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Induction of valproic acid metabolism in rat liver microsomes by carbamazepine and carbamazepine-10,… Panesar, Sukhbinder Kaur 1993

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INDUCTION OF VALPROIC ACID METABOLISM IN RAT LWER MICROSOMES BY CARBAMAZEPINE AND CARBAMAZEPINE-1O,1 1-EPOXIDE by SUKHBINDER KAUR PANESAR B.Sc. (Pharm.), University of British Columbia, 1983 M.Sc., University of British Columbia, 1987 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES FACULTY OF PHARMACEUTICAL SCIENCES Division of Pharmaceutical Chemistry  We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA December 1993 © Sukhbinder Kaur Panesar, 1993  In  presenting  this  thesis  in  partial  fulfilment  of the  requirements  for an  advanced  degree at the University of British Columbia, I agree that the Library shall make it 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 is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  i% 1 t4tt.b6i-  The University of British Coimbia Vancouver, Canada  Date 2c-&/,  DE-6 (2/88)  ,g93  1r c  S7J  4-C-iL-€  ABSTRACT anticonvulsant agent that is Vaiproic acid (VPA) is a commonly used zepine (CBZ) to maximize seizure often given in combination with carbama hepatotoxicity is increased when control. The incidence of VPA associated ts. One or more of VPAs metabolites coadministered with other anticonvulsan may be responsible for the hepatotoxicity. , carbamazepine-10,11-epoxide The consequences of induction by CBZ ) on the metabolism of VPA and (CBZE), phenobarbital (PB) and clofibrate (CFB were examined. Total hepatic (E)-2-ene VPA by rat hepatic microsomes ) of cytochrome P-450 induced by cytochrome P-450 content and the isozyme(s pared. The metabolism of VPA was these agents were determined and com g the microsomal formation of 4monitored following induction by quantitatin and 5-OH VPA. For (E)-2-ene VPA, ene VPA, 3-OH VPA, 4-OH VPA, 4-keto VPA )-2,3’-diene VPA was monitored. the formation of (E)-2,4-diene VPA and (E,E treated intraperitoneally (i.p.) Adult male Long Evans rats (4/group) were or CBZE 50 mg/kg every 12 h for 3, 7, with one of the following: CBZ 100 mg/kg or PB 75 mg/kg for 4 days. The mean 10 or 14 days, CFB 350 mg/kg for 7 days, CBZ treatment groups over the ten hepatic cytochrome P-450 content for the nced 1.5 to 1.8 fold compared to the day treatment period was significantly enha nt did not appear to affect total vehicle control group while CBZE treatme tment resulted in a significant 1.9 hepatic cytochrome P-450 content. PB trea rol, but a significant increase was not fold increase compared to the vehicle cont observed for the CFB treatment group. me P-450b was induced by Immunoblot analysis indicated that cytochro me P-450b constituted 65 % of the PB and also by CBZ and CBZE. Cytochro the PB induced microsomes and total hepatic cytochrome P-450 content in  Ii  Abstract  ranged from 31 to 66 % in the CBZ and CBZE groups over the 14 day treatment period. Pentoxyresorufin, a substrate for cytochrome P-450b, was preferentially metabolized compared to ethoxyresorufin by microsomal protein isolated from PB, CBZ and CBZE treated rats. Mean pentoxyresorufin 0-dealkylation rates for the CBZ, CBZE and PB treatment groups were enhanced 12 to 53 fold when compared to their respective vehicle control groups. The metabolism of VPA and (E)-2-ene VPA was enhanced by PB, CBZ and to a lesser extent by CBZE treatment. CFB pretreatment did not have any significant effects on the metabolism of VPA or (E)-2-ene VPA. An antibody directed against rat cytochrome P-450b was effective in completely inhibiting the metabolism of VPA to 4-ene VPA by microsomal protein isolated from PB or CBZ 3 day treated rats.  The formation of 4-OH VPA and 4-keto VPA was  inhibited greater than 75 % in the presence of the antibody. The metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA by microsomal protein from PB or CBZ 3 day treatment groups was inhibited 89 % and 85 % respectively, in the presence of the anti-rat cytochrome P-450b antibody. Three days of treatment with CBZ at a dose of 100 mg/kg i.p. every 12 h was as effective as typical PB treatment for inducing total hepatic cytochrome P-450 content, cytochrome P-450b, pentoxyresorufin 0-dealkylation, and the in vitro metabolism of VPA and (E)-2-ene VPA.  CBZE, used at an equivalent  molar dose as CBZ, also may be as effective an inducer as PB. Cytochrome P 450b plays an important role in the in vitro metabolism of VPA and (E)-2-ene VPA to 4-ene VPA and (E)-2,4-diene VPA, respectively. These two metabolites are known hepatotoxins and their enhanced formation in the presence of enzyme  inducing  agents  are  likely  responsible  for  VPA  associated  hepatotoxicity. The isozyme of cytochrome P-450 induced by CBZ or CBZE has not previously been identified.  111  TABLE OF CONTENTS Abstract  ii  Table of contents  iv  List of tables  ix  List of figures  xi  List of abbreviations  xv  Dedication  xviii  Acknowledgements  xix  Introduction Vaiproic Acid Metabolism Fatty acid metabolism Mitochondrial n-oxidation of fatty acids Peroxisomal 13-oxidation of fatty acids Omega and omega-i oxidation of fatty acids Metabolism of valproic acid Metabolism of 4-ene VPA Metabolite activity Adverse effects Side effects Biochemical effects Pancreatitis Embryotoxicity/teratogenicity Hepatotoxicity (E)-2-ene VPA Anticonvulsant activity Teratogenicity Metabolism Carbamazepine Carbamazepine-10,11-epoxide Interaction between VPA and CBZ Cytochrome P-450  1 3 3 3 3 5 6 6 9 10 10 10 11 13 13 15 19 20 20 21 21 24 25 27  iv  Induction of cytochrome P-450 by phenobarbital Clinical implications Specific objectives  Table of Contents .28  32 34 35 35 35 35 35 36 37 38 38 38 39 39 39 39 40 40 40 40 40 40 41 41  Experimental Reagents and Materials Vaiproic acid and metabolites Internal standards Carbamazepine and metabolites Reagents Primary antibodies Methods Induction studies Animals Treatment of solvents and compounds Treatment of animals with carbamazepine Treatment of animals with carbamazepine-10,11-epoxide Treatment of animals with phenobarbital Treatment of animals with cloflbrate Treatment of animals with vaiproic acid Analysis Vaiproic acid and metabolites Stock solutions of internal standards for GCMS Preparation of standard curves in phosphate buffer Standard curve for VPA and metabolites Standard curve for (E)-2-ene VPA and metabolites Extraction of VPA and metabolites from standard samples and incubates Carbamazepine and metabolites Preparation of stock solutions for HPLC Preparation of standard curve for CBZ and metabolites. Extraction of CBZ, CBZE and CBZD from urine samples Instrumentation Valproic acid and metabolites Carbamazepine and metabolites Preparation of subcellular fractions from rat livers V  .  .  .  41 43 43 43 43 44 44 46 46  Table of Contents Determination of protein content of various subcellular 47 fractions Determination of cytochrome P-450 content in hepatic 47 microsomes 48 Gel electrophoresis of microsomal protein 48 Immunoblot Quantitation of cytochrome P-450b in microsomal protein from 49 PB, CBZ and CBZE treated rats 50 In vitro microsomal metabolism of VPA and (E)-2-ene VPA In vitro microsomal metabolism of VPA and (E)-2-ene VPA in the presence of anti-rat cytochrome P-450b or anti-rat cytochrome P-450h antibody In vitro microsomal metabolism of VPA and (E)-2-ene VPA in the presence of both anti-rat cytochrome P-450b and antirat cytochrome P-450h antibodies Microsomal 0-dealkylation of ethoxyresorufin and pentoxyresorufin Statistical analysis Results Quantitation and identification of cytochromes P-450 in hepatic microsomes Quantitation of total hepatic microsomal cytochrome P-450 content Identification of the cytochrome P-450 isozymes induced by CBZ and CBZE using SDS-PAGE and Western blot techniques In vitro 0-dealkylation of pentoxyresorufin and ethoxyresorufin catalyzed by hepatic microsomal protein from the various treatment groups Quantitation of cytochrome P-450b in microsomes from CBZ, CBZE and PB treated rats by SDS-PAGE and Western blot techniques In vitro metabolism of VPA and (E)-2-ene VPA Analysis of VPA and metabolites by GCMS In vitro metabolism conditions for VPA and (E)-2-ene VPA In vitro metabolism of VPA vi  50  51 51 51 52 52 52  58  64  71 72 74 74 76  Table of Contents Formation of 3-OH VPA from VPA. 76 76 Formation of 4-OH VPA from VPA 82 Formation of 5-OH VPA from VPA 87 Formation of 4-ene VPA from VPA 91 Formation of 4-keto VPA from VPA  91 95 99  In vitro metabolism of(E)-2-ene VPA Formation of (E,E)-2,3’-diene VPA from (E)-2-ene VPA Formation of (E)-2,4-diene VPA from (E)-2-ene VPA Effect of anti-rat cytochrome P-450b antibody on the in vitro  metabolism of VPA and (E)-2-ene VPA by microsomes from PB and CBZ treated rats Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA Effect of anti-rat cytochrome P-450b antibody on the in  103 103 105  vitro metabolism of (E)-2-ene VPA Effect of anti-rat cytochrome P-450h on the in vitro metabolism of VPA and (E)-2-ene VPA by untreated microsomes Quantitation of CBZ, CBZE and CBZD Analysis of CBZ and metabolites in rat urine by HPLC Urinary recoveries of CBZ, CBZE and CBZD after dosing with  112 113 113 113  CBZ Urinary recoveries of CBZE and CBZD after dosing with CBZE  116 120 120 120 120 121  Discussion Choice of experimental conditions Animal model Choice of vehicle for CBZ and CBZE GCMS analysis of VPA and metabolites Choice of metabolites monitored from the in vitro microsomal metabolism of VPA and (E)-2-ene VPA HPLC analysis of CBZ and metabolites A comparison of the induction of rat hepatic microsomal cytochrome P-450 content by PB, CBZ, CBZE and other inducing agents. Cytochrome P-450 content in hepatic microsomes from .  untreated rats Effect of VPA on cytochrome P-450 content vii  122 122  .  .  123 123 124  Table of Contents 124  Effect of CFB on hepatic cytochrome P-450 content A comparison of the effects of PB, CBZ and CBZE on hepatic cytochrome P-450 content in rats A comparison of the effects of PB, CBZ and CBZE on the induction of cytochrome P-450b Effect of CBZ, CBZE and PB treatment on cytochromes P-450f  and P-450g In vitro metabolism studies of ‘[PA and (E)-2-ene VPA[Effects of inducing agents Interaction between ‘[PA and CBZ Effect of anti-rat cytochromes P-450b and P-450h antibodies on VPA metabolite profiles from rat liver microsomes A comparison of the effects of PB, CBZ and CBZE induction on the in vitro metabolism of VPA Formation of 4-ene ‘[PA Significance of 4-ene VPA formation to the mechanism of VPA hepatotoxicity Formation of the hydroxy metabolites of ‘[PA Effect of anti-rat cytochrome P-450b and P-450h antibodies on (E)-2-ene VPA metabolite profiles from rat liver microsomes A comparison of the effects of PB, CBZ and CBZE induction on the in vitro metabolism of (E)-2-ene VPA in rat liver microsomes Effect of CFB treatment on the metabolism of VPA and (E)-2-  126 130 134 135 135 136 140 140 141 143  146  147 151 151  ene ‘[PA Clinical relevance Summary and conclusions  153  References  155  viii  LIST OF TABLES  Table 1.  Table 2.  Table 3.  Table 4.  Table 5.  Table 6.  Table 7.  Table 8.  Table 9.  Summary comparing the nomenclature of Ryan and Levin (1990) and Nelson et al. (1993) for isozymes of cytochrome P 450 purified from rat liver microsomes  29  Summary of total hepatic cytochrome P-450 content (nmollmg protein, mean ± s.d.) and change in cytochrome P 450 relative to the untreated group or to the respective vehicle control group for the PB, CFB, CBZ and CBZE treatment groups (n=4)  57  Summary of PROD (nmol resorufinlminlmg protein) and changes in PROD relative to the untreated group or to the respective vehicle control group for the PB, CFB, CBZ and CBZE treatment groups (n=4)  68  Comparison of mean PROD activities of CBZE 3, 7, 10 and 14 day treated groups as a percent of the PROD activities of the PB and CBZ 3, 7, 10 and 14 day treatment groups  70  Cytochrome P-450b (pmolJ5 pmol of spectrally determined cytochrome P-450 or as percent of total hepatic cytochrome P-450) in microsomes from rats treated with either PB, CBZ for 3, 7, 10 or 14 days or CBZE for 3, 7, 10 or 14 days  73  A comparison of the metabolism of VPA to 3-OH VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4)  79  A comparison of the metabolism of VPA to 4-OH VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4)  83  A comparison of the metabolism of VPA to 5-OH VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4)  86  A comparison of the metabolism of VPA to 4-ene VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4)  90  ix  Table 10.  Table 11.  List of Tables A comparison of the metabolism of VPA to 4-keto VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle 94 control group (n=4)  A comparison of the metabolism of (E)-2-ene VPA to (E,E) 2,3’-diene VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4)  98  Table 12.  A comparison of the metabolism of (E)-2-ene VPA to (E)-2,4diene by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective 102 vehicle control group (n=4)  Table 13.  Total 12 h urinary recoveries of CBZ, CBZE and CBZD (jig) 115 from rats treated with CBZ 100 mg/kg every 12 h  Table 14.  Urinary recoveries (12 h) of CBZ, CBZE and CBZD as percent of dose administered from rats treated with CBZ 100 117 mg/kgeveryl2h  Table 15.  Total 12 h urinary recoveries of CBZE and CBZD (jig) from 118 rats treated with CBZE 50 mg/kg every 12 h  Table 16.  Urinary recoveries (12 h) of CBZE and CBZD as percent of 119 dose from rats treated with CBZE 50 mg/kg every 12 h  x  LIST OF FIGURES Figure 1.  Summary of vaiproic acid metabolism  .7  Figure 2.  Summary of carbamazepine metabolism  23  Figure 3.  Summary of the extraction procedure for vaiproic acid and metabolites  42  Figure 4.  Summary scheme of the extraction of carbamazepine and metabolites from urine of rats.  45  Figure 5.  Carbon monoxide sodium dithionite-reduced difference spectrum of hepatic microsomal cytochrome P-450  53  Figure 6.  Cytochrome P450 content (nmol of spectrally determined cytochrome P-4501mg protein, mean ± s.d.) of microsomes from control, PB, NS, CFB and CO treated rats (n4)  54  Cytochrome P-450 content (nmol of spectrally determined cytochrome P-450/mg protein, mean ± s.d.) of microsomes from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4).  55  Figure 8.  SDS-PAGE gel of rat liver microsomal fractions from various treatment groups  60  Figure 9.  SDS-PAGE gel of rat liver microsomal fractions from various treatment groups  61  Figure 10.  Immunoblot of rat liver microsomal proteins probed with anti-rat cytochrome P-450b antibody  62  Figure 11.  Immunoblot of rat liver microsomal proteins probed with anti-rat cytochrome P-450b antibody  63  Figure 12.  Microsomal 0-dealkylation of pentoxyresorufin and ethoxyresorufin (mriol resorufinlminlmg protein, mean ± s.d.) by microsomes from control, PB, NS, CFB and CO treated rats (n=4)  65  Microsomal 0-dealkylation of pentoxyresorufin (nmol resorufinlminlmg protein, mean ± s.d.) by microsomes from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  66  Figure 7.  Figure 13.  xi  Figure 14.  Figure 15.  Figure 16.  Figure 17.  Figure 18.  Figure 19.  Figure 20.  Figure 21.  Figure 22.  List of Figures Microsomal 0-dealkylation of ethoxyresorufin (nmol resorufmnlminlmg protein, mean ± s.d.) by microsomes from CBZ, CBZE and P03, 7, 10 and 14 day treated rats (n=4)..... 67 Formation of 3-OH VPA (jig, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4)  77  Formation of 3-OH VPA (jig, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  78  Formation of 4-OH VPA (jig, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4)  80  Formation of 4-OH VPA (jig, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  81  Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes-(2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4)  84  Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  85  Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4)  88  Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitro metabolism of ‘[PA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  89  xii  Figure 23.  Figure 24.  Figure 25.  Figure 26.  List of Figures Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB 92 and CO treated rats (n=4)  Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  93  Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4)  96  Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4)  97  Figure 27.  Formation of (E)-2,4-diene VPA (jig, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, 100 PB, NS, CFB and CO treated rats (n=4)  Figure 28.  Formation of (E)-2,4-diene VPA (jig, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE 101 and PG 3, 7, 10 and 14 day treated rats (n=4)  Figure 29.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 3-OH VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 104 day treated rats  Figure 30.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 4-OH VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 106 day treated rats  Figure 31.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 5-OH V.PA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 107 day treated rats  Figure 32.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 4-ene VPA by microsomes (2 nmol of xlii  List of Figures and CBZ 3 PB from P-450) spectrally determined cytochrome 108 day treated rats Figure 33.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 4-keto VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 109 day treated rats  Figure 34.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of (E)-2-ene VPA to (E,E)-2,3’-diene VPA by microsomes (2 nmol of spectrally determined cytochrome P 110 450) from PB and CBZ 3 day treated rats  Figure 35.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA by microsomes (2 nmol of spectrally determined cytochrome P 111 450) from PB and CBZ 3 day treated rats  Figure 36.  HPLC chromatograms of a) standards of CBZ, CBZE, CBZD and MCBZ, b) extracted blank rat urine sample, c) extracted spiked rat urine sample and d) extracted rat urine sample. Peak 1, CBZD, peak 2, CBZE, peak 3, CBZ and peak 4, 114 MCBZ  Figure 37.  Cytochrome P-450 catalyzed metabolism of VPA to 3-OH 138 VPA, 4-OH VPA, 5-OH VPA, 3-ene VPA and 4-ene VPA  Figure 38.  Structural similarity amongst hypoglycin, 4-pentenoic acid 142 and 4-ene VPA  Figure 39.  The f-oxidation mitochondria  pathway  of  VPA  metabolism  in 149  xiv  LIST OF ABBREVIATIONS  (E)-2,4-diene VPA  2-propyl-(E)-2,4-pentadienoic acid  (E,E)-2,3’-diene VPA  2- [(E)- 1’-propenyl)] -(E)-2-pentenoic acid  2-ene VPA  2-propyl-2-pentenoic acid  2-PGA  2-propyiglutaric acid  2-PSA  2-propylsuccinic acid  3-ene VPA  2-propyl-3-pentenoic acid  3-keto VPA  2-propyl-3-oxopentanoic acid  3-OH VPA  2-propyl-3-hydroxypentanoic acid  TPA 4 4-ene ‘  2-propyl-.4-pentenoic acid  4-keto VPA  2-propyl-4-oxopentanoic acid  4-OH VPA  2-propyl-4-hydroxypentanoic acid  5-OH VPA  2-propyl-5-hydroxypentanoic acid  AUC  area under the serum concentration versus time curve  Bis  N,N’-methylene-bis-acrylamide  BSA  bovine serum albumin  CBZ  carbamazepine  CBZD  trans-lO, 1 1-dihydroxy-lO, 1 1-dihydrocarbamazepine (carbamazepine diol)  CBZE  carbamazepine- 10,1 1-epoxide  CFB  clofibrate  CO  corn oil  CoA  coenzyme A  E  trans  EDTA  ethylenediaminetetraacetic acid  g  gram(s)  xv  List of abbreviations GC  gas chromatography  GCMS  gas chromatography-mass spectrometry  GSH  glutathione  h  hour(s)  HEPES  N- [2-Hydroxyl] piperazine-N- [2-ethanesulfonic acic  HPLC  high performance liquid chromatography  i.d.  internal diameter  i .p.  intraperitoneal  IgG  immunoglobulin, antibody  JVS  Jamaican Vomiting Sickness  k  thousand  kg  kilogram(s) micron(s), micrometer(s)  MCBZ  lO-methoxycarbamazepine  MCPA  methylenecyclopropylacetic acid  MES  maximal electroseizures  pg  microgram(s)  mg  milligram(s)  2-MGA  2-methylgiutaric acid  mm  minute(s) microlitre  mM  millimolar  MSD  mass selective detector  MTBSTFA  N-tert-butyldimethylsilyl-N-methyltrifluoroacetamide  NAC  N-acetylcysteine  NADH  nicotinamide adenine dinucleotide, reduced  NADPH  nicotinamide adenine dinucleotide phosphate, reduced  xvi  List of abbreviations  ND  not detected  NS  normal saline  PA  4-pentenoic acid  PB  phenobarbital  PBS  phosphate buffered saline  PG  propylene glycol  PROD  pentoxyresorufin 0-dealkylation  PTZ  pentylenetetrazole  RS  Reye’s syndrome  s.d.  standard deviation  SDS  sodium dodecyl sulphate  SDS-PAGE  sodium dodecyl suiphate-polyacrylamide gel electrophoresis  SGOT  serum glutamic oxaloacetic transaminase aspartate aminotransferase  SGPT  serum glutamic pyruvic transaminase alanine aminotransferase  SIM  selected ion monitoring  tBDMS  tertiary -butyldimethylsilyl  TEMED  iamine ,N 1V’-tetramethylethylened 1 ,N,N  Vd  volume of distribution  VPA  vaiproic acid (2-propylpentanoic acid)  xvii  DEDICATION  To my parents for their support throughout the years.  xviii  ACKNOWLEDGEMENTS  I would like to acknowledge my supervisor Dr. Frank Abbott for his guidance and support throughout the years. I am also deeply indebted to Dr. Stelvio Bandiera for his aid and guidance, for the use of his laboratory and expertise. My appreciation also to Drs. David Seccombe and James Orr for their constructive and helpful comments. Thanks also to my able chairperson, Dr. James Axelson. I would also like to acknowledge the following people for their help with the animal studies beyond the call of duty and friendship: Anthony Borel, Grace Chan and Michael Gentleman. My extreme gratefulness to Dr. Dianchen Yu for  his help with the GCMS analyses. The figure of the summary of vaiproic acid metabolism in Introduction was generously provided by John Gordon. The metabolism figures in Discussion were generously provided by Wei Tang. My fellow graduate students and colleagues: Bruce Allen, Grace Chan, Rajesh Mahey (now my husband) and Jacqueline Walisser for their help, discussions, company, and friendship.  xix  INTRODUCTION  Epilepsy affects approximately 20 to 40 million people throughout the world (Rail and Schleifer, 1990). The incidence of epilepsy is higher in children than in adults, with approximately 8 in 1000 children under the age of 7 years demonstrating epilepsy. Epilepsy is characterized by abnormal phenomena of motor, sensory, autonomic or psychic origin. It can be designated as primary (idiopathic) or secondary (symptomatic) epilepsy.  Primary epilepsy has no  known identifiable cause while secondary epilepsy may be caused by various factors including trauma, neoplasms, infection or cerebrovascular disease. The International Classification of Epileptic Seizures describes in detail the various types of epilepsy (Dreifuss, 1990). A number of anticonvulsant agents are available for the treatment of epilepsy. Approximately 80% of patients can be effectively treated with a single anticonvulsant while the remainder require multiple anticonvulsants (Duncan, 1991).  Two commonly used anticonvulsant agents are valproic acid (VPA,  Depakene®) and carbamazepine (CBZ, Tegretol®).  These 2 drugs are often  used in combination therapy in efforts to optimize seizure control (Cloyd et al., 1985). Because treatment of epilepsy is a long term endeavour, the opportunities for undesirable interactions with other xenobiotics are immense. An interaction may result in the formation of a metabolite(s) which not only adversely affects seizure control but also proceeds to a resultant toxic reaction. Although the side effects associated with VPA usage are generally of the mild gastrointestinal type, incidents of teratogenicity, pancreatitis (Isom, 1984) and hepatotoxicity (Jager-Roman et al., 1986) have also been reported.  Hepatotoxicity in most  cases has involved young children on multiple anticonvulsant therapy (Dreifuss  1  Introduction et al., 1987).  The mechanism of VPA hepatotoxicity is not well understood,  although one or more of its metabolites are thought to be responsible (Baillie, 1992). The metabolite, 4-ene VPA, is a minor metabolite of VPA and is similar in structure to 2 known hepatotoxic terminal olefins, 4-pentenoic acid and methylenecyclopropylacetic acid. Metabolism of 4-ene VPA via mitochondrial  -  oxidation may result in the formation of chemically reactive intermediates which can alklyate cellular macromolecules.  Thus, it is postulated that  coadministration of other anticonvulsant drugs, a number of which are known to be enzyme inducing agents, will enhance the formation of the potentially toxic metabolite(s). Because of the frequency of the combination of VPA and CBZ and the potential risks of toxicity that might result from induction of VPA metabolism, it is important to characterize this interaction completely. This study will allow us to determine if metabolic formation of the potential hepatotoxin, 4-ene VPA, is increased in the presence of an enzyme inducing agent such as CBZ. Since this thesis focuses on characterizing the effect of CBZ on the in vitro metabolism of VPA, a brief discussion on the metabolism of VPA and the toxicity associated with its usage will be presented. The major serum metabolite of VPA, 2-ene VPA appears to be devoid of the severe toxicities associated with ‘[PA and thus has been considered as a potential alternative anticonvulsant agent (Loscher, 1992). Therefore, it was deemed important to also investigate the effect of CBZ induction on 2-ene VPA metabolism. A brief review on 2-ene VPA as well as a brief summary of the literature on CBZ and the interaction between VPA and CBZ will also be presented.  In addition, a brief review on induction of  cytochrome P-450 by phenobarbital is also presented.  2  Introduction VALPROIC ACID Originally synthesized for use as a solvent over a century ago (Burton, 1881), VPA has only been available for therapeutic use in North America since 1978 although its anticonvulsant effects have been known since the early 1960’s (Meunier et at., 1963). VPA is effective in the treatment of a variety of seizure types including absence, myoclonic, tonic-clonic, partial (Rimmer and Richens, 1985; Duncan, 1991), infantile and photo-convulsive (Rimmer and Richens, 1985) seizures. Because of its broad effectiveness against a number of seizure types, VPA is widely used either as a single agent (monotherapy) or in combination with other anticonvulsants (polytherapy).  Other uses for VPA  include prophylactic treatment of febrile convulsions (Lee et at., 1986), posttrauma epilepsy, status epilepticus, acute mania and alcohol withdrawal (Rimmer and Richens, 1985). Metabolism Since VPA shares in common the metabolic pathways of fatty acids, a brief review on fatty acid metabolism is presented. Fatty acid metabolism Fatty acids consist of a terminal carboxyl group and an alkyl side chain (CH (Devlin, 1986). Fatty acids -COOH 2 3 CH and are of the basic formula, ) are mainly metabolized via n-oxidation as their coenzyme A (CoA) esters. In 3oxidation, 2 carbon fragments are sequentially removed from the carboxyl terminal after dehydrogenation, hydration, oxidation and thiolysis (Devlin, 1986; Stryer, 1981). Mitochondrial f3-oxidation offatty acids The first step in the f3-oxidation cycle is the activation of the fatty acid to a fatty acyl CoA which occurs either in the endoplasmic reticulum or in the 3  Introduction  outer mitochondrial membrane (Devlin, 1986; Montgomery et al., 1990). The activation is performed by an acyl CoA synthase (thiokinase or acyl CoA ligase), of which at least 4 enzymes are known (Montgomery et al., 1990). A short chain enzyme is specific for acetate and propionate, a medium chain enzyme specific for 4 to 10 carbon fatty acids, a long chain enzyme specific for fatty acids longer than 12 carbons and a separate enzyme for arachidonic acid. The 2 enzymes specific for short and medium chain fatty acids are mitochondrial in location whilst the other 2 enzymes are located in the endoplasmic reticulum. Because the mitochondrial membrane is impermeable to CoA and its derivatives, a carrier is necessary for transportation of the fatty acid across the membrane (Devlin, 1986).  This function is performed by carnitine which is  required for the transport of activated fatty acids of chain length 12 to 18 carbons across the mitochondrial membrane. The acyl group is transferred by carnitine palmitoyl transferase I to the hydroxyl group on the carnitine molecule from the sulphur atom of CoA on the outer surface of the membrane. At the inner mitochondrial membrane, the acyl group is transferred from carnitine back to CoA by carnitine palmitoyl transferase II.  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. Inside the mitochondrion, the CoA derivatives are 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 3 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 the third carbon atoms.  4  The 2  Introduction hydrogen atoms are accepted by flavin adenine dinucleotide (FAD) and ultimately, 2 electrons are channelled into the electron transport system. The x,3-unsaturated acyl CoA accepts a molecule of water, a reaction catalyzed by enoyl CoA hydrase to form L-3-hydroxyacyl CoA (Devlin, 1986). L 3-hydroxyacyl CoA is oxidized by 3-hydroxyacy1 CoA dehydrogenase to [3ketoacyl CoA which is further oxidized in the [3-position by f3-ketothiolase. CoA is inserted and cleavage occurs at the [3-carbon to yield acetyl CoA and a saturated acyl CoA with 2 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 [3-oxidized to yield acetoacetyl CoA and subsequently 2 molecules of acetyl CoA. Feroxisomal [3-oxidation offatty acids The [3-oxidation of fatty acids also occurs in peroxisomes, subcellular organelles which are widely distributed in mammalian tissues including kidney, liver, muscle, intestine, heart and spleen (Lazarow, 1987). Peroxisomal [3-oxidation in mammals differs from mitochondrial 13oxidation in 3 ways. First, carnitine is not required for fatty acid entry into the peroxisome (Lazarow, 1987).  Second, the initial dehydrogenation step in  peroxisomes is catalyzed by a cyanide-insensitive oxidase leading to the formation of hydrogen peroxide which is eventually eliminated.  Third, the  enzymes involved in the cycle differ slightly, in that 3 proteins perform the 4 reactions of peroxisomal [3-oxidation whereas in mitochondrial [3-oxidation, 4 proteins are involved. It appears that the role of peroxisomes is to shorten the chain length of relatively long fatty acids for [3-oxidation in the mitochondria, since peroxisomal [3-oxidation is unable to proceed beyond 8 carbons in the shortening of long chain fatty acids.  5  Introduction Omega and omega-i oxidation of fatty acids Metabolism of fatty acids via o- and biotransformation pathways (Devlin, 1986).  0)-i  oxidation represent minor  Primarily, medium chain length  fatty acids undergo metabolism via these oxidative pathways which occur in the endoplasmic reticulum of many tissues. Omega oxidation involves hydroxylation at the methyl carbon on the opposite end from the carboxyl group while o-1 oxidation occurs at the penultimate carbOn atom next to the terminal methyl group.  After hydroxylation, the fatty acid may be further oxidized to a  dicarboxylic acid at which stage f3-oxidation can occur from either end of the molecule.  The co- and 0)-i oxidations are cytochrome P-450 mediated events  (Montgomery et at., 1990).  Metabolism of valproic acid VPA, an 8 carbon, branched chain fatty acid, possesses an unique structure amongst the wide array of anticonvulsant agents. anticonvulsant agents, VPA lacks a nitrogen moiety.  Unlike other  Despite its simple  structure, VPA undergoes extensive biotransformation (figure 1) via several pathways: glucuronidation, 13-oxidation and 1981a; Granneman et at., 1984a).  (0-  and co-i oxidation (Loscher,  Very small quantities (3 to 7%) of the  unchanged drug are recovered in the urine (Schobben et at., 1975; Dickinson et at., 1989). Glucuronidation and f3-oxidation are the 2 major metabolic pathways in both man and rat (Granneman et at., 1984a). The glucuronide conjugate accounts for approximately 11 to 68% of urinary recovery in man (Granneman et at., 1984a; Dickinson et at., 1989). With increasing doses of VPA, glucuronidation, which occurs in the endoplasmic reticulum, increases at the expense of 13-oxidation (Granneman et at., 1984a; Granneman et at., 1984b).  Conjugation with glycine occurs but is a minor  metabolic pathway. 6  /\/c/\  23-diene VPA (E,E)- and (EZ)-isomers  OOH  /  3-keto VPA  24-dIene VPA (E)- and (Z)-isomers  COOH  il.  4-ene VPA  COOH  VPA  HOOC  COOH  COOH  /  2-PSA  COOH  4-keto VPA  O•  HOOC  HO 5-OH VPA COOH  4-OH VPA  OH  N  COO-GIu  VPA. glucuronide  Figure 1. Summary of vaiproic acid metabolism (Yu et al.).  3-OH VPA  COOH  2-ene VPA (B)- and (Z)-Isomers  3-ene VPA (B)- and (Z)-Isomers.  COOH  COOH  2-PGA  COOH  I  Introduction  Mitochondrial 13-oxidation of VPA results in the formation of 2-ene VPA, 3-OH VPA and 3-keto VPA (Granneman et at., 1984a; Li et aL, 1991). In the mitochondria, VPA is activated to VPA-CoA, then dehydrogenated to 2-ene VPA CoA by acyl-CoA dehydrogenase, then hydrated by enoyl-CoA hydrase to 3-OH VPA-CoA and finally dehydrogenated to 3-keto VPA-CoA by 3-hydroxyacyl-CoA dehydrogenase.  The 3-keto VPA metabolite is the terminal product of this  pathway due to prevention of thiolytic cleavage by the enzyme 3-ketoacyl CoA thiolase (Fong and Schulz, 1978) because of the presence of the 2-propyl branch (Li et at., 1991). Peroxisomal 13-oxidation of VPA may also occur since enhanced excretion of 3-keto VPA was observed in rats after pretreatment with clofibrate, a known peroxisomal inducer (Heinemeyer et at., 1985).  VPA is also  hydroxylated to 3-OH VPA microsomally via co-2 oxidation (Prickett and Baillie, 1984). Mean urinary recoveries of the 13-oxidation pathway metabolites account for approximately 25% of the recovered dose in patients (Abbott et at., 1986; Dickinson et at., 1989). Metabolism of VPA via 0)-oxidation results in the formation of 5-OH VPA and 2-propylglutaric acid (2-PGA) (Granneman et at., 1984a). The metabolite, 2PGA, is the end product of co-oxidation and does not undergo further metabolism via 13-oxidation (Kuhara and Matsumoto, 1974). Oxidation of VPA at the co-i  position leads to 4-OH VPA, 4-keto VPA and 2-propylsuccinic acid (2-PSA). The hydroxylated metabolites are not further metabolized to the unsaturated metabolites (Granneman et at., 1984a).  The co- and co-i oxidation pathways  account for approximately 20% of the recovered dose in patients (Abbott et at., 1986). Dehydrogenation of VPA at the y-carbon results in the formation of 4-ene VPA (Granneman et at., 1984a).  4-Ene VPA represents less than 1% of the  recovered dose in man (Abbott et at., 1986; Dickinson et at., 1989).  8  The  Introduction metabolism of VPA to 4-ene VPA is cytochrome P-450 mediated (Rettie et at., 1987). Further metabolism of 4-ene VPA, 3-ene VPA and 2-ene VPA may result in the formation of diunsaturated metabolites. For example, 4-ene VPA and 3ene VPA are metabolized via the 13-oxidation pathway to form 2,4-diene VPA (Rettenmeier et at., 1986a) and 2,3’-diene VPA (Bjorge and Baillie, 1991), respectively.  The major diunsaturated metabolite in humans has been  identified as 2,3’-diene VPA (Acheampong and Abbott, 1985). (E)-2-ene VPA is dehydrogenated to both diunsaturated metabolites and is 13-oxidized to 3-keto VPA (Granneman et at., 1984a; Loscher et at., 1992). Metabolism of 4-ene VPA The metabolism of 4-ene VPA has been studied in rat (Granneman et at, 1984a; Rettenmeier et at., 1985) and rhesus monkey (Rettenmeier et at., 1986a). Eight metabolites of 4-ene VPA were detected by GLC and GCMS from rat bile and the perfusate medium from isolated rat livers (Rettenmeier et at., 1985). The recovered metabolites were identified 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 lactone, 2-PGA and the parent compound, 4-ene VPA. Most metabolites were derived via either 13-oxidation or cytochrome P-450 mediated reactions. metabolic pathways for the biotransformation of 4-ene VPA were assigned:  Six  13-  oxidation on the unsaturated side chain (2,4-diene YPA and 5’-OH-4-ene VPA, cytochrome P-450 mediated), 13-oxidation on the saturated side chain (3’-keto), w-hydroxylation to the primary alcohol and subsequently to the dicarboxylic acid (2-PGA), reduction followed by oxidation to the diacid, epoxidation to the gamma butyrolactone derivative and hydroxylation at the C-3 position to form 3-OH-4-ene VPA. Twenty metabolites of 4-ene VPA were identified in the urine of rhesus monkeys with 59% of the dose recovered in 24 h (Rettenmeier et at., 1986a). In addition to the metabolites identified in rat liver perfusate medium, 9  Introduction other metabolites identified included (E)-3-ene VPA, 4-OH VPA, 4-OH VPA lactone, 4’-OH-4-ene VPA, 4’-OH-4-ene VPA lactone, 3-keto VPA and 2-PSA. Furthermore, 2 isomers of 2,4-diene VPA, a third diene metabolite and the conjugate  glycine  also  of (E)-2,4-diene VPA were  identified.  Again,  glucuronidation and 13-oxidation were identified as the major metabolic pathways and  -  and (0-1 oxidation as minor biotransformation routes.  Metabolite activity The metabolites (E)-2-ene ‘[PA, 4-ene VPA (Loscher, 1981b), 3-ene VPA (Kochen  and  Scheffner,  1980)  (E,E)-2,3’-diene  and  VPA  (Abbott  and  Acheampong, 1988) possess anticonvulsant activity. (E,E)-2,3-diene VPA is as active as (E)-2-ene VPA in mice (Abbott and Acheampong, 1988). (E)-2-ene VPA and 4-ene ‘[PA are the most active of the metabolites, displaying approximately 60 to 90% of the potency of ‘[PA although they are more sedating than ‘[PA in mice (Nau and Loscher, 1984; Loscher and Nau, 1985).  2-Ene VPA was  detectable in mouse plasma and brain 2 days after discontinuation of VPA and may be responsible for the elevated seizure threshold in the absence of detectable VPA brain levels (Loscher and Nau, 1984). Adverse effects Side effects The majority of side effects associated with VPA are mild; nausea, vomiting, diarrhea and abdominal cramps are the most commonly observed (Bruni and Wilder, 1979). Other side effects include transient hair loss, weight gain,  hyperkinesia,  fine  postural  tremor,  drowsiness  and  transient  hallucinations (Dulac et al., 1986). Tremor, weight gain, transient hair loss and limb edema are dose related side effects (Willmore et al., 1991) and occur in 25% of patients on VPA therapy (Smith and Bleck, 1991). Transient and self limiting 10  Introduction neutropenia and thrombocytopenia have also been observed with VPA (Barr et al., 1982) as well as reduced platelet adhesiveness and prolonged bleeding time (Smith and Bleck, 1991). Dementia has also been observed in patients on VPA therapy, although there was prompt remission after withdrawal of the drug (Zaret and Cohen, 1986). Biochemical effects Biochemical effects of VPA include hyperammonemia, inhibition of f3oxidation (Becker and Harris, 1983; Van Den Branden and Roels, 1985; Willmore et al., 1991), inhibition of gluconeogenesis (Becker and Harris, 1983; Rogiers et al., 1985) and hyperglycinemia (Cherruau et al., 1981). Patients on VPA are found to excrete higher amounts of dicarboxylic acids.  This increased excretion is possibly due to impaired 13-oxidation  (Turnbull et al., 1986). Increased amounts of 6 carbon dicarboxylic acids, eg. adipic acid, are also found in the urine of rats treated with VPA (Mortensen et al., 1980). In a 6 year old male with Reye’s syndrome, increased amounts of lactic and adipic acids as well as increased quantities of 2-PGA, the end product of VPA o-oxidation, were recovered in the urine (Kuhara et al., 1985). In isolated rat hepatocytes VPA, (E)-2-ene VPA, 4-ene VPA, 4-OH VPA, 5OH VPA and 2-PSA produced a concentration-dependent inhibition of gluconeogenesis from lactate (Rogiers et al., 1985).  The extent of toxicity in  decreasing order was VPA and 4-ene VPA, 5-OH VPA, 4-OH VPA, (E)-2-ene VPA and 2-PGA. VPA interferes with the folate-dependent one carbon enzyme responsible for glycine cleavage, resulting in hyperglycinemia in patients and animals treated with VPA (Carl, 1986).  Hyperglycinemia and hyperglycinuria are  observed in rats administered VPA chronically for several weeks at doses ranging from  0.3  to  1.2  mmoLlkg (Cherruau et al., 11  1981).  Chronic  Introduction administration of 1% VPA to young rats resulted in significant increases in blood, liver and brain glycine levels (Martin-Gallardo et at., 1985). Hyperammonemia and hyperbilirubinemia are also associated with VPA \TPA inhibits urea  therapy (Matsuda et at., 1986; Ratnaike et at., 19.86). synthesis  in  rat  hepatocytes  (Coude,  1983;  Turnbull  et  at.,  1983).  Coadministration of other anticonvulsants with VPA, particularly phenobarbital or phenytoin, results in increased serum ammonia levels (Warter et at., 1983; Haidukewych et at., 1985; Zaccara et at., 1985; Ratnaike et at., 1986). VPA administration results in carnitine deficiency (Borum and Bennett, 1986; Laub et at., 1986).  VPA forms vaiproylcarnitine derivatives which  themselves are not toxic but cause an increased metabolic need for carnitine to excrete the more toxic metabolites. Patients on VPA therapy display decreased plasma carnitine levels accompanied by elevated blood ammonia levels (Ohtani et at., 1982). successful  in  Oral administration of carnitine 50 mg/kg/day for 4 weeks was correcting  hyperammonemia.  the  Carnitine  VPA  induced  deficiency  may  carnitine be  the  deficiency end  result  and of  hyperammonemia. In mice, VPA exerts an immediate but transient effect on carnitine metabolism (Rozas et at., 1990).  Single doses of VPA in the  therapeutic range for man decreased hepatic levels of free CoA, acetyl CoA and free carnitine in normal infant mice (Thurston et at., 1985). VPA at doses greater than 1 mM may uncouple mitochondrial respiration (Benavides et at., 1982) due to accumulation of valproyl CoA and its further metabolites in the matrix of the hepatic mitochondria (Turnbull et at., 1983) or by altering the integrity of the inner mitochondrial membrane or by actions on the substrate carriers or mitochondrial metabolites (Rumbach et at., 1983). Valproyl CoA in the mitochondrial matrix is a weak inhibitor of 3-oxidation (Sherratt and Vietch, 1984). Inhibition of mitochondrial 13-oxidition by VPA may  12  Introduction be due to sequestration of CoA as valproyl-CoA (Veitch and Van Hoof, 1990). Increased activity of the peroxisomal n-oxidation enzymes in rat liver was observed after chronic administration of VPA (Hone and Suga, 1985; Ponchaut et at., 1991).  Decreased cytochrome P-450 levels were observed in rats  administered VPA (172.8 to 259.2 mg/kg/day) (Cotariu et at., 1985). Pancreatitis Pancreatitis has been associated with \TPA usage (Coulter et at., 1980; Isom, 1984). Until 1991, 24 cases of VPA implicated pancreatitis were reported (Binek et at., 1991; Asconape et at., 1993).  A recent survey of physicians  revealed 15 additional cases of VPA induced pancreatitis (Asconape et at., 1993). A further 6 cases of pancreatitis in addition to hepatic failure with VPA usage have also been reported in the literature (Binek et at., 1991). Most of the cases of VPA implicated pancreatitis (77%) involved patients under the age of 20 years and 68.8% of the 39 cases occurred within the first year of VPA therapy (Asconape et at., 1993).  Furthermore, 76% of the cases were on VPA  polytherapy. VPA pancreatitis manifests itself initially as abdominal pain and vomiting (Parker et at., 1981; Wyllie et at., 1984; Rosenberg et at., 1987). Increased serum amylase and lipase levels have also been observed in some cases (Parker et at., 1981; Wyllie et at., 1984; Rosenberg et at., 1987; Lott et at., 1990). Although 3 patients have died from VPA induced pancreatitis, in most cases the reaction is mild with rapid resolution once VPA is discontinued (Asconape et at., 1993). VPA induced pancreatitis appears to be an idiosyncratic reaction, unrelated to VPA dosage or serum concentrations. Embryotoxicity / teratogenicity VPA possesses the potential for teratogenicity in all species thus far investigated, including man, monkey and rodent (Cotariu and Zaidman, 1991).  13  Introduction Although the exact mechanism by which VPA exerts its teratogenic effects has not  been  established,  possible mechanisms  include  alteration  of fetal  glutathione status, alteration of fetal lipid metabolism, effects on folate or zinc levels or effects on the regulation of embryonic pH (Cotariu and Zaidman, 1991). VPA teratogenicity appears to require quite rigid structural specificities (Nau and Scott, 1987; Nau and Siemes, 1992).  Structural requirements for  teratogenic activity include a free carboxyl group attached to a carbon atom which is substituted with only 2 alkyl chains (Nau and Siemes, 1992). The -H atom is quite important for teratogenic activity; substitution abolishes teratogenic activity as does the introduction of an co-2 double bond (2-ene VPA) (Nau and Scott, 1987). The introduction of a double bond in the o (4-ene VPA) position has no effect on the malformation rate. A decrease in teratogenicity is observed if the alkyl chain length is shortened or lengthened (Nau and Scott, 1987; Nau and Siemes, 1992). Teratogenic activity may be expressed through chiral interactions of the branched chain carboxylic acids with various embryonic constituents important in developmental processes (Nau and Siemes, 1992). Fetal vaiproate syndrome includes a flat nasal bridge, an upturned nose, a long upper lip, downturned mouth, long, thin overlapping fingers and toes and hyperconvex nails (Yerby et al., 1992). A consistent facial phenotype (epicanthal folds, flat nasal bridge etc.) was observed in 7 children who had been exposed to VPA in utero (DiLiberti et al., 1984). A 1 to 2% incidence in humans of VPA induced neural defects is associated with VPA administration early in pregnancy (Nau and Siemes, 1992) and may be due to interferences with zinc and other trace element metabolism (Weinbaum et al., 1986).  Interference with embryonic folate metabolism  (Wegner and Nau, 1992) with a resultant folate deficiency has been implicated  14  Introduction to play an important role in the induction of neural tube defects by VPA (Dansky et al., 1992). Neural tube defects are manifested mainly as spina bifida aperta, but incidents of myelomeningocele have been reported (Nau and Siemes, 1992). The intensity of the teratogenic response as measured by neural tube defects in mice, was dependent on the concentrations of VPA achieved in the mother and fetus (Nau, 1985). In pregnant mice, both the dose of VPA and area under the serum concentration versus time curve (AUC) correlate with embryolethality and fetal weight retardation (Nau, 1985). Although therapeutic doses of VPA in pregnant women do not affect fetal growth, the risk for fetal perinatal distress increases with higher doses of VPA (Jager-Roman et al., 1986). VPA administration to rhesus monkeys at a human therapeutic dose of 20 mg/kg/day during organogenesi did not yield any adverse effects (Mast et al., 1986). However, a dose of 200 mg/kg/day caused low birth weights, craniofacial and skeletal defects and a dose of 300 mg/kg/day was embryolethal (Mast et al., 1986). A later study in rhesus monkeys (20 to 600 mg/kg) also resulted in a dose dependent developmental toxicity which manifested itself as increased embryo/fetal mortality, intrauterine growth retardation and craniofacial and skeletal defects (Hendrickx et al., 1988). In whole rat embryos, VPA at doses greater than 40 mg/kg/day caused abnormal development in 30% of embryos (Lewandowski et aL, 1986). In rats administered VPA 300 mg/kg daily on embryonic days 7 to 18, a decrease in maternal bodyweight and fetal weight and an increase in malformations were observed (Vorhees et at., 1991). Hepatotoxicity Although side effects associated with VPA use are generally mild, a number of cases of fatal hepatotoxicity have been reported (Kochen et at., 1984; 15  Introduction Jager-Roman et al., 1986). Hepatotoxicity associated with VPA usage is of 2 types, either dose-related or idiosyncratic (Dreifuss et al., 1987). Dose-related VPA hepatotoxicity resolves with a decrease in dosage or discontinuation of the drug. Up to 44% of patients on VPA therapy demonstrate dose related increases in liver enzyme levels (Sussman and McLain, 1979) but these increases are not predictive of hepatotoxicity since less than 0.01% of patients develop fatal hepatotoxicity (Dreifuss et al., 1987).  The fatal but rare idiosyncratic  hepatotoxicity is irreversible and dose-independent. A recent survey of reported cases of fatal hepatotoxicity associated with VPA usage in the United States between 1978 and 1984 concluded that age and polytherapy were the major determinants (Dreifuss et al., 1987). The incidence of VPA induced hepatotoxicity decreased with increasing age.  The incidence  was highest in those children under 2 years of age on polytherapy (1/500) compared to the same age group on monotherapy (1/7000). incidence of VPA hepatotoxicity was 1/10,000.  The overall  Many of the patients who  developed hêpatotoxicity suffered from other medical conditions including mental  retardation,  developmental  delay,  congenital  abnormalities  and  metabolic disorders which conspired to place them at higher risk. A follow-up study for the period 1985 to 1986 demonstrated a decline in the incidence of hepatic fatalities related to VPA from 1/10,000 to 1/49,000.  This occurred  despite an overall increased usage of VPA but as a single agent rather than in polytherapy (Dreifuss et al., 1989). VPA hepatotoxicity is characterized clinically by loss of appetite, nausea, vomiting, edema, abdominal distress, lethargy and malaise (Dreifuss et al., 1987).  The onset is usually within 90 days of initiating VPA therapy.  Microvesicular steatosis is the most prominent finding with VPA hepatotoxicity (Zimmerman and Ishak, 1982) and resembles the lesions seen in Jamaican  16  Introduction Vomiting Sickness (JVS), Rey&s Syndrome (RS) and 4-pentenoic acid (PA) toxicity (Lewis et al., 1982; Nau and Loscher, 1984).  Clinically, VPA  hepatotoxicity shares similar manifestations with JVS, RS and PA toxicity. Reye’s syndrome was first described in 21 children (Reye et al., 1963) and the etiology of RS remains unknown.  RS involves encephalopathy and fatty  degeneration of the liver, kidney and occasionally other organs. Other clinical features include fever, convulsions, vomiting, hypoglycemia and increased serum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvic transaminase (SGPT) levels. Hypoglycin A (hypoglycin, L-x-amino-2-methylenecyclopropylpropionic acid), an amino acid found in unripe akee fruit and in the seeds of several varieties of maple trees, is responsible for JVS (Tanaka et al., 1976). Hypoglycin is metabolized to methylenecyclopropylacetic acid (MCPA) which forms an enoyl CoA ester that inhibits fatty acid metabolism and gluconeogenesis (Kean, 1975). The toxicity results from inhibition of several acyl-CoA dehydrogenases (Kean, 1975) and is possibly worsened by sequestration of mitochondrial carnitine and coenzyine  A  by  MCPA  (Bressler  et  al.,  1969).  Hypoglycin  causes  hyperammonemia in rats (Glasgow, 1983) and hypoglycemia is common in patients with JVS (Jelliffe and Stuart, 1954). In addition, encephalopathy and fatty degeneration of the viscera are common features. The incidence of JVS decreases after the age of 10 (Reye et al., 1963). 4-Pentenoic acid is a structural analogue of hypoglycin. produces similar features to JVS and RS including  In rats, PA  hypoglycemia, fatty  degeneration of the liver, hyperammonemia and increased SGOT levels (Glasgow and Chase, 1975). PA inhibits fatty acid 13-oxidation (Hart et al., 1989) via its 13-oxidation metabolites, pent-2,4-dienoyl-CoA (Glasgow, 1983) and 3keto-4-pentenoyl-CoA (Schulz, 1983) through inhibition of the 13-oxidation  17  Introduction  enzyme 3-ketoacyl-CoA-thiolase (Fong and Schulz,  1978;  Schulz,  1983).  Although 3-keto-4-pentenoyl-CoA is a minor metabolite of PA, it is a more effective inhibitor of the enzyme (Schulz,  1983).  The inhibition and  morphological manifestations caused by PA can be partially reversed by L carnitine (Billington et at., 1978; Sugimoto et at., 1990). The formation of toxic metabolites is thought to be responsible for VPA hepatotoxicity. Because of the similarities between VPA hepatotoxicity, JVS, RS and PA toxicity, a metabolite of VPA similar in structure to PA and MCPA may be responsible for VPA associated toxicity. The hepatotoxicity of VPA is thought to be caused by its mono- and/or diunsaturated metabolites (Kochen et at., 1984). An increased formation of the diunsaturated metabolites appears to be characteristic in fatal hepatic failure. 4-Ene VPA was detected in the urine of 6 patients and 4,4’-diene in 3 of the patients who died of VPA related hepatic failure (Scheffner et at., 1988).  The metabolite, 4-ene VPA, is structurally  similar to 4-pentenoic acid and MCPA and may play an important role in VPA associated hepatotoxicity.  One 7 year old patient on phenobarbital and VPA  therapy who died from hepatic failure resembling Reye’s syndrome (Kochen et at., 1983) demonstrated plasma and urine concentrations 4 to 5 times the normal levels of several unsaturated metabolites including 4-ene VPA. The 4,4’diene VPA was also present. The f3-oxidation pathway appeared to be inhibited as evidenced by the absence of 3-keto VPA, the end product of VPA 3-oxidation. In hepatotoxicity studies of VPA and metabolites in young rats, 4-ene VPA and 2,4-diene VPA caused hepatic steatosis and inhibition of 13-oxidation (Granneman et al., 1984c; Kesterson et at., 1984).  These unsaturated  metabolites may be further metabolized to the chemically reactive 3-keto-4-ene VPA which could then alkylate mitochondrial proteins including enzymes involved in 13-oxidation (Rettenmeier et at., 1986b). This mechanism is based on  18  Introduction  observations with 4-pentenoic acid which is transformed to a reactive intermediate that alkylates and destroys the terminal enzyme of the 13-oxidation pathway (Schulz, 1983; Fong and Schulz, 1983). In rat liver homogenates, VPA and 4-ene VPA caused inhibition of decanoic acid f3-oxidation (Bjorge and Baillie, 1985).  VPA depletes free CoA  poois (Fears, 1985) and may cause a transient and mild inhibition of the f3oxidation pathway by sequestration of CoA (Kesterson et aL, 1984). 4-Ene VPA produces a more potent and prolonged inhibition by forming CoA esters which directly  inhibit  enzymes  in  the  13-oxidation  pathway.  VPA inhibits  mitochondrial 13-oxidation by forming vaiproyl CoA which is a weak inhibitor but acts at a different site than 4-pentenoic acid and hypoglycin (Sherratt and Veitch, 1984). VPA, 4-ene VPA and 2,4-diene VPA trap intramitochondrial free CoA (Ponchaut et at., 1992). The glutathione conjugate of (E)-2,4-diene VPA was identified in the bile of rats administered either (E)-2,4-diene VPA or 4-ene VPA while the N-. acetylcysteine conjugate of (E)-2,4-diene VPA was a major urinary metabolite (Kassahun et at., 1991). Since (E)-2,4-diene VPA is a 13-oxidation metabolite of 4-ene VPA, in vivo activation to a CoA ester which readily interacts with glutathione may explain the high urinary recovery.  In patients with VPA  associated hepatic failure, the levels of (E)-2,4-diene VPA recovered in the urine as the N-acetylcysteine conjugate were 3 to 4 times the levels observed in healthy patients (Kassahun et at., 1991).  (E)-2-ENE VPA  The  metabolite  (E)-2-ene VPA has  been touted  as  a  potential  anticonvulsant agent due to its lack of teratogenicity (Nau et at., 1984; Nau and Loscher, 1986; Nau, 1986), lack of embryotoxicity (Loscher et at., 1984;  19  Introduction Lewandowski et al., 1986; Nau, 1986) and apparent lack of hepatotoxicity (Kesterson et al., 1984; Schafer and Luhrs, 1984; Loscher, 1992) in experimental animals. Anticonvulsant activity (E)-2-ene VPA is as potent as VPA with respect to anticonvulsant activity (Loscher et al., 1984).  In experimental seizure models, (E)-2-ene VPA is as  effective as VPA on a weight for weight basis (Loscher and Nau, 1985). In rats (E)-2-ene VPA is 2 to 3 times more potent than VPA in elevating the clonic threshold of pentylenetetrazole (PTZ) induced seizures (Semmes and Shen, 1991). The spectrum of activity of (E)-2-ene VPA is similar to that of VPA without the potential for embryotoxicity even at doses of 600 mg/kg (Loscher et al., 1984).  In 4 different models of anticonvulsant activity, (E)-2-ene VPA  activity was similar to that of VPA. (E)-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 to 300 mg/kg of (E)-2-ene VPA were more sedating than VPA. However, sedation was not observed in rats or gerbils at these doses. The anticonvulsant activity of (E)-2ene VPA is of shorter duration (2 h compared to 5 h) than VPA in mice after doses of 4 mmollkg (Keane et al., 1985). Teratogenicity (E)-2-ene VPA possesses very little teratogenic potential (Nau, 1986). At similar doses to VPA, (E)-2-ene VPA is not teratogenic in rats (Vorhees et al., 1991).  In a murine model, (E)-2-ene VPA did not affect embryonic folate  metabolism at doses of 500 mg/kg i.p. (Wegner and Nau, 1992).  In rats  administered (E)-2-ene VPA at doses of 300 mg/kg daily on embryonic days 7 to  20  Introduction 18, no increases in the percentage of resorptions or malformations were observed (Vorhees et al., 1991).  Increasing the dose to 400 mg/kg decreased  fetal bodyweight by approximately 8% but with no change in resorptions or malformations. Abnormal embryo development or retardation of growth was not observed after administration of 2 doses of 400 mg/kg on day 10 of pregnancy in rats (Klug et aL, 1990). In whole rat embryos, (E)-2-ene VPA did not produce any adverse effects at doses up to 200 mg/kg/day (Lewandowski et al., 1986). Metabolism To date, the metabolism of 2-ene VPA has only been examined in rats. After (E)-2-ene VPA administration, the major metabolites in the serum are the 13-oxidation products, (E,E)-2,3’-diene VPA and 3-keto VPA and 2,4-diene VPA (Loscher et aL, 1992). 3-Ene VPA has also been detected in serum, most likely resulting from isomerization (Vorhees et al., 1991). In addition, in urine up to 2% of the administered dose was recovered as V.PA (Granneman et al., 1984a).  CARBAMAZEPINE Carbamazepine (CBZ,  5-.carbamoyl-5H-dibenz [b,f] azepine,  carbamoyl  iminostilbene), an iminostilbene derivative (Eadie and Tyrer,  1989), is  structurally similar to the tricyclic antidepressants (Kutt, 1989).  CBZ is  effective in the treatment of a wide variety of seizure types including partial, generalized tonic-clonic and mixed seizure disorders (Rail and Schieifer, 1990). After oral administration in man, CBZ undergoes slow and erratic absorption from the gastrointestinal tract (Rail and Schleifer, 1990).  Peak  plasma levels are attained in 2 to 8 h. Therapeutic plasma levels of CBZ are in the range of 3 to 14 ig/mL. CBZ has a relatively long half-life ranging from 8 to 72 h. The drug is highly plasma protein bound (75 to 90%). 21  Introduction Carbamazepine undergoes extensive metabolism (figure 2) via the liver micros omal enzyme system, with less than 1% of the parent drug excreted unchanged in the urine (Bertilsson and Tomson, 1986).  The major route of  metabolism is the epoxide-diol pathway where CBZ is transformed to carbamazepine-10,11-epoxide (CBZE) which is further hydrolyzed to trans 10,1 1-dihydroxy-lO, 1 1-dihydro-CBZ (trans-CBZ-diol, CBZD).  In volunteers,  approximately 22% of a single dose is recovered in the urine as CBZD (Eichelbaum et at., 1984). In chronic therapy, the urinary recovery of CBZD accounts for 30 to 60% of the administered dose.  Other metabolic pathways  include hydroxylation in the 2, 3 and 9 positions and account for approximately 15% of the dose. Carbamazepine induces its own metabolism as well as the metabolism of other drugs via induction of the hepatic microsomal enzyme system (Bertilsson and Tomson, 1986). CBZ induces the metabolism of warfarin (Hansen et al., 1971; Ross and Beeley, 1980), phenytoin (Hansen et at., 1971), theophylline (Rosenberry et at., 1983), clonazepam (Lai et at., 1978), tetracycline (Neuvonen et at., 1975), haloperidol, ethosuximide, oral contraceptives (Fernandez et al., 1985) and VPA (Reunanen et at., 1980). In long term treatment, CBZ induces its own metabolism (Pynnonen, 1979; Bertilsson and Tomson, 1986; Rall and Schleifer, 1990) via induction of the epoxide-diol pathway (Eichelbaum et at., 1984). CBZ autoinduction in man has been reported to occur within 2 or 3 days (Pynnonen, 1979) although maximal CBZ autoinduction may require 3 to 4 weeks (Bleck, 1990). In a group of 77 patients, autoinduction of CBZ metabolism appeared to be complete within one week of initiating CBZ therapy or upon a change in dose (Kudriakova et at., 1992).  CBZ autoinduction is dose dependent in man (Rapeport et at., 1983;  Kudriakova et at., 1992) as evidenced by dose dependent increases in antipyrine  22  I  3.OH.CBZ  CONH  +  2.OH.CBZ  COHH  /  /  /  /  /  •1  /  Figure 2. Summary of carbamazepine metabolism.  9-OH-CBZ  CON H  CH OH 2  I  (CBZ)  Carbamazepine  2 COHH  /  trans-CBZ.dIoI  2 CONH  epoxide hydrolase  CBZ-1O11poxdo  CONH  0  0  S.  Introduction clearance in healthy volunteers (Rapeport et al., 1983). In rats, chronic administration of CBZ (25 mg/kg i.p. every 12 h for 7 days) resulted in decreased protection against maximum electroshock seizures (Farghali-Hassan et al., 1976). In rats administered oral doses of 315 mol/kg (approximately 75 mg/kg/day) for 4 days, CBZ increased liver weight and induced cytochrome P-450, NADPH cytochrome P-450 reductase, aminopyrine N-demethylase and UDP-glucuronyltransferase (Wagner and Schmid, 1987). Regnaud et al. (1988) attempted to determine the dose dependency of CBZ induction in rats. CBZ was administered intraperitoneally at 60, 120 and 200 mg/kg/day for 4 days.  Epoxide hydrolase activity appeared to increase with  increasing dose as did aminopyrine N-demethylase activity. Cytochrome P-450 and aniline hydroxylase activity did not increase at doses higher than 120 mg/kg/day. Carbamazepine-lO, 11 -epoxide  Carbamazepine-10,11-epoxide is an active metabolite and serum levels are usually 15 to 55% and 5 to 81% of the parent compound in adults and children, respectively (Bertilsson and Tomson, 1984).  CBZE possesses  anticonvulsant activity similar to CBZ in various animal models (Tomson and Bertilsson, 1991). In 7 adult patients, no change was observed in seizure control when CBZE was substituted for CBZ (Tomson et al., 1990).  CBZE was  equipotent to CBZ in controlling pain due to trigeminal neuralgia (Tomson and Bertilsson, 1984). CBZE has a shorter half-life than CBZ (approximately 6 h) in man and is metabolized almost completely to CBZD, with 67 to 100% of the epoxide dose recovered as CBZD (Tomson et al., 1983; Spina et al., 1988). When CBZE was administered to rats intraperitoneally at a dose of 100 mg/kg daily for 3 and 7 days, maximal induction of epoxide hydrolase and glutathione transferase appeared to be achieved in 3 days (Jung et al., 1980). 24  Introduction However, CBZE treatment did not alter total hepatic cytochrome P-450 levels.  INTERACTION BETWEEN VPA AND CBZ Because VPA undergoes such extensive biotransformation in the body, coadministration of metabolic inducing agents may result in the increased formation of potentially toxic metabolites.  Interactions of VPA with other  anticonvulsant drugs have been studied most extensively. These interactions may be pharmaceutical, pharmacokinetic or pharmacodynamic (Smith and Bleck, 1991). In polytherapy with other anticonvulsants, (CBZ, phenytoin and phenobarbital) shorter serum VPA half-lives are observed compared to monotherapy (Kutt, 1984). In addition, the ratio of VPA steady-state plasma levels to dose is much lower in children receiving other anticonvulsants (Sackellares et al., 1981). CBZ has been shown to decrease the plasma levels of VPA by inducing VPA metabolism (Kutt, 1984). CBZ affects VPA disposition in both pediatric and adult patients (Kutt, 1984; Baciewicz, 1986).  Lower serum VPA  concentrations, despite higher VPA doses, have been observed in both adult (Reunanen et al., 1980) and pediatric patients (Abbott et al., 1986) on VPA and CBZ compared to VPA alone.  VPA serum half-life decreased and plasma  clearance (Clv) increased when VPA was coadministered with CBZ in adult epileptic patients (Hoffman et al., 1986). Bowdie and coworkers (1979) demonstrated that administration of CBZ at a dose of 200 mg daily in 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.  25  Introduction In 5 adult volunteers, the plasma clearance of VPA was significantly increased following CBZ coadministration (Panesar et al., 1989). The volume of distribution (Vd) of VPA remained unchanged after CBZ, thus providing support for the induction of VPA metabolism by CBZ. In vitro, CBZ does not displace \TPA from its plasma protein binding sites (Mattson et al., 1982) so a change in the Vd was not expected. 4-Ene VPA serum levels were unchanged after CBZ administration but urinary recovery of this metabolite was increased after CBZ. The serum data suggested induction of the CBZ.  0)-  and 0)-i oxidative pathways by  The major unsaturated metabolite, (E)-2-ene VPA, was significantly  reduced in serum and urine suggesting at first that induction of 13-oxidation via peroxisomes had occurred. The 13-oxidation end product, 3-keto VPA, however. was not increased suggesting that (E)-2-ene VPA metabolism was shunted into alternate pathways. Furthermore, urinary recoveries based on the assay of VPA and metabolites could not confirm an increase in VPA metabolism.  Thus, it  would appear that either VPA metabolite elimination occurred via non-renal routes or the assay failed to detect a significant proportion of the VPA metabolites.  The urinary recovery of VPA and metabolites representing  approximately 65% of the dose was consistent with other investigators (Pollack et al., 1986). In a study performed in our laboratory (unpublished data), significant differences were observed between the VPA metabolite profiles of 16 pediatric patients on VPA and CBZ polytherapy and the profiles of 37 pediatric patients on VPA monotherapy.  VPA and unsaturated metabolites were significantly  reduced in the serum of the VPA and CBZ group. The pediatric patient profiles did not show an increase in urinary 4-ene VPA elimination yet decreased serum 4-ene VPA concentrations were observed with CBZ coadministration. Induction of 4-ene VPA metabolism may have occurred, resulting in the formation of  26  Introduction secondary f3-oxidation metabolites such as 3’-keto-4-ene VPA and the postulated reactive 3-keto-4-ene VPA which our assay did not detect. Urinary profiles were generally a reflection of the serum data. Thus, there was apparent induction of VPA metabolism via the  0)-,  0)-i and 13-oxidation pathways by CBZ. Again, the  net recovery of VPA and metabolites in the urine of the VPA and CBZ group did not account for the increased VPA metabolism. An apparent induction of the  (0-  and e-1 oxidation pathways of VPA metabolism was also observed in another pediatric group on combined VPA and CBZ therapy (Kassahun et at., 1990).  CYTOCHROME P-450 The hepatic microsomal mixed-function oxidase systems consists of cytochrome P-450 (a hemoprotein), NADPH-cytochrome P-450 reductase and phospholipids (Lu and Levin, 1974.) Cytochrome P-450 is a fairly ubiquitous enzyme, present in virtually every tissue including lung, small intestine, liver, adrenals, testis, kidney and duodenum (Okey, 1990) with the exception of erythrocytes and striated muscle (Guengerich, 1991). Cytochrome P-450 is also present in the mitochondria and has been shown to be involved in the metabolism  of carcinogens  including aflatoxin  Bi,  benzo[a]pyrene  and  dimethylnitrosoamine (Niranjan et at., 1984). For the purposes of this study, only cytochrome P-450 in hepatic microsomes was measured. Cytochromes P-450 represent the main group of phase 1 enzymes which are responsible for the biotransformation of hydrophobic molecules to more hydrophilic molecules which can undergo further metabolism by phase II enzymes prior to their excretion in either urine or bile (Leroux et at., 1989). However, situations arise where metabolism by cytochrome P-450 results in the formation of a reactive metabolite which may ultimately cause hepatotoxicity as in the case of cocaine (Boelsterli et at., 1992) and VPA (Rettie et at., 1987).  27  Introduction The expression of the constitutive forms of cytochrome P-450 is dependent on the sex, age and strain of the animal (Soucek and Gut, 1992). In addition, levels of cytochrome P-450 may be influenced by growth hormone, the physiological status of the animal, starvation and hypertension. As an example, nutritional deficiences generally result in decreased rates of xenobiotic metabolism in rat liver microsomal fractions (Yang et al., 1992). Xenobiotics capable of inducing cytochrome P-450 can be grouped into 6 categories: 1) the polycyclic aromatic hydrocarbons (eg. 3-methylcholanthrene, 3-napthoflavone), 2) the barbiturates (eg. phenobarbital), 3) steroids (eg. dexamethasone, pregnenolone-l6oc-carbonitrile), 4) simple hydrocarbons with aliphatic chains (eg. ethanol, acetone), 5) the hypolipidemic drugs (eg. clofibrate) and 6) the macrolide antibiotics (eg. triacetyloleandomycin) (Soucek and Gut, 1992).  Induction of cytochrome P-450 can occur either via transcriptional  activation, mRNA stabilization, or by protein stabilization. The nomenclature of Levin and co-workers (Ryan and Levin, 1990) will be used in this thesis when referring to the isozymes of cytochrome P-450.  A  summary comparing their nomenclature to that of Nelson et al., (1993) is provided in table 1.  In humans, orthologues have been identified for rat  cytochromes P-450j and P-450p (Soucek and Gut, 1992). Induction of cytochrome P-450 by phenobarbital Phenobarbital (PB) is an effective inducer of a number of isozymes of cytochrome P-450 in rats and other laboratory animals (Waxman and Azaroff 1992). In addition to induction of cytochrome P-450, PB also induces a number of other enzymes which are involved in the metabolism of xenobiotics including aldehyde dehydrogenase, epoxide hydrolase, NADPH-dependent:cytochrome P 450  reductase,  UDP-glucuronyltransferase  and  glutathione  transferases.  Induction is invoked by the parent compound itself as opposed to its major 28  Introduction Table 1.  Summary comparing the nomenclature of Ryan and Levin (1990) and Nelson et al. (1993) for isozymes of cytochrome P-450 purified from rat liver microsomes.  Ryan and Levin  Nelson et al.  cytochrome P-450a cytochrome P-450b cytochrome P-450e cytochrome P-450f cytochrome P-450g cytochrome P-450h cytochrome P-450j cytochrome P-450k cytochrome P-450p  CYP2A1 CYP2B1 CYP2B2 CYP2C7 CYP2C13 CYP2C11 CYP2E1 CYP2C6 CYP3A1  Adapted from Nelson et al. (1993), Ryan and Levin (1990) and Soucek and Gut (1992).  29  Introduction metabolite, p-hydroxyphenobarbital (Cresteil et al., 1980). Phenobarbital induction results in proliferation of smooth endoplasmic reticulum within liver cells, increased liver weight (Remmer and Merker, 1963), liver tumour promotion and a general stabilization of liver microsomal proteins (Rees, 1979; Waxman and Azaroff, 1992). After administration of PB to rats, a rapid increase in the cytochrome P-450 levels in rough endoplasmic reticulum is observed within 3 h and maximal levels are achieved within 6 h (Ernster and Orrenius, 1965).  Six hours after the administration of PB, the levels of  cytochrome P-450 in the smooth endoplasmic reticulum slowly start to increase and after 12 h surpass the levels achieved in the rough endoplasmic reticulum. Maximal induction with phenobarbital is achieved in 3 days compared to 24 h with 3-methylcholanthrene treatment (Greim et aL, 1981).  With the  barbiturates, the extent of induction in rats is directly related to plasma halflife, i.e. compounds possessing longer plasma half-lives are more effective inducers of cytochrome P-450 (Toannides and Parke, 1975).  Total hepatic  cytochrome P-450 levels in rat liver returned to baseline levels within 5 days after discontinuation of PB injections (Ernster and Orrenius, 1965). The half lives of cytochromes P-450b and P-450e are approximately 37 h (Parkinson et al., 1983). Some isozymes of cytochrome P-450 in rats are modestly induced after PB treatment while others are more dramatically affected (Waxman and Azaroff, 1992). For example in adult rats, PB treatment induces a 2 to 4 fold increase in cytochromes P-450a (Thomas et al., 1981) and P-450k (Waxman et at., 1985) and modestly induces cytochrome P-450p (Heuman et at., 1982). Conversely, up to 40 fold increases in cytochromes P-450e and P-450b have been reported (Thomas et at., 1981; Thomas et at., 1987). In addition to PB, phenothiazine, SKF-525A, Ardor 1254, isosafrole, trans-stilbene oxide (Thomas et at., 1981), acetone and  30  Introduction ethanol (Ryan et at., 1982) also induce cytochromes P-450b and P-450e in rats. Cytochromes P-450b and P-450e share greater than 97% amino acid sequence homology (Fujii-Kuriyama et at., 1982). Induction of cytochrome P-450 by PB in rat liver is due primarily to newly synthesized cytochrome P-450b/e protein that results from increased steady state levels of cytochrome P-450b/e mRNA (Phillips et at., 1981; Waxman and Azaroff, 1992). A 20 fold induction in mRNA was observed after PB induction in rats (Phillips et at., 1981). Stabilization of mRNA or proteins is not believed to be involved in the mechanism of PB induction. Microheterogeneity of cytochromes P-450b and P-450e also exists in some strains of rats (Vlasuk et at., 1982; Wilson et at., 1987; Oertle et at., 1991). In male Sprague-Dawley rats, PB induction resulted in the identification of 6 members of the cytochrome P-450b/e family by monoclonal antibodies and partial sequence analysis of tryptic peptides (Oertle et al., 1991). Three of the 6 proteins belonged to the cytochrome P-450b family whilst the other 3 were identified as members of the cytochrome P-450e family. Cytochrome P-450b metabolizes a wide spectrum of lipophilic drugs in addition to steroids including androgens and androstenedione (Waxman and Azaroff, 1992). Cytochrome P-450e has a similar substrate profile but is not as active as cytochrome P-450b.  The differences in activity between these 2  isozymes may arise due to several factors. The cytochrome P-450e isozyme may be more susceptible to denaturation during the purification process or requires the presence of as yet unknown specific phospholipids upon reconstitution (Christou et at., 1987). Alternatively, it is possible that activation of cytochrome P-450b occurs during the purification process from a membrane environment to a reconstituted system.  31  Introduction CLINICAL IMPLICATIONS Enzyme induction is “the process which increases the rate of synthesis of an enzyme relative to its normal rate of synthesis in the uninduced organism” (Gelboin and Wiebel, 1971).  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 parent compound and its metabolites in addition to their elimination (Gillette, 1979) and possibly result in the formation of potentially toxic metabolites. Such is the case for VPA where induction of metabolism appears to play a major role in the production of toxicity.  In a retrospective study of fatal  hepatotoxicity associated with VPA usage, young children on polytherapy were more susceptible to development of toxicity than those on VPA monotherapy (Dreifuss et at., 1987). In young rats, PB pretreatment was necessary for VPA to demonstrate liver toxicity in young rats (Kesterson et at., 1984). At low doses of 350 mg/kg, VPA did not produce microvesicular steatosis unless rats were pretreated with PB (Lewis et at., 1982). Glucuronidation and  0)-  and 0)-i oxidation of VPA were  reported to increase following PB induction (Watkins et at., 1982; Heinemeyer et at., 1985). The potential hepatotoxin, 4-ene ‘[PA, which was produced via the cytochrome P-450 oxidation of VPA, was observed only with microsomes from phenobarbital treated rats (Rettie et at., 1987). At low concentrations of 4-ene VPA (10 ig/mL), significant alterations to the membrane permeability of guinea pig hepatocytes were not observed unless phenytoin or phenobarbital was also present (Yu et at., 1991). Clofibrate (CFB) pretreatment in rats resulted in enhanced 13-oxidation of  32  Introduction VPA (Heinemeyer et al., 1985). Clofibrate is a known peroxisomal proliferator (Lazarow, 1987) and these observations suggested that peroxisomal 13-oxidation of VPA had occurred. There was also some microsomal induction since excretion of 4-OH VPA in rats was reported to increase following CFB treatment (Heinemeyer et al., 1985).  In rats, 3-methyicholanthrene pretreatment also  induced 3-OH VPA formation. Increased production of 4-ene VPA by microsomal metabolism which might then be readily converted by 13-oxidation to a reactive metabolite could have serious consequences regarding the risk potential of the VPA and CBZ drug combination. CBZ is not likely to be a peroxisomal inducer because it does not contain a carboxyl group, like other known peroxisome proliferators (Lundgren et al., 1987). However, the major metabolite of CBZ is an epoxide which in turn is metabolized by epoxide hydrolase to CBZD (Tybring et al., 1981; Eichelbaum et al., 1985).  Increased epoxide hydrolase activity with  peroxisome proliferation has been reported (Oesch and Schladt, 1987; Moody and Hammock, 1987). Since CBZ induces its own metabolism via induction of the enzymes of the epoxide-diol pathway (Eichelbaum et al., 1985), enhanced f3oxidation may result. Our goal, then, is to detail the VPA and CBZ interaction based on the changes in VPA metabolism resulting from CBZ induction. The rat will be used as the model. Although CBZ is known to be an enzyme inducer, neither the time course of induction nor the extent of induction has previously been determined.  The effects of CBZE on microsomal enzymes will also be  investigated. VPA and (E)-2-ene VPA metabolite profiles will be compared in animals pretreated with CBZ, CBZE, PB and CFB. The hypothesis to be tested is that CBZ enhances VPA toxicity through enhanced production of toxic ‘[PA metabolites.  33  SPECIFIC OBJECTiVES  1.  To determine the time course and the extent of induction of hepatic microsomal enzymes in the rat by CBZ at a given dose.  The effects  produced by CBZ will be compared to the commonly used inducing agent PB and to CFB, a known peroxisomal inducer. 2.  To determine the time course and the extent of induction of hepatic microsomal enzymes in the rat by CBZE at a given dose. These results will be compared to those obtained for CBZ, PB and CFB.  3.  To determine the contribution by CBZE to the overall induction produced by CBZ.  4.  To identify the isozyme(s) of cytochrome P-450 induced by CBZ and CZBE and determine if the same isozyme(s) are induced by PB.  5.  To identify the metabolites from the in vitro metabolism of VPA using microsomal fractions from CBZ, CBZE, PB and CFB treated rats.  6.  To identify the products resulting from the in vitro metabolism of (E)-2ene VPA using microsomal fractions from CBZ, CBZE, PB and CFB treated rats.  34  EXPERIMENTAL  REAGENTS AWD MATERIALS Vaiproic acid and metabolites Vaiproic acid (di-n-propylacetic acid) was obtained from K and K Fine Chemicals, ICN Biochemicals Inc. (Plainview, NY). The metabolites, (E)-2-ene VPA, 3-ene VPA, 4-ene VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3-keto VPA, 4keto VPA, 2-propylgiutaric acid (2-PGA) and 2-propylsuccinic acid (2-PSA) used for the preparation of the calibration curves and for in vitro incubations were synthesized in our laboratory as reported previously (Acheampong et al., 1983). (E,E)-2,3’-diene VPA was synthesized in our laboratory as reported elsewhere (Acheampong and Abbott, 1985) as was (E)-2,4-diene VPA (Lee et al., 1989). Internal standards ]4-ene VPA, 7 H 2 ]E-2-ene VPA, [ 7 H 2 The deuterated internal standards, [ ]4-keto VPA and 7 H 2 13-keto VPA, [ 7 H 2 ]5-OH VPA, [ 7 H 2 ]3-OH VPA, [ 7 H 2 [ ]VPA were synthesized in our laboratory (Zheng, M.Sc. thesis, 1993). 27 H 2 [ Methylgiutaric acid (2-MGA) was obtained from Aldrich Chemical Company. Carbamazepine and metabolites Carbamazepine, carbamazepine- 10,1 1-epoxide, carbamazepine- 10,1 1-diol and lO-methoxycarbamazepine for use as standards in the HPLC analyses were generously supplied by Ciba-Geigy Ltd. (Canada).  Carbamazepine-10,11-  epoxide for use in the animal studies was also supplied by Ciba-Geigy Ltd. (Canada).  Carbamazepine for use in the animal studies was purchased from  Sigma Chemical Company (St. Louis, MO, U.S.A.).  35  Experimental Reagents  Chemicals, solvents and reagents were obtained from the following sources:  BDH CHEMICALS (Vancouver, B.C., Canada). Acetonitrile OmniSolve® grade, ammonium acetate, calcium chloride, citric acid anhydrous, di-potassium hydrogen orthophosphate, di-sodium hydrogen  dichloromethane  orthophosphate,  ethylenediaminetetraacetic  acid  (EDTA),  OmniSolve®  hydrochloric  acid,  grade,  magnesium  chloride, methanol OmniSolve® grade, potassium dihydrogen orthophosphate, potasssium chloride, sodium chloride, sodium dihydrogen orthophosphate, sodium  hydroxide,  sodium  sulphate  anhydrous,  sulphuric  acid  and  trichioroacetic acid.  BlO-RAD LABORATORIES (Richmond, California, U.S..A). Acrylamide 99.9%, ammonium persuiphate 98%, bis (IVN’-methylene bisacrylamide), 2-mercaptoethanol, SDS-PAGE 10 to lOOK molecular weight standards, SDS-PAGE 40 to 250K molecular weight standards, sodium dodecyl sulphate (SDS) and TEMED (N,N,N’,N’-tetramethyethylenediamine).  BOEHRINGER MANNHEIM CANADA LTD. (Laval, Quebec, Canada). Bovine serum albumin, fraction V (BSA), bovine serum albumin, fatty acid free, fraction V, nicotinamide adenine dinucleotide, reduced (NADH) and nicotinamide adenine dinucleotide phosphate, reduced (NADPH).  CALEDON (Georgetown, Ontario, Canada). Ethyl acetate distilled-in-glass grade.  36  Experimental  FISHER SCIENTIFIC LTD. (Vancouver, B.C., Canada). Creatinine.  INTER MEDICO (Markham, Ontario, Canada) 2 anti-rabbit IgG (G+L) horseradish peroxidase conjugated Goat F(ab’) IgG, affinity purified (TAGO).  J.T. BAKER CHEMICAL CO. (Phillipsburg, New Jersey, U.S.A.). Sodium dithionite.  MOLECULAR PROBES, INC. (Eugene, Oregon, U.S.A.). Ethoxyresorufin, pentoxyresorufin and resorufin.  PIERCE CHEMICAL COMPANY (Rockford, illinois, U.S.A.). N-tert-butyldimethylsilyl-N-methyl-trifluoroacetamide (MTBSTFA).  SIGMA CHEMICAL COMPANY (St. Louis, MO, U.S.A.). Clofibrate, 4-chloro-1-naphthol, Folin & Ciocalteu’s Phenol reagent, glycine,  N- [2-Hydroxyethyl] piperizine-N’- [2-ethanesulfonic  acid]  (HEPES),  hydrogen peroxide 30% solution, picric acid saturated solution, propylene glycol, sodium potassium tartrate, sucrose, Trizma base, Trizma HC1 and Tween 20.  Primary antibodies The primary antibodies, namely anti-rat cytochromes P-450b (P-450 2B1), P-450f (P-450 2C7), P-450g (P-450 2C13) and P-450h (P-450 2C11), were 37  Experimental prepared and generously provided by Dr. Stelvio Bandiera’s group.  The  antibodies were raised in female New Zealand rabbits immunized with the electrophoretically homogeneous proteins. IgG was purified from a pooi of heatinactivated high-titer antisera obtained from multiple bleedings from several rabbits using a combination of caprylic acid precipitation followed by ammonium sulphate precipation and a final cleanup on a DEAE-Sephacel column (Bandiera and Dworschak,  1992).  Antibody concentration was  E cm determined spectrophotometrically at 280 nm, 1  =  13 for a 1% solution.  Each antibody was extensively immunoabsorbed in a manner analogous to that developed for anti-rat cytochromes P-450f and P-450g to generate monospecific antibody (anti-rat cytochromes P-450f, P-450g and P-450h).  The anti-rat  cytochrome P-450b antibody was polyspecific. The specificity of each antibody was assessed using Ouchterlony double diffusion analysis, noncompetitive ELISA and immunoblots.  The anti-rat  cytochrome P-450f, P-450g and P-450h antibodies only reacted with the antigen of immunization and did not react with any other purified cytochrome P-450. In the case of the anti-rat cytochrome P-450b antibody, it reacted with cytochrome P-450e and also with a third, noninducible member of the cytochrome P-450 2B family.  METHODS Induction studies Animals Adult male Long Evans rats (190 to 225 g, Charles River, Montreal) were used for the experiments. After arrival, rats were allowed to recover for 3 to 5 days prior to commencing the studies. Rats were fed standard rat chow (Purina  38  Experimental 5001®) ad libitum and allowed drinking water ad libitum.  The rats were  housed on corn cob bedding (Anderson’s®) in a room with controlled light (14 h) and dark (10 h) cycles. Treatment of solvents and compounds  Normal saline (NS), corn oil (CO) and propylene glycol (PG) were filtered via either a 0.2 or 0.45  t  filter (Gelman FP-Vericel) prior to use as vehicles for  phenobarbital (PB), clofibrate (CFB), carbamazepine (CBZ) and carbamazepine 10,11-epoxide (CBZE). Compounds were dissolved or suspended such that the volume of the dose administered to the animal was 0.1 mL/100 g body weight. Vehicle control animals received an equivalent volume per weight of the appropriate vehicle. Treatment of animals with carbamazepine Adult male Long Evans rats (4 per group) were treated i.p. with CBZ suspended in PG at a dose of 100 mg/kg every 12 h for 3, 7, 10 and 14 days. Control rats received an equivalent volume of PG. After administration of the last dose of CBZ, each rat was placed in a separate metabolic cage and urine collected for the 12 h period until sacrifice. Treatment of animals with carbamazepine- 10,11 -epoxide Rats (4 per group) were treated i.p. with CBZE suspended in PG at a dose of 50 mg/kg every 12 h for 3, 7, 10 and 14 days. Again, control rats received an equivalent volume of PG for each dose studied. After administration of the last dose of CBZE, rats were placed in metabolic cages and urine collected for the 12 h period until sacrifice. Treatment of animals with phenobarbital Rats were administered PB dissolved in normal saline (NS) 75 mg/kg i.p.  39  Experimental daily for 4 days. Vehicle control animals received an equivalent volume of NS. Treatment of animals with clofibrate Animals were administered CFB diluted in corn oil (CO) at a dose of 350 mg/kg i.p. daily for 7 days. Vehicle control animals received injections of CO. Treatment of animals with vaiproic acid VPA was administered at a dose of 150 mg/kg twice daily i.p. for 3 days. Vehicle control animals received injections of water.  ANALYSIS. Vaiproic acid and metabolites Stock solutions of internal standards for GCMS To decrease pipetting errors, the required internal standards were mixed such that the amounts required for each sample could be added simultaneously. A 200 tL aliquot contained the following amounts of each internal standard: 3-OHVPA 400 ng, [ ] 7 H 4-ene VPA 100 ng, 2 [ ] 7 H E-2-ene VPA 200 ng, 2 [ 1 7 H 2 3-keto VPA 200 ng, [ ] 7 H ]4-keto VPA 100 ng, 2 7 H 2 5-OH VPA 400 ng, [ [ ] 7 H 2 VPA 100 ng and 2-MGA 50 ng. Stock solutions were kept frozen at 20 °C [ ] 7 H 2 -  until needed. Preparation of standard curves in phosphate buffer A bulk stock solution (hereafter referred to as standard 5) was prepared in 0.2 M phosphate buffer, pH 7.4. For the calibration curve 200, 400, 600, 800 and 1,000 !IL of standard 5 were made up to a final volume of 1 mL with phosphate buffer. One mL of the phosphate buffer served as the blank for the calibration curve. The stock solution was stored at 20 °C until required. -  40  Experimental Standard curve for VPA and metabolites The concentrations of VPA and metabolites thus obtained were as follows: VPA 0, 12, 24, 36, 48 and 60 ig/mL; 3-ene VPA, 3-keto VPA, 4-ene VPA, 4-keto VPA, 2-PSA, 2-PGA, (E,E)-2,3’-diene VPA, (E)-2,4-diene VPA and (E)-2-ene VPA 0, 20, 40, 60, 80 and 100 ng/mL; 5-OH VPA 0, 40, 80, 120, 160 and 200 ng/mL; 3OH VPA 0, 60, 120, 180, 240 and 300 ng/mL and 4-OH VPA 0, 0.24, 0.48, 0.72, 0.96 and 1.2 j.tg/mL. Standard curve for (E)-2-ene VPA and metabolites The concentrations of (E)-2-ene VPA and metabolites were as follows: VPA, 3-keto VPA, 4-ene VPA, 3-OH VPA, 3-ene VPA and (E,E)-2,3’-diene VPA 0, 19.2, 38.4, 57.6, 76.8 and 96 ng/mL; (E)-2,4-diene VPA 0, 0.8, 1.6, 2.4, 3.2 and 4 p,g/mL; and (E)-2-ene VPA 0, 11.52, 23.04, 34.6, 46.08 and 57.6 j.tg/mL. The calibration curves were generated by plotting the ratio of the peak area of metabolite or VPA to that of the respective internal standard versus the concentration of VPA or the particular metabolite. The deuterated compounds served as the internal standards for their respective undeuterated counterparts. Deuterated (E)-2-ene VPA served as the internal standard for 3-ene VPA and the diene metabolites. Deuterated VPA served as the internal standard for 4OH VPA while 2-MGA was used as the internal standard for the 2 dicarboxylic acid metabolites. Standard curves were prepared and injected into the GCMS with each batch of samples. Extraction of VPA and metabolites from standard samples and incubates The extraction procedure for VPA and metabolites is shown in figure 3. Two hundred iiL of internal standard mixture were added to each tube containing the sample for analysis or standard sample (1 mL). The pH of the standard curve and incubates was adjusted to between 1.5 to 2.0  41  The final  Experimental  Microsomal Incubate or Standard Sample lmL  Internal Standard Mixture (200 E.LL) 2-MGA (50 ng) ]VPA (100 ng) 7 H 2 [ 1E-2-ene VPA (200 ng) 7 H 2 [ ]4-ene VPA (100 ng) 7 H 2 [ 1 3-OH VPA (400 ng) 7 H 2 [ ] 5-OH VPA (400 ng) 7 H 2 [ ]3-keto VPA (200 ng) 7 H 2 [ ]4-keto VPA (100 ng) 7 H 2 [  $ Adjust pH to 1.5 2 0 2 Adjust final volume to 3 mL with H -  1  Extract with 3 mL ethyl acetate x 30 mm x 2  Organic layer Dry over anhydrous sodium sulphate Concentrate to 200 .tL  4  Derivatize with MTBSTFA 100 iL Heat for 1 h at 60 °C Inject 1 jiL into GCMS Figure 3.  Summary of the extraction procedure for valproic acid and metabolites.  42  Experimental volume was adjusted to 3 mL with distilled water. Each sample was extracted twice with 3 mL ethyl acetate by gentle rotation for 30 mm. The organic layer (total volume approximately 5 mL) was transferred to a test tube and dried over anhydrous sodium sulphate by vortexing for 1 mm, waiting 10 mm  and  centrifuging for 10 mm at 3,000 rpm. The dried organic layer was transferred to another test tube, concentrated to approximately 200 iL, transferred to a 1 mL conical vial and derivatized with 100 j.tL MTBSTFA for 60 mm  at 60 °C. The  derivatized samples were transferred to autosampler vials and 1 tL injected into the GCMS.  Carbamazepine and metabolites Preparation of stock solutions for HPLC Stock solutions of CBZ, CBZE and CBZD at a concentration of 1 mg/mL in methanol were prepared. A stock solution of lO-methoxycarbamazepine (MCBZ, internal standard) also at a concentration of 1 mg/mL was prepared in methanol and further diluted in distilled water to yield the working solution of 40 ig/mL. All stock and working solutions were stored at 20 °C until required. -  Preparation of standard curve for CBZ and metabolites A bulk standard stock solution was prepared in rat urine from PG treated rats. Standard curves were prepared from 2, 4, 6, 8, 12, 16 and 20 pgImL of CBZ, CBZE and CBZD. Unspiked urine served as the blank for the standard curve.  Peak area ratios of CBZ, CBZE or CBZD to internal standard were  plotted versus concentration to prepare calibration curves. Extraction of CBZ, CBZE and CBZD from urine samples CBZ and its 2 metabolites, CBZE and CBZD were extracted from urine using a procedure based on literature methods of Elyas et al. (1982) and Kumps  43  Experimental et al. (1985) (figure 4). A 400 jiL aliquot of urine (either standard or sample) was placed into a test tube to which were added 4 pg (100 jiL) of the internal standard (MCBZ) and 500 ilL of 300 mM phosphate buffer, pH 6.7. The sample was extracted with 2.5 mL of ethyl acetate by gentle rotation for 10 mm  and  centrifuged at 2,500 rpm for 10 mm. The top organic layer was transferred to a second tube and evaporated under a gentle nitrogen stream in a water bath at 40 °C, reconstituted with 200 jiL of acetonitrile, evaporated and reconstituted again with 200 tL of acetonitrile. The samples were transferred to autosampler vials and for each sample 20 jiL were injected into the liquid chromatograph. Instrumentation Vaiproic acid and metabolites The analyses were performed on a Hewlett-Packard 5890 Series II gas chromatograph interfaced with a Hewlett-Packard 5971A Mass Selective Detector and equipped with a 7673 autosampler. A Hewlett-Packard Vectra® 25T 486 computer, Hewlett-Packard Video Graphics Colour Display and a Hewlett-Packard Laserjet Series II printer accompanied the MSD. 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. Source temperature was 180 °C. Injection mode was splitless and column head pressure was 15 psi. A DB 1701 (0.25  j.i)  bonded phase capillary column, 30 m x 0.25 mm I.D.,  (J & W Scientific, Folsom, California) was used for the analysis. Temperature programming for tBDMS derivatives was initial column oven temperature of 80 °C, increasing by 10 °C/min from 80 to 100 °C, then 2 °C/min from 100 to 130 °C and 30 °C/min from 130 to 260 °C and held at 260 °C for 8 mm. Total run  44  Experimental  Urine (400 j.tL)  4 jig (100 jIL) MCBZ 500 jiL 300 mM phosphate buffer, pH 6.7  I  Extract with 2.5 mL ethyl acetate x 10 mm  Centrifuge at 2,500 rpm for 10 mm  I  Organic layer 2 Evaporate to dryness under N  • I  Reconstitute with 200 jiL acetonitrile x 2 2x2 Evaporate to dryness under N  Reconstitute with 200 p.L acetonitrile Inject 20 jiL into HPLC  Figure 4.  Summary scheme of the extraction of carbamazepine and metabolites from urine of rats. -  45  Experimental time was approximately 29 mm. Selected ion monitoring mode was used for the analyses.  The ions  scanned were mlz 100 (4-OH ‘[PA lactone), mlz 197 (dienes), mlz 199 (enes), mlz 4-ene VPA), mlz 208 [ ] 7 H E-2-ene VPA and 2 ([ ] 7 H 201 (‘[PA), mlz 206 2 VPA), (mlz 213 (3-keto-4’-ene VPA, monoderivative), mlz 215 (3- and 4([ ] 7 H 2 4-keto VPA), ([ ] 7 H keto VPA, monoderivative), rnlz 217 (3-OH VPA), mlz 222 2 3-OH VPA), mlz 317 (2-methyiglutaric acid), mlz 327 (3-keto-4’([ J 7 H mlz 224 2 ene VPA, diderivative), m/z 329 (3-keto VPA, diderivative), mlz 331 (5-OH ‘[PA 5-OH VPA) and mlz 345 ([ 1 7 H 3-keto ‘[PA), mlz 338 2 ([ ] 7 H and 2-PSA), mlz 336 2 (2-PGA). Carbamazepine and metabolites A Hewlett-Packard Series II 1090 Liquid Chromatograph equipped with a HP3396A integrator with a Beckman Ultrasphere® ODS column, particle size 5 urn, 250 mm length, I.D. 4.6 mm was used for the analysis.  Detection  wavelength was 215nm and flow rate was 1 mlJmin. A gradient using water (% A) and acetonitrile (% B) was formed such that the mixture was 85% A and 15% B from 0 to 2 mm, 65% A and 35% B at 24 mm, 65% A and 35% B at 28 mm and 85% A and 15% B at 30 mm. Stop time was 50 mm. Ten minutes were allowed between each run for a total run time of 60 mm. Preparation of subcellular fractions from rat livers Initially, assays were to be performed using mitochondrial, peroxisomal and microsomal fractions and thus differential centrifugation of rat liver homogenates according to the method of Cook et al., (1986) was used. The livers were removed, washed in 0.9% NaC1 and homogenized in 20 mL of 0.25 M sucrose/0.1% ethanoll5 mM Tris/1.15% KC1 solution. centrifuged at 600 g for 10 mm  The homogenate was  (4,200 rpm, J-20 rotor), the supernatant filtered  46  Experimental  through 4 layers of cheesecloth and centrifuged at 7,500 g for 10 mm  (8,000  rpm, J-20 rotor) to obtain the mitochondrial fraction. The 7,500 g supernatant was centrifuged at 17,000 g for 10 mm  (12,000 rpm, J-20 rotor) to obtain the  peroxisomal fraction. The 17,000 g supernatant was centrifuged at 100,000 g for 60 mm  (33,500 rpm, 50.2 Ti rotor) to obtain the microsomal fraction. The  mitochondrial and peroxisomal fractions were each washed once with the above buffer and resuspended in either 0.25 M sucrose for the mitochondrial fraction or 0.25 M sucrose with 0.1% ethanol for the peroxisomal fraction.  The  microsomal pellet was washed once in buffer containing 1.15% KC1 and 10 mM EDTA buffer and then resuspended in an equivalent volume of 0.25 M sucrose. The samples were stored in cryovials at  -  65 °C.  Eventually, only the  microsomal fractions were used. Determination of protein content of various subcellular fractions The protein content of the various subcellular fractions was determined according to the method of Lowry et al. (1951). The analyses were performed on a Hewlett-Packard 8452A diode array spectrophotometer in triplicate. BSA was used as the protein standard. Determination of cytoclirome P-450 content in hepatic microsomes Cytochrome P-450 content in the microsomal fraction from various treated groups was determined on a Hewlett-Packard 8452A diode array spectrophotometer using the method of Omura and Sato (1964).  Microsomal  protein was diluted 1:25 in a buffer containing 0.1 M sodium phosphate, pH 7.4, 20% glycerol and 0.1 M EDTA. cuvette (sample) for 1 mm  Carbon monoxide was bubbled through one  and a small amount of sodium dithionite was added  to each cuvette and mixed thoroughly. After one mm, the sample was scanned over the range of 325 to 625 nm. The amount of cytochrome P-450 in the sample  47  Experimental  - for 1 was calculated using the millimolar extinction coefficient of 91 cm’ mM cytochrome P.450 (Omura and Sato, 1964). The analyses were performed in duplicate. Gel electrophoresis of microsomal protein SDS-polyacrylamide gel electrophoresis was performed according to the method of Laemmli (1970) using a Hoeffer vertical slab gel unit. The separating gel was 7.5% acrylamide-bis, 0.375 M Tris-HC1, 0.1% SDS, 0.042% ammonium persulphate and 0.03% TEMED. The stacking gel was 3% acrylamide-bis, 0.125 M Tris-HC1, 0.1% SDS, 0.08% ammonium persulphate and 0.05% TEMED. Samples were diluted in sample dilution buffer (0.062 M Tris HC1, 1% SDS, 0.001% bromophenol blue, 10% glycerol and 5% f-mercaptoethanol), boiled for 2 mm  and 10 pg of microsomal protein were loaded per lane in a 20 tL volume.  The gels were run at 12.5 mA per gel for approximately 45-50 mm  (time for  proteins to travel through stacking gel) and then the current was increased to 25 mA per gel for approximately 2 h until the dye front reached the bottom of the gel. The gel was then fixed for 1 h in fixative (25% isopropanol and 10% acetic acid in water) to remove SDS, stained for 1 h (25% isopropanol, 10% acetic acid and 0.05% Coomassie Blue) and destained (10% isopropanol and 10% acetic acid) as required. In gels where the amount of cytochrome P-450b was to be quantitated, 5 pmol of spectrally determined microsomal cytochrome P-450 were loaded per lane. Immunoblot Immunoblotting was performed according to the method of Towbin et al. (1979) using a Hoeffer TE 52 Transphor® unit equipped with a power lid. The proteins were resolved using SDS-PAGE and electrophoretically transferred onto a sheet of 0.20  ji  nitrocellulose transfer membrane (BA-S 83, Schleicher &  48  Experimental  Schuell, Keene, NH). The transfer was performed at 0.4 mA for 2 h in a cold cabinet using a precooled buffer containing 20% methanol, 0.02 M Tris, 0.154 M glycine and 0.008% SDS at pH 8.3.  After completion of the transfer, the  nitrocellulose sheet was placed in an utility box containing 50 mL of blocking buffer (1% BSA and 3% skim milk powder (Carnation®) in phosphate buffered saline (PBS)) and stored at 4 °C overnight or until development. For development of the blot, the blocking buffer was discarded and the primary antibody diluted (5 tg/mL, 1:2,500 dilution) in antibody dilution buffer (1% BSA, 3% skim milk powder and 0.05% Tween in PBS) and incubated for 2 h at 37 °C in a shaking water bath (Haake). The primary antibody was discarded and the nitrocellulose sheet washed 3 times for 10 mm with wash buffer (0.05% Tween in PBS). The secondary antibody, goat anti-rabbit peroxidase conjugated antibody, was diluted (1:3,000 dilution) in antibody dilution buffer and incubated for 2 h at 37 °C in a shaking water bath. The nitrocellulose sheet was washed 3 times at 10 mm  each.  The reaction was then visualized using a  substrate solution containing 3 mL of 0.018% 4-chloro-1-naphthol in methanol, 0 and 47 mL of PBS. The reaction was allowed to proceed 2 30 jiL of 30% H until visually satisfactory and terminated by submersing the nitrocellulose sheet in a tray of distilled water. Quantitation of cytochrome P-450b in microsomal protein from PB, CBZ and CBZE treated rats A standard curve for cytochrome P-450b was generated by loading 0.2, 0.5, 1, 2, 3 and 4 pmol of purified cytochrome P-450b per lane.  Microsomal  protein equivalent to 5 pmol of spectrally determined cytochrome P-450 was loaded per lane.  The conditions for gel electrophoresis and immunoblotting  were as noted above.  Rabbit anti-rat cytochrome P-450b antibody at a  concentration of 5 jig/mL was used to probe the blot. The staining intensity of 49  Experimental  the bands from these experiments was quantitated using the VISAGE® 110 Bio Image Analyzer (Bio Image, Ann Arbor, MI) using whole band analysis and optical density. In vitro microsomal metabolism of VPA and (E)-2-ene VPA In vitro metabolism of VPA and (E)-2-ene VPA was performed according  to the method of Rettie et at. (1987). Five hundred jiL 0.2 M phosphate buffer, , 2 nmol of spectrally determined cytochrome P 2 pH 7.4, 10 j.tL 300 mM MgC1 450 (as microsomal protein), 10 .tL 0.01 M NADPH and 10 .tL 0.01 M NADH adjusted with water to a final volume of 1,000 jiL were combined in glass screw capped test tubes and preincubated for 10 mm  at 37 °C.  The reaction was  initiated with either 10 j.tL of 0.04 M VPA or 40 jiL of 0.01 M (E)-2-ene VPA and allowed to proceed for 40 mm at 37 °C. The reaction was terminated with 1 mL of 10% HC1 and the samples extracted as outlined above for GCMS analysis. In vitro microsomal metabolism of YPA and (E)-2-ene YPA in the presence of anti-rat cytochrome P-450b or anti-rat cytochrome P-450h antibody These incubations were performed as above, except that increasing amounts of one of anti-rat cytochrome P-450b antibody, anti-rat cytochrome P 450h antibody or control rabbit IgG was preincubated with the microsomal protein (2 nmol of spectrally determined cytochrome P-450) for 10 mm  prior to  initiation of the reaction by the addition of VPA or (E)-2-ene VPA (Chang, Ph.D. thesis, 1991). The antibodies were employed at concentrations of 0, 0.5, 1, 1.5, 2 and 2.5 mg IgG/nmol of cytochrome P-450.  50  Experimental In vitro microsomal metabolism of VPA and (E)-2-ene VPA in the  presence of both anti-rat cytochrome P-450b and anti-rat cytochrome P-450h antibodies. These incubations were performed as above, except that the microsomal protein was preincubated with both anti-rat cytochrome P-450b and anti-rat cytochrome P-450h antibodies for 10 mm  prior to initiation of the reaction by  the addition of VPA or (E)-2-ene VPA.  The antibodies were each used at a  concentration of 2 mg IgG/nmol of cytochrome P-450. Microsomal 0-dealkylation of ethoxyresorufin and pentoxyresorufin Microsomal 0-dealkylation rates were determined according to the method of Burke et al. (1985) using either pentoxyresorufin or ethoxyresorufin as the substrate.  The assays were performed on a RF-540 Shimadzu  spectrofluorometer equipped with a Shimadzu DR-3 Data Recorder. Excitation wavelength was 530 nm and emission wavelength was 582 nm with a slit width of 5 nm. The reaction mixture contained 1.93 mL of 0.1 M Hepes/5 mM MgC1 , 2 pH 7.8, 10 tL of either 1 mM pentoxyresorufin or 1 mM ethoxyresorufin in DMSO and 50 iL of microsomal protein diluted in sucrose to 2 mg/mL. The reaction was initiated with the addition of 10 iiL of 50 mM NADPH diluted in the above mentioned buffer. minute for 10 mm.  The fluorescence reading was recorded every  Enzyme activity was expressed as either nmol resorufin  formedlminlmg protein or nmol resorufin formedlminlnmol cytochrome P-450. Statistical analysis Statistical analysis was performed using one way ANOVA (Newman Keuls test). The level of statistical significance chosen was p  51  0.05.  RESULTS  The goals of this project were to identify the isozyme(s) of cytochrome P 450 induced by carbamazepine (CBZ) and its major metabolite, carbamazepine in 10,11-epoxide (CBZE) and to determine the effect of these inducers on the VPA. vitro metabolism of vaiproic acid (VPA) and its major metabolite, (E)-2-ene red These effects of CBZ and CBZE on cytochrome P-450 were then to be compa which to those of the classic inducer, phenobarbital (PB) and to clofibrate (CFB) had been reported to induce VPA metabolism in rats (Heinemeyer et aL, 1985).  QUANTITATION AND IDENTIFICATION OF CYTOCHROMES P.450  IN  HEPA TIC MICROSOMES t Quantitation of total hepatic microsomal cytochrome P-450 conten PB (Remmer and Merker, 1963), CBZ (Wagner and Schmid, 1987; to induce Regnaud et al., 1988) and CFB (Gibson, 1992) have been reported rome cytochrome P-450. The effectiveness of these compounds to induce cytoch omes P-450 was compared. Total cytochrome P-450 content in rat hepatic micros the isolated from the various treatment groups was measured according to uced method of Omura and Sato (1964). The terms “untreated animals”, “unind animals animals” and “control animals” are used interchangeably to describe carbon which did not receive any compound or vehicle. A representative m is monoxide sodium dithionite-reduced cytochrome P-450 difference spectru  shown in figure 5. t of A graphic representation of the total hepatic cytochrome P-450 conten rats is microsomes (mean ± s.d.) from untreated, PB, NS, CFB and CO treated and shown in figure 6 and is similarly detailed for microsomes from CBZ, CBZE P-450 PG treated rats in figure 7. The changes in total hepatic cytochrome 52  Results  0.20000  0.14000 LJ  0.08000 02  C  0.02000  —.01000  400 U  Figure 5.  S00 V EL E N G TH  600  Carbon monoxide sodium dithionite-reduced difference spectrum of hepatic microsomal cytochrome P-450.  53  Results  3.0 t2J  0  2.5  S CD  2.0  to  iii  1.5  S0 C)  1.0  0 C)  0.5  0  S 0.0 control Figure 6.  PB  NS  CFB  CO  Cytochrome P-450 content (nmol of spectrally determined from cytochrome P-450/mg protein, mean ± s.d.) of microsomes control, PB, NS, CFB and CO treated rats (n=4). a significanti 0.05), greater than microsomes from untreated animals (p significantly greater than microsomes from appropriate vehicle control animals. Cytochrome P-450 was determined as outlined in the Experimental section.  54  Results  3.0  2.5  S cD  2.0  1.5  S0 .c: C.) 0  10  C.) —  0.5  0  S 0.0 CBZ  Figure 7.  CBZE  PG  Cytochrome P-450 content (nmol of spectrally determined cytochrome P-450/mg protein, mean ± s.d.) of microsomes froma CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). significantly greater than microsomes from untreate animals (0.6 ± 0.04 nmol cytochrome P-4501mg protein, p 0.05), significantly vehicle control animals greater than microsomes from appropriate C significantly greater than treated over the same time period, microsomes from CBZE treated animals over the same time period. Cytochrome P-450 was determined as outlined in the Experimental section.  55  Results  content relative to the control group and to the respective vehicle groups for the PB, CFB, CBZ and CBZE treatment groups are summarized in table 2. The mean cytochrome P-450 content of microsomes prepared from PB (1.5 ± 0.2 nmol cytochrome P-450/mg protein, 2.3 fold increase), CFB (1.2 ± 0.2, 1.9 fold increase), CBZ 3 day (1.3 ± 0.2, 2.1 fold increase), CBZ 7 day (1.5 ± 0.4, 2.3 fold increase), CBZ 10 day (1.5 ± 0.3, 2.3 fold increase) and CBZ 14 day (1.2 ± 0.2, 1.8 fold increase) treated rats were all significantly greater when compared to the mean cytochrome P-450 content of microsomes from untreated animals (0.6 ± 0.04 nmol cytochrome P-4501mg protein).  No significant differences were  observed when either the NS, CO, CBZE 3, 7, 10 and 14 day or PG 3, 7, 10 and 14 day treated groups were compared to the control group. When PB treated animals were compared to the NS (vehicle) treated group, the mean cytochrome P450 content was significantly higher (1.5 ± 0.2 versus 0.8 ± 0.05 nmol cytochrome P-450/mg protein, a 1.9 fold increase, table 2).  There was no statistical difference between the cytochrome P-450 content of microsomes prepared from the CFB and the CO treated rats (1.2 ± 0.2 versus 1.0 ± 0.2 nmol cytochrome P-450/mg protein, table 2). The amount of cytochrome P-450 in microsomes isolated from CBZ 3, 7 and 10 day treated rats was significantly greater than the PG (vehicle) treated rats for the corresponding time period (1.5 to 1.8 fold increase, table 2). The CBZ 14 day treated group was an exception where a significant difference was not observed for the amount of total cytochrome P-450 found when compared to the corresponding PG vehicle treated group. Hepatic microsomes from the CBZE treatment groups did not display any significant increases in the amount of total cytochrome P-450 when compared to their corresponding PG vehicle controls (figure 7, table 2).  56  Results Table 2.  Summary of total hepatic cytochrome P.450 content (nmollmg protein, mean ± s.d.) and change in cytochrome P-450 relative to the untreated group or to the respective vehicle control group for the PB, CFB, CBZ and CBZE treatment groups (n=4). Cytochrome P-450 was determined as outlined in the Experimental section.  Cytochrome P-450 (nmollmg protein)  Treatment nmollmg protein mean ± s.d.  fold increase relative to untreated group  fold increase relative to vehicle group  Untreated  0.6 ± 0.04  PB NS  ab 1.5 ± 02 0.8 ± 0.05  2.3 1.3  1.9  CFB CO  a 1.2 ± 02 1.0±0.2  1.9 1.7  1.2  ab 1.3 ± 02 1.5 ± 0.4 abc 1.5 ± 03 a 1.2 ± 02  2.1 2.3 2.3 1.8  1.8 1.7 1.5 1.4  CBZE 3 day CBZE 7 day CBZE1Oday CBZE 14 day  1.1 ± 0.2 0.9 ± 0.2 0.9±0.1 0.9 ± 0.1  1.7 1.4 1.5 1.4  1.5 1.0 1.0 1.1  PG 3 day PG7day PGlOday PG 14 day  0.7 ± 0.2 0.9±0.2 1.0±0.2 0.8 ± 0.2  1.2 1.5 1.7 1.3  CBZ CBZ CBZ CBZ  (p a b c  3 day 7 day 10 day 14 day  0.05) significantly greater than microsomes from untreated animals significantly greater than microsomes from appropriate vehicle control animals treated over the same time period significantly greater than microsomes from CBZE treated animals over the same time period  57  Results  Comparable amounts of hepatic microsomal cytochrome P-450 were found for all 4 CBZ treatment groups as well as for the PB and CFB treated rats, with no statistical differences observed. Increases ranged from 1.8 to 2.3 fold over the untreated group (table 2). Cytochrome P-450 levels were found to be consistent over the time course of treatment in both the CBZ and CBZE treated groups (table 2). However, the total microsomal cytochrome P-450 levels of the CBZ 7 and CBZ 10 day groups were found to be higher than the corresponding CBZE treated group. Identification of the cytochrome P-450 isozymes induced by CBZ and CBZE using SDS-PAGE and Western blot techniques Previous literature has suggested that CBZ may induce the same isozymes of cytochrome P-450 as PB (Wagner and Schmid, 1987). Based on this information, studies were performed to determine if cytochrome P-450b (Ryan et at., 1982) (cytochrome P-4502B1, Nelson et at., 1993), an isozyme known to be induced by PB, was also induced by either CBZ or CBZE.  CFB served as a  control since it is known to induce cytochrome P-452 (CYP4A1, lauric acid hydroxylase) (Leroux et at., 1989). The antibodies against various isozymes of cytochrome P-450 were prepared by Dr. Bandier&s group.  The polyspecific antibody against rat  cytochrome P-450b also reacted with cytochrome P-450e, another isozyme induced by PB. The nomenclature of Levin and co-workers will be used throughout this thesis when referring to isozymes of cytochrome P-450 (Ryan and Levin, 1990) and a summary comparing their nomenclature to that of Nelson et at. (1993) is provided in table 1 in the introduction. Representative SDS-PAGE gels of microsomal protein isolated from the livers of rats treated with either CBZ or CBZE over the time course of treatment 58  Results are shown in figures 8 and 9. In both gels, lanes 2, 9 and 18 are the molecular weight standards while lane 3 is the purified cytochrome P-450b standard. Lane 5 contains microsomal protein from the PB treatment group while lanes 10, 12, 14 and 16 contain microsomal protein from either the CBZ 3, 7, 10 and 14 day treatment groups, respectively in figure 8 or from the CBZE 3, 7, 10 and 14 day treatment groups, respectively in figure 9.  Lanes 11, 13, 15 and 17  contain microsomal protein from PG 3, 7, 10 and 14 day treated groups, respectively. After the separated proteins were electrophoretically transferred to a nitrocellulose membrane and probed with the antibody directed against rat cytochrome P-450b, microsomal protein from both CBZ and CBZE treated rats reacted positively, indicating that cytochrome P-450b was present (figures 10 and 11). Cytochrome P-450b could be detected in the microsomes after 3 days of treatment with either CBZ or CBZE.  A second band due to reaction of the  antibody with cytochrome P-450e was also observed just above the cytochrome P-450b band. Neither cytochrome P-450b nor cytochrome P-450e was detected in microsomes from untreated, NS, CO and PG treated animals. Surprisingly, a positive reaction was also observed with microsomal protein from the CFB treated group suggestive of the presence of cytochromes P-450b and P-450e. Microsomal protein isolated from VPA treated rats did not react with the anti-rat cytochrome P-450b antibody, indicating that VPA does not influence this particular isozyme of cytochrome P-450 (data not shown). Microsomal protein from none of the treatment groups reacted to any appreciable degree with anti-rat cytochrome P-450f or anti-cytochrome P-450g (data not shown).  59  Results  Mwt (kDa)  —-—-- _  -  97.4-  —  —  .  -  —  21.5  Lane  Figure 8.  —  —  -ian_aii  42.7—  31  —.—  -  —  —— — — 66.2—  -  ____-ø_, —-- —  .•  .  .---  —  2 3 4 5 6 7 8 9 10 1112 131415161718  SDS-PAGE gel of rat liver microsomal fractions from various treatment groups. Lane 2, molecular weight standards; lane 3, purified cytochrome P-450b; lane 4, control; lane 5, PB; lane 6, NS; lane 7, CFB; lane 8, CO; lane 9, molecular weight standards; lane 10, CBZ 3 day; lane 11, PG 3 day; lane 12, CBZ 7 day; lane 13, PG 7 day; lane 14, CBZ 10 day; lane 15, PG 10 day; lane 16, CBZ 14 day; lane 17, PG 14 day and lane 18, molecular weight standards.  60  Results  Mwt (kDa)  97.4  -  --———  —  66.2-‘  ,  —  w  42.7-  31  —  21.5—  Lane  Figure 9.  2  34 5  6 7 8  9 1011 12131415161718  SDS-PAGE gel of rat liver microsomal fractions from various treatment groups. Lane 2, molecular weight standards; lane 3, purified cytochrome P-450b; lane 4, control; lane 5, PB; lane 6, NS; lane 7, CFB; lane 8, CO; lane 9, molecular weight standards; lane 10, CBZE 3 day; lane 11, PG 3 day; lane 12, CBZE 7 day; lane 13, PG 7 day; lane 14, CBZE 10 day; lane 15, PG 10 day; lane 16, CBZE 14 day; lane 17, PG 14 day and lane 18, molecular weight standards.  61  Results  Lane  Figure 10.  3 4  5 6  7 8 9 10 11  12 13 14 15 16  Immunoblot of rat liver microsomal proteins probed with anti-rat cytochrome P-450b antibody. Lane 3, purified cytochrome P-450b; lane 4, control; lane 5, PB; lane 6, NS; lane 7, CFB; lane 8, CO; lane 10, CBZ 3 day; lane 11, PG 3 day; lane 12, CBZ 7 day; lane 13, PG 7 day; lane 14, CBZ 10 day; lane 15, PG 10 day; lane 16, CBZ 14 day.  62  Results  3 4  Figure 11.  5  6 7 8  9 10111213 1415 16  Immunoblot of rat liver microsomal proteins probed with anti-rat cytochrome P-450b antibody. Lane 3, purified cytochrome P-450b; lane 4, control; lane 5, PB; lane 6, NS; lane 7, CFB; lane 8, CO; lane 10, CBZE 3 day; lane 11, PG 3 day; lane 12, CBZE 7 day; lane 13, PG 7 day; lane 14, CBZE 10 day; lane 15, PG 10 day; lane 16, CBZE 14 day.  63  Results In vitro 0-dealkylation of pentoxyresorufin and ethoxyresorufin  catalyzed by hepatic microsomal protein from the various treatment groups To the best of our knowledge, the isozyme(s) of cytochrome P-450 induced by CBZ and CBZE have not previously been identified. Pentoxyresorufin and ethoxyresorufin were used as substrates for the microsomal 0-dealkylation reactions to confirm the identification of cytochrome P-450b isozyme of cytochrome P.450 induced by CBZ and CBZE. Pentoxyresorufin is a preferred substrate for cytochrome P-450b (PB inducible) while ethoxyresorufin is a preferred substrate for cytochrome P-450c (3-methylcholanthrene inducible) (Burke et al., 1985). The  results  from  the  0-dealkylation  of  ethoxyresorufln  and  pentoxyresorufin by microsomes from untreated, PB, NS, CFB and CO treated rats are shown in figure 12.  In the untreated, CFB, or CO treated rats,  ethoxyresorufin was not utilized as a substrate while 0-dealkylation of ethoxyresorufin occurred only to a minor degree in the PB and NS groups. On the other hand it was readily apparent that pentoxyresorufin was utilized as a substrate to varying degrees by microsomes from all 5 treatment groups. The microsomal O-dealkylation of pentoxyresorufin and ethoxyresorufin by microsomes from CBZ, CBZE and PG treated animals over the time course of treatment is illustrated in figures 13 and 14, respectively. Pentoxyresorufin, without question, was the preferred substrate by microsomes from all of the treatment groups.  Induction of ethoxyresorufin 0-dealkylation activity was  observed for the CBZ 3 day treatment group (figure 14). Changes in pentoxyresorufin O-dealkylation (PROD) for the PB, CFB, CBZ and CBZE treatment groups relative to the untreated group and to the vehicle control groups are summarized in table 3.  64  Results  12 ci) 0  10  bO  8  6  0  4  ‘i2  ci) -l  0  2  0 control Figure 12.  NS  PB  CFB  CO  and pentoxyresorufin of 0-dealkylation Microsomal ethoxyresorufln (nmol resoruflnlminlmg protein, mean ± s.d.) by microsomes from control, PB, NS, CFB and CO treated rats (n=4). a siguiflntly greater than microsomes from untreated animals (p 0.05), U significantly greater than microsomes from appropriate vehicle control animals treated over the same time period. Microsomal 0-dealkylation was determined as outlined in the Experimental section.  65  Results  12 .,-1  0  10  0 4 c1  8  .  z 0  6  4  U)  a)  0  2  0 CBZ Figure 13.  CBZE  PG  (nmol of pentoxyresorufin 0-dealkylation Microsomal resorufinlminlmg protein, mean ± s.d.) by microsomes from CBZ CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). animals (0.1 significantly greater than microsomes from untreate4 ‘ significantly 0.05), ± 0.01 nmol resorufinlminlmg protein, p vehicle control animals greater than microsomes from appropriate treated over the same time period, C significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal 0-dealkylation was determined as outlined in the Experimental section.  66  Results  1.0  substrate:  ethoxyresorufin 3 day 7day  0.8-  iIIIIIII[  -‘•  lOday 14 day  04  ‘ :: Figure 14.  ._  CBZ  CBZE  PG  (nmol ethoxyresorufin of 0-dealkylation Microsomal CBZ, from omes resorufinlminlmg protein, mean ± s.d.) by micros omal (nz4). Micros rats ed CBZE and PG 3, 7, 10 and 14 day treat mental the Experi in 0-dealkylation was determined as outlined section.  67  Results Table 3.  Summary of PROD (nmol resorufinlminlmg in PROD relative to the untreated group vehicle control group for the PB, CFB, CBZ PROD was determined groups (n=4). l section. Experimenta  protein) and changes or to the respective and CBZE treatment as outlined in the  Pentoxyresorufin O-dealkylation Activity (PROD)  Treatment  fold increase relative to untreated protein mg formedlminl nmol resorufin group mean ± s.d.  fold increase relative to vehicle group  Untreated  0.1 ± 0.01  PB NS  ab 5 • 6.6 ± 0 0.6±0.1  66 6  12  CFB CO  0.4±0.1 0.1±0.1  4 1  3  CBZ 3 day CBZ7 day CBZlOday CBZ 14 day  abc 3 • 8.9 ± 1 i. bc ± 8 . 9 a 2 i. bc ± 0 . 8 a 4 i. bc ± 9 . 6 a 5  89 98 80 69  34 38 40 53  CBZE 3 day CBZE7day CBZE 10 day CBZE14day  ab 3.9 ± 10 3.2±2.0 ab 2.6 ± 05 8.b 7 0 ± 5 . 2  39 32 25 25  15 12 13 19  PG3day PG7day PGlOday PGl4day  0.3±0.1 0.3±0.2 0.2±0.1 0.1±0.1  (p a b C  3 3 2 1  0.05) significantly greater than microsomes from untreated animals significantly greater than microsomes from appropriate vehicle control animals treated over the same time period significantly greater than microsomes from CBZE treated animals over the same time period  68  Results  There  was  no  statistical  difference  in  the  0-dealkylation  of  pentoxyresorufin by microsomes from the CFB treated animals when compared to the CO treated group, although the rate for the CFB induced microsomes was 3 fold greater than that of the CO induced microsomes (figure 12). The rates of pentoxyresorufln O-dealkylation by microsomes prepared from PB, CBZ and CBZE treated rats were significantly greater when compared to the microsomes from the untreated animals, with the increases ranging from a low of 25 fold for the CBZE 10 and 14 day groups to a high of 98 fold for the CBZ 7 day group (table 3). Similarly, the rates of pentoxyresorufin 0-dealkylation by microsomes from PB, CBZ or CBZE treated rats were significantly greater when compared to the O-dealkylation rates of their respective vehicle control groups with the increases ranging from 12 fold for the PB group to 53 fold for the CBZ 14 day group (table 3). When the inducing agents were compared, the rates of pentoxyresorufin 0-dealkylation by microsomes from CBZ 3 and 7 day treated animals were significantly greater than the mean pentoxyresorufin 0-dealkylation rate for microsomes from PB treated rats. The higher pentoxyresorufin 0-dealkylation rates compared to the PB treated group were only true for the CBZ 3 and 7 day treated groups as the rates for the CBZ 10 and 14 day treated groups appeared to decline (table 3). Pentoxyresorufin O-dealkylation rates for microsomes from CBZE 3, 7, 10 and 14 day treated rats were not statistically different from each other over the course of treatment. When CBZE pentoxyresorufin 0-dealkylation rates over the time course were compared to those of the corresponding CBZ treated group or to the PB treated group, CBZE rates were approximately 32 to 43% of the appropriate CBZ treated group and 38 to 58% of the PB treated group (table 4).  69  Results Table 4.  Comparison of mean PROD activities of CBZE 3, 7, 10 and 14 day treated groups as a percent of the PROD activities of the PB and CBZ 3, 7, 10 and 14 day treatment groups. PROD was determined as outlined in the Experimental section.  PROD (nmol resorufin formed/mm/mg protein) CBZE 7 day  CBZE 10 day  CBZE 14 day  (%)  (%)  (%)  (%)  PB  58  49  39  38  CBZ3day CBZ7day CBZlOday CBZ 14 day  43  -  -  -  CBZE 3 day  33  -  -  -  -  -  70  -  32 -  -  -  36  Results  The rates of pentoxyresorufin 0-dealkylation by microsomes from CBZ 3, 7 and 10 day treated animals were not significantly different from each other. However, the rate of pentoxyresorufin 0-dealkylation by microsomes from CBZ 14 day treated animals was significantly lower than the rates for microsomes from CBZ 3, 7 and 10 day treated rats. Results similar to those discussed above for rates normalized to protein content  were  obtained  when  the  rate  of either  pentoxyresorufin  or  ethoxyresorufin 0-dealkylation was normalized to total cytochrome P-450 (data not shown).  Pentoxyresorufin 0-dealkylation rates for microsomes from PB,  CBZ 3, 7, 10 and 14 day and CBZE 3, 7, 10 and 14 day treated animals were significantly increased when compared to the untreated group (17 to 42 fold) or to their respective vehicle control group (6 to 69 fold).  The rates of  pentoxyresorufin 0-dealkylation of the CBZ treated groups were comparable to the PB treated group with the exception of the CBZ 3 day treated group which was significantly higher (6.8 ± 0.4 versus 4.5 ± 0.4 nmol resorufinlmin/nmol cytochrome. P-450). CBZE pentoxyresorufin 0-dealkylation rates over the time course were approximately 61 to 80% of the PB treated group and 48 to 64% of the appropriate CBZ treated groups (data not shown). Quantitation of cytochrome P-450b in microsomes from CBZ, CBZE and PB treated rats by SDS-PAGE and Western blot techniques While PB is known to induce cytochrome P-450b (Waxman and Azaroff, 1992), induction of cytochrome P-450b by CBZ or CBZE has not been reported previously. Since it had been verified in the previous sections that cytochrome P-450b was inducible by CBZ, this particular isozyme was quantitated in microsomes from CBZ 3, 7, 10 and 14 day, CBZE 3, 7, 10 and 14 day and PB treated rats to determine if the inducing abilities of CBZ and CBZE were quantitatively similar to that of PB. 71  Cytochrome P-450b content was  Results quantitated from Western blots based on the intensity of the bands using the Visage Bio-Image Analyser. The amount of cytochrome P-450b quantitated in microsomes from rats treated with PB, CBZ and CBZE over the time course is shown in table 5 as pmol cytochrome P-450b/5 pmol total cytochrome P-450 (loaded per lane) and as a percentage of total hepatic cytochrome P-450. The percent of cytochrome P 450b present ranged from 31% (CBZE 14 day group) to 66% (CBZ 3 day group). Statistically equivalent amounts of the isozyme were present in the CBZ treated microsomes when compared to PB induced microsomes (table 5).  The  microsomes from the PB treated group contained 3.3 ± 0.1 pmol of cytochrome P-450b/5 pmol of spectrally determined cytochrome P-450, representing 65% of total hepatic cytochrome P-450 content. The microsomes from the CBZ 3 day treated group contained 3.3 ± 0.8 pmol cytochrome P-450b15 pmol of spectrally determined cytochrome P-450 representing 66% of total hepatic cytochrome P 450. Although mean quantities of the isozyme in the CBZE induced microsomes (mean 31 to 53%, over the time course) appeared lower than in microsomes from both the PB (65%) and CBZ induced microsomes (mean 39 to 66% over the time course), no statistical differences were observed between the 3 treatment groups.  IN VITRO METABOLISM OF VPA AND (E)-2-ENE VPA Enzyme induction plays an important role in the formation of toxic metabolites of VPA. Enzyme induction due to polytherapy is associated with a high incidence of VPA induced hepatotoxicity (Dreifuss et al., 1987). The effects of PB and CBZ on the in vitro metabolism of VPA have been briefly investigated whilst the effects of CBZE induction have not been yet investigated.  In the  present work, the effects of induàtion by PB, CBZ, CBZE and CFB on the in  72  Results Table 5.  Cytochrome P-450b (pmolI5 pmol of spectrally determined cytochrome P-450 or as percent of total hepatic cytochrome P-450) in microsomes from rats treated with either PB, CBZ for 3, 7, 10 or 14 days or CBZE for 3, 7, 10 or 14 days. Microsomal protein (5 pmol of spectrally determined cytochrome P-45Oflane) was separated by SDS-PAGE and probed using an anti-rat cytochrome P-450b antibody as outlined in the Experimental section. (n=3, mean ± s.d.).  Treatment  cytochrome P-450b pmoIJ5 pmol P-450  cytochrome P-450b of (% total cytochrome P-450)  PB  3.3±0.1  65±2  CBZ 3 day CBZ7day CBZ 10 day CBZ 14 day  3.3 ± 0.8 2.7±0.8 2.7 ± 0.5 1.9 ± 0.4  66 ± 16 54±16 55 ± 11 39±8  CBZE3day CBZE 7 day CBZE 10 day CBZE 14 day  1.8±0.8 2.7 ± 1.4 1.6 ± 0.7 1.6 ± 1.1  35± 17 53±29 33 ± 14 31 ±23  73  Results vitro metabolism of VPA were investigated. Analysis of VPA and metabolites by GCMS The GCMS assay previously developed in our laboratory (Abbott et al., 1986) was used for the analysis of reaction products extracted from the in vitro microsomal metabolism of VPA and (E)-2-ene VPA. It was possible to separate E-2-ene [ ] 7 H and detect 16 metabolites of VPA. The deuterated compounds, 2 3-keto VPA, [ ] 7 H 3-OH VPA, [ [ ] 7 H 4-ene VPA, 2 [ ] 7 H VPA, 2 ]5-OH VPA, 2 7 H 2 VPA recently synthesized in our laboratory (Zheng, [ ] 7 H 4-keto VPA and 2 [ ] 7 H 2 M.Sc. thesis, 1993) were used as internal standards. In vitro metabolism conditions for VPA and (E)-2-ene VPA The in vitro metabolism of VPA and (E)-2-ene VPA by microsomes from the various treatment groups was investigated to determine the formation of cytochrome P-450 mediated metabolites and the effect of induction on the formation of these metabolites. The method used in. this work was based on a procedure from the literature (Rettie et al., 1987) and is outlined in the Experimental section. The amount of VPA used in the incubations was chosen such that the VPA peak in the GCMS chromatogram did not overlap extensively with that of the 4-ene VPA peak, the metabolite of primary interest. Metabolism of the major serum metabolite, (E)-2-ene VPA, was then studied at an equivalent molar dose to VPA to allow a direct comparison. Cytochrome P 450 was varied over the concentration range of 1 to 6 nmol of spectrally determined cytochrome P-450 per incubation and it was determined that 2 nmol of spectrally determined microsomal cytochrome P-450 per in vitro incubation were adequate to obtain quantifiable amounts of VPA or (E)-2-ene VPA metabolites as measured by our GCMS method. Over this range of cytochrome P-450 investigated, a linear relationship was not observed between the product  74  Results  and the amount of protein.  The use of 2 nmol of spectrally determined  cytochrome P-450 per incubation avoided the use of excessive amounts of microsomal protein since the amount of product recovered did not increase. An optimal incubation time was investigated in order to allow the reaction to proceed to completion.  Incubation times of 20, 30, 40, 50 or 60 mm  were  examined. It was determined that the amount of metabolites formed did not change considerably but to be on the safe side an incubation time of 40 mm was selected. Not all 16 metabolites of VPA that could be quantitated in the GCMS assay were monitored. Firstly, not all of the metabolites are cytochrome P-450 generated products and thus were not detected. Additionally, some metabolites, for example 3-ene VPA, could not be distinguished from the background noise. Therefore, when VPA served as the substrate, the metabolites quantitated were 3-OH VPA, 4-OH VPA, 5-OH VPA, 4-ene VPA and 4-keto VPA.  Only 2  metabolites, (E)-2,4-diene VPA and (E,E)-2,3’-diene VPA, were measured when (E)-2-ene VPA was used as the substrate. Additional metabolites monitored for included the formation of (E)-2-ene VPA from VPA and the formation of 3-ene VPA and 4-ene VPA from (E)-2-ene VPA. These metabolic products, however, were not detected. In order to determine that the metabolites to be quantitated were in fact products of cytochrome P-450 mediated metabolism, the incubations were performed using boiled microsomes, or in the absence of cofactors, or in the absence of substrate to eliminate artifacts. Under these conditions, none of the expected metabolites of either VPA or (E)-2-ene VPA were detected (data not shown).  75  Results In vitro metabolism of YPA  Formation of 3-OH VPA from VPA The amount of 3-OH VPA formed from VPA (0.4 jimol) by microsomes from control, PB, NS, CFB and CO treated animals is shown in figure 15 while that by microsomes from CBZ, CBZE and PG treated animals is illustrated in figure 16. Very small quantities of 3-OH VPA were detected for the PG and CBZE 14 day treated groups. The changes in the formation of 3-OH ‘[PA by the PB, CFB, CBZ and CBZE treated groups relative to the untreated group and to their respective vehicle control group are summarized in table 6. The formation of 3-OH VPA from VPA by microsomes from PB treated animals (0.40 ± 0.09 jig) was significantly increased 7 fold when compared to untreated microsomes ( 0.05 ± 0.02 jig) while 3 to 4 fold increases were observed for CBZ 3 day (0.20 ± 0.02 jig), 7 day (0.10 ± 0.05 jig) and 10 day (0.10 ± 0.04 jig) induced microsomes, respectively (figures 15 and 16, table 6). A 9 fold increase in the formation of 3-OH \TPA was observed for the PB treated group when compared to the NS treated group (0.40 ± 0.09 jig versus 0.04 ±0.01 jig) (figure 15, table 6). Increases of 4 to 20 fold were observed when the CBZ treated groups were compared to the PG treated groups (figure 16, table 6).  The mean value of 3-OH VPA produced by microsomes from PB  treated rats was significantly greater when compared to all other treatment groups. Formation of 4-OH VPA from VPA The amount of 4-OH ‘[PA formed from ‘[PA by microsomes from control, PB, NS, CFB and CO treated rats is shown in figure 17 while that by microsomes from CBZ, CBZE and PG treated rats is illustrated in figure 18. The changes in the formation of 4-OH ‘[PA due to induction by PB, CFB, CBZ  76  Results  0.5 ab  0.4  0.2 C’D  control  Figure 15.  PB  NS  CFB  CO  Formation of 3-OH VPA (gig, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4). a significntly greater than microsomes from untreated animals (p 0.05), U significantly greater than microsomes from appropriate vehicle control animals treated over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  77  Results  0.5  0.4  C)  0.3  X  0.2  0.1.  0.0 CBZ  Figure 16.  CBZE  PG  Formation of 3-OH VPA (.tg, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). a significantly greater tian microsomes from untreated animals (0.05 ± 0.02 j.tg, p 0.05), significantly greater than microsomes from appropriate vehicle control animals treated over the same time period, C significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  78  Results Table 6.  A comparison of the metabolism of VPA to 3-OH VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of 3-OH VPA were performed as outlined in the Experimental section.  Formation of 3-OH VPA  Treatment amount formed mean ± s.d. (hg) Untreated  0.05 ± 0.02  PB NS  0.40 ± 0.09 0.04 ± 0.01  CFB CO  0.10±0.01 0.04 ± 0.01  CBZ CBZ CBZ CBZ  3 day 7’day 10 day 14 day  CBZE CBZE CBZE CBZE  3 day 7 day 10 day 14 day  PG3day PG 7 day PG 10 day PG 14 day  --  fold increase relative to untreated group  fold increase relative to vehicle group  7  9  --  2  2  --  0.20 ± 0.02 0.10 ± 0.05 0.10 ± 0.04  4 3 3  *** --  0.10 ± 0.02 0.10 ± 0.05 0.10 ± 0.01  2 2 --  ***  --  ***  --  0.03 ± 0.01 0.02 ±0.02  --  --  --  increase less than 2 fold trace quantities detected  79  20 6 6 4 9 4 2 --  Results  2.0  1.5 bJ C)  1.0 0  4 0.5  0.0 control  Figure 17.  PB  NS  CFB  CO  Formation of 4-OH VPA (.tg, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4). a signific.ntly greater than microsomes from untreated animals (p 0.05), KJ significantly greater than microsomes from appropriate vehicle control animals treated over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  80  Results  2.0  1.5 C-)  1.0 > 0  4 0.5  0.0 CBZ  Figure 18.  CBZE  PG  Formation of 4-OH VPA (rig, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). a significantly greater tan microsomes from untreated animals (0.09 ± 0.01 rig, p 0.05), significantly greater than microsomes from appropriate vehicle control animals treated over the same time period, c significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  81  Results and CBZE relative to the untreated group and to the appropriate vehicle control groups are noted in table 7. When compared to microsomes from untreated animals (0.09 ± 0.01 jig, table 7), the amount of 4-OH VPA produced by PB induced microsomes was increased 15 fold (1.30 ± 0.08 jig) and was significantly greater than all other treatment groups. Increases for the CBZ treated groups ranged from 7 to 12 fold when compared to the untreated group (table 7). Of particular note was the comparison of amounts of 4-OH VPA produced by microsomes from the CBZ and CBZE treated groups when compared to their appropriate PG control groups (figure 18). The increases ranged from a low of 9 fold for the CBZE 10 and 14 day treatment groups to a high of 52 fold for the CBZ 10 day treatment group (table 7) because very low quantities of 4-OH VPA were detected for the PG treated groups. The CBZ treated group over the time course yielded significantly higher amounts of 4-OH VPA when compared to the appropriate CBZE treated group (figure 18). Formation of 5-OH VPA from VPA The amount of 5-OH VPA produced by microsomes from PB, NS, CFB, CO and untreated animals is depicted in figure 19 while that by microsomes from CBZ, CBZE and PG treated animals are shown in figure 20. A summary of the changes in the formation of 5-OH VPA by induced microsomes is given in table 8. Compared to the untreated group (16 ± 3 ng), the amount of 5-OH VPA produced by the PB treated group (87 ± 7 ng) was significantly increased 5 fold (table 8). Significant increases ranging from 3 to 8 fold were also observed for the CFB, CBZ and CBZE 3 and 14 day treated groups when compared to the untreated group (table 8). 82  Results Table 7.  A comparison of the metabolism of VPA to 4-OH VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of 4-OH VPA were performed as outlined in the Experimental section.  Formation of 4-OH VPA  Treatment amount formed mean ± s.d. (jig) Untreated  0.09 ± 0.01  PB NS  1.30 ± 0.08 0.10±0.01  CFB CO  0.20 ± 0.04 0.10±0.01  CBZ CBZ CBZ CBZ  3 day 7 day 10 day 14 day  CBZE CBZE CBZE CBZE  3 day 7 day 10 day 14 day  PG3 day PG7 day PG 10 day PG 14 day  --  fold increase relative to untreated group  fold increase relative to vehicle group  15  14  --  2  2  --  0.80 0.60 1.10 0.60  ± ± ± ±  0.10 0.30 0.50 0.04  10 7 12 7  40 30 52 29  0.30 0.20 0.20 0.20  ± ± ± ±  0.10 0.10 0.10 0.04  3 3 2 2  12 11 9 9  0.02±0.02 0.01±0.00 0.01 ± 0.00 0.01 ± 0.00  --  --  --  --  increase less than 2 fold  83  Results  160 140 120 100 80 60 40 20 0 control  Figure 19.  PB  NS  CFB  CO  Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4). a significgntly greater than microsomes from untreated animals (p 0.05), ‘ significantly greater than microsomes from appropriate vehicle control animals treated over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  84  Results  160 140 120 100 >  60 40 20 0 CBZE  CBZ  Figure 20.  PG  Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P.450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). a significantly greqer than microsomes from untreated animals (16 ±3 ng, p 0.05), u significantly greater than microsomes from appropriate vehicle control animals treated over the same time period, c significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  85  Results Table 8.  A comparison of the metabolism of VPA to 5-OH VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of 5-OH VPA were performed as outlined in the Experimental section.  Formation of 5-OH VPA  Treatment amount formed mean ± s.d. (ng)  fold increase relative to untreated group  fold increase relative to vehicle group  5  6  Untreated  16 ± 3  PB NS  87±7 15±3  CFB CO  43±10 26±11  CBZ3day CBZ7day CBZlOday CBZl4day  126±8 43±18 44±13 86±6  8 3 3 5  3 4 5 2  CBZE3day CBZE7day CBZE1Oday CBZE 14 day  87±13 27±9 21±6 45 ± 7  5 2  2 3 2  PG3day PG7day POlOday PGl4day  38±10 10±2 10±3 39±8  --  --  3  2  --  --  3 2 --  --  2  increase less than 2 fold  86  --  Results The formation of 5-OH VPA from VPA by microsomes from the PB treated rats was significantly increased 6 fold when compared to the NS group (table 8). When the CBZ treated groups were compared to the appropiate PG treated group over the time course, significant increases ranging from 2 to 5 fold were observed. A significant difference was not observed between the CBZE treated groups when compared to the PG treated groups over the time course (table 8). With the exception of the CBZ 7 day treated group, the formation of 5-OH VPA was significantly higher when the CBZ treated groups were compared to the corresponding CBZE treated group. The amount of 5-OH VPA formed from VPA by CBZ 3 day treated microsomes was significantly greater when compared to PB microsomes and all other treatment groups (table 8). Formation of 4-ene VPA from VPA The amounts of 4-ene VPA produced by microsomes from the untreated, PB, NS, CFB and CO treated rats are depicted in figure 21. Values for the CBZ, CBZE and PG treated groups are illustrated in figure 22. Table 9 summarizes the increases in 4-ene VPA formation by the inducing agents when compared to the untreated and the vehicle control groups. PB, CBZ and CBZE were capable of enhancing 4-ene VPA formation relative to the untreated group (table 9). PB gave the greatest increase (6 fold) in this regard, although the increase for CBZ was 4 to 5 fold (table 9). CBZE 3 day treatment also yielded a 2 fold increase in 4-ene formation. Very small quantities of 4-ene VPA were detected for the CBZE 7, 10 and 14 day and the PG treatment groups. The metabolism of VPA to 4-ene VPA was induced 5 fold by PB treatment when compared to the NS group (17 ± 2 ng versus 3 ± 0.6 ng) while the formation of 4-ene VPA by the CBZ treated groups was increased compared to 87  Results  25  20  10  5  0 control  Figure 21.  PB  NS  CFB  CO  Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4). a signific.ntly greater than microsomes from untreated 0.05), U significantly greater than microsomes from animals (p appropriate vehicle control animals treated over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  88  Results  25  3day 7day 10 day 14 day  20 abc  -  15  abc  abc abc  1o ab  I 5  0  -  -  CBZE  CBZ  Figure 22.  PG  Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by micros omes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day a significantly greter than microsomes from treated rats (n=4). 0.05), u significantly greater than untreated animals (3 ± 1 ng, p microsomes from appropriate vehicle control animals treated over C the same time period, significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  89  Results Table 9.  A comparison of the metabolism of VPA to 4ene VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of 4-ene VPA were performed as outlined in the Experimental section.  Formation of 4-ene VPA  Treatment  amount formed mean ± s.d. (ng)  fold increase relative to untreated group  fold increase relative to vehicle group  6  5  Untreated  3±1  PB NS  17±2 3±1  CFB CO  5±1 4±1  CBZ3day CBZ7day CBZlOday CBZl4day  13±2 13±3 13±2 12±2  5 4 4 4  CBZE3day CBZE 7 day CBZE 10 day CBZE 14 day  7±1  2  PG3 day PG7 day PG 10 day PG 14 day  ***  --  --  2 --  *** ***  *** *** ***  increase less than 2 fold trace quantities detected  90  Results the PG treated groups (table 9).  The production of 4-ene VPA by the CBZ  treatment groups was significantly higher when compared to the CBZE and PG treated groups over the time course. Only the formation of 4-ene VPA by the CBZE 3 day treated group was significantly greater when compared to the PG 3 day treated group. Formation of 4-keto VPA from VPA The mean quantities of 4-keto VPA produced from VPA by microsomes from the various treatment groups are displayed in figures 23 and 24.  The  effects of induction by PB, CFB, CBZ and CBZE on the formation of 4-keto VPA relative to the untreated and vehicle control groups are summarized in table 10. The formation of 4-keto VPA was significantly enhanced by CBZ over the time course.  Very large increases relative to the untreated group were  observed. However, this increase could not be determined because only trace quantities of 4-keto VPA were formed by microsomes from the untreated group. When compared to the vehicle controls, significant increases of 3 to 7 fold were observed for the CBZ treatment groups, plus a 4 fold increase for the CBZE 3 day treatment group (table 10). Despite much higher mean quantities of 4-keto VPA for the PB treated group, it was not significantly greater when compared to the NS treated group (figure 23). The formation of 4-keto VPA by the CBZ 3, 7 and 10 day treated groups was significantly greater when compared to the PB treated group and the corresponding CBZE treated group. In vitro metabolism of (E)-2-ene VPA Since (E)-2-ene VPA is the major metabolite of VPA in the serum and possesses anticonvulsant activity, it was important to study the effects of various inducing agents on its in vitro metabolism. The PB induced in vitro metabolism of (E)-2-ene VPA has only recently been reported (Kassahun and  91  Results  25  20 biJ  15  0 0  10  5  0 control  Figure 23.  PB  NS  CFB  CO  Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and CO treated rats (n=4). a significantly greater than microsomes from untreated animals (p 0.05). Microsomal incubations were performed as outlined in the Experimental section.  92  Results  60  50  40  >  30  0  20  10  0 CBZ  Figure 24.  CBZE  PG  Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitro metabolism of VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). a significantly greater tian microsomes from untreated animals (0.1 ± 0.01 ng, p 0.05), significantly greater than microsomes from appropriate vehicle control animals treated over the same time period, C significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  93  Results  Table 10.  A comparison of the metabolism of VPA to 4-keto VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of 4-keto VPA were performed as outlined in the Experimental section.  Formation of 4-keto VPA  Treatment amount formed mean ± s.d. (ng)  fold increase relative to untreated group  fold increase relative to vehicle group  *  4  Untreated PB NS  16±4 4±2  CFB CO  7±5 4±1  * *  2  *  CBZ3day CBZ7day CBZlOday CBZl4day  37±14 33±8 44±7 25±15  *  7  *  7  *  3 4  CBZE3day CBZE7day CBZE1Oday CBZE14day  20±12 1±3 18±5 8±2  *  PG3day PG7day PGlOday PGl4day  -  *  *  --  --  *  ***  *  18±7 6±3  * *  trace quantities detected *  * *  5±1  increase less than 2 fold  increase> 20 fold  94  4 2  Results Baillie, 1993). If(E)-2-ene VPA is to be utilized as an anticonvulsant agent, it is likely to be used in combination with CBZ, thus necessitating investigation into the effects of induction on its metabolism.  (E)-2-ene VPA is metabolized to  several diunsaturated metabolites, one ((E,E)-2,3’-diene VPA) of which possesses anticonvulsant activity (Abbott and Acheampong,  1988) while  another, ((E)-2,4-diene VPA), is known to produce hepatic steatosis in rats (Granneman et al., 1984c). Formation of (E,E)-2,3 ‘-diene VPA from (E)-2-ene VPA The  production  of (E,E)-2,3’-diene VPA from  (E)-2-ene VPA by  microsomes from control, PB, NS, CFB and CO treated rats is shown in figure 25 while that by microsomes from CBZ, CBZE and PG treated rats is illustrated in figure 26. The changes in the formation of (E,E)-2,3’-diene VPA relative to the untreated and vehicle groups are summarized in table 11. The amount of (E,E)-2,3’-diene VPA formed from (E)-2-ene VPA significantly increased 9 fold and 2 fold, respectively for the PB and CFB treated groups when compared to the untreated group (table 11). Significant increases of 2 to 8 fold were observed for the CBZ treated groups when compared to the untreated group (table 11).  The formation of (E,E)-2,3’-diene VPA by  microsomes from rats treated for 3, 7 and 10 days with CBZE increased significantly 2 to 5 fold when compared to the untreated group (table 11). The formation of (E,E)-2,3’-diene VPA from (E)-2-ene VPA by microsomes from PB treated rats was significantly greater (7 fold, 18 ± 4 ng versus 3 ± 1 ng) than its vehicle control, NS (figure 25, table 11). The metabolism of (E)-2-ene VPA by microsomes from CBZ treated animals over the time course were significantly greater when compared to microsomes from PG treated animals over the same time period with increases ranging from 3 to 9 fold (figure 26). The mean values observed of (E,E)-2,3’-diene VPA from the CBZE treated 95  Results  30  .LJ  >  25  20  ci) ci -4  15  10  5  0 control  Figure 25.  PB  NS  CFB  Co  Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from untreated, PB, NS, CFB and Co treated rats (n=4). a significant1yU greater than significantly 0.05), microsomes from untreated animals (p greater than microsomes from appropriate vehicle control animals treated over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  96  Results  30  bO  25  20 G) cjj  C\2  15  10  5  0 CBZ  Figure 26.  CBZE  PG  Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of from CBZ, CBZE and PG spectrally determined cytochrome P-450) 3, 7, 10 and 14 day treated rats (n=4). a significantly greater tha 0.05), microsomes from untreated animals (2 ± 1 ng, p significantly greater than microsomes from appropriate vehicle control animals treated over the same time period, significantly greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  97  Results Table 11.  A comparison of the metabolism of (E)-2-ene VPA to (E,E)-2,3’diene VPA by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of (E,E)-2,3’-diene VPA were performed as outlined in the Experimental section.  Formation of (E,E)-2,3’-diene VPA  Treatment amount formed mean ± s.d. (ng)  fold increase relative to untreated group  fold increase relative to vehicle group  9  7  Untreated  2±1  PB NS  18±4 3±1  CFB CO  4±1 2±1  CBZ3day CBZ7day CBZlOday CBZl4day  17±2 14±2 11±3 4±1  8 7 5 2  3 4 5 9  CBZE3day CBZE7day CBZE1Oday CBZE14day  10±1 9±1 6±1 2±1  5 5 2  2 3 2 3  PG3day PG7day PG 10 day PG 14 day  6±1 5±1  --  --  2 --  --  3 3  *** ***  increase less than 2 fold  >‘  2  trace quantities detected  98  Results groups significantly increased 2 to 3 fold when compared to the PG treated groups (table 11, figure 26). In addition, (E,E)-2,3’-diene VPA values for the CBZ 3, 7, 10 and 14 day treated groups were significantly higher than the CBZE treated group over the same time period (figure 26). The formation of (E,E)-2,3’-diene VPA from (E)-2ene VPA by PB (18 ± 4 ng) was significantly greater when compared to all other groups, with the only exception being the CBZ 3 day treated group (17±2 ng). Formation of (E)-2,4-diene VPA from (E)-2-ene VPA The amount of (E)-2,4-diene VPA produced from the biotransformation of (E)-2-ene VPA by microsomes from the various treatment groups is depicted in figures 27 and 28.  The changes in the formation of (E)-2,4-diene VPA by  microsomes from the PB, CFB, CBZ and CBZE treated groups relative to the untreated and vehicle control groups are summarized in table 12. When compared to microsomes isolated from untreated control rats, the production of (E)-2,4-diene VPA was significantly increased 13 fold for the PB treated group, 6 fold for the CBZ 3 day treated group, 13 fold for the CBZ 7 day treated group, 11 fold for the CBZ 10 day treated group, 5 fold by the CBZ 14 day treated group and 9 fold for the CBZE 7 day treated group (table 12). No significant differences were observed in the formation of (E)-2,4-diene VPA when the other treatment groups were compared to the control group. The formation of (E).-2,4-diene VPA by microsomes from PB treated animals was significantly higher than the NS treated group (12 fold increase, table 12). The amount of (E)-2,4-diene VPA produced from (E)-2-ene VPA was significantly increased for the CBZ treated groups when compared to the PG treated groups over the same time course with increases of 6 to 12 fold (figure 28, table 12). Of the CBZE treated groups, only the CBZE 7 day treated group was significantly enhanced when compared to the PG 7 day treated group (4 fold 99  Results  4.0  3.5 0  3.0 2.5  a) a)  2.0  .-4  1.5 1.0 0.5 0.0 control  Figure 27.  PB  NS  CFB  CO  Formation of (E)-2,4-diene VPA (tg, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P450) from untreated, PB, NS, CFB and a significanty greater than microsomes CO treated rats significantly greater than 0.05), from untreated animals (p microsomes from appropriate vehicle control animals treated over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  100  Results  r abc abc  gi  I  3day 7 day 10 day l4day  2.5 ab  2.0  4 —‘  1.5  abc  ]  abc  1.0  ::___ Figure 28.  CBZ  CBZE  PG  Formation of (E)-2,4-diene VPA (jig, mean ± s.d.) from the in vitro metabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). a significantly greater tha 0.05), microsomes from untreated animals (0.20 ± 0.02 jig, p vehicle appropriate from microsomes greater than significantly significantly period, time same the over control animals treated greater than microsomes from CBZE treated animals over the same time period. Microsomal incubations were performed as outlined in the Experimental section.  101  Results Table 12.  A comparison of the metabolism of (E)-2-ene VPA to (E)-2,4-diene by microsomes from PB, CFB, CBZ and CBZE treated rats, relative to the untreated group or to the respective vehicle control group (n=4). Microsomal incubations and quantitation of (E)-2,4-diene VPA were performed as outlined in the Experimental section.  Formation of(E)-2,4-diene VPA  Treatment amount formed mean ±s.d. (‘g) Untreated  0.20 ± 0.02  PB NS  2.5 ± 0.60 0.20±0.10  CFB CO  0.40 ± 0.10 0.20 ± 0.10  fold increase relative to untreated group  fold increase relative to vehicle group  13  12  --  --  --  --  CBZ 3 day CBZ 7 day CBZlOday CBZ 14 day  1.2 ± 0.2 2.5 ± 0.6 2.1±1.1 1.0±0.2  6 13 11 5  12 6 6 6  CBZE3day CBZE7day CBZE1Oday CBZE14day  0.5 ±0.2 1.6±0.6 0.4±0.04 0.3±1.0  2 9 2 2  5 4 3 2  PG3day PG 7 day PG 10 day PG 14 day  0.1±0.04 0.5 ± 0.02 0.1 ± 0.10 0.2± 0.05  --  --  2 --  --  increase less than 2 fold  102  Results increase). In addition, a statistical difference was not observed in the amount of (E)-2,4-diene VPA formed from (E)-2-ene \TPA by microsomes from PB, CBZ 7 day and CBZ 10 day treated rats. The amount of (E)-2,4-diene VPA formed by the CBZ treated groups were significantly higher than the corresponding CBZE treated groups. Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA and (E)-2-ene VPA by inicrosomes from PB and CBZ treated rats Cytochrome P-450b has been implicated in the formation of 4-ene \TPA from VPA (Rettie et al., 1988). Since cytochrome P-450b had been identified as an isozyme capable of being induced by CBZ and PB, the effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA and (E)-2-ene VPA was investigated. Thus, the extent of involvement of cytochrome P-450b in  the microsomal metabolism of VPA and (E)-2-ene VPA could be determined. Microsomal protein was incubated with either the anti-rat cytochrome P-450b antibody or control IgO for 10 mm prior to initiation of the reaction by addition of the substrate, either VPA or (E)-2-ene VPA (Chang, Ph.D. thesis, 1991). Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA The effects of anti-rat cytochrome P-450b on the in vitro metabolism of VPA to 3-OH VPA, 4-OH VPA, 5-OH VPA, 4-ene VPA and 4-keto VPA by  microsomes from rats treated with either CBZ for 3 days or with PB is illustrated in figures 29 to 33. The metabolism of \TPA to 3-OH VPA by microsomes prepared from PB and CBZ 3 day treated rats was inhibited 63% and 52%, respectively at the highest antibody concentration of 2.5 mg of IgG/nmol of cytochrome P-450 (figure 29).  103  Results  100 90  o •  PB CBZ3day  W80  70 0 0 ,  60  0  g50 40 >  c  30 20 10 0 0.0  0.5  I  I  1.0  1.5  2.0  2.5  3.0  mg IgG/nmol cyt. P—450  Figure 29.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 3-OH VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  104  Results The formation of 4-OH VPA from VPA by microsomes prepared from PB and CBZ treated rats microsomes was inhibited 81% and 88%, respectively at the highest IgG concentration investigated (figure 30). The biotransformation of VPA to 5-OH VPA using microsomes from PB and CBZ 3 day induced rats was inhibited 60% and 45%, respectively at the highest antibody concentration investigated (figure 31). The metabolism of VPA to 4-ene VPA by microsomes from PB and CBZ 3 day treated rats was completely inhibited in each case at an antibody concentration of 2.5 mg of IgG/nmol of cytochrome P-450 (figure 32). The production of 4-keto VPA from VPA by microsomes from PB and CBZ 3 day induced rats was blocked 86% and 75%, respectively in the presence of the antibody at the highest concentration examined (figure 33). Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of(E)-2ene VPA The effect of anti-rat cytochrome P-450b on the in vitro metabolism of (E) 2-ene VPA to (E,E)-2,3’-diene VPA and (E)-2,4-diene VPA by microsomes from rats treated with CBZ for 3 days or PB is shown in figures 34 and 35, respectively. Metabolism to (E,E)-2,3’-diene VPA by microsomes prepared from CBZ treated rats was inhibited approximately 18% at the highest antibody concentration (2.5 mg of IgG/nmol of cytochrome P-450) examined while it was inhibited 48% in microsomes from PB treated rats (figure 34). Metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA was inhibited 89% and 85% by microsomes from PB and CBZ treated rats, respectively at the highest antibody concentration investigated (figure 35).  105  Results  100 90 0 -4-)  0 C-)  a •  80  PB CBZ3day  70 60  0  50 -c1  40 30  0  20 10 0 0.0  0.5  1.0  1 .5  2.0  2.5  3.0  mg IgG/nmol cyt. P—450  Figure 30.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 4-OH VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  106  Results  1 20 110 0  100 0 s-I  0 C) 0  •  90  PB CBZ3day  80 70 60 50 40  c  30 20 10 0 0.0  I  I  0.5  1.0  1 .5  I  I  2.0  2.5  3.0  mg IgG/nmol cyt. P—450  Figure 31.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 5-OH VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  107  Results  1 20 110 0  100  .  PB CBZ3day  0  90 o  80  t4j  70  C)  0  60 50 >.  40  10  I  I  0.5  1.0  1.5  I  I  2.0  2.5  3.0  mg lgG/nmol cyt. P—450  Figure 32.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 4-ene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  108  Results  1 20 110 0 0 4)  90  PB CBZ3day  0 C)  o —  70 60  124  50  o  40  U.)  ‘  30 20 10 0 0.0  I  I  I  0.5  1.0  1 .5  2.0  2.5  3.0  mg IgG/nmol cyt. P—450  Figure 33.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of VPA to 4-keto VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  109  Results  120 0  0 C)  110 100  90  0  80 70 60 G)  a)  50 40  C)  c’•  30 20  0  PB  •  CBZ3day  10 0 —0.5  I  I  I  0.0  0.5  1.0  1.5  2.0  2.5  3.0  mg IgG/nmol cyt. P—450  Figure 34.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of (E)-2-ene VPA to (E,E)-2,3’-diene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  110  Results  1 20 —  0 -  0  110  100 90  I.  o  ‘—  80 70  60 50 40 30  20  0 —0.5  0.0  0.5  1.0  1.5  2.0  2.5  3.0  mg IgG/nmol cyt. P—450  Figure 35.  Effect of anti-rat cytochrome P-450b antibody on the in vitro metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA by microsomes (2 nmol of spectrally determined cytochrome P-450) from PB and CBZ 3 day treated rats. Microsomes were prepared from 4 pooled livers. Microsomal incubations were performed as outlined in the Experimental section.  111  Results Effect of anti-rat cytochrome P-450h on the in vitro metabolism of VPA and (E)-2-ene VPA by untreated microsomes  Cytochrome P-450h, a male specific isozyme, catalyzes a number of metabolic reactions including progesterone and testosterone 2c- and 16ahydroxylation (Ryan and Levin, 1990). The effect of an antibody specific for cytochrome P-450h was investigated to determine if this isozyme was also involved in the metabolism of VPA or (E)-2-ene VPA, particularily to the metabolites whose formation was not completely inhibited in the presence of the anti-rat cytochrome P-450b antibody. Anti-rat cytochrome P-450h antibody did not exhibit any significant effects on the metabolism of VPA by control microsomes (data not shown). In order to examine the possibility of an additive effect on VPA metabolism, the anti-rat cytochrome P-450h antibody (2 mg of IgG/nmol of cytochrome P-450) was added in the presence of the anti-rat cytochrome P-450b antibody (2 mg of IgG/nmol of cytochrome P-450) to microsomes prepared from CBZ 3 day or PB treated rats. No differences were observed in the inhibition profiles of 3-OH VPA, 4-OH VPA and 5-OH VPA (data not shown). The results obtained were similar to those obtained with the anti-rat cytochrome P-450b antibody alone. In an effort to test that the isozyme(s) of cytochrome P-450 responsible for (E)-2-ene VPA metabolism were the same as those for VPA, the effects of the antibody against rat cytochrome P-450h on the in vitro metabolism of (E)-2-ene VPA to (E,E)-2,3’-diene VPA and (E)-2,4-diene VPA by microsomes prepared from untreated animals was investigated. As in the case of VPA, the antibody did not exhibit any significant inhibitory effects on the biotransformation of (E) 2-ene VPA to its 2 diunsaturated metabolites. When the additive effects of anti-rat cytochrome P-450h antibody (2 mg/nmol cytochrome P-450) and anti-rat cytochrome P-450b antibody (2  112  Results  mg/nmol cytochrome P-450) were examined in either CBZ 3 day or PB microsomes, no significant inhibitory actions were observed.  QUANTITATION OF CBZ, CBZE AND CBZD CBZ is known to induce its own metabolism via the epoxide-diol pathway (Eichelbaum et aL, 1985). The metabolites of CBZ and CBZE were quantitated in 12 h rat urine collections over the 14 day time course to confirm if this pathway was induced with chronic administration. Analysis of CBZ and metabolites in rat urine by HPLC The methods of Elyas et al. (1982) and Kumps et al. (1985) were combined to provide the methodology for CBZ and metabolite analysis by HPLC. Ethyl acetate  was  found  to  be  dichloromethane for CBZD.  a  more  efficient  extraction  solvent  than  The isocratic method of Elyas et al. (1982) was  found to be incapable of adequately separating CBZD from the endogenous compounds present in rat urine so a gradient method was developed. Representative chromatograms of CBZ and metabolite  standards, with  comparison to CBZ and metabolites extracted from a rat urine sample and a spiked urine sample are shown in figure 36.  The assay was linear over the  concentration range examined.  Urinary recoveries of CBZ, CBZE and CBZD after dosing with CBZ The 12 h urinary recoveries of CBZ and metabolites after dosing rats with CBZ at 100 mg/kg are shown in table 13. Very small quantites of CBZ were recovered in all 12 h collections, ranging from mean quantities of 17.3 jig for the CBZ 3 day group to 44.4 jig for the CBZ 7 day group. The recoveries of CBZE (mean 267 to 339 jig) and CBZD (337 to 602 jig) were an order of magnitude higher than the CBZ levels.  113  Results  a  b  2 1  4 a,  3  d  C  3  4  2  4  L/1  Figure 36.  HPLC chromatograms of a) standards of CBZ, CBZE, CBZD and MCBZ, b) extracted blank rat urine sample, c) extracted spiked rat urine sample and d) extracted rat urine sample. Peak 1, CBZD, peak 2, CBZE, peak 3, CBZ and peak 4, MCBZ. Chromatography conditions were as described in the Experimental section.  114  Results Table 13.  Total 12 h urinary recoveries of CBZ, CBZE and CBZD (jig) from rats treated with CBZ 100 mg/kg every 12 h. (n=4, mean ± s.d.). CBZ, CBZE and CBZD were quantitated as described in the Experimental section.  Treatment  CBZ (jig)  CBZE (jig)  CBZ3 day CBZ 7 day CBZ 10 day CBZ 14 day  17.3± 15.5 44.4 ± 25.3 35.9 ± 19.7 38.4 ± 8.3  317±256 270 ± 138 267 ± 94 339 ± 195  115  CBZD (jig) 602 337 551 365  ±461 ± 152 ± 279 ± 110  Results The recoveries of CBZ, CBZE and CBZD expressed as a percent of the dose of CBZ administered are shown in table 14. Only 0.1 to 0.2% of the dose administered was recovered as CBZ.  The recoveries of CBZE and CBZD  represented 1.1 to 1.6% and 1.5 to 3.0% of the dose administered, respectively. Approximately 2.9 to 4.6% of the dose was recovered in the urine in the 12 h following administration of the last dose. No significant differences were observed in the urinary recoveries over the 14 day treatment period investigated. Urinary recoveries of CBZE and CBZD after dosing with CBZE The 12 h urinary recoveries of CBZE and CBZD after administration of CI3ZE at a dose of 50 mg/kg are summarized in table 15.  The mean 12 h  recovery of CBZE ranged from 121 to 504 jig while the mean recovery of CBZD ranged from 177 to 523 jig over the same time period. The recoveries of CBZE and CBZD expressed as a percent of the dose of CBZE administered are shown in table 16. The mean recovery of CBZE as a percent of the dose administered ranged from 0.4 to 2.0% while the recovery of CBZD ranged from 0.6 to 2.0% over the same time period.  The total dose  recovered as CBZE and CBZD ranged from 1.5 to 4.0% of the dose administered. As with the CBZ treatment groups, no significant differences were observed over the 2 week period of CBZE administration.  116  Results Table 14.  Urinary recoveries (12 h) of CBZ, CBZE and CBZD as percent of dose administered from rats treated with CBZ 100 mg/kg every 12 h. (n=4, mean ± s.d.). CBZ, CBZE and CBZD were quantitated as described in the Experimental section.  Treatment  CBZ (% of dose)  CBZE (% of dose)  CBZD (% of dose)  total (% of dose)  CBZ 3 day CBZ7day CBZ 10 day CBZ 14 day  0.1 ± 0.1 0.2±0.1 0.2 ± 0.1 0.2 ± 0.1  1.6 ± 1.3 1.2±0.6 1.1 ± 0.4 1.4 ± 0.8  3.0 ± 2.4 1.5±0.7 2.3 ± 1.1 1.5 ± 0.5  4.6 ± 3.6 2.9±1.1 3.5 ± 1.5 3.0 ± 1.3  117  Results Table 15.  Total 12 h urinary recoveries of CBZE and CBZD (jig) from rats treated with CBZE 50 mg/kg every 12 h. (n=4, mean ± s.d.). CBZE and CBZD were quantitated as described in the Experimental section.  Treatment  CBZE (jig)  CBZE3day CBZE7day CBZE1Oday CBZE14day  247 ±83 504± 174 121±83 249±134  118  CBZD (jig) 447 ±494 523± 139 319±256 177±83  Results Table 16.  Urinary recoveries (12 h) of CBZE and CBZD as percent of dose from rats treated with CBZE 50 mg/kg every 12 h. (n=4, mean ± s.d.). CBZE and CBZD were quantitated as described in the Experimental section.  Treatment  CBZE 3 day CBZE 7 day CBZE1Oday CBZE 14 day  CBZE (% of dose)  CBZD (% of dose)  total (% of dose)  1.1 ± 0.4 2.0 ± 0.6 0.4±0.3 0.9 ± 0.5  2.0 ± 2.2 2.0 ± 0.5 1.1±0.9 0.6 ± 0.3  3.1 ± 2.6 4.0 ± 1.1 1.6±1.1 1.5 ± 0.5  119  DISCUSSION  CHOICE OF EXPERIMENTAL CONDITIONS Animal model Previous work suggested the rat should provide a reasonably good model to study the interaction between VPA and CBZ since the metabolism of both CBZ (Faigle and Feldmann, 1982) and VPA (Granneman et al., 1984a) in the rat are quite similar to that of man. CBZ undergoes biotransformation in rat and human by the same major metabolic pathways: epoxidation of the 10,11 double bond, hydroxylation of the 6-membered aromatic rings, N-glucuronidation of the carbamoyl side chain and substitution on the 6 membered rings by sulphur containing groups (Faigle and Feldmann, 1982; Faigle and Feldmann, 1989). In both rat and man, the 2 major metabolic pathways of VPA metabolism are glucuronidation and 3-oxidation (Granneman et al., 1984a). The male Long Evans rats used in this study weighed from 195 to 230 g and were approximately 7 to 9 weeks of age and, thus, are considered to be adults. In male, untreated Wistar rats, total hepatic cytochrome P-450 content remained unchanged from 1 week to 24 months of age and low levels of cytochromes P-450b and P-450e were detectable (Imaoka et aL, 1991). Choice of vehicle for CBZ and CBZE CBZ possesses very low solubility in water (72 mg/L in phosphate buffer) but is quite soluble in organic solvents including chloroform, dichioromethane (Kutt, 1989), alcohol, acetone and propylene glycol (The Merck Index, 1989). CBZE also has very limited solubility in aqueous solutions but like CBZ, is readily soluble in organic solvents (Kerr and Levy, 1989). Some organic solvents such as acetone and ethanol are known to induce cytochrome P-450 in rats  120  Discussion (Soucek and Gut, 1992). Thus, propylene glycol was chosen as the suspending vehicle for CBZ and CBZE because in humans it did not produce changes in the antipyrine test at an oral dose of 55 mL (Nelson et al., 1987). Although there has been one report in the literature of PG causing appetite suppression in rats (Carl and Smith, 1989), the doses used in the current study (0.1 mLfkg twice daily) were much lower than in the 1989 study (8 mLfkg 3 times daily via gastric intubation). GCMS analysis of VPA and metabolites A modified version of the gas chromatograph-mass spectrometric assay previously developed in our laboratory was employed for the analysis of VPA and its metabolites in the microsomal incubates (Abbott et al., 1986).  The  samples were analyzed on a gas chromatograph equipped with a mass selective detector. With this assay, it was possible to simultaneously quantitate VPA and 16 metabolites using the selected ion monitoring mode.  Briefly, the method  employed a capillary column and the extracted samples were derivatized with MTBSTFA to yield the tBDMS derivatives. The [M-57] ion which was formed from the loss of the t-butyl group was monitored. The modified assay utilized 2MGA as the internal standard for the 2 diacid metabolites of VPA, 2-PSA and 2PGA.  In addition, heptadeuterated analogues of VPA and some of the VPA  3-keto VPA, [ 1 7 H 2-ene VPA (E and Z), 2 [ ] 7 H 4-ene VPA, 2 [ ] 7 H metabolites, 2 5-OH VPA, were used as internal [ ] 7 H 3-OH VPA and 2 [ ] 7 H 4-keto VPA, 2 [ ] 7 H 2 standards.  Correlation coefficients of the calibration curves were 0.990 or  better. The assay has been described at a recent conference (Yu et al., 1992) and a manuscript is currently in preparation (Yu et at.).  121  Discussion Choice of metabolites monitored from the in vitro microsomal metabolism of VPA and (E)-2-ene VPA Initially upon injection of the derivatized extracts of the microsomal incubates, all possible metabolites of VPA and (E)-2-ene VPA were monitored. For VPA, the additional metabolites monitored included 3-keto VPA, 2-ene VPA, 3-ene VPA, 2-PSA and 2-PGA.  Signals from these metabolites could not be  distinguished from background and subsequently only 3-OH VPA, 4-OH VPA, 5OH VPA, 4-keto VPA and 4-ene VPA were monitored and quantitated.  In  extracts of incubates where (E)-2-ene VPA was employed as the substrate, neither 3-ene VPA nor VPA peaks were detectable above the background noise although these metabolites have been reported as products of 2-ene VPA metabolism in rats (Granneman et al., 1984a; Lee, 1991; Vorhees et at., 1991; Loscher et at., 1992). The 3-OH VPA and 3-keto VPA metabolites of (E)-2-ene VPA, that arise from mitochondrial [3-oxidation were not detected from the in vitro microsomal metabolism of (E)-2-ene VPA. Consequently, only the (E)-2,4diene VPA and (E,E)-2,3’-diene VPA metabolites were monitored as the products of rat hepatic microsomal metabolism of (E)-2-ene ‘[PA. HPLC analysis of CBZ and metabolites The HPLC assay used for the analysis of CBZ, CBZE and CBZD from rat urine samples was a combination of 2 procedures from the literature (Elyas et at., 1983; Kumps et at., 1985).  Kumps and co-workers (1985) were able to  separate a number of anticonvulsants and their metabolites (16 compounds in total) in serum. The extraction of CBZD was greatly enhanced by changing the extraction solvent from dichloromethane to ethyl acetate.  Many attempts to  separate the CBZD peak from endogenous compounds extracted from the urine, including varying the composition of the mobile phase, changing columns and employing various gradient systems did not yield the extent of separation 122  Discussion claimed in the literature (Kumps et al., 1985).  Urine may contain more  endogenous compounds compared to serum, leading to a more complicated chromatographic separation.  A COMPARISON OF THE INDUCTION OF RAT HEPATIC MICROSOMAL CYTOCHROME P-450 CONTENT BY PB, CBZ, CBZE AJTD OTHER INDUCING AGENTS Cytochrome P-450 content in hepatic microsomes from untreated rats Cytochromes P-450b and P-450e were not detected on the immunoblots of hepatic microsomal protein from untreated rats. This was not surprising since these isozymes are present in very low quantities in untreated rat liver (Waxmann and Azaroff 1992). In untreated rats, cytochrome P-450b content is reported to be highest in the lung in comparison to liver, kidney, adrenal or small intestine while cytochrome P-450e content is highest in the liver (Christou et at., 1987). With PB induction, the highest levels of both cytochromes P-450b and P-450e are found in the liver. Sex differences in rats have been observed in the induction of these 2 isozymes (Yamazoe et at., 1987). Cytochromes P-450b and P-450e are minor constituents of the cytochrome P-450 pool, representing 5% or less of the total cytochrome P-450 content of hepatic microsomes from untreated male or female Long Evans rats (Thomas et at., 1981).  In uninduced Long Evans, male immature and adult rats,  cytochrome P-450b content was 4% and 2%, respectively, of the total hepatic cytochrome P-450 content (Thomas et at., 1981). The expression of cytochrome P-450b in uninduced male rats from 5 different strains (Sprague-Dawley, Long Evans, Wistar, Brown Norway and Fischer F344) varied considerably (< 2 to 9 pmoLfmg protein) (Wilson et at., 1987). However, the expression of cytochrome P-450e exhibited very little interstrain variability (17 ± 5 pmoll mg protein) with 123  Discussion the exception of the Brown Norway strain (8.5 pmollmg protein). Effect of VPA on cytochrome P-450 content  VPA is generally not considered to be an enzyme inducing agent although there are conflicting reports in the literature. For instance, VPA did not affect total rat hepatic microsomal cytochrome P-450 content when administered at doses of 80 mg/kg or 120 mg/kg i.p. twice daily for 3 days (Sapeika and Kaplan, 1975).  In another study, when VPA was administered to rats at doses of 100  mg/kg or 200 mg/kg i.p. daily for 30 days, a dose dependent increase in mitochondrial carnitine acetyltransferase activity occurred but no changes were observed in the total hepatic cytochrome P-450 content (Singh et at., 1987). Cotariu et at. (1985) observed a decrease in cytochrome P450 content when VPA was administered to male rats whereas Rogiers et at. (1988) observed a significant increase in total cytochrome P-450 content when VPA was administered to rats at a dose of 100 mg/kg i.p. daily for 10 days. In the current study, VPA administration did not result in increased total hepatic cytochrome P-450 content. Furthermore, anti-rat cytochrome P-450b antibody did not react with microsomal protein isolated from the livers of rats treated with VPA. The effects of VPA on cytochrome P-450, thus, remain unclear. Perhaps a longer treatment with VPA is necessary to elicit changes in cytochrome P-450.  We  treated rats for only 3 days similar to the study of Sapeika and Kaplan (1975) who observed no changes while Rogiers et at. (1988) noted increased cytochrome P-450 content with administration of VPA over 10 days at a lower dose. Effect of CFB on hepatic cytochrome P-450 content  Corn oil is commonly used as a vehicle for CFB and other compounds not readily soluble in aqueous solutions and when administered as such it has not been reported to affect hepatic cytochrome P-450 (Thomas et at., 1981). In this  124  Discussion  study, corn oil itself, appeared to cause an increase in the total hepatic cytochrome P-450 content. Increased levels of cytochromes P-450d, P-450e, P 450j and P-450p have been observed when CO (20%) was administered as a dietary source (Yoo et at., 1992). CFB treatment appeared to induce total hepatic cytochrome P-450 content when compared to the untreated group. However, when compared to the CO treated group, the increase was not statistically significant. A higher dose of CFB was initially used, but due to the loss of 2 animals with an “apparent” drug toxicity, the dose of CFB was decreased to 350 mg/kg i.p. daily from 500 mg/kg i.p. daily. Based on reports in the literature, a wide range of CFB doses have been utilized for induction experiments in rats. Bachmann and co-workers (1988) used doses of 200 mg/kg of CFB i.p. daily for 3 days and Heinemeyer and co-workers (1985) used doses of 500 mg/kg i.p. daily for 7 days. A dose of 250 mg/kg daily for 3 days via gastric intubation resulted in significantly increased (33.6%) cytochrome P-450 levels when the CFB treated group was compared to a peanut oil control group (Sharma et at., 1988a). In the current study, only a 22% increase was observed when the CFB treated group was compared to the CO control group. The assumption then, is that either the dose of CFB that we employed andlor the time interval of CFB treatment was inadequate to achieve maximal induction of total hepatic cytochrome P-450 content. Cytochrome P-452 (lauric acid hydroxylase, cytochrome P-450 4A1), is the isozyme of cytochrome P-450 that is induced by CFB (Soucek and Gut, 1992). Cytochrome P-452 did not display any cross reactivity with the isozymes of cytochrome P-450 induced by PB or by polycyclic hydrocarbons (Tamburini et at., 1984).  Thus, the reaction of anti-rat cytochrome P-450b antibody with  microsomal protein from CFB treated rats on some Western blots was somewhat  125  Discussion surprising (figures 10 and 11). This reaction was not observed on all Western blots and may be due to carryover. Cytochrome P-452 constituted approximately 6% of the total cytochrome P-450 pool in untreated male Long Evans rats and after CFB administration increased to 11% (Chinje and Gibson, 1991). Although specific cytochrome P 452 was not quantitated in this study, it has been reported to increase in a dose dependent manner when CFB (50 to 250 mg/kg daily) was administered to rats for 3 days via gastric intubation (Sharma et al., 1988b). Ethoxyresorufin, the preferred substrate for cytochrome P-450c, which is induced by 3-methylcholanthrene, was not utilized as a substrate by microsomes from CBZ treated rats (Burke et al., 1985). Although the microsomal protein from the CFB treated rats appeared to cross react with cytochrome P-450b, pentoxyresorufin was utilized as a substrate only to the same extent as microsomal protein from untreated rats (figure 12) further demonstrating the differences between cytochrome P452 and PB inducible cytochrome P-450b. This finding agrees with the results of Tamburini et al. (1984). A comparison of the effects of PB, CBZ and CBZE on hepatic cytochrome P-450 content in rats To the best of our knowledge, there are no reports in the literature where the time dependency of cytochrome P-450 inductiôn by CBZ in animal models has been examined.  Thus, the time period tested for the induction of  cytochrome P-450 by CBZ was based on the assumption that 2 weeks of induction in rats should be comparable to the reported autoinduction of CBZ metabolism in humans of 4 to 5 weeks (Bertilsson et al., 1980; Pynnonen et al., 1980; Moreland et al., 1982). In the case of CBZE, treatment of rats for either 3 or 7 days did not demonstrate any differences in total hepatic cytochrome P-450 content between the 2 time periods of treatment (Jung et al., 1980). Therefore, 126  Discussion rats were treated for 3, 7, 10 or 14 days in order to determine the time course for induction by CBZ and CBZE. When the CBZ or CBZE treated groups were compared over the 14 day treatment period, statistical differences were not observed in total cytochrome P-450 content. This would indicate that 3 days of treatment with CBZ or CBZE were sufficient to yield maximal induction of cytochrome P-450 at the doses employed in this study. The dose of CBZ employed in the current study was based on reports that an oral dose of 400 mg/kg in rats yielded serum levels of CBZ equivalent to the 8 to 10 ig/mL levels observed in man (Morselli et al., 1971) and that the oral absorption of CBZ is reported to range from 58 to 86% in monkeys (Morselli and Frigerio, 1975). Carbamazepine-10,11-epoxide was dosed at half the CBZ dose based on reported serum CBZE concentrations after dosing with CBZ to be usually 10 to 50% those of the parent drug in humans (Bertilsson and Tomson, 1986).  Carbamazepine and carbarnazepine-10,11-epoxide were suspended in  propylene glycol at a concentration of 100 mg/mL and 50 mg/mL, respectively. While not specifically investigated in this work, the induction properties of CBZ and presumably also of CBZE, suggest dose dependency in humans. CBZ invoked dose dependent induction of antipyrine clearance which returned to control values within 2 weeks after discontinuation of CBZ therapy in adult volunteers (Rapeport et al., 1983). At higher doses of CBZ in patients, increases in dosage resulted in disproportionately low elevations of CBZ plasma concentrations as a result of dose dependent induction (Tomson et al., 1989). In humans, CBZ demonstrated dose dependent induction as evidenced by a curvilinear relationship (Kudriokova et al., 1992).  between  dose  and  steady  state  concentrations  In rats, however, a statistical difference was not  observed between a 60 mg/kg and a 100 mg/kg twice daily dose when the  127  Discussion induction of various enzyme activities was examined (Regnaud et al., 1988). The observed increase in hepatic microsomal cytochrome P-450 content in the PB and CBZ treated groups when compared to the untreated control group were not unexpected since 2 to 3 fold increases in cytochrome P-450 content have been observed after PB treatment in rats (Conney, 1967; Phillips et al., 1981). Regnaud and co-workers (1988) observed significant increases in total hepatic cytochrome P-450 content when CBZ treated rats were compared to control rats. A 48% increase in total hepatic cytochrome P-450 content by CBZ treatment over 4 days in rats compared to the control group was reported by Wagner and Schmid (1987). The total hepatic cytochrome P-450 content of microsomes from each of the CBZ 3, 7 and 10 day treated groups was significantly increased when compared to their corresponding PG treated group while there was no difference in the total hepatic cytochrome P-450 content of the CBZ 14 and PG 14 day treated groups (figure 7).  A plateau may have been reached whereby the  continued presence of CBZ did not result in a further-increase in cytochrome P 450 content; i.e. tolerance to induction by CBZ had developed.  Another  consideration is that since the inducer must be present in high concentrations to produce  induction  (Conney,  1967)  and  because  CBZ  metabolism  is  autoinducible, lower concentrations of CBZ can be expected at the site of action when studied at the longest time point.  In order to invoke induction, the  inducing agent must be able to maintain adequate intracellular concentrations after repeated administration (Conney, 1967). In one case of PB induction in rats, a plateau in cytochrome P.450 levels was achieved after 5 days possibly as the result of the formation of a repressor as opposed to any increase in metabolism of PB (Ernster and Orrenius, 1965; Orrenius et al., 1965). In order to investigate the possibility of autoinduction of CBZ and CBZE  128  Discussion  metabolism over the time course, rat blood samples were collected at sacrifice after 3, 7, 10 and 14 days of treatment. Unfortunately, the concentrations of CBZ and metabolites that were present 12 to 13 h after administration of the last dose were extremely low and thus unquantifiable.  However, CBZ and  metabolites were quantitated in urine collected for 12 h following the last dose of either CBZ or CBZE.  The urinary recoveries of CBZ, CBZE and CBZD  representing the expoxide-diol metabolic pathway did not increase over the 2 week time period and ranged from 2.9 to 4.6% of the administered dose. In a previously reported rat study, the recovery of compounds in the epoxide-diol pathway (CBZ, CBZE and CBZD) in a 24 h urine collection accounted for 7 to 10% of the dose of CBZ administered (Regnaud et al., 1988). Our recoveries are therefore significantly lower and may be due to differences in our assay and/or the strain of rat used.  The low recoveries observed for metabolites in the  epoxide-diol pathway make it difficult to use such data to conclude if autoinduction had in fact occurred. It is possible that other metabolites of CBZ were formed that we did not or could not measure. A possible clue to this is the study by Regnaud and co-workers (1988) where 50% of the CBZ dose was recovered in the urine in the form of thioethers.  These thioethers were not  specifically identified but suggest an increased formation of mercapturate metabolites after repeated administration of CBZ to rats. We were not able to pursue the identification and quantitation of such metabolites in this investigation. The lack of statistical differences apparent between the observed total hepatic cytochrome P-450 values for the CBZE and PG treated groups may be due to the lower dose of CBZE used compared to CBZ.  Previously, CBZE  administration to Sprague-Dawley rats at a dose of 100 mg/kg daily i.p. for either 3 or 7 days did not affect total hepatic cytochrome P-450 when compared  129  Discussion to the control group (Jung et at., 1980). Assuming that CBZE induction is a dose dependent phenomenon as reported for CBZ, the CBZE even at lower serum concentration could play an important role in the induction of cytochrome P-450. If plasma half-life plays an important role in achieving maximal induction for the barbiturates (loannides and Parke, 1975), perhaps this is also true for CBZ and CBZE. Repeated administration of CBZ enhances its own elimination and this may also be true for CBZE (Faigle and Feldmann, 1982). The mean plasma half-life of CBZ after a single dose was 35.6 ± 15.3 h and decreased to 20.9 ± 5.0 h after chronic administration in patients (Eichelbaum et at., 1985). The plasma half-life of CBZE in humans was approximately 6 to 8 h after single doses (Tomson et at., 1983; Spina et at., 1988; Tomson and Bertilsson, 1991).  The  shorter plasma half-life of CBZE could also be responsible for limiting its effectiveness at inducing total hepatic cytochrome P-450. The mean recoveries of CBZE and CBZD in the 12 h urine following administration of CBZE was only 1.5 to 4% of the administered CBZE dose. By comparison, it was reported that 15% and 1% of the dose was recovered as CBZE and CBZD, respectively, in urine collected for 5 days from Sprague Dawley rats dosed with 4 mg of CBZE i.v. (Kerr and Levy, 1989). In man, after single doses of CBZE, the urinary recovery of CBZD varies from 67 to 90% of the dose with urine collections over 3 (Spina et at., 1988) or 5 days (Tomson et at., 1983).  Our results and that of Kerr and Levy (1989) implicate a species  difference in the metabolism of CBZE to CBZD with rats being much less efficient than humans in producing the diol metabolite. A comparison of the effects of PB, CBZ and CBZE on the induction of cytochrome P-450b Previous reports in the literature speculated that CBZ should induce the same isozyme(s) of cytochrome P-450 as does PB (Faigle and Feldmann, 1982;  130  Discussion Wagner and Schmid, 1987). The results of our study would appear to support that speculation. After treatment for just 3 days with either CBZ or CBZE, cytochrome P-450b was clearly detectable as was cytochrome P-450e. The rat anti-cytochrome P-450b antibody used in the present study reacts with cytochrome P-450e, also inducible by PB (Dutton and Parkinson, 1989). Cytochromes P-450b and P-450e are 2 of the major PB inducible isozymes and may be induced up to 40 fold in rats (Thomas et al., 1981; Thomas et al., 1987). Cytochrome P-450e migrates in SDS-PAGE just above the cytochrome P-450b band and has a slightly higher apparent molecular weight of 52,500 daltons versus 52,000 daltons for cytochrome P-450b (Ryan and Levin, 1990). Rat liver cytochromes P-450b and P-450e share greater than 97% amino acid sequence homology as determined from amino acid and cDNA analysis (Fujii-Kuriyama et al., 1982).  Cytochromes P-450b and P-450e are not immunochemically  separable when using polyclonal antibodies unless first separated by SDS-PAGE (Ryan et al., 1982). Cytochrome P-450b was found to comprise 65% of the total hepatic cytochrome P-450 isolated from the PB treated group of rats (table 5). Over the 14 day time course, microsomal cytochrome P-450b content varied from 39 to 66% of the total cytochrome P-450 for the CBZ treated groups and 31 to 53% for the CBZE treated groups. Mean quantities of cytochrome P-450b in the CBZE treated groups appeared to be lower than both the PB and CBZ induced groups but did not test statistically different. The specific content of cytochrome P-450b in the cytochrome P-450 protein of the PB and CBZ 3 day treatment groups was very similar to literature values. After PB treatment of male Long Evans rats, microsomal cytochrome P-450b content ranged from 43 to 57% of the total hepatic cytochrome P-450 content (Thomas et al., 1981). In PB induced adult, male Sprague-Dawley rats, cytochrome P-450b represented 51% of the total  131  Discussion  hepatic cytochrome P-450 (Wilson et at., 1987). Thus, the values for cytochrome P-450b obtained after PB induction in our study are very close to the reported literature values. Furthermore, our results suggest that at the doses used in this study, CBZ is equally as effective as PB in inducing cytochrome P-450b. CBZE, if utilized at an equivalent molar dose to CBZ, also may have provided comparable induction of cytochrome P-450b. Although the induction of cytochrome P-450b in the PB, CBZ and CBZE treatment groups was confirmed by the use of antibodies, quantitation of the monooxygenase enzyme activities by fluorimetric assays can provide further corroboration of the identity of the isozymes induced. include  0.-dealkylation  of  coumarins  Fluorometric assays  (umbelliferones),  phenoxazones  (resorufins), fluorescein (Mayer et at., 1989) and quinolones (Mayer et at., 1990). Of these, pentoxyresorufin and ethoxyresorufin have been demonstrated to be specifically metabolized by the isozymes of cytochrome P-450 induced by PB and 3-methyicholanthrene, respectively (Mayer et at., 1989). Although we did not study the effects of antibodies on microsomal dealkylation, an antibody against cytochrome P-450b inhibited pentoxyresorufin dealkylation more than 90% in hepatic microsomes from PB induced rats (Dutton and Parkinson, 1989). In the present study, pentoxyresorufin was found to be the preferential substrate for the 0-dealkylation of reactions catalyzed by cytochromes P-450 induced not only by PB but by CBZ and CBZE as well (figures 12 to 14). The rates of pentoxyresorufin 0-dealkylation on a per mg of protein basis by microsomes from the PB treated group were increased 12 fold over the vehicle control group (table 3).  Induction with PB is reported to result in an  enhancement of pentoxyresorufin 0-dealkylation activity over a wide range of 20 to 283 fold in rats (Burke et at., 1985; Dutton and Parkinson, 1989; Lubet et at., 1990; Mayer et at., 1990).  The values obtained in this study for the PB  132  Discussion  treated group were slightly lower when compared to the literature values. Pentoxyresorufin 0-dealkylation rates for microsomal protein from the CBZ (34 to 53 fold increase) and the CBZE (12 to 19 fold) treatment groups were also in the reported range for PB. Thus, it appears that 3 days of treatment with CBZ at 100 mg/kg i.p. twice daily will provide induction of total hepatic cytochrome P-450, cytochrome P-450b content and enhancement of pentoxyresorufin 0-dealkylation rates that are very similar to that obtained with typical PB induction in rats. Although we did not examine the following enzyme activities, CBZ administration to rats is reported to result in increased activities of UDP-glucuronyltransferase, NADPH-cytochrome P-450 reductase (Faigle and Feldmann, 1982; Wagner and Schmid, 1987), 4-nitroanisole 0-demethylase (Wagner and Schmid, 1987), aminopyrine N-demethylase and aniline hydroxylase (Wagner and Schmid, 1987; Regnaud et al., 1988).  The increases in enzyme activities reported for  CBZ induction, albeit of lower magnitude, were similar to those observed with PB administration. The rates for pentoxyresorufin 0-dealkylation for the CBZE treated rats ranged from 32 to 43% that of the CBZ treatment groups and appear to reflect the decreased dose of CBZE that was tested.  Furthermore, these rates also  reflect the lower levels of cytochrome P-450b quantitated in microsomal protein from the CBZE treatment groups, with the exception of the CBZE 7 day treatment group.  An example in the literature that was similar to these  findings is the reported induction of microsomal ethoxycoumarin 0-dealkylation by 115% in rats after CBZE administration for 3 days at a dose of 100 mg/kg (Jung et al., 1980). No further increases in ethoxycoumarin 0-dealkylation were observed when CBZE was administered for an additional 4 days.  133  Discussion Effect of CBZ, CBZE and PB treatment on cytochromes P-450f and P 450g The microsomal protein isolated from the CBZ, CBZE and PB treatment groups did not react to any appreciable degree with anti-rat cytochrome P-450f or anti-rat cytochrome P-450g antibody. These results are consistent with what is known about these particular isozymes of cytochrome P-450. Cytochrome P 450f is a constitutive isozyme present at higher concentrations in females than in males and is regulated by age (Leroux et al., 1989). Cytochrome P-450g is a constitutive, male specific isozyme which is regulated by both sex and age These isozymes are normally present in small  (Soucek and Gut, 1992).  quantities in untreated rat hepatic microsomes.  Cytochromes P-450f and P  450g represent approximately 1.4% and 0.8%, respectively of the total hepatic P 450 content of hepatic microsomes from untreated immature male Long Evans rats (Bandiera et al., 1986). In adult rats, the amounts of cytochromes P-450f and P-450g increase to approximately 7% and 17% of total hepatic cytochrome P-450, respectively (Bandiera et al., 1986). Furthermore, cytochromes P-450f and P-450g are relatively refractory to induction by all common classes of P-450 inducers (Bandiera et al., 1986). In summary, hepatic cytochrome P-450b in rats was significantly induced by PB and CBZ and appeared also to be induced by CBZE administration. Three days of induction with CBZ at a dose of 100 mg/kg i.p twice daily was found to be sufficient to yield induction comparable to the usual PB dose of 75 mg/kg i.p. for 4 days.  CBZE at a dose of 50 mg/kg i.p. twice daily was  approximately 50% as effective as CBZ for both the induction of cytochrome P— 450b and the enhancement of pentoxyresorufin 0-dealkylation and is attributed to  the  lower  dose  used.  The  induction  of cytochrome  P-450b  and  pentoxyresorufin 0-dealkylation by CBZ and CBZE has not previously been  134  Discussion reported.  We attempted to confirm this induction by examination of the  metabolic profile of CBZ and CBZE in rats, but the urinary recoveries of metabolites did not provide substantial evidence for the induction of the epoxide-diol metabolic pathway.  IN VITRO METABOLISM STUDIES OF VPA AND (E)-2-ENE VPAIEFFECTS OF INDUCING AGENTS In order to detail the effects of CBZ induction on VPA metabolism that is apparent from patient interaction studies and from in vivo studies in rats, it was important to investigate in vitro effects of CBZ induction on VPA metabolism and one of its major metabolites, (E)-2-ene VPA. Induction by CBZ and CBZE was compared to the classic inducer, PB and to CFB. Since maximal induction of total cytochrome P-450 content, cytochrome P-450b and microsomal pentoxyresorufin 0-dealkylation appeared to be achieved after 3 days of CBZ treatment, only the group treated with CBZ for 3 days will be compared to the PB, CFB and CBZE 3 day treatment groups. Interaction between YPA and CBZ As the number of medications that a patient is prescribed increases, so does the possibility of adverse drug interactions (Mclnnes and Brodie, 1988). CBZ induces the metabolism of VPA in healthy subjects and in epileptic patients (Levy and Pitlick, 1982). In epileptic patients on VPA and CBZ, steady state VPA levels were 37 to 64% lower than predicted from single dose studies of VPA (Levy and Pitlick, 1982). In one volunteer study, CBZ caused increased clearance of VPA accompanied by decreased steady state plasma concentrations (Bowdie et al., 1979). The effect of CBZ on VPA clearance does not result from competition for protein binding sites because CBZ plasma levels are low compared to the amount of albumin present and thus, CBZ should not act as a 135  Discussion  displacing agent (Levy and Pitlick, 1982). In a volunteer study conducted in this laboratory, the volume of distribution of VPA did not change upon CBZ administration suggesting that enzyme induction and not a competition for plasma protein binding sites was responsible for the increased clearance of VPA and the resulting decreased serum levels, AUC and half-life values (Panesar et at., 1989).  The major  unsaturated metabolite, (E)-2-ene VPA, was significantly decreased in serum and urine, suggesting that it too was being cleared as a result of induced metabolism.  The serum levels of 4-ene VPA were unchanged after CBZ  administration but urinary recoveries, mainly as the glucuronide conjugate, were increased after CBZ.  When the urinary recoveries of VPA and its  metabolites were compared before and after CBZ administration, an increase in metabolism elicited by CBZ could not be confirmed.  Either VPA metabolites  were eliminated via non-renal routes or the GCMS assay failed to detect a significant proportion of the VPA metabolites.  Thus, in order to verifSr this  apparent in vivo induction of VPA metabolism, it was deemed important to examine the effects of CBZ on the metabolism of VPA at the in vitro level. Effect of anti-rat cytochromes P-450b and P-450h antibodies on YPA metabolite profiles from rat liver microsomes Anti-rat cytochrome P-450b and P-450h antibodies were employed to determine the extent of involvement of cytochromes P-450b and P-450h in the in vitro microsomal metabolism of VPA and (E)-2-ene VPA.  These antibodies,  prepared and made available to us by Dr. Bandiera’s research group, were used in a concentration range of 0.5 to 2.5 mg of IgG/nmol of spectrally determined cytochrome P-450. The metabolism of VPA to 4-ene VPA by microsomal protein from PB and CBZ 3 day treated rats was completely inhibited in the presence of the anti-rat 136  Discussion  cytochrome P-450b antibody while the formation of 3-OH VPA and 5-OH VPA was only partially inhibited. The formation of 4-OH VPA and 4-keto VPA was inhibited by 75% or greater. These results suggest that cytochrome P-450b is the major isozyme of cytochrome P-450 involved in the metabolism of VPA to 4ene VPA, 4-OH VPA and 4-keto VPA while other isozymes of cytochrome P-450 participate in the formation of 3-OH VPA and 5-OH VPA. The results obtained for the microsomal metabolism of VPA to 4-ene VPA demonstrate that formation of this putative hepatotoxin occurs directly from VPA and not via dehydration of either 4-OH VPA or 5-OH VPA (Kochen and Scheffner, 1980; Granneman et at., 1984a). Metabolic studies of stable isotope analogues of VPA had indicated that the origin of 4-ene VPA was different from that of 3-ene VPA and (E)-2-ene VPA (Rettenmeier et at., 1987). Thus, induction of a particular isozyme of cytochrome P-450, namely cytochrome P-450b, may play an important role in VPA induced hepatotoxicity. The formation of 4-ene VPA occurs via cytochrome P-450b catalyzed oxidation of a nonactivated alkyl substituent to the corresponding olefin (Baillie, 1988). This cytochrome P-450 isozyme functions as a desaturase (figure 37) oxidizing certain alkanes to olefins without an intermediate alcohol due to partitioning between hydroxylation  and  desaturation  reactions  (Guengerich,  1990).  Initial  abstraction of a hydrogen atom generates a transient free-radical intermediate that partitions between recombination (alcohol formation) and elimination (olefin production).  Based on the observed metabolism of deuterium labeled  VPA, the carbon-centered radical was found to be located at C-4 (Rettie et at., 1988).  This partitioning mechanism is similar to the cytochrome P.450  mediated formation of 17f3-hydroxy-4,6-androstadien-3-one from testosterone via dual hydrogen abstraction (Nagata et at., 1986). The 63-hydrogen is abstracted forming a transient radical, followed by abstraction of the 73-hydrogen to form  137  Discussion  COGH  h: k-  +  2 [FeOH]  3 (Fej  2 (FoOHJ  COOH  -—  .. 1  A  0 2 H  I  -,.-  (3-Ene VPA)  ‘I _,  (FeOHI3÷  I  -  000H  3 [FeOH]  GOGH  3 [Fe]  (I)  OH (3-Hiiroxy VPA)  [FeOH] 3 [FeO] COGH  [FeOH]  3 [FeO]  GOGH (VPA)  (Ill)  [FeO] 3 [FeOH] 3 [FeJ  GOGH  [FeO GOGH  HO  3 [FeOH]  [Fe]  HO  GOGH  (II) (4-Hytiroxy VPA)  [FeOHJ (5-Hydroxy VPA) 2 [FeOH] GOGH  2 [FeOH]  [Fe  0 2 H  Figure 37.  COOH  (4-Ene VPA)  Cytochrome P-450 catalyzed metabolism of VPA to 3-OH VPA, 4OH VPA, 5-OH VPA, 3-ene VPA and 4-ene VPA. (Based on Rettie et at., 1988).  138  Discussion the resultant double bond. The free radical intermediate generated solely by cytochrome P-450b which partitions between alcohol and olefin formation explains our results in that the antibody to cytochrome P-450b not only completely blocked 4-ene VPA formation but also the formation of 4-OH VPA and the derived 4-keto VPA (figures 30, 32 and 33). In order to further affirm the specificity of cytochrome P-450b in the metabolism of VPA to 4-ene VPA and 4-OH VPA, one other antibody to cytochrome P-450 was tested. Cytochrome P-450h is a male specific isozyme of cytochrome P-450 which catalyzes a number of metabolic reactions (Ryan and Levin, 1990). It is non-detectable in newborn rats but rapidly increases at 4 to 6 weeks of age and then plateaus (Waxman et at., 1985). The anti-rat cytochrome P-450h antibody did not exhibit any significant effects on the metabolism of VPA by microsomes from untreated, PB or CBZ 3 day treated groups, evidence that VPA or (E)-2-ene VPA are not substrates for cytochrome P-450h. Since the antibody directed against cytochrome P-450b was unable to completely inhibit the metabolism of VPA to 3-OH VPA and 5-OH VPA, other isozymes of cytochrome P-450 may be involved. Another isozyme of cytochrome P-450 inducible by PB is cytochrome P-450p (Soucek and Gut, 1992). Induction of cytochrome P-450p significantly increased the biotransformation of CBZ to CBZE in mouse hepatic microsomes and this reaction was inhibited by gestodene, a cytochrome P-450p inhibitor (Pirmohamed et at., 1992). CBZ and CBZE may also induce cytochrome P-450p. To determine whether cytochrome P-450p is involved in the metabolism of VPA to 3-OH VPA and 5-OH VPA, future inhibition experiments with gestodene or anti-cytochrome P-450p antibody are required.  139  Discussion A comparison of the effects of PB, CBZ and CBZE induction on the in vitro metabolism of VPA  Phenobarbital pretreatment resulted in enhanced metabolism of VPA to 3-OH VPA, 4-OH VPA, 5-OH VPA, 4-ene VPA and 4-keto VPA. Similar results were obtained with the CBZ treated groups. Formation of 4-ene VPA Granneman and co-workers (1984a) were one of the first groups to report that PB induction increased 4-ene VPA formation in VPA treated rats.  Our  work confirms that CBZ and CBZE are capable of this action as well. Rettie and co-workers (1987) were the first to report the in vitro formation of 4-ene VPA from VPA as a result of a cytochrome P-450 mediated reaction. The 4-ene VPA metabolite could only be observed in the presence of PB induced rat liver microsomes.  Similar results were obtained for liver microsomes from PB  induced rabbits, mice and humans with 2.5 to 8.4 fold increases in the formation of 4-ene VPA, depending upon the species (Rettie et al., 1988). A comparative study using microsomes from CBZ, phenytoin and PB induced rats (80 mg/kg for 4 days) indicated that PB was the more effective inducer of this desaturation pathway leading to 4-ene VPA (Rettie et al., 1987). In our study, the formation of 4-ene VPA was increased 6 fold and 5 fold respectively, by the PB and CBZ 3 day treated groups when compared to the untreated group.  Only a 2 fold  increase was observed with the CBZE 3 day treated group. The 4-ene VPA metabolite is not easily detected because of the low serum levels. For example, in a survey of 49 patients, 4-ene VPA was only detected in the serum of patients receiving ‘[PA in combination with CBZ (Tennison et al., 1988). This was probably due in part to increased doses of VPA in the VPA and CBZ group plus the induction effects of CBZ in forming increased 4-ene VPA. Extremely sensitive assays are required in order to reliably quantifr significant 140  Discussion changes in 4-ene VPA serum concentrations.  Using a GCMS assay, the  formation clearance of 4-ene VPA was determined to increase 2 fold in patients who were on combined therapy with VPA and either CBZ or phenytoin (Levy et al., 1990). Significance of 4-ene VPA formation to the mechanism of VPA hepatotoxicity There are currently 2 major hypotheses regarding the mechanism(s) of VPA induced hepatotoxicity.  The first proposed mechanism for VPA toxicity  entails the depletion of free CoA due to sequestration by VPA to form valproyl CoA and derivatives (Becker and Harris, 1983; Thurston and Hauhart, 1992). The reduced concentrations of free CoA result in increased levels of fatty acids and decreased 13-oxidation. A second proposed mechanism that our laboratory favours, is that hepatotoxicity of VPA is mediated through the formation of a toxic metabolite and 4-ene VPA has been implicated as the most likely candidate (Zimmerman and Ishak, 1982; Rettenmeier et al., 1985; Rettenmeier et al., 1986b).  The 4-ene VPA metabolite is similar in structure to the  hepatotoxin 4-pentenoic acid (PA) and to MCPA, the metabolite of hypoglycin (figure 38).  VPA hepatotoxicity shares many similar manifestations with  Jamaican Vomiting Sickness (JVS), Reye’s Syndrome (RS) and PA toxicity (Lewis et al., 1982; Nau and Loscher, 1984). PA is bioactivated to the reactive electrophilic species, 3-keto-4-pentenoic acid, which alkylates 3-ketoacyl CoA thiolase, the terminal enzyme of mitochondrial 13-oxidation (Schulz, 1983; Fong and Schulz, 1983).  It has been postulated that analogous to PA, 4-ene VPA  undergoes 13-oxidation to (E)-2,4-diene VPA which is ultimately bioactivated to the electrophilic metabolite, 3-keto-4-ene VPA (Rettenmeier et al., 1985). This metabolite has just recently been identified in rats although it is present in very trace quantities and specialized techniques were required for its identification (Kassahun et al., 1993). 141  Discussion  CCH —CHr—CH 2 H — COOH 2  (4-Pentenoic add)  NH2 C=C 2 H  —CH—COOH 2 CH—CH  —  (Hypoglycin)  C= C 2 H  —  CH  —  — COOH 2 CH  (Methylene-cydopropytacetic add)  7 H 3 C C= CH— CH— CH—COOH 2 H  (4-EneVPA)  Figure 38.  Structural similarity amongst hypoglycin, 4-pentenoic acid and 4ene VPA.  142  Discussion It has been established that VPA and 4-ene VPA undergo metabolic activation in rat liver, both in vivo and in vitro, to electrophilic intermediates that bind covalently to cellular macromolecules (Porubek et at., 1989).  In  support of a reactive metabolite mechanism, Kassahun et at. (1991) found that in rats administered 4-ene ‘[PA, the major metabolite recovered in bile was the gluthathione (GSH) conjugate of (E)-2,4-diene ‘[PA. GSH is a tripeptide which reacts with a variety of electrophilic compounds aiding in the minimization of cellular injury (Reed, 1990; DeLeve and Kaplowitz, 1991).  Urinary N  acetylcysteine (NAC) conjugates are the end products of GSH conjugation. The NAC conjugate of (E)-2,4-diene ‘[PA was a prominent urinary metabolite in rats that had received 4-ene VPA and was detected in the urine of patients receiving ‘[PA. It was proposed that the toxic effect exerted by (E)-2,4-diene ‘[PA could be due to localized mitochondrial depletion of GSH, leading to oxidative stress and subsequent cellular injury (Kassahun et at., 1991).  Thus, patients who are  subjected to increased serum levels of 4-ene VPA andlor have deficiencies in their glutathione defense mechanisms could be at an increased risk to VPA induced hepatotoxicity. In the retrospective study by Dreifuss and co-workers (1987), ‘[PA polytherapy, especially when in combination with CBZ, was implicated as a major factor in the increased incidence of VPA hepatotoxicity. Formation of the hydroxy metabolites of VPA The formation of the hydroxy metabolites was significantly enhanced by CBZ and PB induction while smaller increases were observed with CBZE treatment. The hydroxy metabolites of VPA (3-OH ‘[PA, 4-OH ‘[PA and 5-OH VPA) have been demonstrated in human embryo homogenates of liver, lung, brain and adrenals (Rettie et at., 1986) and rat liver microsomes (Prickett and Baillie, 1984).  The formation of 3-OH VPA from VPA was thought to be  mediated primarily via the 13-oxidation pathway (Bjorge and Baillie, 1991; Li et 143  Discussion at., 1991) but evidence suggests that it may partly arise via cytochrome P-450 mediated o-2 oxidation (Prickett and Baillie, 1984). The o-2 oxidation pathway, inducible by PB, is a minor metabolic route for the rat hepatic microsomal metabolism of simple alkanes, such as n-hexane (Frommer et at., 1972) and n heptane (Frommer et at., 1974). Phenobarbital treatment in rats was reported to primarily enhance the o 1 oxidation pathway of VPA metabolism with the urinary recoveries of 4-OH VPA, 4-keto VPA and 2-PSA being increased by a factor of 3.8 (Granneman et at., 1984a).  The urinary excretion of 5-OH VPA was also increased.  In one  pediatric patient who died from hepatic failure associated with VPA and PB polytherapy, increased quantities of 2-PGA, the endproduct of the (0-oxidation of VPA, were recovered in the urine (Kuhara et al., 1990). In the present study, PB treatment was more effective at inducing w-1 (4-OH VPA) and co-2 (3-OH VPA) hydroxylation while CBZ was more efficient for the induction of  (0-  oxidation (5-OH VPA). This could, therefore, have implications regarding the incidence of VPA induced hepatic failure depending on the associated drugs and the metabolic pathway responsible for the ultimate hepatotoxic response. Despite the presence of 3-OH VPA, 3-ene VPA was not detected in the microsomal incubates from the metabolism of VPA. In rats, 3-ene VPA was not observed as a metabolite of 3-OH VPA (Granneman et at., 1984a). These results indicate that 3-ene VPA probably arises through a different mechanism than the 4-ene VPAI4-OH VPA pathway described in figure 37. Alternatively, the degree of 3-ene VPA formation may have been below the level of detection of our assay. The oxidation of fatty acids involving hydroxylation at the o, o-1 or o-2 positions  occurs  microsomally  and  can  be  succeeded  by  further  biotransformation via alcohol or aldehyde dehydrogenases to the dicarboxylic  144  Discussion  acids or keto acids (Bjorkhem, 1972a). Dehydrogenation reactions can occur in either  the  soluble  or  microsomal  fraction,  although  the  microsomal  dehydrogenase activity is not as efficient as that of the soluble fraction (Bjorkhem, 1972b). This may explain why 4-keto VPA was detected in the in vitro microsomal metabolism of VPA although it was not expected, since the formation of 4-keto VPA is the second step in the o-1 oxidation pathway. Based on the theory that partitioning occurs between desaturation and hydroxylation as illustrated in figure 37, if larger quantities of the alcohol are formed (i.e. 4OH VPA) then a larger concentration of substrate is present for alcohol dehydrogenases to form 4-keto VPA. CBZ was a more effective inducer of the formation of 4-keto VPA than was PB which may also relate to the lower quantities of 4-ene VPA observed with the CBZ treated group. In a discussion of the effects of induction by CBZ and CBZE on the metabolism of VPA and (E)-2-ene VPA, the vehicle, PG, needs to be considered. PG appeared to invoke an inhibitory effect on the microsomal metabolism of VPA to some metabolites. There is some precedence in the literature for this inhibition by PG from studies in mice. Administration of PG (4 mLfkg i.p.) to mice within 3 h of receiving acetaminophen, provided protection against acetaminophen induced liver toxicity (Hughes et al., 1991). This protection by PG was perhaps afforded by inhibition of the formation of the toxic metabolite N-acetyl-p-benzoquinoneimine by cytochrome P-450, a mechanism by which ethanol also provides protection against acetaminophen toxicity (Wong et al., 1980; Tredger et at., 1985).  In rats PG (4 mL/kg twice daily i.p. for 3 days)  increased hexobarbital sleeping time from 30 to 53 mm (Dean and Stock, 1974). In microsomes prepared from rats treated with PG (4 mL/kg twice daily i.p. for 3 days), aminopyrine demethylase activity was observed to decrease.  This  depressant effect of PG on aminopyrine demethylase activity was abolished  145  Discussion  when PB was co-administered. We cannot be certain that induction by CBZ and CBZE produced the same effects as PB.  Alternate vehicles which lack the  inhibitory effects may need to be considered for further studies of CBZ and CBZE induction. Effect of anti-rat cytochrome P-450b and P-450h antibodies on (E)-2-ene VPA metabolite profiles from rat liver microsomes The major serum metabolite of VPA, (E)-2-ene VPA, has been touted as a possible anticonvulsant agent (Nau et at., 1984).  (E)-2-ene VPA possesses  anticonvulsant activity similar to VPA without exhibiting the toxicities (teratogenicity, embryotoxicity and hepatotoxicity) associated with the parent compound (Honack et at., 1992).  In rats, (E)-2-ene VPA is metabolized to  several diunsaturated metabolites, (E,E)-2,3’-diene VPA and 2,4-diene VPA, as well as 3-keto VPA (Loscher et at., 1992).  (E,E)-2,3’-diene VPA, the major  diunsaturated metabolite in the serum also possesses anticonvulsant activity (Acheampong and Abbott,  1985).  However, the  second diunsaturated  metabolite, (E)-2,4-diene VPA, produces hepatic steatosis in rats (Granneman et at., 1984c; Kesterson et at., 1984). The effect of the anti-rat cytochrome P-450b antibody on the metabolism of (E)-2-ene VPA was investigated to determine the extent of this isozyme’s involvement.  The metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA in  microsomes from PB and CBZ 3 day treated rats was inhibited by almost 90% while the formation of (E,E)-2,3’-diene VPA was partially inhibited in the presence of the anti-rat cytochrome P-450b antibody. The anti-rat cytochrome P-450h antibody did not exert any inhibitory effects on the metabolism of (E)-2ene VPA.  These results clearly indicate that cytochrome P-450b plays an  important role in the metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA analogous to the formation of 4-ene VPA from VPA. However, other cytochrome 146  Discussion  P-450 isozymes appear to be responsible for the formation of (E,E)-2,3’-diene VPA.  Further details of the in vitro metabolism of (E)-2-ene VPA and the  significance of our findings with respect to the prospective use of this compound as an anticonvulsant agent are described below. A comparison of the effects of PB, CBZ and CBZE induction on the in vitro metabolism of (E)-2-ene YPA in rat liver microsomes The production of (E,E)-2,3’-diene VPA by microsomes isolated from the PB, CBZ 3 day and CBZE 3 day treatment microsomes was significantly enhanced over controls as was the formation of (E)-2,4-diene VPA by the PB and CBZ 3 day treatment groups.  The extent of induction by CBZ and PB as  measured by the degree of formation of these 2 metabolites was very similar. In our in vitro studies of (E)-2-ene VPA metabolism, the 4-ene VPA metabolite was not detected. We were also unable to distinguish the peaks for VPA or 3-ene VPA from the background in the SIM chromatograms of the extracted incubates. VPA, albeit in small quantities, has been detected in rats after administration of (E)-2-ene VPA (Granneman et at., 1984a; Loscher et at., 1992).  The metabolite 3-ene VPA was detected in rat plasma after  administration of 300 mg of (E)-2-ene VPA daily for 12 days (Vorhees et at., 1992).  Thus, our results suggest that microsomal cytochrome P-450 enzymes  are not responsible for the reduction of (E)-2-ene VPA to VPA nor for the isomerization of (E)-2-ene ‘[PA to 3-ene VPA. After dosing with (E)-2-ene VPA (250 mg/kg i.p. for 7 days), ‘[PA, (E) and (Z)-2-ene VPA, (E,E)-2,3’-diene VPA, (E)-2,4-diene VPA and 3-keto VPA were detected in rat plasma (Loscher et at., 1992). The major urinary metabolite of (E)-2-ene VPA detected in rats was the 13-oxidation product 3-keto VPA (Granneman et at., 1984a). The detection of (E)-2,3’-diene VPA as a microsomal metabolite of (E)-2ene VPA in our study was unusual because its formation has been attributed to 147  Discussion  f3-oxidation mechanisms in mitochondria (Bjorge and Baillie, 1991) that are summarized in figure 39. The (E)-2-ene VPA metabolite reversibly isomerizes to 3-ene VPA, that in turn is metabolized by the 3-oxidation pathway to (E,E)-2,3diene VPA (Bjorge and Baillie, 1991).  However, our in vitro results indicate  that similiar to the formation of 3-OH VPA from VPA, (E,E)-2,3’-diene from (E) 2-ene VPA also appears to arise as a product of cytochrome P-450b metabolism, although in very small quantities. The induced formation of (E)-2,4-diene VPA by microsomes from the CBZ 3 day treated group was comparable to that achieved by the PB treated group, with increases of 12 fold compared to vehicle controls and confirms that CBZ is as effective as PB for the induction of this desaturation pathway. A significant increase in the formation of (E)-2,4-diene VPA occurred only in the CBZE 7 day treatment group which also corresponded to the maximal increase observed for cytochrome P-450b by CBZE. Similar results have recently been reported for the effects of PB on the induction of (E)-2-ene VPA metabolism to (E)-2,4-diene VPA in vitro (Kassahun and Baillie, 1993). The (E)-2,4-diene VPA was a major metabolite and was increased approximately 3 fold by PB. The metabolite, (E)-2,4-diene VPA, is a known to cause steatosis, although doses as high as 100 mg/kg daily must be administered in order to invoke hepatotoxicity in rats (Granneman et at., 1984c; Kesterson et at., 1984). Elevated plasma and urinary levels of (E)-2,4-diene VPA were observed in 6 cases of fatal VPA hepatotoxicity (Kochen et at., 1984). In patients with VPA associated hepatic failure, the urinary recovery of (E)-2,4-diene VPA as the NAC conjugate was increased 3 to 4 fold (Kassahun et at., 1991).  Therefore, the  significant enhancement of (E)-2,4-diene VPA formation by CBZ and PB on the metabolism of (E)-2-ene VPA raises important questions regarding the potential  148  Discussion  COOH  Coraffon -  VPA gIucunde  (VPA) p-oddalion  COOL-f  COOH  H000  COOH +  OH (3HroVPA)  ((Z)-3--Ene VPA)  ((E)-3-Erie VPA)  ((E)—2-Ene VPA)  I  COOH COOH 0  ÷  (3-Keto VPA) ((E,E)-2, 3-Oiene VPA)  Figure 39.  ((E,Z)-2,3-Diene VPA)  The 13-oxidation pathway of VPA metabolism in mitochondria.  149  Discussion  safety of this drug.  This is especially true should (E)-2-ene VPA be used in  situations of polytherapy. Consideration should also be given to the relative amounts of (E)-2,4diene VPA that is formed from either VPA or (E)-2-ene VPA administration. A comparison of the amounts of (E)-2,4-diene VPA and 4-ene VPA formed from (E)-2-ene VPA and VPA, respectively, revealed that the formation of (E)-2,4diene VPA greatly exceeded that of 4-ene VPA.  For example, with the PB  treatment group, 17 ng of 4-ene VPA were formed compared to 2.5 pg of (E)-2,4diene VPA. For the CBZ treatment groups over the 2 week treatment period, 11 to 13 ng of 4-ene VPA were produced in comparison to 1 to 2.5 tg of the (E)-2,4diene VPA. Thus, the desaturation catalyzed by cytochrome P-450b in (E)-2-ene VPA metabolism appears to be considerably more efficient than for VPA. One possible explanation is that because of the conjugated structure of the (E)-2,4diene VPA product, radical intermediates are being stabilized by the double bond during the metabolism of (E)-2-ene VPA. The in vitro results from our study indicating the enhanced formation of (E)-2,4-diene VPA from (E)-2-ene VPA by CBZ and PB induction suggest that the relative lack of toxicity reported for (E)-2-ene VPA should be perceived with caution. If (E)-2,4-diene VPA is hepatotoxic in rats as reported (Kesterson et al., 1984), then it is surprising that (E)-2-ene VPA is claimed to be less hepatotoxic than VPA. One rationale for such a hypothesis may be related to the organelle in which the (E)-2,4-diene VPA is formed. With (E)-2-ene VPA, the desaturation to (E)-2,4-diene VPA occurs microsomally in comparison to mitochondrial formation from 4-ene VPA after the administration of VPA. In mitochondria, the site of initial liver toxicity, the (E)-2,4-diene is readily conjugated by OSH (Kassahun et at., 1991) and may cause depletion of GSH while (E)-2,4-diene VPA produced in microsomes can react with the much larger pooi of cytosolic  150  Discussion OSH (Reed, 1990). Effect of CFB treatment on the metabolism of VPA and (E)-2-ene VPA CFB treatment did not appear to have any significant effects on the metabolism of either VPA or (E)-2-ene VPA although the CO vehicle may have exerted an effect which overshadowed or minimized any CFB invoked effects. When olive oil, was used as a vehicle for CFB, an increased urinary excretion of 4-OH VPA, 2-PGA and 3-keto VPA after VPA administration to CFB treated rats was observed (Heinemeyer et al., 1985). The cytochrome P-450 isozymes inducible by CFB possess the unique ability to -oxidize fatty acids (Bains et al., 1985). The mechanism by which CFB affects fatty acid metabolism is thought to be as follows. Administration of CFB elicits an increase in cytochrome P-452, leading to increased hydroxylation of fatty acids.  o  These fatty acids undergo further cytosolic.  oxidation to long chain dicarboxylic acids which then enter the peroxisomes. The resultant increased peroxisomal 13-oxidation provides an increased load of shorter chain fatty acids for mitochondrial 13-oxidation (Sharma et at., 1988a). In rats, CFB treatment was reported to increase the excretion of 4-OH VPA and the f3-oxidation metabolite 3-keto VPA (Heinemeyer et at., 1985.) However, in a more recent study, CFB pretreatment in rats did not significantly affect the metabolism of VPA, suggesting a minimal role for peroxisomal mediated 13oxidation (Bachmann et at., 1988). Based on the results of our study, neither VPA nor (E)-2-ene VPA were substrates for cytochrome P-452.  CLINICAL RELEVANCE Cytochrome P-450b was the major isozyme in rats induced by PB, CBZ and to a lesser extent by CBZE. The in vitro microsomal formation of 4-ene VPA from VPA and (E)-2,4-diene VPA from (E)-2-ene VPA was enhanced by 151  Discussion pretreatment with all 3 drugs and was inhibited completely in the presence of the anti-rat cytochrome P-450b antibody.  Thus, both of these metabolites  appear to be formed by a common desaturation mechanism.  Both of these  metabolites are capable of producing hepatotoxicity in rats (Granneman et at., 1984c; Kesterson et at., 1984). Elevated levels of (E)-2,4-diene VPA and 4-ene VPA have been observed in cases of VPA associated hepatotoxicity (Kochen et at., 1983; Scheffner et at., 1988).  The demonstrated inducibility of (E)-2-ene  VPA to (E)-2,4-diene VPA suggests that the ‘toxicity free’ nature of (E)-2-ene VPA needs to be re-evaluated, particularly when the drug is administered in combination with inducing agents such as CBZ. Alternatively, if (E)-2-ene VPA proves to be free of hepatotoxicity, the proposed role of (E)-2,4-diene VPA in the hepatotoxicity of VPA will need reassessment. Future studies in rats should examine the induction of cytochrome P-450 by an equivalent molar dose of CBZE to that of CBZ and thus establish the apparent dose dependent properties of CBZE.  In previous studies of VPA  metabolism in patients, CBZ significantly reduced serum (E)-2-ene VPA concentrations but not those of (E)-2,4-diene VPA. The results from this study suggest that enhanced metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA or inhibition of 3-oxidation occurs. Any further studies must include mitochondrial and peroxisomal effects of CBZ and CBZE, in light of the evidence that 1 mitochondrjal cytochromes P.450 are involved in the activation of aflatoxin B (Niranjan et at., 1984; Shayiq and Avadhani, 1989) and may also play a role in VPA metabolism. In addition, the effects of induction on phase II metabolism of VPA and metabolites, including glutathione conjugates should be investigated.  152  SUMMARY AND CONCLUSIONS  CBZ was compared to PB with respect to induction of total hepatic cytochrome  cytochrome  P-450,  P-450b  the  and  catalysis  of  pentoxyresorufin 0-dealkylation in rats. For maximal induction of these parameters, an i.p. dose of 100 mg/kg twice daily for 3 days of CBZ appeared to be equivalent to the normal protocol for PB of 75 mg/kg i.p. for 4 days. 2.  Except for an apparent dose-related effect, CBZE produced a similar induction  profile  to  that  of  CBZ.  Cytochrome  P-450b  and  pentoxyresorufin 0-dealkylation values for the CBZE treatment groups were approximately 50% of the CBZ treatment groups. 3.  Evidence for autoinduction of CBZ metabolism was sought by examining the urinary recoveries of metabolites that constitute the epoxide-diol pathway.  No differences in metabolite recoveries were found after  chronic administration of CBZ. 4.  Studies with anti-rat cytochrome P-450b antibody indicated that cytochrome P-450b was the primary isozyme that catalyzes the biotransformation of VPA to 4-ene VPA, 4-OH VPA and 4-keto ‘[PA. In addition, cytochrome P-450e may also be involved.  Results from the  inhibition experiments indicated that all 3 metabolites probably arise from a common intermediate. 5.  Inhibition studies with anti-rat cytochrome P-450b antibody on the formation of (E)-2,4-diene VPA from (E)-2-ene VPA strongly suggest that cytochrome  P-450b  is  the  primary isozyme  biotransformation.  153  responsible  for this  Summary and Conclusions  6.  The amount of (E)-2,4-diene VPA formed from (E)-2-ene VPA per nmol of cytochrome P-450 was approximately 100 fold greater than the conversion of VPA to 4-ene VPA as measured in microsomes from either CBZ 3 day or PB treated rats.  The enhanced production of (E)-2,4-diene VPA, a  metabolite known to be hepatotoxic in rats, could have  severe  consequences regarding the relative safety of (E)-2-ene VPA if used therapeutically and in combination with other drugs. 7.  Anti-rat cytochrome P-450h antibody did not inhibit the metabolism of VPA or (E)-2-ene VPA in microsomes from CBZ 3 day or PB treated rats. This result further illustrates the specificity of cytochrome P-450b in the metabolism of both VPA and (E)-2-ene VPA to potentially hepatotoxic metabolites.  8.  A major metabolite of VPA, (E,E)-2,3’-diene VPA, recently shown to be a mitochondrial metabolite of 3-ene VPA, was identified in this study as a microsomal metabolite of (E)-2-ene VPA.  Inhibition studies indicated  cytochrome P-450b to be only partially responsible for the conversion of (E)-2-ene VPA to (E,E)-2,3’-thene VPA. 9.  In addition to cytochrome P-450b, other isozymes are likely to be involved in the metabolism of VPA to 3-OH VPA and to 5-OH VPA by micros omes from CBZ 3 day and PB induced animals.  10.  The treatment of rats with CFB produced no obvious effects on the vitro microsomal metabolism of XTPA or (E)-2-ene VPA. reported effects of CFB on the  in vivo  in  Thus, the  metabolism of VPA likely occur  via  non-cytochrome P-450 catalyzed mechanisms. 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