beta-oxidation  in  mammals  mitochondrial beta-oxidation in three ways. dehydrogenation step  differs  F i r s t , the  from initial  in peroxisomes i s catalyzed by a cyanide-  insensitive oxidase system leading to the formation of hydrogen peroxide which  i s eventually  11  eliminated.  Second, the enzymes  involved in the cycle d i f f e r s l i g h t l y and peroxisomes appear to be s p e c i f i c  for longer  chain fatty  acids.  Third, i t appears  that the role of peroxisomes may be to shorten the chain length of r e l a t i v e l y  long fatty  acids such  oxidized in the mitochondria. observations that  that they  This  can then  be  hypothesis i s based  on  peroxisomes are unable to proceed beyond 8  carbons in shortening long chain fatty acids.  co-Oxidat ion  1.5.  co-Oxidation i s a minor and  occurs  (Devlin,  primarily  in medium  co-Oxidation  1986).  reticulum of  pathway for  many tissues  chain occurs  and involves  fatty acid metabolism length  fatty  acids  in the endoplasmic hydroxylation at the  methyl carbon on the opposite end from the carboxyl group, co-1 Oxidation occurs Hydroxylation 450,  at the carbon atom next to the methyl carbon.  involves "mixed function oxidases", cytochrome P-  oxygen and NADPH.  After hydroxylation, the fatty acid may  be further oxidized to a dicarboxylic acid at which point betaoxidation can occur from either end of the molecule.  2.  Metabolism of valproic acid VPA  undergoes  through at  extensive  least four  biotransformation  major metabolic  in the body  pathways:  glucuronid-  ation, beta-oxidation, co-oxidation, and co-1 oxidation (Loscher, 1981;  Granneman  mitochondria  and  et a l . ,  1984a).  Beta-oxidation  occurs  in  in peroxisomes while co- and co-1 oxidation are  12  endoplasmic  reticulum events (Van Den Branden and Roels, 1985).  The metabolism  of VPA  Glucuronidation  and  metabolic pathways 1984a).  in both  are  rats and doses  the expense  1984a; Granneman  is illustrated  beta-oxidation  With increasing  increased at  the major  in humans  of  man  the  in figure 1. two  primary  (Granneman  et a l . ,  VPA,  glucuronidation i s  of beta-oxidation (Granneman et a l . ,  et a l . ,  1984b).  In the male rhesus monkey,  metabolic pathways in order of importance were ester  glucuronidation, w-oxidation, beta-oxidation, and w-1 oxidation (Rettenmeier et a l . ,  1986a).  Conjugation on the carboxyl group leads to the formation of the glucuronide  ester.  microsomes (Granneman  Glucuronidation occurs et a l . ,  of an oral dose (1 g) of VPA the glucuronide conjugate Beta-oxidation of 3-OH  VPA,  1984a).  in man  mainly in the  Approximately  15 - 20 %  i s excreted in the urine as  (Granneman et a l . , 1984b).  VPA leads to the formation of 2-ene VPA,  and 3-keto  VPA.  Heinemeyer  and  co-workers  have  suggested that 3-keto VPA may be a product of peroxisomal betaoxidation  since  clofibrate, a  i t s excretion  peroxisomal  in  rats  proliferator  is  enhanced  (Heinemeyer  by  et a l . ,  1985). w-Oxidation  results  propylglutaric acid oxidation and  i s not  in the  (2-PGA).  5-OH  VPA and 2-  2-PGA i s the end product of co-  metabolized further  (Kuhara and Matsumoto, 1974). PGA  formation of  by  beta-oxidation  After oral administration of 2-  to rats, the intact compound was recovered in urine.  13  CMJ-CHJ-O.  CHj—CH-CHj CHC00O.M  C  "J-  C  M  .CHCOOH CHj-CHj-CH^  I- 2 W  MLPROIC MID  JCHCOOH  CHpCH-CHj < . « ' - d l t w »P>  *-tnt »P>  CH,—Otf-CHj  CH=CH-CH,  ,CHCOOH  CHj-CHj-CH^  CH=CH-C  CH.-CH-CH  ON  CHj-CHj-c(tj  eH,-CH -CH^ ?  OH  CHCOOH  3-twt »P*  CMJ-CMJ-CH^  S-OH  C-COOH  CHCOOH  CNJ-CH-CHJ  W  CHCOOH CH,_CH -CH  CHJ-CHJ-CH^  CHj— CH=CII  ?  4-OH »P>  CH,-CH -CH^  c—com  HOOC-CHj-CH,  ?(C).3'(C)H1UI»  t-«nt m ;CHC00H  0 CHj-C-CH  i-PropyloluUrU i c l d  i  ?  CHCOOH CHj-CHj-CH^  /JH CHj-CHj-CH^ CH-COOH CHj-CHj-CH^  «-Ktto VP*. 3-OH  HOOC-CH, COOH /vvun  CH,-CH,-CH,-CHr * * COOH l-P>op»lMlon1c tc\i  C  CHCOOH  CH,-CH -o£  H  3-  C  H  m  ?-  C  \ CHCOOH  CHj-CHj-CH^  ?  ?-Propirl»ucclnlc t H d  Figure  ^C-COOH CH—CHj—CH  ?  1.  3-*eto VP*  Human metabolism of v a l p r o i c  acid.  m  co-1 Oxidation VPA,  4-keto  on carbon 4 leads to the formation of 4-OH  VPA  (Acheampong  et  al.,  1983),  and  2-  propylsuccinic acid (2-PSA). The formation peroxisomal  or  of 2-ene  dienes.  3-ene VPA  mitochondrial  (Granneman  of 4-ene  3-ene  Further metabolism results in  VPA and  VPA,  may be  et  VPA  either  a l . , 1984a).  and  2-ene  VPA  the formation of the diunsaturated metabolites, the  The unsaturated  metabolite, 4-ene VPA i s metabolized  further v i a dehydrogenation to 2,4-diene VPA and s i m i l a r i l y 3ene VPA  to 2,3'-diene  VPA.  The two diunsaturated metabolites  possibly may be formed from 2-ene VPA. metabolite has  been  identified  as  The major diunsaturated 2-[(E)-1'-propenyl](E)-2-  pentenoic acid (2,3'-diene VPA) (Acheampong and Abbott, Hydroxylation of microsomal process l i v e r microsomes 3-OH VPA  VPA in the 3,  4, and  1985).  5 positions  is a  as determined by incubation of VPA with rat (Prickett and B a i l l i e ,  may also  1984).  The metabolite  through co-2 oxidation as well as  be formed  beta-oxidat ion.  2.1.  Metabolism of 4-ene VPA  The metabolism male rhesus i.v. bolus  of 4-ene  monkey. dose of  pharmacokinetic  VPA has been studied in the adult  A biexponential curve was found after an 14 mg/kg (Rettenmeier  profile  was similar  to that of VPA.  ene VPA plasma levels were approximately Twenty metabolites  were i d e n t i f i e d  15  et a l . , 1986b).  The  Free 4-  2.5 times that of VPA.  in the  urine with 59 % of  the dose  being  recovered  i n 24  oxidation  were  identified  a s t h e major m e t a b o l i c  and  to-1 o x i d a t i o n were m i n o r Eight  metabolites  GCMS from livers were  rat bile  VPA,  2-PGA,  metabolites  the  o f 4-ene  et a l . ,  VPA,  were  on  derived  VPA,  alcohol  and  reduction the  mediated  chain  either  reactions. of  2.2.  rat  metabolites  subsequently  followed  to  VPA  gamma  4-ene VPA.  A l l  beta-oxidation  S i x metabolic  4-ene VPA side chain  (3-keto),  (2,4-diene  to the d i a c i d ,  derivative,  beta-  VPA, a n d 3-  beta-oxidation  co-hydroxylation  or  pathways  were a s s i g n e d :  the dicarboxylic  by o x i d a t i o n  gamma b u t y r o l a c t o n e  3 position  co-  GLC a n d  isolated  4,5-dihydroxy  metabolite,  via  the unsaturated  side  from  by  The r e c o v e r e d  OH-4-ene VPA, c y t o c h r o m e P-450 m e d i a t e d ) , saturated  pathways,  detected  medium  1985).  parent  biotransformation  oxidation  VPA were  5-OH  and t h e  c y t o c h r o m e P-450 for  and beta-  a s 2 , 4 - d i e n e VPA, 3-OH-4-ene VPA, 3 - k e t o - 4 ' - e n e  5-OH-4'-ene  lactone,  Glucuronidation  routes.  and p e r f u s a t e  (Rettenmeier  identified  h.  on t h e  t o the primary acid  (2-PGA),  epoxidation to  a n d h y d r o x y l a t i o n a t t h e C-  t o 3-OH-4-ene VPA.  Metabolites VPA m e t a b o l i t e s  plasma o f  humans.  are present  in  The q u a n t i t i e s  various  amounts  a r e as f o l l o w s :  i n the 2-ene VPA  3 - 7 %, 3 - k e t o VPA 5 - 11 %, 3-OH VPA 0.5 - 2%, 4-OH VPA 0.2 1 %, 5-OH VPA 0.2 - 2%, a n d 2-PGA 0.2 - 2.5 % (Nau a n d L o s c h e r , 1984;  Losher,  1981c).  These v a l u e s  16  were d e t e r m i n e d  i n the  plasma by on VPA  gas chromatography from 26 e p i l e p t i c patients either  alone or  in combination  Values determined GCMS assay  0.16 -  follows in  0.02 -  1.22, 3-ene  other  anticonvulsants.  from the serum of 34 pediatric patients by a  were as  2,4-diene VPA  with  uq/mL:  0.58, 2,3'-diene  4-OH VPA 0.5 -  0 - 1.78,  7.29, 4-ene VPA  VPA 0.25 - 1.86, 2-ene c i s 0.06 - 0.40, 2-  ene trans  0.95 - 11.3, VPA 11.8 to 105, 3-keto 0.29 - 15.6, 4-  keto 0.01  - 1.29, 5-OH VPA 0 - 1.25, 2-PSA 0 - 0.44, and 2-PGA  0 - 1.23 (Abbott et a l . , 1986a). The metabolites 2-ene VPA, 3-OH VPA, 3-keto VPA, 4-ene VPA, 5-OH VPA,  2-PGA, 3-ene  thresholds  for  VPA, and  maximal  4-OH VPA  a l l elevated  electroconvulsions  p e n t y l e n e t e t r a z o l (PTZ) convulsions in mice  but  the  (MEC)  and  were  less  potent than VPA (Loscher, 198ld). The two unsaturated metabolites 2-ene VPA and 4-ene VPA are the most active of the metabolites, displaying approximately 60 - 90  %  of the potency of VPA although they are more sedating  than VPA 1985).  in mice These two  (Nau and  Loscher, 1984;  metabolites  may  be  Loscher  responsible  and  Nau,  for the  delayed effects of VPA observed after the drug i s withdrawn and no longer detectable in the plasma. The spectrum of a c t i v i t y of 2-ene VPA i s similar to that of VPA without 600 mg/kg. mg i . p . , models of  the potential (Loscher et a l . ,  for embryotoxicity even at doses of 1984).  2-ene i s more sedating  At high doses of 200 - 300 than VPA.  In four d i f f e r e n t  anticonvulsant a c t i v i t y , i t s a c t i v i t y was similar to  17  that of VPA.  2-Ene VPA was more potent in general tonic clonic  seizures in rats.  gerbils and  In the  mice, doses but side doses.  in p e t i t  mal recurrent  maximal electroseizures  seizures in  (MES) and PTZ tests in  of 200 - 300 mg/kg of 2-ene VPA were more sedating  effects were not observed  in rats or gerbils at these  The anticonvulsant a c t i v i t y of 2-ene VPA i s of shorter  duration (2 h compared to 5 h) than VPA in mice after 4 mmol/kg doses (Keane et a l . , 1985). Both VPA plasma to and  and 2-ene  the l i v e r  Loscher,  However, 2-ene  transferred rapidly  from the  in mice after oral doses of 50 mg/kg (Nau  1985;  concentrations were  VPA are  Loscher  and  Nau,  higher compared  VPA plasma  1984).  VPA  liver  to plasma concentrations.  concentrations  were  greater  than  l i v e r levels possibly due to the greater plasma protein binding of  2-ene  VPA  (97  Liver/plasma drug suggesting the Valpromide, the than VPA  (2 -  %)  level  compared ratios  p o s s i b i l i t y of acid amide 5 times)  to  VPA  were an active  (36  %)  in mice.  concentration-dependent transport mechanism.  derivative of VPA, was more potent  and 2-ene  VPA but  displayed greater  sedation and t o x i c i t y in three seizure tests (MES, MEC,and PTZ) after i.p. injections in mice (Loscher et a l . , 1984).  18  C.  ADVERSE EFFECTS, METABOLIC DISTURBANCES, AND  1.  Adverse effects The majority  nausea,  TOXICITIES  of side e f f e c t s associated with VPA are mild;  vomiting,  diarrhea  and  abdominal  cramps  are  most  commonly observed (Bruni and Wilder, 1979).  Other side e f f e c t s  include transient  hyperkinesia, fine  hair loss,  weight gain,  postural tremor, drowsiness, and transient hallucinations, with the l a t t e r  three being  Transient and have also mg oral  self l i m i t i n g  been observed dose of  healthy males al.,  dose related  (Dulac  neutropenia and  et  a l . , 1986).  thrombocytopenia  with VPA (Barr et a l . , 1982).  VPA increased  growth  hormone  An 800  secretion  in  but not in chronic schizophrenics (Monteleone et  1986). A 21  year old e p i l e p t i c man displayed VPA-induced dementia  (VPA trough  level 116.3  remitted after  /ig/mL) which  completely and promptly  withdrawal of the drug (Zaret and Cohen, 1986).  The dementia may have resulted through a direct central nervous system e f f e c t , an indirect central nervous system effect due to VPA-induced hyperammonemia,  or a  paradoxical effect secondary  to VPA. Therapeutic doses adverse effects distress i s al.,  1986).  of VPA  are dose  related and  associated with VPA  therapeutic dose  do not  the risk  growth but  for perinatal  high doses of VPA (Jager-Roman et  administration to of 20  affect f e t a l  rhesus monkeys at a human  mg/kg/day during organogenesis did not  19  result in  any adverse  i t resulted  in low  defects, while (Mast et a l . , folds, f l a t  effects but at a dose of 200 mg/kg/day,  b i r t h weights,  c r a n i o f a c i a l and  skeletal  at a  dose of 300 mg/kg/day i t was embryolethal  1986).  A consistent f a c i a l phenotype (epicanthal  nasal bridge  etc.) was observed in seven children  who had been exposed to VPA in utero (DiLiberti et a l . , 1984). In  whole  rat  mg/kg/day caused 2-ene VPA  embryos,  VPA  than  40  1986).  VPA i s associated with  an increased  f i r s t trimester  interferences with  zinc and  humans  et  (Weinbaum  concentrations reached  (Nau, 1986).  greater  adverse effects at doses up to 200 mg/kg/day  (Lewandowski et a l . ,  correlate with  doses  abnormal development in 30 % of embryos while  had no  defects after  in  risk of  f e t a l exposure  neural tube  possibly due to  other trace element metabolism in  a l . , 1986). in the  the incidence  Peak  or  steady  state  mother and gestational material of neural  tube defects  in mice  In mice, the dose of VPA and area under the serum  concentration  versus  time  curve  (AUC)  correlate  with  embryolethality and f e t a l weight retardation (Nau, 1985). VPA i s  also thought  manner similar  to zinc  Administration of the incidence  to cause  renal damage  deficiency  (Vormann  et  in rats  in a  a l . , 1986).  VPA 300 mg/kg/day to pregnant rats increased  of f e t a l  hydronephrosis and  hydrops in animals  fed with a zinc deficient diet and induced f e t a l l i v e r necrosis independent of were toxic  zinc status.  on VPA  Plasma  were decreased  20  zinc l e v e l s in rats that  although there  was not any  conclusive  evidence  hepatotoxic  doses of VPA (Daffron and Kasarskis, 1984).  2.  Metabolic  responsible  for  deficiency  with the folate-dependent glycine  concentrations of  is  caused  by  cleavage,  one carbon enzyme  leading  to  increased  glycine in patients and animals treated with  1986).  folate with  zinc  disturbances  VPA interferes  VPA (Carl,  that  VPA i n i t i a l l y  causes a  r e d i s t r i b u t i o n of  decreases in l i v e r concentrations and increases in  brain and plasma levels, but the effects revert to normal after several weeks. Hyperglycinemia and administered  VPA  several weeks of 1 in  % VPA  the  hyperglycinuria are  at doses  ranging from 0.3 to 1.2 mmol/kg for  (Cherruau et a l . , 1981).  cleavage  system  respectively (Martin-Gallardo blood, l i v e r ,  and  brain  This action  disorder, non-ketotic VPA therapy  i s also  (Matsuda et  a l . , 1986;  other  in the  et a l . ,  glycine  liver  1985).  levels  were  and  brain,  Consequently, significantly  hyperglycinemia.  ammonia  with  administration  of VPA simulates in rats the metabolic  erythrocyte  levels are  Chronic  to young rats resulted in 50 % and 35 % reductions  glycine  elevated.  observed in rats  associated with  levels  as  well  increased serum as  hyperbilirubinemia  Ratnaike et a l . , 1986).  p a r t i c u l a r i l y increased anticonvulsants,  21  and  Serum ammonia  when VPA i s coadministered  especially  phenobarbital  or  phenytoin  (Ratnaike  et  a l . , 1986;  Warter  et  a l . , 1983;  Haidukewych et a l . , 1985; Zaccara et a l . , 1985). VPA  administration results in a VPA-induced deficiency of  carnitine (Borum  and Bennett,  forms v a l p r o y l c a r n i t i n e VPA, leading  to an  excrete the  1986; Laub  derivatives which  increased metabolic  more toxic  metabolites.  et a l . , 1986).  are less toxic than need for c a r n i t i n e to  Patients on VPA display  decreased plasma c a r n i t i n e l e v e l s accompanied by elevated ammonia l e v e l s  (Ohtani et  VPA  a l . , 1982).  blood  Oral administration of  carnitine 50 mg/kg/day for 4 weeks was successful in correcting the  VPA  induced  hyperammonemia.  carnitine  normal and  as  the  carnitine  In  levels  one were  d i d not a i d in reversing  (Laub et al.., 1986). of VPA  decreased  in the therapeutic range for man w i l l  plasma  decreased hepatic  beta-hydroxybutyrate  l e v e l s of  free CoA,  VPA i n h i b i t s Turnbull  urea synthesis et a l . ,  in  1983).  levels,  acetyl CoA,  carnitine in normal infant mice (Thurston  1983;  well  induced hyperammonemia.  c a r n i t i n e supplementation  Single doses cause  of the  developed hepatotoxicity,  the t o x i c i t y  as  It i s possible that the decrease in c a r n i t i n e  levels i s a result patient who  deficiency  and  and free  et a l . , 1985).  rat hepatocytes  (Coude,  I t also i n h i b i t s pyruvate and  palmitate oxidation by 30 - 50 % in i s o l a t e d rat hepatocytes at doses ranging  from  0.1  -  1  mM  (Turnbull  a l . , 1983).  Oxidative phosphorylation  in i s o l a t e d  inhibited along  glycine cleavage system (Hayasaka et  with the  22  liver  et  mitochondria  is  al.,  1986).  Fatty acid synthesis and fatty acid oxidation are  also inhibited  by  VPA  i n isolated  rat hepatocytes.  VPA  competitively i n h i b i t s the pyruvate c a r r i e r in rat brain and in l i v e r mitochondria i s hypoglycemic al.,  (Benavides et a l . , 1982).  At 100 mg/kg, VPA  and hypoketonaemic in fasted rats (Turnbull et  1983). At doses  greater than 1 mM, VPA may uncouple mitochondrial  respiration (Benavides mitochondrial system valproyl CoA  may be  and i t s  hepatic mitochondria i n t e g r i t y of  et a l . ,  1982).  This  mediated by  effect  the accumulation  1983).  of  further metabolites in the matrix of the  (Turnbull et a l . , 1983) or by a l t e r i n g the  the inner mitochondrial membrane or by actions on  the substrate c a r r i e r s or mitochondrial metabolites al.,  on the  (Rumbach et  Valproyl CoA in the mitochondrial matrix  i n h i b i t o r of beta-oxidation  i s a weak  (Sherratt and Vietch, 1984).  At therapeutic concentrations of VPA, mitochondrial but not peroxisomal Branden  and  beta-oxidation Roels,  peroxisomes seem  in rats  1985).  to take  At  i s inhibited low  VPA  (Van Den  concentrations,  over part of the mitochondrial  beta-  oxidation . Peroxisomal beta-oxidation  increased 4  fold in rat l i v e r  and 2 fold in mouse l i v e r after administration of 1 % VPA for 2 weeks (Horie  and Suga,  hepatic beta-oxidation  1985).  The inducing effect of VPA on  was similar  hypolipidemic drugs.  23  to c l o f i b r a t e  and  other  I.P. administration of 1.2  and 1.8  of VPA to rats for seven days at doses  mmol/kg/day (172.8  and 259.2  mg/kg/day)  has  resulted in decreased cytochrome P-450 levels (Cotariu et a l . , 1985). Treatment of 4-ene  VPA,  isolated rat hepatocytes with VPA, 2-ene VPA,  4-OH  VPA,  concentration-dependent lactate (Rogiers  5-OH  VPA  and  inhibition  et a l . ,  1985).  2-PSA  of  OH VPA,  4-OH VPA,  2-ene VPA,  S a l i c y l a t e and  al., by  1985). adult  of t o x i c i t y  This effect of VPA  octanoate can  provide  partial  of metabolic  protection processes by  CoA formation in rat hepatocytes  (Brown et  The acyl CoA metabolite of VPA i s synthesized only hepatic  tissue  so  that  sequestration  (Coenzyme A) results in the depletion of acetyl CoA. in CoA  of  treatment.  against the VPA-induced i n h i b i t i o n i n h i b i t i n g valproyl  from  VPA and 4-ene VPA, 5-  and 2-PGA.  could be reversed with glucagon  in a  gluconeogenesis  The extent  these compounds in decreasing order was:  resulted  of  CoASH  Decreases  are due to accumulation of acid soluble (nonacetyl) CoA  esters (valproyl and further derivatives) as shown by decreased plasma beta-hydroxybutyrate CoA,  and  carnitine  suckling mice 1985).  in  l e v e l s , decreased free CoA, acetyl  fasting  after single  epileptic  doses of  This i s accompanied by  VPA  children (Thurston  an increase  in acid  and in et  al.,  soluble  short chain fatty acid CoA esters and acid insoluble long chain c a r n i t i n e esters (Thurston et a l . , 1985).  24  Of are  co-oxidized  the  slightly  more  contributors  in  Beta-oxidation intermediates tissue  on  VPA  are This  amounts of  6 carbon  also  found  i n the  al.,  1980). may  4,4'-diene not  (Simula  lead  to  et  VPA  the  major  al.,  1985).  to chemically  reactive  m a c r o m o l e c u l e s and  of  was  (Turnbull  et  rats  form  fatty  recovered  i n the  of  of  is possibly  due  Increased  adipic  w i t h VPA  excretion  In of  was  still  lactic  this  and  end  year  et  old  product  (Kuhara  the  are  inhibiting  a six  the  increased  acid,  (Mortensen  a c i d m e t a b o l i s m by  amounts o f  urine  eg.  co-oxidation.  glucuronidation  amounts  a l . , 1986).  treated  markedly  Increased  higher  excretion  inducing  2-PGA  excrete  increased  R e y e ' s syndrome, t h e  metabolism.  et  of  al.,  m a j o r pathway  of  adipic acids  were  been a s s o c i a t e d  with  patient.  Toxicity Pancreatitis  14  urine  but  1985). However,  VPA  are  dicarboxylic acids,  i n t e r f e r e with  beta-oxidation  3.  may  found  beta-oxidation  impaired  co-oxidation,  but  and  residues.  to  also  mice  alkylate cellular  acids.  male w i t h  4-ene VPA  toxicity  4-ene VPA  dicarboxylic  VPA  in  induced  which  bound  Patients  toxic  VPA of  metabolites,  usage cases  reported  and  (Isom J.B., of  hepatotoxicity 1984;  Coulter  p a n c r e a t i t i s where VPA  (Wyllie  hepatotoxicity  et  al.,  throughout  1984). the  25  world  have et may  a l . , 1980). be  Sixty  Since  implicated eight  have  c a s e s of  have been o b s e r v e d  1979, been fatal  i n which  VPA may  be incriminated  (Kochen  and  Sprunck,  1984).  The  hepatotoxic aspects of VPA have been reviewed by Powell-Jackson et a l . ,  (1984).  From a  f a t a l hepatotoxicity States between  recent survey  associated with  1978 and  The incidence  with increasing usually show  1984).  cases of  i n the United that age and  (Driefuss and S a n t i l l i ,  of VPA induced hepatotoxicity decreased Liver function  transient increases  and Loscher, in hepatic  age.  VPA usage  1984, i t was concluded  polytherapy were the major determinants 1986).  of reported  tests (SGOT  and SGPT)  with VPA administration (Nau  However, serum transaminases  are increased  f a i l u r e in patients on VPA (Cotariu et a l . , 1985).  Micro-vesicular steatosis i s common in cases of hepatotoxicity, and resembles  the lesions  seen in Jamaican Vomiting Sickness,  Reye's Syndrome and 4-pentenoic 1984;  Lewis et a l . ,  3.1.  Hepatotoxicity  1982).  The hepatotoxicity mono- and/or 1984).  acid t o x i c i t y (Nau and Loscher,  of VPA  i s thought  diunsaturated metabolites  (Kochen  and  Sprunck,  An increased formation of the diunsaturated metabolites  appears to be c h a r a c t e r i s t i c  in f a t a l hepatic f a i l u r e .  metabolites are s t r u c t u r a l l y related hepatotoxin, and al.,  to be caused by i t s  to the hypoglycin A  to 4-pentenoic  These  acid,  a  metabolite (Jezequel et  1984). The t o x i c i t y  to be  a result  associated with of i n h i b i t i o n  26  VPA and 4-ene VPA i s thought  of the beta-oxidation  pathway  (Kesterson et a l . , 1984). ene  VPA  cause  (Bjorge and free CoA  inhibition  Baillie,  of  1985).  decanoic VPA  pools (Fears, 1985).  i n h i b i t i o n of CoA  In rat l i v e r homogenates, VPA and  (Kesterson et a l . , 1984).  and prolonged  i n h i b i t i o n of  which d i r e c t l y  beta-oxidation  w i l l cause depletion of the  VPA  the beta-oxidation  acid  4-  causes a transient and mild pathway by  sequestration of  4-Ene VPA produces a more potent the pathway by forming CoA esters  i n h i b i t enzymes  in the beta-oxidation pathway.  A f a t a l case of hepatic f a i l u r e due to suppression of the betaoxidation pathway as shown by the lack of the 3-keto metabolite has been reported by Kochen and co-workers in 4-Ene VPA  i s similar in structure to 4-pentenoic acid, the  causative agent  in Reye-like  metabolite responsible - Loscher,  syndrome and to the hypoglycin A  for Jamaican Vomiting  Sickness  (Nau  and  1984).  In rat 4-ene VPA  hepatic microsomal preparations, the ethyl ester of was more active than 4-ene VPA  NADPH- and (33 %  1983.  time-dependent loss of cytochrome P-450 over 30 min  compared to  action of  8%)  4-ene VPA  mechanism to  (Prickett  and B a i l l i e ,  1986).  This  ethyl ester i s thought to be by a similar  that of allyisopropyl-acetamide and other related  monosubstituted o l e f i n s . cytochrome  in i t s e f f e c t s on the  P-450  covalently bind  to to the  These  compounds  chemically heme moiety  ultimately lead to i t s destruction.  27  are  reactive  converted species  of cytochrome  by  which  P-450  and  Of a l l the monounsaturated the  most  changes  decreased  in  metabolites, 4-ene  factors  beta-hydroxybutyrate  VPA causes  (dicarboxylic aciduria reduction)  which  and  indicate  i n h i b i t i o n of  the beta-oxidation pathway in rats (Granneman et  al.,  However,  1984c).  these changes are not as pronounced as  with hypoglycin  A.  In hypoglycin  adipic, suberic  and sebacic  A toxicity,  excretion  acids i s increased.  of hypoglycin  A, methylenecyclopropylacetic  i n h i b i t fatty  acid  oxidation  through  of  A metabolite  acid, appears  effects  on  acyl  to CoA  dehydrogenases.  E.  DRUG INTERACTIONS  Several properties drugs. with  VPA  drugs 1981).  hepatic metabolism susceptible to not induce  body, the result  for It  binding has the  of other  metabolites.  Interactions i n h i b i t i n g the  i t s own metabolism i s  inducing agents. and  VPA  i t s e l f does  Richens,  1985).  extensive biotransformation in the  coadministration of the  (Drug  c a p a b i l i t y of  enzymes (Rimmer  undergoes such  in  sites  drugs, but  l i v e r enzyme  the hepatic  Because VPA  promote interactions with other  i s strongly plasma protein bound and thus competes  other  Newsletter,  of VPA  increased  metabolic formation  Interactions of  VPA  with  drugs have been studied most extensively.  28  inducing of other  agents  possible  may  toxic  anticonvulsant  1.  Drugs which a l t e r VPA  concentrations  Salicylates  VPA  (Levy  and  displace  Koch,  1982).  coadministration (11.5 clearance of  from plasma protein binding sites epileptic  children,  ASA  - 16.9 mg/kg/Q6H) resulted in decreased  free VPA  clearance ( F a r r e l l  In  without  et a l . ,  s i g n i f i c a n t changes on t o t a l  1982).  Free fraction  VPA  of VPA  was  found to increase in rank order at ASA concentrations exceeding 50 mg/L  in  in  resulted in  vitro  studies.  ASA  coadministration  also  longer serum h a l f - l i v e s of both free and t o t a l  VPA  (Orr et a l . , 1982). The urinary subjects (one at steady  VPA  ASA  VPA  conjugate  13  metabolites  healthy male volunteer and 6 pediatric  showed  s i g n i f i c a n t differences  significantly and  4-ene VPA  beta-oxidation decrease)  pathway  while  the lack  decreased  in  7  patients)  (Abbott et a l . ,  while  VPA  and 3-keto glucuronide  were s i g n i f i c a n t l y  increased.  was  inhibited  significantly  glucuronidation  Suppression  by induction  significantly  The (66  increased  of the beta-oxidation pathway was  % by  balanced  of the glucuronidation pathway, which may  explain  of change in t o t a l clearance in spite of the increase  in the free fraction of VPA. VPA  and  After ASA coadministration, 2-ene trans VPA  were  30 %.  of  state before and after administration of antipyretic  doses of 1986b).  profiles  ASA may  i n h i b i t beta-oxidation of  by decreasing the amount of valproyl-Coenzyme A formed. Cimetidine in doses of 1 g per day in patients can cause up  to  a  20  %  decrease  in  VPA  29  clearance  accompanied  by  a  s i g n i f i c a n t l y prolonged  elimination h a l f - l i f e  (Webster et a l . ,  1984). In animal studies, free fatty acids ( I n t r a l i p i d ) increased R  free levels  of VPA  s l i g h t extent  (Kutt, 1984).  in concentrations 48 %  and 82  and also  of 100  - 118  inhibited VPA  metabolism  to a  Administration of free fatty acids and 200 j/g/mL resulted in 19 -  nq/mL  % increases  in the free f r a c t i o n of VPA,  respectively (Patel and Levy, 1979). Other anticonvulsants induce VPA  metabolism as  (CBZ, phenytoin, and phenobarbital) supported by longer serum VPA half-  l i f e in monotherapy compared to polytherapy ratio of  (Kutt, 1984).  The  VPA steady-state plasma levels to dose i s much lower  in children receiving other anticonvulsants in addition to VPA (Sackellares  et  a l . , 1981).  Antacids  may  affect  the  absorption rate of VPA (Kutt, 1984).  2.  E f f e c t s of VPA on other drugs Increased serum  VPA  levels of  phenobarbital are observed when  i s administered concommitantly, necessitating reduction of  phenobarbital dosage Phenobarbital serum 50 %.  in up  to 80  h a l f - l i f e may  % of patients (Kutt, 1984). be increased  by as much as  The mechanism appears to be i n h i b i t i o n of phenobarbital  metabolism  by  hydroxylation. to i n h i b i t  VPA,  possibly  inhibition  of  phenobarbital  In rat hepatic microsomes, VPA has been shown  the parahydroxylation of phenobarbital (Taburet and  Aymard, 1983).  VPA  also  has  30  been  found  to competitively  inhibit liver  glucuronidation microsomes. is  phenobarbital.  VPA  so  parahydroxyphenobarbital  This  glucuronidation  extent,  of  that  a  is  of  major  affects  extreme  pathway  importance  for  both  primidone metabolism  reductions  in  in  primidone  rat since  VPA  and  to a lesser  dosages  are  not  required. VPA w i l l  cause either  a transient increase or decrease in  t o t a l phenytoin  l e v e l s , with  previous values  within days  in serum  phenytoin levels  elevations.  Increases  those patients competes with especially  who  are  phenytoin  at  higher  in  the altered  or weeks (Kutt, 1984). are observed  Decreases  more frequently  phenytoin serum levels may  close to for  saturation  plasma  than  occur in  kinetics.  protein  concentrations  Phenytoin plasma protein binding may % to 85 % or l e s s .  levels returning to  binding  around  VPA sites, ug/mL.  100  decrease from the usual  90  Phenytoin i s primarily bound to one s i t e on  the albumin molecule (warfarin binding site) while VPA  binds to  both the warfarin and the diazepam s i t e s (Kober et a l . , 1980) VPA 1984).  also VPA  increases antipyrine  45  %  (Kutt,  displaces tolbutamide from i t s binding sites on the  albumin molecule plasma protein levels of  h a l f - l i f e by  (Fernandez et binding sites  diazepam are  allows increased  a l . , 1985).  that  free  elevated (Hariton et a l . , 1985).  This  penetration  diazepam.  31  with  of  diazepam  VPA competes for  the  such  blood-brain-barrier  by  F.  INTERACTION BETWEEN VALPROIC ACID AND CARBAMAZEPINE  This i s a c l i n i c a l l y drugs are  important interaction  frequently used  seizure c o n t r o l .  One  in combination  to  as these  obtain  two  maximal  drug i s often added to the other in an  e f f o r t to obtain better seizure c o n t r o l .  Since CBZ i s known to  induce the hepatic microsomal enzyme systems, a brief review on enzyme induction follows.  1.  Enzyme induction Brown, M i l l e r  and M i l l e r  f i r s t reported the phenomenon of  increased hepatic  enzyme a c t i v i t y  small amounts  polycyclic  1967).  of  in rats a f t e r injections of  aromatic  hydrocarbons  (Conney,  Remmer and coworkers discovered the inducing effects of  barbiturates on hepatic microsomal drug metabolism. Enzyme induction increases the normal rate 1971).  rate of  Increased  enhanced  Induction  enhancement  in  duration and Induction can  synthesis  1967).  or  be  inhibition  intensity of  enzyme  An enhancement of enzyme a c t i v i t y  the  metabolic  attained  of  a l t e r a t i o n s in the amount  of  alter the  which  an enzyme r e l a t i v e to i t s  l e v e l s in mammals may  obtained without  present.  "the process  in the uninduced organism" (Gelboin,  enzyme  degradation (Conney,  described as  synthesis of  of synthesis  either through  " can be  may be  liver  rate  enzymes  and  drug actions  steady state  32  thus  results  directly  in man  of enzyme in an affects  and  animals.  concentrations  of the  parent compound  and  i t s metabolites  elimination ( G i l l e t t e , single enzyme at several  in  addition  to  their  1979).  Several compounds can induce a  and conversely,  a single inducing agent can act  steps  in  the  induction  process  (Estabrook  and  Lindenlaub, 1979).  1.1.  Metabolism L i p i d soluble  urine because to undergo as their  compounds are  excreted very slowly into the  of their chemical properties and thus, they tend  extensive biotransformation and are excreted mainly metabolites ( G i l l e t t e ,  1986).  Polar  unchanged.  compounds,  Some  1979;  however,  xenobiotics  endogenous substances  Astrom  may  are  system(s).  endogenous  counterparts  usually  excreted resemble  be metabolized by the  Foreign compounds are  DePierre,  structurally  and therefore, may  same enzyme  and  which do  metabolized  by  not have relatively i  nonspecific enzymes. more polar  than the  The  products of  parent compound  metabolism are usually and  thus  renal  and/or  b i l i a r y excretion i s enhanced. 1.2.  Types of reactions There are  enhance the  four types removal of  of general a  foreign  reactions which compound  from  act  the  to  body:  oxidation, reduction, hydrolysis, and conjugation (Goldstein et al.,  1974).  biotransformation of  The  reactions  responsible  foreign compounds may  33  for  the  be divided into two  phases.  Phase one  reactions include oxidation, reduction and  hydrolysis ( G i l l e t t e , take place require  at  the  both  1979). hepatic  NADPH  and  mainly  enzyme have conjugation  acylation, and  reactions  generally  endoplasmic  reticulum  site  oxygen  reactions are catalysed by of t h i s  Oxidative  (Gillette,  These  cytochrome P-450 and several forms  been i d e n t i f i e d . reactions  mercapturic  1979).  and  Phase two reactions are  including  acid,  glucuronidation  sulphate,  and  and  dihydrodiol  formation.  1.3.  Factors influencing enzyme induction  The duration the rate  and intensity  of drug  number of  metabolism by  factors can  including d i e t , changes and  of drug action i s dependent on  influence the  disease state,  ingestion  Conney, 1982).  of  greatly influence  the e f f e c t  1978).  fats  induction.  The amount  hydrocarbon  hydroxylase  (Conney, 1967).  rate of  nutritional  foreign  Nutritional  Saturated  the body  factors, of  tend to  act as  activity  hormonal  (Conney,  especially  inducing  of hepatic  drug metabolism  status,  compounds  1967;  fats,  agents  can  (Goldberg,  promoters  of  cytochrome P-450 is  A  decreased  enzyme  and a r y l in  severe  h e p a t i t i s or active c i r r h o s i s ( F a r r e l l et a l . , 1979).  1.4.  Properties of enzyme inducing agents  The compounds are extremely  which act as inducers of microsomal enzymes  diverse in structure, e f f e c t ,  34  and  ability  to  induce  (Conney,  1967).  microsomal enzymes  Examples of compounds known to induce  in man  include anticonvulsant  phenobarbital, phenytoin, and carbamazepine), agents  (eg. phenylbutazone),  rifampin), anticoagulants products  (eg. dioxane), oral  sedatives (Goldberg,  anti-inflammatory  antifungals,  (eg. warfarin),  agents (eg.  antibiotics  (eg.  alcohol, i n d u s t r i a l  contraceptives, pesticides,  1980).  Usually, enzyme  are l i p i d soluble at physiological pH.  and  inducing agents  A high l i p i d  solubility  enables the inducing agent to reach the l i p o p h i l i c membranes of the endoplasmic  reticulum  with  increased  ease  as  well  as  allowing the formation of a stronger inducer-enzyme complex and ultimately (Estabrook compound  a  longer  duration  and Lindenlaub, required  to  of  1979).  cause  pharmacological  activity  The quantity of the inducing  induction  varies  considerably  (Conney, 1967).  1.5.  Types of enzyme inducing agents  There are four known groups of enzyme inducing agents which are  represented  by  3-methylcholanthrene,  i s o s a f r o l e , and polychlorinated biphenyls. biphenyls induce both  cytochrome P-450s  phenobarbital  Isosafrole induces the only  chemical  and  in  The polychlorinated  which are  3-methylcholanthrene  a novel  also induced by (Rees,  1979).  species of cytochrome P-450 and i s  i t s group to date.  methylcholanthrene represent  phenobarbital,  the majority  (Conney, 1967).  35  Phenobarbital and 3of inducing  agents  1.5.1.  Phenobarbital and 3-methylcholanthrene  Phenobarbital type of metabolic reduction,  pathways  and  including  types  more limited  a l i p h a t i c , and  of 3  phenobarbital must  group of  to 6  observed within animals  to 10  aromatic  fold in  inducing agents  24 h.  leads  reticulum in  1967).  to  d a i l y for type  hydrocarbons)  The course and  of  the  at least  (polycyclic  w i l l double  Administration  cell.  3-  For a  the microsomal enzymes,  maximal increases  proliferation  the l i v e r  The  these two types also d i f f e r .  be administered  h and  oxidation,  reactions including aromatic,  3-methylcholanthrene  hydrocarbons) of within 3  (Conney,  n-hydroxylation (Conney, 1967).  maximal increase  The  glucuronidation,  (polycyclic  intensity of . induction of  days.  agents influence a variety  de-esterification  methylcholanthrene affect a  of inducing  aromatic  enzyme a c t i v i t y  of 5 of  three  to 10  fold are  phenobarbital to smooth  endoplasmic  3-Methylcholanthrene does  not  a l t e r the amount of smooth endoplasmic reticulum.  1.6.  Clinical Enzyme  induction  activation of an increased action  if  implications can  lead  foreign compounds. rate of the  to  either  deactivation  or  Enzyme induction results in  drug metabolism and thus, decreased drug  metabolite  is  not  active  (Conney,  However, i f  the metabolite  the drug may  result in an i n t e n s i f i e d drug action.  1967).  i s active, increased metabolism of  36  Chronic administration heteroinduction  (Conney,  of a  drug may  1969).  result in auto- and  Drugs which induce their  metabolism during chronic administration include phenylbutazone, tolbutamide and Enzyme induction  may  toxic metabolites. which are  The  lead  own  phenobarbital,  CBZ. to the formation  t o x i c i t y of  r e l a t i v e l y non-toxic  organic  until  of p o t e n t i a l l y thiophosphates,  metabolized  to  potent  cholinesterase i n h i b i t o r s , i s increased after pretreatment with enzyme inducing both the  Enzyme inducing agents may  i n i t i a t i o n and  carcinogenesis  1.7.  agents.  promotion of  influence  experimentally  (Park and Breckenridge,  induced  1981).  Markers of enzyme induction Several indices are u t i l i z e d to measure the rate and  of enzyme rate or  induction. half-lives  system (Goldberg, commonly used carbon  1978).  metabolism  breath  of drugs  drug into of  in  aminopyrine in  Salivary clearances  in  is  the  most  administration  humans i s  can also  often  employed. the  the s a l i v a i s often pH dependent.  The  compounds  plasma  37  be used  of  although  endogenous  Changes  hydroxylation  The carbon-14 labelled  after  hydroxycortisol, D-glucaric acid, and utilized.  by the  Antipyrine clearance  i t s convenience.  content  carbon-14 labelled  measurement of clearance  of drugs metabolized  due to  dioxide  secretion of  These include  extent  including  6-beta-  1-xylulose have also been  enzymes  such  as  gamma-  glutamyltransferase induction  2.  a l s o be  used  as  markers  of  enzyme  ( M o r e l a n d e t a l . , 1982).  Carbamazepine CBZ  is  similar as  may  an i m i n o s t i l b e n e  t o the t r i c y c l i c  shown  d e r i v a t i v e which  antidepressants  i s structurally  (Goodman e t a l . , 1980)  below.  i  CONH  2  Carbamazepine  CBZ  is  utilized  including  i n the treatment  partial,  generalized  of a v a r i e t y of s e i z u r e  t o n i c - c l o n i c , and mixed  types  seizure  disorders. After  oral  from the  levels  levels  of  CBZ  are attained  CBZ a r e  relatively highly  CBZ  is  man,  long  i n the  half-life  plasma p r o t e i n is  extensively  unchanged oxidized  to  ranging  bound  3 -  from  absorbed  Therapeutic 14 jug/mL.  8-72  Peak plasma  CBZ h a s a  hours.  The d r u g  (75 - 9 0 % ) .  metabolized  in  l e s s than  man  via  the  liver  1 % of the parent  ( B e r t i l s s o n and Tomson,  10,11-epoxide,  38  i s slowly  (Goodman e t a l . , 1980).  range of  i n the urine the  CBZ  i n 2 - 8 hours.  m i c r o s o m a l enzyme s y s t e m , w i t h excreted  in  gastrointestinal tract  plasma  is  administration  followed  by  drug  1986). further  metabolism to  the dihydroxy  analogue which  i s glucuronidated  p r i o r to excretion. CBZ induces  the hepatic  autoinduction and  microsomal enzyme system, causing  heteroinduction (Goodman  et a l . , 1980).  In  long term treatment, CBZ induces i t s own metabolism (Bertilsson and Tomson,  1986; Pynnonen,  autoinduction may 1979).  occur within  CBZ induces  clonazepam,  1979; Goodman  the metabolism  ethosuximide,  phenytoin, warfarin,  two or  three days  of other  doxycycline,  (Pynnonen,  drugs including  oral  contraceptives,  and haloperidol (Fernandez et a l . , 1985).  Concurrent therapy of erythromycin intoxication (Baciewicz, appears to  et a l . , 1980). CBZ  be capable  and CBZ often results in CBZ  1986; Goulden of causing  et a l . ,  1986).  either enzyme  CBZ  induction or  enzyme i n h i b i t i o n , depending on the individual. CBZ has been shown to decrease the plasma levels of VPA inducing VPA  metabolism (Kutt,  extensive biotransformation  1984).  in the  Since  VPA  by  undergoes  body, the effect of CBZ on  VPA metabolic pathways should be investigated. Bowdle  and  coworkers  (1979)  demonstrated  that  CBZ  administration (200 mg po daily) to healthy volunteers resulted in increased  VPA clearance  and  l e v e l s after  2 weeks.  change  constant, Ke, suggest that to plasma  was not  A  observed.  decreased in the  VPA  steady  elimination  state rate  I_n v i t r o binding experiments  CBZ does not have an effect on the binding of VPA  proteins (Mattson  et a l . ,  39  1980;  Mattson  et  al.,  1982).  VPA addition (50 pg/mL and 100 ug/mL) to CBZ  12 ug/mL)  resulted in elevation of free CBZ  (6, 8, and  levels.  VPA elevates plasma levels of CBZE, the major metabolite of CBZ  (Bertilsson  and  Tomson,  inhibited elimination derivative of both VPA  1986).  of CBZE.  This  Valpromide,  may  be  the  acid  VPA, appears to have a similar e f f e c t .  and valpromide  cause elevations  valpromide appear to be more c l i n i c a l l y  (Pisani et  a l . , 1986).  i n h i b i t i n g epoxide  hydrolase, the  to  amide  Although  in CBZE l e v e l s , the  e f f e c t s of  Valpromide  due  has  the  significant  potential  for  enzyme responsible for CBZE  metabolism, while VPA has l i t t l e effect on the enzyme. Administration of e p i l e p t i c patients  lead to  concentrations of CBZ for  valpromide in  were s t i l l  increase in  CBZ  mean  to  serum  (2 - 8.5 ug/mL) and CBZE when substituted  valproate • (Meijer  valproate caused  a marked  combination with  et  a l . , 1984).  the levels  higher than  Switching  to decrease  back  to  although CBZE levels  the previous values for several weeks.  P a c i f i c i et a l . , (1985) postulated that these increases in CBZE were due  to i n h i b i t i o n  Rhesus monkey effect  of  of epoxide  hepatic microsomes  valproic  acid  and  hydrolase were used  valpromide  by  valpromide.  to determine in  vitro  on  the the  hydration of styrene oxide (1 mmol/L) and  benzo-(a)-pyrene-4,5-  oxide (0.2 mmol/L) by epoxide hydrolase.  Valpromide (0.2 - 0.8  mmol/L) inhibited  both reactions  while VPA  had l i t t l e effect on both reactions.  40  (0.2 - 1.0 mmol/L)  In isolated perfused rat l i v e r s , therapeutic concentrations of VPA  caused a  decrease  in the i n t r i n s i c clearance of CBZ as  well as a decrease in the i n t r i n s i c (Chang and Levy, 1985). preparations from inhibit  CBZ  formation clearance of CBZE  Similar results were obtained in l i v e r  animals  pretreated  metabolism and  with  plasma protein  CBZ.  VPA  binding in  will rhesus  monkeys (Levy et a l . , 1984).  3.  Clinical  significance  This interaction important  one  responsible  between  since  for  some  to determine  hepatotoxin, 4-ene enzyme inducing  but  with VPA i f the  CBZ  is a  clinically  metabolites  fatal,  agent such  as CBZ.  This of  study  the  of will  potential of an  Also, we w i l l be able to  specific  induction of  be  occurrences  usage.  formation  may  VPA, i s increased in the presence  characterize i f CBZ induces general, overall  and  of VPA's  the rare,  hepatatoxicity associated allow us  VPA  pathways  VPA metabolism.  or  causes  a  The purpose of  t h i s study i s to investigate the effects of CBZ on the kinetics of VPA  and i t s metabolites i n normal,  steady state. determined  As  well,  the  in one volunteer.  41  kinetics  healthy of  subjects at  Dg-VPA  will  be  OBJECTIVES  1.  The GCMS  developed in  assay for  valproic  our laboratory  proposed modifications internal standards.  w i l l be  include  As  acid  and  i t s metabolites  further modified.  experimentation  well, several  with  derivatizing  The various  reagents  w i l l be tested and compared to the reagent currently used.  2.  The interaction between VPA and CBZ  volunteers w i l l obtained  before  analyzed. VPA w i l l before  be characterized. and  after  be determined. and  after  and  administration  urine  of  CBZ  samples will  be  CBZ  Kinetic parameters w i l l be administration  for  VPA.  determined As  well,  metabolic clearances w i l l be calculated for each  metabolic pathway  before and  fraction metabolized also be calculated.  administration.  The  by each pathway before and after CBZ  will  after CBZ  This w i l l allow further elucidation of the  effect of  CBZ on  enable us  to determine  is  Serum  The e f f e c t s of CBZ on the metabolism and kinetics of  formation and  CBZ  in five normal healthy  particular metabolic  concentrated on  i f the  pathways.  This  will  induction of VPA metabolism by  s p e c i f i c metabolic  pathways, or  is a  general o v e r a l l e f f e c t .  3.  The effect of substituting hexadeuterated VPA  be determined of stable  in one  healthy volunteer.  i s o t o p i c a l l y labelled  42  VPA are  The  for VPA  will  pharmacokinetics  to be  determined in  t h i s volunteer. i n the  This  w i l l allow the i d e n t i f i c a t i o n of  metabolic pathways  due to  isotope e f f e c t s .  u r i n e samples w i l l again be analyzed f o r t h i s  43  shifts  Serum and  investigation.  II.  EXPERIMENTAL  A.  REAGENTS AND MATERIALS  1.  Valproic acid and metabolites Valproic acid  and K  (di-n-propylacetic acid) was obtained from K  Fine Chemicals, ICN Pharmaceutical  metabolites, 2-ene VPA, 5-OH  VPA, 3-ene  VPA, 3-keto  and 2-propylsuccinic calibration  curves  VPA, 4-ene  The  VPA, 3-OH VPA, 4-OH  VPA, 4-keto VPA, 2-propylglutaric acid, acid used  were  for the  synthesized  (Acheampong et  a l . , 1983).  our laboratory  as reported  preparation  as  reported  of the elsewhere  2,3'-Diene VPA was synthesized in elsewhere (Acheampong  1985) as was 2,4-diene VPA (unpublished  2.  (Plainview, NY).  and Abbott,  data).  Internal Standards 3-Octanone (99%) and 2-methylglutaric  from the acid  A l d r i c h Chemical  (caprylic  Biochemicals et a l . ,  Corporation  1984) and  synthesized as acid,  acid)  HA)  (Rochester,  was  synthesized in  Company (Milwaukee,  was  purchased  D -2-ene VPA  from  thesis, 1985).  44  by  WI).  Octanoic  the  National  a l . , 1986a)  were  Hexanoic acid (n-caproic Eastman  Di-n-butylacetic laboratory  obtained  Dg-VPA (Acheampong  (Abbott et  3  purchased  our  from  (Cleveland, Ohio).  previously reported.  N.Y.).  acid were  Andrew  Organic acid  Chemicals  (DNBA)  Acheampong  was  (Ph.D.  3.  Reagents Chemicals and drugs were obtained from the following  sources: ALDRICH CHEMICAL COMPANY (Milwaukee, WI, U.S.A.). tBDMCS, t ert-butyldimethylsilylchloride, MTBSTFA, N-(f  97% purity.  ert-butyldimethylsilyl)-N-methyltrifluoroacetamide  ,98 % purity. D i a z a l d , N-methyl-N-nitroso-p-toluenesulfonamide. R  DMAP, dimethylaminopyridine, 99% p u r i t y . 2-Methylglutaric a c i d . Pyridine.  BDH CHEMICALS (Canada). Anhydrous sodium sulphate. C i t r i c acid  anhydrous.  Sodium hydroxide. Water, g l a s s - d i s t i l l e d - g r a d e .  CALEDON (Georgetown,  Ontario).  A c e t o n i t r i l e HPLC grade. Dichloromethane HPLC grade. Ethyl acetate d i s t i l l e d - i n - g l a s s grade. Methanol d i s t i l l e d - i n - g l a s s grade. Methanol HPLC grade.  45  FISCHER SCIENTIFIC LTD (Canada). Creatinine. Hydrochloric  acid.  PIERCE CHEMICAL COMPANY (Rockford, I l i n o i s , MSTFA,  U.S.A.).  N-methyl-N-trimethylsilyltrifluoracetamide.  SIGMA CHEMICAL COMPANY (St. Louis, MO, U.S.A.). P i c r i c acid saturated  4.  solution.  Drugs  ABBOTT PHARMACEUTICALS (Canada). Valproic acid 50 mg/mL syrup (Depakene ). R  CIBA-GEIGY Ltd. (Canada). Carbamazepine 200 mg tablets (Tegretol *). 1  Carbamazepine standard. Carbamazepine-10,11-epoxide. 1O-Methoxycarbamazepine  standard.  B.  DRUG INTERACTION STUDY  1.  Volunteer Details Five  healthy  male  participated in the study been  obtained.  volunteers  (21 -  48  years old)  for which human ethics approval had  The volunteers  46  were  not on  any  chronic  medications and were asked to abstain from smoking and drinking during the  study.  They were  also  asked  not  to  take  any  medications and to inform the study supervisors i f the occasion arose that  other medication was required.  Blood chemistry and  l i v e r function tests were performed prior to, during, and after the study in a l l volunteers. Volunteer weights follows: mg; RM  and t o t a l  daily dosages  of VPA were as  BA 70 kg, 1100 mg; FS 70 kg, 1400 mg; MS 75 kg, 1200 65 kg,  received VPA mg/kg/day  1000 mg and WT 80 kg, 1200 mg. 15 mg/kg/day  (mean  VPA  dose  Medication administration kinetic studies  16.4  volunteer (FS) received 20  mg/kg/day)  times were  were performed  Administration of study days  and one  the drug(s)  and reinstated  Four volunteers  in  0800 h  on days  form.  and 2000 h.  The  9 and  25.  7 -  was interrupted  after 48  syrup  23 -  on the kinetic  h for the f i r s t  kinetic  study.  Blood was c o l l e c t e d prior to the morning dose on days 7  and 23  after overnight fasts and 0.5, 1, 1.5, 2, 2.5, 3, 5, 7,  9, 12, 24, 30, 36, and 48 h after the dose.  Urine samples were  collected in  12  h,  24  -  2 hour  blocks overnight, overnight (36  blocks for the f i r s t  in 6  - 48  h).  h  blocks  to 200  the sodium  h,  and  On day 16 the evening dose of CBZ  mg for a t o t a l daily dose of 300 mg.  days 23 - 25, the study from days 7 - 9 FS also  36  On day 9, CBZ 100 mg twice d a i l y was  added to the dosing regimen. was increased  between  convenient  On  was repeated.  received six doses of Dg-VPA in l i q u i d form where salt was formed by adding excess a l k a l i  47  (1.70 mL of  3N NaOH)  and then  adjusting the pH to nL).  (approximately 300  The solution  7.4 - 7.8 with 4N HC1 was then  administered  neat. The doses were taken at the following times: h; day  9, 0800 h; day 25, 2000 h; day 26, 0800 and 2000 h, and  day 27, the  day 8, 2000  0800 h.  same  The kinetic  protocol  as  study was performed according to  above  on  days  27  -  29 following  administration of four doses of the deuterated drug. the decline  of Dg-VPA  was observed  On day 9,  only for 12 hours after 2  v  doses had been administered. Blood samples  were c o l l e c t e d  vacutainers. For  ease of  number of venepunctures, catheters placed with .sterile  in s t e r i l e ,  nonheparinized  blood c o l l e c t i o n and to decrease the  the volunteers had indwelling f l e x i b l e  into an  arm vein.  normal saline  The catheter was flushed  (without preservatives)  and  then  locked with 1 mL of heparin 100 U/mL solution between sampling. Samples were serum. The stored at  allowed to c l o t and then centrifuged to y i e l d the serum was  -20 °C  recorded and  u n t i l analysis.  a homogenous  c o l l e c t e d following concommitantly of  the  5 %  transferred to  aliquot  s t e r i l e vacutainers, and  Total was  urine volumes saved.  Saliva  were was  stimulation with 5 % c i t r i c acid solution,  with the blood c o l l e c t i o n for the f i r s t  kinetic study.  12 hours  The procedure was to administer 4 mL of  c i t r i c acid which was held in the mouth for 2 min and then  removed.  The  Approximately  5  saliva to 10  was  collected  between  2 - 3  mL aliquots were c o l l e c t e d .  48  min.  Saliva and  urine samples  were also  stored under  the same  conditions as  serum samples.  C.  ANALYSIS  1.  Valproic acid and metabolites  1.1.  Stock solutions of internal standards Stock solutions  of a l l internal standards were prepared in  methanol ( d i s t i l l e d - i n - g l a s s d i l u t e d to distilled  100 mL water  methylglutaric butylacetic  to y i e l d of  acid  (OA) and  internal standards prepared  nq/mL  r e f r i g e r a t o r when  The  be  concentrations  in  D -2-ene, 3  acid  working  One  as  DgVPA,  (HA), and  nq/mL  2-  di-n-  concentrations  3-octanone (OCT) were 500  of  and 1  hundred uh- of each of the required  were added  fresh  for  hexanoic  (DNBA).  mg/mL, respectively.  were  100  that 1 mL could  the working  (MGA),  acid  octanoic acid  grade) such  to each  needed  and  tube. were  Stock solutions stored  in the  not required for use. To decrease pipetting  errors the required internal standards were mixed such that the amounts required for each sample could be added  1.2.  simultaneously.  Preparation of urine and serum standards A set of references was prepared for both urine and serum.  The 4-OH  VPA lactone, 5-OH VPA lactone, 3-keto VPA ethyl ester  and 2,4-diene VPA ethyl ester were dissolved in 3N NaOH (over 3 days with  constant  s t i r r i n g ) and the other metabolites, namely  49  2-ene VPA,  3-ene VPA,  2,3'-diene VPA,  4-ene VPA,  4-keto VPA,  2-PSA,  and VPA were dissolved in methanol  in-glass grade).  A  bulk stock  solution of  prepared in either control urine or serum.  2-PGA,  (distilled-  standard  5  was  For the c a l i b r a t i o n  curve 0, 200, 400, 600, 800 and 1000 ML of standard 5 were made up to  1 mL with either control urine or serum depending on the  samples.  The  obtained in 800,  and  concentrations  urine were  1000  500 ug/mL,  uq/mL,  of  as follows:  VPA  and  4-OH  2-PGA, 2,3'-diene  trans VPA  20, 40,  1.2, 1.8,  2.4, and  60, 80, 3  metabolites  100,  200,  VPA, 2,4-diene  18,  and 250  and  nq/mL  concentrations were 2,3'-diene VPA,  and  30  uq/mh,  2-PSA 4,  5-OH  8, 12,  as follows:  and 2,4-diene  VPA  50,  16, and  1.6, and  0.9, 1.2, 2.0  (iq/mL,  and  4-OH  and 1.5  vq/mL, 100,  20  and  2-ene  4-keto  150,  200,  Serum  uq/mL.  VPA, 2-ene  trans VPA,  VPA 4, 8, 12, 16, and 20 *zg/mL,  2-ene c i s VPA 0. 1 2, 0.24, 0.36, 0.48, and 0.6 0.3, 0.6,  400,  4-ene VPA 0.3, 0.6, 0.9, 1.2, and  uq/mh,  VPA  24,  thus  2-ene c i s VPA 0.6,  uq/mh,  3-ene VPA 0.2, 0.4, 0.6, 0.8, and 1.0  12,  300,  VPA,  1.5 yg/mL, 6,  VPA  3-keto VPA 200, 400, 600,  VPA  and 100  and  jug/mL, 3-ene  uq/mL,  4-ene VPA  VPA 0.4, 0.8, 1.2,  VPA 25, 50, 75, 100, and 125  uq/mL,  3-keto  VPA 20, 40, 60, 80, and 100 /zg/mL, 4-keto VPA, 2-PSA, and 2-PGA 0.2, 0.4, 10  0.6, 0.8, and 1.0 ag/mL and 5-OH VPA 2, 4, 6, 8, and  uq/mL.  The c a l i b r a t i o n area r a t i o s  curves were generated by p l o t t i n g the peak  of metabolite  versus concentration  or VPA  to  the  internal  standard  of VPA or the particular metabolite.  50  D3-  2-ene c i s VPA was used as the internal standard for VPA and a l l metabolites except  2-PSA  internal standard  for the  Hexanoic acid metabolites  was  used  derived  Standard curves  and  2-PGA.  MGA  two dicarboxylic  as  from  the the  were prepared  internal  human  was  used  as  the  acid metabolites. standard  metabolism  of  for a l l Dg-VPA.  and injected with each batch of  samples.  1.3.  Extraction  patient  and  of  standards  and  samples  As i l l u s t r a t e d added to  in figure  2, the  internal standards  were  the standard ( t o t a l volume 1 mL) or to the b i o l o g i c a l  sample (serum/urine,  t o t a l volume  1 mL),  adjusted to pH > 12  NaOH (approximately 100 uL) and heated for one hour at  with 3N 60 °C  derivatization  to hydrolyze  available, the and the  If one mL was not  sample was d i l u t e d to 1 mL with d i s t i l l e d water  dilution  calculations.  the conjugates present.  factor  taken  After cooling  into  consideration  to room temperature,  for the  the samples  were a c i d i f i e d to pH < 2 with 4N HC1 (approximately 120 uD and allowed to  e q u i l i b r a t e for  15 minutes.  The samples were then  extracted with 3 mL ethyl acetate ( d i s t i l l e d - i n - g l a s s grade) by gentle rotation  for 30  minutes.  twice at the slowest speed possible stubborn emulsions. for 30  Serum samples were extracted to prevent the formation of  Serum samples also required centrifugation  minutes at 2500 rpm and 14 °C to separate the emulsions  formed during  extraction.  The  51  organic  phase  (top  layer,  Serum/Urine 1 mL Internal  octanoic acid 2-methylglutaric acid VPA-D  Standards  100 uL  6  2- ene  VPA-D3  3 - octanone pH > 12.5 Heat for 1 h at 55-60 °C  I  pH 2 with 4N HCl  I  Extract with ethyl acetate, 3 mL  I  Organic  I  layer  Dry over anhydrous sodium sulphate  i  Dry to 200 ML under N  J  2  Derivatize tBDMCS reagent with 5 % catalyst 60 uL  \ Heat for 4 h at 60 °C  I  Inject Figure 2.  1 ML into GCMS  Extraction and d e r i v a t i z a t i o n procedure for valproic acid and metabolites from serum or urine.  52  approximately 2 samples)  was  mL for urine  transferred  samples  and  to another  tube  4  mL  and  for serum dried  over  anhydrous sodium sulphate by vortexing for one minute, followed by centrifugation was then  for 10 minutes at 2500 rpm.  transferred to a  approximately 200 concentrated reagent.  sample  added and  was  For tBDMCS  containing 5 four hours  uL under  tube  and  concentrated  a gentle  nitrogen  derivatized  with  derivatives,  % catalyst  at 60  third  The supernatant  (DMAP) was  60  uL  stream.  to The  the appropriate of  the  reagent  added and then heated for  °C. For TMS derivatives, 30 ULL of MSTFA was  heated at  60 °C  for 20  - 30  minutes.  For tBDMS  derivatives from  MTBSTFA, 60 uL of reagent were added but only  30  heating  minutes  of  d e r i v a t i z a t i o n at  time  were  required  for adequate  60 °C. One ttL of the derivatized, extracted  sample was then injected into the GCMS.  1.4.  Preparation of tBDMCS reagent with 5 % catalyst The d e r i v a t i z i n g  reagent used in the analysis was prepared  by dissolving 50 mg of dimethylaminopyridine (catalyst) in 1 mL of dry pyridine. and mixed  This mixture was then added to 1 g of tBDMCS  thoroughly by  t i g h t l y capped  to prevent  vortexing.  The reagent  reaction with  and was prepared fresh as required.  53  was  atmospheric  stored moisture  2.  Carbamazepine  and carbamazepine-10,11-epoxide in  serum 2.1.  Preparation of stock solutions  A concentrated stock solution of CBZ 25 mg in 25 mL of HPLC grade methanol  was prepared.  methanol to y i e l d stock  This  solutions  of  2, 4,  further  d i l u t e d in  solutions of 100, 200, 300, 400, 600,  and 800 jxg/mL concentrations. diluted in blank serum  was  These samples were then further  and in methanol to give the  6, 8,  12 and  16 uq/mL  of  CBZ  working f o r the  c a l i b r a t i o n curves. A concentrated concentration of  stock solution 1.5 m'g/mL  of CBZE  in HPLC  was prepared  grade methanol.  at a  This was  diluted to y i e l d stock solutions of 50, 100, 150, 200, 300, and 400 uq/mL. and in uq/mL  The working solutions were then prepared in methanol  blank serum  at concentrations  of 1, 2, 3, 4, 6, and 8  of CBZE.  A  concentrated  stock  carbamazepine (MCBZ, grade methanol  solution  of 1 mg/mL  internal standard)  and was  further diluted  10-methoxy-  was prepared to y i e l d  in HPLC  the working  solution of 40 Mg/mL in HPLC grade methanol. A l l stock  solutions and working solutions were kept frozen  u n t i l required. Standard curves  were prepared  and 16 uq/mL of CBZ and 1, 2, Four Mg sample.  (100 ML) Peak area  of the  to contain  2, 4, 6, 8, 12,  3, 4, 6, and 8 uq/mh of CBZE.  internal standard were added to each  ratios of CBZ or CBZE to internal standard  54  were plotted  versus concentration  of CBZ  or CBZE  to prepare  c a l i b r a t i o n curves.  2.2.  Extraction of serum samples for CBZ and CBZE The  HPLC  assay  developed by  Elyas et  analysis of  carbamazepine  a l . (1982)  to the  and  i t s epoxide  was modified  serum samples (figure 3).  collected prior days 7  for  and used  as for  Volunteer serum samples  morning dose, 3 h and 5 h post dose on  and 23 were analyzed for CBZ and CBZE l e v e l s .  A 250 uL  aliquot of serum (either standard or patient sample) was placed into a  test tube  and 125 uL of 4N NaOH.  internal standard, extracted with rotation for rpm for  discarded and second tube. a  3 mL  gentle  The  The sample was then centrifuged at 2500 top layer  the bottom  layer  (aqueous) was (organic)  aspirated  transferred  and to  a  The organic layer was evaporated to dryness under  nitrogen  reconstituted  The sample was then  of dichloromethane (HPLC grade) by gentle  10 min.  7 min.  were added four u.g (100 uL) of the  to which  with  stream 200  uL  in  a  water  bath  of  acetonitrile  at  (HPLC  40  °C,  grade),  evaporated and reconstituted again with 200 uL of a c e t o n i t r i l e . Ten ttL were injected into the. l i q u i d chromatograph.  Total run  time was under 10 minutes.  3.  Determination of urinary creatinine Urinary  creatinine  colourimetric method.  levels This  were  involved  55  the  determined chemical  by  a  reaction  Plasma/Serum 250 iiL 4 uq 10-MCBZ (I.S.) 125 uL 4N NaOH 3 mL dichloromethane Extract for 10 min Centrifuge at 2500 rpm for 10 min  I  Organic layer (discard aqueous layer)  \  Evaporate to dryness under N  i  2  Reconstitute with 200 ML a c e t o n i t r i l e  I Evaporate to dryness under N  2  I Reconstitute with 200 ixL a c e t o n i t r i l e  \ Inject 10 uL into l i q u i d  chromatograph  Modified from A.A. Elyas et a l J . Chromatogr. 1982;231:93-101  Figure 3.  Extraction procedure for carbamazepine and carbamazepine-10,11-epoxide from serum.  56  between p i c r i c complex.  acid and  creatinine to  form  a  red coloured  The absorbance was measured at 500 nm on a Spectronic  20 spectrophotometer. range of  100 -  analyzed with were diluted  Standard  curves were  400 mg  creatinine per  each batch  of patient  prepared over  100 mL  of  samples.  urine  a  and  Urine samples  1/200 (0.5 mL in 100 mL d i s t i l l e d water) and 2 mL  of the d i l u t e d sample analyzed.  4. Instrumentation 4.1.  Valproic acid and metabolites The assay  system.  was performed  on a  Hewlett-Packard 5987A  GCMS  Operating conditions for tBDMS derivatives were source  and injection  port temperatures  temperature of  of 240  °C and  an  interface  270 °C. Helium (carrier gas) flow was 1 mL/min  and the operating electron  ionization  energy  for the mass  spectrometer was 70 eV. An OV 0.32 mm  1701 (0.25 u) bonded phase c a p i l l a r y column, 25 m x I.D., (Quadrex S c i e n t i f i c , New Haven, Connecticut) was  used for the analysis. derivatives was increasing by 100 to  Temperature  i n i t i a l column 30 °C/min  230 °C  and held  oven  programming temperature  for tBDMS of  50 °C,  from 50 to 100 °C, then 8 °C/min from at 260  °C for two minutes post run.  Total run time was approximately 18 minutes. The selected scanned were m/z 197  ion monitoring  mode  was  used.  The  ions  m/z 100 (4-OH VPA lactone), m/z 128 (3-octanone),  (dienes), m/z  199 (enes),  57  m/z 201  (VPA and octanoic  a c i d ) , m/z  202 (D -2-ene  and 4-keto VPA), m/z VPA and  207 (D -VPA), m/z 215 (36  317 (2-methylglutaric a c i d ) , m/z 331  2-PSA) and m/z 345 (2-PGA).  for determining VPA.  VPA), m/z  3  metabolites from  The same program was used  the metabolism of deuterated  The ions selected for scanning included m/z  lactone), m/z  128 (3-octanone), m/z  202 (deuterated  (5-OH  173  103 (4-OH VPA  (hexanoic acid),  m/z  2,4-diene VPA), m/z 203 (deuterated 2,3'-diene  VPA), m/z 204 (deuterated 4-ene VPA), m/z 205 ( c i s and trans 2ene VPA, m/z 221  3-ene VPA),  m/z 207  (D -VPA), m/z 218 (4-keto VPA), g  (3-keto VPA), m/z 229 (DNBA), m/z 334 (2-PSA), m/z  336  (5-OH), and m/z 348 (2-PGA).  4.2.  Carbamazepine and carbamazepine-10,11-epoxide A Whatman PartiSphere 5 C18 column, p a r t i c l e size 5 urn, 110  mm length, Model  O.D.  110A  7.94  pump  wavelength, at Injector were  mm,and I.D. 4.70 mm was used.  and  215 nm)  160  Absorbance  and  a  Waters  detector Associates  used with an Altex Model C-R1A  A Beckman (variable Model  UGK  Recorder. Mobile  phase used was acetonitrile.water (35:65) at a flow rate of 1.1 mL/min.  5.  Statistical  analysis  S t a t i s t i c a l analysis was performed using Student's paired t test comparing CBZ.  results before  S t a t i s t i c a l analysis  and after the administration of  was performed  58  using the Michigan  Interactive Data  Analysis System  (MIDAS) on  Significance level chosen was p <  6.  the MTS  system.  0.05.  Pharmacokinetic model development and calculations The pharmacokinetic model employed to study the interaction  between VPA and CBZ was based on the model used to describe the interaction between M.Sc.  thesis,  Levy et The  VPA and  1985).  ASA  (Abbott et a l . , 1986b; Kassam  This model was based on one reported by  a l . (1983) for the clobazam-carbamazepine interaction.  model,  shown  elimination from  in  the  figure  central  4,  is  a  linear  compartment.  model  It  with  allows  the  c a l c u l a t i o n of formation and elimination (metabolic) clearances of VPA  metabolites.  through a  these,  the  fraction  metabolized  p a r t i c u l a r route may also be determined.  i n t e r v a l , T, was As  From  shown  The dosing  12 h for a l l calculations. in  concentration of  Figure  VPA  in  4,  Cp  S S  is  the  the central compartment.  steady Cp  S S  state can be  calculated from the following equation:  Equation 1.  C  p s s  = VPA  AUCT  /T  T = 12 h  ko i s defined as the dosing rate of the drug which was every 12 h for  t h i s study.  over t h i s  Hence,  i n t e r v a l , and  AUC values reported are calculated  urinary recoveries  same i n t e r v a l .  59  are also over the  .^remainder (not measured)  m2 3-ene VPA 2,3-diene VPA  L  -m3 4-ene VPA 2,4-diene VPA  L  c  m4 4-OH VPA 4-KetO VPA 2-PSA  m2  m3  c  5-OH VPA 2-PGA  Figure 4.  m4  L  * m5 cl  Pharmacokinetic model applied in the valproic acidcarbamazepine study in healthy volunteers.  60  Total body clearance of VPA (Clp), i s defined as the sum of all  formation  clearances  (unmeasured clearances, C l  Cl c  p  =  Cl  +  f l  Cl  +  f 2  Cl  r e m a  (Clf)  plus  i  as shown below:  +  f 3  n c  :  e r  Cl  )  +  f 4  Cl  unknown  +  f 5  Cl  clearances  f 6  +  Cl  r  +  *remainder  cl  remainder  was not  m  a  v  b e  d r u  measured.  9  lost through the b i l e and feces which  Clp can  be calculated from the dose given  and the AUC value for the drug over the dosing  Equation 2.  Clp = Dose/AUC  T  m , m, 1  interval:  m,  2  and 1115 represent the sum of the individual  m,  3  4  metabolites in a given pathway. m1'  m2'  c  m3'  c  m4,  c  concentrations of  C  n  d  m5  c  the metabolites  described previously. serum AUC  a  for each  These  a  r  e  t  h  steady  e  in the  state  respective pathways  are calculated  from  the  total  pathway divided by the dosing interval (12  h).  Equation 3.  Cl  m 1  C i =  , Cl  m 2  ,  or elimination determined  AUCj/T  m  Cl  m 3  , Cl  m 4  ^  Cl  m 5  , and C l  m 6  are the metabolite  clearances for pathways 1 through 6.  These are  from the t o t a l serum AUC for a given pathway and the  61  t o t a l amount  recovered in  the urine  for a given pathway over  the same time period.  Equation 4.  c  The formation for the  ^mi  clearances C l f  metabolites are  steady  nti/AUCi  =  C l f 2 ' d f 3 » Clf4* and C l j g  l f  determined  from  state  concentrations  of  multiplied by  the metabolite  clearance  the r a t i o of average  metabolite  to  for  parent  that  drug  particular  pathway.  Equation 5.  c l  However, the  f i s s = miss • c  formation clearance  c l  mi/ p c  can also  V P A  ss  be calculated from  the following equation:  Equation 6.  c l  fiss  =  m  i/  A U C  vPA  This equation i s derived from the following sequence: c l  fiss  and C l and C  mi  and C  p  =  c  m i  miss • ^mi/ PvPAss c  =  c  mi/ADC^  = AUC/T =  AUC  VPA  therefore, C l f j  s s  /T = m  =  i/  A U C  mi/ pss c  mi/T/Cp mi/AUC  VPA  62  x  A U C  mi/  T  The f  m3'  f  fraction  m4'  f  m5'  f  metabolized by a p a r t i c u l a r  m6  a  n  d  f  Clr  w  a  s  determined  following equation:  Equation 7.  f  m i  =  63  Cl  f  i  s  s  /Cl  p  s  s  pathway,  f i'^m2' m  a c c o r d i n g to  the  III.  A.  RESULTS  ASSAY DEVELOPMENT  The assay developed  for the analysis  in our laboratory  this study  (Abbott et  of  was further  a l . , 1986a).  uses selected  ion monitoring  fragment from  tBDMS derivatives.  ene VPA  were the  VPA  mode  and  14  metabolites  modified for use in  I t i s a GCMS assay which to  monitor  the  [M-57]  +  3-0ctanone, Dg-VPA and D 3 - 2 -  internal standards  used.  Peak area ratios  were calculated using D -VPA for VPA and D3~2-ene trans VPA for 6  the metabolites. concentration  The  ranges  determination ( r ) 2  l e v e l of  c a l i b r a t i o n curves were linear over the measured  the  coefficient  was generally greater than 0.99.  detection of  reproducible and  and  the  the assay relative  metabolites was less than 8 %.  was 0.1 ng/mL. standard  of  The lower  The assay i s  deviation  for most  The r e l a t i v e standard deviation  for 2-PSA and 4-OH VPA c a l i b r a t i o n curves exceeded 10 %.  There  was a problem with r e p r o d u c i b i l i t y in d e r i v a t i z i n g 2-PGA.  1.  Modifications to the assay The modifications to the assay included the addition of two  new internal acid.  standards,  Octanoic acid  measuring  Dg-VPA  octanoic  was added  and  monitoring chromatograms  acid as an  i t s metabolites. of VPA  64  and 2-methylglutaric internal standard for The  selected  ion  and metabolites including MGA  and OA as internal standards are shown in figure 5. did not  interfere with  being measured. to overcome  The  was based  chemical properties c a l i b r a t i o n curves  or any  of the metabolites  diacid internal standard, MGA, was added  the r e p r o d u c i b i l i t y  The choice  and 7.  either VPA  OA and MGA  problems of  on s i m i l a r i t i e s of MGA  to the  for 2-PSA  2-PSA and 2-PGA.  in structure, size, and  diacid metabolites.  The  and 2-PGA are shown in figures 6  These curves were reproducible and had approximately 7  % intra-assay  deviation.  D3~2-ene c i s  VPA was  used as the  internal standard for VPA and the remaining metabolites.  2.  Analysis of deuterated samples When  assaying  metabolites  administration, a l l of the octanone were  deuterated  internal standards  discovered to  compounds being analyzed.  after  cause some  except for 3-  interference with the  Octanoic acid was investigated as an  alternative internal  standard for quantitation of  metabolites,  deuterated  obviously not the deuterated  since  be used.  VPA  internal  deuterated  standards  could  Unfortunately, OA d i d not resolve from  analogue of 2,3'-diene VPA.  3-0ctanone usually  serves as an index of the extent of evaporation in each sample. Di-n-butylacetic acid therefore tested deuterated VPA been used in  our  (DNBA) and  as internal  standards  and i t s metabolites.  as internal  hexanoic acid  (HA) were  for quantitation of  These two compounds have  standards in a GCMS assay for 4-ene VPA  laboratory  (Singh  65  et  al.,  1987).  s—  I  I  1  I  1  1  1  i—//  3  4  6  6  7  B  9  10  TIME  Figure 5.  i  •  1  I  13  14  15  16  (min)  Selected ion chromatograms of tBDMS d e r i v a t i v e s of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 3-octanone, 2 = D -2-ene c i s VPA, 3 = D -2-ene trans VPA, 4 = 4-OH VPA lactones, 5 = VPA, 6 = D -VPA, 7 = 4-ene VPA, 8 = 3-ene VPA, 9 = 2-ene c i s VPA, 10 = 2-ene trans VPA, 11 = (E)-2,4-diene VPA, 12 = (E,E)-2,3'diene VPA, 13 = octanoic acid, 14 = 3-keto VPA, 15 = 4-keto VPA, 16 = MGA, 17 = 5-OH VPA, 18 = 2-PSA, 19 = 2-PGA. ( = internal standard). a  a  3  a  3  a  6  a  a  a  0.12-n  CONCENTRATION, JUG/ML  F i g u r e 6.  Calibration curve f o r 2-PSA i n serum using m e t h y l g l u t a r i c a c i d as the i n t e r n a l s t a n d a r d .  67  2-  0.14  F i g u r e 7.  -i  Calibration curve f o r 2-PGA i n serum using m e t h y l g l u t a r i c a c i d as the i n t e r n a l s t a n d a r d .  68  2-  HA was  ultimately used  for the quantitation  of  metabolites, i . e .  for the calculation of  DNBA and HA were  both completely resolved from deuterated VPA  and i t s metabolites and  and metabolites  area  ratios.  showed no i n t e r f e r i n g peaks using the  selected ion monitoring mode. of VPA  peak  deuterated  with  The selected ion chromatograms HA  and DNBA  as  the  internal  standards are shown in figure 8.  3.  Preparation of internal MGA was  not readily  concentration of  100  NaOH in an attempt alkaline solution problems when  soluble  water  at ,a  i n i t i a l l y prepared  in 3N  to overcome these s o l u b i l i t y problems. of the internal  soluble in a mixture  standard,  however,  An  posed  the glucuronide conjugates of VPA  was not desired.  as such  in d i s t i l l e d  and was  uq/mL  hydrolysis of  and metabolites  was used  standards  of ethyl  by Granneman  Although MGA  was  readily  acetate and hexane (70:30) and and coworkers  (1984a) for the  analysis of VPA and metabolites, this solvent mixture was not suitable with successful was HPLC grade  our extraction procedure. to produce  methanol, which  a 10  A method which proved  fold stock solution of MGA in  could then be diluted in d i s t i l l e d  water to obtain the desired working concentration of 100 for the assay.  Subsequently,  a l l of  the other  standard solutions were made up in a similar manner.  69  uq/mL  internal  m/r  197  14 —  m/i  215  m/z 340  o T - / / T -  10  13  —r—  i  14  15  16  TIME (min)  Figure 8.  Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 3-octanone, 2 = hexanoic acid, 3 = 4-OH VPA lactones, 4 = VPA, 5 = 4-ene VPA, 6 = 3-ene VPA, 7 = 2-ene c i s VPA, 8 = 2-ene trans VPA, 9 = (E)-2,4-diene VPA, 10 = (E,E)-2,3'-diene VPA, 11 = di-n-butylacetic acid, 12 = 3-keto VPA, 13 = 4-keto VPA, 14 = 5-OH VPA, 15 = 2-PSA, 16 = 2-PGA. ( = internal standard). a  a  a  a  4.  Derivatizing reagents Various d e r i v a t i z i n g  (TMS  esters),  reagents were  tested including MSTFA  tBDMCS in pyridine reagent  (tBDMS e s t e r s ) ,  and MTBSTFA  with 5  (tBDMS esters).  MTBSTFA are commercially available  and  %  Both  catalyst MSTFA and  are highly  reactive  reagents.  4.1.  Comparison of TMS and tBDMS derivatives TMS and  compared.  tBDMS derivatives Aliquots of  and then  (30 uh)  urine samples  patient urine were extracted  to the assay procedure MSTFA reagent  of patient  for 20  derivatized either  min or  the tBDMCS  were  according with the  in pyridine  reagent with 5 % catalyst (60 iiL) for 4 h. The GCMS was set on selected ion monitoring mode. derivatives, the methyl function 57]  +  peak  +  resulting from  was monitored.  the loss  of a  For tBDMS derivatives, the [M-  was monitored which resulted from the loss of the t-  butyl group. the  [M-15] peak  With TMS  Several  differences were  chromatography.  derivatives  but  approximately derivatives.  The  TMS  30 The  same  esters  min  longer  column  required  compared  noted with respect to  to  run time  was a  18  used  retention min  for both time  of  for the tBDMS  i s required  with  TMS  derivatives in order to achieve adequate resolution of peaks, particularily  of  derivatives required for tBDMS  4-ene  VPA  from  VPA.  heating for only 30  derivatives.  Conversely,  TMS  min compared to 4 h  TMS derivatives were stable for one to  71  two days, while tBDMS derivatives were stable for several weeks when stored at -20 °C. fold  greater  The  sensitivity  metabolites than the TMS Selected VPA  and  tBDMS derivatives possessed 5 to 10  ion  are  shown  In comparing 9), 4-OH  100)  as  present  the  monoderivative (peaks  16, m/z  (figure 10)  4-OH  100)  are evident.  and di-TMS  compared to  figures  lactone the  217).  9  and  10,  of  the  TMS  isomers (peaks 1, isomers  of  With tBDMS  lactone  3-keto VPA  more  the tBDMS  derivatized.  isomers  4-OH  derivatives (peaks 1,  the monoderivative. readily  as  217)  Although TMS results  s t a b i l i t y did use  VPA  m/z  mono-  3-OH  The VPA  derivative  i s very poorly  di-TMS derivative are sharp  in contrast to tBDMS derivatives.  with  formed more  respect  to  readily,  3-OH  VPA,  and the  yield longer  time, decreased s e n s i t i v i t y , and decreased  not make for  The  di-TMS  derivative were 3-OH  esters are  chromatographic run  the  m/z VPA  i s present both as the  The peaks of the 3-OH  (peaks 15, m/z  reagent to  and tBDMS derivatives of in  as  VPA  yielded only  chromatograms  better  unsaturated  derivative (peak 14, and peaks 17, 2 isomers).  tBDMS reagent also  The  the  chromatography  VPA  well  only the  and  esters.  derivatives (figure are  VPA  chromatograms of TMS  metabolites  respectively.  for  the  MSTFA a routine  metabolites.  72  more a t t r a c t i v e analysis  of  derivatizing VPA  and  its  m/2 207  m/z 202  m/z 201  m/z 199  \  I  Ik  m/z 197 m/z 215 m/z 100  8  10  12  14  16  18  20  22  TIME (min)  Figure 9.  Selected ion chromatograms of TMS derivatives of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 4-OH VPA lactones, 2 = (E,E)-2,3'-diene VPA, 3,4 = dienes VPA, 5 = 2-ene trans VPA, 6 = 2-ene c i s VPA, 7 = 3ene VPA, 8 = 4-ene VPA, 9 = VPA, 10 = D -2-ene trans VPA, 11 = D3~2-ene trans VPA, 12 = D -VPA, 13 = 4-keto VPA, 14 = 3-keto VPA TMS monoderivative, 15 = 3-0H VPA (2 isomers), 1 6 = 4 OH VPA (2 isomers), 17 = 3-keto VPA TMS diderivative (2 isomers), 18 = 5-OH VPA, 1 9 = 2 PSA, 20 = 2-PGA. a  3  a  a  6  73  j, ^1  10 TIME  Figure 10.  12  14  m/z  345  j^ni/z  331  m/z  317  —i 16  (min)  Selected ion chromatograms of tBDMS d e r i v a t i v e s of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 4-OH VPA lactones, 2 = 3octanone, 3 = (E)-2,4-diene VPA, 4 = (E,E)-2,3'-diene VPA, 5 = 4-ene VPA, 6 = 3-ene VPA, 7 = 2-ene c i s VPA, 8 = 2-ene trans VPA, 9 = VPA, 10 = D -2-ene c i s VPA, 11 = D -2-ene trans VPA, 12 = Dr-VPA, 13 = 3-keto VPA, 14 = 4-keto VPA, 15 = 3-OH VPA, 16 = adipic acid, 17 = 5-OH VPA, 18 = 2-PSA, 19 = 2-PGA. ( = internal standard). a  a  a  3  a  a  3  4.2.  Comparison of tBDMCS reagent and MTBSTFA reagent The two reagents were compared with respect to s t a b i l i t y of  the derivative  formed and  derivatization. esters, there Using the  heating time  Although  both  were several  of VPA  these  reagents  form  tBDMS  differences noted between the two.  MTBSTFA reagent,  tBDMS derivatives  required for complete  the selected  ion chromatograms of  and metabolites from a patient urine  sample are shown in figure 11.  Chromatographic  conditions were  the same as those used for the derivatives using the reagent in pyridine with  5 %  derivative of  3-keto VPA  keto  catalyst.  monoderivative  MTBSTFA  (peaks 14, m/z  (peak  12,  m/z  provides  a  di-tBDMS  329) as well as the 3215).  Changes  in  d e r i v a t i z a t i o n were not observed for the other metabolites. The heating MTBSTFA was the same heated at  time required  determined by  sample for 60 °C  reagent for  for adequate  d e r i v a t i z i n g extracted  d i f f e r e n t lengths  after addition  0.33,  1,  2, 3,  5, 8,  observed in  metabolite to  D3~2-ene c i s VPA with However,  derivative formed longer heating  h had  (peaks 14,  m/z  peak  the amount  with MTBSTFA  Samples were  12, and area  16 h. ratio  There  was  VPA  or  of  an increase in length of of  was found  3-keto  VPA  di-tBDMS  to increase  with a  time with a corresponding decrease in the mono-  tBDMS derivative than 5  the  of time.  aliquots of  uL of the d e r i v a t i z i n g  of 40  l i t t l e change  heating time.  d e r i v a t i z a t i o n by  (figure 12).  the di-tBDMS 329).  Any  samples heated for longer  derivative of  After  75  12 h  3-keto VPA  present  of heating, a considerable  m/z  201  -16  m/z  207  16  IT.  cr>  m/z m/z  10Q  —17  m/z  331  326  I m/z  TIME (min)  Figure 1 1 .  216  m/z  i  i  12  13  i 14  —i  16  Selected ion chromatograms of tBDMS d e r i v a t i v e s of v a l p r o i c acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 4-OH VPA lactones, 2 = Dg-VPA, 3 = VPA, 4 = 4-ene VPA, 5 = 3-ene VPA, 6 = 2-ene c i s VPA, 7 = 2 - ene trans VPA, 8 = D -2-ene c i s VPA, 9 = Do-2-ene trans VPA, 10 = (E)-2,4-diene VPA, 11 = (E,E)-2,3'-diene VPA, 1 2 = 3- keto VPA, 13 = 4-keto VPA, 14 = 3-keto VPA tBDMS diderivatives ( 2 isomers), 15 = 5 - O H VPA, 1 6 = 2-PSA, 1 7 = 2 PGA. ( = internal standard). a  a  a  3  a  346  10 ->  9-  7-  2  6  \  < UJ  cr < * <  •X  5  2-  -2  /  \ :A  -0  AY  "T~ 0  2-ENE TRANS VPA  5 - O H VPA  4-KETO VPA  2 - E N E CIS VPA  •E  •a2  6  4  2,4-DIENE VPA 8  -1 10  TIME, WEEKS  Figure 12.  Peak area r a t i o of tBDMS derivatives of 2-ene trans VPA, 5-OH VPA, 4-keto VPA, 2~ene c i s VPA, and 2,4diene VPA from MTBSTFA reagent versus heating time.  77  portion tBDMS  of  the d e r i v a t i z e d  study  the s t a b i l i t y  MTBSTFA, s a m p l e s intervals.  Figure  storage,  of  converted all  completely  -20  °C  13 i l l u s t r a t e s  of  3-keto  the  converted  Although  the  derivatization 3 - k e t o VPA  however, t h e mono-  heating  formed  from  MTBSTFA  time  derivative  However,  r e g a r d l e s s of  time  for  complete  (30 m i n ) , t h e c o n v e r s i o n  d i d not the  was  ( f i g u r e 14).  to the d i d e r i v a t i v e  time  was  A f t e r seven weeks o f  mono-tBDMS  reaction  much s h o r t e r  heating  use.  Upon s t o r a g e ,  VPA  MTBSTFA  at  f o r 2-ene c i s VPA,  t o t h e di-TBDMS d e r i v a t i v e ,  monoderivative  increased to  is  reinjected  this  VPA  3-keto  and  a t -20 ° C , few c h a n g e s  t o the d i d e r i v a t i v e .  l e n g t h of the i n i t i a l  feasible  at  2 , 3 ' - d i e n e VPA.  derivative  gradually  upon  make t h i s  use  recommended h i g h l y f o r a n a l y s i s o f o t h e r  B.  as the d i -  tBDMS d e r i v a t i v e formed by  A f t e r s e v e n weeks o f s t o r a g e  Dg-VPA, and tBDMS  of the  were s t o r e d  were o b s e r v e d .  with  was p r e s e n t  derivative.  To  the  3 - k e t o VPA  of  of the  s t o r a g e and  reagent  MTBSTFA  metabolites  more  could  be  o f VPA.  ANALYSIS OF SERUM AND URINE SAMPLES AFTER ADMINISTRATION OF DEUTERATED VPA  Serum and u r i n e Dg-VPA were  samples  subjected  to  f r o m t h e one v o l u n t e e r similar  nondeuterated  VPA and m e t a b o l i t e s .  analyze  data  this  as  steady  78  who  a n a l y s i s as those  state  received  containing  However, i t i s d i f f i c u l t concentrations  were  to not  Figure  13.  Change i n peak a r e a r a t i o o f 3 - k e t o VPA mono- and d i - d e r i v a t i v e t o D3~2ene c i s VPA w i t h increased heating time.  79  UO-i  Figure 14.  Change in peak area r a t i o of 3-keto VPA mono- and di-derivative to D -2-ene c i s VPA with storage. 3  80  achieved in either part data to h will  of the  study.  Even normalizing the  VPA or the t o t a l amount recovered in the urine over 12 not provide  are, thus, values  viable information.  serum trough  (table  2)  concentrations (table  and  comparing quantities  The results presented  12  after  h  urinary  1), serum  recoveries  undeuterated  and  AUC  (table  deuterated  3) VPA  administration following 2 weeks of CBZ therapy. Serum concentrations of Dg-VPA and VPA were 25.74 and 33.85 mg/L after CBZ administration (table 1). The concentrations of 4-OH VPA, higher  2-ene c i s VPA, 2-ene after  Dg-VPA  administration.  trans VPA  administration  The concentrations  and 5-OH than  VPA were  after  VPA  of the other metabolites,  namely, 4-ene VPA, 3-ene VPA, 3-keto VPA, 4-keto VPA, 2-PSA, 2PGA, 2,3'-diene  VPA and  administration.  The  demonstrated  a  concentrations.  2,4-diene VPA were lower after Dg-VPA  AUC  values  similar  trend  The  h  12  for VPA  (table  urinary  2)  and to  recoveries  metabolites serum of  trough VPA  and  metabolites were lower following Dg-VPA administration.  C.  ANALYSIS OF CARBAMAZEPINE AND  CARBAMAZEPINE-10,11-EPOXIDE  IN SERUM  Serum samples CBZ and  from the  f i v e volunteers  were analyzed for  CBZE concentrations by modifying an HPLC method chosen  from the d a i l y dose  l i t e r a t u r e (Elyas of CBZ  et a l . ,  1982).  Since the  total  was increased to 300 mg from 200 mg for the  81  Table 1.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene trans VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum trough concentrations (mg/L) for VPA and metabolites after administration of VPA and Dg-VPA following 2 weeks of carbamazepine therapy for FS.  VPA  D/--VPA  3.417 0.345 0. 179 0.063 6.482 33.85 6.318 0.308 0.148 0.018 0. 123 1 .561 0.822  14.52 0.087 0.119 0.100 8.043 25.74 1 .450 0.242 0.257 0.015 0.016 1 .441 0.259  82  Table 2.  Metabolite 4-OH 4-ene 3-ene 2-ene c i s 2-ene trans VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum AUC values (mg.h/L) for VPA and metabolites obtained after VPA and Dg-VPA administration following 2 weeks of carbamazepine therapy for FS.  VPA  Dg-VPA  48.60 4.547 2. 178 0.784 81 .87 567. 1 88.42 4.605 3.425 0.275 2.010 20.90 10.79  212.3 1 .283 1 .476 1 .346 90.21 399.2 20.86 2.884 3.535 0.179 0.153 16.57 2.796  83  Table 3.  Amount (ymol) of VPA and metabolites recovered in the urine over 12 h after VPA and D6-VPA administration following 2 weeks of carbamazepine therapy for FS.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene trans VPA (unchanged) VPA (conjugate) 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  VPA 663.9 6.632 0.682 2.527 89.15 53.74 1263. 883.4 71 .29 1 003. 22.43 187.8 48.71 1 4.74  84  D -VPA 6  408.5 2.455 0. 164 1.211 41 .74 48.89 677.8 581 .2 52.27 44.90 7.985 4.773 1 5.44 2.224  second week,  the samples  chosen for analysis were post dose  the  C in m  sample, and those acquired  3 and 5 h  on both the  seventh and fourteenth day  following the commencement of CBZ  administration. The l i q u i d  chromatogram  of the internal  methoxycarbamazepine (MCBZ),  extracted  sample i s shown in figure 15.  from  Figures  standard, a  spiked  16 and  10serum  17 are the  chromatograms of CBZ, CBZE, and MCBZ from a spiked serum sample and from a patient sample 3 h post dose after seven days of CBZ 200 mg d a i l y , respectively. The serum for each Table 5  CBZ concentrations  in the six samples analysed  of the five volunteers  are presented  summarizes the corresponding serum  in table  CBZE l e v e l s .  percent r a t i o of CBZE to CBZ i s shown in table 6. the metabolite  4. The  The r a t i o of  to the parent compound was calculated to detect  i f any changes in the r a t i o had occurred during the two weeks. The c o e f f i c i e n t  of determination  curves for CBZ and CBZE was  ( r ) for the standard 2  0.9978 and 0.9975, respectively.  Slopes for the two curves were 0.0813 and 0.0895, respectively.  D.  INTERACTION BETWEEN VALPROIC ACID AND CARBAMAZEPINE  1.  Analysis of serum samples for VPA The  semi-logarithmic  versus time  plots  of  before and after CBZ  shown in figures 18  - 22.  serum  concentration  for a l l five volunteers are  The kinetic  85  VPA  data  for VPA i s  Table  4.  Serum CBZ concentrations (nq/mL) in healthy volunteers after 7 and 14 days of CBZ administration. A t o t a l daily dose of 200 mg was taken for the f i r s t week and 300 mg for the second week. Serum samples are C ^ (prior to morning dose), 3 h and 5 h post dose. m  Volunteer BA  Time  c  c  3.07 (13.0) 3.54 (15.0) 3.58 (15.2)  3.04 (12.9) 3.71 (15.7) 3.62 (15.3)  4.53 (18.4) 5. 1 1 (21.7) 4.67 (19.8)  5h  2.79 (11.8) 3.65 (15.5) 3.52 (14.9)  3.77 (16.0) 4.43 (18.8) 4. 13 (17.5)  5h  3.68 (15.6) 4.34 (18.4) 4.19 (17.8)  4.40 (18.6) 4.70 (19.9) 4.74 (20. 1 )  2.51 (10.6) 2.92 (12.4) 2.96 (12.5)  4.18 (17.7) 4.62 (19.6) 3.86 (16.4)  n  ?  h  n  5h MS  c  9i  RM  WT c  w  5h * numbers  1 4 days  2.37 (10.0)* 2.80 (11.9) 2.71 (11.5)  si 5h  FS  7 Says  n  n  i n brackets indicate  86  c o n c e n t r a t i o n i n Mmol/mL  Table  5.  Serum CBZE concentrations (uq/mL) in healthy volunteers after 7 and 14 days of CBZ administration. A t o t a l d a i l y dose of 200 mg was taken for the f i r s t week and 300 mg for the second week. Serum samples are C ^ (prior to morning dose), 3 h and 5 h post dose. m  Volunteer BA  Time  c  si  0.38 0.38 0.41  n  5h FS  c  ?h 5h  n  MS 5h RM c  w  5h WT  c  ?h 5h  7 days  n  (1 .49)* (1.49) (1 .62)  n  14 days 0.51 (2.02) 0.55 (2.18) 0.54 (2.15)  0.85 (3.38) 0.85 (3.38) 0.87 (3.45)  1.20 (4.75) 1.34 (5.32) 1.34 (5.32)  0.50 0.60 0.61  (1.99) (2.38) (2.40)  0.63 (2.50) 0.78 (3.08) 0.74 (2.94)  0.56 (2.21) 0.62 (2.44) 0.57 (2.26)  0.75 (2.98) 0.77 (3.04) 0.82 (3.26)  0.42 6.42 0.45  0.64 (2.54) 0.69 (2.75) 0.54 (2.16)  (1.67) (1.65) (1.80)  numbers in brackets indicate concentration in Mmol/mL  87  Table  6.  Percent r a t i o of serum CBZE to serum CBZ concentrations in healthy volunteers after 7 and 14 days of CBZ administration. A total daily dose of 200 mg was taken for the f i r s t week and 300 mg for the second week. Serum samples are min ( P i ° to morning dose), 3 h and 5 h post dose. c  r  r  Serum CBZE/CBZ (%) Volunteer  Time  7 days  14 days  5h  15.9 13.4 15.1  16.6 15.5 15.2  5h  28.0 22.9 24.0  27.7 26.2 28.7  18.0 16.5 17.2  16.7 17.5 17.9  15.1 14.2 13.6  17.1 16.3 17.3  16.8 14.3 15.3  15.3 15.0 14.1  BA  FS  MS  h 5h  C m  n  RM 5h WT C  W  5h  88  SO  Oft?  ITCH  o  Figure  15.  L i q u i d chromatogram of 1O-methoxycarbamazepine from a spiked serum sample. Column; P a r t i S p h e r e C ^ (11cm x 4.70mm). Mobile phase; 35% A c e t o n i t r i l e : 6 5 % Water. Flow r a t e 1.1mL/min.  89  ro o.  in  CM  ro CD  ON  QJ  a. ID  jid_L  o  r  F i g u r e 16.  L i q u i d chromatogram of 1O-methoxycarbamazepine, carbamazepine and carbamazepine-10,11-epoxide from a s p i k e d serum sample. Column; PartiSphere C i o (11cm x 4.70mm). Mobile phase; 35% Acetonitrile:65% Water. Flow rate 1.1mL/min. Peak 1) carbamazepine-10,11-epoxide, Peak 2) carbamazepine, Peak 3) 1O-methoxycarbamazepine.  90  en ID  a.  Figure  17.  Liquid chromatogram of a patient serum sample 3 h post dose, after one week of carbamazepine 200 mg daily. Column; PartiSphere C ^ (11cm x 4.70mm). Mobile phase; 35% Acetonitrile:t>5% Water. Flow rate 1.1mL/min. Peak 1) carbamazepine-10,11epoxide, Peak 2) carbamazepine, Peak 3) 10methoxycarbamazepine.  91  Figure 18.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for BA before CBZ ( • ) and after CBZ ( O ) administration.  1-1  1  1  1  1  0  10  20  30  40  Time, h  Figure 19.  Semilogarithmic plot of serum VPA concentration (mg/L) versus time for FS before CBZ ( • ) and after CBZ ( O ) administration.  !  50  10  20  30  —r— 40  —I 50  Time, h  Figure 2 0 .  Semilogarithmic p l o t of serum VPA c o n c e n t r a t i o n (mg/L) versus time for MS before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  10  20  30  40  Time, h  Figure  21.  S e m i l o g a r i t h m i c p l o t o f serum VPA c o n c e n t r a t i o n (mg/L) v e r s u s t i m e f o r RM b e f o r e CBZ ( • ) a n d a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  50  100  Time, h Figure  22.  Semilogarithmic p l o t of serum VPA c o n c e n t r a t i o n (mg/L) versus time for WT before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  summarized in table 7. under the  1  serum concentration  values were  31.4  %)  (Clp, 40.8 were  administration.  2  versus time curve (AUC, 29.5 %)  s i g n i f i c a n t l y decreased  Plasma clearance (k,  Serum h a l f - l i f e (t / » 24.7 %) and area  after CBZ administration.  %) and  elimination rate constant  significantly  increased  after  CBZ  There was l i t t l e change in the mean volume of  d i s t r i b u t i o n (Vd). These trends are summarized in figures 23 27.  2.  Analysis of urine samples for VPA The mean  slightly  amount of  from  806.1  administration while of dose %.  VPA to  decreased 5.32  of VPA % from  unchanged VPA  767.3  over  Mmol  12  (4.80  h  %)  decreased after  CBZ  the amount recovered expressed as percent  administered also  The amount  recovered  decreased 4.77% from 19.09 to 18.18  recovered as the glucuronide conjugate 589.2 to 557.9 Mmol while the amount of  recovered decreased  3.39 %  from 216.9 to 209.5  Mmol after CBZ administration.  3.  Analysis of serum samples for VPA metabolites Representative semi-logarithmic  concentration versus are shown  in figures  the Appendix in mean  time before  section.  and individual  administration.  28 -  39.  plots of  serum metabolite  and after CBZ administration Individual plots are shown in  Figures 40 - 51 i l l u s t r a t e the changes serum AUC  values before and after CBZ  Mean metabolite serum trough l e v e l s before and  97  Table 7.  Valproic acid kinetic parameters for five healthy volunteers before and after administration of carbamazepine.  Parameter  k (h~ )  1/2  Volunteer  % change  BA FS MS RM WT MEAN s .d.  0.047 0.046 0.046 0.045 0.071 0.051 +0.011  0.065 0.062 0.063 0.059 0.085 0.067* +0.011  + 38.3 + 34.8 + 37.0 + 31.1 + 19.7 + 31.4  (h)  BA FS MS RM WT MEAN s.d.  14.71 15.13 15.14 15.35 9.800 1 4.03 +2.374  10.67 1 1 .23 1 1 .02 11.71 8. 160 10.56* + 1 .392  -27.5 -25.8 -27.2 -23.7 -16.7 -24.7  BA FS MS RM WT MEAN  786. 1 796.8 710.7 583.3 498.3 675.0 +130.5  501.4 567. 1 483.6 468.2 358.2 475.7* +75.73  -36.2 -28.8 -32.0 -19.7 -28. 1 -29.6  BA FS MS RM WT MEAN S.d.  0.700 0.879 0.844 0.857 1 .200 0.897 +0.184  1 .097 1 .230 1 .240 1 .068 1 .670 1.263* +0.241  + 56.7 + 39.9 + 46.9 + 24.6 + 39.2 + 40.8  BA FS MS RM WT MEAN s.d.  0.247 0.274 0.246 0.293 0.213 0.255 +0.030  0.241 0.286 0.263 0.278 0.246 0.263 +0.020  -2.43 + 4.38 + 6.91 -5.12 + 15.5 + 3.14  S.d.  p  After CBZ  1  AUC (mg.h/L)  Cl  Before CBZ  (L/h)  Vd (L/kg)  * s i g n i f i c a n t l y different from before CBZ value at p < 0.05.  98  Figure 23.  Plot of VPA after (Day (n=5).  h a l f - l i f e ( t . / , h) before (Day 7) and 23) CBZ administration in volunteers 2  99  1.7 M  WT  DAYS  Figure 24.  Plot of VPA clearance (CI , L/h) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  100  0.090-1  0.085H  -H WT  0.080H  > 0.075 OC  P  2  i<  0.070  00 O  o  MEAN 0.065 H  <  cr O  0.060 H  < z 0.055 H  0.050 H  0.045  DAYS  Figure 25.  Plot of VPA elimination rate constant (Ke, h~ ) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5). 1  101  DAYS  Figure 26.  Plot of VPA AUC (mg.h/L) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  102  0.30-1  0.29  0.28H  -X  FS  "H  RM  x^  0.27-1 MEAN MS  0.26-^  £  0.25-1  0.24 H O >  0.23  0.22  XT 0.21  gure 27.  -r-  10  15 DAYS  20  25  Plot of VPA volume of d i s t r i b u t i o n (Vd, L/kg) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5).  103  100q  i 0  1 10  1 20  1 30  1 40  [ 50  Time, h  Figure  28.  Representative semilogarithmic p l o t of 4-OH VPA c o n c e n t r a t i o n (mg/L) versus time before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  Figure 29.  Representative semilogarithmic p l o t of 4-ene VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  Time, h Figure  30.  Representative semilogarithmic p l o t of 3-ene VPA c o n c e n t r a t i o n (mg/L) v e r s u s time before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  1  0.01-  T -  10  Figure  31.  —r—  20  Time, h  30  i  40  50  Representative semilogarithmic p l o t of 2-ene c i s VPA c o n c e n t r a t i o n (mg/L) versus time before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  Figure 32.  Representative semilogarithmic plot of 2-ene trans VPA concentration (mg/L) versus time before CBZ ( • ) and after CBZ ( O ) administration.  100  Figure  33.  Representative s e m i l o g a r i t h m i c p l o t of 3-keto VPA c o n c e n t r a t i o n (mg/L) versus time before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  Figure  34.  Representative s e m i l o g a r i t h m i c p l o t of 4-keto VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  Time, h  Figure 3 5 .  Representative s e m i l o g a r i t h m i c p l o t of 5-OH VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e CBZ ( • and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  )  1  O)  0.1:  E C  Time, h  Figure  36.  Representative s e m i l o g a r i t h m i c p l o t of 2-PSA c o n c e n t r a t i o n (mg/L) versus time b e f o r e CBZ ( • and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  )  Time, h  Figure  37.  Representative s e m i l o g a r i t h m i c p l o t of 2-PGA c o n c e n t r a t i o n (mg/L) versus time b e f o r e CBZ ( • and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  )  Figure  38.  Representative s e m i l o g a r i t h m i c p l o t of 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n (mg/L) versus time before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  I  10  -T— 20  30  40  Time, h  Figure 3 9 .  Representative s e m i l o g a r i t h m i c p l o t of 2 , 4 - d i e n e VPA c o n c e n t r a t i o n (mg/L) versus time before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n .  50  F i g u r e 40.  P l o t of 4-OH VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n in v o l u n t e e r s (n=5). 116  6-i  F i g u r e 41  P l o t of 4-ene VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  1 17  6.5  DAYS  F i g u r e 42.  P l o t of 3-ene VPA AUC (mg.h/L) b e f o r e CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  118  1.20-1  1.15-  1.10  H  1.05 H  o  B  H  RM  CO  QC 3 O  X CN  0.95  or LU  >  Q  0.90  u Z> <  0.85-I 0.80H  — X 0.75  FS  H •S  0.70  10  15  -r  20  -  WT -1 25  DAYS  F i g u r e 43.  P l o t of 2-ene c i s VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  119  260-,  DAYS  F i g u r e 44.  P l o t of 2-ene t r a n s VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  120  90-1  80 H  70-  6 cn  -X  ZD  O X  MEAN  60H  CN  ui > O o ZD  <  •  MS  &  WT  50  40  30  1  10  15  20  I  25  DAYS F i g u r e 45.  P l o t of 3-keto VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  121  5-i  BA FS 4.5  .X-  in on ZD O X  3.5H  CN  RM  DC LJ > O  o ZD <  MEAN  MS  S  7^  3H  WT  2.5  10  15  ~r20  25  DAYS  Figure  46.  P l o t of 4-keto VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n in v o l u n t e e r s (n=5).  122  F i g u r e 47.  P l o t of 5-OH VPA AUC (mg.h/L) b e f o r e CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  123  0.35  BA  0.30 H  FS  0.25H  X  d to  CC ZD O X  0.20-  CN  CC  ^  Ld >  o o  ZD <  < -  _—K  MEAN  0.15  'S  IK-  0.10  WT MS  H  RM  0.05 10  15  -r— 20  I  25  DAYS  F i g u r e 48.  P l o t of 2-PSA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  124  2.2-.  FS  1.8 H  *  4  X O  1.6  to  cn ZD  o  El  1.4  RM  CN ^^•X  >  O O ZD <  MEAN  A  BA  1.2  MS ffi WT  0.8 H  0.6  10  15  20  2 5  DAYS  Figure 49.  Plot of 2-PGA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  125  50-i  45 H  E...  40 "ffi A  WT BA  35H  CO cc  30  CN  X-  UJ  > O  o ZD 20<  X  FS  . — KI  RM  X-  15 H  10-  10  15  DAYS  Figure 50.  i  20  25  Plot of 2,3'-diene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in volunteers (n=5).  126  11  "1  5  I  I  10  15  1  20  1  25  DAYS  F i g u r e 51.  P l o t of 2 , 4 - d i e n e VPA AUC (mg.h/L) before CBZ (Day 7) and a f t e r CBZ (Day 23) a d m i n i s t r a t i o n in v o l u n t e e r s (n=5). 127  after CBZ  for the five healthy volunteers are shown in table 8  while serum  AUC values  are summarized in table 9. Individual  serum trough and AUC values for the volunteers are found in the Appendix section.  Figures 52 and 53 are  graphical depictions  of the changes in mean AUC values. A s i g n i f i c a n t decrease  in serum trough levels (table 8) for  2-ene trans VPA (15.21 to 11.53 mg/L) was observed. trough concentrations 2-PGA, 2,3'-diene  Mean serum  for a l l metabolites except for 4-OH VPA,  VPA and  2,4-diene VPA  decreased after  CBZ  observed  in mean 2-ene c i s VPA (14.6 %) and  administration. The decreases 2-ene trans  VPA (21.6  were s t a t i s t i c a l l y for  AUC  values  CBZ.  mentioned above,  values after CBZ administration  significant. of  decreased after  %) AUC  the  There was a general tendency  monounsaturated  In addition  4-ene VPA  two diunsaturated VPA,  6.24  %  administration.  2,4-diene  There was  were reduced by 5.60 %  However, the AUC values of the  metabolites were  and  to be  to 2-ene c i s and trans VPA  AUC values  and 3-ene VPA values by 15.4 %.  metabolites  both increased, 2,3'-diene  VPA,  a general  22.7  %,  tendency  following  CBZ  for the  AUC  values of  polar metabolites, namely, 4-OH VPA (11.5 % ) , 4-keto  VPA (29.3  % ) , 5-OH  (22.3 % ) ,  to be  only polar  VPA (2.69  % ) , 2-PSA  increased after  metabolite whose  CBZ.  (23.9 % ) , and 2-PGA The 3-keto VPA was the  AUC value was not increased after  CBZ administration.  128  Table 8.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene trans VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Mean serum valproic acid and metabolites trough concentrations (mg/L) before and after administration of carbamazepine in f i v e volunteers. Numbers in parentheses represent range.  Before CBZ 2.693 0.391 0.302 0.082 15 .21 44 .02 6.299 0.299 1 .014 0.014 0.090 2.391 0.562  After CBZ  (1 .015- 4.681 ) (0. 276- 0.475) (0. 1 520.507) (0. 062- 0. 103) (8. 035- 20 .53) (27 .37- 53 .22) (4. 501- 8. 151 ) (0. 212- 0.391 ) (0. 512- 1 .247) (0. 002- 0.030) (0. 057- 0. 120) (0. 852- 3.624) (0. 510- 0.626)  2.893 0.389 0.248 0.069 11 .53* 27 .01* 5.815 0.288 0.824 0.013 0.094 2.434 0.647  (1 .913- 3.781 ) (0. 334- 0.556) (0. 1 19-0.381 ) (0. 055- 0.095) (6. 482- 15 .33) (16 .64- 33 .85) (4. 421- 7.337) (0. 184- 0.438) (0. 148- 1 .374) (0. 008- 0.019) (0. 057- 0. 136) (1 .059- 3.685) (0. 386- 0.923)  % change + 7.43 -0.51 -17.9 -15.8 -24.2 -38.6 -7.68 -3.68 -18.7 -7.14 + 4.44 + 1 .80 + 15.1  * s i g n i f i c a n t l y different from before CBZ value at p < 0.05.  129  Table 9.  Mean serum AUC (mg.h/L) for VPA and metabolites over 12 h before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  Serum AUC (mg.h/L) Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene trans VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Before CBZ  After CBZ  35.62 (18.60-61.48) 4.555 (3.347-5.620) 3.452 (1.756-6.041) 0.970 (0.751-1.158) 179. 1 (93.41-243.7) 675.0 (498.3-796.8) 65.00 (55.40-77.63) 2.961 (2.139-3.973) 13.78 (11.04-19.82) 0. 1 42 (0.058-0.184) 1 .047 (0.773-1.365) 25. 18 (9.973-45.06) 6.307 (3.688-8.064)  39.70 4.300 2.920 0.828 140.4* 475.7* 62.80 3.830* 14.15 0.176 1.280 26.75 7.740  22 .60-56.15) 3. 364-4.852) 1 .064-4.554) 0. 717-0.999) 81 .87- 195.3) 358.2- 567.1) 28 .65- 88.42) 3. 105-4.834) 3.425-23.77) 0. 072- 0.306) 0. 823-2.010) 12 .15- 37.67) 5. 154- 10.79)  % change +1 1 .5 -5. 60 -15 .4 -14 .6 -21 .6 -29 .5 -3. 38 + 29 .3 + 2.69 + 23 .9 + 22.3 + 6.24 + 22 .7  * s i g n i f i c a n t l y different from before CBZ value at p < 0.05.  130  Figure 52.  Histograms of AUC (mg.h/L)values of p o l a r m e t a b o l i t e s before CBZ ( £ 2 ) a f t e r CBZ ( a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5). a  n  d  _j  200-i  Figure  53.  Histograms of AUC (mg.h/L)values of unsaturated metabolites before CBZ ( £ 2 ) and a f t e r CBZ ( 0 a d m i n i s t r a t i o n i n v o l u n t e e r s (n=5).  Tables 10 and 11 summarize the AUC values as t o t a l of polar and unsaturated  metabolites.  The mean  sum of the AUC values  for the polar metabolites  increased about  121.9  12  mg.h/L  over  the  s t a t i s t i c a l l y significant 183.0  mg.h/L  h  3 %  dosing  decrease of  from 118.6 to  interval  16.6 %  while  from  a  219.5 to  in the sum of the AUC values for the unsaturated  metabolites was observed. To determine affected by  if a  CBZ, AUC  metabolic pathways  particular  pathway  values were  was  specifically  separated with  depicted in figures 54  respect  - 58:  to  pathway 1  includes 2-ene VPA and 3-keto VPA; pathway 2 includes 3-ene VPA and 2,3'-diene VPA;  pathway  pathway 5  VPA; pathway 3 includes 4-ene VPA and 2,4-diene 4 includes  includes 5-OH  glucuronide conjugation cleared through conjugate  4-OH VPA,  as  VPA and and C l  the kidneys. well  as  distinguished separately of the conjugate present  4-keto VPA  2-PGA.  Pathway  i s unchanged  r  In  the free  was  also  VPA  i s VPA  which i s  drug  as  these  were not  in the serum samples since the amount i s very  small.  A mean decrease of in pathway 1 AUC  CBZ administration (table 12) and the VPA pathway  significantly  Pathways 2,  6  the serum, VPA includes the  16.7 % from 245.0 to 204.1 mg.h/L was observed values after  and 2-PSA; and  3, 4,  and 5  decreased  as  discussed  were apparently  previously.  increased after CBZ  therapy by 3.63, 10.9, 12.9, and 3.98 %, respectively. Average steady state concentrations of VPA and metabolites, used later  in the pharmacokinetic  133  calculations,  are shown in  Table  10.  Sum of serum AUC (mg.h/L) f o r p o l a r m e t a b o l i t e s of v a l p r o i c a c i d over 12 h f o r the f i v e healthy volunteers before and a f t e r administration of carbamazepine.  Serum AUC (mg.h/L) Volunteer  Before CBZ  A f t e r CBZ  BA FS MS RM WT MEAN  144.5 97.23 104.0 88.60 158.3 118.6 +41.45  1 37 147, 107 118 101 121 +20.01  S.d.  Table  11.  Before CBZ  A f t e r CBZ  BA FS MS RM WT MEAN  264.7 124.9 240.4 1 60.2 307.5 219.5 +41.45  197.9 121.1 212.2 1 36.0 247.7 183.0* +26.23  significantly  -5.12 + 51.5 + 3.27 + 33.7 -35.8 + 2.78  Sum of serum AUC (mg.h/L) for unsaturated m e t a b o l i t e s of v a l p r o i c a c i d over 12 h f o r the and after five healthy volunteers before a d m i n i s t r a t i o n of carbamazepine.  Volunteer  S.d.  % change  different  % change -25.2 -3.04 -11 .7 -15.1 -19.4 -16.6  from before CBZ value at p < 0 . 0 5 .  134  Table 12.  Pathway  2  1 2 3 4 5 VPA  Mean serum AUC (mg.h/L) over 12 h for valproic acid and metabolites expressed as pathways before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  After CBZ  Before CBZ 245.0 28.63 10.86 38.72 14.83 675.0  (153.5-322. 1 ) (11.73-49.90) (8.023- 13.10) (20.80-64.42) (12.02-21.14) (498.3-796.8)  204.1 (171.1-226.7) 29.67 (13.21-41.59) 12.05 (9.492- 15.34) 43.70 (26.06-59.37) 15.42 (5.535-24.99) 475.7* (358.2-567.1)  % change -16.7 + 3.63 + 10.9 + 12.9 + 3.98 -29.5  Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2,3'-diene VPA. Pathway 3 includes 4-ene VPA and 2,4-diene VPA. Pathway 4 includes 4-OH VPA, 4-keto VPA and 2-PSA. Pathway includes 5-OH VPA and 2-PGA. VPA includes free and conjugated VPA. s i g n i f i c a n t l y different from before CBZ value at p < 0.05.  135  CH -CH -CH 3  CH  2  2  CH CH  3  2  \  (CHCOOH  2  VALPROIC ACID  CHo-CHo-CH  CH - C H 3  CH  — 2  /  CCOOH  2  2-ENE VPA  OH CH -CH -CH 3  2  CHCOOH CH  CH ~CH  3  2  2  3-OH VPA  CH -CH -C 3  2  CHCOOH CH  CH CH _  3  2  2  3-KETO VPA  Figure 54.  Pathway 1 (beta-oxidation of valproic acid)  136  CH -CH -CH 3  2  2  \ CH  CH  3  _ 2  CH  CHCOOH  2  VALPROIC  ACID  CH-5-CH=CH  \ CH  CH  3  2  3-ENE  CH  CHCOOH  2  VPA  CHo-CH=CH CCOOH CH ~CH 3  2  CH  2,3'-DIENE  Figure  55.  Pathway  2  VPA  (dehydrogenation  137  of  valproic  acid)  CH -CH -CH 3  2  2  ^HCOOH  CH  3  CH CH 2  / 2  VALPROIC ACID  CHo=CH-CH  9  \ CH  3  C H  CHCOOH  /  2  C H  2  4-ENE VPA  I CHo=CH-CH  CCOOH CH  3  CH ~CH 2  2  2,4-DIENE VPA  Figure 56. Pathway 3 (dehydrogenation of valproic acid)  138  CH -CH -CH 3  2  2  ^CHCOOH CH —CH —CH 3  2  2  VALPROIC ACID  OH  I  CHo-CH-CHp N  CH -CH -CH 3  2  /  CHCOOH  2  4-OH VPA  PJ CH CH -C-CH  22  3  CHCOOH CH  CH ~CH  3  2  2  4-KETO VPA  I HOOC-CH  2  \  CH CH ~CH  CHCOOH  _  3  2  2  2-PSA  Figure 57. Pathway 4 (CJ-1 oxidation of valproic a c i d ) .  139  CH^ CH2 CH -  _  2  \  CHCOOH  / CH  CH2 CH _  3  2  VALPROIC  OH  ACID  |  I CH2 CH2 CH2 _  _  \  CHCOOH  CH3-CH2 CH 2 —  5-OH V P A  I  HOOC-CH -CH 2  2  ^CHCOOH CH^  CH2 OH2  2-PGA  Figure  58.  Pathway  5 (co-oxidation  140  of valproic  acid)  table 13 dosing  and were  interval  were also  calculated by d i v i d i n g the AUC value by the  of 12  h.  Average steady state  concentrations  expressed in pathways as previously defined and are  summarized in table 14.  4.  Analysis of urine samples for VPA metabolites The amounts  of mean  VPA and  metabolites  recovered  in the  urine in jumol before and after CBZ over a 12 h period are shown in table  15.  Individual urinary recoveries for the volunteers  are in the Appendix  section.  s i g n i f i c a n t l y increased  by 12.5  after CBZ administration. OH VPA,  4-ene VPA,  The mean  urinary recovery  administration. 2,3'-diene VPA (5.07  %)  to 1 .859 Aimol/12 h)  4-keto VPA, 5-OH VPA, 2-PSA, and 2,4-diene  increased although  25.2 %  % ( 1 .652  of 2-ene c i s VPA  Twelve hour urinary recoveries of 4-  VPA also  decreased by  The recovery  they d i d not reach s i g n i f i c a n c e . of 2-ene  trans  VPA  significantly  (117.3 to 87.82 umol/12 h) following CBZ  The mean (13.8 % ) ,  also decreased  12 h recoveries for 2-PGA (39.3 % ) , 3-ene VPA  (43.7 % ) ,  and 3-keto VPA  after CBZ administration although the  decreases d i d not reach s i g n i f i c a n c e . the changes in mean metabolite  Figures 59 and 60 depict  amounts recovered  When the mean amount of metabolites  over 12 h.  recovered  in urine are  expressed as the pathways previously defined, a decrease in the t o t a l amounts  recovered  for pathways 1 and 2 (8.46 and 14.3 %,  respectively) was observed (table 16) after CBZ administration.  141  Table  13.  Mean ' a v e r a g e serum steady s t a t e c o n c e n t r a t i o n s ' (mg/L) f o r v a l p r o i c a c i d and m e t a b o l i t e s b e f o r e and after administration of carbamazepine. Numbers i n p a r e n t h e s e s r e p r e s e n t r a n g e (n=5). 9  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Before 2. 968 0. 380 0. 288 0. 081 14 .93 56 .25 5. 417 0. 247 1 .1 48 0. 012 0. 087 2. 098 0. 526  CBZ  After  (1 . 550- 5. 123) ( 0 . 279- 0. 468) ( 0 . 146- 0. 503) ( 0 . 063- 0. 097) ( 7 . 784- 20 .31 ) (41 .53- 66 .40) (4. 617- 6. 469) ( 0 . 1 78-0. 331 ) ( 0 . 920- 1 .652) ( 0 . 005- 0. 015) ( 0 . 064- 0. 1 14) ( 0 . 831- 3. 755) ( 0 . 307- 0. 672)  3. 308 0. 359 0. 244 0. 069 1 1.70* 39 .64* 5. 236 0. 319* 1 .179 0. 015 0. 107 2. 229 0. 645  CBZ  (1 . 883- 4. 679) (0. 280- 0. 404) (0. 089- 0. 380) ( 0 . 060- 0. 083) (6. 823- 16 .28) (29 .85- 47 .26) ( 2 . 389- 7. 368) ( 0 . 259- 0. 403) ( 0 . 285- 1 .981 ) ( 0 . 006- 0. 025) ( 0 . 069- 0. 168) (1 . 013- 3. 139) (0. 430- 0. 899)  average steady s t a t e c o n c e n t r a t i o n i s c a l c u l a t e d t h e AUC v a l u e by t h e d o s i n g i n t e r v a l .  a  * significantly  different  from b e f o r e  142  CBZ  % change  by  value at p <  + 11.5 -5.52 -15.3 -14.8 -21.6 -29.5 -3.34 + 29.2 + 2.70 + 25.0 + 23.0 + 6.24 + 22.6 dividing 0.05.  Table 14.  Pathway" 1 2 3 4 5 VPA  Mean 'average steady state serum concentrations (mg/L) of valproic acid and metabolites expressed as pathways before and after administration of carbamazepine. Numbers in parentheses represent range (n=5). a  Before CBZ 20.42 2.386 0.906 3.227 1.235 56.25  After CBZ  (12.79-26.84) (0.977-4.158) (0.669-1.092) (1.733-5.368) (1.002-1.762) (41.53-66.40)  17.01 (14.26-18.89) 2.473 (1.101-3.466) 1.004 (0.791-1.278) 3.642 (2.172-4.948) 1.285 (0.453-2.083) 39.64*(29.85-47.26)  % change -16.7 +3.65 +10.8 +12.9 +4.05 -29.5  average steady state concentration i s calculated by dividing the AUC value by the dosing i n t e r v a l .  a  k Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2,3'-diene VPA. Pathway 3 includes 4-ene VPA and 2,4-diene VPA. Pathway 4 includes 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 includes 5-OH VPA and 2-PGA. VPA includes free and conjugated VPA. * s i g n i f i c a n t l y d i f f e r e n t from before CBZ value at p < 0.05.  143  Table  15.  Mean v a l p r o i c a c i d and m e t a b o l i t e s (Mmol) r e c o v e r e d in urine o v e r 12 h b e f o r e and a f t e r c a r b a m a z e p i n e administration. Numbers i n p a r e n t h e s e s r e p r e s e n t r a n g e (n=5).  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA ( t o t a l ) 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  B e f o r e CBZ 295.0 1 .957 0.769 1 .652 117.3 806.1 567.8 44.98 642.3 8.046 185.7 53.54 9.984  * significantly  After  (250.6- 342.0) (1 .056- 4.472) (0.212- 2.555) (0.771- 2.477) (65.07- 154.4) (464.9- 1564.) (29.25- 1642.) (25.21- 74.22) (303.9- 1323.) (5.103- 12.48) (74.44- 528.1 ) (34.27- 84.85) (5.418- 13.99)  different  CBZ  354.7 (217.0- 663.9) 2.818 (1.390- 6.632) 0.433 (0.200- 0.682) 1 .859*(1.047- 2.620) 87.82* (53.35- 102.9) 767.3 (461.9- 1317.) 539.0 (282.3- 883.4) 52.28 (30.60- 71.29) 760. 1 (501.8- 1099.) 10.26 (6.159- 22.43) 112.6 (77.94- 187.8) 46. 13 (22.35- 69.42) 1 0.28 (6.559- 14.74)  f r o m b e f o r e CBZ v a l u e a t p <  144  % change  + 20 .3 + 44 .0 -43 .7 + 12 .5 -25 .2 -4. 80 -5. 07 + 16 .2 + 21 .8 + 27 .5 -39 .3 -13 .8 + 2. 94 0.05.  Table  16.  Pathway  3  1 2 3 4 5 6 Clr a  Pathway Pathway Pathway Pathway Pathway Pathway Pathway  Mean valproic acid and metabolites (umol) recovered in the urine over 12 h expressed as pathways before and after carbamazepine administration. Numbers in parentheses represent range (n=5).  Before CBZ  After CBZ  686.8 (147.3- 1778.) 54.31 (34.64-85.24) 1 1 .94(6.474- 18.46) 348.0 (289.2-373.5) 810.0 (379. 1- 1488.) 589.2 (94.20- 1514.) 216.9 (50.18-550.0)  628 .7 46. 57 13. 10 417 .3 872 .8 557 .9 209 .5  (385.2-975.1) (22.55-69.83) (7.949-21.37) (254.6-757.6) (586.2- 1226.) (187.0- 1263.) (53.74-346.3)  1 includes 2-ene VPA and 3-keto VPA. 2 includes 3-ene VPA and 2,3'-diene VPA. 3 includes 4-ene VPA and 2,4-diene VPA. 4 includes 4-OH VPA, 4-keto VPA and 2-PSA. 5 includes 5-OH VPA and 2-PGA. 6 i s VPA glucuronide conjugate. C l r i s free (unchanged) VPA.  145  % change -8. 46 -14 .3 + 9.72 + 19 . 1 + 7.75 -5. 32 -3. 39  9*  I  F i g u r e 60.  Histograms of mean recovery (nmol) of p o l a r metabolites before CBZ ( g r j j f t e r CBZ ( a d m i n i s t r a t i o n in v o l u n t e e r s (n=5). a  n  d  a  )  The  12 h urinary recoveries of t o t a l metabolites in pathways 3,  4, and  5 increased  9.72, 19.1, and 7.75 %, respectively after  CBZ administration. significant.  None  of the changes were  statistically  Urinary recoveries expressed as pathways for the  individual volunteers are in the Appendix section. The  mean  12  h  urinary  increased approximately administration  while  18).  6 %  (1726 to  the mean  metabolites decreased 17 and  recovery  19.4 %  The decrease  of  polar  1829  Mmol)  recovery  of  metabolites after  unsaturated  from 185.2 to 149.3 Mmol (tables  in the urinary  unsaturated metabolites was s t a t i s t i c a l l y  recovery  of the  significant.  When expressed as a percent of the dose administered 19, figures 4-ene VPA, PSA, and 26.6,  2-ene c i s VPA, 3-keto VPA, 4-keto VPA, 5-OH VPA, 2-  2,4-diene VPA increased 15.0, 43.5, 12.4, 4.10, 17.4,  22.4  and 2.89  % respectively, after CBZ administration.  of 4-ene  The recoveries VPA  (table  61 and 62), the mean urinary recovery of 4-OH VPA,  The recovery  diene  CBZ  increased by 43.5 %.  of 3-ene VPA, 2-ene trans VPA, 2-PGA, and 2,3'-  decreased  respectively, after dose recovered.  VPA s i g n i f i c a n t l y  41.2, 24.6,  4.77,  CBZ administration  The  recovery of  44.0, and  15.2 %  when expressed as % of  2-ene trans  VPA  decreased  significantly. The percent of dose administered which was recovered as VPA and metabolites  in the urine over  (table 20) was approximately  12 h  before and after CBZ  the same (64.6 % versus 66.5 % ) .  148  Table 17.  Volunteer BA FS MS RM WT MEAN s.d.  Table 18.  Volunteer BA FS MS RM WT MEAN S.d.  Sum of polar metabolites of VPA (ttmol) recovered in the urine over 12 h for the five healthy volunteers before and after administration of carbamazepine.  Before CBZ  After CBZ  1005 3486 1 120 1623 1 395 1726 + 1013  1213. 2832. 1 534. 2289. 1 278 1829. +704.8  % change + 20.8 -18.8 + 36.9 + 41 .0 -8.39 + 5.98  Sum of unsaturated metabolites of VPA (Mmol) recovered in the urine over 12 h for the five healthy volunteers before and after administration of carbamazepine.  Before CBZ  After CBZ  107.0 210.0 180,,6 175,,3 253, 4 185, 2 +53.63  * s i g n i f i c a n t l y different  92.56 162.4 177.9 1 29.2 184.6 149.3* +38.26  % change -13.5 -22.7 -1 .50 -26.3 -27. 1 -19.4  from before CBZ value at p < 0.05.  149  Table  19.  Mean v a l p r o i c a c i d a n d m e t a b o l i t e s r e c o v e r e d i n t h e urine over 12 h a s p e r c e n t o f VPA d o s e b e f o r e and a f t e r carbamazepine administration. Numbers i n p a r e n t h e s e s r e p r e s e n t r a n g e (n=5).  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Before 7.305 0.046 0.017 0.041 2.863 19.09 12.68 1 .092 14.87 0.1 96 4.883 1 .307 0.242  * significantly  CBZ  ( 5 . 534( 0 . 028(0. 006(0. 020(1 . 704(11 .44(0. 842(0. 605( 7 . 622(0. 134(1 . 786(0. 897(0. 142-  different  After  9.027) 0.092) 0.053) 0.071 ) 3.705) 32.17) 33.78) 1 .527) 27.22) 0.281 ) 15.21) 2.036) 0.303)  8.399 0.066* 0.010 0.046* 2.158* 18.18 1 3.20 1 .282 18.83 0.240 2.733 1 .1 08 0.249  CBZ  (5. 208- 13 .66) (0. 036- 0. 136) (0. 006- 0. 014) (0. 027- 0. 075) (1 .397- 2. 653) (13 .26- 27 .09) (6. 774- 21 .58) (0. 734- 1 .679) (12 .81- 31 .65) (0. 168- 0. 461 ) (1 .870- 3. 863) (0. 644- 1 .666) (0. 172- 0. 303)  % change + 15 .0 + 43 .5 -41 .2 + 12 .4 -24 .6 -4. 77 + 4. 10 + 17 .4 + 26 .6 + 22 .4 -44 .0 -15 .2 + 2.89  from b e f o r e CBZ v a l u e a t p < 0.05.  150  Table 20.  Percent of v a l p r o i c a c i d dose recovered i n the urine as VPA and m e t a b o l i t e s over 12 h f o r the five healthy volunteers before and a f t e r carbamazepine administration.  Volunteer  Before CBZ  BA FS MS RM WT MEAN  41 .3 108.2 52.4 70.3 60.0 64.6 26.5  S .d.  151  A f t e r CBZ 47.5 88.7 62.6 82.9 50.8 66.5 18.6  39 t  MEAN RECOVERY OF UNSATURATED METABOLITES AS PERCENT OF DOSE  C  to  < — 3  o  X  [SJrr CO c SJCo rr 3 CT O rr L > J  ro ro  CU  M-  CD  >-i 3 rt g oi QJ to 01  0) ,—s CO O 3 l-h CD l-h II rr X oi ro TJ 3 i-i n ro ro co • O 01 3 CO 01 N ro n  ^-  _ •  a. ro  CO cn  o  O <  • ron T J wro  Co o 3  3 H-rt 3 M-  01  O l-h  •<  » 3 O  I—*  '  rr O n Qj r-h 0) 0 rr oi c M'  O 3 3  ro 3 01 t r OJ  CO  rr  O  n o> rr  I-I  (B o ca M  ro a  £91  MEAN R E C O V E R Y OF POLAR METABOLITES lO  c ro  NJ  •  I  < — 3  o „ ft> ti)>— 1—' ^ r r  c O O ) rr tr O 3 rr — O id L  V  C D Q) n> i-l  0)  = rr 3  Ul QJ ro cn ,—~  3  o  0)  ct> rD MI rr m ro *o 3 •—•  II M  •  1  !(  1  ro cu  O w 3  CO(t 10 N  a ^ __o» • 01 •  1  n o o< n>  —- ro »-j  ant: 3 3 3 H-ft Or3 o ^ 01 rr. O  rr  O J o rr oi TJ O  CD O  t-  3 tr 0» M- M l  3  O  n rD  n to  A S PERCENT OF D O S E  Table 21  depicts 12  percent of The data  the t o t a l  3.39 %) (22.0 to  recoveries expressed  as a  amount of VPA and metabolites recovered.  i s graphically  recovery of  h urinary  2-ene trans  presented in  figures 63 and 64.  VPA s i g n i f i c a n t l y  decreased  The  (4.81 to  while the recovery of 5-OH VPA s i g n i f i c a n t l y increased 28.2 %)  4-OH VPA,  after CBZ administration.  3-ene VPA,  decreased after  2-PGA, 2,3'-diene  CBZ administration.  The recoveries of  VPA and 2,4-diene VPA  The  recoveries  of the  other metabolites (4-ene VPA, 2-ene c i s VPA, 3-keto VPA, 4-keto VPA, 5-OH  VPA, and  2-PSA) increased  after CBZ administration  when expressed as a percent of the t o t a l amount recovered. Table 22  shows the  12 h  urinary recovery  of metabolites  expressed as a percent of VPA (unchanged + conjugate) recovered over 12  h.  The recoveries  VPA, 3-keto  of 4-OH VPA, 4-ene VPA, 2-ene c i s  VPA, 4-keto VPA, 5 OH VPA, 2-PSA and 2,4-diene VPA  increased after CBZ administration.  The remaining metabolites,  3-ene  VPA,  VPA,  2-PGA  and  recoveries (figures  2,3'-diene  65 and  exhibited  decreased  66). S t a t i s t i c a l analysis did not  demonstrate any s i g n i f i c a n t differences. Mean urinary before and  creatinine concentrations  (data  not  shown)  after CBZ were 123.6 mg/100 mL and 91.76 mg/100 mL,  respectively.  S t a t i s t i c a l analysis  difference in these two values.  1 54  demonstrated  a  lack  of  Table 21.  Mean v a l p r o i c a c i d and m e t a b o l i t e s (/xmolar b a s i s ) recovered over 12 h expressed as percent of t o t a l recovered before and after carbamazepine administration in five volunteers. Numbers in parentheses represent range.  Compound  Before CBZ  4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene a  actual  12.7 0.07 0.02 0.06 4.81 29.7 18.5 1 .74 22.0 0.31 7.39 2.27 0.39  (5.11-15.9) (0.05-0.08) (0.01-0.05) (0.04-0.10) (2.55-7.27) (22.4-40.4) (1.20-31.2) (1.16-2.13) (14.6-28.3) (0.24-0.40) (3.14-21.6) ( 1 .00-3.99) (0.27-0.53)  After 12.6 0.09 0.02 0.07 3.39* 28.0 19.1 1 .96 28.2* 0.36 4.08 1 .83 0.38  CBZ  (8.66-15.7) (0.07-0.15) (0.01-0.02) (0.06-0.09) (2.07-4.80) (16.0-34.4) (13.3-26.0) ( 1 .45-2.56) (20.5-38.2) (0.21-0.52) (3.31-4.66) (0.77-3.28) (0.34-0.48)  % change -0.79 + 28.6 0.00 + 16.7 -29.5 -5.72 + 3.24 + 12.6 + 28.2 + 16.1 -44.8 -19.4 -2.56  change was -27.3 %. i  * significantly different  from before CBZ value at p < 0.05.  155  a  Table 22.  Mean v a l p r o i c a c i d and m e t a b o l i t e s (/xmolar b a s i s ) recovered over 12 h i n the u r i n e expressed as percent of VPA recovered before and after carbamazepine a d m i n i s t r a t i o n in five volunteers. Numbers i n parentheses represent range.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Before CBZ 44.3 0.23 0.08 0.22 17.3 64.4 6.27 79. 1 1.11 27.0 8.27 1 .41  (17.2- 62.9) ( 0 . 1 9 - 0.29) (0.03- 0.16) (0.15- 0.39) (8.57- 32.4) (4.54- 105. ) (2.86- 8.74) ( 3 6 . 1 - 107. ) (0.66- 1.51) (8.45- 82.0) (3.37- 17.8) ( 0 . 9 0 - 2.37)  156  After 45.9 0.35 0.06 0.27 12.9 77.7 7.60 1 15. 1 .30 16.0 6.35 1 .45  CBZ  ( 3 3 . 2 - 54.0) ( 0 . 2 6 - 0.50) (0.04- 0.08) ( 0 . 1 9 - 0.57) ( 6 . 7 7 - 19.9) ( 4 3 . 2 - 162. ) ( 4 . 6 8 - 12.6) ( 5 9 . 5 - 238. ) ( 0 . 9 3 - 1 .70) ( 9 . 6 3 - 27.4) ( 3 . 7 0 - 10.6) ( 1 . 1 2 - 2.13)  % change + 3.61 + 52.2 -25.0 + 22.7 -25.4 + 20.7 + 21.2 + 45.4 + 17.1 -40.7 -23.2 + 2.84  I.  si  MEAN R E C O V E R Y OF UNSATURATED METABOLITES  c ro as OJ  * CD r r I-I X »-h 3 * fD (-"• CO r r fD O (0 O rr i Q I-I r r < O 3 O fD i O M- O r r i l-h CU fD 0)  ro  tz)  O 01 3  (T  h-*  •< a •->• l-h »-h fD  r3o rt  l-h n O  3  n a g(0  —. 0)  3  M-  o  3 O l-h C rr 3 3-3 r r fD fD 0) 3 n O i fD i - l C 3 o >-»• O 3 3 3 < CD VI fD 0* i n i - l fD r r r r fD X C •-I i-t CU 0> rr fD r r y->-fD (0 O l-h 3 O fD  •  c  at? tr ro (fl a n a fD3 fD  tr rr-h o 3 01 r r O fit 0) o l-h W tr tsj 0) o ro < o ro —TJ rr to M < fD (0 o -NJo ^fD < CD c ^ 3 M 3 rt r r 0) fD 3 O fD Q i l - h 01 i-i rt C  ro  •o IA  o o  (Jl  cn  AS PERCENT OF TOTAL RECOVERED  89 I  MEAN R E C O V E R Y OF POLAR METABOLITES A S PERCENT OF TOTAL R E C O V E R E D  IA o o tn  150-1  CO Ul  vo  Figure 65.  Histograms of mean unsaturated m e t a b o l i t e s recovered in the u r i n e expressed as a percent of VPA recovered before CBZ ( \£2 ) a f t e r CBZ ( ) a d m i n i s t r a t i o n in f i v e v o l u n t e e r s . a  n  d  091  MEAN R E C O V E R Y OF U N S A T U R A T E D METABOLITES 03 C  n ro  CTl CT\  • D> CT rr X QJ (D 3* M3  rft  ro cn  »-»• O rt 3 I C O ro n uQ  cn <-t  co ro 3 cn ro o  OJ N rt  O 3 •->•  \Jro  3  cn ro  3 ^ c n o> ro 3 Mi OJ  a  < Oi CU o 1 OJ cn Ql Mill) tl <  i-  rt  o H-" re g c n ro ro 3 rt rt rt O o 0»  ro w 3ro Ocr ro M •-I  cn  -—•  • _  •  rt t~> O rr  M> n>  TJ n  > ro o H o ro < o ro o n < ro ro «. a ro (->•  a  3  A S P E R C E N T OF VPA R E C O V E R E D  5.  Pharmacokinetic a n a l y s i s  5.1.  Pathway a n a l y s i s Mean formation  table  23.  are in  Individual  the Appendix  pathway 6 was not  i s an  pathways  significant. and  the serum.  increased  in  The  after 3,  %,  volunteers  clearance  formation r a t e s  CBZ  5  in  for  value s i n c e VPA conjugate  The  for  administration.  and  Increases observed  37.4  are shown  formation  apparent c l e a r a n c e  were  increases  a l l pathways  formation c l e a r a n c e s f o r the section.  measured in  pathways  49.0,  clearances for  6  were  were 3 2 . 6 ,  all The  statistically  18.9,  50.0,  59.3,  r e s p e c t i v e l y , f o r pathways 1 to 6 a f t e r CBZ  administration. Mean m e t a b o l i t e in t a b l e  24.  clearances for a l l  Individual  Appendix s e c t i o n . are apparent were not  Metabolite  of  VPA  i n c r e a s e of  c l e a r a n c e s of 37.4,  metabolite significant.  i n the  serum.  increased  36.7  d i d not reach s t a t i s t i c a l  and 35.0  6 and  of  There were  c l e a r a n c e s f o r pathways 1,  the r  The m e t a b o l i t e %  after  significance.  Mean m e t a b o l i t e Cl  r  increased  %, r e s p e c t i v e l y .  clearance  in  CBZ An  41 % in mean plasma c l e a r a n c e of VPA  CBZ t h e r a p y .  pathways 5,  are  the conjugate and unchanged VPA  (total)  approximately  was observed a f t e r  clearances  illustrated  c l e a r a n c e s f o r pathway 6 and C l  clearances since  a d m i n i s t r a t i o n but  32.8,  metabolite  determined s e p a r a t e l y  clearance  pathways are  pathway  The 6  was  or  after  3, and 4 of 6.53,  161  CBZ  increase in  by the  statistically  decreases observed i n the 2,  elimination  29.8,  metabolite 3.11,  and  Table 23.  Pathway  Mean formation c l e a r a n c e s ( C l « , L / h ) f o r pathways 1 - 6 before and a f t e r CBZ a d m i n i s t r a t i o n f o r five healthy v o l u n t e e r s . a  Before CBZ  13  1 2 3 4 5 6 Sum  A f t e r CBZ  C  0.1528 + 0.1245 0.0122 + 0.0068 0.0026 + 0.0010 0.0856 + 0.0217 0.2006+0.1216 0.1178+0.0948 0.6225 + 0.2654  C  0.2026 + 0.0693 0.0145 + 0.0083 0.0039 + 0.0012* 0.1364 + 0.0494 0.2989+0.0982* 0.1619+0.0974* 0.8869 + 0.2237  % change +32.6 +18.9 +50.0 +59.3 +49.0 +37.4 +42.5  C l f c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e over 12 h from a given pathway by VPA AUC. a  b  Pathway Pathway Pathway Pathway Pathway Pathway  1 2 3 4 5 6  i n c l u d e s 2-ene VPA and 3-keto VPA. i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. i n c l u d e s 5-OH VPA and 2-PGA. i s VPA g l u c u r o n i d e c o n j u g a t e .  c  v a l u e s represent mean + standard d e v i a t i o n  * significantly different  from before CBZ value at p < 0 . 0 5 .  162  Table 2 4 .  Pathway 1 2 3 4 5 6 ci Sum D P r  C 1  C  b  Mean m e t a b o l i t e c l e a r a n c e s ( C l , L / h ) for pathways before and after CBZ a d m i n i s t r a t i o n for five healthy v o l u n t e e r s . a  m  Before C B Z 0.5438 + 0.3325 + 0.1574 + 1 .7180 + 9.9855 + 0 . 1 178 + 0.0509 12.905 + 0.8967 +  A f t e r CBZ  d  0 .0715 0 .1641 0 .0538 0 .8426 0 .7483 0 .0948 0 .0512 8 .9030 0 .1857  0.5083 0.2335 0.1525 1.5824 13.259 0.1619 0.0686 15.966 1.2630  + + + + + + + + +  d  0.2857 0.0786 0.0422 0.6407 12.700 0.0974* 0.0490 13.440 0.2433  % change -6.53 -29.8 -3.11 -7.89 + 32.8 + 37.4 + 35.0 + 23.7 + 40.8  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered in the u r i n e over 12 h from a given pathway by the corresponding AUC.  a  m  b  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s f r e e (unchanged) d r u g . r  c  d  Cl  p  = Dose/AUC(  V P A )  .  v a l u e s represent mean + standard d e v i a t i o n .  * significantly different  from before CBZ value at p < 0 . 0 5 .  163  7.89  %  , respectively  although  the  changes  did  not  reach  statistical significance. The  mean  fraction  c a l c u l a t e d before table  25.  Appendix s e c t i o n .  data f o r  None  by  significant.  by  each  pathway  was  and a f t e r CBZ a d m i n i s t r a t i o n and i s shown in  Individual  metabolized  metabolized  a  of  the  the  particular  volunteers  changes pathway  in  are mean  were  in  the  fraction  statistically  The mean f r a c t i o n metabolized for pathways 1,  and 6 decreased 0.72,  15.5,  f r a c t i o n metabolized  v i a pathways 3,  4, and 5 was i n c r e a s e d by  %, r e s p e c t i v e l y .  The mean f r a c t i o n of VPA  10.7,  15.5,  and 8.03  and 3.52  ( t o t a l ) metabolized decreased 4.76  5.2.  Metabolite After  pathways,  The mean  % a f t e r CBZ a d m i n i s t r a t i o n .  analysis  calculating the  %, r e s p e c t i v e l y .  2,  Clf,  Cl  same parameters  m  and  f  were a l s o  for  m  the  metabolic  c a l c u l a t e d for  each  metabolite. Table 26 metabolites. PGA  summarizes the  14  administration.  2,3'-diene 3.85,  38.8,  the  The mean formation c l e a r a n c e s of 3-ene VPA and 2-  decreased  VPA, 2-ene  mean formation c l e a r a n c e s for  % and  The formation  c i s VPA, VPA  21.2  41.7,  CBZ a d m i n i s t r a t i o n .  73.2, The  50,  respectively,  c l e a r a n c e s of  3-keto VPA,  and 2 , 4 - d i e n e  %,  after  CBZ  4-OH VPA, 4-ene  4-keto VPA, 5-OH VPA, 2-PSA,  VPA i n c r e a s e d  58.9,  101,  16.7 and 50 %, r e s p e c t i v e l y ,  54.0, after  i n c r e a s e i n formation c l e a r a n c e s for  164  Table 25.  Pathway" 1 2 3 4 5 6 ci Sum r  a  b  f  Mean f r a c t i o n metabolized (f ) by each pathway before and after CBZ a d m i n i s t r a t i o n for five volunteers. a  m  Before CBZ 0.1677 0.0129 0.0028 0.0953 0.2242 0.1337 0.0573 0.6939  A f t e r CBZ  C  + + + + + +  0.1359 0.0042 0.0008 0.0152 0.1399 0.1086 0.0601 + 0.2861  0.1665 0.0109 0.0031 0.1101 0.2422 0.1290 0.0529 0.7147  + 0.0713 + + + + + +  0.0043 0.0008 0.0413 0.0961 0.0791 0.0351 0.2034  i s c a l c u l a t e d by d i v i d i n g C l f by C l p .  m  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s f r e e (unchanged) VPA. r  c  C  v a l u e s represent mean + standard d e v i a t i o n  165  % change -0.72 -15.5 + 10.7 + 15.5 +8.03 -3.52 -7.68 + 3.00  Table 26.  Mean m e t a b o l i t e formation c l e a r a n c e s ( C l j , L / h ) f o r the VPA metabolites before and after CBZ administration. Numbers i n parentheses represent range (n=5). a  Metabolite 4-OH 4-ene 3-ene 2 -ene c i s 2 -ene t r a n s 3- k e t o 4- k e t o 5 -OH 2 -PSA 2 -PGA 2,3'-diene 2 ,4-diene  Before CBZ  A f t e r CBZ  0. 073 ( 0 . 051- 0 .096) 0. 400 ( 0 . 191- 0 .797)f 0. 150 ( 0 . 052- 0 .455)J> 0. 359 (0. 139- 0 . 6 0 3 ) 0. 026 (0. 012- 0 .044) 0. 127 ( 0 . 008- 0 .326) 0. 012 ( 0 . 006- 0 .015) b  0. 149 0. 002 0. 052 0. 012 0. 002  (0. 062- 0 .266) (0. 004- 0 .003) ( 0 . 017- 0 .157) ( 0 . 006- 0 .024) ( 0 . 001- 0 .003)  0 . 116 ( 0 . 0740 . 8 0 6 * ( 0 . 3940 . 129 ( 0 . 0610. 5 5 3 M 0 . 297-  0. 0. 0.  0. 0. 0.  0. 0.  % change  0 . 187)^ 0 . 166)^  0. 171 )£ 0. 7 9 5 ) 027 (0. 015- 0. 040) 175 ( 0 . 107- 0 . 253) 017*(0. 013- 0 . 020) 258*(0. 160- 0. 376) 003 (0. 002- 0. 006) 041 ( 0 . 029- 0. 058) 014 ( 0 . 007- 0. 027) 0 0 3 M 0 . 002- 0. 004) b  + 58.9 + 101. -14.0 + 54.0 + 3.85 + 37.8 + 41.7 + 73.2 + 50.0 -21.2 + 16.7 + 50.0  C l f c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e over 12 h by VPA AUC.  a  b  x 10" . 3  * significantly different  from before CBZ value at p < 0 . 0 5 .  166  4-ene VPA,  2-ene  statistically  cis  c i s VPA  27.  and 5-OH  7.18  c i s VPA  % after  The f r a c t i o n  fraction  were  27.4  % and 84.7 %,  other m e t a b o l i t e s  The  4-  mean  decreased  by  CBZ a d m i n i s t r a t i o n .  VPA decreased 3 9 . 3 ,  after  CBZ of  following administration  t r a n s VPA,  by 4 7 . 6 ,  reaching s i g n i f i c a n c e .  of the  metabolized  observed in  VPA  metabolized of 3-ene VPA, 2-ene t r a n s VPA, 2-  PGA, and 2 , 3 ' - d i e n e respectively,  5-OH  CBZ a d m i n i s t r a t i o n with the i n c r e a s e s f o r  clearances  to 52.2  and  c l e a r a n c e s f o r 4-ene VPA, 2-ene  VPA i n c r e a s e d  and 2-ene  metabolite  VPA  c l e a r a n c e s f o r these m e t a b o l i t e s are shown  Mean m e t a b o l i t e  respectively after ene VPA  4-keto  significant.  Mean m e t a b o l i t e in t a b l e  VPA,  the f r a c t i o n and 4-ene  f r a c t i o n metabolized  25.0,  administration the  of CBZ  other by 4.32  (table  to 50 %.  and 15.4 %, 28).  metabolites  The  increased The changes  metabolized for 2-ene c i s VPA, 2-ene  VPA were s t a t i s t i c a l l y of 2 , 4 - d i e n e  the same before and a f t e r  44.1,  VPA remained  CBZ a d m i n i s t r a t i o n .  167  significant.  The  approximately  Table 27.  Metabolite 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Mean m e t a b o l i t e m e t a b o l i t e clearances ( C l , L / h ) for the VPA m e t a b o l i t e s before and a f t e r CBZ administration. Numbers in parentheses represent range (n=5). a  m  Before CBZ 1 602 . 0. 063 0. 046 0. 248 0. 107 1 427 . 2. 482 8. 193 1 1 .53 32 .05 0. 375 0. 231  (0. 780(0. 027(0. 009(0. 097(0. 044(0. 083(1. 333(2. 453(4. 692(9. 895(0. 131(0. 122-  * significantly different  A f t e r CBZ  2.696) 0.143) 0.175) 0.397) 0.204) 4.379) 3.719) 19.02) 26.99) 93.95) 0.635) 0.330)  1 .487 (0. 6180.093* (0. 0410.024 (0. 0130 . 3 1 6 * ( 0 . 1830.097 (0. 0531 .369 (0. 7382.188 (1. 51715.13 (3. 37810.43 (3. 85815.31 (11 .980.255 (0. 1140. 197 (0. 132-  2. 186) 0.207) 0.044) 0.458) 0.155) 1.671) 2.718) 46.86) 13.69) 16.46) 0.326) 0.273)  % change -7.18 + 47.6 -47.8 + 27.4 -9.35 -4.06 -11.8 + 84.7 -9.54 -52.2 -32.8 -14.7  from before CBZ value at p < 0.05.  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e over 12 h by i t s c o r r e s p o n d i n g AUC value. a  m  168  Table 28.  Mean f r a c t i o n ( f ) of m e t a b o l i t e metabolized before and a f t e r carbamazepine a d m i n i s t r a t i o n . Numbers i n parentheses represent range (n=5). a  m  Metabolite 4- OH 4- ene 3- ene 2- ene c i s 2- ene t r a n s 3- keto 4- keto 5- OH 2- PSA 2- PGA 2, 3 ' - d i e n e 2, 4-diene a  b  f  m  Before CBZ 0. 081 0. 449 0. 168 0. 403 0. 028 0. 139 0. 012 0. 165 0. 002 0. 059 0. 013 0. 002  (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0. (0.  A f t e r CBZ  061 - 0. 100) 273- 0. 9 0 7 ) 060- 0. 518)^ 199- 0. 7 0 4 ) 017- 0. 037) 009- 0. 371 ) 007- 0. 017) 085- 0. 302) 001- 0. 003) 022- 0. 184) 009- 0. 019) 001- 0. 003) b  b  0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0. 0.  093 (0. 648* (0. 102 (0. 452*(0. 021* (0. 145 (0. 014 (0. 209 (0. 003 (0. 033 (0. 01 1 (0. 002 (0.  058359057270014074008142002023006002-  % change 0.152) 1. 3 4 6 ) 0.138) 0.744) 0 .026) 0 .237) 0 .018) 0 .352) 0 .005) 0 .047) 0 .016) 0 .003)  u b b b  + 14.8 + 44 .3 -39 .3 + 12 .2 -25 .0 + 4. 32 + 16 .7 + 26 .7 + 50 .0 -44 . 1 -15 .4 0. 00  i s c a l c u l a t e d by d i v i d i n g C l f by C l p .  x 10" . 3  * significantly different  from before CBZ value at p < 0.05.  169  IV.  DISCUSSION  A.  GCMS ANALYSIS OF VALPROIC ACID AND METABOLITES  1.  Assay The gas chromatograph-mass s p e c t r o m e t r i c assay developed in  our l a b o r a t o r y was used f o r the a n a l y s i s of VPA and m e t a b o l i t e s (Abbott et  al.,  1986a).  With  this assay, i t  simultaneously quantitate  VPA and  serum u s i n g  ion monitoring  the s e l e c t e d  was p o s s i b l e to  14 m e t a b o l i t e s mode.  in u r i n e and Briefly,  the  method employs a c a p i l l a r y column and the e x t r a c t e d samples may be d e r i v a t i z e d to tBDMS, m e t h y l , or TMS d e r i v a t i v e s . d e r i v a t i v e s were used in t h i s S e v e r a l attempts This included  study.  were made  to f u r t h e r  improve the a s s a y .  i n v e s t i g a t i o n of four new i n t e r n a l  two commercially a v a i l a b l e d e r i v a t i z i n g  1.1.  standards and  reagents.  I n t e r n a l standards Due to problems with r e p r o d u c i b l e d e r i v a t i z a t i o n  d i a c i d metabolites,  2-PSA and  2-methylglutaric  acid,  and  similar  chemically  reproducibility with i t s  use.  in  was i n v e s t i g a t e d . to  these  the standard  For e i g h t  2-PSA  and 2-PGA.  The  of the two  2-PGA, a new i n t e r n a l  two  MGA i s  greatly  serum standard c u r v e s , the % and 6.76  relative  1 70  standard,  structurally  metabolites  curves was  standard d e v i a t i o n v a l u e s were 5.28 for  The tBDMS  and  the  enhanced relative  %, r e s p e c t i v e l y ,  standard d e v i a t i o n values  for eight  u r i n e standard  respectively, internal  for  2-PSA and  standard  metabolites  curves  2-PGA.  by other  While the r e p r o d u c i b i l i t y  It  3-keto VPA  as an  sensitivity  and  particular  in the standard curves of the  two  enhanced with  s t a n d a r d , 3-keto VPA remains  internal  standard  reproducibility  in order of  the  to i n c r e a s e the assay  was i n v e s t i g a t e d as an i n t e r n a l  analysis  of  samples  However,  containing  octanoic acid  deuterated analogue of 2 , 3 ' - d i e n e  other  internal  butylacetic acid, found not  %,  been used as an  2-PSA and 2-PGA, was g r e a t l y  hexadeuterated VPA.  two  9.32  for  this  metabolite.  the  from the  MGA has  and  may be necessary to use a d e u t e r a t e d analogue of  Octanoic a c i d use in  %  1984a).  the a d d i t i o n of MGA as an i n t e r n a l an enigma.  9.41  workers in the a n a l y s i s of VPA and  (Granneman et a l . ,  diacid metabolites,  were  standards,  were i n v e s t i g a t e d  to i n t e r f e r e  with  any  metabolites d i d not  VPA.  hexanoic  standard for of  resolve  Subsequently,  acid  and  di-n-  for t h i s purpose and were of  the  metabolites  being  measured. As a  result  of  these i n v e s t i g a t i o n s , D3~2-ene,  octanone, o c t a n o i c a c i d , internal  standards  and i t s m e t a b o l i t e s . 3-octanone were biological  and 2 - m e t h y l g l u t a r i c  in the  a n a l y s i s of  Hexanoic a c i d ,  used as i n t e r n a l  samples  containing  metabolites.  171  Dg-VPA,  3-  a c i d were used as  samples c o n t a i n i n g VPA  di-/i-butylacetic acid,  and  standards for the a n a l y s i s of hexadeuterated  VPA  and  its  1.2.  Choice of d e r i v a t i z i n g Derivatization  of  f o r GCMS a n a l y s i s . for  this  the u r i n e  groups  groups ( M e r r i t t derivatives  are a p p l i c a b l e  including  are  and  (tBDMS)  1980).  derivatives  u r i n e samples  reaction  chromatographic  polarity, intense  TMS d e r i v a t i v e s and  p a t t e r n but  are  have abundant  [M-15]  peak.  +  i s very  large  The most  versatile number and  to a  of  VPA  and  were compared with respect time, easily  sensitivity, prepared,  molecular ions  s u s c e p t i b l e to  mass.  with a  +  is  formed by  molecular  ion.  derivatization  Although time,  tBDMS d e r i v a t i v e s proved to measured. for a l l  the l o s s  be l e s s  their  mass  relatively  fragmentation The  TMS d e r i v a t i v e s decreased  The tBDMS  172  these  from  require  Also,  a  [Mthe  shorter  compared  to  TMS d e r i v a t i v e s  some of the m e t a b o l i t e s  derivatives yielded  m e t a b o l i t e s except f o r  of  group  stability  was a major drawback. s e n s i t i v e for  tBDMS  retention  spectra  t-butyl  to  low  A prominent and intense  of the  in  and  have  hydrolysis.  The  d e r i v a t i v e s are a l s o l e s s complex. 57]  tert-  metabolites  The TMS group d i r e c t s the  larger  used  and  d e r i v a t i v e s are ten times more s t a b l e and have longer times due  amino  commonly  (TMS)  of  derivatives.  pediatric patient  stability.  a  carboxyl,  trimethylsilyl  tBDMS  time,  to  hydroxyl,  and McEwen,  butyldimethylsilyl TMS  or serum samples was r e q u i r e d  S i l y l a t i o n r e a c t i o n s are the most  purpose and  functional  reagent  greater  3-keto VPA and t h i s  being  sensitivity is  important  as some  VPA m e t a b o l i t e s  r e s u l t s were acids  similar  (Woollard,  A new  to  those obtained  commercially a v a i l a b l e i s more  our l a b o r a t o r y  was a l s o  derivatizing  in t r a c e amounts. for  reagent.  due to  reactive  than  the reagent  investigated  for  s u i t a b l e replacement  prepared  in  derivatization  t h i s reagent  for the  tBDMCS in  was not pyridine  with 5 % c a t a l y s t .  S t a b l e i s o t o p e s in pharmacokinetic Stable  istopes  and oxygen-17  such as  and 18  studies  c a r b o n - 1 3 , deuterium,  have had  i n c r e a s e d usage  nitrogen-15, over the  decade due  to the development of more s o p h i s t i c a t e d  t o o l s such  as  gas  isotopes  radioactive  chromatography-mass do  not  be a d m i n i s t e r e d  pregnant women.  radioactive  have  the  to neonates,  Oxygen  and  as  isotope  labelled  children,  adults,  nitrogen  e q u i v a l e n t s of s u f f i c i e n t l y  (GCMS).  restrictions  Stable  last  analytical  spectrometry same  i s o t o p e s in human s t u d i e s .  compounds may and t o  reagent,  the formation of the 3-keto VPA d i d e r i v a t i v e  considered a  Stable  fatty  use as an a l t e r n a t e  MTBSTFA r e q u i r e s s h o r t e r  and on longer h e a t i n g t i m e s ,  1.3.  chain  tBDMS d e r i v a t i z i n g  upon storage  reagent  long  These  1983).  MTBSTFA, which  times but  are present  do  not  long h a l f - l i v e s  have  for use  in metabolism s t u d i e s . In  pharmacokinetic  compounds may being the  be used  most commonly  studies,  stable  as i n t e r n a l used s t a b l e  173  isotope  standards  with  labelled deuterium  isotope f o r t h i s purpose.  The assay  used in  this  s t a n d a r d s , Dg-VPA analogue of  study  " p u l s e " dose 1984).  In  single  dose  deuterated  14 deuterium  atoms has  standard (von Unruh et a l . ,  of a  as i n  of VPA  may  as a  (Acheampong  be  single et  al.,  t h i s s t u d y , hexadeuterated VPA was a d m i n i s t e r e d as a to  a  volunteer  at  steady  state  Hexadeuterated VPA was found to be k i n e t i c a l l y VPA.  a l s o been  drug  l a b e l l e d analogue  the example  An  1980).  c h r o n i c a l l y administered  administering a  internal  1986b).  3  used as an i n t e r n a l  determined by  two  and D - 2 - e n e VPA (Abbott et a l . ,  VPA c o n t a i n i n g  The k i n e t i c s  has  Other  pharmacokinetic  studies  VPA  t e t r a d e u t e r a t e d analogue (von Unruh et a l . ,  1980)  been found to be k i n e t i c a l l y e q u i v a l e n t Hexadeuterated VPA participating  in  for ultimate  use of  be  1982)  the s t a b l e  tested  in  VPA  a  which a l s o have  the  volunteers  study i n  preparation  in p e d i a t r i c  of  used  and mono- and  isotope l a b e l l e d  of VPA  parameters  have  to  to VPA.  t h i s VPA+CBZ i n t e r a c t i o n  pharmacokinetic s t u d i e s pharmacokinetic  was to  VPA.  bioequivalent  of  heptadeuterated analogues (Kochen et a l . ,  on  could  analogue  patients. be  in  Thus,  determined  in  p a t i e n t s without d i s c o n t i n u i n g the d r u g . The s e n s i t i v i t y of the present assay d i d not permit u s i n g a s i n g l e pulse d e c l i n e of was to over  dose of metabolites  f o l l o w the 48  the deuterated  h  while  hexadeuterated VPA.  from  serum.  d e c l i n e of  drug and  the  An a l t e r n a t i v e procedure  u n l a b e l l e d VPA  maintaining  following  dosing  and m e t a b o l i t e s  every  12  h  with  The s e n s i t i v i t y of the assay was improved  174  s i n c e the  m e t a b o l i t e s were at  s u c c e s s f u l in of Dg-VPA  "steady s t a t e "  in the  first  Because m u l t i p l e very expensive the  T h i s was  the one v o l u n t e e r who was a d m i n i s t e r e d two doses part  of the  study ( c o n t r o l )  doses in the second p a r t of the study  be  levels.  dosing of  (induced).  hexadeuterated VPA  procedure f o r use in p a t i e n t s ,  preferred  method  sensitive analytical  once  and four  the  would be a  pulse dosing may  development  technique such  as negative  of  a  more  ion chemical  i o n i z a t i o n becomes a v a i l a b l e to assay VPA and m e t a b o l i t e s . It  was  difficult  presence of deuterated  to  analyze deuterated m e t a b o l i t e s  unlabelled metabolites, 4-ene  VPA  c o n c e n t r a t i o n s of the l a b e l l e d  and  e s p e c i a l l y in the case of  VPA.  the u n l a b e l l e d  metabolites since  in the  In  this  study,  the  m e t a b o l i t e s were higher steady s t a t e  than  was not achieved  with the deuterated d r u g .  In a d d i t i o n , u n l a b e l l e d and l a b e l l e d  metabolites  measured  cannot  i n t e r f e r e n c e by VPA and  be  the i n t e r n a l  metabolites.  exchange of  The  simultaneously  standards used deuterated  three deuterium  due  to  i n the assay f o r  4-keto  VPA  undergoes  atoms f o r three hydrogen atoms in  the workup procedure. Since steady were not was  achieved in  difficult  metabolism. p r o f i l e was % lower  state concentrations  to  either  the  analyze  However,  an  the isotope  hexadeuterated  c o n t r o l or induced s t a t e , effect effect  observed as shown i n t a b l e  serum trough  of  of in  CBZ the  on  VPA it  D^-VPA  metabolic  1 with approximately  66  c o n c e n t r a t i o n s of l a b e l l e d 4-ene VPA and  175  2 , 4 - d i e n e VPA VPA.  o c c u r r i n g a f t e r a d m i n i s t r a t i o n of  The serum  metabolites  trough c o n c e n t r a t i o n s  after  comparable to  the  hexadeuterated  of the  VPA  concentrations  of  hexadeuterated other  labelled  administration  unlabelled  were  metabolites  f o l l o w i n g VPA a d m i n i s t r a t i o n .  B.  EFFECT OF CARBAMAZEPINE ON VALPROIC ACID METABOLISM  CBZ induces in e p i l e p t i c  the metabolism  patients  (Levy  patients  on  % lower  than p r e d i c t e d  of VPA in healthy  and P i t l i c k ,  s u b j e c t s and  1982).  In  epileptic  VPA and CBZ, steady s t a t e VPA l e v e l s were 37 to 64 from s i n g l e  dose s t u d i e s of VPA.  The  r a t i o of VPA plasma c o n c e n t r a t i o n to VPA dose was 38 % lower the presence patients.  of CBZ In one  c l e a r a n c e from  compared to  volunteer  the body  a monotherapy group in  study,  CBZ caused  metabolism does not r e s u l t  binding s i t e s . amount of  CBZ  i n c r e a s e d VPA  1979).  are low  and t h u s ,  (Levy and  The e f f e c t  from c o m p e t i t i o n f o r  plasma l e v e l s  albumin p r e s e n t ,  d i s p l a c i n g agent  adult  accompanied by decreased steady s t a t e  plasma c o n c e n t r a t i o n s (Bowdle et a l . , on VPA  in  Pitlick,  it  compared  should  1982).  of CBZ protein to  the  not act as a  As  well,  the  a s s o c i a t i o n constant between CBZ and albumin i s not very h i g h .  1.  Inducing p r o p e r t i e s of carbamazepine CBZ i s known to induce the metabolism of other compounds as  w e l l as  i t s own  metabolism.  176  Repeated a d m i n i s t r a t i o n  of CBZ  enhances i t s  own  Serum steady  state  levels  two t o  three  are u s u a l l y  elimination  (Faigle  p r e d i c t e d from times  observed in c h r o n i c CBZ therapy et a l . ,  1982).  i t s own  metabolism a f t e r  state life  serum  At doses  for a  Feldmann,  1982).  s i n g l e dose s t u d i e s  higher than the a c t u a l values (Rawlins et a l . ,  1975;  Moreland  of 300-1200 mg/day, CBZ w i l l 60 days  levels progressively  shortens  and  few weeks  (Greim,  1981).  d e c l i n e and after  induce  CBZ steady  the serum h a l f -  initiation  of  therapy  (Perucca and R i c h e n s , 1982). CBZ induces the microsomal monooxygenase system (cytochrome P-450 and  NADPH-cytochrome c  reductase)  UDP-glucuronyltransferase (Faigle induction  pattern  equimolar d o s e s .  of The  CBZ  is  and  slightly  and Feldmann, similar  effects  of  to  CBZ,  induces  1982).  The  phenobarbital however,  at  are  less  drastic. CBZ induces agent  (Serlin  Hansen et  al.,  and  Beeley,  1980).  1971).  1983;  Ross  Decreases i n w a r f a r i n  hypoprothrombin a c t i v i t y  an a n t i c o a g u l a n t and B e e l e y , half-life,  are observed  1980; plasma  (Ross  and  A 56 year o l d man was s t a b i l i z e d on 6 mg/day of  (prothrombin  trigeminal  of w a r f a r i n ,  and B r e c k e n r i d g e ,  levels,  warfarin  the metabolism  neuralgia  time 2 (Ross  -  3  and  d i s c o n t i n u a t i o n of  CBZ t h e r a p y ,  to f i v e  control value.  times the  times c o n t r o l ) Beeley,  and CBZ for  1980).  After  the prothrombin time i n c r e a s e d After  the dose of  warfarin  was decreased to 4 mg/day, the prothrombin time returned to to three  times c o n t r o l .  177  two  CBZ s t i m u l a t e s (Hansen et  al.,  the metabolism  1971).  study decreased  significantly  administration.  A  daily  i n c r e a s e d metabolism comedication and  10.6  of  of to  600  p e r s i s t s for  4  concentrations, of  loss  2.75  a d d i t i o n of  CBZ  Patients r a t e and  excrete  controls  (Perucca  c o n t r o l , and a decreased serum weeks (Rosenberry et a l . , in  control  of  to 6.50  antipyrine  at a  child's h. faster  amounts of D - g l u c a r i c a c i d compared to 1984b).  CBZ  has  et a l . ,  between  1981).  When measuring i n d u c t i o n of 84  %  1984).  of  the  potency  of  At doses of 400 -  600  the ages  In nine newly diagnosed e p i l e p t i c of 6  c l e a r a n c e i n c r e a s e d from 65 to CBZ  the  1983).  CBZ in humans, a n t i p y r i n e metabolism i n c r e a s e d a f t e r  190 days (Greim,  patients  through an  theophylline  et a l . ,  (Perucca  of  10 to 21 days a f t e r  CBZ to  with CBZ e l i m i n a t e  larger  days  r e s u l t e d i n subtherapeutic  CBZ r e s u l t e d  clearance,  phenobarbital  80 -  14  in  theophylline  i n three  treated  CBZ  results  to  asthma and an i n c r e a s e i n t h e o p h y l l i n e h a l f - l i f e  mg/day of  after  the  of asthma  h  D i s c o n t i n u a t i o n of  antipyrine  h  1981).  The  regimen of an asthmatic  patients  phenytoin in one 6.4  mg  within  in  induces the metabolism of t h e o p h y l l i n e  unknown mechanism.  half-life  from  of phenytoin  (Greim,  phenytoin  half-life  dose  the e f f e c t  CBZ i s withdrawn CBZ a l s o  Serum  of  therapy  antipyrine  (Moreland  decreased  et  178  y e a r s , mean  antipyrine  143 mL/kg/h d u r i n g f i v e weeks of  al.,  from 6.24  to 14  1982). + 1.23  The  h to 2.78  half-life + 0.59  of  h, and  urinary 1.77  6-beta-hydroxycortisol  n g / d a y / k g to 17.85 In e p i l e p t i c  shortened  administration A daily  + 6.75  patients,  half-life  excretion  of  +  ng/day/kg.  CBZ  administration  doxycycline  i s not s u f f i c i e n t  dose of  i n c r e a s e d from 5.10  so  results  that  one  (Neuvonen et a l . ,  in  a  dose/day  1975).  200 mg of CBZ i n c r e a s e d the metabolism of  clonazepam a f t e r only four days (Greim,  1981).  When v o l u n t e e r s  who had a t t a i n e d steady s t a t e c o n c e n t r a t i o n s of clonazepam were a d m i n i s t e r e d CBZ l e v e l s and  + 11.5  daily,  decreased  observed (Lai  of clonazepam  .  An i n c r e a s e urinary  mg  h a l f - l i v e s were  mean h a l f - l i f e 22.5  200  clonazepam et a l . ,  decreased from  32.1  serum  1978).  The  + 16.6 h to  Clonazepam serum l e v e l s decreased by 19 to 37 %. of two  to four  excretion  administration,  of  thus  f o l d was  also  D-glucaric  indicating  observed  acid  the  in  following  occurrence  of  the CBZ  enzyme  induction.  2.  Volunteer  2.1.  data  CBZ and CBZE c o n c e n t r a t i o n s in serum Carbamazepine was  f o r the f i r s t week. dose  therapeutic  the  of 200  mg d a i l y  manufacturer.  concentrations  study,  serum  After  one  a dose  of 200 mg d a i l y  week and i n c r e a s e d to 300 mg d a i l y  The dose by  a d m i n i s t e r e d at  samples were week  of  In  of CBZ  i s the recommended order  179  doses  to  had been  analyzed f o r  daily  f o r the  of  both 200  second  starting  ascertain achieved CBZ mg  in  that the  and  CBZE.  CBZ,  serum  concentrations of -  3.68  CBZ f o r the  nq/mL (10.0  -  CBZ c o n c e n t r a t i o n s w i t h i n the 1980)  or  12 -  for  ug/mL or  13.0  4.6  of  five  3 -  4.  2.37 Serum  v o l u n t e e r s were not  14 jug/mL (Goodman et 1982).  After  al.,  two weeks  where the dose of CBZ had been i n c r e a s e d to  the  9.4  (Brodie et  second week,  -  18.4  serum CBZ  /umol/mL)  for a l l  al.,  1983)  (Brodie et a l . ,  values  1986)  have been r e p o r t e d  and 7.05  -  1 3.59  -  4.53 in  CBZ c o n c e n t r a t i o n s and 2.7  -  10.8  in e p i l e p t i c  CBZ c o n c e n t r a t i o n s  1983)  (3.07  300  f i v e v o l u n t e e r s were  range.  Atg/mL (Agbato et a l . ,  monotherapy.  and VPA  range  region of the t h e r a p e u t i c  -  on CBZ  of the  50 Mmol/mL ( E l y a s et a l . ,  mg d a i l y  the lower  ^mol/mL) as shown in t a b l e  f o r three  therapeutic  of CBZ t h e r a p y ,  of  15.6  f i v e v o l u n t e e r s ranged from  of  5.0  -  ng/mL  patients  10.3  ng/mL  /ug/mL in p a t i e n t s  on CBZ  comedication have a l s o been observed (Schoeman et  al.,  1984b). CBZE serum the parent  c o n c e n t r a t i o n s are  drug c o n c e n t r a t i o n s  c o n c e n t r a t i o n s of ug/mL  (1.49  -  administration after  3.38  and  ( E l y a s et  this  Mmol/mL)  0.51  -  1.20  study  observed in  epileptic  c o n c e n t r a t i o n s of and VPA  CBZE ranging patients  0.75  -  2.6  were c o a d m i n i s t e r e d  1983).  Although  al.,  ug/mL  10  -  50 % of  1982).  The  ranged from 0.38  after  two weeks of CBZ a d m i n i s t r a t i o n  C o n c e n t r a t i o n s of  al.,  CBZE i n  approximately  one (2.02  week -  -  0.85  of  4.75  CBZ  /xmol/mL)  as summarized in t a b l e  from 0.9  -  2.1  (Agbato et  5.  jig/mL have been  al.,  1986).  CBZE  ng/mL have been r e p o r t e d when CBZ in e p i l e p t i c  the  180  patients  concentrations  of  (Brodie  the  et  epoxide  metabolite  observed  in t h i s  study were  observed i n  patients,  parent  c o n c e n t r a t i o n s as  drug  CBZE to CBZ i n t a b l e The r a t i o as  one week  of CBZ  %  it  reported r a t i o s  the  first  administration,  week  of  patients,  50 % of  the percent  15.3  ratio  - 27.7  v a l u e s are  CBZ  ratio  the  range  after  two  slightly  CBZ epoxide  -  three  1984a). of  five  the  epileptic After  the  range r e p o r t e d for  five  epileptic volunteers  weeks of CBZ a d m i n i s t r a t i o n . of  lower to  After  lower than  - 42.76 % in  administration,  6 is  % was observed a f t e r  slightly  17.44  the c o n c e n t r a t i o n s  were  of  ranged from 15.1  VPA and CBZ, while only one of the this  the  ratio  induction.  and CBZ (Schoeman et a l . ,  w i t h i n the  Thus, although volunteers  These  the  of CBZE to CBZ of  v o l u n t e e r s were  was w i t h i n  range of  both VPA  c h i l d r e n on  shown by  10 -  may serve as an i n d i c a t o r of  of CBZ.  c h i l d r e n on  represent  6.  and a s i m i l a r  two weeks  still  between CBZE and CBZ c o n c e n t r a t i o n s in t a b l e  important  28.0  they  lower than the v a l u e s  CBZ  than  CBZ  ratio  and  CBZE  typically was  in found  more  or  the in less  c o n s i s t e n t with reported v a l u e s .  2.2.  VPA data  2.2.1.  Protocol  Healthy male study  although  Volunteers  were  examination and  v o l u n t e e r s were chosen to p a r t i c i p a t e females required  were to  blood chemistry  181  not  intentionally  complete  and  pass  in  this  excluded. a  medical  a n a l y s i s before being allowed  to p a r t i c i p a t e alcohol, the  in the  study.  They were asked to a b s t a i n  smoking, and any other medications for the d u r a t i o n  of  study. T h i s study  Dg-VPA c o u l d CBZ.  was i n i t i a l l y a l s o be  However,  a limited  obtained  a d m i n i s t e r e d and of  the absence and presence of  in  of VPA  then dosing  VPA  accumulation  in  the deuterated  morning dose(s)  decline  designed so that the k i n e t i c s of  t h i s was performed in only one v o l u n t e e r  supply of  only the  and  its  u r i n e was  a c c u r a t e l y determine were c o l l e c t e d  the  over a  drug.  or  VPA  and  CBZ  was d i s c o n t i n u e d metabolites  Initially, VPA was  a  for  in  the  chosen, but  volunteer  who  48 h  parameters.  48 h.  The  serum  and  Although samples  p e r i o d , many of the  20 mg/kg/day  untoward symptoms  subsequently withdrew  volunteers  was(were)  followed over 48 h i n order to more kinetic  dose of  due to  On the study d a y s ,  f o r example AUC, are based on the dosing i n t e r v a l  five  from  calculations, of  12 h.  in two d i v i d e d doses of were observed  from the  in  study.  who completed the s t u d y , one v o l u n t e e r  Of  r e c e i v e d 15 mg/kg/day.  dose i s  dose  although  recommended  much  higher  starting doses  are  by  actually  the  the  received  20 mg/kg/day while four v o l u n t e e r s the  one  This  manufacturer,  administered  in  patients.  2.2.2.  Serum VPA data  From the volunteers  in  serum data t h i s study,  summarized in the e f f e c t  182  table 7  for  the  five  of CBZ on VPA metabolism  was q u i t e  obvious.  enhanced  as  was  administration. a f t e r CBZ  Plasma the  clearance  elimination  Serum h a l f - l i f e  comedication.  of  rate  VPA  was  constant  greatly  after  CBZ  of VPA decreased s i g n i f i c a n t l y  The decreases  i n steady s t a t e  serum  VPA c o n c e n t r a t i o n s and AUC v a l u e s a f t e r CBZ a d m i n i s t r a t i o n were also  significant. Steady s t a t e  CBZ and  39.64  c o n c e n t r a t i o n s of +  9.79  mg/L  56.25 +  after  CBZ  administration  were  Serum steady  state  obtained by d i v i d i n g the AUC value by 12 h. VPA c o n c e n t r a t i o n s of 55.9  + 3.0 mg/L a f t e r  s i n g l e 600 mg doses  have been r e p o r t e d for v o l u n t e e r s by Loscher 34.2  +  after 8.1  7.9 mg/L  ( P o l l a c k et  250 mg VPA twice d a i l y  mg/L a f t e r a s i n g l e  al.,  1986)  10.15 mg/L before  (1978).  and 34.4  (Bowdle et a l . ,  1000 mg dose ( B i a l e r  Values of + 5.1  mg/L  1979), and 107.5 + et a l . ,  1985)  have  a l s o been r e p o r t e d . Trough serum CBZ and  27.01  obtained. on VPA  +  c o n c e n t r a t i o n s of 10.37  mg/L  44.02 +  after  Trough c o n c e n t r a t i o n s  CBZ  16.65  mg/L before  administration  of VPA in p e d i a t r i c  monotherapy ranged from 11.8  -  105 mg/L (Abbott  were  patients et  al.,  1986a) . AUC v a l u e s as shown in t a b l e +  130.5  mg.h/L  administration. 114.5 mg.h/L absence of al.,  to  475.7  +  7 for VPA decreased from 675.0 75.73  mg.h/L  after  CBZ  AUC values of 726.6 + 62.0 m g . h / L , and 531.0  in v o l u n t e e r s any other  have been  drugs (Perucca  1985).  183  r e p o r t e d f o r VPA in et a l . ,  1984a; B i a l e r  +  the et  The VPA w i t h VPA  serum h a l f - l i f e  monotherapy in  l i t e r a t u r e values 14.86  +  2.36  et a l . ,  1985)  11.83  4.02  +  1985), and  of  +1.0  et a l . ,  in v o l u n t e e r s .  +  2.7  + 2.374 h obtained  are i n agreement  h (Perucca  et a l . ,  1984b) and 14.9  1985), 8.5  h (Orr et a l . ,  + 1.6  1982)  with  1984a),  + 2.4 h  In p e d i a t r i c p a t i e n t s ,  h (Cloyd et a l . ,  10.4  14.03  the v o l u n t e e r s  13.0  h (Bialer  v a l u e s of  (Bialer  values of  h ( H a l l et  al.,  have been observed  i n VPA monotherapy. The v a l u e s  f o r VPA  a d m i n i s t r a t i o n were half-life 1.6  values  half-life  10.56  of 7.10  +  in  1.392  + 1.77  our v o l u n t e e r s a f t e r CBZ  h.  In p e d i a t r i c  (Otten et a l . ,  1984)  h have been observed in the presence of other  drugs  (Cloyd et a l . ,  were s l i g h t l y half-life  of  1985).  higher than VPA a f t e r  Our h a l f - l i f e  p a t i e n t s was reported to be 12.9 + 1.8 Clearance values mL/h/kg) + 3.35  of  0.897  +  and 7.5 +  antiepileptic  values in v o l u n t e e r s  those observed  administration  patients,  in p a t i e n t s .  of  ASA  in  pediatric  h (Orr et a l . ,  0.184  L/h  before CBZ a d m i n i s t r a t i o n and 1.263  1982).  (12.46  + 0.241  The  +  L/h  2.56 (17.54  mL/h/kg) a f t e r CBZ a d m i n i s t r a t i o n were obtained and are  summarized in  table  l i t e r a t u r e values and 16.2  of  7.  These  14.95  v a l u e s are  in agreement  with  + 2.86 mL/h/kg (Cloyd et a l . ,  1985)  + 6.6 mL/h/kg ( H a l l et a l . ,  1985)  f o r VPA monotherapy.  In p e d i a t r i c p a t i e n t s on VPA monotherapy, mean c l e a r a n c e values of 22  mL/h/kg have  two v o l u n t e e r s clearance values  been observed  administered s i n g l e of 0.418  L / h and  184  (Farrell  et a l . ,  doses  of  0.664  L/h  VPA were  1982). (600  In mg),  obtained  (Abbott  et  al.,  1982).  Literature  v a l u e s for  VPA c l e a r a n c e  when c o a d m i n i s t e r e d with other a n t i c o n v u l s a n t agents were 26.74 + 10.43  mL/h/kg (Cloyd  (Otten et a l . ,  1984)  The volume  monotherapy of  in agreement  with r e p o r t e d  L / k g (Cloyd  et a l . , 0.192  obtained in 0.263 +  +  18.0  + 3.6  mL/h/kg  patients. values  obtained  + 0.86  for  VPA  of 0.25  L/kg  ( H a l l et a l . ,  +  0.06  ( P o l l a c k et  al.,  1985).  Vd values  study a f t e r CBZ a d m i n i s t r a t i o n  and were  i n agreement  in  L/kg ( t a b l e 7) were  l i t e r a t u r e values  1985), 0.216  our v o l u n t e e r  and  0.255 + 0.03  0.049 L/kg  0.020 L/kg  1985)  in p e d i a t r i c  of d i s t r i b u t i o n  v o l u n t e e r s on  1986), and  et a l . ,  with r e p o r t e d  were data.  Values r e p o r t e d in the l i t e r a t u r e f o r Vd of VPA in the presence of other 1985)  a n t i c o n v u l s a n t s were  and 0 . 1 8 9 + 0.038 L / k g  The e l i m i n a t i o n + 0.011  to 0.067  + 0.011  h  decreased from  0.0623 +  was based  on  p o s s i b l y by  changes  available  for  VPA from  _ 1  as  shown i n  this  table  interaction,  7.  the  Ke  0.0168 to 0.0573 + 0.0168 h "  in  the  VPA  volume  of  thus i n c r e a s i n g  metabolism. have shown  i t s protein  However, that jun  1  value (Bowdle  interaction  distribution, binding  free  Mattson  coworkers  and  drug  CBZ does not d i s p l a c e  The  current  volunteer  study supports these r e s u l t s with volume of d i s t r i b u t i o n  185  the  the amount of  vitro,  binding s i t e s .  0.051 In  CBZ d i s p l a c i n g VPA from i t s plasma p r o t e i n  albumin and  1982)  al.,  1984).  These authors suggested that t h i s  s i t e s on  (1980;  L/kg (Cloyd et  r a t e constant for VPA i n c r e a s e d from  investigating  1979).  + 0.06  (Otten et a l . ,  p r e v i o u s study  et a l . ,  0.26  of VPA  i n c r e a s i n g only  slightly  pharmacokinetic r e s u l t s between VPA competition  and CBZ for  following  CBZ c o m e d i c a t i o n .  h i g h l y suggest  i s based  that  the  These  interaction  on enzyme i n d u c t i o n rather  protein binding  sites.  VPA i s  than  not known to  induce i t s own metabolism (Rimmer and R i c h e n s , 1985). The r e s u l t s coworkers f o r was much  obtained may  differ  s e v e r a l reasons:  lower  (mean  v o l u n t e e r s were  mg/day  of the  were only  c o l l e c t e d over  used; CBZ  comedication was  those of Bowdle and  the dose of VPA i n t h e i r  dose 500  not a l l  from  12 h;  versus  same sex;  1181  study  mg/day);  b i o l o g i c a l samples  a l e s s s e n s i t i v e GC assay was  initiated  after  four days  of VPA  d o s i n g ; and CBZ was administered at a dose of 200 mg once d a i l y throughout  the  study.  The  approximations and may not be  2.2.3.  Serum m e t a b o l i t e  U n l i k e the beta-oxidation  all  VPA was  between  VPA and  a f f e c t e d by  metabolic  and  pathways  ASA,  Vd  are  where  only  the a d d i t i o n of ASA to  1986b), t h i s  From the serum m e t a b o l i t e  VPA  Ke  reliable.  the dosing regimen (Abbott et a l . , with CBZ.  for  data  interaction of  values  profiles,  are  i s not the case it  appears that  influenced  by  CBZ  coadministration.  2.2.3.1. Serum  Metabolite trough  summarized in  table  serum c o n c e n t r a t i o n s concentrations 8.  For  186  for  many of  VPA  metabolites  the m e t a b o l i t e s ,  are serum  c o n c e n t r a t i o n values volunteers.  have  previously  Serum c o n c e n t r a t i o n s f o r  not been r e p o r t e d by any l a b o r a t o r y A comparison data  is  of our  summarized in  with respect there are  to VPA  only two  studies is volunteers  in t h i s  metabolites s t u d i e s for  interaction  The c o n c e n t r a t i o n s  epileptic  are  although, again,  than  table  13.  recently  state  reported  30 i s  comparison  compared were higher  assay.  In  i s obvious  and  one  as  of  the  D e s p i t e the small number of  from two  patients  3-keto  results  patient  (Abbott  VPA  observed  in  are  studies.  et a l . ,  1986b)  (1981a)  for  concentrations  in  pediatric  patients  study  of these r e s u l t s with a  (Pollack  et  our r e s u l t s  dose  187  al.,  1986).  which c o u l d be  T h i s may be a r e f l e c t i o n  reported r e s u l t s the  were  AUC value by 12 h and are shown in  i n our s t u d y .  addition,  concentrations  of the seven m e t a b o l i t e s  assays s i n c e  and the  (1981a).  a comparison  volunteer  C o n c e n t r a t i o n s of three  GCMS assay  lack of p u b l i s h e d data  metabolite  d i v i d i n g the  Table  the d i f f e r e n t  literature  the range of values i s c o n s i s t e n t with 3-keto  steady  c a l c u l a t e d by  to p a t i e n t  the serum  VPA r e s u l t s found in the Loscher study Serum  have  VPA and 2-ene t r a n s VPA are higher  Mean  higher  in  than our own.  in the range r e p o r t e d by Loscher  patients.  v o l u n t e e r s are  in  with r e s u l t s of 4-OH  reported  s t u d y , the v o l u n t e e r  i n v o l u n t e e r s than in p e d i a t r i c although they  The  laboratory.  generally consistent  other  29.  been  some VPA m e t a b o l i t e s  v o l u n t e e r data table  from t h i s  not  of  were obtained  of  with a  were obtained with a GLC VPA  in  our  study  was  Table 29.  Comparison of serum VPA and m e t a b o l i t e c o n c e n t r a t i o n s (nq/mh) in v o l u n t e e r s with p a t i e n t data. Numbers in parentheses represent range.  Compound  Volunteers  Pediatric" Patients (n=34)  3  (n=5) 4-OH 4-ene 3-ene 2-ene c i s 2-ene trans VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene N.R.  2.69 0.39 0.30 0.08 15.2 44.0 6.30 0.30 1.01 0.01 0.09 2.39 0.56  (1 .02-4.68) (0.28-0.48) (0.15-0.51) (0.06-0.10) (8.04-20.5) (27.4-53.2) (4.50-8.15) (0.21-0.39) (0.51-1.25) (0.00-0.03) (0.06-0.12) (0.85-3.62) (0.51-0.63)  0.38 0.67 0.94 0.19 5.53 46.4 3.59 0.40 0.18 0.04 0.20 2.95 0.20  (0.01-1.78) (0.16-1.22) (0.25-1.86) (0.06-0.40) (0.95-11.3) (11.8-105.) (0.29-15.6) (0.01-1.29) (0.01-1.25) (0.01-0.44) (0.01-1.23) (0.50-7.29) (0.02-0.58)  Epileptic Patients (n = 26) 0.70  (0.30- 3.50) N.R. N.R. N.R. 6.40 (3.BO- 18.0) 105. O S . 0 - 234. ) 5.40 (1.40- 13.8) N.R. 1 .70 (0.50- 4.20) N.R. N.R. N.R. N.R.  values not reported  3  mean dose 16.4 mg/kg/day, volunteer  b  VPA monotherapy  c  VPA monotherapy and combined with other a n t i c o n v u l s a n t s  (Abbott  study  et a l . , 1986a), mean dose 27.2 mg/kg/day (Loscher,  1981)  Table 30.  Comparison of mean serum steady state VPA and metabolite concentrations (uq/mL) from two volunteer studies. Numbers in parentheses represent range.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene our study with mg/day a  Study 1 2.97 0.38 0.29 0.08 14.9 56.3 5.42 0.25 1.15 0.01 0.09 2.10 0.53  a  (n=5)  (1 .55-5.12) (0.28-0.47) (0.15-0.50) (0.06-0.10) (7.78-20.3) (41.5-66.4) (4.62-6.47) (0.18-0.33) (0.92-1.65) (0.01-0.02) (0.06-0. 1 1 ) (0.83-3.76) (0.31-0.67)  b  (n= 5)  0.92 ( 0 . 6 7 - 1 . 45) 0.62 ( 0 . 1 2 - 1 . 44) 0.19 ( 0 . 0 0 - 0 . 64) N.R. 4.07 ( 3 . 1 4 - 6 . 01) 34.2 (27.7-43 .4) 2.55 ( 1 . 5 2 - 3 . 73) N.R. 0.31 ( 0 . 0 0 - 0 . 90) N.R. N.R. N.R. N.R.  m e t a b o l i t e s measured by GCMS, mean dose 1181  metabolites measured.using dose 500 mg/day b  N.R.  Study 2  not r e p o r t e d  189  GLC ( P o l l a c k et a l . , 1986), mean  considerably higher, 500 mg d a i l y  2.2.3.2.  with a  in the v o l u n t e e r  Metabolite  Serum  mean dose of  AUC  1181 mg d a i l y versus  study by P o l l a c k  (1986).  AUC v a l u e s  values  for  the  monounsaturated  metabolites  decreased a f t e r CBZ a d m i n i s t r a t i o n accompanied by i n c r e a s e d AUC v a l u e s for the p o l a r m e t a b o l i t e s . general o v e r a l l  T h i s i n d i c a t e s that there  i n d u c t i o n due to CBZ.  is  The s i g n i f i c a n t i n c r e a s e  in 4-keto VPA AUC values a f t e r CBZ a d m i n i s t r a t i o n suggests that the  co-1  oxidation  pathway  is  particularly  AUC  values  sensitive  to  i n d u c t i o n by CBZ. The  decrease  m e t a b o l i t e s may  in  suggest that  CBZ a d m i n i s t r a t i o n . 2,3'-diene of 3-ene  VPA  the  monounsaturated  these pathways  are  i n h i b i t e d by  However, the i n c r e a s e i n the AUC values of  and 2 , 4 - d i e n e  VPA and  for  VPA suggests enhanced metabolism  4-ene VPA to the secondary diene  metabolites  and thus c o u l d e x p l a i n the decrease in the AUC v a l u e s f o r  these  two monounsaturated m e t a b o l i t e s . The decrease in AUC v a l u e s for 2-ene t r a n s VPA i s to e x p l a i n .  If  enhanced a f t e r i n AUC  further  CBZ a d m i n i s t r a t i o n ,  v a l u e s for  o x i d a t i o n pathway administration, keto VPA were  metabolism  3-keto  VPA.  of 2-ene  trans  difficult VPA  was  one would expect i n c r e a s e s Conversely,  if  the  beta-  i s s i g n i f i c a n t l y blocked or i n h i b i t e d by CBZ  one  AUC v a l u e s .  approximately  would expect However, the  same  190  s i g n i f i c a n t decreases  in 3-  the AUC v a l u e s f o r 3-keto VPA before  and  after  CBZ  administration.  It  metabolized to  i s p o s s i b l e t h a t 2-ene t r a n s VPA i s  the d i e n e s ,  but the  further  i n c r e a s e in the AUC values  of the diene m e t a b o l i t e s does not balance the sharp decrease in the AUC  value for 2-ene t r a n s VPA.  VPA may  be metabolized  t h i s assay  VPA AUC  v a l u e s may  reflect  trans  measured by  The decrease in  the  shift  to  other  pathways.  The i n c r e a s e  in the  sum of  the AUC  m e t a b o l i t e s and  the decrease  the unsaturated  m e t a b o l i t e s suggests  ene VPA,  2-ene  m e t a b o l i t e s not  or e l i m i n a t e d by some other organ.  2-ene t r a n s oxidative  to other  Alternatively,  values f o r the p o l a r  i n the sum of the AUC v a l u e s for further  metabolism of  3-ene VPA, and 4-ene VPA to secondary p r o d u c t s .  would e x p l a i n  the i n c r e a s e  diunsaturated metabolites, AUC  values  determine  if  were  in serum 2,3'-diene  separated  a particular  s i n c e one m e t a b o l i t e  another  decreases,  Consequently,  significant  f o r the  This two  and 2 , 4 - d i e n e VPA.  into  metabolic  pathway was  However,  the  AUC values  2-  pathways  specifically  to  induced.  in the pathway may i n c r e a s e while  net  change  changes were  would not  be  observed  minimal. in  AUC  v a l u e s f o r the pathways a f t e r CBZ a d m i n i s t r a t i o n .  2.2.4.  Urinary  The assay of m e t a b o l i t e  data as o u t l i n e d p r e v i o u s l y measures the t o t a l amount present and  does not  distinguish  conjugated and unconjugated m e t a b o l i t e .  191  between  the  2.2.4.1.  Recovery expressed as a percent of dose  Twelve hour purposes.  urinary recoveries  The  expressed as  total  a percent  66.5  % a f t e r CBZ  over  12  1986).  recovery  h in  (VPA  of the  and  for c a l c u l a t i o n  metabolites)  dose was 64.6  administration.  v o l u n t e e r s has  were used  when  % before CBZ and  Approximately 65 % recovery  been observed  ( P o l l a c k et  al.,  In the rhesus monkey, 82 % of the a d m i n i s t e r e d dose was  recovered i n  the u r i n e  over 24 h (Rettenmeier  et a l . ,  1986a).  A f t e r a d m i n i s t r a t i o n of carbon-14 l a b e l l e d VPA to r a t s ,  70 % of  the dose was recovered i n the u r i n e Recovery of dose i s  VPA and  shown in  another v o l u n t e e r recovery of  VPA as  a d m i n i s t r a t i o n and supported by  table  35.2  %  1974).  m e t a b o l i t e s expressed as a percent of 19.  A comparison of our r e s u l t s with  study i s  summarized  a percent 18.18  r e s u l t s of  study ( P o l l a c k et a l . ,  (Kuhara et a l . ,  of dose  table  was 19.09  31.  21.5 %  in a  Mean  % before CBZ  % a f t e r CBZ a d m i n i s t r a t i o n .  1986).  recovery observed  in  steady state  This is volunteer  T h i s i s , however, lower than  by Granneman  and coworkers  (1984b)  a f t e r a s i n g l e 1000 mg dose of VPA and much lower than the % recovery Of the  i n the  rhesus monkey  (Rettenmeier  et a l . ,  the  61.1  1986a).  s i x m e t a b o l i t e s which can be compared, three of the  m e t a b o l i t e s are reasonably c o n s i s t e n t . VPA and r e s u l t of c o u l d be from VPA.  3-ene VPA  are c o n s i d e r a b l y  the d i f f e r e n c e s  Our r e c o v e r i e s of lower and  i n the a s s a y s .  higher due to some i n t e r f e r e n c e  t h i s may  six  4-ene be a  The reported values i n the chromatography  Our recovery of 5-OH VPA was c o n s i d e r a b l y h i g h e r .  192  Table 31.  Comparison of u r i n a r y recovery of VPA and m e t a b o l i t e s expressed as a percent of dose i n two volunteer s t u d i e s . Numbers i n parentheses represent range.  Compound  Study 1  4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene N.R.  not  7.305 0.046 0.017 0.041 2.863 19.09 12.68 1 .092 14.87 0. 196 4.883 1 .307 0.242  a  (n=5)  (5. 534-9.027) (0. 028-0.092) (0. 006-0.053) (0. 020-0.071) (1 . 704-3.705) (1 1.44-32.17) (0. 842-33.78) (0. 605-1.527) (7. 622-27.22) (0. 134-0.281 ) (1 . 786-15.21) (0. 897-2.036) (0. 142-0.303)  Study 2  b  5. 83 (1.93- 14.5) 0. 36 (0.00- 1 .80) 2. 78 (0.45- 6.64) N.R. 2. 82 (0.00- 8.31 ) 21 .5 (6.70- 44.7) 28 . 1 (17.8- 39.6) N.R. 2. 26 (0.41- 8.00) N.R. N.R. N.R. N.R.  reported  a  our v o l u n t e e r  b  mean dose 500 mg/day  study, mean dose 1181 mg/day, GCMS assay (Pollack  193  (n=5)  et a l . , 1986), GLC assay  Approximately 14.5 a f t e r CBZ  This is  recovered as  before CBZ  e x c r e t e d as  in agreement  the g l u c u r o n i d e  et a l . ,  2.2.4.2.  the dose  a d m i n i s t r a t i o n was  conjugate.  (Bialer  % of  and 14.2  %  the VPA g l u c u r o n i d e  with 15  - 20 % of the dose  conjugate in a s i n g l e dose study  1985).  Recovery expressed as a percent of t o t a l  excreted  U r i n a r y recovery expressed as a percent of the t o t a l amount excreted over  12 h  pediatric patients the d i e n e s , higher.  pediatric  i s summarized  4-OH VPA,  5-OH VPA,  were lower. patients  study.  as w e l l ,  in t a b l e  21.  in table and  A comparison with 32.  2-ene  Our v a l u e s trans  VPA  for were  The v a l u e s for 4-ene VPA, 3-ene VPA, 4-keto VPA, VPA,  and 2-PSA  state  i s shown  However,  on VPA  Ultimately,  one  must  this  i s a comparison between  monotherapy and  a volunteer  steady  the doses i n p a t i e n t s are h i g h e r , and  consider  the  differences  in  metabolism  between these two age groups.  2.2.5.  E f f e c t of CBZ on VPA metabolism  The percent metabolites  of dose  before  a d m i n i s t r a t i o n was  recovered in  (64.6  %)  almost the  the u r i n e  and  after  same.  This  as  (66.5 is  VPA  and  %)  CBZ  difficult  to  explain since  the amount of VPA present in the serum decreased  drastically.  Recovery of  d i d not  increase after  recovery of  2-ene trans  the VPA  CBZ  g l u c u r o n i d e conjugate a l s o  administration.  VPA in  194  The  decreased  the u r i n e was c o n s i s t e n t with  Table 32.  Comparison of recovery of VPA and m e t a b o l i t e s as a percent of the t o t a l amount r e c o v e r e d . Numbers i n parentheses represent range.  Compound  Study 1  4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene N.R.  a  (n=5)  12.7 (5.11-15.9) 0.07 (0.05-0.08) 0.02 (0.01-0.05) 0.06 (0.04-0.10) 4.81 (2.55-7.27) 29.7 (22.4-40.4) 18.5 (1.20-31 .2) 1 .74 (1.16-2.13) 22.0 (14.6-28.3) 0.31 (0.24-0.40) 7.39 (3.14-21.6) 2.27 ( 1 .00-3.99) 0.39 (0.27-0.53)  not r e p o r t e d  a  volunteer  s t u d y , mean dose 16.4 mg/kg/day  b  pediatric patients,  (Abbott et a l . , 1986b),  195  Study 2 5.00 0.28 0.08  b  (n=7)  (3.10-6.90) (0.09-0.47) (0.06-1.00) N.R. 1 .51 (0.99-2.03) 50.5 (37.9-63.1) 20.2 (10.5-29.9) 3.00 (1.50-4.50) 3.10 (1.30-4.90) 1 .46 (0.00-4.36) 1 1 .9(5.70-18.1) 0.67 (0.40-0.94) 0.09 (0.03-0.15)  the decreased  amounts present in the serum.  problem, s i n c e expressed as  T h i s a l s o poses a  the amount of 3-keto VPA recovered in the  a percent of dose d i d not i n c r e a s e to balance the  decrease i n  2-ene trans  pretreatment  resulted  although 3-keto (Heinemeyer  et  recoveries  of  VPA l e v e l s .  in  increased  VPA recovery al.,  In  various  excretion  d i d not  1985).  The  rats,  phenobarbital of  increase  increase  metabolites  2-ene  observed  is  which at t h i s point  in  consistent  recovery  of  4-ene  increased  volunteers.  As w e l l ,  2.2.6.  the  the  recovery  of  there  statistically  significant.  CBZ  well  as  4-ene  administration  in  VPA the  4-keto  i s c o n s i s t e n t with i n d u c t i o n of  into  also decreased.  It  pathways  of one a  The  was  not  the  extremely  m e t a b o l i t e might  second  were apparent  1 (beta-oxidation)  pathway are  as  i n c r e a s e in the r e c o v e r i e s of  the recovery  Consequently,  pathway  pathway  pathways  metabolites  helpful since while  with  pathway.  Metabolite  Grouping  VPA,  after  2-PSA i n the u r i n e  co-1 o x i d a t i o n  the  i s an enigma.  glucuronide,  VPA and  VPA  significantly  enhancement of metabolism except for the b e t a - o x i d a t i o n  The  urine  metabolite  increase decreased.  changes although decrease i n  none were  the recovery  of  was unexpected, s i n c e the AUC values  is possible  that the m e t a b o l i t e s  undergoing b i o t r a n s f o r m a t i o n  are not c o n s i d e r e d in t h i s a s s a y .  196  However,  in  this  to m e t a b o l i t e s which in rats  it  has been  r e p o r t e d that  p h e n o b a r b i t a l pretreatment  e x c r e t i o n of of  co-1  4-OH VPA,  oxidation  decreased with glucuronide  did  the  the  recoveries  the e x c e p t i o n conjugate  recoveries  are c o n s i s t e n t  of 5-OH  of  of  et  1  of pathways  and  3,  4,  induction pathways  VPA and VPA  al.,  a f t e r CBZ  pathways  other  VPA, 4-ene  (Granneman  VPA decreased  increased recoveries  al.,  4-keto VPA, and 2-PSA due to  while  G l u c u r o n i d a t i o n of  r e s u l t s in i n c r e a s e d  1984a).  a d m i n i s t r a t i o n as  2,  accompanied  and 5.  by  These r e s u l t s  with the r e s u l t s observed in r a t s  (Granneman et  1984a).  2.2.7.  Pharmacokinetic a n a l y s i s  The c l e a r a n c e elimination As w e l l ,  from  model was the c e n t r a l  the r a t i o  parent drug  can be  c o n c e n t r a t i o n s of based on  one used  CBZ  induction (Faigle  compartment seemed  obtained from metabolite  to  to d e c r i b e  the r a t i o  appropriate.  to c l e a r a n c e of of steady  parent d r u g .  The  state  model was  the i n t e r a c t i o n between CBZ and  1983).  Pathway a n a l y s i s  The formation after  a l i n e a r model with  of c l e a r a n c e of m e t a b o l i t e  clobazam (Levy et a l . ,  2.2.7.1.  chosen s i n c e  rates for  administration of  and  approximately  the  hepatic  Feldmann,  a l l metabolic (table  23)  microsomal  1982).  The  pathways i n c r e a s e d  most  likely  due  monooxygenase increases  ranged  to  system from  19 % to 59 % with the i n c r e a s e s in pathways 3,  197  5,  and 6  reaching s i g n i f i c a n c e .  pathways  (co-  and co-1 o x i d a t i o n )  formation c l e a r a n c e unexpected.  of the  1986).  could possibly itself  may  1985).  A  was expected but the i n c r e a s e d  h e p a t i c microsomal  CBZ although  possible agent  peroxisomes and  beta-oxidation  c l e a r a n c e of  is  enzyme i n d u c i n g  formation of  induce peroxisomal  The formation  totally  thought to occur mainly i n the  peroxisomal b e t a - o x i d a t i o n  enhance the  increased after  i n c r e a s e i n the microsomal  b e t a - o x i d a t i o n pathway was  Beta-oxidation is  m i t o c h o n d r i a although (Devlin,  The  VPA  (Horie and Suga,  the g l u c u r o n i d e conjugate  the enzymes r e s p o n s i b l e f o r  this  are only s l i g h t l y a f f e c t e d by CBZ ( F a i g l e and Feldmann, 1982). Metabolite  clearances  decreased a f t e r the amount from the  (table  CBZ a d m i n i s t r a t i o n ,  of m e t a b o l i t e s  i n these  c e n t r a l compartment.  pathway 5  24)  and of  administration.  The  VPA ( t o t a l  for  indicating a  metabolite  of these  1  -  4  decrease in  pathways being  + conjugate)  However, a l l  pathways  eliminated  clearance  of  i n c r e a s e d a f t e r CBZ  changes d i d not reach  significance. The f r a c t i o n metabolized demonstrated changes The f r a c t i o n  ( t a b l e 25)  by each pathway,  which were not s t a t i s t i c a l l y  metabolized by  pathways 1,  2, and  again,  significant. 6  decreased  a f t e r CBZ a d m i n i s t r a t i o n while the f r a c t i o n metabolized through pathways 3,  4, and 5 i n c r e a s e d a f t e r CBZ a d m i n i s t r a t i o n .  The formation administration metabolite  c l e a r a n c e of  accompanied  clearance.  by  This  198  pathway a could  1  slight  i n c r e a s e d a f t e r CBZ decrease  explain  the  in  the  decreased  recovery  in  decreased,  the  so  urine.  it  is d i f f i c u l t  were e l i m i n a t e d . pathways 2  and  The 3  i n c r e a s e d amounts since less  was  for pathway Pathway 5  eliminated,  The  4 may  rate.  The i n c r e a s e  metabolite  CBZ  formation c l e a r a n c e .  serum.  In t h i s  but case,  for  these  c l e a r a n c e for the  urinary clearance  i n c r e a s e d serum c o n c e n t r a t i o n s . in the  by i t s  of pathway supporting  i n serum  It  c l e a r a n c e s of  decrease in the m e t a b o l i t e  c l e a r a n c e d i d not  also  administration,  administration,  is explained  the u r i n e ,  values  concentrations  were i n c r e a s e d  Increased amounts  recovered in  in the  serum  e x p l a i n the  AUC values  CBZ  Although the m e t a b o l i t e  after  c o m e d i c a t i o n , which rate.  after  were present  recovery i n c r e a s e d .  AUC  to e x p l a i n how these m e t a b o l i t e s  decreased  decreased  serum  r e c o v e r i e s and m e t a b o l i t e  pathways were i n c r e a s e d . pathway 4  However,  serum a f t e r  CBZ  increased  formation  5 metabolites  were a l s o  the  increased  AUC values  suggests  metabolic that  the  i n c r e a s e to the same degree as the  is possible  that b i l i a r y  or  other  organ e l i m i n a t i o n of VPA and m e t a b o l i t e s was a l s o enhanced.  2.2.7.2.  Individual  The formation except 2-PGA  clearances (table  26)  for  and 3-ene VPA i n c r e a s e d a f t e r  The m e t a b o l i t e 4-ene VPA,  metabolites  c l e a r a n c e s ( t a b l e 27)  2-ene c i s  administration.  VPA, and  all  metabolites  CBZ a d m i n i s t r a t i o n .  for a l l m e t a b o l i t e s  5-OH VPA  except  decreased a f t e r  CBZ  T h i s i s c o n s i s t e n t with i n c r e a s e d e x c r e t i o n of  4-ene VPA and 5-OH VPA a f t e r  p h e n o b a r b i t a l pretreatment  199  in  rats  (Granneman et metabolite  al.,  after  1984a).  The  CBZ a d m i n i s t r a t i o n decreased f o r 3-ene VPA, 2-  ene t r a n s VPA, 2-PGA, 2 , 3 ' - d i e n e difficult  slightly after u r i n e was  mean  AUC  value  CBZ a d m i n i s t r a t i o n ,  conjugate  administration.  The  was  increased after  an i n c r e a s e d  metabolite  enhances formation (Rettie  VPA was at  serum at  further  VPA.  4-ene  VPA  pretreatment  and p o s s i b l y CBZ does c l e a r a n c e of  4-ene  of 2-ene VPA i s s t a t e d  (Keane et  c i s and  al.,  1985),  to our  t r a n s VPA p e r s i s t in the  the parent d r u g .  The  support  of  CBZ  than the formation c l e a r a n c e .  than VPA  long as  decreased  after  Phenobarbital  effect  a l s o observed for  2,4-diene  dienes may  increased  The m e t a b o l i t e  that 2-ene  l e a s t as  VPA  is  recovery of 4-ene VPA  clearance  clearance.  1987).  in duration  l e v e l s were  also  100 f o l d g r e a t e r  results indicate  4-ene  It  CBZ a d m i n i s t r a t i o n accompanied by  Although the a n t i e p i l e p t i c be s h o r t e r  for  of 4-ene VPA i n r a t s ,  et a l . ,  least  time.  The  formation  significantly  this  VPA.  the amount recovered in the  increased considerably.  glucuronide  VPA and  VPA, and 2 , 4 - d i e n e  to e x p l a i n these v a l u e s at  A l t h o u g h , the  also  f r a c t i o n metabolized of each  Sustained plasma  3-ene VPA, 4-ene VPA, s u s t a i n e d plasma  2,3'-diene  l e v e l s of  the  the theory that they a r i s e from 2-  ene VPA. It  is  p o s s i b l e that  not s u f f i c i e n t enzymes. s i m i l a r to  to cause  However, that of  if  two weeks  of CBZ a d m i n i s t r a t i o n  maximal i n d u c t i o n the mechanism  phenobarbital  200  of  of the  induction  were  microsomal of CBZ  is  ( F a i g l e and Feldmann, 1982),  then two  weeks of  cause maximal of phenytoin  treatment with  induction. w i t h i n 4 to  14 days (Greim,  study was  to 300  mg d a i l y  for the  that  induction  administration  200 mg d a i l y  of  occurred 200  2.2.8.  interaction  importance s i n c e in e f f o r t s  (Kesterson  through  cellular  The  of  (1979).  of 200 mg/day  is  al.,  microsomal enzymes,  1984).  interesting  T h i s combination may are known  the f a t t y a c i d b e t a 4-ene  VPA,  F u r t h e r metabolism of  pathway may  and  1985).  4-ene VPA  clinical  coadministered  hepatotoxin,  intermediates  it  of,  of f a t t y a c i d s by forming v a l p r o y l CoA  macromolecules  formation of  CBZ  some VPA m e t a b o l i t e s  potential  et a l . ,  reactive  et  and  i n h i b i t i o n of  the b e t a - o x i d a t i o n  chemically  VPA  dangerous s i n c e  hepatotoxic  VPA v i a  is  CBZ doses  seizure c o n t r o l .  i n h i b i t s beta-oxidation  It  days  volunteers  these two drugs are f r e q u e n t l y  o x i d a t i o n pathway.  (Simula  in  seven  1978).  between  to optimize  be p o t e n t i a l l y  esters  mg/day  and coworkers  C l i n i c a l s i g n i f i c a n c e of VPA and CBZ i n t e r a c t i o n  The  to be  The dose used  Bowdle within  i n c r e a s e d with  w i t h i n 4 days (Lai et a l . ,  1981).  f o r one week and then i n c r e a s e d  second week.  CBZ  Clonazepam metabolism  to  CBZ 600 mg/day i n c r e a s e d metabolism  in our  reported  CBZ should be s u f f i c i e n t  was  capable  forming Since of  l e a d to  tissue  CBZ  importance  formation  of  of  alkylating  bound  induces  4-ene  the  residues hepatic  to e s t a b l i s h i f  the  was enhanced a f t e r CBZ a d m i n i s t r a t i o n .  to note that although the formation of  201  4-ene  VPA was  induced by  clearance.  CBZ a d m i n i s t r a t i o n ,  The t o x i c i t y  so was i t s  metabolite  of the 4-ene VPA g l u c u r o n i d e conjugate  has not been determined. It  has  been  a l t h o u g h CBZ metabolic  induces  4-ene  volunteers. 2,3'-diene  VPA  in  this  of  VPA  that  to  its  amounts  of  the  potential  were  present  in  the  serum  not  increased  c o u l d be  amounts of  in the of some  become a c t i v a t e d  serum d a t a .  study  increased  This  of  2 , 4 - d i e n e VPA and  serum of  the  importance,  and cause  r e c o v e r i e s of the m e t a b o l i t e s  support the  volunteer  biotransformation  were present  This  molecules may  volunteers since  these  t i s s u e damage.  The  i n some i n s t a n c e s d i d not  may be  due to  enhancement of  e x c r e t i o n which was not measured.  Unfortunately, determine done in  VPA  However,  a f t e r CBZ.  biliary  the  byproducts,  hepatotoxin  urinary  determined  this  five  volunteers  complex i n t e r a c t i o n  were  not  and f u r t h e r  sufficient  study must be  order to a c c u r a t e l y c h a r a c t e r i z e t h i s i n t e r a c t i o n .  may be more i n f o r m a t i v e  to perform the study in p a t i e n t s  than v o l u n t e e r s .  202  to  It  rather  SUMMARY AND CONCLUSIONS  1.  The assay  m o d i f i e d with internal  for v a l p r o i c  a c i d and  r e s p e c t to the  internal  standards,  were employed. to  octanoic acid  Modifications  quantitate  m e t a b o l i t e s was standards u s e d .  acid,  were made to the assay i n order  metabolites  after  Two new i n t e r n a l  and d i - / i - b u t y l a c e t i c  acid,  were  administration  were chosen  of  s t a n d a r d s , hexanoic a c i d  used i n s t e a d  of the  standards used i n the assay f o r VPA and m e t a b o l i t e s . standards  Two new  and 2 - m e t h y l g l u t a r i c  hexadeuterated VPA.  internal  further  as they d i d not  internal These two  interfere  with  compared  with  the compounds being measured.  2.  TMS (MSTFA)  respect to  tBDMS  sensitivity,  D e s p i t e the and 3-OH  and  fact  VPA i s  that  stability,  A new,  were  and chromatographic  more e a s i l y  derivatized,  tBDMS  commercially a v a i l a b l e  tBDMS d e r i v a t i z i n g to the reagent  used f o r the a n a l y s i s of VPA and m e t a b o l i t e s .  major drawback  was the  were  stable  f o r at  formation of  least  203  currently  The d e r i v a t i v e s  seven  weeks.  a diderivative  VPA upon i n c r e a s e d h e a t i n g time and s t o r a g e .  reagent  MTBSTFA r e q u i r e d  l e s s h e a t i n g time f o r adequate d e r i v a t i z a t i o n . extremely  esters  time  respects.  (MTBSTFA) was t e s t e d as an a l t e r n a t i v e  formed were  time.  MSTFA r e q u i r e d l e s s d e r i v a t i z a t i o n  found to be s u p e r i o r i n a l l  3.  derivatives  of  The  3-keto  4.  P r e l i m i n a r y data  from one v o l u n t e e r . s i n c e steady study.  on the metabolism of Dg-VPA was obtained It  s t a t e was  However,  r e c o v e r i e s were i l l u s t r a t e d by  was not p o s s i b l e to o b t a i n k i n e t i c not a c h i e v e d  in e i t h e r  part  of  data the  serum c o n c e n t r a t i o n s , AUC v a l u e s , and u r i n a r y obtained.  An  isotope  effect  occurred  as  the approximately 66 % decrease i n serum trough  c o n c e n t r a t i o n s of 4-ene VPA and 2 , 4 - d i e n e VPA.  5.  To  study  the  effect  pharmacokinetic parameters a f t e r CBZ a d m i n i s t r a t i o n results indicated a f t e r CBZ  of CBZ  CBZ  for VPA  in f i v e ,  administration.  VPA  metabolism, before  and  healthy male v o l u n t e e r s .  The  T h i s was  of VPA  from the plasma  accompanied by decreased  serum h a l f - l i f e ,  comedication.  to induce  on  were o b t a i n e d  increased clearance  plasma c o n c e n t r a t i o n s , a f t e r CBZ  of  and AUC values of VPA  T h i s was c o n s i s t e n t with the  the h e p a t i c  microsomal enzyme  ability  systems in a  manner s i m i l a r to p h e n o b a r b i t a l .  6.  Twelve m e t a b o l i t e s of VPA were a l s o measured in the serum.  Serum trough  and steady  were determined  state concentrations  before and  after  amount of 4-ene VPA, a p o t e n t i a l in the  serum a f t e r  two d i u n s a t u r a t e d VPA, were  CBZ  and AUC  values  administration.  The  h e p a t o t o x i n , was not i n c r e a s e d  a d m i n i s t r a t i o n of CBZ. metabolites,  i n c r e a s e d in  2,3'-diene  the serum  204  The amounts of VPA  and  the  2,4-diene  of the v o l u n t e e r s a f t e r CBZ  administration.  The amount of 2-ene t r a n s VPA i n the serum was  s i g n i f i c a n t l y decreased amount of  after  CBZ  administration,  3-keto VPA d i d not i n c r e a s e .  monounsaturated m e t a b o l i t e s  while  the  The AUC v a l u e s f o r  decreased a f t e r  the  CBZ a d m i n i s t r a t i o n  while the AUC v a l u e s of the p o l a r m e t a b o l i t e s i n c r e a s e d .  7.  U r i n a r y metabolic  v o l u n t e e r s before determined  p r o f i l e s were  and a f t e r  individually  determined for  CBZ a d m i n i s t r a t i o n .  and  grouped  into  the  These  five were  pathways.  The  r e c o v e r i e s were a l s o expressed as a percent of dose, percent of t o t a l recovered,  percent of VPA r e c o v e r e d , and as sum of p o l a r  and sum of unsaturated m e t a b o l i t e s . OH VPA,  4-keto VPA,  and 2-PSA  Increased r e c o v e r i e s of  after  CBZ  4-  administration  were  the k i n e t i c s of VPA m e t a b o l i t e s  were  c o n s i s t e n t with enhanced co-1 o x i d a t i o n .  8.  For the  determined i n  first  time,  the absence  study.  The  metabolite  c l e a r a n c e , and f r a c t i o n m e t a b o l i z e d , were  f o r the  following  and presence of CBZ i n a c o n t r o l l e d parameters,  formation  metabolic pathways and for the i n d i v i d u a l  The formation  c l e a r a n c e s of a l l  clearance, determined  metabolites.  pathways i n c r e a s e d as a r e s u l t  of CBZ a d m i n i s t r a t i o n .  9.  The r e s u l t s  caused a  obtained from  general i n d u c t i o n  s p e c i f i c a l l y affect  of  a particular  205  t h i s study VPA  i n d i c a t e that  metabolism  pathway.  and  did  CBZ not  The i n c r e a s e in AUC  v a l u e s f o r the p o l a r m e t a b o l i t e s was c o n s i s t e n t with microsomal induction.  10.  The e f f e c t  of CBZ  c l e a r l y understood. beta-oxidation, extent.  or  As w e l l ,  on the  b e t a - o x i d a t i o n pathway i s not  CBZ may cause a metabolic s h i f t away from actually  inhibit  beta-oxidation  to  some  peroxisomal b e t a - o x i d a t i o n may be i n v o l v e d .  206  V.  REFERENCES  Abbott F . S . , Kassam J . , Acheampong A . , Ferguson S . , Panesar S . , Burton R . , F a r r e l l K. and Orr J. Capillary GC-MS of v a l p r o i c a c i d metabolites in serum and u r i n e u s i n g tertbutyldimethylsilyl derivatives. J . Chromatogr. 1986a; 375: 285. Abbott F . S . , Kassam J . , Orr J . 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Yu H - Y . , Sugiyama Y. and Hanano M. Changes in pharmacokinetics of v a l p r o i c a c i d in guinea p i g s from b i r t h to m a t u r i t y . E p i l e p s i a 1985; 26: 243. Zaccara G . , Paganini M . , Campostrini R . , Moroni F . , Valenza T . , Messori A . , Bartelli M., Arnetoli G. and Zappoli R. E f f e c t of a s s o c i a t e d a n t i e p i l e p t i c treatment on v a l p r o a t e induced hyperammonemia. T h e r . Drug M o n i t . 1985; 7: 185. Zaret B . S . and Cohen R.A. dementia: A case r e p o r t .  Reversible valproic acid-induced E p i l e p s i a 1986; 27: 234.  223  APPENDIX Page 1.  2.  3.  4.  5.  6.  7.  8.  9.  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) for BA before and a f t e r CBZ administration.  229  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) for FS before and a f t e r CBZ administration.  230  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) for MS before and a f t e r CBZ administration.  231  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) for RM before and a f t e r CBZ administration.  232  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) for WT before and a f t e r CBZ admin i s t r a t i o n .  233  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r BA before and a f t e r a d m i n i s t r a t i o n of CBZ.  234  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) for FS before and a f t e r a d m i n i s t r a t i o n of CBZ.  235  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r MS before and a f t e r CBZ administration.  236  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r RM before and a f t e r a d m i n i s t r a t i o n of CBZ.  237  224  10.  11.  12.  13.  14.  15.  16.  17.  18.  19.  Serum A U C for V P A and m e t a b o l i t e s over (mg.h/L) f o r WT before and a f t e r C B Z administration.  12 h 238  Serum A U C for V P A and m e t a b o l i t e s over 1 2 h expressed as pathways (mg.h/L) for B A b e f o r e and a f t e r C B Z a d m i n i s t r a t i o n .  239  Serum A U C for V P A and m e t a b o l i t e s over 1 2 h expressed as pathways (mg.h/L) for FS b e f o r e and a f t e r C B Z a d m i n i s t r a t i o n .  240  Serum A U C f o r V P A and m e t a b o l i t e s over 1 2 h expressed as pathways (mg.h/L) for MS b e f o r e and a f t e r C B Z a d m i n i s t r a t i o n .  241  Serum A U C f o r V P A and m e t a b o l i t e s over 1 2 h expressed as pathways (mg.h/L) for RM b e f o r e and a f t e r C B Z a d m i n i s t r a t i o n .  242  Serum A U C f o r V P A and m e t a b o l i t e s over 12 h expressed as pathways (mg.h/L) for WT before and a f t e r C B Z a d m i n i s t r a t i o n .  243  and m e t a b o l i t e s recovered in u r i n e over 12 h f o r B A before and a f t e r a d m i n i s t r a t i o n of C B Z .  (M^OI)  244  V P A and m e t a b o l i t e s recovered in u r i n e over 1 2 h f o r FS before and a f t e r a d m i n i s t r a t i o n of C B Z .  (Mmol)  V P A and m e t a b o l i t e s recovered in u r i n e over 1 2 h for MS before and a f t e r a d m i n i s t r a t i o n of C B Z .  (Mmol)  V P A and m e t a b o l i t e s recovered in u r i n e over 1 2 h f o r RM before and a f t e r a d m i n i s t r a t i o n of C B Z .  (umol)  VPA  225  245  246  247  20.  21.  22.  23.  24.  25.  VPA and m e t a b o l i t e s recovered in u r i n e over 12 h f o r WT before and a f t e r a d m i n i s t r a t i o n of CBZ.  (Mmol) 248  VPA and m e t a b o l i t e s recovered over 12 h i n u r i n e (Mmol) expressed as pathways f o r BA b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n .  the  VPA and m e t a b o l i t e s recovered over 12 h i n u r i n e (Mmol) expressed as pathways f o r FS before and a f t e r CBZ a d m i n i s t r a t i o n .  the  VPA and m e t a b o l i t e s recovered over 12 h in u r i n e (Mmol) expressed as pathways f o r MS before and a f t e r CBZ a d m i n i s t r a t i o n .  the  VPA and m e t a b o l i t e s recovered over 12 h in u r i n e (Mmol) expressed as pathways f o r RM before and a f t e r CBZ a d m i n i s t r a t i o n .  the  VPA and m e t a b o l i t e s recovered over 12 h i n u r i n e (Mmol) expressed as pathways for WT before and a f t e r CBZ a d m i n i s t r a t i o n .  the  249  250  251  252  253  26.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l m ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for BA ( L / h ) .  254  27.  Pathway formation c l e a r a n c e s ( C l f ) before and a f t e r CBZ a d m i n i s t r a t i o n for BA ( L / h ) .  255  28.  F r a c t i o n m e t a b o l i z e d ( f m ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r BA.  256  29.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l m ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for FS ( L / h ) .  257  30.  Pathway formation c l e a r a n c e s ( C l f ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for FS ( L / h ) .  258  31.  F r a c t i o n m e t a b o l i z e d (fm) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r F S .  259  a  a  a  a  a  a  226  32.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l m ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n f o r MS ( L / h ) .  260  33.  Pathway formation c l e a r a n c e s ( C l f ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for MS ( L / h ) .  261  34.  F r a c t i o n metabolized ( f m ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r MS.  262  35.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l m ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for RM ( L / h ) .  263  36.  Pathway formation c l e a r a n c e s ( C l f ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for RM ( L / h ) .  264  37.  F r a c t i o n metabolized ( f m ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r RM.  265  38.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l m ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for WT ( L / h ) .  266  39.  Pathway formation c l e a r a n c e s ( C l f ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for WT ( L / h ) .  267  40.  F r a c t i o n metabolized ( f m ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r WT.  268  41.  S e m i l o g a r i t h m i c p l o t of serum 4-OH VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for a) F S , b) MS, c) RM, d) WT.  42.  43.  a  a  a  a  a  a  a  a  a  S e m i l o g a r i t h m i c p l o t of serum 4-ene VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) MS, d) RM. S e m i l o g a r i t h m i c p l o t of serum 3-ene VPA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) MS, d) WT.  227  269  270  271  44.  45.  46.  47.  48.  49.  50.  51.  52.  S e m i l o g a r i t h m i c p l o t of serum 2-ene c i s VPA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n f o r a) BA, b) F S , c) RM, d) WT. S e m i l o g a r i t h m i c p l o t of serum 2-ene t r a n s VPA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n f o r a) BA, b) F S , c) MS, d) WT. S e m i l o g a r i t h m i c p l o t of serum 3-keto VPA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n f o r a) BA, b) F S , c) MS, d) WT. S e m i l o g a r i t h m i c p l o t of serum 4-keto VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n f o r a) F S , b) MS, c) RM, d) WT. S e m i l o g a r i t h m i c p l o t of serum 5-OH VPA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n f o r a) BA, b) F S , c) MS, d) WT. S e m i l o g a r i t h m i c p l o t of serum 2-PSA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n f o r a) F S , b) MS, c) RM, d) WT. S e m i l o g a r i t h m i c p l o t of serum 2-PGA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n f o r a) BA, b) F S , c) RM, d) WT. S e m i l o g a r i t h m i c p l o t of serum 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n (mg/L) versus time b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) RM, d) WT. S e m i l o g a r i t h m i c p l o t of serum 2 , 4 - d i e n e VPA c o n c e n t r a t i o n (mg/L) versus time before and a f t e r CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) RM, d) WT.  228  272  273  274  275  276  277  278  279  280  Appendix 1.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) f o r BA before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  % change  3.917 0.475 0.507 0.079 17.11 46.46 8.151 0.391 1 .240 0.016 0.088 3.314 0.510  3.781 0.375 0.381 0.066 1 3.63 26.57 7.367 0.434 1 .374 0.016 0.099 3.549 0.602  -3.47 -21 .0 -24.9 -16.5 -20.3 -42.8 -9.62 + 11.0 + 10.8 + 4.50 + 12.5 + 7.09 + 18.0  229  I  Appendix 2.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) f o r FS before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  1 .063 0.424 0. 184 0.066 8.035 53.05 4.501 0.310 1 .247 0.013 0. 120 1 .461 0.582  3.417 0.345 0. 1 79 0.063 6.482 33.85 6.318 0.308 0. 148 0.018 0. 1 23 1 .561 0.822  230  % change + 221 . -18.6 -2.72 -4.55 -19.3 -36.2 + 40.4 -0.65 -88. 1 + 38.5 + 2.50 + 6.84 + 41 .2  Appendix 3.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) f o r MS before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  2.787 0.383 0.255 0. 102 20.53 53.22 7.267 0.338 1 .038 .0.030 0. 105 2.704 0.512  3.356 0.334 0.237 0.066 1 3.33 26. 14 5.235 0.229 0.727 0.011 0.057 2.317 0.386  231  % change + 20.4 -12.8 -7.06 -35.3 -35. 1 -50.9 -28.0 -32.2 -30.0 -63.3 -45.7 -14.3 -24.6  Appendix 4.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) f o r RM before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  1.015 0.276 0. 152 0. 103 1 1 .34 40.01 5.356 0.212 0.51 1 0.002 0.081 0.852 0.577  1 .997 0.556 0.119 0.095 8.892 31 .87 5.762 0.281 1 . 1 52 0.013 0. 1 36 1.059 0.923  232  % change + 96.7 + 101 . -21.7 -7.77 -21 .6 -20.3 + 7.58 + 32.5 + 125. + 550. + 67.9 + 24.3 + 60.0  Appendix 5.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum VPA and m e t a b o l i t e trough c o n c e n t r a t i o n s (mg/L) f o r WT b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  4.681 0.400 0.41 1 0.062 1 9.04 27.37 6.220 0.245 1 .032 0.010 0.057 3.624 0.626  1.913 0.338 0.324 0.055 1 5.33 1 6.64 4.421 0.184 0.720 0.008 0.056 3.685 0.500  233  % change -59.1 -15.5 -21.2 -11.3 -19.5 -39.2 -28.9 -24.9 -30.2 -20.0 -1 .75 + 1 .68 -20. 1  Appendix 6.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r BA b e f o r e and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  % change  42.28 5.620 6.041 1 . 1 24 209.2 786. 1 76.94 3.973 19.82 0.174 1.321 36.52 6.237  34.06 4.852 4.554 0.814 144.1 501 .4 72.89 4.834 23.77 0.306 1 .225 36.60 6.985  -19.4 -13.7 -24.6 -14.1 -31.1 -36.2 -5.26 + 21 .7 + 19.9 + 75.6 -7.27 ' +0.22 + 12.0  234  Appendix 7.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r FS before and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  21.81 4.439 2.071 0.808 93.41 796.8 59.24 3.501 11.13 0.184 1 .365 16.61 7.520  48.60 4.547 2.178 0.784 81 .87 567.1 88.42 4.605 3.425 0.275 2.010 20.90 1 0.79  235  % chang< + 123. + 2.43 + 5.17 -2.82 -12.4 -28.8 + 49.3 + 31 .5 -69.2 + 49.5 + 47.3 + 25.8 + 43.5  Appendix 8.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r MS before and a f t e r CBZ administration.  Before CBZ  A f t e r CBZ  33.92 4.335 2.559 1 .009 211.1 710.7 55.40 2.370 1 1 .40 0. 183 0.733 17.74 3.688  37.09 4.338 2.904 0.825 172.5 483.6 53.38 3.203 12.65 0.110 0.927 26.44 5. 154  236  % change  •  + 9.35 + 0.07 + 13.5 -18.3 -18.3 -31 .0 -3.65 + 35. 1 + 11.0 -39.9 + 20.0 + 49.0 + 39.8  Appendix 9.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum AUC f o r VPA and m e t a b o l i t e s over 12 h (mg.h/L) f o r RM before and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  18.60 3.347 1 .756 1 . 1 58 137.9 583.3 55.79 2. 139 11.04 0.058 0.978 9.973 6.025  22.60 3.364 1 .064 0.999 1 08.3 468.2 70.83 3.388 20.21 0.072 1 .405 12.15 10.13  237  % change + 21.5 +0.51 -39.4 -14.0 -21 .5 -19.7 + 26.9 + 58.4 + 83. 1 + 24.6 + 44.7 + 21 .8 + 68. 1  Appendix  10.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  Serum AUC f o r VPA and m e t a b o l i t e s over (mg.h/L) f o r WT before and a f t e r CBZ admini s t r a t i o n .  Before CBZ  A f t e r CBZ  61 .48 5.036 4.835 0.751 243.7 498.3 77.63 2.822 1 5.50 0.113 0.798 45.06 8.064  56. 15 4.409 3.923 0.717 195.3 358.2 30.72 3. 1 05 10.67 0.119 0.823 37.67 5.659  238  12 h  % change -8.67 -12.5 -18.9 -4.60 -19.9 -28.1 -60.4 + 10.0 -31.2 +5.13 + 3.16 -16.4 -29.8  Appendix  Pathway  2 3 4 5 VPA  a  3  11.  Serum AUC for VPA and m e t a b o l i t e s over 12 h expressed as pathways (mg.h/L) f o r BA before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  287.3 42.56 1 1 .86 46.43 21.14 786. 1  217.8 41.15 1 1 .84 39.20 24.99 501 .4  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. VPA i n c l u d e s unconjugated and conjugated VPA.  239  % change -23.2 -3.31 -15.6 -36.2 + 8.20 -36.2  Appendix  Pathway  2 3 4 5 VPA  3  3  12.  Serum AUC for VPA and m e t a b o l i t e s over 12 h expressed as pathways (mg.h/L) for FS before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  1 53.5 1 8.68 1 1 .96 25.49 1 2.50 796.8  171.1 23.08 15.34 53.48 5.435 567. 1  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. VPA i n c l u d e s unconjugated and conjugated VPA.  240  % change + 9.40 + 23.6 + 28.3 + 110. -56.5 -28.8  Appendix 13.  Pathway 1 2 3 4 5 VPA  3  Serum AUC for VPA and m e t a b o l i t e s over 12 h expressed as pathways (mg.h/L) f o r MS before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  267.5 20.30 8.023 36.47 12.17 710.7  226.7 29.34 9.492 40.40 13.58 483.6  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. .Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA, Pathway i n c l u d e s 5-OH VPA and 2-PGA. VPA i n c l u d e s unconjugated and conjugated VPA.  241  % change -24.8 -44.5 + 18.3 + 10.8 + 11.6 -31.9  Appendix  Pathway  2 3 4 5 VPA  a  3  14.  Serum AUC for VPA and m e t a b o l i t e s over 12 h expressed as pathways (mg.h/L) for RM before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  1 94.9 1 1 .73 9.372 20.80 1 2.02 583.3  180. 1 13.21 13.49 26.06 21 .62 468.2  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. VPA i n c l u d e s unconjugated and conjugated VPA.  242  % change -8.61 + 12.6 + 43.9 + 25.3 + 79.9 -19.7  Appendix  Pathway 1 2 3 4 5 VPA  3  3  15.  Serum AUC for VPA and m e t a b o l i t e s over 12 h expressed as pathways (mg.h/L) for WT before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  322.1 49.90 13.10 64.42 16.30 498.3  224.7 41.59 10.07 59.37 11.49 358.2  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. VPA i n c l u d e s unconjugated and conjugated VPA.  243  % change -28.4 -16.6 -23.1 -7.84 -29.5 -28.1  Appendix  16.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA ( t o t a l ) 3-keto 4-keto 5-OH . 2-PSA 2-PGA 2,3'-diene 2,4-diene  VPA and m e t a b o l i t e s recovered i n u r i n e (jumol) over 12 h f o r BA b e f o r e and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  250.6 1 .056 0.369 0.771 65.07 464.9 336.4 33.51 303.9 5. 1 03 75. 16 34.27 5.418  232.8 1 .387 0.417 1 .047 53.35 506.4 340.5 46.43 501 .8 7.369 84.38 29.80 6.599  244  % chang< -7.10 + 31 .3 + 13.0 + 35.6 -18.0 +8.93 + 1 .22 + 38.6 + 65. 1 + 44.4 + 12.3 -13.0 + 21.1  Appendix  17.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA ( t o t a l ) 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  VPA and m e t a b o l i t e s recovered in u r i n e (/xmol) over 12 h f o r FS before and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  269.0 4.472 2.555 2.261 134. 1 1 564. 1642. 74.22 1 323. 12.49 165.4 52.65 13.99  663.9 6.632 0.682 2.527 89. 15 1317. 883.4 71 .29 1003. 22.43 187.8 48.71 14.74  245  % change + 147. + 48.3 -73.3 + 11.8 -33.5 -15.8 -46.2 -3.95 -24.2 + 79.6 + 13.5 -7.48 + 5.36  Appendix  18.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA ( t o t a l ) 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  VPA and m e t a b o l i t e s recovered i n u r i n e (ymol) over 12 h f o r MS before and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  342.0 1 .743 0.313 1 .586 117.4 880.7 355.3 25.21 317.6 5.779 74.44 50.72 8.670  410.4 2.293 0.453 1 .829 102.9 898.0 439.7 54.81 533.9 8.344 86.49 60.39 1 0.03  246  % change + 20.0 + 31.6 + 44.7 + 15.3 -12.4 + 1 .96 + 23.8 + 117. + 68. 1 + 44.4 + 16.2 + 19.1 + 15.7  Appendix  19.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA ( t o t a l ) 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  VPA and m e t a b o l i t e s recovered in u r i n e (Mmol) over 12 h f o r RM before and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  313.4 1 .250 0.212 2.477 115.6 644.2 29.25 50.36 692.0 9.740 528. 1 45.23 1 0.53  249.5 2.110 0.200 2.620 92. 10 461 .9 749. 1 58.29 1099. 6. 159 126.5 22.35 9.859  247  % change -20.4 + 68.8 -5.66 + 5.77 -20.3 -28.3 + 2461 + 15.7 + 58.8 -36.8 -76.0 -50.6 -6.37  Appendix 20.  Compound 4-OH 4-ene 3-ene 2-ene c i s 2-ene t r a n s VPA 3-keto 4-keto 5-OH 2-PSA 2-PGA 2,3'-diene 2,4-diene  VPA and m e t a b o l i t e s recovered i n u r i n e (Mmol) over 12 h f o r WT before and a f t e r a d m i n i s t r a t i o n of carbamazepine.  Before CBZ  A f t e r CBZ  299.9 1 .265 0.394 1.165 1 54.4 476.5 475.9 41 .62 485. 1 7. 1 25 85.28 84.85 1 1 .28  217.0 1 .667 0.413 1 .271 101.6 653.4 282.3 30.60 663.0 6.997 77.94 69.42 1 0.20  248  % change -27.6 + 31.8 + 4.82 + 9.10 -34.2 + 37. 1 -40.7 -26.5 + 36.7 -1 .80 -8.60 -18.2 -9.57  Appendix  Pathway 1 2 3 4 5 6 CI.  3  3  21.  VPA and m e t a b o l i t e s recovered over 12 h in the u r i n e (nmol) expressed as pathways f o r BA before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  402.2 34.64 6.474 289.2 379. 1 393. 1 71 .86  394.9 30.22 7.946 286.6 586.1 436. 1 70.38  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA, Pathway 5 i n c l u d e s - 5 - O H VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  249  % change -1 .82 -12.8 + 22.7 -0.90 + 54.6 + 10.9 -2.06  Appendix  Pathway' 1 2 3 4 5 6  VPA and m e t a b o l i t e s r e c o v e r e d o v e r 12 h i n t h e u r i n e (/umol) e x p r e s s e d a s p a t h w a y s f o r FS b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n .  Before  CBZ  After  1778. 55.21 18.46 355.7 1 488. 1514. 50. 18  CI,  a  22.  CBZ  975. 1 49.39 21 .37 756.6 1191. 1263. 53.74  Pathway 1 i n c l u d e s 2-ene VPA and 3 - k e t o VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4 - O H VPA, 4 - k e t o VPA a n d 2-PSA, Pathway 5 i n c l u d e s 5 - O H VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s u n c h a n g e d VPA. r  250  % change -45.2 -10.5 + 15.8 + 113. -20.0 -16.6 + 7.09  Appendix  Pathway  2 3 4 5 6  a  3  23.  VPA and m e t a b o l i t e s recovered over 12 h in the u r i n e (jumol) expressed as pathways f o r MS before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  474.4 51 .03 10.41 373.0 392.0 615.8 264.9  544.4 60.84 12.32 473.6 620.4 551 .7 346.3  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  251  % change + 14.8 + 19.2 + 18.3 + 26.9 + 58.3 -10.4 + 30.7  Appendix 24.  Pathway  Before CBZ 147.3 45.44 1 1 .78 373.5 1220. 94.20 550.0  2 3 4 5 6  3  3  VPA and m e t a b o l i t e s recovered over 12 h in the u r i n e (/nmol) expressed as pathways f o r RM before and a f t e r CBZ a d m i n i s t r a t i o n .  A f t e r CBZ  % change  843.8 22.55 1 1 .97 313.9 1 225. 187.0 274.9  + 473. -50.4 + 1 .61 -15.9 + 0.40 + 98.5 -50.0  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  252  Appendix  Pathway' 1 2 3 4 5 6 Cl,  a  25.  VPA and m e t a b o l i t e s r e c o v e r e d over 12 h in the u r i n e (/mol) expressed as pathways f o r WT before and a f t e r CBZ a d m i n i s t r a t i o n .  Before CBZ  A f t e r CBZ  631 .5 85.24 12.55 348.6 570.4 329.0 147.5  385.2 69.83 1 1 .87 254.6 740.9 351 .4 302.3  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway i n c l u d e s 5-OH VPA and 2-PGA. Pathway i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  253  % change -39.0 -18.1 -5.42 -26.9 + 29.9 + 6.81 + 105.  Appendix 26.  Pathway  Pathway m e t a b o l i t e c l e a r a n c e s ( C l ) before and a f t e r CBZ a d m i n i s t r a t i o n f o r BA ( L / h ) . a  m  Before CBZ  A f t e r CBZ  0.2176 0.1140 0.0766 0.9953 2.9183 0.0721 0.0132 4.4071 0.6997  2 3 4 5 6 Sum  0.2825 0.1029 0.0943 1.1674 3.7993 0.1253 0.0202 5.5919 1.0969  % change +29.8 -9.74 + 23. 1 + 17.3 + 30.2 + 73.8 + 53.0 + 26.9 + 56.8  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e over 12 h from a g i v e n pathway by the c o r r e s p o n d i n g AUC. a  m  k Pathway 1 i n c l u d e s • 2 - e n e VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  c  Cl  p  = Dose/AUC  ( v p A  )  254  Appendix 2 7 .  Pathway 1 2 3 4 5 6 c i Sum  13  Pathway formation c l e a r a n c e s ( C l f ) before and a f t e r CBZ a d m i n i s t r a t i o n for BA ( L / h ) . a  A f t e r CBZ  Before CBZ 0.0795 0.0062 0.001 2 0.0588 0.0785 0.0721 0.0132 0.3095  r  0.1227 0.0084 0.0022 0.0913 0.1894 0.1253 0.0202 0.5595  % change + 54.3 + 35.5 +83.3 + 55.3 + 141. + 73.8 + 53.0 + 80.8  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s r e c o v e r e d in the u r i n e from a given pathway by VPA AUC ( 1 2  a  f  Pathway 1 i n c l u d e s 2 - e n e VPA and 3 - k e t o VPA. Pathway 2 i n c l u d e s 3 - e n e VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4 - e n e VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4 - k e t o VPA and 2 - P S A . Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  255  h).  Appendix 28.  Pathway* 1 2 3 4 5 6 ci Sum r  :  3  Fraction metabolized ( f ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r BA. a  m  Before CBZ  A f t e r CBZ  0.1136 0.0088 0.0017 0.0840 0.1122 0.1030 0.0188 0.4421  0.1119 0.0077 0.0020 0.0832 0.1727 0.1143 0.0184 0.5102  i s c a l c u l a t e d by d i v i d i n g C l f by C l p .  m  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA, Pathway i n c l u d e s 5-OH VPA and 2-PGA. Pathway i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  256  % change -1 . SO -12.5 + 17.6 -0.95 + 53.9 + 11.0 -2.13 + 15.4  Appendix 29.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l ) before and a f t e r CBZ a d m i n i s t r a t i o n for FS ( L / h ) . a  m  Pathway"  Before CBZ  A f t e r CBZ  1 2 3 4 5 6 ci Sum Cl  1.8166 0.4142 0.2170 2.2264 19.243 0.2738 0.0091 24.200 0.8785  0.8919 0.2998 0.1960 2.2639 35.536 0.3209 0.0137 39.522 1.2344  r  c  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered in the u r i n e from a g i v e n pathway by the c o r r e s p o n d i n g AUC (12 h ) . a  m  b  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  c  Clp = D o s e / A U C  ( v p A  )  257  % change -50.9 -27.6 -9.68 + 1 .68 + 84.7 + 17.2 + 50.5 + 63.3 + 40.5  Appendix 30.  Pathway 1 2 3 4 5 6 ci Sum r  13  Pathway formation c l e a r a n c e s ( C l r ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n for FS ( L / h ) . a  Before CBZ  A f t e r CBZ  0.3499 0.0097 0.0033 0.0712 0.3018 0.2738 0.0091 1 .0188  0.2691 0.0122 0.0053 0.2135 0.3406 0.3209 0.0137 1.1753  % change -23. 1 +25.8 +60.6 +200. + 12.8 + 17.2 + 50.5 + 15.4  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered in the u r i n e from a given pathway by VPA AUC (12  a  f  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway i n c l u d e s 5-OH VPA and 2-PGA. Pathway i s VPA g l u c u r o n i d e c o n j u g a t e . Pathway Cl i s unchanged VPA. r  258  h).  Appendix 31  Pathway* 1 2 3 4 5 6 ci Sum r  f  3  F r a c t i o n metabolized ( f ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r F S . a  m  Before CBZ  A f t e r CBZ  0.3982 0.0111 0.0037 0.0811 0.3435 0.3117 0.0103 1.1596  0.2180 0.0099 0.0043 0.1730 0.2759 0.2599 0.0111 0.9521  i s c a l c u l a t e d by d i v i d i n g C l f by C l p .  m  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s • 4 - O H VPA, 4-keto VPA and 2-PSA, Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  259  % change -45.3 -10.8 + 16.2 + 113. -19.7 -16.6 + 7.77 -17.9  Appendix 32.  Pathway  Pathway m e t a b o l i t e c l e a r a n c e s ( C l ) before and a f t e r CBZ a d m i n i s t r a t i o n f o r MS ( L / h ) . a  m  Before CBZ  A f t e r CBZ  0.2731 0.3521 0.1827 1.6348 5.2382 0.1249 0.0537 7.8595 0.8442  2 3 4 5 6 Sum  0.3720 0.2904 0. 1823 1 .8726 7.3998 0.1644 0. 1032 10.385 1.2407  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e from a given pathway by the corresponding AUC (12 h ) . a  m  b  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  c  Cl  p  = Dose/AUC(  V P A  )  260  % change + 36.2 -17.5 -0.22 + 14.5 + 41 .3 + 31.6 + 92.2 + 32. 1 + 47.0  Appendix 3 3 .  Pathway 1 2 3 4 5 6 ci Sum r  13  Pathway formation c l e a r a n c e s ( C l c ) before and a f t e r CBZ a d m i n i s t r a t i o n f o r MS ( L / h ) . a  Before CBZ  A f t e r CBZ  0. 1028 0.0101 0.0021 0.0839 0.0897 0. 1249 0.0537 0.4672  0.1744 0.0176 0.0036 0.1565 0.2078 0.1644 0. 1032 0.8275  % change +69.6 + 74.3 + 71.4 + 86.5 + 1 32. + 31.6 + 92.2 + 77. 1  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered in the u r i n e from a given pathway by VPA AUC (12  a  f  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  261  h).  Appendix 34.  Pathway 1 2 3 4 5 6 ci Sum r  a  f  0  F r a c t i o n metabolized ( f ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r MS. a  m  Before CBZ  A f t e r CBZ  0.1217 0.0119 0.0024 0.0994 0.1063 0.1479 0.0636 0.5532  0.1406 0.0142 0.0029 0.1261 0.1674 0.1325 0.0832 0.6669  i s c a l c u l a t e d by d i v i d i n g C l  m  f  by C l . p  b Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  262  % change + 15.5 + 19.3 + 20.8 + 26.9 + 57.5 -10.4 + 30.8 + 20.6  Appendix 3 5 .  Pathway m e t a b o l i t e c l e a r a n c e s ( C l ) before and a f t e r CBZ a d m i n i s t r a t i o n f o r RM ( L / h ) . a  m  Pathway"  Before CBZ  A f t e r CBZ  1 2 3 4 5 6 ci Sum Cl ^ P  0.1098 0.5427 0.1763 2.8686 16.855 0.0233 0 . 1359 20.712 0.8572  0.7317 0.2390 0.1245 1.9230 9.1529 0.0576 0.0846 12.313 1.0679  r  c  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered in the u r i n e from a given pathway by the c o r r e s p o n d i n g AUC (12 h ) . a  m  b  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  c  Clp = D o s e / A U C  ( v p A  )  263  % change + 566. -56.0 -29.4 -33.0 -45.7 + 1 47. -37.7 -40.6 + 24.6  Appendix 36.  Pathway* 1 2 3 4 5 6 ci Sum r  5  Pathway formation c l e a r a n c e s ( C l f ) before and a f t e r CBZ a d m i n i s t r a t i o n for RM ( L / h ) . a  Before CBZ  A f t e r CBZ  0.0367 0.0109 0.0028 0.1023 0.3473 0.0233 0. 1359 0.6592  0.2815 0.0067 0.0036 0.1070 0.4226 0.0576 0.0846 0.9636  % change  -  +667. -38.5 +28.6 + 4.59 + 21.7 + 1 47. -37.7 + 46.2  C l f c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e from a given pathway by VPA AUC (12  a  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s • 4 - e n e VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  264  h).  Appendix 37.  Pathway 1 2 3 4 5 6 Cl Sum r  f  13  F r a c t i o n metabolized ( f ) b e f o r e and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r RM. a  m  Before CBZ  A f t e r CBZ  % change  0.0428 0.0127 0.0033 0.1193 0.4051 0.0271 0.1585 0.7688  0.2636 0.0063 0.0034 0.1002 0.3957 0.0539 0.0792 0.9023  + 516. -50.4 + 3.03 -16.0 -2.32 + 98.9 -50.0 + 17.4  i s c a l c u l a t e d by d i v i d i n g C l f by C l p .  m  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  265  Appendix 38.  Pathway m e t a b o l i t e c l e a r a n c e s ( C l ) before and a f t e r CBZ a d m i n i s t r a t i o n f o r WT ( L / h ) . a  m  Pathway"  Before CBZ  A f t e r CBZ  1 2 3 4 5 6 ci Sum CI  0.3020 0.2393 0.1343 0.8647 5.6723 0.0951 0.0427 7.3504 1.2041  0.2635 0.2352 0.1654 0.6850 10.409 0.1414 0.1216 12.021 1.6750  r  c  % change -12.7 -1 .71 + 23.2 -20.8 + 83.5 + 48.7 + 185. + 63.5 + 39. 1  C l c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered i n the u r i n e from a g i v e n pathway by the c o r r e s p o n d i n g AUC (12 h ) .  a  m  b Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. #  r  c  Cl  p  = Dose/AUC(  V p A  )  266  Appendix 3 9 .  Pathway 1 2 3 4 5 6 ci Sum r  0  Pathway formation c l e a r a n c e s ( C l c ) before and a f t e r CBZ a d m i n i s t r a t i o n f o r WT ( L / h ) . a  Before CBZ  A f t e r CBZ  0.1952 0.0240 0.0035 0.1118 0.1855 0.0951 0.0427 0.6578  0 . 1653 0.0273 0.0046 0.1136 0.3340 0.1414 0.1216 0.9078  % change -15.3 + 13.8 + 31 .4 + 1 .61 +80.0 + 48.7 + 185. + 38.0  C l f c a l c u l a t e d by d i v i d i n g the amount of m e t a b o l i t e s recovered in the u r i n e from a given pathway by VPA AUC (12  a  D  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway 3 i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway 4 i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA. Pathway 5 i n c l u d e s 5-OH VPA and 2-PGA. Pathway 6 i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  267  h).  Appendix 40.  Pathway 1 2 3 4 5 6 ci Sum r  f  1  F r a c t i o n metabolized ( f ) before and a f t e r CBZ a d m i n i s t r a t i o n by each pathway f o r WT. a  m  Before CBZ  A f t e r CBZ  0.1621 0.0199 0.0029 0.0928 0.1541 0.0791 0.0354 0.5463  0.0987 0.0163 0.0028 0.0678 0.1994 0.0844 0.0726 0.5420  i s c a l c u l a t e d by d i v i d i n g C l j by C l p ,  m  Pathway 1 i n c l u d e s 2-ene VPA and 3-keto VPA. Pathway 2 i n c l u d e s 3-ene VPA and 2 , 3 ' - d i e n e VPA. Pathway i n c l u d e s 4-ene VPA and 2 , 4 - d i e n e VPA. Pathway i n c l u d e s 4-OH VPA, 4-keto VPA and 2-PSA, Pathway i n c l u d e s 5-OH VPA and 2-PGA. Pathway i s VPA g l u c u r o n i d e c o n j u g a t e . Cl i s unchanged VPA. r  268  % change -39.1 -18.1 -3.45 -26.9 + 29.4 + 6.70 + 105. -0.79  693  Concentration, mg/L  Concentration, mg/L  >  X)  TJ ro 3 Qj  cr B --'=)  3  n  a oo3  tn ro  3  M.  W Q) (D H i-h 3 O  rt rtkQ  o ro n n> •—- »-t 0» r-|  50 ^ 3 Qj —'  O  51  rt O  3*  M-  — O 3 \  • ^ n r  •  M-  rt  *o  tB ^  rt  < 0) (B  MI  o  3  oi cn c ro 3 01 c 0) 3 i 3 o> ro ' i  rt o 3  cr ro  l-h  o i-h n o ro i-t  OJ LO  O  \x  Concentration, mg/L  Concentration, mg/L  QLZ  Concentration, mg/L  Concentration, mg/L  Concentration, mg/L  Concentration, mg/L  > TJ TJ  CD  3  to  o o 3 a. o  cr co 3  CO  ro 3  ro CO D» 3 o rr L Q n 0) 01 1 rr i— 3 M - rr to O =r 3 3 Cu  O  3  O  3 O C O  .  CO —' rr  —^_  ^  CS)  < O cu ro i-n QJ n 3 cn cn ro M- c  (  3 *»  0) ro rr  o 3  to >  ro  cr 3  ro ro  o  l-h i-l o ro i-t  a*  I  //  A  // t  / /  1 1  / 1  SERUM 4- ENE VPA FOR R.M.  3 cn c cn rr 3  Appendix 43.  Semilogarithmic p l o t of serum 3-ene VPA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) MS, d) WT.  Appendix 44.  Semilogarithmic p l o t of serum 2-ene c i s VPA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) RM, d) WT.  a)  b)  20  Time, h  Appendix  45.  Time, h  30  Semilogarithmic p l o t of serum 2-ene t r a n s VPA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n for a) BA, b) FS, c) MS, d) WT.  Appendix 46.  Semilogarithmic p l o t of serum 3-keto VPA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n for a) BA, b) F S , c) MS, d) WT.  Appendix 4 7 .  Semilogarithmic p l o t of serum 4 - k e t o VPA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n for a) F S , b) MS, c) RM, d) WT.  9LZ  Concentration, mg/L  Concentration, mg/L  Concentration, mg/L  Concentration, mg/L  > TJ ro  3  CO  (TWO O ro Qi 3 3 U O co ai ro M l 3 O  -^3  rr rr uo  n u fi -—i-i Oi  B  2  —  rr  -  o  co Qi  —  rr O 3  ^  3 O  £  3 ^ i £ > TJ \ i-  •  to—-  <  III (P QJ  3  rr O Ml  H  to oi  3 oi n  c  CO rr g rr i->. 1 3 Oi a* ro  rr  i O  H - L T I  O ro  3  O l-h i-l O ro i-i  a >  <  TJ >  Appendix 49.  Semilogarithmic p l o t of serum 2-PSA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n f o r a) F S , b) MS, c) RM, d) WT.  -J  CD  Time, h  Appendix  50.  Semilogarithmic p l o t of serum 2-PGA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n f o r a) BA, b) F S , c) RM, d) WT.  Time, h  Appendix  51.  Semilogarithmic p l o t of serum 2 , 3 ' - d i e n e VPA c o n c e n t r a t i o n (mg/L) versus time before ( • ) and a f t e r ( O ) CBZ a d m i n i s t r a t i o n f o r a) BA, b) FS, c) RM, d) WT.  082  Concentration, mg/L  Concentration, mg/L  Concentration, mg/L  Concentration, mg/L  > TJ  ro  a  tn to  cr cu o CO a o ro Oi  3 3  s  o to 0) rtroKQ r-r. i-t 3 0) ro  0> r-l rt M W^ rt 3 O 3-  a— o 3 •->o• — i n TI  H  •  n c o 1  cd tsi  rt  < OJ ro Qj l-t 3 to cn  o i-h cn ro  8-  c to rt rt t- 3 -» 3 to OJ ro  cr i  ro a. o ro n 3 ro ro  ~<  TJ  CO  >  a  

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