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

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T H E E F F E C T O F C A R B A M A Z E P I N E ON V A L P R O I C A C I D M E T A B O L I S M B y S U K H B I N D E R K A U R P A N E S A R T H E S I S S U B M I T T E D I N P A R T I A L F U L F I L L M E N T T H E R E Q U I R E M E N T S F O R T H E D E G R E E OF M A S T E R OF S C I E N C E i n T H E F A C U L T Y OF G R A D U A T E S T U D I E S D i v i s i o n o f P h a r m a c e u t i c a l C h e m i s t r y F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s We a c c e p t t h i s t h e s i s a s c o n f o r m i n g t o t h e r e q u i r e d s t a n d a r d T H E © U N I V E R S I T Y OF B R I T I S H C O L U M B I A A p r i l 1 9 8 7 S u k h b i n d e r K a u r P a n e s a r , 1 9 8 7 In presenting this thesis in part ia l fulfilment of the requirements for an advanced degree at the University of Br i t i sh Columbia, I agree that the Library shal l make i t freely available for reference and study. I further agree that permission for extensive copying of this 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 this thesis for f inancial gain shal l not be allowed without my written permission. B e W M e n t o f P h a r m a c e u t i c a l C h e m i s t r y  F a c u l t y o f P h a r m a c e u t i c a l S c i e n c e s The U n i v e r s i t y o f B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date A p r i l 30, 1987 ABSTRACT Modifications to the GCMS assay for valproic acid and 12 metabolites were attempted with respect to internal standards and derivatizing reagents. 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 derivatizing reagents, MSTFA and MTBSTFA, were tested as alternatives to the reagent previously used. TMS (MSTFA) and tBDMS derivatives were compared with respect to sensitivity, stability, and chromatographic time. The derivatives formed 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 on the metabolism of Dg-VPA was obtained in one volunteer. 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 and carbamazepine are frequently coadministered in efforts to optimize seizure control. VPA is extensively metabolized while CBZ is known to induce the hepatic microsomal enzyme system, and thus, this is a potentially toxic interaction. Pharmacokinetic parameters for VPA were obtained before and after CBZ administration in five, healthy male volunteers. Increased plasma clearance of VPA i i accompanied by decreased plasma concentrations, serum half-l i f e , and AUC values were observed after CBZ comedication. This was consistent with the a b i l i t y of CBZ to induce the hepatic microsomal enzyme systems in a manner similar to phenobarbital. Serum trough and steady state concentrations and AUC values for 12 metabolites were determined before and after CBZ administration. The AUC values for the monounsaturated metabolites decreased after CBZ administration while the AUC values of the polar metabolites increased. The amount of 4-ene VPA, a potential hepatotoxin, was not increased in the serum after administration of CBZ. The amounts of the two diunsaturated metabolites, 2,3'-diene VPA and 2,4-diene VPA, were increased in the serum of the volunteers after CBZ administration. The amount of 2-ene trans VPA in the serum was significantly decreased after CBZ administration, while the amount of 3-keto VPA did not increase. Urinary metabolic profiles were determined individually and grouped in pathways for the five volunteers before and after CBZ administration. Increased recoveries of 4-ene VPA, 4-keto VPA, and 2-PSA after CBZ administration were consistent with enhanced CJ-1 oxidation. Formation clearance, metabolic clearance, and fraction metabolized were determined for the metabolic pathways and for the individual metabolites. CBZ adminstration resulted in increased formation clearances for a l l pathways. The results obtained from this study indicate that CBZ caused a general induction of VPA metabolism and did not specifically affect a particular pathway. The effect of CBZ on the beta-oxidation pathway is not clearly understood. CBZ may cause a metabolic shift away from beta-oxidation, or actually inhibit beta-oxidation to some extent. As well, peroxisomal beta-oxidation may be involved. iv TABLE OF CONTENTS Page ABSTRACT i i LIST OF TABLES ix LIST OF FIGURES x i i i LIST OF ABBREVIATIONS xx I. INTRODUCTION 1 A. Mechanism of a c t i o n 2 B. Pharmacokinetics 5 1. Absorption and d i s t r i b u t i o n 5 2. Plasma p r o t e i n b i n d i n g 6 3. E l i m i n a t i o n 7 C. Metabolism 8 1 . F a t t y a c i d metabolism 8 1.1. B e t a - o x i d a t i o n 9 1.2. Ketone bodies 10 1.3. Role of c a r n i t i n e i n f a t t y a c i d metabolism 1 1 1.4. Peroxisomal b e t a - o x i d a t i o n 11 1.5. cj-Oxidat ion 12 2. Metabolism of v a l p r o i c a c i d 12 2.1. Metabolism of 4-ene VPA 15 2.2. M e t a b o l i t e s 16 D. Adverse e f f e c t s , m e t a b o l i c d i s t u r b a n c e s , and t o x i c i t i e s 19 1. Adverse e f f e c t s 19 2. Me t a b o l i c d i s t u r b a n c e s 21 3. T o x i c i t y 25 3.1. H e p a t o t o x i c i t y 26 E. Drug i n t e r a c t i o n s 28 1. Drugs which a l t e r VPA c o n c e n t r a t i o n s 29 2. E f f e c t s of VPA on other drugs 30 F. I n t e r a c t i o n between v a l p r o i c a c i d and carbamazepine 32 v 1. Enzyme induction 32 1.1. Metabolism 33 1.2. Types of reactions 33 1.3. Factors influencing enzyme induction 34 1.4. Properties of enzyme inducing agents 34 1.5. Types of enzyme inducing agents 35 1.5.1. Phenobarbital and 3-methylcholanthrene 36 1.6. Cl i n i c a l implications 36 1.7. Markers of enzyme induction 37 OBJECTIVES 42 II. EXPERIMENTAL 44 A. Reagents and materials 44 1. Valproic acid and metabolites 44 2. Internal standards 44 3. Reagents 45 4. Drugs 46 B. Drug interaction study 46 1. Volunteer Details 46 C. Analysis 49 1. Valproic acid and metabolites 49 1.1. Stock solutions of internal standards 49 1.2. Preparation of urine and serum standards 49 1.3. Extraction and derivatization of standards and patient samples 51 1.4. Preparation of tBDMCS reagent with 5 % catalyst 53 2. Carbamazepine and carbamazepine-10,11-epoxide in serum 54 2.1. Preparation of stock solutions 54 2.2. Extraction of serum samples for CBZ and CBZE 55 3. Determination of urinary creatinine 55 4. Instrumentation 57 4.1. Valproic acid and metabolites 57 4.2. Carbamazepine and carbamazepine - 10,11-epoxide 58 5. Stat i s t i c a l analysis 58 6. Pharmacokinetic model development and calculations 59 vi III. RESULTS 64 A. Assay development 64 1. Modifications to the assay 64 2. Analysis of deuterated samples 65 3. Preparation of internal standards 69 4. Derivatizing reagents 71 4.1. Comparison of TMS and tBDMS derivatives 71 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 carbamazepine-10,11-epoxide in serum 81 D. Interaction between valproic acid and carbamazepine 85 1. Analysis of serum samples for VPA 85 2. Analysis of urine samples for VPA 97 3. Analysis of serum samples for VPA metabolites 97 4. Analysis of urine samples for VPA metabolites 141 5. Pharmacokinetic analysis 161 5.1. Pathway analysis 161 5.2. Metabolite analysis 164 IV. DISCUSSION 170 A. GCMS analysis of valproic acid and metabolites 170 1. Assay 170 1.1. Internal standards 170 1.2. Choice of derivatizing reagent 172 1.3. Stable isotopes in pharmacokinetic studies 173 B. Effect of carbamazepine on valproic acid metabolism 176 1. Inducing properties of carbamazepine 176 2. Volunteer data 179 2.1. CBZ and CBZE concentrations in serum 179 2.2. VPA data 181 2.2.1. Protocol 181 2.2.2. Serum VPA data 182 2.2.3. Serum metabolite data 186 2.2.3.1. Metabolite serum concentrations 186 2.-2.3.2. Metabolite AUC values 190 v i i 2.2.4. Urinary data 191 2.2.4.1. Recovery expressed as a percent of dose 191 2.2.4.2. Recovery expressed as a percent of total excreted 194 2.2.5. Effect of CBZ on VPA metabolism 194 2.2.6. Metabolite pathways 196 2.2.7. Pharmacokinetic analysis 197 2.2.7.1. Pathway analysis 197 2.2.7.2. Individual metabolites 199 2.2.8. Cli n i c a l significance of VPA and CBZ interaction 201 SUMMARY AND CONCLUSIONS 203 REFERENCES 207 APPENDIX 224 v i i i LIST OF TABLES Serum trough concentrations (mg/L) for VPA and metabolites after administration of VPA and Dg-VPA 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 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 C m i n (prior to morning dose), 3 h, and 5 h post close. Serum CBZE concentrations (uq/mL) 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 C m i n (prior to morning dose), 3 h, and 5 h post dose. Percent ratio 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 C m^ n (prior to morning dose) 3h, and 5 h post dose. Valproic acid kinetic parameters for five healthy volunteers before and after administration of carbamazepine. ix 8. Mean serum valproic acid and metabolites trough concentrations (mg/L) before and after administration of carbamazepine in five 129 volunteers. Numbers in parentheses represent range. 9. Mean serum AUC (mg.h/L) for VPA and metabolites over 12 h before and after carbamazepine administration. Numbers in parentheses 130 represent range (n=5). 10. Sum of serum AUC (mg.h/L) for polar metabolites of valproic acid over 12 h for the five healthy volunteers before and after 134 administration of carbamazepine. 11. Sum of serum AUC (mg.h/L) for unsaturated metabolites of valproic acid over 12 h for the five healthy volunteers before and after 134 administration of carbamazepine. 12. Mean serum AUC (mg.h/L) over 12 h for valproic acid and metabolites expressed as pathways before and after carbamazepine administration. 135 Numbers in parentheses represent range (n=5). 13. Mean 'average serum steady state concentrations' (mg/L) for valproic acid and metabolites before and after administration of 142 carbamazepine. Numbers in parentheses represent range (n=5). 14. Mean 'average steady state serum concentrations' (mg/L) of valproic acid and metabolites expressed as pathways before and 143 after administration of carbamazepine. Numbers in parentheses represent range (n=5). 15. Mean valproic acid and metabolites (nxmol) recovered in urine over 12 h before and after carbamazepine administration. Numbers in 144 parentheses represent range (n=5). x 16. Mean valproic acid and metabolites (Mmol) recovered in the urine over 12 h expressed as pathways before and after carbamazepine 145 administration. Numbers in parentheses represent range (n=5). 17. Sum of polar metabolites of VPA (/nmol) recovered in the urine over 12 h for the five healthy volunteers before and after 149 administration of carbamazepine. 18. Sum of unsaturated metabolites of VPA (Mmol) recovered in the urine over 12 h for the five healthy volunteers before and after 149 administration of carbamazepine. 19. Mean valproic acid and metabolites recovered in the urine over 12 h as percent of VPA dose before and after carbamazepine administration. 150 Numbers in parentheses represent range (n=5). 20. Percent of valproic acid dose recovered in the urine as VPA and metabolites over 12 h for the five healthy volunteers before and after 151 carbamazepine administration. 21. Mean valproic acid and metabolites (umolar basis) recovered over 12 h expressed as percent of total recovered before and after 155 carbamazepine administration in five volunteers. Numbers in parentheses represent range. 22. Mean valproic acid and metabolites (Mmolar basis) recovered over 12 h in the urine expressed as percent of VPA recovered before 156 and after carbamazepine administration in five volunteers. Numbers in parentheses represent range. 23. Mean formation clearances (Clf, L/h) for pathways 1 - 6 before and after CBZ administration for five healthy volunteers. 162 xi 24. Mean metabolite clearances ( C l m , L/h) for pathways before and after CBZ administration for five healthy volunteers. 163 25. Mean fraction metabolized ( f m ) by each pathway before and after CBZ administration for five volunteers. 165 26. Mean metabolite formation clearances (Clf, L/h) for the VPA metabolites before and after CBZ administration. Numbers in parentheses 166 represent range (n=5). 27. Mean metabolite metabolite clearances (Cl^, L/h) for the VPA metabolites before and after CBZ administration. Numbers in parentheses 168 represent range (n=5). 28. Mean fraction ( f m ) of metabolite metabolized before and after carbamazepine administration. Numbers in parentheses represent range (n=5). 169 29. Comparison of serum VPA and metabolite concentrations (nq/mL) in volunteers with patient data. Numbers in parentheses 188 represent range. 30. Comparison of mean serum steady state VPA and metabolite concentrations (uq/mh) from two volunteer studies. Numbers in parentheses 189 represent range. 31. Comparison of urinary recovery of VPA and metabolites expressed as a percent of dose in two volunteer studies. Numbers in parentheses 193 represent range. 32. Comparison of recovery of VPA and metabolites as a percent of the total amount recovered. Numbers in parentheses represent range. 195 x i i LIST OF FIGURES Page 1. Human metabolism of valproic acid. 14 2. Extraction and derivatization scheme for valproic acid and metabolites from urine and 52 serum. 3. Extraction scheme for carbamazepine and carbamazepine-10,11-epoxide from serum. 56 4. Pharmacokinetic model applied to valproic acid-carbamazepine study. 60 5. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient 66 urine sample. 6. Calibration curve for 2-PSA in serum using 2-methylglutaric acid as the internal standard. 67 7. Calibration curve for 2-PGA in serum using 2-methylglutaric acid as the internal standard. 68 8. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient 70 urine sample. 9. Selected ion chromatograms of TMS derivatives of valproic acid and metabolites from a patient 73 urine sample. 10. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites 74 from a patient urine sample. xi i i 11. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites 76 from a patient urine sample. 12. Peak area ratio of tBDMS derivatives of 2-ene trans VPA, 5-OH VPA, 4-keto VPA, 2-ene cis 77 VPA, and 2,4-diene VPA from MTBSTFA reagent versus heating time. 13. Change in peak area ratio of 3-keto VPA mono-and diderivative to D3-2ene cis VPA with 79 increased heating time. 14. Change in peak area ratio of 3-keto VPA mono-and diderivative to D3~2-ene cis VPA with 80 storage. 15. Liquid chromatogram of 1O-methoxycarbamazepine from a spiked serum sample. 89 16. Liquid chromatogram of 10-methoxycarbamazepine, carbamazepine and 90 carbamazepine-10,11-epoxide from a spiked serum sample. 17. Liquid chromatogram of a patient serum sample 3 h post dose, after one week of carbamazepine 91 200 mg daily. 18. Semilogarithmic plot of serum VPA concentration (mg/L) versus time for BA before 92 CBZ and after CBZ administration. 19. Semilogarithmic plot of serum VPA concentration (mg/L) versus time for FS before 93 CBZ and after CBZ administration. 20. Semilogarithmic plot of serum VPA concentration (mg/L) versus time for MS before 94 CBZ and after CBZ administration. xiv 21. Semilogarithmic plot of serum VPA concentration (mg/L) versus time for RM before 95 CBZ and after CBZ administration. 22. Semilogarithmic plot of serum VPA concentration (mg/L) versus time for WT before 96 CBZ and after CBZ administration. 23. Plot of VPA h a l f - l i f e ( t , / 2 , h) before (Day 7) and after (Day 23) CBZ administration in 99 volunteers (n=5). 24. Plot of VPA clearance ( C l p , L/h) before (Day 7) and after (Day 23) CBZ administration in 100 volunteers (n=5). 25. Plot of VPA elimination rate constant (Ke, h - 1) before (Day 7) and after (Day 23) 101 CBZ administration in volunteers (n=5). 26. Plot of VPA AUC (mg.h/L) before (Day 7) and after (Day 23) CBZ administration in 102 volunteers (n=5). 27. Plot of VPA volume of distribution (Vd, L/kg) before (Day 7) and after (Day 23) CBZ 103 administration in volunteers (n=5). 28. Representative semilogarithmic plot of 4-OH VPA concentration (mg/L) versus time before 104 CBZ and after CBZ administration. 29. Representative semilogarithmic plot of 4-ene VPA concentration (mg/L) versus time before 105 CBZ and after CBZ administration. 30. Representative semilogarithmic plot of 3-ene VPA concentration (mg/L) versus time before 106 CBZ and after CBZ administration. xv 31. Representative semilogarithmic plot of 2-ene cis VPA concentration (mg/L) versus time 107 before CBZ and after CBZ administration. 32. Representative semilogarithmic plot of 2-ene trans VPA concentration (mg/L) versus time 108 before CBZ and after CBZ administration. 33. Representative semilogarithmic plot of 3-keto VPA concentration (mg/L) versus time before 109 CBZ and after CBZ administration. 34. Representative semilogarithmic plot of 4-keto VPA concentration (mg/L) versus time before 110 CBZ and after CBZ administration. 35. Representative semilogarithmic plot of 5-OH VPA concentration (mg/L) versus time before 11 1 CBZ and after CBZ administration. 36. Representative semilogarithmic plot of 2-PSA concentration (mg/L) versus time before CBZ 112 and after CBZ administration. 37. Representative semilogarithmic plot of 2-PGA concentration (mg/L) versus time before CBZ 113 and after CBZ administration. 38. Representative semilogarithmic plot of 2,3'-diene VPA concentration (mg/L) versus time 114 before CBZ and after CBZ administration. 39. Representative semilogarithmic plot of 2,4-diene VPA concentration (mg/L) versus time 115 before CBZ and after CBZ administration. 40. Plot of 4-OH VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in 116 volunteers (n=5). xvi 41. Plot of 4-ene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in 117 volunteers (n=5). 42. Plot of 3-ene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in 118 volunteers (n=5). 43. Plot of 2-ene cis VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration 119 in volunteers (n=5). 44. Plot of 2-ene trans VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) 120 administration in volunteers (n=5). 45. Plot of 3-keto VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration 121 in volunteers (n=5). 46. Plot of 4-keto VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration 122 in volunteers (n=5). 47. Plot of 5-OH VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in 123 volunteers (n=5). 48. Plot of 2-PSA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in 124 volunteers (n=5). 49. Plot of 2-PGA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration in 125 volunteers (n=5). 50. Plot of 2,3'-diene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration 126 in volunteers (n=5). xvi i 51. Plot of 2,4-diene VPA AUC (mg.h/L) before CBZ (Day 7) and after CBZ (Day 23) administration 127 in volunteers (n=5). 52. Histograms of AUC (mg.h/L)values of polar metabolites before CBZ and after CBZ 131 administration in volunteers (n=5). 53. Histograms of AUC (mg.h/L)values of unsaturated metabolites before CBZ and after 132 CBZ administration in volunteers (n=5). 54. Pathway 1 (beta-oxidation of valproic acid). 136 55. Pathway 2 (dehydrogenation of valproic acid). 137 56. Pathway 3 (dehydrogenation of valproic acid). 138 57. Pathway 4 (CJ-1 oxidation of valproic acid). 139 58. Pathway 5 (a>-oxidation of valproic acid). 140 59. Histograms of mean recovery (Mmol) of unsaturated metabolites before CBZ and after 146 CBZ administration in volunteers (n=5). 60. Histograms of mean recovery (Mmol) of polar metabolites before CBZ and after CBZ 147 administration in volunteers (n=5). 61. Histograms of mean recovery (Mmol) of unsaturated metabolites expressed as percent 152 of dose before CBZ and after CBZ administration in volunteers (n=5). 62. Histograms of mean recovery (Mmol) of polar metabolites expressed as percent of dose 153 before CBZ and after CBZ administration in volunteers (n=5). xvi i i 63. Histograms of mean unsaturated metabolites recovered in the urine expressed as a percent 157 of the total amount recovered before CBZ and after CBZ administration in five volunteers. 64. Histograms of mean polar metabolites recovered in the urine expressed as a percent of the 158 total amount recovered before CBZ and after CBZ administration in five volunteers. 65. Histograms of mean unsaturated metabolites recovered in the urine expressed as a percent 159 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 160 recovered before CBZ and after CBZ administration in five volunteers. xix LIST OF ABBREVIATIONS 2,3'-Diene VPA 2,4-Diene VPA 2-Ene VPA 2-PGA 2- PSA 3- Ene VPA 3-Keto VPA 3- OH VPA 4- Ene VPA 4-Keto VPA 4- OH VPA 5- OH VPA [2H3]-2-Ene VPA [2H6]-VPA AUC CBZ CBZE c i p D3-2-Ene VPA D6-VPA Degrees ° DMAP DNBA 2-[(E)-1'-propenyl]-(E)-2-pentenoic acid 2-Propyl-(E)-2,4-pentadienoic acid 2-Propyl-2-pentenoic acid 2-Propylglutaric acid 2-Propylsuccinic acid 2-Propyl-3-pentenoic acid 2-Propyl-3-oxopentanoic acid 2-Propyl-3-hydroxypentanoic acid 2-Propyl-4-pentenoic acid 2-Propyl-4-oxopentanoic acid 2-Propyl-4-hydroxypentanoic acid 2-Propyl-5-hydroxypentanoic acid [3,5,5,2H]-3-heptene-4-carboxylic ac id [ 2Hg]-Valproic acid Area under the serum concentration versus time curve Carbamazepine Carbamazepine-10,11-epoxide Plasma clearance [3,5,5,2H]-3~heptene-4-carboxylie acid [ 2Hg]-Valproic acid Degrees Celsius 4-Dimethylaminopyridine Di-/z-butylacetic acid xx LIST OF ABBREVIATIONS (CONT'D) GCMS Gas chromatography-mass spectrometry GLC Gas liquid chromatography HA Hexanoic acid HPLC High performance liquid chromatography Ke Elimination rate constant MCBZ 1O-Methoxycarbamazepine MGA 2-Methylglutaric acid MSTFA N-methyl-N-trimethylsilyltrifluoroacetamide MTBSTFA N-tert-butyldimethylsilyl-N-methyl-tr i fluoroacetamide OA Octanoic acid SD Standard deviation SIM Selected ion monitoring t i / 2 H a l f - l i f e tBDMCS Tertiarybutyldimethylsilylchlorosilyl tBDMS Tertiarybutyldimethylsilyl TMS Trimethylsilyl Vd Volume of distribution VPA Valproic acid (2-propylpentanoic acid) xxi ACKNOWLEDGEMENT The author would like to acknowledge the supervision provided by Dr. F. S. Abbott. The author is deeply indebted to Dr. Andrew Acheampong for the synthesis of compounds used in this study, Dr. James Orr for assistance in pharmacokinetic modelling, Dr. Kevin Farrell for assistance and advice in the volunteer study, and Mr. Roland Burton for his technical and computer help. The author also wishes to acknowledge the following people for their contributions to this project: Ms. Phyllis Abbott, Mr. Bruce Allen, Mr. Shawn Black, Ms. Grace Chan, Ms. Leanne Embree, Ms. Barbara Fraser, Mr. Rajesh Mahey, Ms. Barbara McErlane, Ms. Radana Vaughn, Ms. Janice Woodley, and Mr. Matthew Wright. X X > I DEDICATION To my parents for their support. I. INTRODUCTION The anticonvulsant activity of valproic acid (VPA) was serendipitously discovered by Meunier and coworkers in 1963, almost 100 years after Burton had synthesized the compound to u t i l i z e as a solvent (Burton, 1881). The results of the f i r s t c l i n i c t r i a l on the effectiveness of VPA's anticonvulsant activity were reported in 1964 by Carraz and coworkers. VPA has been available in North America for use as an anticonvulsant agent since 1978. It is effective in the treatment of a variety of seizure types including absence, myoclonic, tonic-clonic, infantile, partial, and photo-convulsive (Rimmer and Richens, 1985). VPA is equally effective as diazepam in the prophylactic treatment of febrile convulsions (Lee et a l . , 1986)., It is also effective in the prophylaxis of post-trauma epilepsy and in status epilepticus (Rimmer and Richens, 1985). VPA has been employed successfully in a variety of other conditions including acute mania and alcohol withdrawal. Unlike other antiepileptic drugs, VPA is a branched, short chain fatty acid as illustrated below. CH3-CH2-CH2 \ CH-COOH / CH3~CH2 Valproic acid 1 VPA is extensively metabolized in the liver and a number of metabolites have been identified (Acheampong et a l . , 1983). Some of the metabolites also possess anticonvulsant activity while others may be responsible for the adverse effects associated with VPA usage (Granneman et a l . , 1984a). Since anticonvulsant therapy in most instances is a long term treatment, the potential for interactions with other drugs is greatly increased. The majority of these interactions are with other anticonvulsant agents since polytherapy is frequently initiated in an effort to optimize seizure control. The interaction between VPA and carbamazepine (CBZ) is the subject of this investigation as these two drugs are frequently coadministered in an attempt to maximize seizure control. Because CBZ is capable of inducing the metabolism of other compounds in addition to its own, this combination of drugs is a very important one to investigate. A. MECHANISM OF ACTION Although the mechanism of action of VPA is not well understood, i t s anticonvulsant activity is thought to be mediated through effects on gamma-aminobutyric acid (GABA). VPA may exert i t s effects by increasing GABA levels in the brain through competitive inhibition of GABA-transaminase (Johnston D., 1984; Wilder and Bruni, 1982; Glaser et a l . , 2 1980; Alkadhi and Banks, 1984; Perlman and Goldstein, 1984), by enhancing the neuronal responsiveness to GABA, or by a direct membrane effect (Johnston, 1984). A positive correlation between increases in GABA levels and anticonvulsant activity has been 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 a chloride ionophore or a related site to enhance GABA mediated inhibition (Meldrum, 1986). VPA also limits sustained repetitive fi r i n g without altering GABA responsiveness (MacDonald et a l . , 1985). The major locus of action of VPA appears to be suprahypophyseal, although there is the possibility of direct pituitary action (Jones et a l . , 1984). VPA will provide protection, at the same dose, against seizures induced in mice by picrotoxin, bicucculline, and pentylenetetrazole (Wilder and Bruni, 1982). Seizure control has been maintained for up to two weeks after withdrawal of the drug in a chronically infused model (Glaser et a l . , 1980). One hypothesis is that VPA and ethanol may act through the same membrane disordering mechanism (Perlman and Goldstein, 1984). After therapeutic concentrations in rats, VPA causes structural changes in the internal mitochondrial membrane by altering the conformation of the membrane proteins (Rumbach et a l . , 1986). At low doses, VPA has a direct effect on nerve membranes in frog sciatic nerve (Van Dougen et a l . , 1986). Decreased sodium and potassium conductances accompanied by a 3 reduced excitability have also been observed with VPA in this particular model. VPA acts indirectly as an inhibitor of GABA-transaminase (GABA-T) at doses as low as 125 mg/kg in mice after i.p. administration (Loscher, 1981a). VPA is superior to specific GABA-T inhibitors with respect to anticonvulsant potency at doses of 170 mg/kg in mice (Loscher, 1981b). The metabolite 2-ene VPA is more potent than VPA in inhibiting GABA-aminotransferase in vitro (Nau and Loscher, 1984). The hydroxy metabolites, 3-OH VPA and 4-OH VPA, also significantly inhibit the enzyme ijri vitro. The enzyme is also inhibited in vivo in nerve endings from mouse brain by 2-ene VPA, 3-ene VPA, 3-OH VPA, and 4-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 which may possess anticonvulsant activity (Javors and Erwin, 1980), and 2) stimulation of the beta-oxidation pathway by VPA which results in a shift towards metabolic acidosis. The ketone bodies formed are utilized by the brain and result in a greater tolerance to transient stimulation (Schreiber, 1981). VPA decreases indirectly evoked contractions of rat diaphragm and cat gastrocnemicis muscle in a dose related manner (Mansuri et a l . , 1984). This effect is thought to be due to a neuromuscular blockade caused by VPA. VPA did not have an effect on directly evoked responses. 4 B. PHARMACOKINETICS 1. Absorption and distribution After oral administration in man, VPA is rapidly and almost completely absorbed from the gastrointestinal tract (Bruni and Wilder, 1979; Delgado et a l . , 1983; Pinder et a l . , 1977). Peak plasma levels are attained within one to four hours. A delay in absorption may be observed when food is ingested concommitantly (Bruni and Wilder, 1979). Plasma therapeutic levels of VPA are in the range of 50 - 100 ug/mL (Bruni and Wilder, 1979; Delgado et a l . , 1983; Pinder et a l . , 1977). A sustained release preparation of VPA (matrix tablet) yielded a more prolonged and uniform absorption rate in dogs when compared to the regular dosage form (Bialer et a l . , 1984a). Sustained plasma levels were also obtained with this dosage form. The sustained release preparation had an oral bioavailability of 0.84 with respect to the regular dosage form. The area under the blood curve was not effected by the ingestion of food. Enteric coated tablets have been found to be almost 100 % bioavailable when compared to the standard dosage forms in man (Hoffman et a l . , 1986). Similar pharmacokinetic parameters have been reported for a regular and an enteric coated dosage form in epileptic patients (Albright et a l . , 1984). The only difference noted was a lag time in the absorption of the enteric coated form. The bioavailabilities 5 of a 1 g standard tablet, 1 g enteric coated tablet, and a 0.8 g gelatin capsule were not significantly different in man (Bialer et a l . , 1984b). VPA undergoes rapid distribution in the body, and is detectable in the brain within a few minutes (Delgado-Escueta et a l . , 1983). VPA is extensively bound to plasma albumin (85 - 95 %) and thus has a small apparent volume of distribution (Vd) of 0.15 - 0.40 L/kg which corresponds to the extracellular space. 2. Plasma protein binding Albumin is the only serum protein to significantly bind VPA (Kober et a l . , 1980). VPA binds at two different sites, the diazepam (Ka = 3.1 x 104 M_1) and warfarin (Ka = 1.7 x 104 M"1) sites, on the albumin molecule in human serum. In dog, VPA is 78.5 % plasma protein bound, in rat 63.4 %, and in mouse 11.9 % (Loscher, 1978). In dog, VPA binding is independent of concentration in the 5-70 Mg/mL range but decreases at higher concentrations. The binding of VPA is saturable and at high doses the free fraction may be as high as 50 % (Chadwick, 1985). However, serum VPA concentrations of 500 to 700 nM are required to saturate the binding sites on the albumin molecules (Holtzman, 1983). The degree of VPA binding is affected by pregnancy, old age, uremia, hepatic disease and hypoalbuminemic states (Levy and Moreland, 1984). The free fraction is also affected by diurnal variations in plasma free fatty acids 6 (Perucca, 1984). VPA can be displaced from i t s binding sites and consequently, the free fraction increases when drugs which are displacing agents are coadministered. The administration of fat emulsion (Intralipid R) to rhesus monkeys lead to an elevation in free VPA levels (Kutt, 1984). 3. Elimination VPA is rapidly eliminated from the body, with a plasma h a l f - l i f e of 12 - 15 h in healthy volunteers. In patients, plasma half-lives of 8 - 15 h in monotherapy, and 6 - 10 h in polytherapy are observed (Levy and Morland, 1984). In dogs and rats, after 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 is decreased in neonates and in the elderly (Gram and Bentsen, 1985; Nau et. a l . , 1981). In the elderly, free levels of VPA are increased with an accompanying reduction in intrinsic clearance (Perucca et a l . , 1984). This is possibly due to decreased protein binding or decreased drug metabolizing capacity which results in decreased hepatic clearance of free drug. The h a l f - l i f e of VPA in neonates varies from 10 - 70 h (Nau et a l . , 1981; Nau et a l . , 1982a, 1982b; Nau et a l . , 1984) with a mean h a l f - l i f e of 47 h (Nau et a l . , 1981). In guinea pigs under 10 days of age, longer half-l i f e and higher clearance values for VPA are observed (Yu et a l . , 1985). 7 In a 24 day old neonate, the h a l f - l i f e of VPA was 17.2 h, clearance (Cl) was 0.18 mL/min/kg, and Vd was 0.28 L/kg (Irvine-Meek et a l . , 1982). In the same patient at six months of age, the h a l f - l i f e of VPA decreased to 7.5 h and the Cl increased to 0.53 mL/min/kg without a significant change in Vd. Hepatic enzyme maturation and/or polytherapy may be responsible for these changes. C. METABOLISM A brief review of fatty acid metabolism is presented since VPA may share some common metabolic pathways with the fatty ac ids. 1. Fatty acid metabolism Fatty acids consist of a terminal carboxyl group and an alkyl side chain and have the basic formula, CH3-(CH2)n-COOH (Devlin, 1986). Fatty acids may be stored as triacylglycerols, formed into complex lipids which are utilized in the synthesis of c e l l structures, or used in the tricarboxylic acid cycle. Fatty acids are mainly metabolized via beta-oxidation. In beta-oxidation, 2 carbon fragments are sequentially removed from the carboxyl terminal after dehydrogenation, hydration, oxidation, and thiolysis (Devlin, 1986; Stryer, 1981). 8 1.1. Beta-oxidation The f i r s t step in the beta-oxidation cycle is the activation of the fatty acid by the formation of a fatty acyl coenzyme A (CoA), which occurs either in the endoplasmic reticulum or in the outer mitochondrial membrane (Devlin, 1986). The mitochondrial membrane is impermeable to CoA and its derivatives, so a carrier is necessary for transportation across the membrane. Carnitine is responsible for transporting the activated acyl groups across the mitochondrial membrane. The acyl group is transferred to the hydroxyl group on the carnitine molecule from the sulphur atom of CoA on the outer surface of the membrane by carnitine palmitoyl transferase I. At the inner mitochondrial membrane, the acyl group is transferred from carnitine back to CoA by carnitine palmitoyl transferase II. Inside the mitochondrion, the CoA derivatives may be oxidized by one of a group of acyl CoA dehydrogenases (Devlin, 1986). These enzymes are specific for a certain chain length; palmitoyl CoA dehydrogenase for medium and long chain fatty acids, while the other three enzymes, octanoyl CoA, and 2 butyryl dehydrogenases, are specific for shorter chain fatty acids. The function of these dehydrogenases is to remove 2 hydrogen atoms to form an enoyl CoA with a trans double bond between the second and third carbon atoms. The 2 hydrogen atoms are accepted by flavin adenine dinucleotide (FAD) and 9 ultimately, 2 electrons are channelled into the electron transport system. The alpha,beta-unsaturated acyl CoA accepts a molecule of water, a reaction catalyzed by enoyl CoA hydrase to form L-beta-hydroxyacyl CoA. L-beta-hydroxyacyl CoA is oxidized by beta-hydroxyacyl CoA dehydrogenase to beta-ketoacyl CoA which is then further oxidized in the beta-position by beta-ketothiolase. CoA is inserted and cleavage occurs at the beta-carbon to yield acetyl CoA and a saturated acyl CoA with two fewer carbons than the original substrate. The steps described above are repeated until a 4 carbon butyryl CoA remains as the intermediate. Butyryl CoA is beta-oxidized to yield acetoacetyl CoA and subsequently 2 molecules of acetyl CoA. 1.2. Ketone bodies Ketone bodies are composed of acetoacetic acid and its reduction product beta-hydroxybutyric acid (Devlin, 1986). Acetoacetate undergoes a slow, spontaneous, nonenzymatic decarboxylation to acetone or is reduced to beta-hydroxybutyrate depending on the intramitochondrial NADH/NAD+ ratio. Under normal conditions the serum concentrations of the two constituents are less than 0.2 mM. During starvation, the concentrations may be as high as 3 - 5 mM. In diabetic ketoacidosis, the concentrations of ketone bodies may be as 10 high as 20 mM. Ketone bodies replace glucose as the major fuel for respiration for the CNS during prolonged starvation. 1.3. Role of carnitine in fatty acid metabolism Carnitine is required for the transport of activated fatty acids of chain length 12 to 18 carbons across the mitochondrial membrane. However, short chain fatty acids can directly diffuse across the membrane and become activated to the CoA derivatives in the matrix compartment of the mitochondrion, i.e. the oxidation of short chain fatty acids is independent of carnitine. Deficiency of carnitine leads to aching muscle cramps which are precipitated by fasting, exercise, or a high fat diet. These are a l l instances where fatty acid oxidation is the major energy-yielding source. 1.4. Peroxisomal beta-oxidation Beta-oxidation of fatty acids was i n i t i a l l y thought to be a mitochondrial event, but recent work has demonstrated that peroxisomes are capable of performing beta-oxidation (Devlin, 1986). Peroxisomes are subcellular organelles which are present in the kidney, li v e r , and other various tissues. Peroxisomal beta-oxidation in mammals differs from mitochondrial beta-oxidation in three ways. First, the i n i t i a l dehydrogenation step in peroxisomes is catalyzed by a cyanide-insensitive oxidase system leading to the formation of hydrogen peroxide which is eventually eliminated. Second, the enzymes 11 involved in the cycle differ slightly and peroxisomes appear to be specific for longer chain fatty acids. Third, i t appears that the role of peroxisomes may be to shorten the chain length of relatively long fatty acids such that they can then be oxidized in the mitochondria. This hypothesis is based on observations that peroxisomes are unable to proceed beyond 8 carbons in shortening long chain fatty acids. 1.5. co-Oxidat ion co-Oxidation is a minor pathway for fatty acid metabolism and occurs primarily in medium chain length fatty acids (Devlin, 1986). co-Oxidation occurs in the endoplasmic reticulum of many tissues and involves hydroxylation at the methyl carbon on the opposite end from the carboxyl group, co-1 Oxidation occurs at the carbon atom next to the methyl carbon. Hydroxylation involves "mixed function oxidases", cytochrome P-450, oxygen and NADPH. After hydroxylation, the fatty acid may be further oxidized to a dicarboxylic acid at which point beta-oxidation can occur from either end of the molecule. 2. Metabolism of valproic acid VPA undergoes extensive biotransformation in the body through at least four major metabolic pathways: glucuronid-ation, beta-oxidation, co-oxidation, and co-1 oxidation (Loscher, 1981; Granneman et a l . , 1984a). Beta-oxidation occurs in mitochondria and in peroxisomes while co- and co-1 oxidation are 12 endoplasmic reticulum events (Van Den Branden and Roels, 1985). The metabolism of VPA in humans is illustrated in figure 1. Glucuronidation and beta-oxidation are the two primary metabolic pathways in both rats and man (Granneman et a l . , 1984a). With increasing doses of VPA, glucuronidation is increased at the expense of beta-oxidation (Granneman et a l . , 1984a; Granneman et a l . , 1984b). In the male rhesus monkey, the major 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. Glucuronidation occurs mainly in the microsomes (Granneman et a l . , 1984a). Approximately 15 - 20 % of an oral dose (1 g) of VPA in man is excreted in the urine as the glucuronide conjugate (Granneman et a l . , 1984b). Beta-oxidation of VPA leads to the formation of 2-ene VPA, 3-OH VPA, and 3-keto VPA. Heinemeyer and co-workers have suggested that 3-keto VPA may be a product of peroxisomal beta-oxidation since its excretion in rats is enhanced by clofibrate, a peroxisomal proliferator (Heinemeyer et a l . , 1985). w-Oxidation results in the formation of 5-OH VPA and 2-propylglutaric acid (2-PGA). 2-PGA is the end product of co-oxidation and is not metabolized further by beta-oxidation (Kuhara and Matsumoto, 1974). After oral administration of 2-PGA to rats, the intact compound was recovered in urine. 13 CMJ-CHJ-O. CHC00O.M C " J - C M I - W 2 ON C M J - C M J - C H ^ S-OH W CHCOOH HOOC-CHj-CH, ;CHC00H i -Propy lo luUrU i c l d MLPROIC MID CH,—Otf-CHj CHj-CHj-CH^ OH CNJ -CH-CHJ C HJ - C HJ - C H ^ ,CHCOOH CHCOOH 4-OH »P> 0 C H j - C - C H ? C H j - C H j - C H ^ «-Ktto VP*. CHCOOH CHj—CH-CHj .CHCOOH C H = C H - C H , CH j -CH j -CH^ *-tnt »P> CH. -CH-CH JCHCOOH CHpCH-CHj < . « ' - d l t w »P> e H , - C H ? - C H ^ 3-twt »P* C H = C H - C CHCOOH C-COOH CH,_CH ? -CH C H , - C H ? - C H ^ t-«nt m c—com CHj-CHj-c(tj CHj— CH=CII ^C-COOH CH—CHj—CH ?(C).3'(C)H1UI» m i /JH C H j - C H j - C H ^ C H j - C H j - C H ^ 3-OH m CH-COOH COOH /vvun CH, -CH, -CH , -CHr * * COOH l-P>op»lMlon1c tc\i HOOC-CH, CHCOOH C H , - C H ? - o £ ?-Propirl»ucclnlc t H d C H 3 - C H ? - C \ C H j - C H j - C H ^ CHCOOH 3-*eto VP* Figure 1 . Human metabolism of va lp ro ic a c i d . co-1 Oxidation on carbon 4 leads to the formation of 4-OH VPA, 4-keto VPA (Acheampong et a l . , 1983), and 2-propylsuccinic acid (2-PSA). The formation of 2-ene VPA and 3-ene VPA may be either peroxisomal or mitochondrial (Granneman et a l . , 1984a). Further metabolism of 4-ene VPA, 3-ene VPA and 2-ene VPA results in the formation of the diunsaturated metabolites, the dienes. The unsaturated metabolite, 4-ene VPA is metabolized further via dehydrogenation to 2,4-diene VPA and similarily 3-ene VPA to 2,3'-diene VPA. The two diunsaturated metabolites possibly may be formed from 2-ene VPA. The major diunsaturated metabolite has been identified as 2-[(E)-1'-propenyl](E)-2-pentenoic acid (2,3'-diene VPA) (Acheampong and Abbott, 1985). Hydroxylation of VPA in the 3, 4, and 5 positions is a microsomal process as determined by incubation of VPA with rat liver microsomes (Prickett and B a i l l i e , 1984). The metabolite 3-OH VPA may also be formed through co-2 oxidation as well as beta-oxidat ion. 2.1. Metabolism of 4-ene VPA The metabolism of 4-ene VPA has been studied in the adult male rhesus monkey. A biexponential curve was found after an i.v. bolus dose of 14 mg/kg (Rettenmeier et a l . , 1986b). The pharmacokinetic profile was similar to that of VPA. Free 4-ene VPA plasma levels were approximately 2.5 times that of VPA. Twenty metabolites were identified in the urine with 59 % of 15 the dose being recovered i n 24 h. G l u c u r o n i d a t i o n and beta-o x i d a t i o n were i d e n t i f i e d as the major metabolic pathways, co-and to-1 o x i d a t i o n were minor r o u t e s . E i g h t m e t a b o l i t e s of 4-ene VPA were d e t e c t e d by GLC and GCMS from r a t b i l e and p e r f u s a t e medium from i s o l a t e d r a t l i v e r s (Rettenmeier et a l . , 1985). The recovered m e t a b o l i t e s were i d e n t i f i e d as 2,4-diene VPA, 3-OH-4-ene VPA, 3-keto-4'-ene VPA, 5-OH-4'-ene VPA, 5-OH VPA, 4,5-dihydroxy VPA gamma l a c t o n e , 2-PGA, and the parent m e t a b o l i t e , 4-ene VPA. A l l me t a b o l i t e s were d e r i v e d v i a e i t h e r b e t a - o x i d a t i o n or cytochrome P-450 mediated r e a c t i o n s . Six metabolic pathways f o r the b i o t r a n s f o r m a t i o n of 4-ene VPA were a s s i g n e d : beta-o x i d a t i o n on the unsaturated s i d e c h a i n (2,4-diene VPA, and 3-OH-4-ene VPA, cytochrome P-450 mediated), b e t a - o x i d a t i o n on the s a t u r a t e d s i d e c h a i n (3-keto), co-hydroxylation to the primary a l c o h o l and subsequently to the d i c a r b o x y l i c a c i d (2-PGA), r e d u c t i o n f o l l o w e d by o x i d a t i o n to the d i a c i d , e p o x i d a t i o n to the gamma bu t y r o l a c t o n e d e r i v a t i v e , and h y d r o x y l a t i o n at the C-3 p o s i t i o n t o 3-OH-4-ene VPA. 2.2. M e t a b o l i t e s VPA m e t a b o l i t e s are present i n v a r i o u s amounts i n the plasma of humans. The q u a n t i t i e s are as f o l l o w s : 2-ene VPA 3 - 7 %, 3-keto VPA 5 - 11 %, 3-OH VPA 0.5 - 2%, 4-OH VPA 0.2 -1 %, 5-OH VPA 0.2 - 2%, and 2-PGA 0.2 - 2.5 % (Nau and Loscher, 1984; Losher, 1981c). These val u e s were determined i n the 16 plasma by gas chromatography from 26 epileptic patients either on VPA alone or in combination with other anticonvulsants. Values determined from the serum of 34 pediatric patients by a GCMS assay were as follows in uq/mL: 4-OH VPA 0 - 1.78, 2,4-diene VPA 0.02 - 0.58, 2,3'-diene 0.5 - 7.29, 4-ene VPA 0.16 - 1.22, 3-ene VPA 0.25 - 1.86, 2-ene cis 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 VPA, and 4-OH VPA a l l elevated the thresholds for maximal electroconvulsions (MEC) and pentylenetetrazol (PTZ) convulsions in mice but 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 in mice (Nau and Loscher, 1984; Loscher and Nau, 1985). These two metabolites may be responsible for the delayed effects of VPA observed after the drug is withdrawn and no longer detectable in the plasma. The spectrum of activity of 2-ene VPA is similar to that of VPA without the potential for embryotoxicity even at doses of 600 mg/kg. (Loscher et a l . , 1984). At high doses of 200 - 300 mg i.p., 2-ene is more sedating than VPA. In four different models of anticonvulsant activity, i t s activity was similar to 17 that of VPA. 2-Ene VPA was more potent in general tonic clonic seizures in gerbils and in petit mal recurrent seizures in rats. In the maximal electroseizures (MES) and PTZ tests in mice, doses of 200 - 300 mg/kg of 2-ene VPA were more sedating but side effects were not observed in rats or gerbils at these doses. The anticonvulsant activity of 2-ene VPA is 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 and 2-ene VPA are transferred rapidly from the plasma to the liver in mice after oral doses of 50 mg/kg (Nau and Loscher, 1985; Loscher and Nau, 1984). VPA liver concentrations were higher compared to plasma concentrations. However, 2-ene VPA plasma concentrations were greater than liver levels possibly due to the greater plasma protein binding of 2-ene VPA (97 %) compared to VPA (36 %) in mice. Liver/plasma drug level ratios were concentration-dependent suggesting the possibility of an active transport mechanism. Valpromide, the acid amide derivative of VPA, was more potent than VPA (2 - 5 times) and 2-ene VPA but displayed greater sedation and toxicity 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 TOXICITIES 1. Adverse effects The majority of side effects associated with VPA are mild; nausea, vomiting, diarrhea and abdominal cramps are most commonly observed (Bruni and Wilder, 1979). Other side effects include transient hair loss, weight gain, hyperkinesia, fine postural tremor, drowsiness, and transient hallucinations, with the latter three being dose related (Dulac et a l . , 1986). Transient and self limiting neutropenia and thrombocytopenia have also been observed with VPA (Barr et a l . , 1982). An 800 mg oral dose of VPA increased growth hormone secretion in healthy males but not in chronic schizophrenics (Monteleone et a l . , 1986). A 21 year old epileptic man displayed VPA-induced dementia (VPA trough level 116.3 /ig/mL) which completely and promptly remitted after withdrawal of the drug (Zaret and Cohen, 1986). The dementia may have resulted through a direct central nervous system effect, an indirect central nervous system effect due to VPA-induced hyperammonemia, or a paradoxical effect secondary to VPA. Therapeutic doses of VPA do not affect fetal growth but adverse effects are dose related and the risk for perinatal distress is associated with high doses of VPA (Jager-Roman et a l . , 1986). VPA administration to rhesus monkeys at a human therapeutic dose of 20 mg/kg/day during organogenesis did not 19 result in any adverse effects but at a dose of 200 mg/kg/day, i t resulted in low birth weights, craniofacial and skeletal defects, while at a dose of 300 mg/kg/day i t was embryolethal (Mast et a l . , 1986). A consistent facial phenotype (epicanthal folds, f l a t nasal bridge etc.) was observed in seven children who had been exposed to VPA in utero (DiLiberti et a l . , 1984). In whole rat embryos, VPA in doses greater than 40 mg/kg/day caused abnormal development in 30 % of embryos while 2-ene VPA had no adverse effects at doses up to 200 mg/kg/day (Lewandowski et a l . , 1986). VPA is associated with an increased risk of neural tube defects after f i r s t trimester fetal exposure possibly due to interferences with zinc and other trace element metabolism in humans (Weinbaum et a l . , 1986). Peak or steady state concentrations reached in the mother and gestational material correlate with the incidence of neural tube defects in mice (Nau, 1986). In mice, the dose of VPA and area under the serum concentration versus time curve (AUC) correlate with embryolethality and fetal weight retardation (Nau, 1985). VPA is also thought to cause renal damage in rats in a manner similar to zinc deficiency (Vormann et a l . , 1986). Administration of VPA 300 mg/kg/day to pregnant rats increased the incidence of fetal hydronephrosis and hydrops in animals fed with a zinc deficient diet and induced fetal liver necrosis independent of zinc status. Plasma zinc levels in rats that were toxic on VPA were decreased although there was not any 20 conclusive evidence that zinc deficiency is caused by hepatotoxic doses of VPA (Daffron and Kasarskis, 1984). 2. Metabolic disturbances VPA interferes with the folate-dependent one carbon enzyme responsible for glycine cleavage, leading to increased concentrations of glycine in patients and animals treated with VPA (Carl, 1986). VPA i n i t i a l l y causes a redistribution of folate with decreases in liver concentrations and increases in brain and plasma levels, but the effects revert to normal after several weeks. Hyperglycinemia and hyperglycinuria are observed in rats administered VPA at doses ranging from 0.3 to 1.2 mmol/kg for several weeks (Cherruau et a l . , 1981). Chronic administration of 1 % VPA to young rats resulted in 50 % and 35 % reductions in the glycine cleavage system in the liver and brain, respectively (Martin-Gallardo et a l . , 1985). Consequently, blood, liver, and brain glycine levels were significantly elevated. This action of VPA simulates in rats the metabolic disorder, non-ketotic hyperglycinemia. VPA therapy is also associated with increased serum and erythrocyte ammonia levels as well as hyperbilirubinemia (Matsuda et a l . , 1986; Ratnaike et a l . , 1986). Serum ammonia levels are particularily increased when VPA is coadministered with other anticonvulsants, especially phenobarbital or 21 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, 1986; Laub et a l . , 1986). VPA forms valproylcarnitine derivatives which are less toxic than VPA, leading to an increased metabolic need for carnitine to excrete the more toxic metabolites. Patients on VPA display decreased plasma carnitine levels accompanied by elevated blood ammonia levels (Ohtani et a l . , 1982). Oral administration of carnitine 50 mg/kg/day for 4 weeks was successful in correcting the VPA induced carnitine deficiency as well as the hyperammonemia. It is possible that the decrease in carnitine levels is a result of the induced hyperammonemia. In one patient who developed hepatotoxicity, carnitine levels were normal and carnitine supplementation did not aid in reversing the toxicity (Laub et al.., 1986). Single doses of VPA in the therapeutic range for man will cause decreased plasma beta-hydroxybutyrate levels, and decreased hepatic levels of free CoA, acetyl CoA, and free carnitine in normal infant mice (Thurston et a l . , 1985). VPA inhibits urea synthesis in rat hepatocytes (Coude, 1983; Turnbull et a l . , 1983). It also inhibits pyruvate and palmitate oxidation by 30 - 50 % in isolated rat hepatocytes at doses ranging from 0.1 - 1 mM (Turnbull et a l . , 1983). Oxidative phosphorylation in isolated liver mitochondria is inhibited along with the glycine cleavage system (Hayasaka et 22 a l . , 1986). Fatty acid synthesis and fatty acid oxidation are also inhibited by VPA in isolated rat hepatocytes. VPA competitively inhibits the pyruvate carrier in rat brain and in liver mitochondria (Benavides et a l . , 1982). At 100 mg/kg, VPA is hypoglycemic and hypoketonaemic in fasted rats (Turnbull et a l . , 1983). At doses greater than 1 mM, VPA may uncouple mitochondrial respiration (Benavides et a l . , 1982). This effect on the mitochondrial system may be mediated by the accumulation of valproyl CoA and its further metabolites in the matrix of the hepatic mitochondria (Turnbull et a l . , 1983) or by altering the integrity of the inner mitochondrial membrane or by actions on the substrate carriers or mitochondrial metabolites (Rumbach et a l . , 1983). Valproyl CoA in the mitochondrial matrix is a weak inhibitor of beta-oxidation (Sherratt and Vietch, 1984). At therapeutic concentrations of VPA, mitochondrial but not peroxisomal beta-oxidation in rats is inhibited (Van Den Branden and Roels, 1985). At low VPA concentrations, peroxisomes seem to take over part of the mitochondrial beta-oxidation . Peroxisomal beta-oxidation increased 4 fold in rat liver and 2 fold in mouse liver after administration of 1 % VPA for 2 weeks (Horie and Suga, 1985). The inducing effect of VPA on hepatic beta-oxidation was similar to clofibrate and other hypolipidemic drugs. 23 I.P. administration of VPA to rats for seven days at doses of 1.2 and 1.8 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 isolated rat hepatocytes with VPA, 2-ene VPA, 4-ene VPA, 4-OH VPA, 5-OH VPA and 2-PSA resulted in a concentration-dependent inhibition of gluconeogenesis from lactate (Rogiers et a l . , 1985). The extent of toxicity of these compounds in decreasing order was: VPA and 4-ene VPA, 5-OH VPA, 4-OH VPA, 2-ene VPA, and 2-PGA. This effect of VPA could be reversed with glucagon treatment. Salicylate and octanoate can provide partial protection against the VPA-induced inhibition of metabolic processes by inhibiting valproyl CoA formation in rat hepatocytes (Brown et a l . , 1985). The acyl CoA metabolite of VPA is synthesized only by adult hepatic tissue so that sequestration of CoASH (Coenzyme A) results in the depletion of acetyl CoA. Decreases in CoA are due to accumulation of acid soluble (nonacetyl) CoA esters (valproyl and further derivatives) as shown by decreased plasma beta-hydroxybutyrate levels, decreased free CoA, acetyl CoA, and carnitine in fasting epileptic children and in suckling mice after single doses of VPA (Thurston et a l . , 1985). This is accompanied by an increase in acid soluble short chain fatty acid CoA esters and acid insoluble long chain carnitine esters (Thurston et a l . , 1985). 24 Of the c o-oxidized m e t a b o l i t e s , 4-ene VPA and 4,4'-diene VPA are s l i g h t l y more t o x i c i n mice but are not the major c o n t r i b u t o r s i n VPA induced t o x i c i t y (Simula et a l . , 1985). B e t a - o x i d a t i o n of 4-ene VPA may l e a d to c h e m i c a l l y r e a c t i v e i n t e r m e d i a t e s which a l k y l a t e c e l l u l a r macromolecules and form t i s s u e bound r e s i d u e s . P a t i e n t s on VPA are found t o excrete higher amounts of d i c a r b o x y l i c a c i d s . T h i s i n c r e a s e d e x c r e t i o n i s p o s s i b l y due to impaired b e t a - o x i d a t i o n ( T u r n b u l l et a l . , 1986). Increased amounts of 6 carbon d i c a r b o x y l i c a c i d s , eg. a d i p i c a c i d , are a l s o found i n the u r i n e of r a t s t r e a t e d with VPA (Mortensen et a l . , 1980). VPA may i n t e r f e r e with f a t t y a c i d metabolism by i n h i b i t i n g b e t a - o x i d a t i o n but i n d u c i n g c o-oxidation. In a s i x year o l d male with Reye's syndrome, the e x c r e t i o n of the end product of c o-oxidation, 2-PGA was markedly i n c r e a s e d (Kuhara et a l . , 1985). However, g l u c u r o n i d a t i o n was s t i l l the major pathway of metabolism. Increased amounts of l a c t i c and a d i p i c a c i d s were a l s o recovered i n the u r i n e of t h i s p a t i e n t . 3. T o x i c i t y P a n c r e a t i t i s and h e p a t o t o x i c i t y have been a s s o c i a t e d with VPA usage (Isom J.B., 1984; C o u l t e r et a l . , 1980). Since 1979, 14 cases of p a n c r e a t i t i s where VPA may be i m p l i c a t e d have been re p o r t e d ( W y l l i e et a l . , 1984). S i x t y e i g h t cases of f a t a l h e p a t o t o x i c i t y throughout the world have been observed i n which 25 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 recent survey of reported cases of fatal hepatotoxicity associated with VPA usage in the United States between 1978 and 1984, i t was concluded that age and polytherapy were the major determinants (Driefuss and S a n t i l l i , 1986). The incidence of VPA induced hepatotoxicity decreased with increasing age. Liver function tests (SGOT and SGPT) usually show transient increases with VPA administration (Nau and Loscher, 1984). However, serum transaminases are increased in hepatic failure in patients on VPA (Cotariu et a l . , 1985). Micro-vesicular steatosis is common in cases of hepatotoxicity, and resembles the lesions seen in Jamaican Vomiting Sickness, Reye's Syndrome and 4-pentenoic acid toxicity (Nau and Loscher, 1984; Lewis et a l . , 1982). 3.1. Hepatotoxicity The hepatotoxicity of VPA is thought to be caused by its mono- and/or diunsaturated metabolites (Kochen and Sprunck, 1984). An increased formation of the diunsaturated metabolites appears to be characteristic in fatal hepatic failure. These metabolites are structurally related to 4-pentenoic acid, a hepatotoxin, and to the hypoglycin A metabolite (Jezequel et a l . , 1984). The toxicity associated with VPA and 4-ene VPA is thought to be a result of inhibition of the beta-oxidation pathway 26 (Kesterson et a l . , 1984). In rat liver homogenates, VPA and 4-ene VPA cause inhibition of decanoic acid beta-oxidation (Bjorge and B a i l l i e , 1985). VPA will cause depletion of the free CoA pools (Fears, 1985). VPA causes a transient and mild inhibition of the beta-oxidation pathway by sequestration of CoA (Kesterson et a l . , 1984). 4-Ene VPA produces a more potent and prolonged inhibition of the pathway by forming CoA esters which directly inhibit enzymes in the beta-oxidation pathway. A fatal case of hepatic failure due to suppression of the beta-oxidation pathway as shown by the lack of the 3-keto metabolite has been reported by Kochen and co-workers in 1983. 4-Ene VPA is similar in structure to 4-pentenoic acid, the causative agent in Reye-like syndrome and to the hypoglycin A metabolite responsible for Jamaican Vomiting Sickness (Nau and - Loscher, 1984). In rat hepatic microsomal preparations, the ethyl ester of 4-ene VPA was more active than 4-ene VPA in its effects on the NADPH- and time-dependent loss of cytochrome P-450 over 30 min (33 % compared to 8%) (Prickett and B a i l l i e , 1986). This action of 4-ene VPA ethyl ester is thought to be by a similar mechanism to that of allyisopropyl-acetamide and other related monosubstituted olefins. These compounds are converted by cytochrome P-450 to chemically reactive species which covalently bind to the heme moiety of cytochrome P-450 and ultimately lead to its destruction. 27 Of a l l the monounsaturated metabolites, 4-ene VPA causes the most changes in factors (dicarboxylic aciduria and decreased beta-hydroxybutyrate reduction) which indicate inhibition of the beta-oxidation pathway in rats (Granneman et a l . , 1984c). However, these changes are not as pronounced as with hypoglycin A. In hypoglycin A toxicity, excretion of adipic, suberic and sebacic acids is increased. A metabolite of hypoglycin A, methylenecyclopropylacetic acid, appears to inhibit fatty acid oxidation through effects on acyl CoA dehydrogenases. E. DRUG INTERACTIONS Several properties of VPA promote interactions with other drugs. VPA is strongly plasma protein bound and thus competes with other drugs for binding sites (Drug Interactions Newsletter, 1981). It has the capability of inhibiting the hepatic metabolism of other drugs, but i t s own metabolism is susceptible to liver enzyme inducing agents. VPA i t s e l f does not induce the hepatic enzymes (Rimmer and Richens, 1985). Because VPA undergoes such extensive biotransformation in the body, the coadministration of metabolic inducing agents may result in the increased formation of possible toxic metabolites. Interactions of VPA with other anticonvulsant drugs have been studied most extensively. 28 1. Drugs which alter VPA concentrations Salicylates displace VPA from plasma protein binding sites (Levy and Koch, 1982). In epileptic children, ASA coadministration (11.5 - 16.9 mg/kg/Q6H) resulted in decreased clearance of free VPA without significant changes on total VPA clearance (Farrell et a l . , 1982). Free fraction of VPA was found to increase in rank order at ASA concentrations exceeding 50 mg/L in in vitro studies. ASA coadministration also resulted in longer serum half-lives of both free and total VPA (Orr et a l . , 1982). The urinary profiles of VPA and 13 metabolites in 7 subjects (one healthy male volunteer and 6 pediatric patients) at steady state before and after administration of antipyretic doses of ASA showed significant differences (Abbott et a l . , 1986b). After ASA coadministration, 2-ene trans VPA and 3-keto VPA were significantly decreased while VPA glucuronide conjugate and 4-ene VPA were significantly increased. The beta-oxidation pathway was significantly inhibited (66 % decrease) while glucuronidation significantly increased by 30 %. Suppression of the beta-oxidation pathway was balanced by induction of the glucuronidation pathway, which may explain the lack of change in total clearance in spite of the increase in the free fraction of VPA. ASA may inhibit beta-oxidation of VPA 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 clearance accompanied by a 29 significantly prolonged elimination h a l f - l i f e (Webster et a l . , 1984). In animal studies, free fatty acids (Intralipid R) increased free levels of VPA and also inhibited VPA metabolism to a slight extent (Kutt, 1984). Administration of free fatty acids in concentrations of 100 nq/mL and 200 j/g/mL resulted in 19 -48 % and 82 - 118 % increases in the free fraction of VPA, respectively (Patel and Levy, 1979). Other anticonvulsants (CBZ, phenytoin, and phenobarbital) induce VPA metabolism as supported by longer serum VPA half-l i f e in monotherapy compared to polytherapy (Kutt, 1984). The ratio of VPA steady-state plasma levels to dose is 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. Effects of VPA on other drugs Increased serum levels of phenobarbital are observed when VPA is administered concommitantly, necessitating reduction of phenobarbital dosage in up to 80 % of patients (Kutt, 1984). Phenobarbital serum h a l f - l i f e may be increased by as much as 50 %. The mechanism appears to be inhibition of phenobarbital metabolism by VPA, possibly inhibition of phenobarbital hydroxylation. In rat hepatic microsomes, VPA has been shown to inhibit the parahydroxylation of phenobarbital (Taburet and Aymard, 1983). VPA also has been found to competitively 30 inhibit glucuronidation of parahydroxyphenobarbital in rat liver microsomes. This is of extreme importance since glucuronidation is a major pathway for both VPA and phenobarbital. VPA affects primidone metabolism to a lesser extent, so that reductions in primidone dosages are not required. VPA w i l l cause either a transient increase or decrease in total phenytoin levels, with the altered levels returning to previous values within days or weeks (Kutt, 1984). Decreases in serum phenytoin levels are observed more frequently than elevations. Increases in phenytoin serum levels may occur in those patients who are close to saturation kinetics. VPA competes with phenytoin for plasma protein binding sites, especially at higher concentrations around 100 ug/mL. Phenytoin plasma protein binding may decrease from the usual 90 % to 85 % or less. Phenytoin is primarily bound to one site on the albumin molecule (warfarin binding site) while VPA binds to both the warfarin and the diazepam sites (Kober et a l . , 1980) VPA also increases antipyrine h a l f - l i f e by 45 % (Kutt, 1984). VPA displaces tolbutamide from i t s binding sites on the albumin molecule (Fernandez et a l . , 1985). VPA competes for plasma protein binding sites with diazepam such that free levels of diazepam are elevated (Hariton et a l . , 1985). This allows increased penetration of the blood-brain-barrier by diazepam. 31 F. INTERACTION BETWEEN VALPROIC ACID AND CARBAMAZEPINE This is a c l i n i c a l l y important interaction as these two drugs are frequently used in combination to obtain maximal seizure control. One drug is often added to the other in an effort to obtain better seizure control. Since CBZ is known to induce the hepatic microsomal enzyme systems, a brief review on enzyme induction follows. 1. Enzyme induction Brown, Miller and Miller f i r s t reported the phenomenon of increased hepatic enzyme activity in rats after injections of small amounts of polycyclic aromatic hydrocarbons (Conney, 1967). Remmer and coworkers discovered the inducing effects of barbiturates on hepatic microsomal drug metabolism. Enzyme induction may be described as "the process which increases the rate of synthesis of an enzyme relative to its normal rate of synthesis in the uninduced organism" (Gelboin, 1971). Increased enzyme levels in mammals may be attained either through enhanced synthesis or inhibition of enzyme degradation (Conney, 1967). An enhancement of enzyme activity " can be obtained without alterations in the amount of enzyme present. Induction of the liver enzymes results in an enhancement in metabolic rate and thus directly affects duration and intensity of drug actions in man and animals. Induction can alter the steady state concentrations of the 32 parent compound and i t s metabolites in addition to their elimination (Gillette, 1979). Several compounds can induce a single enzyme and conversely, a single inducing agent can act at several steps in the induction process (Estabrook and Lindenlaub, 1979). 1.1. Metabolism Lipid soluble compounds are excreted very slowly into the urine because of their chemical properties and thus, they tend to undergo extensive biotransformation and are excreted mainly as their metabolites (Gillette, 1979; Astrom and DePierre, 1986). Polar compounds, however, are usually excreted unchanged. Some xenobiotics may structurally resemble endogenous substances and therefore, may be metabolized by the same enzyme system(s). Foreign compounds which do not have endogenous counterparts are metabolized by relatively i nonspecific enzymes. The products of metabolism are usually more polar than the parent compound and thus renal and/or biliary excretion is enhanced. 1.2. Types of reactions There are four types of general reactions which act to enhance the removal of a foreign compound from the body: oxidation, reduction, hydrolysis, and conjugation (Goldstein et a l . , 1974). The reactions responsible for the biotransformation of foreign compounds may be divided into two 33 phases. Phase one reactions include oxidation, reduction and hydrolysis (Gillette, 1979). Oxidative reactions generally take place at the hepatic endoplasmic reticulum site and require both NADPH and oxygen (Gillette, 1979). These reactions are catalysed by cytochrome P-450 and several forms of this enzyme have been identified. Phase two reactions are mainly conjugation reactions including glucuronidation and acylation, and mercapturic acid, sulphate, and dihydrodiol formation. 1.3. Factors influencing enzyme induction The duration and intensity of drug action is dependent on the rate of drug metabolism by the body (Conney, 1967). A number of factors can influence the rate of drug metabolism including diet, disease state, nutritional status, hormonal changes and ingestion of foreign compounds (Conney, 1967; Conney, 1982). Nutritional factors, especially fats, can greatly influence the effect of inducing agents (Goldberg, 1978). Saturated fats tend to act as promoters of enzyme induction. The amount of hepatic cytochrome P-450 and aryl hydrocarbon hydroxylase activity is decreased in severe hepatitis or active cirrhosis (Farrell et a l . , 1979). 1.4. Properties of enzyme inducing agents The compounds which act as inducers of microsomal enzymes are extremely diverse in structure, effect, and ab i l i t y to 34 induce (Conney, 1967). Examples of compounds known to induce microsomal enzymes in man include anticonvulsant agents (eg. phenobarbital, phenytoin, and carbamazepine), anti-inflammatory agents (eg. phenylbutazone), antifungals, antibiotics (eg. rifampin), anticoagulants (eg. warfarin), alcohol, industrial products (eg. dioxane), oral contraceptives, pesticides, and sedatives (Goldberg, 1980). Usually, enzyme inducing agents are l i p i d soluble at physiological pH. A high l i p i d solubility enables the inducing agent to reach the lipophilic membranes of the endoplasmic reticulum with increased ease as well as allowing the formation of a stronger inducer-enzyme complex and ultimately a longer duration of pharmacological activity (Estabrook and Lindenlaub, 1979). The quantity of the inducing compound required to cause 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, phenobarbital, isosafrole, and polychlorinated biphenyls. The polychlorinated biphenyls induce cytochrome P-450s which are also induced by both phenobarbital and 3-methylcholanthrene (Rees, 1979). Isosafrole induces a novel species of cytochrome P-450 and is the only chemical in its group to date. Phenobarbital and 3-methylcholanthrene represent the majority of inducing agents (Conney, 1967). 35 1.5.1. Phenobarbital and 3-methylcholanthrene Phenobarbital type of inducing agents influence a variety of metabolic pathways including glucuronidation, oxidation, reduction, and de-esterification (Conney, 1967). The 3-methylcholanthrene types (polycyclic aromatic hydrocarbons) affect a more limited group of reactions including aromatic, aliphatic, and n-hydroxylation (Conney, 1967). The course and intensity of . induction of these two types also d i f f e r . For a maximal increase of 3 to 10 fold in the microsomal enzymes, phenobarbital must be administered daily for at least three days. The 3-methylcholanthrene type (polycyclic aromatic hydrocarbons) of inducing agents wi l l double enzyme activity within 3 to 6 h and maximal increases of 5 to 10 fold are observed within 24 h. Administration of phenobarbital to animals leads to proliferation of the smooth endoplasmic reticulum in the liver c e l l . 3-Methylcholanthrene does not alter the amount of smooth endoplasmic reticulum. 1.6. C l i n i c a l implications Enzyme induction can lead to either deactivation or activation of foreign compounds. Enzyme induction results in an increased rate of drug metabolism and thus, decreased drug action i f the metabolite is not active (Conney, 1967). However, if the metabolite is active, increased metabolism of the drug may result in an intensified drug action. 36 Chronic administration of a drug may result in auto- and heteroinduction (Conney, 1969). Drugs which induce their own metabolism during chronic administration include phenobarbital, phenylbutazone, tolbutamide and CBZ. Enzyme induction may lead to the formation of potentially toxic metabolites. The toxicity of organic thiophosphates, which are relatively non-toxic until metabolized to potent cholinesterase inhibitors, is increased after pretreatment with enzyme inducing agents. Enzyme inducing agents may influence both the initiation and promotion of experimentally induced carcinogenesis (Park and Breckenridge, 1981). 1.7. Markers of enzyme induction Several indices are utilized to measure the rate and extent of enzyme induction. These include measurement of clearance rate or half-lives of drugs metabolized by the hydroxylation system (Goldberg, 1978). Antipyrine clearance is the most commonly used due to i t s convenience. The carbon-14 labelled carbon dioxide content in breath after administration of carbon-14 labelled aminopyrine in humans is often employed. Salivary clearances of drugs can also be used although the secretion of drug into the saliva is often pH dependent. The metabolism of endogenous compounds including 6-beta-hydroxycortisol, D-glucaric acid, and 1-xylulose have also been ut i l i z e d . Changes in plasma enzymes such as gamma-37 g l u t a m y l t r a n s f e r a s e may a l s o be used as markers of enzyme i n d u c t i o n (Moreland et a l . , 1982). 2. Carbamazepine CBZ i s an i m i n o s t i l b e n e d e r i v a t i v e which i s s t r u c t u r a l l y s i m i l a r t o the t r i c y c l i c a n t i d e p r e s s a n t s (Goodman et a l . , 1980) as shown below. i CONH 2 Carbamazepine CBZ i s u t i l i z e d i n the treatment of a v a r i e t y of s e i z u r e types i n c l u d i n g p a r t i a l , g e n e r a l i z e d t o n i c - c l o n i c , and mixed s e i z u r e d i s o r d e r s . A f t e r o r a l a d m i n i s t r a t i o n i n man, CBZ i s slowly absorbed from the g a s t r o i n t e s t i n a l t r a c t (Goodman et a l . , 1980). Peak plasma l e v e l s are a t t a i n e d i n 2 - 8 hours. Therapeutic plasma l e v e l s of CBZ are i n the range of 3 - 14 jug/mL. CBZ has a r e l a t i v e l y long h a l f - l i f e ranging from 8 - 7 2 hours. The drug i s h i g h l y plasma p r o t e i n bound (75 - 90%). CBZ i s e x t e n s i v e l y metabolized i n man v i a the l i v e r microsomal enzyme system, with l e s s than 1 % of the parent drug e x c r e t e d unchanged i n the u r i n e ( B e r t i l s s o n and Tomson, 1986). CBZ i s o x i d i z e d to the 10,11-epoxide, f o l l o w e d by f u r t h e r 38 metabolism to the dihydroxy analogue which is glucuronidated prior to excretion. CBZ induces the hepatic microsomal enzyme system, causing autoinduction and heteroinduction (Goodman et a l . , 1980). In long term treatment, CBZ induces i t s own metabolism (Bertilsson and Tomson, 1986; Pynnonen, 1979; Goodman et a l . , 1980). CBZ autoinduction may occur within two or three days (Pynnonen, 1979). CBZ induces the metabolism of other drugs including clonazepam, ethosuximide, doxycycline, oral contraceptives, phenytoin, warfarin, and haloperidol (Fernandez et a l . , 1985). Concurrent therapy of erythromycin and CBZ often results in CBZ intoxication (Baciewicz, 1986; Goulden et a l . , 1986). CBZ appears to be capable of causing either enzyme induction or enzyme inhibition, depending on the individual. CBZ has been shown to decrease the plasma levels of VPA by inducing VPA metabolism (Kutt, 1984). Since VPA undergoes extensive biotransformation in the 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 decreased VPA steady state levels after 2 weeks. A change in the elimination rate constant, Ke, was not observed. I_n vitro binding experiments suggest that CBZ does not have an effect on the binding of VPA to plasma proteins (Mattson et a l . , 1980; Mattson et a l . , 39 1982). VPA addition (50 pg/mL and 100 ug/mL) to CBZ (6, 8, and 12 ug/mL) resulted in elevation of free CBZ levels. VPA elevates plasma levels of CBZE, the major metabolite of CBZ (Bertilsson and Tomson, 1986). This may be due to inhibited elimination of CBZE. Valpromide, the acid amide derivative of VPA, appears to have a similar effect. Although both VPA and valpromide cause elevations in CBZE levels, the effects of valpromide appear to be more c l i n i c a l l y significant (Pisani et a l . , 1986). Valpromide has the potential for inhibiting epoxide hydrolase, the enzyme responsible for CBZE metabolism, while VPA has l i t t l e effect on the enzyme. Administration of valpromide in combination with CBZ to epileptic patients lead to a marked increase in mean serum concentrations of CBZ (2 - 8.5 ug/mL) and CBZE when substituted for valproate • (Meijer et a l . , 1984). Switching back to valproate caused the levels to decrease although CBZE levels were s t i l l higher than the previous values for several weeks. Pacifici et a l . , (1985) postulated that these increases in CBZE were due to inhibition of epoxide hydrolase by valpromide. Rhesus monkey hepatic microsomes were used to determine the effect of valproic acid and valpromide in vitro on 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 (0.2 - 1.0 mmol/L) had l i t t l e effect on both reactions. 40 In isolated perfused rat livers, therapeutic concentrations of VPA caused a decrease in the intrinsic clearance of CBZ as well as a decrease in the intrinsic formation clearance of CBZE (Chang and Levy, 1985). Similar results were obtained in liver preparations from animals pretreated with CBZ. VPA wi l l inhibit CBZ metabolism and plasma protein binding in rhesus monkeys (Levy et a l . , 1984). 3. Cli n i c a l significance This interaction between VPA and CBZ is a c l i n i c a l l y important one since some of VPA's metabolites may be responsible for the rare, but fatal, occurrences of hepatatoxicity associated with VPA usage. This study w i l l allow us to determine i f the formation of the potential hepatotoxin, 4-ene VPA, is increased in the presence of an enzyme inducing agent such as CBZ. Also, we w i l l be able to characterize if CBZ induces specific pathways or causes a general, overall induction of VPA metabolism. The purpose of this study is to investigate the effects of CBZ on the kinetics of VPA and its metabolites in normal, healthy subjects at steady state. As well, the kinetics of Dg-VPA will be determined in one volunteer. 41 OBJECTIVES 1. The GCMS assay for valproic acid and its metabolites developed in our laboratory will be further modified. The proposed modifications include experimentation with various internal standards. As well, several derivatizing reagents will be tested and compared to the reagent currently used. 2. The interaction between VPA and CBZ in five normal healthy volunteers will be characterized. Serum and urine samples obtained before and after administration of CBZ w i l l be analyzed. The effects of CBZ on the metabolism and kinetics of VPA w i l l be determined. Kinetic parameters will be determined before and after CBZ administration for VPA. As well, formation and metabolic clearances will be calculated for each metabolic pathway before and after CBZ administration. The fraction metabolized by each pathway before and after CBZ will also be calculated. This w i l l allow further elucidation of the effect of CBZ on particular metabolic pathways. This will enable us to determine i f the induction of VPA metabolism by CBZ is concentrated on specific metabolic pathways, or is a general overall effect. 3. The effect of substituting hexadeuterated VPA for VPA will be determined in one healthy volunteer. The pharmacokinetics of stable isotopically labelled VPA are to be determined in 42 t h i s volunteer . This w i l l allow the i d e n t i f i c a t i o n of s h i f t s in the metabolic pathways due to isotope e f f e c t s . Serum and ur ine samples w i l l again be analyzed for t h i s i n v e s t i g a t i o n . 43 II. EXPERIMENTAL A. REAGENTS AND MATERIALS 1. Valproic acid and metabolites Valproic acid (di-n-propylacetic acid) was obtained from K and K Fine Chemicals, ICN Pharmaceutical (Plainview, NY). The metabolites, 2-ene VPA, 3-ene VPA, 4-ene VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3-keto VPA, 4-keto VPA, 2-propylglutaric acid, and 2-propylsuccinic acid used for the preparation of the calibration curves were synthesized as reported elsewhere (Acheampong et a l . , 1983). 2,3'-Diene VPA was synthesized in our laboratory as reported elsewhere (Acheampong and Abbott, 1985) as was 2,4-diene VPA (unpublished data). 2. Internal Standards 3-Octanone (99%) and 2-methylglutaric acid were obtained from the Aldrich Chemical Company (Milwaukee, WI). Octanoic acid (caprylic acid) was purchased from the National Biochemicals Corporation (Cleveland, Ohio). Dg-VPA (Acheampong et a l . , 1984) and D3-2-ene VPA (Abbott et a l . , 1986a) were synthesized as previously reported. Hexanoic acid (n-caproic acid, HA) was purchased from Eastman Organic Chemicals (Rochester, N.Y.). Di-n-butylacetic acid (DNBA) was synthesized in our laboratory by Andrew Acheampong (Ph.D. thesis, 1985). 44 3. Reagents Chemicals and drugs were obtained from the following sources: ALDRICH CHEMICAL COMPANY (Milwaukee, WI, U.S.A.). tBDMCS, t ert-butyldimethylsilylchloride, 97% purity. MTBSTFA, N-(f ert-butyldimethylsilyl)-N-methyltrifluoroacetamide ,98 % purity. Diazald R, N-methyl-N-nitroso-p-toluenesulfonamide. DMAP, dimethylaminopyridine, 99% purity. 2-Methylglutaric acid. Pyridine. BDH CHEMICALS (Canada). Anhydrous sodium sulphate. Citr i c acid anhydrous. Sodium hydroxide. Water, glass-distilled-grade. CALEDON (Georgetown, Ontario). Acetonitrile HPLC grade. Dichloromethane HPLC grade. Ethyl acetate distilled-in-glass grade. Methanol distilled-in-glass grade. Methanol HPLC grade. 45 FISCHER SCIENTIFIC LTD (Canada). Creatinine. Hydrochloric acid. PIERCE CHEMICAL COMPANY (Rockford, Ilinois, U.S.A.). MSTFA, N-methyl-N-trimethylsilyltrifluoracetamide. SIGMA CHEMICAL COMPANY (St. Louis, MO, U.S.A.). Picric acid saturated solution. 4. Drugs ABBOTT PHARMACEUTICALS (Canada). Valproic acid 50 mg/mL syrup (DepakeneR). 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 volunteers (21 - 48 years old) participated in the study for which human ethics approval had been obtained. The volunteers were not on any chronic 46 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 liver function tests were performed prior to, during, and after the study in a l l volunteers. Volunteer weights and total daily dosages of VPA were as follows: BA 70 kg, 1100 mg; FS 70 kg, 1400 mg; MS 75 kg, 1200 mg; RM 65 kg, 1000 mg and WT 80 kg, 1200 mg. Four volunteers received VPA 15 mg/kg/day and one volunteer (FS) received 20 mg/kg/day (mean VPA dose 16.4 mg/kg/day) in syrup form. Medication administration times were 0800 h and 2000 h. The kinetic studies were performed on days 7 - 9 and 23 - 25. Administration of the drug(s) was interrupted on the kinetic study days and reinstated after 48 h for the f i r s t kinetic study. Blood was collected 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 2 hour blocks for the f i r s t 12 h, convenient blocks overnight, in 6 h blocks between 24 - 36 h, and overnight (36 - 48 h). On day 9, CBZ 100 mg twice daily was added to the dosing regimen. On day 16 the evening dose of CBZ was increased to 200 mg for a total daily dose of 300 mg. On days 23 - 25, the study from days 7 - 9 was repeated. FS also received six doses of Dg-VPA in liquid form where the sodium salt was formed by adding excess alkali (1.70 mL of 47 3N NaOH) and then adjusting the pH to 7.4 - 7.8 with 4N HC1 (approximately 300 nL). The solution was then administered neat. The doses were taken at the following times: day 8, 2000 h; day 9, 0800 h; day 25, 2000 h; day 26, 0800 and 2000 h, and day 27, 0800 h. The kinetic study was performed according to the same protocol as above on days 27 - 29 following administration of four doses of the deuterated drug. On day 9, the decline of Dg-VPA was observedv only for 12 hours after 2 doses had been administered. Blood samples were collected in sterile, nonheparinized vacutainers. For ease of blood collection and to decrease the number of venepunctures, the volunteers had indwelling flexible catheters placed into an arm vein. The catheter was flushed with .sterile normal saline (without preservatives) and then locked with 1 mL of heparin 100 U/mL solution between sampling. Samples were allowed to clot and then centrifuged to yield the serum. The serum was transferred to sterile vacutainers, and stored at -20 °C until analysis. Total urine volumes were recorded and a homogenous aliquot was saved. Saliva was collected following stimulation with 5 % c i t r i c acid solution, concommitantly with the blood collection for the f i r s t 12 hours of the kinetic study. The procedure was to administer 4 mL of 5 % c i t r i c acid which was held in the mouth for 2 min and then removed. The saliva was collected between 2 - 3 min. Approximately 5 to 10 mL aliquots were collected. Saliva and 48 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 (distilled-in-glass grade) such that 1 mL could be diluted to 100 mL to yield the working concentrations in d i s t i l l e d water of 100 nq/mL for D3-2-ene, DgVPA, 2-methylglutaric acid (MGA), hexanoic acid (HA), and di-n-butylacetic acid (DNBA). The working concentrations of octanoic acid (OA) and 3-octanone (OCT) were 500 nq/mL and 1 mg/mL, respectively. One hundred uh- of each of the required internal standards were added to each tube. Stock solutions were prepared fresh as needed and were stored in the refrigerator when 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 simultaneously. 1.2. 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 stirring) and the other metabolites, namely 49 2-ene VPA, 3-ene VPA, 4-ene VPA, 4-keto VPA, 2-PSA, 2-PGA, 2,3'-diene VPA, and VPA were dissolved in methanol ( d i s t i l l e d -in-glass grade). A bulk stock solution of standard 5 was prepared in either control urine or serum. For the calibration 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 concentrations of metabolites and VPA thus obtained in urine were as follows: 3-keto VPA 200, 400, 600, 800, and 1000 uq/mL, VPA and 4-OH VPA 100, 200, 300, 400, and 500 ug/mL, 2-PGA, 2,3'-diene VPA, 2,4-diene VPA, and 2-ene trans VPA 20, 40, 60, 80, and 100 uq/mh, 2-ene cis VPA 0.6, 1.2, 1.8, 2.4, and 3 uq/mh, 4-ene VPA 0.3, 0.6, 0.9, 1.2, and 1.5 yg/mL, 3-ene VPA 0.2, 0.4, 0.6, 0.8, and 1.0 vq/mL, 4-keto VPA 6, 12, 18, 24, and 30 uq/mh, 5-OH VPA 50, 100, 150, 200, and 250 nq/mL and 2-PSA 4, 8, 12, 16, and 20 uq/mL. Serum concentrations were as follows: 4-OH VPA, 2-ene trans VPA, 2,3'-diene VPA, and 2,4-diene VPA 4, 8, 12, 16, and 20 *zg/mL, 2-ene cis VPA 0. 1 2, 0.24, 0.36, 0.48, and 0.6 uq/mL, 4-ene VPA 0.3, 0.6, 0.9, 1.2, and 1.5 jug/mL, 3-ene VPA 0.4, 0.8, 1.2, 1.6, and 2.0 (iq/mL, 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, 0.6, 0.8, and 1.0 ag/mL and 5-OH VPA 2, 4, 6, 8, and 10 uq/mL. The calibration curves were generated by plotting the peak area ratios of metabolite or VPA to the internal standard versus concentration of VPA or the particular metabolite. D 3 -50 2-ene cis VPA was used as the internal standard for VPA and a l l metabolites except 2-PSA and 2-PGA. MGA was used as the internal standard for the two dicarboxylic acid metabolites. Hexanoic acid was used as the internal standard for a l l metabolites derived from the human metabolism of Dg-VPA. Standard curves were prepared and injected with each batch of samples. 1.3. Extraction and derivatization of standards and patient samples As illustrated in figure 2, the internal standards were added to the standard (total volume 1 mL) or to the biological sample (serum/urine, total volume 1 mL), adjusted to pH > 12 with 3N NaOH (approximately 100 uL) and heated for one hour at 60 °C to hydrolyze the conjugates present. If one mL was not available, the sample was diluted to 1 mL with d i s t i l l e d water and the dilution factor taken into consideration for the calculations. After cooling to room temperature, the samples were acidified to pH < 2 with 4N HC1 (approximately 120 uD and allowed to equilibrate for 15 minutes. The samples were then extracted with 3 mL ethyl acetate (distilled-in-glass grade) by gentle rotation for 30 minutes. Serum samples were extracted twice at the slowest speed possible to prevent the formation of stubborn emulsions. Serum samples also required centrifugation for 30 minutes at 2500 rpm and 14 °C to separate the emulsions formed during extraction. The organic phase (top layer, 51 Serum/Urine 1 mL Internal Standards 100 uL pH > 12.5 octanoic acid 2-methylglutaric acid VPA-D6 2- ene VPA-D3 3 - octanone Heat for 1 h at 55-60 °C I pH 2 with 4N HCl I Extract with ethyl acetate, 3 mL I Organic layer I Dry over anhydrous sodium sulphate i Dry to 200 ML under N 2 J Derivatize tBDMCS reagent with 5 % catalyst 60 uL \ Heat for 4 h at 60 °C I Inject 1 ML into GCMS Figure 2. Extraction and derivatization procedure for valproic acid and metabolites from serum or urine. 52 approximately 2 mL for urine samples and 4 mL for serum samples) was transferred to another tube and dried over anhydrous sodium sulphate by vortexing for one minute, followed by centrifugation for 10 minutes at 2500 rpm. The supernatant was then transferred to a third tube and concentrated to approximately 200 uL under a gentle nitrogen stream. The concentrated sample was derivatized with the appropriate reagent. For tBDMCS derivatives, 60 uL of the reagent containing 5 % catalyst (DMAP) was added and then heated for four hours at 60 °C. For TMS derivatives, 30 ULL of MSTFA was added and heated at 60 °C for 20 - 30 minutes. For tBDMS derivatives from MTBSTFA, 60 uL of reagent were added but only 30 minutes of heating time were required for adequate derivatization at 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 derivatizing reagent used in the analysis was prepared by dissolving 50 mg of dimethylaminopyridine (catalyst) in 1 mL of dry pyridine. This mixture was then added to 1 g of tBDMCS and mixed thoroughly by vortexing. The reagent was stored tightly capped to prevent reaction with atmospheric moisture and was prepared fresh as required. 53 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. This was further diluted in methanol to yield stock solutions of 100, 200, 300, 400, 600, and 800 jxg/mL concentrations. These samples were then further diluted in blank serum and in methanol to give the working solutions of 2, 4, 6, 8, 12 and 16 uq/mL of CBZ for the calibration curves. A concentrated stock solution of CBZE was prepared at a concentration of 1.5 m'g/mL in HPLC grade methanol. This was diluted to yield stock solutions of 50, 100, 150, 200, 300, and 400 uq/mL. The working solutions were then prepared in methanol and in blank serum at concentrations of 1, 2, 3, 4, 6, and 8 uq/mL of CBZE. A concentrated stock solution of 1 mg/mL 10-methoxy-carbamazepine (MCBZ, internal standard) was prepared in HPLC grade methanol and was further diluted to yield the working solution of 40 Mg/mL in HPLC grade methanol. A l l stock solutions and working solutions were kept frozen until required. Standard curves were prepared to contain 2, 4, 6, 8, 12, and 16 uq/mL of CBZ and 1, 2, 3, 4, 6, and 8 uq/mh of CBZE. Four Mg (100 ML) of the internal standard were added to each sample. Peak area ratios of CBZ or CBZE to internal standard 54 were plotted versus concentration of CBZ or CBZE to prepare calibration curves. 2.2. Extraction of serum samples for CBZ and CBZE The HPLC assay for carbamazepine and i t s epoxide as developed by Elyas et a l . (1982) was modified and used for analysis of serum samples (figure 3). Volunteer serum samples collected prior to the morning dose, 3 h and 5 h post dose on days 7 and 23 were analyzed for CBZ and CBZE levels. A 250 uL aliquot of serum (either standard or patient sample) was placed into a test tube to which were added four u.g (100 uL) of the internal standard, and 125 uL of 4N NaOH. The sample was then extracted with 3 mL of dichloromethane (HPLC grade) by gentle rotation for 10 min. The sample was then centrifuged at 2500 rpm for 7 min. The top layer (aqueous) was aspirated and discarded and the bottom layer (organic) transferred to a second tube. The organic layer was evaporated to dryness under a gentle nitrogen stream in a water bath at 40 °C, reconstituted with 200 uL of acetonitrile (HPLC grade), evaporated and reconstituted again with 200 uL of acetonitrile. Ten ttL were injected into the. liquid chromatograph. Total run time was under 10 minutes. 3. Determination of urinary creatinine Urinary creatinine levels were determined by a colourimetric method. This involved the chemical reaction 55 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 2 i Reconstitute with 200 ML acetonitrile I Evaporate to dryness under N 2 I Reconstitute with 200 ixL acetonitrile \ Inject 10 uL into liquid chromatograph Modified from A.A. Elyas et al J. Chromatogr. 1982;231:93-101 Figure 3. Extraction procedure for carbamazepine and carbamazepine-10,11-epoxide from serum. 56 between picric acid and creatinine to form a red coloured complex. The absorbance was measured at 500 nm on a Spectronic 20 spectrophotometer. Standard curves were prepared over a range of 100 - 400 mg creatinine per 100 mL of urine and analyzed with each batch of patient samples. Urine samples were diluted 1/200 (0.5 mL in 100 mL d i s t i l l e d water) and 2 mL of the diluted sample analyzed. 4. Instrumentation 4.1. Valproic acid and metabolites The assay was performed on a Hewlett-Packard 5987A GCMS system. Operating conditions for tBDMS derivatives were source and injection port temperatures of 240 °C and an interface temperature of 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 1701 (0.25 u) bonded phase capillary column, 25 m x 0.32 mm I.D., (Quadrex Scientific, New Haven, Connecticut) was used for the analysis. Temperature programming for tBDMS derivatives was i n i t i a l column oven temperature of 50 °C, increasing by 30 °C/min from 50 to 100 °C, then 8 °C/min from 100 to 230 °C and held at 260 °C for two minutes post run. Total run time was approximately 18 minutes. The selected ion monitoring mode was used. The ions scanned were m/z 100 (4-OH VPA lactone), m/z 128 (3-octanone), m/z 197 (dienes), m/z 199 (enes), m/z 201 (VPA and octanoic 57 acid), m/z 202 (D3-2-ene VPA), m/z 207 (D6-VPA), m/z 215 (3-and 4-keto VPA), m/z 317 (2-methylglutaric acid), m/z 331 (5-OH VPA and 2-PSA) and m/z 345 (2-PGA). The same program was used for determining metabolites from the metabolism of deuterated VPA. The ions selected for scanning included m/z 103 (4-OH VPA lactone), m/z 128 (3-octanone), m/z 173 (hexanoic acid), m/z 202 (deuterated 2,4-diene VPA), m/z 203 (deuterated 2,3'-diene VPA), m/z 204 (deuterated 4-ene VPA), m/z 205 (cis and trans 2-ene VPA, 3-ene VPA), m/z 207 (Dg-VPA), m/z 218 (4-keto VPA), m/z 221 (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, particle size 5 urn, 110 mm length, O.D. 7.94 mm,and I.D. 4.70 mm was used. A Beckman Model 110A pump and 160 Absorbance detector (variable wavelength, at 215 nm) and a Waters Associates Model UGK Injector were used with an Altex Model C-R1A Recorder. Mobile phase used was acetonitrile.water (35:65) at a flow rate of 1.1 mL/min. 5. Sta t i s t i c a l analysis St a t i s t i c a l analysis was performed using Student's paired t test comparing results before and after the administration of CBZ. Statistical analysis was performed using the Michigan 58 Interactive Data Analysis System (MIDAS) on the MTS system. Significance level chosen was p < 0.05. 6. 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 VPA and ASA (Abbott et a l . , 1986b; Kassam M.Sc. thesis, 1985). This model was based on one reported by Levy et a l . (1983) for the clobazam-carbamazepine interaction. The model, shown in figure 4, is a linear model with elimination from the central compartment. It allows the calculation of formation and elimination (metabolic) clearances of VPA metabolites. From these, the fraction metabolized through a particular route may also be determined. The dosing interval, T, was 12 h for a l l calculations. As shown in Figure 4, Cp S S is the steady state concentration of VPA in the central compartment. Cp S S can be calculated from the following equation: Equation 1. C p s s = VPA A U C T/T T = 12 h ko is defined as the dosing rate of the drug which was every 12 h for this study. Hence, AUC values reported are calculated over this interval, and urinary recoveries are also over the same interval. 59 .^remainder (not measured) cm2 3-ene VPA 2,3-diene VPA -m3 4-ene VPA 2,4-diene VPA cm4 4-OH VPA 4-KetO VPA 2-PSA 5-OH VPA 2-PGA Lm2 Lm3 Lm4 *clm5 Figure 4. Pharmacokinetic model applied in the valproic acid-carbamazepine study in healthy volunteers. 60 Total body clearance of VPA (Clp), is defined as the sum of a l l formation clearances (Clf) plus unknown clearances (unmeasured clearances, C l r e m a i n c : e r ) as shown below: C l p = C l f l + C l f 2 + C l f 3 + C l f 4 + C l f 5 + C l f 6 + C l r + c*remainder c lremainder m a v b e d r u 9 lost through the bile and feces which was not measured. Clp can be calculated from the dose given and the AUC value for the drug over the dosing interval: Equation 2. Clp = Dose/AUCT m1 , m2, m3, m4, and 1115 represent the sum of the individual metabolites in a given pathway. cm1' cm2' cm3' Cm4, a n d cm5 a r e t h e steady state concentrations of the metabolites in the respective pathways described previously. These are calculated from the total serum AUC for each pathway divided by the dosing interval (12 h). Equation 3. C m i = AUCj/T C l m 1 , C l m 2 , C l m 3 , C l m 4 ^ C l m 5 , and C l m 6 are the metabolite or elimination clearances for pathways 1 through 6. These are determined from the total serum AUC for a given pathway and the 61 total amount recovered in the urine for a given pathway over the same time period. Equation 4. c^mi = nti/AUCi The formation clearances C l f l f Clf2' df3» Clf4* and Cljg for the metabolites are determined from the ratio of average steady state concentrations of metabolite to parent drug multiplied by the metabolite clearance for that particular pathway. Equation 5. c l f i s s = cmiss • c l m i / c p V P A s s However, the formation clearance can also be calculated from the following equation: Equation 6. c l f i s s = m i / A U C v P A This equation is derived from the following sequence: c l f i s s = cmiss • c^mi/cPvPAss and C l m i = mi/ADC^ and C m i = AUC/T and C p = AUC V P A/T therefore, C l f j s s = m i / A U C m i / c p s s x A U C m i / T = mi/T/Cp mi/AUCVPA 62 The f rac t ion metabolized by a p a r t i c u l a r pathway, f m i ' ^m2 ' f m3' fm4' fm5' fm6 a n d f C l r w a s determined according to the fo l lowing equation: Equation 7. f m i = C l f i s s / C l p s s 63 III. RESULTS A. ASSAY DEVELOPMENT The assay for the analysis of VPA and 14 metabolites developed in our laboratory was further modified for use in this study (Abbott et a l . , 1986a). It is a GCMS assay which uses selected ion monitoring mode to monitor the [M-57]+ fragment from tBDMS derivatives. 3-0ctanone, Dg-VPA and D 3 - 2 -ene VPA were the internal standards used. Peak area ratios were calculated using D6-VPA for VPA and D3~2-ene trans VPA for the metabolites. The calibration curves were linear over the concentration ranges measured and the coefficient of determination (r 2) was generally greater than 0.99. The lower level of detection of the assay was 0.1 ng/mL. The assay is reproducible and the relative standard deviation for most metabolites was less than 8 %. The relative standard deviation for 2-PSA and 4-OH VPA calibration curves exceeded 10 %. There was a problem with reproducibility in derivatizing 2-PGA. 1. Modifications to the assay The modifications to the assay included the addition of two new internal standards, octanoic acid and 2-methylglutaric acid. Octanoic acid was added as an internal standard for measuring Dg-VPA and i t s metabolites. The selected ion monitoring chromatograms of VPA and metabolites including MGA 64 and OA as internal standards are shown in figure 5. OA and MGA did not interfere with either VPA or any of the metabolites being measured. The diacid internal standard, MGA, was added to overcome the reproducibility problems of 2-PSA and 2-PGA. The choice was based on similarities in structure, size, and chemical properties of MGA to the diacid metabolites. The calibration curves for 2-PSA and 2-PGA are shown in figures 6 and 7. These curves were reproducible and had approximately 7 % intra-assay deviation. D3~2-ene cis VPA was used as the internal standard for VPA and the remaining metabolites. 2. Analysis of deuterated samples When assaying metabolites after deuterated VPA administration, a l l of the internal standards except for 3-octanone were discovered to cause some interference with the compounds being analyzed. Octanoic acid was investigated as an alternative internal standard for quantitation of deuterated metabolites, since deuterated internal standards could obviously not be used. Unfortunately, OA did not resolve from the deuterated 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 (DNBA) and hexanoic acid (HA) were therefore tested as internal standards for quantitation of deuterated VPA and its metabolites. These two compounds have been used as internal standards in a GCMS assay for 4-ene VPA in our laboratory (Singh et a l . , 1987). 65 s — I I 1 I 1 1 1 i — / / i • 1 I 3 4 6 6 7 B 9 10 13 14 15 16 TIME (min) Figure 5. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 a = 3-octanone, 2 a = D3-2-ene cis VPA, 3 a = D3-2-ene trans VPA, 4 = 4-OH VPA lactones, 5 = VPA, 6 a = D6-VPA, 7 = 4-ene VPA, 8 = 3-ene VPA, 9 = 2-ene cis VPA, 10 = 2-ene trans VPA, 11 = (E)-2,4-diene VPA, 12 = (E,E)-2,3'-diene VPA, 13a = octanoic acid, 14 = 3-keto VPA, 15 = 4-keto VPA, 16a = MGA, 17 = 5-OH VPA, 18 = 2-PSA, 19 = 2-PGA. ( a = internal standard). 0.12-n CONCENTRATION, JUG/ML Figure 6. C a l i b r a t i o n curve for 2-PSA in serum using 2-methylglutar ic ac id as the in terna l standard. 67 0.14 -i Figure 7. C a l i b r a t i o n curve for 2-PGA in serum using 2-methylglutar ic ac id as the in te rna l standard. 68 HA was ultimately used for the quantitation of deuterated metabolites, i.e. for the calculation of peak area ratios. DNBA and HA were both completely resolved from deuterated VPA and its metabolites and showed no interfering peaks using the selected ion monitoring mode. The selected ion chromatograms of VPA and metabolites with HA and DNBA as the internal standards are shown in figure 8. 3. Preparation of internal standards MGA was not readily soluble in d i s t i l l e d water at ,a concentration of 100 uq/mL and was i n i t i a l l y prepared in 3N NaOH in an attempt to overcome these solubility problems. An alkaline solution of the internal standard, however, posed problems when hydrolysis of the glucuronide conjugates of VPA and metabolites was not desired. Although MGA was readily soluble in a mixture of ethyl acetate and hexane (70:30) and was used as such by Granneman and coworkers (1984a) for the analysis of VPA and metabolites, this solvent mixture was not suitable with our extraction procedure. A method which proved successful was to produce a 10 fold stock solution of MGA in HPLC grade methanol, which could then be diluted in d i s t i l l e d water to obtain the desired working concentration of 100 uq/mL for the assay. Subsequently, a l l of the other internal standard solutions were made up in a similar manner. 69 m/r 197 o 14 — m/i 215 m/z 340 T - / / T -10 13 —r— 14 i 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 a = 3-octanone, 2 a = hexanoic acid, 3 = 4-OH VPA lactones, 4 = VPA, 5 = 4-ene VPA, 6 = 3-ene VPA, 7 = 2-ene cis VPA, 8 = 2-ene trans VPA, 9 = (E)-2,4-diene VPA, 10 = (E,E)-2,3'-diene VPA, 11a = di-n-butylacetic acid, 12 = 3-keto VPA, 13 = 4-keto VPA, 14 = 5-OH VPA, 15 = 2-PSA, 16 = 2-PGA. ( a = internal standard). 4. Derivatizing reagents Various derivatizing reagents were tested including MSTFA (TMS esters), tBDMCS in pyridine reagent with 5 % catalyst (tBDMS esters), and MTBSTFA (tBDMS esters). Both MSTFA and MTBSTFA are commercially available and are highly reactive reagents. 4.1. Comparison of TMS and tBDMS derivatives TMS and tBDMS derivatives of patient urine samples were compared. Aliquots of patient urine were extracted according to the assay procedure and then derivatized either with the MSTFA reagent (30 uh) for 20 min or the tBDMCS in pyridine reagent with 5 % catalyst (60 iiL) for 4 h. The GCMS was set on selected ion monitoring mode. With TMS derivatives, the [M-15]+ peak resulting from the loss of a methyl function was monitored. For tBDMS derivatives, the [M-57] + peak was monitored which resulted from the loss of the t-butyl group. Several differences were noted with respect to the chromatography. The same column was used for both derivatives but TMS esters required a retention time of approximately 30 min compared to 18 min for the tBDMS derivatives. The longer run time is required with TMS derivatives in order to achieve adequate resolution of peaks, particularily of 4-ene VPA from VPA. Conversely, TMS derivatives required heating for only 30 min compared to 4 h for tBDMS derivatives. TMS derivatives were stable for one to 71 two days, while tBDMS derivatives were stable for several weeks when stored at -20 °C. The tBDMS derivatives possessed 5 to 10 fold greater sensitivity for VPA and the unsaturated metabolites than the TMS esters. Selected ion chromatograms of TMS and tBDMS derivatives of VPA and metabolites are shown in figures 9 and 10, respectively. In comparing the chromatography of the TMS derivatives (figure 9), 4-OH VPA lactone isomers (peaks 1, m/z 100) are present as well as the isomers of 4-OH VPA monoderivative (peaks 16, m/z 217). With tBDMS derivatives (figure 10) only the 4-OH VPA lactone isomers (peaks 1, m/z 100) are evident. The 3-keto VPA is present both as the mono-and di-TMS derivative (peak 14, and peaks 17, 2 isomers). The tBDMS reagent yielded only the monoderivative. The 3-OH VPA also chromatograms more readily as the di-TMS derivative compared to the tBDMS derivative were 3-OH VPA is very poorly derivatized. The peaks of the 3-OH di-TMS derivative are sharp (peaks 15, m/z 217) in contrast to tBDMS derivatives. Although TMS esters are formed more readily, and yield better results with respect to 3-OH VPA, the longer chromatographic run time, decreased sensitivity, and decreased sta b i l i t y did not make MSTFA a more attractive derivatizing reagent to use for the routine analysis of VPA and its metabolites. 72 m/2 207 m/z 202 m/z 201 m/z 199 \ I Ik m/z 197 m/z 100 m/z 215 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 cis VPA, 7 = 3-ene VPA, 8 = 4-ene VPA, 9 = VPA, 10a = D3-2-ene trans VPA, 11a = D3~2-ene trans VPA, 12a = D6-VPA, 13 = 4-keto VPA, 14 = 3-keto VPA TMS monoderivative, 15 = 3-0H VPA (2 isomers), 16=4-OH VPA (2 isomers), 17 = 3-keto VPA TMS diderivative (2 isomers), 18 = 5-OH VPA, 19=2-PSA, 20 = 2-PGA. 73 1^ m/z 345 j, j^ni/z 331 m/z 317 — i 16 10 12 14 TIME (min) Figure 10. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 4-OH VPA lactones, 2 a = 3-octanone, 3 = (E)-2,4-diene VPA, 4 = (E,E)-2,3'-diene VPA, 5 = 4-ene VPA, 6 = 3-ene VPA, 7 = 2-ene cis VPA, 8 = 2-ene trans VPA, 9 = VPA, 10a = D3-2-ene cis VPA, 11a = D3-2-ene trans VPA, 12a = 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. ( a = internal standard). 4.2. Comparison of tBDMCS reagent and MTBSTFA reagent The two reagents were compared with respect to stability of the derivative formed and heating time required for complete derivatization. Although both these reagents form tBDMS esters, there were several differences noted between the two. Using the MTBSTFA reagent, the selected ion chromatograms of tBDMS derivatives of VPA 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 % catalyst. MTBSTFA provides a di-tBDMS derivative of 3-keto VPA (peaks 14, m/z 329) as well as the 3-keto monoderivative (peak 12, m/z 215). Changes in derivatization were not observed for the other metabolites. The heating time required for adequate derivatization by MTBSTFA was determined by derivatizing extracted aliquots of the same sample for different lengths of time. Samples were heated at 60 °C after addition of 40 uL of the derivatizing reagent for 0.33, 1, 2, 3, 5, 8, 12, and 16 h. There was l i t t l e change observed in the peak area ratio of VPA or metabolite to D3~2-ene cis VPA with an increase in length of heating time. However, the amount of 3-keto VPA di-tBDMS derivative formed with MTBSTFA was found to increase with a longer heating time with a corresponding decrease in the mono-tBDMS derivative (figure 12). Any samples heated for longer than 5 h had the di-tBDMS derivative of 3-keto VPA present (peaks 14, m/z 329). After 12 h of heating, a considerable 75 cr> m/z 201 m/z 207 m/z 10Q I -16 IT. 16 m/z 331 m/z 326 m/z 216 —17 i 12 i 13 i 14 m/z 346 — i 16 TIME (min) Figure 11. Selected ion chromatograms of tBDMS derivatives of valproic acid and metabolites from a patient urine sample. Peak numbers correspond to: 1 = 4-OH VPA lactones, 2 a = Dg-VPA, 3 = VPA, 4 = 4-ene VPA, 5 = 3-ene VPA, 6 = 2-ene cis VPA, 7 = 2- ene trans VPA, 8 a = D3-2-ene cis VPA, 9 a = Do-2-ene trans VPA, 10 = (E)-2,4-diene VPA, 11 = (E,E)-2,3'-diene VPA, 12 = 3- keto VPA, 13 = 4-keto VPA, 14 = 3-keto VPA tBDMS diderivatives (2 isomers), 15 = 5-OH VPA, 16 = 2-PSA, 17 = 2-PGA. ( a = internal standard). 10 -> 9 -7-2 6 < U J c r < * 5 < 2--2 •X 2-ENE TRANS VPA \ / \ :A AY •a- •E "T~ 0 2 4 TIME, WEEKS 6 5-OH VPA - 0 4-KETO VPA 2-ENE CIS VPA 2,4-DIENE VPA 8 -1 10 Figure 12. Peak area ratio of tBDMS derivatives of 2-ene trans VPA, 5-OH VPA, 4-keto VPA, 2~ene cis VPA, and 2,4-diene VPA from MTBSTFA reagent versus heating time. 77 p o r t i o n of the d e r i v a t i z e d 3-keto VPA was present as the d i -tBDMS d e r i v a t i v e . To study the s t a b i l i t y of the tBDMS d e r i v a t i v e formed by MTBSTFA, samples were s t o r e d at -20 °C and r e i n j e c t e d at i n t e r v a l s . A f t e r seven weeks of storage at -20 °C, few changes were observed. F i g u r e 13 i l l u s t r a t e s t h i s f o r 2-ene c i s VPA, Dg-VPA, and 2,3'-diene VPA. Upon storage, however, the mono-tBDMS d e r i v a t i v e of 3-keto VPA formed from MTBSTFA was g r a d u a l l y converted to the d i d e r i v a t i v e . A f t e r seven weeks of sto r a g e , a l l of the 3-keto VPA mono-tBDMS d e r i v a t i v e was completely converted to the di-TBDMS d e r i v a t i v e , r e g a r d l e s s of the l e n g t h of the i n i t i a l h e a t i n g time ( f i g u r e 14). Although the MTBSTFA r e a c t i o n time f o r complete d e r i v a t i z a t i o n i s much s h o r t e r (30 min), the co n v e r s i o n of the 3-keto VPA monoderivative to the d i d e r i v a t i v e upon storage and with i n c r e a s e d h e a t i n g time d i d not make t h i s reagent more f e a s i b l e to use. However, the use of MTBSTFA c o u l d be recommended h i g h l y f o r a n a l y s i s of other m e t a b o l i t e s of VPA. B. ANALYSIS OF SERUM AND URINE SAMPLES AFTER ADMINISTRATION OF DEUTERATED VPA Serum and u r i n e samples from the one v o l u n t e e r who r e c e i v e d Dg-VPA were sub j e c t e d to s i m i l a r a n a l y s i s as those c o n t a i n i n g nondeuterated VPA and m e t a b o l i t e s . However, i t i s d i f f i c u l t to analyze t h i s data as steady s t a t e c o n c e n t r a t i o n s were not 78 F i g u r e 13. Change i n peak area r a t i o of 3-keto VPA mono- and d i - d e r i v a t i v e to D3~2ene c i s VPA with i n c r e a s e d h e a t i n g time. 79 UO-i Figure 14. Change in peak area ratio of 3-keto VPA mono- and di-derivative to D3-2-ene cis VPA with storage. 80 achieved in either part of the study. Even normalizing the data to VPA or the total amount recovered in the urine over 12 h wi l l not provide viable information. The results presented are, thus, serum trough concentrations (table 1), serum AUC values (table 2) and 12 h urinary recoveries (table 3) comparing quantities after undeuterated and deuterated 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, 2-ene cis VPA, 2-ene trans VPA and 5-OH VPA were higher after Dg-VPA administration than after VPA administration. The concentrations of the other metabolites, namely, 4-ene VPA, 3-ene VPA, 3-keto VPA, 4-keto VPA, 2-PSA, 2-PGA, 2,3'-diene VPA and 2,4-diene VPA were lower after Dg-VPA administration. The AUC values for VPA and metabolites demonstrated a similar trend (table 2) to serum trough concentrations. The 12 h urinary recoveries of VPA and metabolites were lower following Dg-VPA administration. C. ANALYSIS OF CARBAMAZEPINE AND CARBAMAZEPINE-10,11-EPOXIDE IN SERUM Serum samples from the five volunteers were analyzed for CBZ and CBZE concentrations by modifying an HPLC method chosen from the literature (Elyas et a l . , 1982). Since the total daily dose of CBZ was increased to 300 mg from 200 mg for the 81 Table 1. Serum trough concentrations (mg/L) for VPA and metabolites after administration of VPA and Dg-VPA following 2 weeks of carbamazepine therapy for FS. Compound VPA D/--VPA 4-OH 3.417 14.52 4-ene 0.345 0.087 3-ene 0. 179 0.119 2-ene cis 0.063 0.100 2-ene trans 6.482 8.043 VPA 33.85 25.74 3-keto 6.318 1 .450 4-keto 0.308 0.242 5-OH 0.148 0.257 2-PSA 0.018 0.015 2-PGA 0. 123 0.016 2,3'-diene 1 .561 1 .441 2,4-diene 0.822 0.259 82 Table 2. 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. Metabolite VPA Dg-VPA 4-OH 48.60 212.3 4-ene 4.547 1 .283 3-ene 2. 178 1 .476 2-ene cis 0.784 1 .346 2-ene trans 81 .87 90.21 VPA 567. 1 399.2 3-keto 88.42 20.86 4-keto 4.605 2.884 5-OH 3.425 3.535 2-PSA 0.275 0.179 2-PGA 2.010 0.153 2,3'-diene 20.90 16.57 2,4-diene 10.79 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 VPA D6-VPA 4-OH 663.9 408.5 4-ene 6.632 2.455 3-ene 0.682 0. 164 2-ene cis 2.527 1.211 2-ene trans 89.15 41 .74 VPA (unchanged) 53.74 48.89 VPA (conjugate) 1263. 677.8 3-keto 883.4 581 .2 4-keto 71 .29 52.27 5-OH 1 003. 44.90 2-PSA 22.43 7.985 2-PGA 187.8 4.773 2,3'-diene 48.71 1 5.44 2,4-diene 1 4.74 2.224 84 second week, the samples chosen for analysis were the Cmin sample, and those acquired 3 and 5 h post dose on both the seventh and fourteenth day following the commencement of CBZ administration. The liquid chromatogram of the internal standard, 10-methoxycarbamazepine (MCBZ), extracted from a spiked serum sample is shown in figure 15. Figures 16 and 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 daily, respectively. The serum CBZ concentrations in the six samples analysed for each of the five volunteers are presented in table 4. Table 5 summarizes the corresponding serum CBZE levels. The percent ratio of CBZE to CBZ is shown in table 6. The ratio of the metabolite to the parent compound was calculated to detect if any changes in the ratio had occurred during the two weeks. The coefficient of determination (r 2) for the standard curves for CBZ and CBZE was 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 plots of serum VPA concentration versus time before and after CBZ for a l l five volunteers are shown in figures 18 - 22. The kinetic data for VPA is 85 Table 4. Serum CBZ concentrations (nq/mL) 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 C m^ n (prior to morning dose), 3 h and 5 h post dose. Volunteer Time 7 Says 1 4 days BA c s i n 5h 2.37 2.80 2.71 (10.0)* (11.9) (11.5) 3.07 3.54 3.58 (13.0) (15.0) (15.2) FS c ? h n 5h 3.04 3.71 3.62 (12.9) (15.7) (15.3) 4.53 5. 1 1 4.67 (18.4) (21.7) (19.8) MS c 9 i n 5h 2.79 3.65 3.52 (11.8) (15.5) (14.9) 3.77 4.43 4. 13 (16.0) (18.8) (17.5) RM 5h 3.68 4.34 4.19 (15.6) (18.4) (17.8) 4.40 4.70 4.74 (18.6) (19.9) (20. 1 ) WT c w 5h 2.51 2.92 2.96 (10.6) (12.4) (12.5) 4.18 4.62 3.86 (17.7) (19.6) (16.4) * numbers i n brackets i n d i c a t e c o n c e n t r a t i o n i n Mmol/mL 86 Table 5. Serum CBZE concentrations (uq/mL) 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 C m^ n (prior to morning dose), 3 h and 5 h post dose. Volunteer Time 7 days 14 days BA c s i n 5h 0.38 0.38 0.41 (1 .49)* (1.49) (1 .62) 0.51 (2.02) 0.55 (2.18) 0.54 (2.15) FS c ? h n 5h 0.85 0.85 0.87 (3.38) (3.38) (3.45) 1.20 (4.75) 1.34 (5.32) 1.34 (5.32) MS 5h 0.50 0.60 0.61 (1.99) (2.38) (2.40) 0.63 (2.50) 0.78 (3.08) 0.74 (2.94) RM c w 5h 0.56 0.62 0.57 (2.21) (2.44) (2.26) 0.75 (2.98) 0.77 (3.04) 0.82 (3.26) WT c ? h n 5h 0.42 6.42 0.45 (1.67) (1.65) (1.80) 0.64 (2.54) 0.69 (2.75) 0.54 (2.16) numbers in brackets indicate concentration in Mmol/mL 87 Table 6. Percent ratio 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 cmin (P ri° r to morning dose), 3 h and 5 h post dose. Serum CBZE/CBZ (%) Volunteer Time 7 days 14 days BA 15.9 16.6 13.4 15.5 5h 15.1 15.2 FS 28.0 22.9 27.7 26.2 5h 24.0 28.7 MS C m h n 18.0 16.5 16.7 17.5 5h 17.2 17.9 RM 15.1 17.1 14.2 16.3 5h 13.6 17.3 WT C W 16.8 14.3 15.3 15.0 5h 15.3 14.1 88 SO Oft? ITCH o Figure 15. L iqu id chromatogram of 1O-methoxycarbamazepine from a spiked serum sample. Column; Part iSphere C ^ (11cm x 4.70mm). Mobile phase; 35% Acetoni t r i le :65% Water. Flow rate 1.1mL/min. 89 ro o. in ro CD ON CM QJ a. ID rjid_L o Figure 16. L iqu id chromatogram of 1O-methoxycarbamazepine, carbamazepine and carbamazepine-10,11-epoxide from a spiked serum sample. Column; Part iSphere C i o (11cm x 4.70mm). Mobile phase; 35% Acetoni t r i le :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-10,11-epoxide, Peak 2) carbamazepine, Peak 3) 10-methoxycarbamazepine. 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 50 Time, h Figure 19. Semilogarithmic plot of serum VPA concentration (mg/L) versus time for FS before CBZ ( • ) and after CBZ ( O ) administration. 10 20 30 Time, h —r— 40 —I 50 Figure 2 0 . Semilogarithmic plot of serum VPA concentrat ion (mg/L) versus time for MS before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . 10 20 30 Time, h 40 50 F i g u r e 2 1 . 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 f o r RM before CBZ ( • ) and a f t e r CBZ ( O ) a d m i n i s t r a t i o n . 100 Time, h Figure 22. Semilogarithmic plot of serum VPA concentrat ion (mg/L) versus time for WT before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . summarized in table 7. Serum h a l f - l i f e (t 1/ 2» 24.7 %) and area under the serum concentration versus time curve (AUC, 29.5 %) values were significantly decreased after CBZ administration. Plasma clearance (Clp, 40.8 %) and elimination rate constant (k, 31.4 %) were significantly increased after CBZ administration. There was l i t t l e change in the mean volume of distribution (Vd). These trends are summarized in figures 23 -27. 2. Analysis of urine samples for VPA The mean amount of VPA recovered over 12 h decreased slightly from 806.1 to 767.3 Mmol (4.80 %) after CBZ administration while the amount recovered expressed as percent of dose administered also decreased 4.77% from 19.09 to 18.18 %. The amount of VPA recovered as the glucuronide conjugate decreased 5.32 % from 589.2 to 557.9 Mmol while the amount of unchanged VPA 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 plots of serum metabolite concentration versus time before and after CBZ administration are shown in figures 28 - 39. Individual plots are shown in the Appendix section. Figures 40 - 51 illustrate the changes in mean and individual serum AUC values before and after CBZ administration. Mean metabolite serum trough levels before and 97 Table 7. Valproic acid kinetic parameters for five healthy volunteers before and after administration of carbamazepine. Parameter Volunteer Before CBZ After CBZ % change k (h~ 1) 1/2 (h) AUC (mg.h/L) C l p (L/h) Vd (L/kg) BA 0.047 0.065 + 38.3 FS 0.046 0.062 + 34.8 MS 0.046 0.063 + 37.0 RM 0.045 0.059 + 31.1 WT 0.071 0.085 + 19.7 MEAN 0.051 0.067* + 31.4 s .d. +0.011 +0.011 BA 14.71 10.67 -27.5 FS 15.13 1 1 .23 -25.8 MS 15.14 1 1 .02 -27.2 RM 15.35 11.71 -23.7 WT 9.800 8. 160 -16.7 MEAN 1 4.03 10.56* -24.7 s.d. +2.374 + 1 .392 BA 786. 1 501.4 -36.2 FS 796.8 567. 1 -28.8 MS 710.7 483.6 -32.0 RM 583.3 468.2 -19.7 WT 498.3 358.2 -28. 1 MEAN 675.0 475.7* -29.6 S.d. +130.5 +75.73 BA 0.700 1 .097 + 56.7 FS 0.879 1 .230 + 39.9 MS 0.844 1 .240 + 46.9 RM 0.857 1 .068 + 24.6 WT 1 .200 1 .670 + 39.2 MEAN 0.897 1.263* + 40.8 S.d. +0.184 +0.241 BA 0.247 0.241 -2.43 FS 0.274 0.286 + 4.38 MS 0.246 0.263 + 6.91 RM 0.293 0.278 -5.12 WT 0.213 0.246 + 15.5 MEAN 0.255 0.263 + 3.14 s.d. +0.030 +0.020 * significantly different from before CBZ value at p < 0.05. 98 Figure 23. Plot of VPA h a l f - l i f e ( t . / 2 , h) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5). 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 2 i< 00 O o < cr 0.090-1 0.085H 0.080H > 0.075 OC P 0.070 0.065 H O 0.060 H < z 0.055 H 0.050 H 0.045 -H WT MEAN DAYS Figure 25. Plot of VPA elimination rate constant (Ke, h~1) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5). 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 ^ 0.27-1 0.26-^  £ 0.25-1 0.24 H O > 0.23 0.22 0.21 -X F S "H RM x-M E A N M S XT - r -10 15 20 25 DAYS gure 27. Plot of VPA volume of distribution (Vd, L/kg) before (Day 7) and after (Day 23) CBZ administration in volunteers (n=5). 103 100q i 1 1 1 1 [ 0 10 2 0 3 0 4 0 5 0 Time, h Figure 28. Representative semilogarithmic p lo t of 4-OH VPA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . Figure 29. Representative semilogarithmic p lo t of 4-ene VPA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . Time, h Figure 30. Representative semilogarithmic p lo t of 3-ene VPA concentration (mg/L) versus time before CBZ ( • ) and after CBZ ( O ) admin is t ra t ion . 1 0.01- T -10 20 —r— 30 i 40 50 Time, h Figure 31. Representative semilogarithmic p lo t of 2-ene c i s VPA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . 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 semilogarithmic p lo t of 3-keto VPA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . Figure 34. Representative semilogarithmic p lo t of 4-keto VPA concentrat ion (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . Time, h Figure 3 5 . Representative semilogarithmic p lo t of 5-OH VPA concentration (mg/L) versus time before CBZ ( • and af ter CBZ ( O ) admin is t ra t ion . ) 1 O) 0.1: E C Time, h Figure 36. Representative semilogarithmic p lo t of 2-PSA concentration (mg/L) versus time before CBZ ( • ) and after CBZ ( O ) admin is t ra t ion . Time, h Figure 37. Representative semilogarithmic plot of 2-PGA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . Figure 38. Representative semilogarithmic p lo t of 2 ,3 ' -d iene VPA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . I 10 -T— 20 30 40 50 Time, h Figure 3 9 . Representative semilogarithmic p lo t of 2 , 4 -d iene VPA concentration (mg/L) versus time before CBZ ( • ) and af ter CBZ ( O ) admin is t ra t ion . Figure 40. Plot of 4-OH VPA AUC (mg.h/L) before CBZ (Day 7) and af ter CBZ (Day 23) administrat ion in volunteers (n=5). 116 6 - i Figure 41 Plot of 4-ene and af ter CBZ (n=5). VPA AUC (mg.h/L) before CBZ (Day 7) (Day 23) administrat ion in volunteers 1 17 6.5 DAYS Figure 42. Plot of 3-ene VPA AUC (mg.h/L) before CBZ (Day 7) and a f ter CBZ (Day 23) administrat ion in volunteers (n=5). 118 o 1.20-1 1.15-1.10 H 1.05 H H CO QC 3 O X 0.95 CN or LU > 0.90 Q u Z> < 0.85-I 0.80H 0.75 H 0.70 B RM — X FS •S WT 10 15 - r -20 - 1 25 D A Y S Figure 43. Plot of 2-ene c i s VPA AUC (mg.h/L) before CBZ (Day 7) and af ter CBZ (Day 23) administrat ion in volunteers (n=5). 119 260-, DAYS Figure 44. Plot of 2-ene trans VPA AUC (mg.h/L) before CBZ (Day 7) and af ter CBZ (Day 23) administrat ion in volunteers (n=5). 120 9 0 - 1 80 H 6 70-6 0 H cn ZD O X CN ui > O o ZD 50 < 40 30 -X M E A N • M S 10 1 15 DAYS 20 & WT I 25 Figure 45. Plot of 3-keto VPA AUC (mg.h/L) before CBZ (Day 7) and af ter CBZ (Day 23) administrat ion in volunteers (n=5). 121 5 - i 4.5 in on ZD O X CN DC LJ > O o ZD < 3.5H 3H 2.5 BA FS .X- MEAN RM MS 7^ S WT 10 15 DAYS ~ r -20 25 Figure 46. Plot of 4-keto VPA AUC (mg.h/L) before CBZ (Day 7) and a f ter CBZ (Day 23) administrat ion in volunteers (n=5). 122 Figure 47. Plot of 5-OH VPA AUC (mg.h/L) before CBZ (Day 7) and a f ter CBZ (Day 23) administrat ion in volunteers (n=5). 123 0.35 0.30 H X d 0.25H 0.20-to CC ZD O X CN CC L d > o o ZD 0.15 < 0.10 H 0.05 BA FS ^ < - _ — K MEAN I K -'S WT MS RM 10 15 D A Y S - r— 20 I 25 Figure 48. Plot of 2-PSA AUC (mg.h/L) before CBZ (Day 7) and a f ter CBZ (Day 23) administrat ion in volunteers (n=5). 124 2.2-. 1.8 H X O 1.6 to cn ZD o 1.4 CN > O 1.2 O ZD < 0.8 H 0.6 4 * FS El RM ^ ^ • X MEAN A BA MS ffi WT 10 15 DAYS 20 2 5 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 40 35H CO cc 30 CN U J > O o ZD 20-< 15 H 10-E . . . " f f i WT A BA X-X FS X-.— KI RM 10 15 i 20 25 DAYS Figure 50. Plot of 2,3'-diene VPA AUC 7) and after CBZ (Day volunteers (n=5). (mg.h/L) before CBZ (Day 23) administration in 126 11 "1 I I 1 1 5 10 15 20 25 DAYS Figure 51. Plot of 2,4-diene VPA AUC (mg.h/L) before CBZ (Day 7) and a f ter CBZ (Day 23) administrat ion in volunteers (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 significant decrease in serum trough levels (table 8) for 2-ene trans VPA (15.21 to 11.53 mg/L) was observed. Mean serum trough concentrations for a l l metabolites except for 4-OH VPA, 2-PGA, 2,3'-diene VPA and 2,4-diene VPA decreased after CBZ administration. The decreases observed in mean 2-ene cis VPA (14.6 %) and 2-ene trans VPA (21.6 %) AUC values after CBZ administration were s t a t i s t i c a l l y significant. There was a general tendency for AUC values of the monounsaturated metabolites to be decreased after CBZ. In addition to 2-ene cis and trans VPA mentioned above, 4-ene VPA AUC values were reduced by 5.60 % and 3-ene VPA values by 15.4 %. However, the AUC values of the two diunsaturated metabolites were both increased, 2,3'-diene VPA, 6.24 % and 2,4-diene VPA, 22.7 %, following CBZ administration. There was a general tendency for the AUC values of polar metabolites, namely, 4-OH VPA (11.5 % ) , 4-keto VPA (29.3 % ) , 5-OH VPA (2.69 % ) , 2-PSA (23.9 % ) , and 2-PGA (22.3 % ) , to be increased after CBZ. The 3-keto VPA was the only polar metabolite whose AUC value was not increased after CBZ administration. 128 Table 8. Mean serum valproic acid and metabolites trough concentrations (mg/L) before and after administration of carbamazepine in five volunteers. Numbers in parentheses represent range. Compound Before CBZ After CBZ % change 4-OH 2. 693 (1 . 015- 4. 681 ) 2. 893 (1 . 913- 3. 781 ) + 7.43 4-ene 0. 391 (0. 276- 0. 475) 0. 389 (0. 334- 0. 556) -0.51 3-ene 0. 302 (0. 1 52-0. 507) 0. 248 (0. 1 19-0. 381 ) -17.9 2-ene cis 0. 082 (0. 062- 0. 103) 0. 069 (0. 055- 0. 095) -15.8 2-ene trans 15 .21 (8. 035- 20 .53) 11 .53* (6. 482- 15 .33) -24.2 VPA 44 .02 (27 .37- 53 .22) 27 .01* (16 .64- 33 .85) -38.6 3-keto 6. 299 (4. 501- 8. 151 ) 5. 815 (4. 421- 7. 337) -7.68 4-keto 0. 299 (0. 212- 0. 391 ) 0. 288 (0. 184- 0. 438) -3.68 5-OH 1 . 014 (0. 512- 1 . 247) 0. 824 (0. 148- 1 . 374) -18.7 2-PSA 0. 014 (0. 002- 0. 030) 0. 013 (0. 008- 0. 019) -7.14 2-PGA 0. 090 (0. 057- 0. 120) 0. 094 (0. 057- 0. 136) + 4.44 2,3'-diene 2. 391 (0. 852- 3. 624) 2. 434 (1 . 059- 3. 685) + 1 .80 2,4-diene 0. 562 (0. 510- 0. 626) 0. 647 (0. 386- 0. 923) + 15.1 * significantly 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 Before CBZ After CBZ % change 4-OH 35.62 4-ene 4.555 3-ene 3.452 2-ene cis 0.970 2-ene trans 179. 1 VPA 675.0 3-keto 65.00 4-keto 2.961 5-OH 13.78 2-PSA 0. 1 42 2-PGA 1 .047 2,3'-diene 25. 18 2,4-diene 6.307 (18.60-61.48) 39.70 (3.347-5.620) 4.300 (1.756-6.041) 2.920 (0.751-1.158) 0.828 (93.41-243.7) 140.4* (498.3-796.8) 475.7* (55.40-77.63) 62.80 (2.139-3.973) 3.830* (11.04-19.82) 14.15 (0.058-0.184) 0.176 (0.773-1.365) 1.280 (9.973-45.06) 26.75 (3.688-8.064) 7.740 22 .60-56.15) + 1 1 .5 3. 364-4.852) -5. 60 1 . 064-4.554) -15 .4 0. 717-0.999) -14 .6 81 .87- 195.3) -21 .6 358.2-567.1) -29 .5 28 .65- 88.42) -3. 38 3. 105-4.834) + 29 .3 3. 425-23.77) + 2. 69 0. 072-0.306) + 23 .9 0. 823-2.010) + 22 .3 12 .15- 37.67) + 6. 24 5. 154- 10.79) + 22 .7 * significantly different from before CBZ value at p < 0.05. 130 Figure 52. Histograms of AUC (mg.h/L)values of polar metabolites before CBZ ( £ 2 ) a n d a f te r CBZ ( administrat ion in volunteers (n=5). _j 200-i Figure 53. Histograms of AUC (mg.h/L)values of unsaturated metabolites before CBZ ( £ 2 ) and a f te r CBZ ( 0 administrat ion in volunteers (n=5). Tables 10 and 11 summarize the AUC values as total of polar and unsaturated metabolites. The mean sum of the AUC values for the polar metabolites increased about 3 % from 118.6 to 121.9 mg.h/L over the 12 h dosing interval while a st a t i s t i c a l l y significant decrease of 16.6 % from 219.5 to 183.0 mg.h/L in the sum of the AUC values for the unsaturated metabolites was observed. To determine i f a particular pathway was specifically affected by CBZ, AUC values were separated with respect to metabolic pathways depicted in figures 54 - 58: 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; and pathway 5 includes 5-OH VPA and 2-PGA. Pathway 6 is VPA glucuronide conjugation and C l r is unchanged VPA which is cleared through the kidneys. In the serum, VPA includes the conjugate as well as the free drug as these were not distinguished separately in the serum samples since the amount of the conjugate present is very small. A mean decrease of 16.7 % from 245.0 to 204.1 mg.h/L was observed in pathway 1 AUC values after CBZ administration (table 12) and the VPA pathway was also significantly decreased as discussed previously. Pathways 2, 3, 4, and 5 were apparently 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 calculations, are shown in 133 Table 10. Sum of serum AUC (mg.h/L) for polar metabolites of va lp ro ic ac id over 12 h for the f i ve healthy volunteers before and a f ter administrat ion of carbamazepine. Serum AUC (mg.h/L) Before CBZ After CBZ Volunteer % change BA FS MS RM WT MEAN S.d. 144.5 97.23 104.0 88.60 158.3 118.6 +41.45 1 37 147, 107 118 101 121 -5.12 + 51.5 + 3.27 + 33.7 -35.8 + 2.78 +20.01 Table 11. Sum of serum AUC (mg.h/L) for metabolites of va lp ro ic ac id over f i ve healthy volunteers before administrat ion of carbamazepine. unsaturated 12 h for the and a f te r Volunteer Before CBZ After CBZ % change BA FS MS RM WT MEAN S.d. 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 -25.2 -3.04 -11 .7 -15.1 -19.4 -16.6 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. 134 Table 12. 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). Pathway2 Before CBZ After CBZ % change 1 245.0 (153.5-322. 1 ) 204.1 (171.1-226.7) -16.7 2 28.63 (11.73-49.90) 29.67 (13.21-41.59) + 3.63 3 10.86 (8.023-13.10) 12.05 (9.492-15.34) + 10.9 4 38.72 (20.80-64.42) 43.70 (26.06-59.37) + 12.9 5 14.83 (12.02-21.14) 15.42 (5.535-24.99) + 3.98 VPA 675.0 (498.3-796.8) 475.7* (358.2-567.1) -29.5 Pathway 1 Pathway 2 Pathway 3 Pathway 4 Pathway includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2,3'-diene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA. includes 5-OH VPA and 2-PGA. VPA includes free and conjugated VPA. significantly different from before CBZ value at p < 0.05. 135 CH3-CH2-CH2 CH3 CH2 CH2 \ ( CHCOOH VALPROIC ACID C H o - C H o - C H / CH 3 - C H 2 — C H 2 CCOOH 2-ENE VPA OH CH3-CH2-CH CH3 CH2~CH2 CHCOOH 3-OH VPA CH3-CH2-C CH3 CH 2 _CH 2 CHCOOH 3-KETO VPA Figure 54. Pathway 1 (beta-oxidation of valproic acid) 136 C H 3 - C H 2 - C H 2 \ C H C O O H C H 3 C H 2 _ C H 2 V A L P R O I C A C I D C H-5 - C H = C H C H 3 C H 2 C H 2 \ C H C O O H 3 - E N E V P A C H o - C H = C H C H 3 ~ C H 2 C H C C O O H 2 , 3 ' - D I E N E V P A F i g u r e 5 5 . P a t h w a y 2 ( d e h y d r o g e n a t i o n o f v a l p r o i c a c i d ) 1 3 7 CH3-CH2-CH2 ^HCOOH / CH3 CH2 CH2 VALPROIC ACID C H o = C H - C H 9 \ C H C O O H / C H 3 C H 2 C H 2 4-ENE VPA I C H o = C H - C H CCOOH CH3 CH2~CH2 2,4-DIENE VPA Figure 56. Pathway 3 (dehydrogenation of valproic acid) 138 CH3-CH2-CH2 ^CHCOOH CH3—CH2—CH2 VALPROIC ACID OH I C H o - C H - C H p N C H C O O H / C H 3 - C H 2 - C H 2 4-OH VPA PJ CH 2 CH3-C- 2 CHCOOH CH3 CH2~CH2 4-KETO VPA I HOOC-CH2 \ CHCOOH CH3_CH2~CH2 2-PSA Figure 57. Pathway 4 (CJ-1 oxidation of valproic acid). 139 C H ^ - C H 2 _ C H 2 \ C H C O O H / C H 3 CH2 _ C H 2 V A L P R O I C A C I D O H | I CH2_CH2_CH2 \ C H C O O H CH3-CH2—CH 2 5 - O H V P A I H O O C - C H 2 - C H 2 ^ C H C O O H C H ^ CH2 OH2 2 - P G A F i g u r e 5 8 . P a t h w a y 5 ( c o - o x i d a t i o n o f v a l p r o i c a c i d ) 140 table 13 and were calculated by dividing the AUC value by the dosing interval of 12 h. Average steady state concentrations were also 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. The recovery of 2-ene cis VPA significantly increased by 12.5 % ( 1 .652 to 1 .859 Aimol/12 h) after CBZ administration. Twelve hour urinary recoveries of 4-OH VPA, 4-ene VPA, 4-keto VPA, 5-OH VPA, 2-PSA, and 2,4-diene VPA also increased although they did not reach significance. The mean urinary recovery of 2-ene trans VPA significantly decreased by 25.2 % (117.3 to 87.82 umol/12 h) following CBZ administration. The mean 12 h recoveries for 2-PGA (39.3 % ) , 2,3'-diene VPA (13.8 % ) , 3-ene VPA (43.7 % ) , and 3-keto VPA (5.07 %) also decreased after CBZ administration although the decreases did not reach significance. Figures 59 and 60 depict the changes in mean metabolite amounts recovered over 12 h. When the mean amount of metabolites recovered in urine are expressed as the pathways previously defined, a decrease in the total amounts recovered for pathways 1 and 2 (8.46 and 14.3 %, respectively) was observed (table 16) after CBZ administration. 141 Table 13. Mean 'average serum steady s t a t e c o n c e n t r a t i o n s ' 9 (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 before and a f t e r a d m i n i s t r a t i o n of carbamazepine. Numbers i n parentheses represent range (n=5). Compound Before CBZ A f t e r CBZ % change 4-OH 2. 968 (1 . 550- 5. 123) 3. 308 (1 . 883- 4. 679) + 11.5 4-ene 0. 380 (0. 279- 0. 468) 0. 359 (0. 280- 0. 404) -5.52 3-ene 0. 288 (0. 146- 0. 503) 0. 244 (0. 089- 0. 380) -15.3 2-ene c i s 0. 081 (0. 063- 0. 097) 0. 069 (0. 060- 0. 083) -14.8 2-ene t r a n s 14 .93 (7. 784- 20 .31 ) 1 1 .70* (6. 823- 16 .28) -21.6 VPA 56 .25 (41 .53- 66 .40) 39 .64* (29 .85- 47 .26) -29.5 3-keto 5. 417 (4. 617- 6. 469) 5. 236 (2. 389- 7. 368) -3.34 4-keto 0. 247 (0. 1 78-0. 331 ) 0. 319* (0. 259- 0. 403) + 29.2 5-OH 1 . 1 48 (0. 920- 1 . 652) 1 . 179 (0. 285- 1 . 981 ) + 2.70 2-PSA 0. 012 (0. 005- 0. 015) 0. 015 (0. 006- 0. 025) + 25.0 2-PGA 0. 087 (0. 064- 0. 1 14) 0. 107 (0. 069- 0. 168) + 23.0 2,3'-diene 2. 098 (0. 831- 3. 755) 2. 229 (1 . 013- 3. 139) + 6.24 2,4-diene 0. 526 (0. 307- 0. 672) 0. 645 (0. 430- 0. 899) + 22.6 a 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 by d i v i d i n g the AUC value by the dosing i n t e r v a l . * 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. 142 Table 14. Mean 'average steady state serum concentrations a (mg/L) of valproic acid and metabolites expressed as pathways before and after administration of carbamazepine. Numbers in parentheses represent range (n=5). Pathway" Before CBZ After CBZ % change 1 20.42 (12.79-26.84) 17.01 (14.26-18.89) -16.7 2 2.386 (0.977-4.158) 2.473 (1.101-3.466) +3.65 3 0.906 (0.669-1.092) 1.004 (0.791-1.278) +10.8 4 3.227 (1.733-5.368) 3.642 (2.172-4.948) +12.9 5 1.235 (1.002-1.762) 1.285 (0.453-2.083) +4.05 VPA 56.25 (41.53-66.40) 39.64*(29.85-47.26) -29.5 a average steady state concentration is calculated by dividing the AUC value by the dosing interval. 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. * significantly different 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) recovered i n u r i n e over 12 h 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). Compound Before CBZ A f t e r CBZ % change 4-OH 295.0 (250.6- 342.0) 354.7 (217.0- 663.9) + 20 .3 4-ene 1 .957 (1 .056-4.472) 2.818 (1.390- 6.632) + 44 .0 3-ene 0.769 (0.212- 2.555) 0.433 (0.200- 0.682) -43 .7 2-ene c i s 1 .652 (0.771- 2.477) 1 .859* (1.047- 2.620) + 12 .5 2-ene t r a n s 117.3 (65.07- 154.4) 87.82* (53.35- 102.9) -25 .2 VPA ( t o t a l ) 806.1 (464.9- 1564.) 767.3 (461.9- 1317.) -4. 80 3-keto 567.8 (29.25- 1642.) 539.0 (282.3- 883.4) -5. 07 4-keto 44.98 (25.21- 74.22) 52.28 (30.60- 71.29) + 16 .2 5-OH 642.3 (303.9- 1323.) 760. 1 (501.8- 1099.) + 21 .8 2-PSA 8.046 (5.103- 12.48) 10.26 (6.159- 22.43) + 27 .5 2-PGA 185.7 (74.44- 528.1 ) 112.6 (77.94- 187.8) -39 .3 2,3'-diene 53.54 (34.27- 84.85) 46. 13 (22.35- 69.42) -13 .8 2,4-diene 9.984 (5.418- 13.99) 1 0.28 (6.559- 14.74) + 2. 94 * 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. 144 Table 16. 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). Pathway3 Before CBZ After CBZ % change 1 686.8 (147.3-1778.) 628 .7 (385.2-975.1) -8. 46 2 54.31 (34.64-85.24) 46. 57 (22.55-69.83) -14 .3 3 1 1 .94 (6.474-18.46) 13. 10 (7.949-21.37) + 9. 72 4 348.0 (289.2-373.5) 417 .3 (254.6-757.6) + 19 . 1 5 810.0 (379. 1-1488.) 872 .8 (586.2-1226.) + 7. 75 6 589.2 (94.20-1514.) 557 .9 (187.0-1263.) -5. 32 Clr 216.9 (50.18-550.0) 209 .5 (53.74-346.3) -3. 39 a 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. Pathway 6 is VPA glucuronide conjugate. Pathway Clr is free (unchanged) VPA. 145 9* I Figure 60. Histograms of mean recovery (nmol) of polar metabolites before CBZ ( gr j j a n d a f t e r CBZ ( administrat ion in volunteers (n=5). ) The 12 h urinary recoveries of total metabolites in pathways 3, 4, and 5 increased 9.72, 19.1, and 7.75 %, respectively after CBZ administration. None of the changes were s t a t i s t i c a l l y significant. Urinary recoveries expressed as pathways for the individual volunteers are in the Appendix section. The mean 12 h urinary recovery of polar metabolites increased approximately 6 % (1726 to 1829 Mmol) after CBZ administration while the mean recovery of unsaturated metabolites decreased 19.4 % from 185.2 to 149.3 Mmol (tables 17 and 18). The decrease in the urinary recovery of the unsaturated metabolites was s t a t i s t i c a l l y significant. When expressed as a percent of the dose administered (table 19, figures 61 and 62), the mean urinary recovery of 4-OH VPA, 4-ene VPA, 2-ene cis VPA, 3-keto VPA, 4-keto VPA, 5-OH VPA, 2-PSA, and 2,4-diene VPA increased 15.0, 43.5, 12.4, 4.10, 17.4, 26.6, 22.4 and 2.89 % respectively, after CBZ administration. The recovery of 4-ene VPA significantly increased by 43.5 %. The recoveries of 3-ene VPA, 2-ene trans VPA, 2-PGA, and 2,3'-diene VPA decreased 41.2, 24.6, 4.77, 44.0, and 15.2 % respectively, after CBZ administration when expressed as % of dose recovered. The recovery of 2-ene trans VPA decreased significantly. The percent of dose administered which was recovered as VPA and metabolites in the urine over 12 h before and after CBZ (table 20) was approximately the same (64.6 % versus 66.5 % ) . 148 Table 17. 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. Volunteer Before CBZ After CBZ % change BA FS MS RM WT MEAN s.d. 1005 3486 1 120 1623 1 395 1726 + 1013 1213. 2832. 1 534. 2289. 1 278 1829. +704.8 + 20.8 -18.8 + 36.9 + 41 .0 -8.39 + 5.98 Table 18. 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. Volunteer Before CBZ After CBZ % change BA FS MS RM WT MEAN S.d. 107.0 210.0 180, 175, 253, 185, ,6 ,3 4 2 +53.63 92.56 162.4 177.9 1 29.2 184.6 149.3* +38.26 -13.5 -22.7 -1 .50 -26.3 -27. 1 -19.4 * significantly different from before CBZ value at p < 0.05. 149 Table 19. 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 recovered i n the u r i n e over 12 h as percent of VPA dose 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). Compound Before CBZ A f t e r CBZ % change 4-OH 7.305 (5. 534- 9.027) 8.399 (5. 208- 13 .66) + 15 .0 4-ene 0.046 (0. 028- 0.092) 0.066* (0. 036- 0. 136) + 43 .5 3-ene 0.017 (0. 006- 0.053) 0.010 (0. 006- 0. 014) -41 .2 2-ene c i s 0.041 (0. 020- 0.071 ) 0.046* (0. 027- 0. 075) + 12 .4 2-ene t r a n s 2.863 (1 . 704- 3.705) 2.158* (1 . 397- 2. 653) -24 .6 VPA 19.09 (11 .44- 32.17) 18.18 (13 .26- 27 .09) -4. 77 3-keto 12.68 (0. 842- 33.78) 1 3.20 (6. 774- 21 .58) + 4. 10 4-keto 1 .092 (0. 605- 1 .527) 1 .282 (0. 734- 1 . 679) + 17 .4 5-OH 14.87 (7. 622- 27.22) 18.83 (12 .81- 31 .65) + 26 .6 2-PSA 0.1 96 (0. 134- 0.281 ) 0.240 (0. 168- 0. 461 ) + 22 .4 2-PGA 4.883 (1 . 786- 15.21) 2.733 (1 . 870- 3. 863) -44 .0 2,3'-diene 1 .307 (0. 897- 2.036) 1 .1 08 (0. 644- 1 . 666) -15 .2 2,4-diene 0.242 (0. 142- 0.303) 0.249 (0. 172- 0. 303) + 2. 89 * 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. 150 Table 20. Percent of va lpro ic a c i d dose recovered in the urine as VPA and metaboli tes over 12 h for the f ive healthy volunteers before and af ter carbamazepine admin is t ra t ion . Volunteer Before CBZ After CBZ BA FS MS RM WT MEAN S .d. 41 .3 108.2 52.4 70.3 60.0 64.6 26.5 47.5 88.7 62.6 82.9 50.8 66.5 18.6 151 39 t C to MEAN RECOVERY OF UNSATURATED METABOLITES AS PERCENT OF DOSE < — 3 X o [SJrr CO c SJCo rr 3 L > J C T O rr 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 ^ - o _ CO O • cn < • ro T J n w r o •< Co o » 3 3 3 H - r t O 3 I—* M - O ' 01 l-h rr O n Qj r-h 0) 0 rr oi c M ' ro 3 O 01 3 t r OJ CO rr 3 O n I-I o> (B rr ro o a ca M £ 9 1 lO c ro NJ • < — 3 I o „ ft> >— 1—' ^ r r ti) c O O ) rr 3 L V tr O rr — O id CD n> Q) 0) i-l = rr 3 Ul QJ ro cn ,—~ 0) o 3 ct> rD MI II rr M m ro *o 3 •—• 1 1 !( • ro cu O w 3 CO 10 N (t 1 a n ^ o __o» o • 01 < • n> —- ro »-j a n t : 3 3 3 H-ft O 3 r-o ^ 01 rr. rr O OJ o rr oi TJ CD O O t-3 tr 0» M- M l 3 O n rD n to MEAN RECOVERY OF POLAR METABOLITES AS PERCENT OF DOSE Table 21 depicts 12 h urinary recoveries expressed as a percent of the total amount of VPA and metabolites recovered. The data is graphically presented in figures 63 and 64. The recovery of 2-ene trans VPA significantly decreased (4.81 to 3.39 %) while the recovery of 5-OH VPA significantly increased (22.0 to 28.2 %) after CBZ administration. The recoveries of 4-OH VPA, 3-ene VPA, 2-PGA, 2,3'-diene VPA and 2,4-diene VPA decreased after CBZ administration. The recoveries of the other metabolites (4-ene VPA, 2-ene cis VPA, 3-keto VPA, 4-keto VPA, 5-OH VPA, and 2-PSA) increased after CBZ administration when expressed as a percent of the total 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 of 4-OH VPA, 4-ene VPA, 2-ene cis VPA, 3-keto VPA, 4-keto VPA, 5 OH VPA, 2-PSA and 2,4-diene VPA increased after CBZ administration. The remaining metabolites, 3-ene VPA, 2-PGA and 2,3'-diene VPA, exhibited decreased recoveries (figures 65 and 66). Statistical analysis did not demonstrate any significant differences. Mean urinary creatinine concentrations (data not shown) before and after CBZ were 123.6 mg/100 mL and 91.76 mg/100 mL, respectively. St a t i s t i c a l analysis demonstrated a lack of difference in these two values. 1 54 Table 21. Mean va lp ro ic ac id and metabolites (/xmolar basis) recovered over 12 h expressed as percent of to ta l recovered before and a f ter carbamazepine administrat ion in f i v e volunteers . Numbers in parentheses represent range. Compound Before CBZ After CBZ % change 4-OH 12.7 (5.11-15.9) 12.6 (8.66-15.7) -0.79 4-ene 0.07 (0.05-0.08) 0.09 (0.07-0.15) + 28.6 3-ene 0.02 (0.01-0.05) 0.02 (0.01-0.02) 0.00 a 2-ene c i s 0.06 (0.04-0.10) 0.07 (0.06-0.09) + 16.7 2-ene trans 4.81 (2.55-7.27) 3.39* (2.07-4.80) -29.5 VPA 29.7 (22.4-40.4) 28.0 (16.0-34.4) -5.72 3-keto 18.5 (1.20-31.2) 19.1 (13.3-26.0) + 3.24 4-keto 1 .74 (1.16-2.13) 1 .96 ( 1 .45-2.56) + 12.6 5-OH 22.0 (14.6-28.3) 28.2* (20.5-38.2) + 28.2 2-PSA 0.31 (0.24-0.40) 0.36 (0.21-0.52) + 16.1 2-PGA 7.39 (3.14-21.6) 4.08 (3.31-4.66) -44.8 2 ,3 ' -d iene 2.27 ( 1 .00-3.99) 1 .83 (0.77-3.28) -19.4 2,4-diene 0.39 (0.27-0.53) 0.38 (0.34-0.48) -2.56 a actual change i was -27.3 %. * s i g n i f i c a n t l y d i f f e ren t from before CBZ value at p < 0.05. 155 Table 22. Mean va lpro ic ac id and metabolites (/xmolar basis) recovered over 12 h in the urine expressed as percent of VPA recovered before and a f ter carbamazepine administrat ion in f i ve vo lunteers . Numbers in parentheses represent range. Compound Before CBZ After CBZ % change 4-OH 44.3 (17.2- 62.9) 45.9 (33.2- 54.0) + 3.61 4-ene 0.23 (0.19- 0.29) 0.35 (0.26- 0.50) + 52.2 3-ene 0.08 (0.03- 0.16) 0.06 (0.04- 0.08) -25.0 2-ene c i s 0.22 (0.15- 0.39) 0.27 (0.19- 0.57) + 22.7 2-ene trans 17.3 (8.57- 32.4) 12.9 (6.77- 19.9) -25.4 3-keto 64.4 (4.54- 105. ) 77.7 (43.2- 162. ) + 20.7 4-keto 6.27 (2.86- 8.74) 7.60 (4.68- 12.6) + 21.2 5-OH 79. 1 (36.1- 107. ) 1 15. (59.5- 238. ) + 45.4 2-PSA 1.11 (0.66- 1.51) 1 .30 (0.93- 1 .70) + 17.1 2-PGA 27.0 (8.45- 82.0) 16.0 (9.63- 27.4) -40.7 2 ,3 ' -d iene 8.27 (3.37- 17.8) 6.35 (3.70- 10.6) -23.2 2,4-diene 1 .41 (0.90- 2.37) 1 .45 (1.12- 2.13) + 2.84 156 I. s i c ro as OJ MEAN RECOVERY OF UNSATURATED METABOLITES AS PERCENT OF TOTAL RECOVERED * CD r r I-I X »-h 3 * fD (-"• CO r r fD O (0 ro O r r i Q I-I r r < O 3 O fD i O M - O r r i l -h tz) CU fD 0) n a g O (0 01 —. 0) M -3 3 3 o (T • O l -h h-* C r r •< 3 3 - 3 r r fD fD a 0) •->• n c 3 l -h O i fD i - l »-h 3 o C fD >-»• O 3 3 3 < CD VI ro fD 0* 3 i n i - l fD r r r t r r fD X C i-t at? •-I l -h CU 0> n r r tr fD r r O y->-fD (0 ro 3 O l -h (fl a 3 O fD tr n a 3 ro fD fD r-h 3 01 r r o O fit 0) l -h W tr ro tsj 0) o < o ro —TJ to r r M < fD o NJo (0 < - ^ f D CD c ^ 3 M 3 r t C r r 0) ro fD 3 O fD Q i l -h 01 i - i r t cn •o I A o o ( J l 89 I MEAN RECOVERY OF POLAR METABOLITES AS PERCENT OF TOTAL RECOVERED IA o o tn 150-1 CO Ul vo Figure 65. Histograms of mean unsaturated metabol i tes recovered in the urine expressed as a percent of VPA recovered before CBZ ( \£2 ) a n d a f t e r CBZ ( ) administrat ion in f i ve vo lunteers . 0 9 1 03 C n ro CTl CT\ • D> CT rr X QJ (D 3* M-3 rft ro cn »-»• O rt 3 I C O ro n uQ cn <-t co ro 3 OJ N cn rt ro o O 3 \Jro 3 •->• cn ro 3 ^cn o> ro 3 Mi OJ a < Oi CU o cn i-1 OJ Ql < Mill) tl o rt H-" re g c n ro ro 3 rt rt rt O o 0» ro w ro cr ro M 3 O •-I rt t~> cn -—• • _ O rr • M> n> TJ n > ro o H o ro < o ro o n < ro ro a «. ro (->• a 3 MEAN RECOVERY OF UNSATURATED METABOLITES AS PERCENT OF VPA RECOVERED 5. Pharmacokinetic ana lys is 5.1. Pathway ana lys is Mean formation clearances for a l l pathways are shown in table 23. Indiv idual formation clearances for the volunteers are in the Appendix s e c t i o n . The formation clearance for pathway 6 i s an apparent clearance value since VPA conjugate was not measured in the serum. The formation rates for a l l pathways were increased a f te r CBZ admin is t ra t ion . The increases in pathways 3, 5 and 6 were s t a t i s t i c a l l y s i g n i f i c a n t . Increases observed were 32.6, 18.9, 50.0, 59.3, 49.0, and 37.4 %, r e s p e c t i v e l y , for pathways 1 to 6 a f te r CBZ admin is t ra t ion . Mean metabolite clearances for a l l pathways are i l l u s t r a t e d in table 24. Indiv idual metabolite clearances are in the Appendix s e c t i o n . Metaboli te clearances for pathway 6 and C l r are apparent clearances since the conjugate and unchanged VPA were not determined separately in the serum. The metabolite clearance of VPA ( tota l ) increased 36.7 % af ter CBZ administ rat ion but d id not reach s t a t i s t i c a l s i g n i f i c a n c e . An increase of approximately 41 % in mean plasma clearance of VPA was observed a f ter CBZ therapy. Mean metabolite or e l iminat ion clearances of pathways 5, 6 and C l r increased af ter CBZ by 32.8, 37.4, and 35.0 %, r e s p e c t i v e l y . The increase in the metabolite clearance of pathway 6 was s t a t i s t i c a l l y s i g n i f i c a n t . There were decreases observed in the metabolite clearances for pathways 1, 2, 3, and 4 of 6.53, 29.8, 3.11, and 161 Table 23. Mean formation clearances ( C l « , L / h ) a for pathways 1 - 6 before and a f ter CBZ administrat ion for f ive healthy vo lunteers . Pathway13 Before CBZ C Af ter CBZ C % change 1 0.1528 + 0.1245 0.2026 + 0.0693 +32.6 2 0.0122 + 0.0068 0.0145 + 0.0083 +18.9 3 0.0026 + 0.0010 0.0039 + 0.0012* +50.0 4 0.0856 + 0.0217 0.1364 + 0.0494 +59.3 5 0 .2006+0.1216 0 .2989+0.0982* +49.0 6 0 .1178+0.0948 0 .1619+0.0974* +37.4 Sum 0.6225 + 0.2654 0.8869 + 0.2237 +42.5 a C l f ca lcu la ted by d i v i d i n g the amount of metaboli tes recovered in the urine over 12 h from a given pathway by VPA AUC. b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. c values represent mean + standard deviat ion * s i g n i f i c a n t l y d i f f e ren t from before CBZ value at p < 0.05. 162 Table 2 4 . Mean metabolite clearances ( C l m , L / h ) a for pathways before and a f ter CBZ administrat ion for f ive healthy vo lunteers . Pathway b Before CBZ d Af ter CBZ d % change 1 0 .5438 + 0 .0715 0 .5083 + 0.2857 - 6 . 5 3 2 0 .3325 + 0 .1641 0 .2335 + 0 .0786 - 2 9 . 8 3 0 .1574 + 0 .0538 0 .1525 + 0 .0422 - 3 . 1 1 4 1 .7180 + 0 .8426 1.5824 + 0 .6407 - 7 . 8 9 5 9 .9855 + 0 .7483 13.259 + 12.700 + 32 .8 6 0 . 1 178 + 0 .0948 0 .1619 + 0 . 0 9 7 4 * + 37 .4 c i r 0.0509 0 .0512 0 .0686 + 0 .0490 + 3 5 . 0 Sum 12.905 + 8 .9030 15.966 + 13.440 + 2 3 . 7 C 1 D C 0.8967 + 0 .1857 1.2630 + 0 .2433 + 4 0 . 8 P a C l m ca lcu la ted by d i v i d i n g the amount of metaboli tes recovered in the urine over 12 h from a given pathway by the corresponding AUC. b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r i s free (unchanged) drug. c C l p = D o s e / A U C ( V P A ) . d values represent mean + standard dev ia t ion . * s i g n i f i c a n t l y d i f f e ren t from before CBZ value at p < 0 . 0 5 . 163 7.89 % , respect ive ly although the changes d id not reach s t a t i s t i c a l s i g n i f i c a n c e . The mean f rac t ion metabolized by each pathway was ca lcu la ted before and a f ter CBZ administrat ion and i s shown in table 25. Indiv idual data for the volunteers are in the Appendix s e c t i o n . None of the changes in mean f rac t ion metabolized by a p a r t i c u l a r pathway were s t a t i s t i c a l l y s i g n i f i c a n t . The mean f rac t ion metabolized for pathways 1, 2, and 6 decreased 0.72, 15.5, and 3.52 %, r e s p e c t i v e l y . The mean f rac t ion metabolized v ia pathways 3, 4, and 5 was increased by 10.7, 15.5, and 8.03 %, r e s p e c t i v e l y . The mean f rac t ion of VPA ( tota l ) metabolized decreased 4.76 % af ter CBZ admin is t ra t ion . 5.2. Metabol i te ana lys is After c a l c u l a t i n g C l f , C l m and f m for the metabolic pathways, the same parameters were a lso ca lcu la ted for each metabol i te . Table 26 summarizes the mean formation clearances for the metabol i tes . The mean formation clearances of 3-ene VPA and 2-PGA decreased 14 % and 21.2 %, respec t i ve ly , a f ter CBZ admin is t ra t ion . The formation clearances of 4-OH VPA, 4-ene VPA, 2-ene c i s VPA, 3-keto VPA, 4-keto VPA, 5-OH VPA, 2-PSA, 2 ,3 ' -d iene VPA and 2,4-diene VPA increased 58.9, 101, 54.0, 3.85, 38.8, 41.7, 73.2, 50, 16.7 and 50 %, r e s p e c t i v e l y , a f ter CBZ admin is t ra t ion . The increase in formation clearances for 164 Table 25. Mean f rac t ion metabolized ( f m ) a by each pathway before and af ter CBZ administrat ion for f i v e vo lunteers . Pathway" Before CBZC Af ter CBZ C % change 1 0.1677 + 0.1359 0.1665 + 0.0713 -0 .72 2 0.0129 + 0.0042 0.0109 0.0043 - 1 5 . 5 3 0.0028 + 0.0008 0.0031 + 0.0008 + 10.7 4 0.0953 + 0.0152 0.1101 + 0.0413 + 15.5 5 0.2242 + 0.1399 0.2422 + 0.0961 +8.03 6 0.1337 + 0.1086 0.1290 + 0.0791 -3 .52 c i r 0.0573 0.0601 0.0529 + 0.0351 -7 .68 Sum 0.6939 + 0.2861 0.7147 + 0.2034 + 3.00 a f m i s ca lcu la ted by d i v i d i n g C l f by C l p . b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 i s VPA glucuronide conjugate. C l r i s free (unchanged) VPA. c values represent mean + standard deviat ion 165 Table 26. Mean metabolite formation clearances ( C l j , L / h ) a for the VPA metaboli tes before and a f ter CBZ admin is t ra t ion . Numbers in parentheses represent range (n=5). Metabol i te Before CBZ After CBZ % change 4 -OH 0. 073 (0. 051- 0 .096) 0. 116 (0 . 074- 0. 187)^ + 58.9 4 -ene 0. 400 (0. 191- 0 .797)f 0. 806* (0 . 394- 0. 166)^ + 101. 3 -ene 0. 150 (0. 052- 0 .455)J> 0. 129 (0 . 061- 0. 171 )£ -14.0 2 -ene c i s 0. 359 (0. 139- 0 .603) b 0. 5 5 3 M 0 . 297- 0. 795) b + 54.0 2 -ene trans 0. 026 (0. 012- 0 .044) 0. 027 (0. 015- 0. 040) + 3.85 3 -keto 0. 127 (0. 008- 0 .326) 0. 175 (0 . 107- 0. 253) + 37.8 4 -keto 0. 012 (0. 006- 0 .015) 0. 017*(0. 013- 0. 020) + 41.7 5 -OH 0. 149 (0. 062- 0 .266) 0. 258*(0. 160- 0. 376) + 73.2 2 -PSA 0. 002 (0. 004- 0 .003) 0. 003 (0. 002- 0. 006) + 50.0 2 -PGA 0. 052 (0 . 017- 0 .157) 0. 041 (0 . 029- 0. 058) -21.2 2 , 3 ' - d i e n e 0. 012 (0 . 006- 0 .024) 0. 014 (0 . 007- 0. 027) + 16.7 2 ,4-diene 0. 002 (0. 001- 0 .003) 0. 003M0. 002- 0. 004) + 50.0 a C l f ca lcu la ted by d i v i d i n g the amount of metaboli tes recovered in the urine over 12 h by VPA AUC. b x 10" 3 . * s i g n i f i c a n t l y d i f f e ren t from before CBZ value at p < 0.05. 166 4-ene VPA, 2-ene c i s VPA, 4-keto VPA and 5-OH VPA were s t a t i s t i c a l l y s i g n i f i c a n t . Mean metabolite c learances for these metabolites are shown in table 27. Mean metabolite clearances for 4-ene VPA, 2-ene c i s VPA and 5-OH VPA increased by 47.6, 27.4 % and 84.7 %, respect ive ly a f ter CBZ administrat ion with the increases for 4-ene VPA and 2-ene c i s VPA reaching s i g n i f i c a n c e . The mean metabolite clearances of the other metabolites decreased by 7.18 to 52.2 % af ter CBZ admin is t ra t ion . The f r a c t i o n metabolized of 3-ene VPA, 2-ene trans VPA, 2-PGA, and 2 ,3 ' -d iene VPA decreased 39.3, 25.0, 44.1, and 15.4 %, r e s p e c t i v e l y , a f ter CBZ administrat ion (table 28). The f rac t ion metabolized of the other metabolites increased fol lowing administrat ion of CBZ by 4.32 to 50 %. The changes observed in the f rac t ion metabolized for 2-ene c i s VPA, 2-ene trans VPA, and 4-ene VPA were s t a t i s t i c a l l y s i g n i f i c a n t . The f rac t ion metabolized of 2,4-diene VPA remained approximately the same before and af ter CBZ admin is t ra t ion . 167 Table 27. Mean metabolite metabolite clearances ( C l m , L / h ) a for the VPA metaboli tes before and a f ter CBZ admin is t ra t ion . Numbers in parentheses represent range (n=5). Metabol i te Before CBZ After CBZ % change 4-OH 1 . 602 (0. 780- 2.696) 1 .487 (0. 618- 2. 186) -7.18 4-ene 0. 063 (0. 027- 0.143) 0.093* (0. 041- 0.207) + 47.6 3-ene 0. 046 (0. 009- 0.175) 0.024 (0. 013- 0.044) -47.8 2-ene c i s 0. 248 (0. 097- 0.397) 0.316*(0. 183- 0.458) + 27.4 2-ene trans 0. 107 (0. 044- 0.204) 0.097 (0. 053- 0.155) -9.35 3-keto 1 . 427 (0. 083- 4.379) 1 .369 (0. 738- 1.671) -4.06 4-keto 2. 482 (1. 333- 3.719) 2.188 (1. 517- 2.718) -11.8 5-OH 8. 193 (2. 453- 19.02) 15.13 (3. 378- 46.86) + 84.7 2-PSA 1 1 .53 (4. 692- 26.99) 10.43 (3. 858- 13.69) -9.54 2-PGA 32 .05 (9. 895- 93.95) 15.31 (11 .98- 16.46) -52.2 2 ,3 ' -d iene 0. 375 (0. 131- 0.635) 0.255 (0. 114- 0.326) -32.8 2,4-diene 0. 231 (0. 122- 0.330) 0. 197 (0. 132- 0.273) -14.7 * s i g n i f i c a n t l y d i f f e ren t from before CBZ value at p < 0.05. a C l m ca lcu la ted by d i v i d i n g the amount of metaboli tes recovered in the urine over 12 h by i t s corresponding AUC va lue . 168 Table 28. Mean f rac t ion ( f m ) a of metabolite metabolized before and af ter carbamazepine admin is t ra t ion . Numbers in parentheses represent range (n=5). Metabol i te Before CBZ After CBZ % change 4- OH 0. 081 (0. 061 - 0. 100) 0. 093 (0. 058- 0 .152) u + 14 .8 4- ene 0. 449 (0. 273- 0. 907) b 0. 648* (0. 359- 1 .346) b + 44 .3 3- ene 0. 168 (0. 060- 0. 518)^ 0. 102 (0. 057- 0 .138) b -39 .3 2- ene c i s 0. 403 (0. 199- 0. 704) b 0. 452*(0. 270- 0 .744) b + 12 .2 2- ene trans 0. 028 (0. 017- 0. 037) 0. 021* (0. 014- 0 .026) -25 .0 3- keto 0. 139 (0. 009- 0. 371 ) 0. 145 (0. 074- 0 .237) + 4. 32 4- keto 0. 012 (0. 007- 0. 017) 0. 014 (0. 008- 0 .018) + 16 .7 5- OH 0. 165 (0. 085- 0. 302) 0. 209 (0. 142- 0 .352) + 26 .7 2- PSA 0. 002 (0. 001- 0. 003) 0. 003 (0. 002- 0 .005) + 50 .0 2- PGA 0. 059 (0. 022- 0. 184) 0. 033 (0. 023- 0 .047) -44 . 1 2, 3 ' -d iene 0. 013 (0. 009- 0. 019) 0. 01 1 (0. 006- 0 .016) -15 .4 2, 4-diene 0. 002 (0. 001- 0. 003) 0. 002 (0. 002- 0 .003) 0. 00 a f m i s ca lcu la ted by d i v i d i n g C l f by C l p . b x 10" 3 . * s i g n i f i c a n t l y d i f f e ren t 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 spectrometric assay developed in our laboratory was used for the ana lys is of VPA and metabolites (Abbott et a l . , 1986a). With t h i s assay, i t was poss ib le to simultaneously quant i tate VPA and 14 metaboli tes in urine and serum using the se lected ion monitoring mode. B r i e f l y , the method employs a c a p i l l a r y column and the extracted samples may be der iva t i zed to tBDMS, methyl, or TMS d e r i v a t i v e s . The tBDMS der iva t i ves were used in t h i s study. Several attempts were made to further improve the assay. This included invest iga t ion of four new in te rna l standards and two commercially ava i l ab le d e r i v a t i z i n g reagents. 1.1. Internal standards Due to problems with reproducible d e r i v a t i z a t i o n of the two d i a c i d metabol i tes , 2-PSA and 2-PGA, a new in terna l standard, 2-methylglutar ic a c i d , was invest iga ted . MGA i s s t r u c t u r a l l y and chemical ly s imi la r to these two metabolites and the r e p r o d u c i b i l i t y in the standard curves was great ly enhanced with i t s use. For eight serum standard curves, the r e l a t i v e standard dev ia t ion values were 5.28 % and 6.76 %, respec t ive ly , for 2-PSA and 2-PGA. The r e l a t i v e standard deviat ion values 1 70 for eight urine standard curves were 9.41 % and 9.32 %, r e s p e c t i v e l y , for 2-PSA and 2-PGA. MGA has been used as an in te rna l standard by other workers in the ana lys is of VPA and metabolites (Granneman et a l . , 1984a). While the r e p r o d u c i b i l i t y in the standard curves of the two d i a c i d metabol i tes , 2-PSA and 2-PGA, was great ly enhanced with the addi t ion of MGA as an in terna l standard, 3-keto VPA remains an enigma. It may be necessary to use a deuterated analogue of 3-keto VPA as an in terna l standard in order to increase the s e n s i t i v i t y and r e p r o d u c i b i l i t y of the assay for th is p a r t i c u l a r metabol i te . Octanoic ac id was invest igated as an in terna l standard for use in the ana lys is of samples containing metaboli tes of hexadeuterated VPA. However, octanoic ac id d id not resolve from the deuterated analogue of 2 ,3 ' -d iene VPA. Subsequently, two other in terna l standards, hexanoic ac id and d i - n -buty lace t ic a c i d , were invest igated for th is purpose and were found not to in te r fe re with any of the metaboli tes being measured. As a resu l t of these i n v e s t i g a t i o n s , D3~2-ene, Dg-VPA, 3-octanone, octanoic a c i d , and 2-methylglutar ic ac id were used as in te rna l standards in the ana lys is of samples containing VPA and i t s metabol i tes . Hexanoic a c i d , d i - / i - b u t y l a c e t i c a c i d , and 3-octanone were used as in terna l standards for the ana lys is of b i o l o g i c a l samples containing hexadeuterated VPA and i t s metabol i tes . 171 1.2. Choice of d e r i v a t i z i n g reagent Der iva t i za t ion of the urine or serum samples was required for GCMS a n a l y s i s . S i l y l a t i o n react ions are the most v e r s a t i l e for t h i s purpose and are app l icab le to a large number of funct iona l groups inc luding hydroxyl , carboxy l , and amino groups (Merr i t t and McEwen, 1980). The most commonly used der iva t ives are t r i m e t h y l s i l y l (TMS) and t e r t -b u t y l d i m e t h y l s i l y l (tBDMS) d e r i v a t i v e s . TMS and tBDMS der iva t ives of VPA and metabolites in ped ia t r i c pat ient urine samples were compared with respect to react ion time, chromatographic time, s e n s i t i v i t y , and s t a b i l i t y . TMS der iva t ives are e a s i l y prepared, have low p o l a r i t y , and have abundant molecular ions with a r e l a t i v e l y intense [M-15] + peak. The TMS group d i r e c t s the fragmentation pattern but i s very suscept ib le to h y d r o l y s i s . The tBDMS der iva t ives are ten times more stable and have longer retent ion times due to a larger mass. The mass spectra of these der iva t ives are a lso less complex. A prominent and intense [M-57] + i s formed by the loss of the t-butyl group from the molecular i o n . Although TMS der iva t ives require a shorter d e r i v a t i z a t i o n time, the i r decreased s t a b i l i t y compared to tBDMS der iva t ives was a major drawback. A l s o , TMS der iva t ives proved to be less sens i t i ve for some of the metabolites being measured. The tBDMS der iva t ives y ie lded greater s e n s i t i v i t y for a l l metabolites except for 3-keto VPA and t h i s is important 172 as some VPA metabolites are present in trace amounts. These r e s u l t s were s imi la r to those obtained for long chain fat ty ac ids (Woollard, 1983). A new commercially ava i l ab le tBDMS d e r i v a t i z i n g reagent, MTBSTFA, which is more react ive than the reagent prepared in our laboratory was a lso invest igated for use as an a l ternate d e r i v a t i z i n g reagent. MTBSTFA requires shorter d e r i v a t i z a t i o n times but due to the formation of the 3-keto VPA d ider iva t i ve upon storage and on longer heating times, th is reagent was not considered a su i tab le replacement for the tBDMCS in pyr id ine reagent with 5 % c a t a l y s t . 1.3. Stable isotopes in pharmacokinetic studies Stable istopes such as carbon-13, deuterium, n i t rogen-15, and oxygen-17 and 18 have had increased usage over the l as t decade due to the development of more sophis t ica ted a n a l y t i c a l too ls such as gas chromatography-mass spectrometry (GCMS). Stable isotopes do not have the same r e s t r i c t i o n s as rad ioact ive isotopes in human s tud ies . Stable isotope l abe l l ed compounds may be administered to neonates, c h i l d r e n , a d u l t s , and to pregnant women. Oxygen and nitrogen do not have rad ioact ive equivalents of s u f f i c i e n t l y long h a l f - l i v e s for use in metabolism s tud ies . In pharmacokinetic s tud ies , stable isotope labe l l ed compounds may be used as in terna l standards with deuterium being the most commonly used stable isotope for t h i s purpose. 173 The assay used in t h i s study has two deuterated in terna l standards, Dg-VPA and D 3 -2-ene VPA (Abbott et a l . , 1986b). An analogue of VPA containing 14 deuterium atoms has a lso been used as an in terna l standard (von Unruh et a l . , 1980). The k i n e t i c s of a c h r o n i c a l l y administered drug may be determined by administer ing a l a b e l l e d analogue as a s ing le "pulse" dose as in the example of VPA (Acheampong et a l . , 1984). In t h i s study, hexadeuterated VPA was administered as a s ing le dose to a volunteer at steady state on VPA. Hexadeuterated VPA was found to be k i n e t i c a l l y bioequivalent to VPA. Other pharmacokinetic studies of VPA have used a tetradeuterated analogue (von Unruh et a l . , 1980) and mono- and heptadeuterated analogues (Kochen et a l . , 1982) which a lso have been found to be k i n e t i c a l l y equivalent to VPA. Hexadeuterated VPA was to be tested in the volunteers p a r t i c i p a t i n g in th is VPA+CBZ in te rac t ion study in preparat ion for ult imate use of the stable isotope l abe l l ed analogue in pharmacokinetic studies of VPA in ped ia t r i c pa t i en ts . Thus, pharmacokinetic parameters of VPA could be determined in pat ients without d iscont inuing the drug. The s e n s i t i v i t y of the present assay did not permit using a s ing le pulse dose of the deuterated drug and fol lowing the dec l ine of metabolites from serum. An a l te rna t ive procedure was to fol low the decl ine of un labe l led VPA and metabolites over 48 h while maintaining dosing every 12 h with hexadeuterated VPA. The s e n s i t i v i t y of the assay was improved 174 since the metabolites were at "steady state" l e v e l s . This was successfu l in the one volunteer who was administered two doses of Dg-VPA in the f i r s t part of the study (control ) and four doses in the second part of the study ( induced). Because mult ip le dosing of hexadeuterated VPA would be a very expensive procedure for use in pa t ien ts , pulse dosing may be the preferred method once the development of a more sens i t i ve a n a l y t i c a l technique such as negative ion chemical ion iza t ion becomes ava i lab le to assay VPA and metabol i tes. It was d i f f i c u l t to analyze deuterated metabolites in the presence of unlabel led metabol i tes, e s p e c i a l l y in the case of deuterated 4-ene VPA and VPA. In th is study, the concentrat ions of the unlabel led metabolites were higher than the l abe l l ed metabolites since steady state was not achieved with the deuterated drug. In a d d i t i o n , unlabel led and l a b e l l e d metabolites cannot be measured simultaneously due to inter ference by the in terna l standards used in the assay for VPA and metabol i tes. The deuterated 4-keto VPA undergoes exchange of three deuterium atoms for three hydrogen atoms in the workup procedure. Since steady state concentrat ions of hexadeuterated VPA were not achieved in e i ther the cont ro l or induced s ta te , i t was d i f f i c u l t to analyze the e f fec t of CBZ on D^-VPA metabolism. However, an isotope e f fec t in the metabolic p r o f i l e was observed as shown in table 1 with approximately 66 % lower serum trough concentrat ions of l a b e l l e d 4-ene VPA and 175 2,4-diene VPA occurr ing a f te r administrat ion of hexadeuterated VPA. The serum trough concentrat ions of the other l abe l l ed metaboli tes af ter hexadeuterated VPA administrat ion were comparable to the concentrat ions of unlabel led metabolites fo l lowing VPA admin is t ra t ion . B. EFFECT OF CARBAMAZEPINE ON VALPROIC ACID METABOLISM CBZ induces the metabolism of VPA in healthy subjects and in e p i l e p t i c pat ients (Levy and P i t l i c k , 1982). In e p i l e p t i c pat ients on VPA and CBZ, steady state VPA l e v e l s were 37 to 64 % lower than predicted from s ingle dose studies of VPA. The r a t i o of VPA plasma concentrat ion to VPA dose was 38 % lower in the presence of CBZ compared to a monotherapy group in adult p a t i e n t s . In one volunteer study, CBZ caused increased VPA clearance from the body accompanied by decreased steady state plasma concentrat ions (Bowdle et a l . , 1979). The e f fec t of CBZ on VPA metabolism does not resul t from competit ion for prote in binding s i t e s . CBZ plasma leve ls are low compared to the amount of albumin present , and thus, i t should not act as a d i s p l a c i n g agent (Levy and P i t l i c k , 1982). As w e l l , the assoc ia t ion constant between CBZ and albumin is not very h igh . 1. Inducing propert ies of carbamazepine CBZ i s known to induce the metabolism of other compounds as well as i t s own metabolism. Repeated administrat ion of CBZ 176 enhances i t s own e l iminat ion (Faigle and Feldmann, 1982). Serum steady state l eve ls predicted from s ing le dose studies are usual ly two to three times higher than the actual values observed in chronic CBZ therapy (Rawlins et a l . , 1975; Moreland et a l . , 1982). At doses of 300-1200 mg/day, CBZ w i l l induce i t s own metabolism af ter 60 days (Greim, 1981). CBZ steady state serum l e v e l s progress ive ly decl ine and the serum h a l f -l i f e shortens for a few weeks a f ter i n i t i a t i o n of therapy (Perucca and Richens, 1982). CBZ induces the microsomal monooxygenase system (cytochrome P-450 and NADPH-cytochrome c reductase) and s l i g h t l y induces UDP-glucuronyltransferase (Faigle and Feldmann, 1982). The induction pattern of CBZ is s imi la r to phenobarbital at equimolar doses. The e f fec ts of CBZ, however, are less d r a s t i c . CBZ induces the metabolism of war far in , an anticoagulant agent (Ser l in and Breckenridge, 1983; Ross and Beeley, 1980; Hansen et a l . , 1971). Decreases in warfarin h a l f - l i f e , plasma l e v e l s , and hypoprothrombin a c t i v i t y are observed (Ross and Beeley, 1980). A 56 year o ld man was s t a b i l i z e d on 6 mg/day of warfarin (prothrombin time 2 - 3 times control ) and CBZ for t r igeminal neuralgia (Ross and Beeley, 1980). Af ter d iscont inuat ion of CBZ therapy, the prothrombin time increased to f ive times the cont ro l va lue . Af ter the dose of warfarin was decreased to 4 mg/day, the prothrombin time returned to two to three times c o n t r o l . 177 CBZ st imulates the metabolism of phenytoin in pat ients (Hansen et a l . , 1971). Serum h a l f - l i f e of phenytoin in one study decreased s i g n i f i c a n t l y from 10.6 to 6.4 h a f ter CBZ admin is t ra t ion . A d a i l y dose of 600 mg CBZ resu l ts in increased metabolism of phenytoin within 4 to 14 days of comedication and the e f fec t p e r s i s t s for 10 to 21 days af ter CBZ i s withdrawn (Greim, 1981). CBZ a lso induces the metabolism of theophyl l ine through an unknown mechanism. The addi t ion of CBZ to the theophyl l ine regimen of an asthmatic resul ted in subtherapeutic theophyl l ine concentrat ions, loss of asthma c o n t r o l , and a decreased serum h a l f - l i f e of 2.75 h in three weeks (Rosenberry et a l . , 1983). Discont inuat ion of CBZ resul ted in cont ro l of the c h i l d ' s asthma and an increase in theophyl l ine h a l f - l i f e to 6.50 h. Pat ients treated with CBZ el iminate ant ipyr ine at a faster rate and excrete larger amounts of D-g lucar ic ac id compared to cont ro ls (Perucca et a l . , 1984b). When measuring induction of ant ipyr ine c learance, CBZ has 84 % of the potency of phenobarbital (Perucca et a l . , 1984). At doses of 400 - 600 mg/day of CBZ in humans, ant ipyr ine metabolism increased af ter 80 - 190 days (Greim, 1981). In nine newly diagnosed e p i l e p t i c pat ients between the ages of 6 to 14 years , mean ant ipyr ine clearance increased from 65 to 143 mL/kg/h during f i ve weeks of CBZ therapy (Moreland et a l . , 1982). The h a l f - l i f e of ant ipyr ine decreased from 6.24 + 1.23 h to 2.78 + 0.59 h, and 178 urinary 6 -beta-hydroxycort iso l excret ion increased from 5.10 + 1.77 ng/day/kg to 17.85 + 6.75 ng/day/kg. In e p i l e p t i c p a t i e n t s , CBZ administrat ion resu l ts in a shortened h a l f - l i f e of doxycycl ine so that one dose/day administrat ion is not s u f f i c i e n t (Neuvonen et a l . , 1975). A d a i l y dose of 200 mg of CBZ increased the metabolism of clonazepam a f te r only four days (Greim, 1981). When volunteers who had at ta ined steady state concentrat ions of clonazepam were administered CBZ 200 mg d a i l y , decreased clonazepam serum l e v e l s and h a l f - l i v e s were observed (Lai et a l . , 1978). The mean h a l f - l i f e of clonazepam decreased from 32.1 + 16.6 h to 22.5 + 11.5 . Clonazepam serum l e v e l s decreased by 19 to 37 %. An increase of two to four f o l d was a lso observed in the urinary excret ion of D-g lucar ic ac id fol lowing CBZ admin is t ra t ion , thus ind ica t ing the occurrence of enzyme induct ion . 2. Volunteer data 2 .1 . CBZ and CBZE concentrat ions in serum Carbamazepine was administered at a dose of 200 mg d a i l y for the f i r s t week and increased to 300 mg d a i l y for the second week. The dose of 200 mg d a i l y is the recommended s tar t ing dose by the manufacturer. In order to ascer ta in that therapeutic concentrat ions of CBZ had been achieved in the study, serum samples were analyzed for both CBZ and CBZE. Af ter one week of d a i l y doses of 200 mg CBZ, serum 179 concentrat ions of CBZ for the f i ve volunteers ranged from 2.37 - 3.68 nq/mL (10.0 - 15.6 ^mol/mL) as shown in table 4. Serum CBZ concentrat ions for three of the f i v e volunteers were not within the therapeutic range of 3 - 14 jug/mL (Goodman et a l . , 1980) or 12 - 50 Mmol/mL (Elyas et a l . , 1982). Af ter two weeks of CBZ therapy, where the dose of CBZ had been increased to 300 mg d a i l y for the second week, serum CBZ values (3.07 - 4.53 ug/mL or 13.0 - 18.4 /umol/mL) for a l l f i ve volunteers were in the lower region of the therapeutic range. CBZ concentrat ions of 4.6 - 9.4 Atg/mL (Agbato et a l . , 1986) and 2.7 - 10.8 ng/mL (Brodie et a l . , 1983) have been reported in e p i l e p t i c pat ients on CBZ monotherapy. CBZ concentrat ions of 5.0 - 10.3 ng/mL (Brodie et a l . , 1983) and 7.05 - 1 3.59 /ug/mL in pat ients on CBZ and VPA comedication have a lso been observed (Schoeman et a l . , 1984b). CBZE serum concentrat ions are approximately 10 - 50 % of the parent drug concentrat ions (Elyas et a l . , 1982). The concentrat ions of CBZE in th is study ranged from 0.38 - 0.85 ug/mL (1.49 - 3.38 Mmol/mL) a f te r one week of CBZ administ rat ion and 0.51 - 1.20 ug/mL (2.02 - 4.75 /xmol/mL) a f ter two weeks of CBZ administrat ion as summarized in table 5. Concentrat ions of CBZE ranging from 0.9 - 2.1 jig/mL have been observed in e p i l e p t i c pat ients (Agbato et a l . , 1986). CBZE concentrat ions of 0.75 - 2.6 ng/mL have been reported when CBZ and VPA were coadministered in e p i l e p t i c pat ients (Brodie et a l . , 1983). Although the concentrat ions of the epoxide 180 metabolite observed in t h i s study were lower than the values observed in pa t i en ts , they s t i l l represent 10 - 50 % of the parent drug concentrat ions as shown by the percent r a t i o of CBZE to CBZ in table 6. The ra t io between CBZE and CBZ concentrat ions in table 6 is important as i t may serve as an ind icator of induct ion . Af ter one week of CBZ admin is t ra t ion , the ra t io ranged from 15.1 -28.0 % and a s imi la r range of 15.3 - 27.7 % was observed af ter two weeks of CBZ. These values are s l i g h t l y lower than the reported ra t ios of CBZE to CBZ of 17.44 - 42.76 % in e p i l e p t i c ch i ld ren on both VPA and CBZ (Schoeman et a l . , 1984a). Af ter the f i r s t week of CBZ admin is t ra t ion , three of the f i ve volunteers were within the r a t i o range reported for e p i l e p t i c ch i ld ren on VPA and CBZ, while only one of the f ive volunteers was within t h i s range a f te r two weeks of CBZ admin is t ra t ion . Thus, although the concentrat ions of CBZ and CBZE in the volunteers were s l i g h t l y lower than t y p i c a l l y found in p a t i e n t s , the CBZ epoxide to CBZ r a t i o was more or less consis tent with reported va lues . 2 .2. VPA data 2 . 2 . 1 . Protocol Healthy male volunteers were chosen to p a r t i c i p a t e in th is study although females were not i n t e n t i o n a l l y excluded. Volunteers were required to complete and pass a medical examination and blood chemistry ana lys is before being allowed 181 to p a r t i c i p a t e in the study. They were asked to abstain from a l c o h o l , smoking, and any other medications for the durat ion of the study. This study was i n i t i a l l y designed so that the k i n e t i c s of Dg-VPA could a lso be obtained in the absence and presence of CBZ. However, th is was performed in only one volunteer due to a l im i ted supply of the deuterated drug. On the study days, only the morning dose(s) of VPA or VPA and CBZ was(were) administered and then dosing was discont inued for 48 h. The dec l ine of VPA and i t s metabolites in the serum and accumulation in urine was followed over 48 h in order to more accurate ly determine the k ine t ic parameters. Although samples were c o l l e c t e d over a 48 h pe r iod , many of the c a l c u l a t i o n s , for example AUC, are based on the dosing in te rva l of 12 h. I n i t i a l l y , a dose of 20 mg/kg/day in two d iv ided doses of VPA was chosen, but untoward symptoms were observed in one volunteer who subsequently withdrew from the study. Of the f ive volunteers who completed the study, one volunteer received 20 mg/kg/day while four volunteers received 15 mg/kg/day. This dose is the recommended s ta r t ing dose by the manufacturer, although much higher doses are ac tua l l y administered in p a t i e n t s . 2 .2 .2 . Serum VPA data From the serum data summarized in table 7 for the f ive volunteers in th is study, the e f fec t of CBZ on VPA metabolism 182 was quite obvious. Plasma clearance of VPA was great ly enhanced as was the e l iminat ion rate constant a f ter CBZ admin is t ra t ion . Serum h a l f - l i f e of VPA decreased s i g n i f i c a n t l y a f ter CBZ comedication. The decreases in steady state serum VPA concentrat ions and AUC values a f ter CBZ administrat ion were a lso s i g n i f i c a n t . Steady state concentrat ions of 56.25 + 10.15 mg/L before CBZ and 39.64 + 9.79 mg/L a f ter CBZ administrat ion were obtained by d i v i d i n g the AUC value by 12 h. Serum steady state VPA concentrat ions of 55.9 + 3.0 mg/L af ter s ing le 600 mg doses have been reported for volunteers by Loscher (1978). Values of 34.2 + 7.9 mg/L (Pollack et a l . , 1986) and 34.4 + 5.1 mg/L af ter 250 mg VPA twice d a i l y (Bowdle et a l . , 1979), and 107.5 + 8.1 mg/L a f te r a s ingle 1000 mg dose (Bia ler et a l . , 1985) have a lso been reported. Trough serum concentrat ions of 44.02 + 16.65 mg/L before CBZ and 27.01 + 10.37 mg/L a f ter CBZ administrat ion were obtained. Trough concentrat ions of VPA in p e d i a t r i c pat ients on VPA monotherapy ranged from 11.8 - 105 mg/L (Abbott et a l . , 1986a) . AUC values as shown in table 7 for VPA decreased from 675.0 + 130.5 mg.h/L to 475.7 + 75.73 mg.h/L a f ter CBZ admin is t ra t ion . AUC values of 726.6 + 62.0 mg.h/L, and 531.0 + 114.5 mg.h/L in volunteers have been reported for VPA in the absence of any other drugs (Perucca et a l . , 1984a; B ia le r et a l . , 1985). 183 The VPA serum h a l f - l i f e values of 14.03 + 2.374 h obtained with VPA monotherapy in the volunteers are in agreement with l i t e r a t u r e values of 13.0 + 1 . 0 h (Perucca et a l . , 1984a), 14.86 + 2.36 h (Bia ler et a l . , 1984b) and 14.9 + 2.4 h (Bialer et a l . , 1985) in vo lunteers . In ped ia t r i c p a t i e n t s , values of 11.83 + 4.02 h (Cloyd et a l . , 1985), 8.5 + 1.6 h (Hal l et a l . , 1985), and 10.4 + 2.7 h (Orr et a l . , 1982) have been observed in VPA monotherapy. The values for VPA h a l f - l i f e in our volunteers af ter CBZ adminis t ra t ion were 10.56 + 1.392 h. In p e d i a t r i c pa t ien ts , h a l f - l i f e values of 7.10 + 1.77 (Otten et a l . , 1984) and 7.5 + 1.6 h have been observed in the presence of other a n t i e p i l e p t i c drugs (Cloyd et a l . , 1985). Our h a l f - l i f e values in volunteers were s l i g h t l y higher than those observed in pa t i en ts . The h a l f - l i f e of VPA a f ter administrat ion of ASA in ped ia t r i c pat ients was reported to be 12.9 + 1.8 h (Orr et a l . , 1982). Clearance values of 0.897 + 0.184 L/h (12.46 + 2.56 mL/h/kg) before CBZ administrat ion and 1.263 + 0.241 L/h (17.54 + 3.35 mL/h/kg) a f ter CBZ administrat ion were obtained and are summarized in table 7. These values are in agreement with l i t e r a t u r e values of 14.95 + 2.86 mL/h/kg (Cloyd et a l . , 1985) and 16.2 + 6.6 mL/h/kg (Hal l et a l . , 1985) for VPA monotherapy. In p e d i a t r i c pat ients on VPA monotherapy, mean clearance values of 22 mL/h/kg have been observed ( F a r r e l l et a l . , 1982). In two volunteers administered s ing le doses of VPA (600 mg), c learance values of 0.418 L/h and 0.664 L/h were obtained 184 (Abbott et a l . , 1982). L i te ra tu re values for VPA clearance when coadministered with other ant iconvulsant agents were 26.74 + 10.43 mL/h/kg (Cloyd et a l . , 1985) and 18.0 + 3.6 mL/h/kg (Otten et a l . , 1984) in p e d i a t r i c pa t i en ts . The volume of d i s t r i b u t i o n values obtained for VPA in volunteers on monotherapy of 0.255 + 0.03 L/kg (table 7) were in agreement with reported l i t e r a t u r e values of 0.25 + 0.06 L/kg (Cloyd et a l . , 1985), 0.216 + 0.86 L/kg (Pollack et a l . , 1986), and 0.192 + 0.049 L/kg (Hal l et a l . , 1985). Vd values obtained in our volunteer study a f ter CBZ administrat ion were 0.263 + 0.020 L/kg and were in agreement with reported data. Values reported in the l i t e r a t u r e for Vd of VPA in the presence of other ant iconvulsants were 0.26 + 0.06 L/kg (Cloyd et a l . , 1985) and 0 . 1 8 9 + 0.038 L/kg (Otten et a l . , 1984). The e l iminat ion rate constant for VPA increased from 0.051 + 0.011 to 0.067 + 0.011 h _ 1 as shown in table 7. In the previous study invest iga t ing t h i s i n t e r a c t i o n , the Ke value decreased from 0.0623 + 0.0168 to 0.0573 + 0.0168 h" 1 (Bowdle et a l . , 1979). These authors suggested that th is in te rac t ion was based on changes in the VPA volume of d i s t r i b u t i o n , poss ib ly by CBZ d i s p l a c i n g VPA from i t s plasma prote in binding s i t e s on albumin and thus increasing the amount of free drug ava i l ab le for metabolism. However, Mattson and coworkers (1980; 1982) have shown that jun v i t r o , CBZ does not d isplace VPA from i t s protein binding s i t e s . The current volunteer study supports these resu l ts with volume of d i s t r i b u t i o n of VPA 185 increasing only s l i g h t l y fol lowing CBZ comedication. These pharmacokinetic r e s u l t s highly suggest that the in te rac t ion between VPA and CBZ is based on enzyme induction rather than competit ion for prote in binding s i t e s . VPA i s not known to induce i t s own metabolism (Rimmer and Richens, 1985). The r e s u l t s obtained may d i f f e r from those of Bowdle and coworkers for several reasons: the dose of VPA in the i r study was much lower (mean dose 500 mg/day versus 1181 mg/day); volunteers were not a l l of the same sex; b i o l o g i c a l samples were only c o l l e c t e d over 12 h; a l ess sens i t i ve GC assay was used; CBZ comedication was i n i t i a t e d a f ter four days of VPA dosing; and CBZ was administered at a dose of 200 mg once d a i l y throughout the study. The values for Ke and Vd are approximations and may not be r e l i a b l e . 2 .2 .3 . Serum metabolite data Unlike the in terac t ion between VPA and ASA, where only beta-oxidat ion of VPA was a f fected by the addi t ion of ASA to the dosing regimen (Abbott et a l . , 1986b), th is is not the case with CBZ. From the serum metabolite p r o f i l e s , i t appears that a l l VPA metabolic pathways are inf luenced by CBZ coadmin is t ra t ion . 2 . 2 . 3 . 1 . Metabol i te serum concentrat ions Serum trough concentrat ions for VPA metabolites are summarized in table 8. For many of the metabol i tes, serum 186 concentrat ion values have prev iously not been reported in vo lunteers . Serum concentrat ions for some VPA metabolites have not been reported by any laboratory other than our own. A comparison of our volunteer data to pat ient l i t e r a t u r e data i s summarized in table 29. The lack of publ ished data with respect to VPA metabolites in the serum is obvious as there are only two studies for comparison and one of the studies is from t h i s laboratory . Despite the small number of volunteers in th is in te rac t ion study, the volunteer resu l ts are general ly consistent with resu l ts from two pat ient s tud ies . The concentrat ions of 4-OH VPA and 2-ene trans VPA are higher in volunteers than in p e d i a t r i c pat ients (Abbott et a l . , 1986b) although they are in the range reported by Loscher (1981a) for e p i l e p t i c p a t i e n t s . Mean 3-keto VPA concentrat ions in volunteers are higher than observed in p e d i a t r i c pat ients although, aga in , the range of values i s consistent with 3-keto VPA resu l ts found in the Loscher study (1981a). Serum steady state metabolite concentrat ions were ca lcu la ted by d iv id ing the AUC value by 12 h and are shown in table 13. Table 30 is a comparison of these resu l ts with a recent ly reported volunteer study (Pollack et a l . , 1986). Concentrations of three of the seven metabolites which could be compared were higher in our study. This may be a r e f l e c t i o n of the d i f f e ren t assays since our r e s u l t s were obtained with a GCMS assay and the reported resu l ts were obtained with a GLC assay. In a d d i t i o n , the dose of VPA in our study was 187 Table 29. Comparison of serum VPA and metabolite concentrat ions (nq/mh) in volunteers with patient data . Numbers in parentheses represent range. Compound Volunteers 3 P e d i a t r i c " E p i l e p t i c Pat ients Pat ients (n=5) (n=34) (n = 26) 4-OH 2.69 (1 .02-4.68) 0.38 (0.01-1.78) 0.70 (0.30- 3.50) 4-ene 0.39 (0.28-0.48) 0.67 (0.16-1.22) N.R. 3-ene 0.30 (0.15-0.51) 0.94 (0.25-1.86) N.R. 2-ene c i s 0.08 (0.06-0.10) 0.19 (0.06-0.40) N.R. 2-ene trans 15.2 (8.04-20.5) 5.53 (0.95-11.3) 6.40 (3.BO- 18.0) VPA 44.0 (27.4-53.2) 46.4 (11.8-105.) 105. O S . 0 - 234. ) 3-keto 6.30 (4.50-8.15) 3.59 (0.29-15.6) 5.40 (1.40- 13.8) 4-keto 0.30 (0.21-0.39) 0.40 (0.01-1.29) N.R. 5-OH 1.01 (0.51-1.25) 0.18 (0.01-1.25) 1 .70 (0.50- 4.20) 2-PSA 0.01 (0.00-0.03) 0.04 (0.01-0.44) N.R. 2-PGA 0.09 (0.06-0.12) 0.20 (0.01-1.23) N.R. 2 ,3 ' -d iene 2.39 (0.85-3.62) 2.95 (0.50-7.29) N.R. 2,4-diene 0.56 (0.51-0.63) 0.20 (0.02-0.58) N.R. N.R. values not reported 3 mean dose 16.4 mg/kg/day, volunteer study b VPA monotherapy (Abbott et a l . , 1986a), mean dose 27.2 mg/kg/day c VPA monotherapy and combined with other ant iconvulsants (Loscher, 1981) Table 30. Comparison of mean serum steady state VPA and metabolite concentrat ions (uq/mL) from two volunteer s t u d i e s . Numbers in parentheses represent range. Compound Study 1 a (n=5) Study 2 b (n= 5) 4-OH 2.97 (1 .55-5.12) 0.92 (0.67-1. 45) 4-ene 0.38 (0.28-0.47) 0.62 (0.12-1. 44) 3-ene 0.29 (0.15-0.50) 0.19 (0.00-0. 64) 2-ene c i s 0.08 (0.06-0.10) N.R. 2-ene trans 14.9 (7.78-20.3) 4.07 (3.14-6. 01) VPA 56.3 (41.5-66.4) 34.2 (27.7-43 .4) 3-keto 5.42 (4.62-6.47) 2.55 (1.52-3. 73) 4-keto 0.25 (0.18-0.33) N.R. 5-OH 1.15 (0.92-1.65) 0.31 (0.00-0. 90) 2-PSA 0.01 (0.01-0.02) N.R. 2-PGA 0.09 (0.06-0. 1 1 ) N.R. 2 ,3 ' -d iene 2.10 (0.83-3.76) N.R. 2,4-diene 0.53 (0.31-0.67) N.R. a our study with metabolites measured by GCMS, mean dose 1181 mg/day b metabolites measured.using GLC (Pollack et a l . , 1986), mean dose 500 mg/day N.R. not reported 189 considerably h igher , with a mean dose of 1181 mg d a i l y versus 500 mg da i l y in the volunteer study by Pol lack (1986). 2 .2 .3 .2 . Metabolite AUC values Serum AUC values for the monounsaturated metabolites decreased a f ter CBZ administrat ion accompanied by increased AUC values for the polar metabol i tes . This ind icates that there is general o v e r a l l induction due to CBZ. The s i g n i f i c a n t increase in 4-keto VPA AUC values a f te r CBZ administrat ion suggests that the co-1 oxidat ion pathway is p a r t i c u l a r l y sens i t i ve to induction by CBZ. The decrease in AUC values for the monounsaturated metaboli tes may suggest that these pathways are inh ib i ted by CBZ admin is t ra t ion . However, the increase in the AUC values of 2 ,3 ' -d iene VPA and 2,4-diene VPA suggests enhanced metabolism of 3-ene VPA and 4-ene VPA to the secondary diene metabolites and thus could explain the decrease in the AUC values for these two monounsaturated metabol i tes . The decrease in AUC values for 2-ene trans VPA i s d i f f i c u l t to e x p l a i n . If further metabolism of 2-ene trans VPA was enhanced a f ter CBZ admin is t ra t ion , one would expect increases in AUC values for 3-keto VPA. Conversely, i f the beta-oxidat ion pathway is s i g n i f i c a n t l y blocked or inh ib i ted by CBZ admin is t ra t ion , one would expect s i g n i f i c a n t decreases in 3-keto VPA AUC values. However, the AUC values for 3-keto VPA were approximately the same before and af ter CBZ 190 admin is t ra t ion . It i s poss ib le that 2-ene trans VPA i s further metabolized to the d ienes, but the increase in the AUC values of the diene metabolites does not balance the sharp decrease in the AUC value for 2-ene trans VPA. A l t e r n a t i v e l y , 2-ene trans VPA may be metabolized to other metabolites not measured by t h i s assay or el iminated by some other organ. The decrease in 2-ene trans VPA AUC values may r e f l e c t the s h i f t to other oxidat ive pathways. The increase in the sum of the AUC values for the polar metaboli tes and the decrease in the sum of the AUC values for the unsaturated metaboli tes suggests further metabolism of 2-ene VPA, 3-ene VPA, and 4-ene VPA to secondary products . This would expla in the increase in serum AUC values for the two diunsaturated metabol i tes, 2 ,3 ' -d iene and 2,4-diene VPA. AUC values were separated into metabolic pathways to determine i f a p a r t i c u l a r pathway was s p e c i f i c a l l y induced. However, s ince one metabolite in the pathway may increase while another decreases, the net change would be minimal. Consequently, s i g n i f i c a n t changes were not observed in AUC values for the pathways af ter CBZ admin is t ra t ion . 2 .2 .4 . Urinary data The assay as out l ined prev iously measures the t o t a l amount of metabolite present and does not d i s t i n g u i s h between the conjugated and unconjugated metabol i te . 191 2 . 2 . 4 . 1 . Recovery expressed as a percent of dose Twelve hour ur inary recover ies were used for c a l c u l a t i o n purposes. The t o t a l recovery (VPA and metabolites) when expressed as a percent of the dose was 64.6 % before CBZ and 66.5 % a f ter CBZ admin is t ra t ion . Approximately 65 % recovery over 12 h in volunteers has been observed (Pollack et a l . , 1986). In the rhesus monkey, 82 % of the administered dose was recovered in the urine over 24 h (Rettenmeier et a l . , 1986a). Af ter administrat ion of carbon-14 l a b e l l e d VPA to ra ts , 70 % of the dose was recovered in the urine (Kuhara et a l . , 1974). Recovery of VPA and metabolites expressed as a percent of dose i s shown in table 19. A comparison of our resu l ts with another volunteer study i s summarized in table 31. Mean recovery of VPA as a percent of dose was 19.09 % before CBZ administrat ion and 18.18 % a f ter CBZ admin is t ra t ion . This is supported by resu l ts of 21.5 % in a steady state volunteer study (Pollack et a l . , 1986). This i s , however, lower than the 35.2 % recovery observed by Granneman and coworkers (1984b) a f te r a s ing le 1000 mg dose of VPA and much lower than the 61.1 % recovery in the rhesus monkey (Rettenmeier et a l . , 1986a). Of the six metabolites which can be compared, three of the s ix metabolites are reasonably c o n s i s t e n t . Our recoveries of 4-ene VPA and 3-ene VPA are considerably lower and th is may be a resu l t of the d i f fe rences in the assays. The reported values could be higher due to some inter ference in the chromatography from VPA. Our recovery of 5-OH VPA was considerably higher . 192 Table 31. Comparison of ur inary recovery of VPA and metabolites expressed as a percent of dose in two volunteer s tud ies . Numbers in parentheses represent range. Compound Study 1 a (n=5) Study 2 b (n=5) 4-OH 7.305 (5. 534-9.027) 5. 83 (1.93- 14.5) 4-ene 0.046 (0. 028-0.092) 0. 36 (0.00- 1 .80) 3-ene 0.017 (0. 006-0.053) 2. 78 (0.45- 6.64) 2-ene c i s 0.041 (0. 020-0.071) N.R. 2-ene trans 2.863 (1 . 704-3.705) 2. 82 (0.00- 8.31 ) VPA 19.09 (1 1 .44-32.17) 21 .5 (6.70- 44.7) 3-keto 12.68 (0. 842-33.78) 28 . 1 (17.8- 39.6) 4-keto 1 .092 (0. 605-1.527) N.R. 5-OH 14.87 (7. 622-27.22) 2. 26 (0.41- 8.00) 2-PSA 0. 196 (0. 134-0.281 ) N.R. 2-PGA 4.883 (1 . 786-15.21) N.R. 2 ,3 ' -d iene 1 .307 (0. 897-2.036) N.R. 2,4-diene 0.242 (0. 142-0.303) N.R. N.R. not reported a our volunteer study, mean dose 1181 mg/day, GCMS assay b mean dose 500 mg/day (Pollack et a l . , 1986), GLC assay 193 Approximately 14.5 % of the dose before CBZ and 14.2 % a f te r CBZ administrat ion was excreted as the VPA glucuronide conjugate. This i s in agreement with 15 - 20 % of the dose recovered as the glucuronide conjugate in a s ing le dose study (Bia ler et a l . , 1985). 2 .2 .4 .2 . Recovery expressed as a percent of t o t a l excreted Urinary recovery expressed as a percent of the t o t a l amount excreted over 12 h is shown in table 21. A comparison with ped ia t r i c pat ients is summarized in table 32. Our values for the d ienes, 4-OH VPA, 5-OH VPA, and 2-ene trans VPA were higher . The values for 4-ene VPA, 3-ene VPA, 4-keto VPA, VPA, and 2-PSA were lower. However, t h i s is a comparison between ped ia t r i c pat ients on VPA monotherapy and a volunteer steady state study. U l t imate ly , the doses in pat ients are higher , and as w e l l , one must consider the d i f fe rences in metabolism between these two age groups. 2 .2 .5 . E f f e c t of CBZ on VPA metabolism The percent of dose recovered in the urine as VPA and metabolites before (64.6 %) and af ter (66.5 %) CBZ administrat ion was almost the same. This is d i f f i c u l t to explain s ince the amount of VPA present in the serum decreased d r a s t i c a l l y . Recovery of the VPA glucuronide conjugate a lso d id not increase a f ter CBZ admin is t ra t ion . The decreased recovery of 2-ene trans VPA in the urine was consistent with 194 Table 32. Comparison of recovery of VPA and metaboli tes as a percent of the t o t a l amount recovered. Numbers in parentheses represent range. Compound Study 1 a (n=5) Study 2 b (n=7) 4-OH 12.7 (5.11-15.9) 5.00 (3.10-6.90) 4-ene 0.07 (0.05-0.08) 0.28 (0.09-0.47) 3-ene 0.02 (0.01-0.05) 0.08 (0.06-1.00) 2-ene c i s 0.06 (0.04-0.10) N.R. 2-ene trans 4.81 (2.55-7.27) 1 .51 (0.99-2.03) VPA 29.7 (22.4-40.4) 50.5 (37.9-63.1) 3-keto 18.5 (1.20-31 .2) 20.2 (10.5-29.9) 4-keto 1 .74 (1.16-2.13) 3.00 (1.50-4.50) 5-OH 22.0 (14.6-28.3) 3.10 (1.30-4.90) 2-PSA 0.31 (0.24-0.40) 1 .46 (0.00-4.36) 2-PGA 7.39 (3.14-21.6) 1 1 .9 (5.70-18.1) 2 ,3 ' -d iene 2.27 ( 1 .00-3.99) 0.67 (0.40-0.94) 2,4-diene 0.39 (0.27-0.53) 0.09 (0.03-0.15) N.R. not reported a volunteer study, mean dose 16.4 mg/kg/day b p e d i a t r i c p a t i e n t s , (Abbott et a l . , 1986b), 195 the decreased amounts present in the serum. This a lso poses a problem, since the amount of 3-keto VPA recovered in the urine expressed as a percent of dose d id not increase to balance the decrease in 2-ene trans VPA l e v e l s . In r a t s , phenobarbital pretreatment resul ted in increased excret ion of 2-ene VPA although 3-keto VPA recovery d id not increase s i g n i f i c a n t l y (Heinemeyer et a l . , 1985). The increase observed in the recover ies of various metabolites i s consistent with enhancement of metabolism except for the beta-oxidat ion pathway which at t h i s point i s an enigma. The recovery of 4-ene VPA, as well as 4-ene VPA glucuronide, increased a f te r CBZ administrat ion in the vo lunteers . As we l l , the increase in the recover ies of 4-keto VPA and 2-PSA in the urine is consistent with induction of the co-1 oxidat ion pathway. 2 .2 .6 . Metaboli te pathways Grouping metabolites into pathways was not extremely h e l p f u l s ince the recovery of one metabolite might increase while the recovery of a second metabolite decreased. Consequently, there were apparent changes although none were s t a t i s t i c a l l y s i g n i f i c a n t . The decrease in the recovery of pathway 1 (beta-oxidation) was unexpected, since the AUC values a lso decreased. It i s poss ib le that the metabolites in th is pathway are undergoing biotransformation to metabolites which are not considered in t h i s assay. However, in rats i t has been 196 reported that phenobarbital pretreatment r e s u l t s in increased excret ion of 4-OH VPA, 4-keto VPA, and 2-PSA due to induction of co-1 oxidat ion while the recoveries of other pathways decreased with the exception of 5-OH VPA, 4-ene VPA and VPA glucuronide conjugate (Granneman et a l . , 1984a). Glucuronidat ion of VPA decreased a f ter CBZ administrat ion as d id the recover ies of pathways 1 and 2, accompanied by increased recover ies of pathways 3, 4, and 5. These resu l ts are consistent with the r e s u l t s observed in rats (Granneman et a l . , 1984a). 2 .2 .7 . Pharmacokinetic ana lys is The clearance model was chosen since a l inear model with e l iminat ion from the cent ra l compartment seemed appropr ia te . As w e l l , the ra t io of clearance of metabolite to clearance of parent drug can be obtained from the r a t i o of steady state concentrat ions of metabolite to parent drug. The model was based on one used to decribe the in te rac t ion between CBZ and clobazam (Levy et a l . , 1983). 2 . 2 . 7 . 1 . Pathway ana lys is The formation rates for a l l metabolic pathways increased a f te r CBZ administrat ion (table 23) most l i k e l y due to induct ion of the hepatic microsomal monooxygenase system (Fa ig le and Feldmann, 1982). The increases ranged from approximately 19 % to 59 % with the increases in pathways 3, 5, 197 and 6 reaching s i g n i f i c a n c e . The increase in the microsomal pathways (co- and co-1 oxidat ion) was expected but the increased formation clearance of the beta-oxidat ion pathway was t o t a l l y unexpected. Beta-oxidat ion i s thought to occur mainly in the mitochondria although peroxisomal beta-oxidat ion is poss ib le (Devl in , 1986). A hepatic microsomal enzyme inducing agent could poss ib ly enhance the formation of peroxisomes and VPA i t s e l f may induce peroxisomal beta-oxidat ion (Horie and Suga, 1985). The formation clearance of the glucuronide conjugate increased af ter CBZ although the enzymes responsible for th is are only s l i g h t l y a f fected by CBZ (Faigle and Feldmann, 1982). Metabol i te clearances (table 24) for pathways 1 - 4 decreased af ter CBZ admin is t ra t ion , ind ica t ing a decrease in the amount of metabolites in these pathways being el iminated from the cent ra l compartment. The metabolite clearance of pathway 5 and of VPA ( to ta l + conjugate) increased a f te r CBZ admin is t ra t ion . However, a l l of these changes d id not reach s i g n i f i c a n c e . The f rac t ion metabolized (table 25) by each pathway, aga in , demonstrated changes which were not s t a t i s t i c a l l y s i g n i f i c a n t . The f r a c t i o n metabolized by pathways 1, 2, and 6 decreased a f te r CBZ administrat ion while the f rac t ion metabolized through pathways 3, 4, and 5 increased a f ter CBZ admin is t ra t ion . The formation clearance of pathway 1 increased a f te r CBZ administrat ion accompanied by a s l i g h t decrease in the metabolite c learance. This could explain the decreased 198 recovery in the u r ine . However, serum AUC values a lso decreased, so i t i s d i f f i c u l t to explain how these metabolites were e l iminated . The recover ies and metabolite clearances of pathways 2 and 3 decreased a f ter CBZ admin is t ra t ion , but increased amounts were present in the serum. In th is case, since less was e l iminated, serum concentrat ions for these pathways were increased. Although the metabolite clearance for pathway 4 decreased a f te r CBZ admin is t ra t ion , the urinary recovery increased. The decrease in the metabolite clearance for pathway 4 may explain the increased serum concentrat ions. Pathway 5 AUC values were increased in the serum af ter CBZ comedication, which is explained by i t s increased formation ra te . Increased amounts of pathway 5 metaboli tes were a lso recovered in the ur ine , supporting the increased metabolic ra te . The increase in serum AUC values suggests that the metabolite clearance d id not increase to the same degree as the formation c learance . It i s poss ib le that b i l i a r y or other organ e l iminat ion of VPA and metabolites was a lso enhanced. 2 .2 .7 .2 . Indiv idual metaboli tes The formation clearances (table 26) for a l l metabolites except 2-PGA and 3-ene VPA increased af ter CBZ admin is t ra t ion . The metabolite clearances ( table 27) for a l l metaboli tes except 4-ene VPA, 2-ene c i s VPA, and 5-OH VPA decreased af ter CBZ admin is t ra t ion . This i s consis tent with increased excret ion of 4-ene VPA and 5-OH VPA af ter phenobarbital pretreatment in rats 199 (Granneman et a l . , 1984a). The f rac t ion metabolized of each metabolite a f ter CBZ administrat ion decreased for 3-ene VPA, 2-ene trans VPA, 2-PGA, 2 ,3 ' -d iene VPA, and 2,4-diene VPA. It i s d i f f i c u l t to expla in these values at th is t ime. Although, the mean AUC value for 4-ene VPA decreased s l i g h t l y a f te r CBZ admin is t ra t ion , the amount recovered in the urine was increased cons iderab ly . The recovery of 4-ene VPA glucuronide conjugate was a lso increased af ter CBZ admin is t ra t ion . The formation clearance of 4-ene VPA s i g n i f i c a n t l y increased af ter CBZ administrat ion accompanied by an increased metabolite c learance. Phenobarbital pretreatment enhances formation of 4-ene VPA in r a t s , and poss ib ly CBZ does a lso (Rett ie et a l . , 1987). The metabolite clearance of 4-ene VPA was at least 100 f o l d greater than the formation c learance. Although the a n t i e p i l e p t i c e f f ec t of 2-ene VPA is stated to be shorter in duration than VPA (Keane et a l . , 1985), our resu l ts indicate that 2-ene c i s and trans VPA pers is t in the serum at least as long as the parent drug. Sustained plasma leve ls were a lso observed for 3-ene VPA, 4-ene VPA, 2 ,3 ' -d iene VPA and 2,4-diene VPA. The sustained plasma leve ls of the dienes may further support the theory that they a r ise from 2-ene VPA. It i s poss ib le that two weeks of CBZ administrat ion were not s u f f i c i e n t to cause maximal induct ion of the microsomal enzymes. However, i f the mechanism of induction of CBZ is s imi la r to that of phenobarbital (Faigle and Feldmann, 1982), 200 then two weeks of treatment with CBZ should be s u f f i c i e n t to cause maximal induct ion . CBZ 600 mg/day increased metabolism of phenytoin within 4 to 14 days (Greim, 1981). The dose used in our study was 200 mg d a i l y for one week and then increased to 300 mg d a i l y for the second week. Bowdle and coworkers reported that induction occurred within seven days of administrat ion of CBZ 200 mg/day in volunteers (1979). Clonazepam metabolism increased with CBZ doses of 200 mg/day within 4 days (Lai et a l . , 1978). 2 .2 .8 . C l i n i c a l s i g n i f i c a n c e of VPA and CBZ in te rac t ion The in terac t ion between VPA and CBZ i s of, c l i n i c a l importance since these two drugs are frequently coadministered in e f f o r t s to optimize seizure c o n t r o l . This combination may be p o t e n t i a l l y dangerous since some VPA metabolites are known to be hepatotoxic through i n h i b i t i o n of the fa t ty ac id beta-oxidat ion pathway. The poten t ia l hepatotoxin, 4-ene VPA, i n h i b i t s beta-oxidat ion of fa t ty ac ids by forming va lp roy l CoA esters (Kesterson et a l . , 1984). Further metabolism of 4-ene VPA v ia the beta-oxidat ion pathway may lead to formation of chemical ly react ive intermediates capable of a l k y l a t i n g c e l l u l a r macromolecules and forming t issue bound residues (Simula et a l . , 1985). Since CBZ induces the hepatic microsomal enzymes, i t was of importance to e s t a b l i s h i f the formation of 4-ene VPA was enhanced af ter CBZ admin is t ra t ion . It i s in te res t ing to note that although the formation of 4-ene 201 VPA was induced by CBZ admin is t ra t ion , so was i t s metabolite c learance . The t o x i c i t y of the 4-ene VPA glucuronide conjugate has not been determined. It has been determined in t h i s volunteer study that although CBZ induces the biotransformation of VPA to i t s metabolic byproducts, increased amounts of the po ten t ia l hepatotoxin 4-ene VPA were not present in the serum of vo lunteers . However, increased amounts of 2,4-diene VPA and 2 ,3 ' -d iene VPA were present in the serum of the volunteers a f te r CBZ. This could be of some importance, since these molecules may become act iva ted and cause t issue damage. The ur inary recover ies of the metabolites in some instances d id not support the serum data . This may be due to enhancement of b i l i a r y excret ion which was not measured. Unfor tunate ly , f i ve volunteers were not s u f f i c i e n t to determine th is complex in te rac t ion and further study must be done in order to accurate ly character ize th is i n t e r a c t i o n . It may be more informative to perform the study in pat ients rather than vo lunteers . 202 SUMMARY AND CONCLUSIONS 1. The assay for va lp ro ic a c i d and metabolites was further modif ied with respect to the in terna l standards used. Two new in te rna l standards, octanoic ac id and 2-methylglutar ic a c i d , were employed. Modi f ica t ions were made to the assay in order to quant i tate metabolites a f ter administrat ion of hexadeuterated VPA. Two new in terna l standards, hexanoic ac id and d i-/i -bu ty lace t i c a c i d , were used instead of the in te rna l standards used in the assay for VPA and metabol i tes. These two in te rna l standards were chosen as they d id not in te r fe re with the compounds being measured. 2. TMS (MSTFA) and tBDMS der iva t ives were compared with respect to s e n s i t i v i t y , s t a b i l i t y , and chromatographic t ime. Despite the fact that MSTFA required less d e r i v a t i z a t i o n time and 3-OH VPA i s more e a s i l y d e r i v a t i z e d , tBDMS esters were found to be superior in a l l respects . 3. A new, commercially ava i lab le tBDMS d e r i v a t i z i n g reagent (MTBSTFA) was tested as an a l te rna t ive to the reagent current ly used for the ana lys is of VPA and metabol i tes. MTBSTFA required l ess heating time for adequate d e r i v a t i z a t i o n . The der iva t ives formed were extremely stable for at least seven weeks. The major drawback was the formation of a d i d e r i v a t i v e of 3-keto VPA upon increased heating time and storage. 203 4. Prel iminary data on the metabolism of Dg-VPA was obtained from one volunteer . It was not poss ib le to obtain k inet ic data s ince steady state was not achieved in e i ther part of the study. However, serum concentrat ions, AUC va lues , and urinary recover ies were obtained. An isotope e f fec t occurred as i l l u s t r a t e d by the approximately 66 % decrease in serum trough concentrat ions of 4-ene VPA and 2,4-diene VPA. 5. To study the e f fec t of CBZ on VPA metabolism, pharmacokinetic parameters for VPA were obtained before and a f ter CBZ administrat ion in f i v e , healthy male vo lunteers . The r e s u l t s indicated increased clearance of VPA from the plasma a f te r CBZ admin is t ra t ion . This was accompanied by decreased plasma concentrat ions, serum h a l f - l i f e , and AUC values of VPA a f te r CBZ comedication. This was consistent with the a b i l i t y of CBZ to induce the hepatic microsomal enzyme systems in a manner s imi la r to phenobarbi ta l . 6. Twelve metabolites of VPA were a lso measured in the serum. Serum trough and steady state concentrat ions and AUC values were determined before and a f ter CBZ admin is t ra t ion . The amount of 4-ene VPA, a potent ia l hepatotoxin, was not increased in the serum af ter administrat ion of CBZ. The amounts of the two diunsaturated metabol i tes, 2 ,3 ' -d iene VPA and 2,4-diene VPA, were increased in the serum of the volunteers a f ter CBZ 204 admin is t ra t ion . 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 a f te r CBZ admin is t ra t ion , while the amount of 3-keto VPA did not increase . The AUC values for the monounsaturated metabolites decreased a f ter CBZ administrat ion while the AUC values of the polar metabolites increased. 7. Urinary metabolic p r o f i l e s were determined for the f ive volunteers before and a f ter CBZ admin is t ra t ion . These were determined i n d i v i d u a l l y and grouped into pathways. The recover ies were a lso expressed as a percent of dose, percent of t o t a l recovered, percent of VPA recovered, and as sum of polar and sum of unsaturated metabol i tes . Increased recover ies of 4-OH VPA, 4-keto VPA, and 2-PSA af ter CBZ administrat ion were consistent with enhanced co-1 ox ida t ion . 8. For the f i r s t time, the k i n e t i c s of VPA metaboli tes were determined in the absence and presence of CBZ in a c o n t r o l l e d study. The fol lowing parameters, formation c learance , metabolite c learance, and f r a c t i o n metabolized, were determined for the metabolic pathways and for the i n d i v i d u a l metabol i tes . The formation clearances of a l l pathways increased as a resu l t of CBZ admin is t ra t ion . 9. The r e s u l t s obtained from th is study ind icate that CBZ caused a general induction of VPA metabolism and d id not s p e c i f i c a l l y a f fec t a p a r t i c u l a r pathway. 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Serum VPA and metaboli te trough concentrat ions (mg/L) for WT before and af ter CBZ admin i s t ra t i o n . 233 6. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for BA before and a f ter adminis t ra t ion of CBZ. 234 7. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for FS before and af ter adminis t ra t ion of CBZ. 235 8. Serum AUC for VPA and metabolites over (mg.h/L) for MS before and a f ter CBZ admin is t ra t ion . 12 h 236 9. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for RM before and a f ter adminis t ra t ion of CBZ. 237 224 1 0 . Serum AUC for V P A and metabolites over 12 h (mg.h/L) for WT before and a f ter CBZ admin is t ra t ion . 238 1 1 . Serum AUC for V P A and metabolites over 12 h expressed as pathways (mg.h/L) for BA before and a f ter CBZ admin is t ra t ion . 2 3 9 12. Serum AUC for V P A and metabolites over 12 h expressed as pathways (mg.h/L) for FS before and a f ter CBZ admin is t ra t ion . 240 13. Serum AUC for V P A and metabolites over 12 h expressed as pathways (mg.h/L) for MS before and a f ter CBZ admin is t ra t ion . 241 14. Serum AUC for V P A and metabolites over 12 h expressed as pathways (mg.h/L) for RM before and af ter C B Z admin is t ra t ion . 242 15. Serum AUC for V P A and metaboli tes over 12 h expressed as pathways (mg.h/L) for WT before and a f te r CBZ admin is t ra t ion . 243 16. V P A and metaboli tes recovered in urine ( M ^ O I ) over 12 h for BA before and a f te r administ ra t ion of C B Z . 244 17. V P A and metaboli tes recovered in urine (Mmol) over 12 h for FS before and a f te r administ ra t ion of C B Z . 2 4 5 18. V P A and metaboli tes recovered in urine (Mmol) over 12 h for MS before and a f ter administ ra t ion of C B Z . 246 19. V P A and metaboli tes recovered in urine (umol) over 12 h for RM before and a f te r adminis t ra t ion of C B Z . 247 2 2 5 20. VPA and metabol i tes recovered in urine (Mmol) over 12 h for WT before and a f ter admin is t ra t ion of CBZ. 248 21. VPA and metabol i tes recovered over 12 h in the urine (Mmol) expressed as pathways for BA before and a f te r CBZ admin is t ra t ion . 249 22. VPA and metabol i tes recovered over 12 h in the urine (Mmol) expressed as pathways for FS before and a f te r CBZ admin is t ra t ion . 250 23. VPA and metabol i tes recovered over 12 h in the urine (Mmol) expressed as pathways for MS before and a f te r CBZ admin is t ra t ion . 251 24. VPA and metabol i tes recovered over 12 h in the urine (Mmol) expressed as pathways for RM before and a f te r CBZ admin is t ra t ion . 252 25. VPA and metabol i tes recovered over 12 h in the urine (Mmol) expressed as pathways for WT before and a f te r CBZ admin is t ra t ion . 253 26. Pathway metabol i te clearances (Clm) a before and a f te r CBZ administrat ion for BA ( L / h ) . 254 27. Pathway formation clearances ( C l f ) a before and a f te r CBZ administ rat ion for BA ( L / h ) . 255 28. F rac t ion metabolized (fm) a before and a f te r CBZ admin is t ra t ion by each pathway for BA. 256 29. Pathway metabol i te clearances (Clm) a before and a f te r CBZ administrat ion for FS ( L / h ) . 257 30. Pathway formation clearances ( C l f ) a before and a f te r CBZ administ rat ion for FS ( L / h ) . 258 31. F rac t ion metabolized (fm) a before and a f te r CBZ admin is t ra t ion by each pathway for FS. 259 226 32. Pathway metabolite clearances (Clm) a before and a f te r CBZ administrat ion for MS ( L / h ) . 260 33. Pathway formation clearances ( C l f ) a before and a f te r CBZ administrat ion for MS ( L / h ) . 261 34. F rac t ion metabolized (fm) a before and a f te r CBZ administ rat ion by each pathway for MS. 262 35. Pathway metabolite clearances (Clm) a before and a f te r CBZ administrat ion for RM ( L / h ) . 263 36. Pathway formation clearances ( C l f ) a before and a f ter CBZ administrat ion for RM ( L / h ) . 264 37. F rac t ion metabolized (fm) a before and a f te r CBZ administ ra t ion by each pathway for RM. 265 38. Pathway metabolite clearances (Clm) a before and a f te r CBZ administrat ion for WT ( L / h ) . 266 39. Pathway formation clearances ( C l f ) a before and a f ter CBZ administrat ion for WT ( L / h ) . 267 40. F rac t ion metabolized (fm) a before and a f te r CBZ administ ra t ion by each pathway for WT. 268 41. Semilogarithmic plot of serum 4-OH VPA concentrat ion (mg/L) versus time before and af ter CBZ administrat ion for a) FS, b) MS, c) 269 RM, d) WT. 42. Semilogarithmic plot of serum 4-ene VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 270 MS, d) RM. 43. Semilogarithmic plot of serum 3-ene VPA concentrat ion (mg/L) versus time before and a f ter CBZ administrat ion for a) BA, b) FS, c) 271 MS, d) WT. 227 44. Semilogarithmic plot of serum 2-ene c i s VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 272 RM, d) WT. 45. Semilogarithmic plot of serum 2-ene trans VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 273 MS, d) WT. 46. Semilogarithmic plot of serum 3-keto VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 274 MS, d) WT. 47. Semilogarithmic plot of serum 4-keto VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) FS , b) MS, c) 275 RM, d) WT. 48. Semilogarithmic plot of serum 5-OH VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 276 MS, d) WT. 49. Semilogarithmic plot of serum 2-PSA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) FS , b) MS, c) 277 RM, d) WT. 50. Semilogarithmic plot of serum 2-PGA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 278 RM, d) WT. 51. Semilogarithmic plot of serum 2 ,3 ' -d iene VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 279 RM, d) WT. 52. Semilogarithmic plot of serum 2,4-diene VPA concentrat ion (mg/L) versus time before and a f te r CBZ administrat ion for a) BA, b) FS, c) 280 RM, d) WT. 228 Appendix 1. Serum VPA and metabolite trough concentrat ions (mg/L) for BA before and a f ter CBZ admin is t ra t ion . Compound Before CBZ After CBZ % change 4-OH 3.917 3.781 -3.47 4-ene 0.475 0.375 -21 .0 3-ene 0.507 0.381 -24.9 2-ene c i s 0.079 0.066 -16.5 2-ene trans 17.11 1 3.63 -20.3 VPA 46.46 26.57 -42.8 3-keto 8.151 7.367 -9.62 4-keto 0.391 0.434 + 11.0 5-OH 1 .240 1 .374 + 10.8 2-PSA 0.016 0.016 + 4.50 2-PGA 0.088 0.099 + 12.5 2 ,3 ' -d iene 3.314 3.549 + 7.09 2,4-diene 0.510 0.602 + 18.0 229 I Appendix 2. Serum VPA and metabolite trough concentrat ions (mg/L) for FS before and a f te r CBZ admin is t ra t ion . Compound Before CBZ Af ter CBZ % change 4-OH 1 .063 3.417 + 221 . 4-ene 0.424 0.345 -18.6 3-ene 0. 184 0. 1 79 -2.72 2-ene c i s 0.066 0.063 -4.55 2-ene trans 8.035 6.482 -19.3 VPA 53.05 33.85 -36.2 3-keto 4.501 6.318 + 40.4 4-keto 0.310 0.308 -0.65 5-OH 1 .247 0. 148 -88. 1 2-PSA 0.013 0.018 + 38.5 2-PGA 0. 120 0. 1 23 + 2.50 2 ,3 ' -d iene 1 .461 1 .561 + 6.84 2,4-diene 0.582 0.822 + 41 .2 230 Appendix 3. Serum VPA and metaboli te trough concentrat ions (mg/L) for MS before and a f te r CBZ admin is t ra t ion . Compound Before CBZ After CBZ % change 4-OH 2.787 3.356 + 20.4 4-ene 0.383 0.334 -12.8 3-ene 0.255 0.237 -7.06 2-ene c i s 0. 102 0.066 -35.3 2-ene trans 20.53 1 3.33 -35. 1 VPA 53.22 26. 14 -50.9 3-keto 7.267 5.235 -28.0 4-keto 0.338 0.229 -32.2 5-OH 1 .038 0.727 -30.0 2-PSA .0.030 0.011 -63.3 2-PGA 0. 105 0.057 -45.7 2 ,3 ' -d iene 2.704 2.317 -14.3 2,4-diene 0.512 0.386 -24.6 231 Appendix 4. Serum VPA and metaboli te trough concentrat ions (mg/L) for RM before and a f te r CBZ admin is t ra t ion . Compound Before CBZ After CBZ % change 4-OH 1.015 1 .997 + 96.7 4-ene 0.276 0.556 + 101 . 3-ene 0. 152 0.119 -21.7 2-ene c i s 0. 103 0.095 -7.77 2-ene trans 1 1 .34 8.892 -21 .6 VPA 40.01 31 .87 -20.3 3-keto 5.356 5.762 + 7.58 4-keto 0.212 0.281 + 32.5 5-OH 0.51 1 1 . 1 52 + 125. 2-PSA 0.002 0.013 + 550. 2-PGA 0.081 0. 1 36 + 67.9 2 ,3 ' -d iene 0.852 1.059 + 24.3 2,4-diene 0.577 0.923 + 60.0 232 Appendix 5. Serum VPA and metaboli te trough concentrat ions (mg/L) for WT before and af ter CBZ admin is t ra t ion . Compound Before CBZ After CBZ % change 4-OH 4.681 1.913 -59.1 4-ene 0.400 0.338 -15.5 3-ene 0.41 1 0.324 -21.2 2-ene c i s 0.062 0.055 -11.3 2-ene trans 1 9.04 1 5.33 -19.5 VPA 27.37 1 6.64 -39.2 3-keto 6.220 4.421 -28.9 4-keto 0.245 0.184 -24.9 5-OH 1 .032 0.720 -30.2 2-PSA 0.010 0.008 -20.0 2-PGA 0.057 0.056 -1 .75 2 ,3 ' -d iene 3.624 3.685 + 1 .68 2,4-diene 0.626 0.500 -20. 1 233 Appendix 6. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for BA before and af ter administrat ion of carbamazepine. Compound Before CBZ Af ter CBZ % change 4-OH 42.28 34.06 -19.4 4-ene 5.620 4.852 -13.7 3-ene 6.041 4.554 -24.6 2-ene c i s 1 . 1 24 0.814 -14.1 2-ene trans 209.2 144.1 -31.1 VPA 786. 1 501 .4 -36.2 3-keto 76.94 72.89 -5.26 4-keto 3.973 4.834 + 21 .7 5-OH 19.82 23.77 + 19.9 2-PSA 0.174 0.306 + 75.6 2-PGA 1.321 1 .225 -7.27 2 ,3 ' -d iene 36.52 36.60 ' +0.22 2,4-diene 6.237 6.985 + 12.0 234 Appendix 7. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for FS before and a f te r administ ra t ion of carbamazepine. Compound Before CBZ After CBZ % chang< 4-OH 21.81 48.60 + 123. 4-ene 4.439 4.547 + 2.43 3-ene 2.071 2.178 + 5.17 2-ene c i s 0.808 0.784 -2.82 2-ene trans 93.41 81 .87 -12.4 VPA 796.8 567.1 -28.8 3-keto 59.24 88.42 + 49.3 4-keto 3.501 4.605 + 31 .5 5-OH 11.13 3.425 -69.2 2-PSA 0.184 0.275 + 49.5 2-PGA 1 .365 2.010 + 47.3 2 ,3 ' -d iene 16.61 20.90 + 25.8 2,4-diene 7.520 1 0.79 + 43.5 235 Appendix 8. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for MS before and a f ter CBZ admin is t ra t ion . Compound Before CBZ After CBZ % change 4-OH 33.92 37.09 + 9.35 4-ene 4.335 4.338 + 0.07 3-ene 2.559 2.904 + 13.5 2-ene c i s 1 .009 0.825 -18.3 2-ene trans 211.1 172.5 • -18.3 VPA 710.7 483.6 -31 .0 3-keto 55.40 53.38 -3.65 4-keto 2.370 3.203 + 35. 1 5-OH 1 1 .40 12.65 + 11.0 2-PSA 0. 183 0.110 -39.9 2-PGA 0.733 0.927 + 20.0 2 ,3 ' -d iene 17.74 26.44 + 49.0 2,4-diene 3.688 5. 154 + 39.8 236 Appendix 9. Serum AUC for VPA and metabolites over 12 h (mg.h/L) for RM before and a f te r adminis t ra t ion of carbamazepine. Compound Before CBZ Af ter CBZ % change 4-OH 18.60 22.60 + 21.5 4-ene 3.347 3.364 +0.51 3-ene 1 .756 1 .064 -39.4 2-ene c i s 1 . 1 58 0.999 -14.0 2-ene trans 137.9 1 08.3 -21 .5 VPA 583.3 468.2 -19.7 3-keto 55.79 70.83 + 26.9 4-keto 2. 139 3.388 + 58.4 5-OH 11.04 20.21 + 83. 1 2-PSA 0.058 0.072 + 24.6 2-PGA 0.978 1 .405 + 44.7 2 ,3 ' -d iene 9.973 12.15 + 21 .8 2,4-diene 6.025 10.13 + 68. 1 237 Appendix 10. Serum AUC for VPA and metaboli tes over 12 h (mg.h/L) for WT before and a f ter CBZ admini s t ra t i o n . Compound Before CBZ After CBZ % change 4-OH 61 .48 56. 15 -8.67 4-ene 5.036 4.409 -12.5 3-ene 4.835 3.923 -18.9 2-ene c i s 0.751 0.717 -4.60 2-ene trans 243.7 195.3 -19.9 VPA 498.3 358.2 -28.1 3-keto 77.63 30.72 -60.4 4-keto 2.822 3. 1 05 + 10.0 5-OH 1 5.50 10.67 -31.2 2-PSA 0.113 0.119 +5.13 2-PGA 0.798 0.823 + 3.16 2 ,3 ' -d iene 45.06 37.67 -16.4 2,4-diene 8.064 5.659 -29.8 238 Appendix 11. Serum AUC for VPA and metabol i tes over 12 h expressed as pathways (mg.h/L) for BA before and a f te r CBZ admin is t ra t ion . Pathway 3 Before CBZ Af ter CBZ % change 2 3 4 5 VPA 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 -23.2 -3.31 -15.6 -36.2 + 8.20 -36.2 a Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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 unconjugated and conjugated VPA. 239 Appendix 12. Serum AUC for VPA and metabol i tes over 12 h expressed as pathways (mg.h/L) for FS before and a f te r CBZ admin is t ra t ion . Pathway 3 Before CBZ After CBZ % change 2 3 4 5 VPA 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 + 9.40 + 23.6 + 28.3 + 110. -56.5 -28.8 3 Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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 unconjugated and conjugated VPA. 240 Appendix 13. Serum AUC for VPA and metabol i tes over 12 h expressed as pathways (mg.h/L) for MS before and af ter CBZ admin is t ra t ion . Pathway 3 Before CBZ Af ter CBZ % change 1 2 3 4 5 VPA 267.5 20.30 8.023 36.47 12.17 710.7 226.7 29.34 9.492 40.40 13.58 483.6 -24.8 -44.5 + 18.3 + 10.8 + 11.6 -31.9 Pathway 1 Pathway 2 Pathway 3 .Pathway 4 Pathway includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2 ,3 ' -d iene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA, includes 5-OH VPA and 2-PGA. VPA includes unconjugated and conjugated VPA. 241 Appendix 14. Serum AUC for VPA and metaboli tes over 12 h expressed as pathways (mg.h/L) for RM before and a f te r CBZ admin is t ra t ion . Pathway 3 Before CBZ Af ter CBZ % change 2 3 4 5 VPA 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 -8.61 + 12.6 + 43.9 + 25.3 + 79.9 -19.7 a Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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 unconjugated and conjugated VPA. 242 Appendix 15. Serum AUC for VPA and metaboli tes over 12 h expressed as pathways (mg.h/L) for WT before and a f ter CBZ admin is t ra t ion . Pathway 3 Before CBZ Af ter CBZ % change 1 322.1 224.7 -28.4 2 49.90 41.59 -16.6 3 13.10 10.07 -23.1 4 64.42 59.37 -7.84 5 16.30 11.49 -29.5 VPA 498.3 358.2 -28.1 3 Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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 unconjugated and conjugated VPA. 243 Appendix 16. VPA and metaboli tes recovered in urine (jumol) over 12 h for BA before and a f ter administrat ion of carbamazepine. Compound Before CBZ Af ter CBZ % chang< 4-OH 250.6 232.8 -7.10 4-ene 1 .056 1 .387 + 31 .3 3-ene 0.369 0.417 + 13.0 2-ene c i s 0.771 1 .047 + 35.6 2-ene trans 65.07 53.35 -18.0 VPA ( to ta l ) 464.9 506.4 +8.93 3-keto 336.4 340.5 + 1 .22 4-keto 33.51 46.43 + 38.6 5-OH . 303.9 501 .8 + 65. 1 2-PSA 5. 1 03 7.369 + 44.4 2-PGA 75. 16 84.38 + 12.3 2 ,3 ' -d iene 34.27 29.80 -13.0 2,4-diene 5.418 6.599 + 21.1 244 Appendix 17. VPA and metaboli tes recovered in urine (/xmol) over 12 h for FS before and a f te r administ rat ion of carbamazepine. Compound Before CBZ After CBZ % change 4-OH 269.0 663.9 + 147. 4-ene 4.472 6.632 + 48.3 3-ene 2.555 0.682 -73.3 2-ene c i s 2.261 2.527 + 11.8 2-ene trans 134. 1 89. 15 -33.5 VPA ( tota l ) 1 564. 1317. -15.8 3-keto 1642. 883.4 -46.2 4-keto 74.22 71 .29 -3.95 5-OH 1 323. 1003. -24.2 2-PSA 12.49 22.43 + 79.6 2-PGA 165.4 187.8 + 13.5 2 ,3 ' -d iene 52.65 48.71 -7.48 2,4-diene 13.99 14.74 + 5.36 245 Appendix 18. VPA and metaboli tes recovered in urine (ymol) over 12 h for MS before and a f ter administ ra t ion of carbamazepine. Compound Before CBZ After CBZ % change 4-OH 342.0 410.4 + 20.0 4-ene 1 .743 2.293 + 31.6 3-ene 0.313 0.453 + 44.7 2-ene c i s 1 .586 1 .829 + 15.3 2-ene trans 117.4 102.9 -12.4 VPA ( tota l ) 880.7 898.0 + 1 .96 3-keto 355.3 439.7 + 23.8 4-keto 25.21 54.81 + 117. 5-OH 317.6 533.9 + 68. 1 2-PSA 5.779 8.344 + 44.4 2-PGA 74.44 86.49 + 16.2 2 ,3 ' -d iene 50.72 60.39 + 19.1 2,4-diene 8.670 1 0.03 + 15.7 246 Appendix 19. VPA and metaboli tes recovered in urine (Mmol) over 12 h for RM before and a f te r administ ra t ion of carbamazepine. Compound Before CBZ After CBZ % change 4-OH 313.4 249.5 -20.4 4-ene 1 .250 2.110 + 68.8 3-ene 0.212 0.200 -5.66 2-ene c i s 2.477 2.620 + 5.77 2-ene trans 115.6 92. 10 -20.3 VPA ( tota l ) 644.2 461 .9 -28.3 3-keto 29.25 749. 1 + 2461 4-keto 50.36 58.29 + 15.7 5-OH 692.0 1099. + 58.8 2-PSA 9.740 6. 159 -36.8 2-PGA 528. 1 126.5 -76.0 2 ,3 ' -d iene 45.23 22.35 -50.6 2,4-diene 1 0.53 9.859 -6.37 247 Appendix 20. VPA and metabolites recovered in urine (Mmol) over 12 h for WT before and a f te r adminis t ra t ion of carbamazepine. Compound Before CBZ After CBZ % change 4-OH 299.9 217.0 -27.6 4-ene 1 .265 1 .667 + 31.8 3-ene 0.394 0.413 + 4.82 2-ene c i s 1.165 1 .271 + 9.10 2-ene trans 1 54.4 101.6 -34.2 VPA 476.5 653.4 + 37. 1 3-keto 475.9 282.3 -40.7 4-keto 41 .62 30.60 -26.5 5-OH 485. 1 663.0 + 36.7 2-PSA 7. 1 25 6.997 -1 .80 2-PGA 85.28 77.94 -8.60 2 ,3 ' -d iene 84.85 69.42 -18.2 2,4-diene 1 1 .28 1 0.20 -9.57 248 Appendix 21. VPA and metabolites recovered over 12 h in the urine (nmol) expressed as pathways for BA before and a f te r CBZ admin is t ra t ion . Pathway 3 Before CBZ After CBZ % change 1 2 3 4 5 6 CI. 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 -1 .82 -12.8 + 22.7 -0.90 + 54.6 + 10.9 -2.06 3 Pathway 1 Pathway 2 Pathway 3 Pathway 4 Pathway 5 Pathway 6 includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2 ,3 ' -d iene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA, includes-5-OH VPA and 2-PGA. is VPA glucuronide conjugate. C l r i s unchanged VPA. 249 Appendix 22. VPA and m e t a b o l i t e s recovered over 12 h i n the u r i n e (/umol) 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 . Pathway' Before CBZ A f t e r CBZ % change 1 2 3 4 5 6 C I , 1778. 55.21 18.46 355.7 1 488. 1514. 50. 18 975. 1 49.39 21 .37 756.6 1191. 1263. 53.74 -45.2 -10.5 + 15.8 + 113. -20.0 -16.6 + 7.09 a Pathway 1 Pathway 2 Pathway 3 Pathway 4 Pathway 5 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'-diene VPA. i n c l u d e s 4-ene VPA and 2,4-diene VPA. i n c l u d e s 4 - O H VPA, 4-keto VPA and 2-PSA, 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 conjugate. C l r i s unchanged VPA. 250 Appendix 23. VPA and metabolites recovered over 12 h in the urine (jumol) expressed as pathways for MS before and a f te r CBZ admin is t ra t ion . Pathway 3 Before CBZ After CBZ % change 2 3 4 5 6 474.4 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 + 14.8 + 19.2 + 18.3 + 26.9 + 58.3 -10.4 + 30.7 a Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r is unchanged VPA. 251 Appendix 24. VPA and metabolites recovered over 12 h in the urine (/nmol) expressed as pathways for RM before and af ter CBZ admin is t ra t ion . Pathway 3 Before CBZ Af ter CBZ % change 2 3 4 5 6 147.3 45.44 1 1 .78 373.5 1220. 94.20 550.0 843.8 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 3 Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 i s VPA glucuronide conjugate. C l r i s unchanged VPA. 252 Appendix 25. VPA and metabolites recovered over 12 h in the urine (/mol) expressed as pathways for WT before and af ter CBZ admin is t ra t ion . Pathway' Before CBZ After CBZ % change 1 2 3 4 5 6 C l , 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 -39.0 -18.1 -5.42 -26.9 + 29.9 + 6.81 + 105. a Pathway 1 Pathway 2 Pathway Pathway Pathway Pathway includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2 ,3 ' -d iene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA. includes 5-OH VPA and 2-PGA. is VPA glucuronide conjugate. C l r i s unchanged VPA. 253 Appendix 26. Pathway metabolite c learances ( C l m ) a before and af ter CBZ administ rat ion for BA (L /h ) . Pathway Before CBZ After CBZ % change Sum 2 3 4 5 6 0.2176 0.1140 0.0766 0.9953 2.9183 0.0721 0.0132 4.4071 0.6997 0.2825 0.1029 0.0943 1.1674 3.7993 0.1253 0.0202 5.5919 1.0969 +29.8 -9.74 + 23. 1 + 17.3 + 30.2 + 73.8 + 53.0 + 26.9 + 56.8 a C l m ca lcu la ted by d i v i d i n g the amount of metabolites recovered in the urine over 12 h from a given pathway by the corresponding AUC. k Pathway 1 includes•2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r i s unchanged VPA. c C l p = D o s e / A U C ( v p A ) 254 Appendix 2 7 . Pathway formation clearances ( C l f ) a before and af ter CBZ administ rat ion for BA ( L / h ) . Pathway1 3 Before CBZ After CBZ % change 1 0 . 0 7 9 5 0 . 1 2 2 7 + 5 4 . 3 2 0 . 0 0 6 2 0 . 0 0 8 4 + 3 5 . 5 3 0.001 2 0. 0 0 2 2 +83.3 4 0 . 0 5 8 8 0 . 0 9 1 3 + 5 5 . 3 5 0 . 0 7 8 5 0 . 1 8 9 4 + 1 4 1 . 6 0.0721 0 . 1 2 5 3 + 7 3 . 8 c i r 0 . 0 1 3 2 0 . 0 2 0 2 + 5 3 . 0 Sum 0 . 3 0 9 5 0 . 5 5 9 5 + 8 0 . 8 a C l f ca lcu la ted recovered in the by d i v i d i n g urine from a the amount of metabolites given pathway by VPA AUC ( 1 2 h ) . Pathway 1 includes 2-ene VPA and 3 -keto VPA. Pathway 2 includes 3-ene VPA and 2,3 ' - d i e n e VPA. Pathway 3 includes 4 -ene VPA and 2,4 -d iene VPA. Pathway 4 includes 4-OH VPA, 4 -ke to VPA and 2 -PSA. Pathway 5 includes 5-OH VPA and 2 -PGA. Pathway 6 i s VPA glucuronide conjugate. C l r i s unchanged VPA. 2 5 5 Appendix 28. Fract ion metabolized ( f m ) a before and af ter CBZ administrat ion by each pathway for BA. Pathway*3 Before CBZ After CBZ % change 1 0.1136 0.1119 -1 . SO 2 0.0088 0.0077 - 1 2 . 5 3 0.0017 0.0020 + 17.6 4 0.0840 0.0832 - 0 . 9 5 5 0.1122 0.1727 + 53.9 6 0.1030 0.1143 + 11.0 c i r 0.0188 0.0184 - 2 . 1 3 Sum 0.4421 0.5102 + 15.4 : m is ca lcu la ted by d i v i d i n g C l f by C lp . Pathway 1 Pathway 2 Pathway Pathway Pathway Pathway includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2 ,3 ' -d iene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA, includes 5-OH VPA and 2-PGA. is VPA glucuronide conjugate. C l r is unchanged VPA. 256 Appendix 29. Pathway metaboli te clearances ( C l m ) a before and a f ter CBZ administ ra t ion for FS ( L / h ) . Pathway" Before CBZ After CBZ % change 1 1.8166 0.8919 -50.9 2 0.4142 0.2998 -27.6 3 0.2170 0.1960 -9.68 4 2.2264 2.2639 + 1 .68 5 19.243 35.536 + 84.7 6 0.2738 0.3209 + 17.2 c i r 0.0091 0.0137 + 50.5 Sum 24.200 39.522 + 63.3 C l c 0.8785 1.2344 + 40.5 a C l m ca lcu la ted by d i v i d i n g the amount of metabolites recovered in the ur ine from a given pathway by the corresponding AUC (12 h ) . b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r is unchanged VPA. c Clp = D o s e / A U C ( v p A ) 257 Appendix 30. Pathway formation clearances ( C l r ) a before and a f ter CBZ administ rat ion for FS ( L / h ) . Pathway13 Before CBZ After CBZ % change 1 0.3499 0.2691 - 2 3 . 1 2 0.0097 0.0122 +25.8 3 0.0033 0.0053 +60.6 4 0.0712 0.2135 +200. 5 0.3018 0.3406 + 12.8 6 0.2738 0.3209 + 17.2 c i r 0.0091 0.0137 + 50.5 Sum 1 .0188 1.1753 + 15.4 a C l f ca lcu la ted recovered in the by d i v i d i n g urine from a the amount of metaboli tes given pathway by VPA AUC (12 h ) . Pathway 1 Pathway 2 Pathway Pathway Pathway Pathway includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2 ,3 ' -d iene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA. includes 5-OH VPA and 2-PGA. i s VPA glucuronide conjugate. C l r i s unchanged VPA. 258 Appendix 31 Frac t ion metabolized ( f m ) a before and a f te r CBZ administ ra t ion by each pathway for FS. Pathway*3 Before CBZ After CBZ % change 1 0.3982 0.2180 -45 .3 2 0.0111 0.0099 -10 .8 3 0.0037 0.0043 + 16.2 4 0.0811 0.1730 + 113. 5 0.3435 0.2759 -19 .7 6 0.3117 0.2599 -16 .6 c i r 0.0103 0.0111 + 7.77 Sum 1.1596 0.9521 -17 .9 f m i s ca lcu la ted by d i v i d i n g C l f by C l p . Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r is unchanged VPA. 259 Appendix 32. Pathway metabolite clearances ( C l m ) a before and a f te r CBZ administrat ion for MS ( L / h ) . Pathway Before CBZ Af ter CBZ % change Sum 2 3 4 5 6 0.2731 0.3521 0.1827 1.6348 5.2382 0.1249 0.0537 7.8595 0.8442 0.3720 0.2904 0. 1823 1 .8726 7.3998 0.1644 0. 1032 10.385 1.2407 + 36.2 -17.5 -0.22 + 14.5 + 41 .3 + 31.6 + 92.2 + 32. 1 + 47.0 a C l m ca lcu la ted by d i v i d i n g the amount of metabol i tes recovered in the ur ine from a given pathway by the corresponding AUC (12 h ) . b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 i s VPA glucuronide conjugate. C l r i s unchanged VPA. c C l p = D o s e / A U C ( V P A ) 260 Appendix 33. Pathway formation clearances ( C l c ) a before and a f te r CBZ administrat ion for MS ( L / h ) . Pathway13 Before CBZ Af ter CBZ % change 1 0. 1028 0.1744 +69.6 2 0.0101 0.0176 + 74.3 3 0.0021 0.0036 + 71.4 4 0.0839 0.1565 + 86.5 5 0.0897 0.2078 + 1 32. 6 0. 1249 0.1644 + 31.6 c i r 0.0537 0. 1032 + 92.2 Sum 0.4672 0.8275 + 77. 1 a C l f ca lcu la ted recovered in the by d i v i d i n g urine from a the amount of metabol i tes given pathway by VPA AUC (12 h ) . Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r i s unchanged VPA. 261 Appendix 34. F rac t ion metabolized ( f m ) a before and af ter CBZ administrat ion by each pathway for MS. Pathway 0 Before CBZ Af ter CBZ % change 1 0.1217 0.1406 + 15.5 2 0.0119 0.0142 + 19.3 3 0.0024 0.0029 + 20.8 4 0.0994 0.1261 + 26.9 5 0.1063 0.1674 + 57.5 6 0.1479 0.1325 -10.4 c i r 0.0636 0.0832 + 30.8 Sum 0.5532 0.6669 + 20.6 a f m i s c a l c u l a t e d by d iv id ing C l f by C l p . b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' - d i ene 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. Pathway 6 is VPA glucuronide conjugate. C l r is unchanged VPA. 262 Appendix 3 5 . Pathway metabolite c learances ( C l m ) a before and a f ter CBZ administ rat ion for RM ( L / h ) . Pathway" Before CBZ Af ter CBZ % change 1 0 .1098 0 .7317 + 566 . 2 0 .5427 0 .2390 - 5 6 . 0 3 0 .1763 0 .1245 - 2 9 . 4 4 2 .8686 1.9230 - 3 3 . 0 5 16 .855 9 .1529 - 4 5 . 7 6 0 .0233 0 .0576 + 1 4 7 . c i r 0 . 1359 0 .0846 - 3 7 . 7 Sum 20 .712 12 .313 - 4 0 . 6 Cl c ^ P 0.8572 1.0679 + 24 .6 a C l m c a l c u l a t e d by d i v i d i n g the amount of metaboli tes recovered in the urine from a given pathway by the corresponding AUC (12 h ) . b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 i s VPA glucuronide conjugate. C l r i s unchanged VPA. c Clp = D o s e / A U C ( v p A ) 263 Appendix 36. Pathway formation clearances ( C l f ) a before and a f ter CBZ administrat ion for RM (L /h ) . Pathway*5 Before CBZ Af ter CBZ % change 1 0.0367 0.2815 +667. 2 0.0109 0.0067 - - 38 .5 3 0.0028 0.0036 +28.6 4 0.1023 0.1070 + 4.59 5 0.3473 0.4226 + 21.7 6 0.0233 0.0576 + 1 47. c i r 0. 1359 0.0846 -37 .7 Sum 0.6592 0.9636 + 46.2 a C l f c a l c u l a t e d recovered in the by d i v i d i n g urine from a the amount of metabolites given pathway by VPA AUC (12 h ) . Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 i s VPA glucuronide conjugate. C l r i s unchanged VPA. 264 Appendix 37. Fract ion metabolized ( f m ) a before and af ter CBZ administrat ion by each pathway for RM. Pathway13 Before CBZ Af ter CBZ % change 1 0.0428 0.2636 + 516. 2 0.0127 0.0063 -50.4 3 0.0033 0.0034 + 3.03 4 0.1193 0.1002 -16.0 5 0.4051 0.3957 -2.32 6 0.0271 0.0539 + 98.9 C l r 0.1585 0.0792 -50.0 Sum 0.7688 0.9023 + 17.4 f m i s ca lcu la ted by d i v i d i n g C l f by C l p . Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r i s unchanged VPA. 265 Appendix 38. Pathway metabolite c learances ( C l m ) a before and af ter CBZ adminis t ra t ion for WT ( L / h ) . Pathway" Before CBZ Af ter CBZ % change 1 0.3020 0.2635 -12.7 2 0.2393 0.2352 -1 .71 3 0.1343 0.1654 + 23.2 4 0.8647 0.6850 -20.8 5 5.6723 10.409 + 83.5 6 0.0951 0.1414 + 48.7 c i r 0.0427 0.1216 + 185. Sum 7.3504 12.021 + 63.5 CI c 1.2041 1.6750 + 39. 1 a C l m ca lcu la ted by d i v i d i n g the amount of metabolites recovered in the urine from a given pathway by the corresponding AUC (12 h ) . b Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. # Pathway 6 is VPA glucuronide conjugate. C l r i s unchanged VPA. c C l p = Dose /AUC( V p A ) 266 Appendix 39. Pathway formation clearances ( C l c ) a before and af ter CBZ adminis t ra t ion for WT ( L / h ) . Pathway 0 Before CBZ Af ter CBZ % change 1 0.1952 0. 1653 -15 .3 2 0.0240 0.0273 + 13.8 3 0.0035 0.0046 + 31 .4 4 0.1118 0.1136 + 1 .61 5 0.1855 0.3340 +80.0 6 0.0951 0.1414 + 48.7 c i r 0.0427 0.1216 + 185. Sum 0.6578 0.9078 + 38.0 a C l f ca lcu la ted by d i v i d i n g the amount of metabolites recovered in the urine from a given pathway by VPA AUC (12 h ) . D Pathway 1 includes 2-ene VPA and 3-keto VPA. Pathway 2 includes 3-ene VPA and 2 ,3 ' -d iene 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. Pathway 6 is VPA glucuronide conjugate. C l r is unchanged VPA. 267 Appendix 40. Fract ion metabolized ( f m ) a before and a f te r CBZ administrat ion by each pathway for WT. Pathway1 Before CBZ After CBZ % change 1 0.1621 0.0987 -39.1 2 0.0199 0.0163 -18.1 3 0.0029 0.0028 - 3 . 4 5 4 0.0928 0.0678 -26 .9 5 0.1541 0.1994 + 29.4 6 0.0791 0.0844 + 6.70 c i r 0.0354 0.0726 + 105. Sum 0.5463 0.5420 -0 .79 f m i s ca lcu la ted by d i v i d i n g C l j by Clp , Pathway 1 Pathway 2 Pathway Pathway Pathway Pathway includes 2-ene VPA and 3-keto VPA. includes 3-ene VPA and 2 ,3 ' -d iene VPA. includes 4-ene VPA and 2,4-diene VPA. includes 4-OH VPA, 4-keto VPA and 2-PSA, includes 5-OH VPA and 2-PGA. i s VPA glucuronide conjugate. C l r i s unchanged VPA. 268 693 > X) TJ ro 3 Qj c r B n tn --'=) o ro a 3 3 3 o M . W Q) (D H - i-h 3 O rt rtkQ o ro n n> •—- »-t 0» r-| rt M -50 ^  rt 3 O 3* O M -Qj — O —' 3 51 \ * -• ^ n r o • tB ^ rt < o 0) (B M I 3 oi cn c 3 01 0) ro c 3 i 3 o> ro ' i rt O cr \x o ro 3 l-h o i-h n o ro i-t OJ LO Concentration, mg/L Concentration, mg/L Concentration, mg/L Concentration, mg/L QLZ Concentration, mg/L > TJ TJ CD 3 to cr co 3 a. CO D» CO ro 3 3 to o o 3 o ro 3 o rr L Q n 0) 01 1 rr i— M - rr O =r 3 3 O Cu O 3 3 O C O . CO —' rr CS) < O cu ro i-n QJ n 3 cn cn M - c 3 cn cn rr ro c 3 0) ro rr o 3 3 *» I ro cr 3 ro ro o l-h i-l o ro i-t a* to > Concentration, mg/L Concentration, mg/L Concentration, mg/L — ^ _ ^ ( / / SERUM 4-A / / t / / 1 1 ENE VPA FOR R.M. / 1 Appendix 43. Semilogarithmic plot of serum 3-ene VPA concentration (mg/L) versus time before ( • ) and af ter ( O ) CBZ administrat ion for a) BA, b) FS, c) MS, d) WT. Appendix 44. Semilogarithmic plot of serum 2-ene c i s VPA concentration (mg/L) versus time before ( • ) and af ter ( O ) CBZ administrat ion for a) BA, b) FS, c) RM, d) WT. a) b) Time, h 20 30 Time, h Appendix 45. Semilogarithmic plot of serum 2-ene trans VPA concentration (mg/L) versus time before ( • ) and af ter ( O ) CBZ administ rat ion for a) BA, b) FS, c) MS, d) WT. Appendix 46. Semilogarithmic plot of serum 3-keto VPA concentration (mg/L) versus time before ( • ) and af ter ( O ) CBZ administrat ion for a) BA, b) FS, c) MS, d) WT. Appendix 47. Semilogarithmic plot of serum 4 -keto VPA concentration (mg/L) versus time before ( • ) and af ter ( O ) CBZ administrat ion for a) FS, b) MS, c) RM, d) WT. 9LZ Concentration, mg/L Concentration, mg/L > TJ ro 3 CO ro 3 ( T W O -^3 O Qi 3 U O co ai ro - M l 3 O rr rr uo n u f i B -—- i-i rr 3 Oi rr 2 — co O - o 3 Q i ^ O — 3 ^ i £ > TJ £ \ i -• t o—- rr < O III (P M l Q J H 3 to oi 3 oi n c CO rr g rr i->. 1 3 O i a* ro i rr O H - L T I O ro 3 O l-h i-l O ro i-i a > < TJ > Concentration, mg/L Concentration, mg/L Appendix 49. Semilogarithmic plot of serum 2-PSA concentrat ion (mg/L) versus time before ( • ) and a f ter ( O ) CBZ administrat ion for a) FS, b) MS, c) RM, d) WT. - J CD Time, h Appendix 50. Semilogarithmic plot of serum 2-PGA concentrat ion (mg/L) versus time before ( • ) and a f te r ( O ) CBZ administrat ion for a) BA, b) FS, c) RM, d) WT. Time, h Appendix 51. Semilogarithmic plot of serum 2 ,3 ' -d iene VPA concentration (mg/L) versus time before ( • ) and af ter ( O ) CBZ administrat ion for a) BA, b) FS, c) RM, d) WT. 082 Concentration, mg/L > TJ ro a tn to cr cu o a o O i 3 s to 0) ro - r-r. 3 CO ro 3 ro o rt KQ i-t 0) 0> r-l rt M -W ^ rt 3 O 3-o •->• a o — 3 — i n TI H n c 1 o • cd rt tsi < o OJ ro i-h Qj l-t 3 to cn cn to rt t--» 3 OJ ro ro c rt 3 to cr i ro a. o ro n 3 ro ro ~ < TJ CO > Concentration, mg/L Concentration, mg/L Concentration, mg/L 8-a 

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