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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 METABOLISMIN RAT LWER MICROSOMES BY CARBAMAZEPINE ANDCARBAMAZEPINE-1O,1 1-EPOXIDEbySUKHBINDER KAUR PANESARB.Sc. (Pharm.), University of British Columbia, 1983M.Sc., University of British Columbia, 1987A THESIS SUBMITTED IN PARTIAL FULFILMENT OFTHE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYinTHE FACULTY OF GRADUATE STUDIESFACULTY OF PHARMACEUTICAL SCIENCESDivision of Pharmaceutical ChemistryWe accept this thesis as conformingto the required standardTHE UNIVERSITY OF BRITISH COLUMBIADecember 1993© Sukhbinder Kaur Panesar, 1993In presenting this thesis in partial fulfilment of the requirements for an advanceddegree at the University of British Columbia, I agree that the Library shall make itfreely available for reference and study. I further agree that permission for extensivecopying of this thesis for scholarly purposes may be granted by the head of mydepartment or by his or her representatives. It is understood that copying orpublication of this thesis for financial gain shall not be allowed without my writtenpermission.(Signature)__________________________Department of %1it4tt.b6i- c1r S7J 4-C-iL-€The University of British CoimbiaVancouver, CanadaDate 2c-&/, ,g93DE-6 (2/88)ABSTRACTVaiproic acid (VPA) is a commonly used anticonvulsant agent that isoften given in combination with carbamazepine (CBZ) to maximize seizurecontrol. The incidence of VPA associated hepatotoxicityis increased whencoadministered with other anticonvulsants. One or more of VPAs metabolitesmay be responsible for the hepatotoxicity.The consequences of induction by CBZ, carbamazepine-10,11-epoxide(CBZE), phenobarbital (PB) and clofibrate (CFB) on the metabolism of VPA and(E)-2-ene VPA by rat hepatic microsomes were examined. Totalhepaticcytochrome P-450 content and the isozyme(s) of cytochrome P-450 induced bythese agents were determined and compared. The metabolism of VPA wasmonitored following induction by quantitating the microsomal formation of 4-ene VPA, 3-OH VPA, 4-OH VPA, 4-keto VPA and 5-OH VPA. For (E)-2-ene VPA,the formation of (E)-2,4-diene VPA and (E,E)-2,3’-diene VPA was monitored.Adult male Long Evans rats (4/group) were treated intraperitoneally (i.p.)with one of the following: CBZ 100 mg/kg or CBZE 50 mg/kg every 12 h for 3, 7,10 or 14 days, CFB 350 mg/kg for 7 days, or PB 75 mg/kg for 4 days. The meanhepatic cytochrome P-450 content for the CBZ treatmentgroups over the tenday treatment period was significantly enhanced 1.5 to 1.8fold compared to thevehicle control group while CBZE treatment did not appear to affect totalhepatic cytochrome P-450 content. PB treatment resultedin a significant 1.9fold increase compared to the vehicle control, but a significant increase was notobserved for the CFB treatment group.Immunoblot analysis indicated that cytochrome P-450b was induced byPB and also by CBZ and CBZE. Cytochrome P-450b constituted 65 % of thetotal hepatic cytochrome P-450 content in the PB induced microsomes andIiAbstractranged from 31 to 66 % in the CBZ and CBZE groups over the 14 day treatmentperiod. Pentoxyresorufin, a substrate for cytochrome P-450b, was preferentiallymetabolized compared to ethoxyresorufin by microsomal protein isolated fromPB, CBZ and CBZE treated rats. Mean pentoxyresorufin 0-dealkylation ratesfor the CBZ, CBZE and PB treatment groups were enhanced 12 to 53 fold whencompared to their respective vehicle control groups.The metabolism of VPA and (E)-2-ene VPA was enhanced by PB, CBZand to a lesser extent by CBZE treatment. CFB pretreatment did not have anysignificant effects on the metabolism of VPA or (E)-2-ene VPA. An antibodydirected against rat cytochrome P-450b was effective in completely inhibitingthe metabolism of VPA to 4-ene VPA by microsomal protein isolated from PB orCBZ 3 day treated rats. The formation of 4-OH VPA and 4-keto VPA wasinhibited 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 3day treatment groups was inhibited 89 % and 85 % respectively, in the presenceof the anti-rat cytochrome P-450b antibody.Three days of treatment with CBZ at a dose of 100 mg/kg i.p. every 12 hwas as effective as typical PB treatment for inducing total hepatic cytochromeP-450 content, cytochrome P-450b, pentoxyresorufin 0-dealkylation, and the invitro metabolism of VPA and (E)-2-ene VPA. CBZE, used at an equivalentmolar dose as CBZ, also may be as effective an inducer as PB. Cytochrome P450b plays an important role in the in vitro metabolism of VPA and (E)-2-eneVPA to 4-ene VPA and (E)-2,4-diene VPA, respectively. These two metabolitesare known hepatotoxins and their enhanced formation in the presence ofenzyme inducing agents are likely responsible for VPA associatedhepatotoxicity. The isozyme of cytochrome P-450 induced by CBZ or CBZE hasnot previously been identified.111TABLE OF CONTENTSAbstract iiTable of contents ivList of tables ixList of figures xiList of abbreviations xvDedication xviiiAcknowledgements xixIntroduction 1Vaiproic Acid 3Metabolism 3Fatty acid metabolism 3Mitochondrial n-oxidation of fatty acids 3Peroxisomal 13-oxidation of fatty acids 5Omega and omega-i oxidation of fatty acids 6Metabolism of valproic acid 6Metabolism of 4-ene VPA 9Metabolite activity 10Adverse effects 10Side effects 10Biochemical effects 11Pancreatitis 13Embryotoxicity/teratogenicity 13Hepatotoxicity 15(E)-2-ene VPA 19Anticonvulsant activity 20Teratogenicity 20Metabolism 21Carbamazepine 21Carbamazepine-10,11-epoxide 24Interaction between VPA and CBZ 25Cytochrome P-450 27ivTable of ContentsInduction of cytochrome P-450 by phenobarbital .28Clinical implications 32Specific objectives 34Experimental 35Reagents and Materials 35Vaiproic acid and metabolites 35Internal standards 35Carbamazepine and metabolites 35Reagents 36Primary antibodies 37Methods 38Induction studies 38Animals 38Treatment of solvents and compounds 39Treatment of animals with carbamazepine 39Treatment of animals with carbamazepine-10,11-epoxide 39Treatment of animals with phenobarbital 39Treatment of animals with cloflbrate 40Treatment of animals with vaiproic acid 40Analysis 40Vaiproic acid and metabolites 40Stock solutions of internal standards for GCMS 40Preparation of standard curves in phosphate buffer 40Standard curve for VPA and metabolites 41Standard curve for (E)-2-ene VPA and metabolites 41Extraction of VPA and metabolites from standard samplesand incubates 41Carbamazepine and metabolites 43Preparation of stock solutions for HPLC 43Preparation of standard curve for CBZ and metabolites. . . . 43Extraction of CBZ, CBZE and CBZD from urinesamples 43Instrumentation 44Valproic acid and metabolites 44Carbamazepine and metabolites 46Preparation of subcellular fractions from rat livers 46VTable of ContentsDetermination of protein content of various subcellularfractions 47Determination of cytochrome P-450 content in hepaticmicrosomes 47Gel electrophoresis of microsomal protein 48Immunoblot 48Quantitation of cytochrome P-450b in microsomal protein fromPB, CBZ and CBZE treated rats 49In vitro microsomal metabolism of VPA and (E)-2-ene VPA 50In vitro microsomal metabolism of VPA and (E)-2-ene VPA inthe presence of anti-rat cytochrome P-450b or anti-ratcytochrome P-450h antibody 50In vitro microsomal metabolism of VPA and (E)-2-ene VPA inthe presence of both anti-rat cytochrome P-450b and anti-rat cytochrome P-450h antibodies 51Microsomal 0-dealkylation of ethoxyresorufin andpentoxyresorufin 51Statistical analysis 51Results 52Quantitation and identification of cytochromes P-450 in hepaticmicrosomes 52Quantitation of total hepatic microsomal cytochrome P-450content 52Identification of the cytochrome P-450 isozymes induced byCBZ and CBZE using SDS-PAGE and Western blottechniques 58In vitro 0-dealkylation of pentoxyresorufin andethoxyresorufin catalyzed by hepatic microsomal proteinfrom the various treatment groups 64Quantitation of cytochrome P-450b in microsomes from CBZ,CBZE and PB treated rats by SDS-PAGE and Westernblot techniques 71In vitro metabolism of VPA and (E)-2-ene VPA 72Analysis of VPA and metabolites by GCMS 74In vitro metabolism conditions for VPA and (E)-2-ene VPA 74In vitro metabolism of VPA 76viTable of ContentsFormation of 3-OH VPA fromVPA. 76Formation of 4-OH VPA from VPA 76Formation of 5-OH VPA from VPA 82Formation of 4-ene VPA from VPA 87Formation of 4-keto VPA from VPA 91In vitro metabolism of(E)-2-ene VPA 91Formation of (E,E)-2,3’-diene VPA from (E)-2-ene VPA 95Formation of (E)-2,4-diene VPA from (E)-2-ene VPA 99Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA and (E)-2-ene VPA by microsomesfrom PB and CBZ treated rats 103Effect of anti-rat cytochrome P-450b antibody on the invitro metabolism of VPA 103Effect of anti-rat cytochrome P-450b antibody on the invitro metabolism of (E)-2-ene VPA 105Effect of anti-rat cytochrome P-450h on the in vitro metabolismof VPA and (E)-2-ene VPA by untreated microsomes 112Quantitation of CBZ, CBZE and CBZD 113Analysis of CBZ and metabolites in rat urine by HPLC 113Urinary recoveries of CBZ, CBZE and CBZD after dosing withCBZ 113Urinary recoveries of CBZE and CBZD after dosing withCBZE 116Discussion 120Choice of experimental conditions 120Animal model 120Choice of vehicle for CBZ and CBZE 120GCMS analysis of VPA and metabolites 121Choice of metabolites monitored from the in vitro microsomalmetabolism of VPA and (E)-2-ene VPA 122HPLC analysis of CBZ and metabolites 122A comparison of the induction of rat hepatic microsomal cytochromeP-450 content by PB, CBZ, CBZE and other inducing agents. . . . 123Cytochrome P-450 content in hepatic microsomes fromuntreated rats 123Effect of VPA on cytochrome P-450 content 124viiTable of ContentsEffect of CFB on hepatic cytochrome P-450 content 124A comparison of the effects of PB, CBZ and CBZE on hepaticcytochrome P-450 content in rats 126A comparison of the effects of PB, CBZ and CBZE on theinduction of cytochrome P-450b 130Effect of CBZ, CBZE and PB treatment on cytochromes P-450fand P-450g 134In vitro metabolism studies of ‘[PA and (E)-2-ene VPA[Effects ofinducing agents 135Interaction between ‘[PA and CBZ 135Effect of anti-rat cytochromes P-450b and P-450h antibodies onVPA metabolite profiles from rat liver microsomes 136A comparison of the effects of PB, CBZ and CBZE induction onthe in vitro metabolism of VPA 140Formation of 4-ene ‘[PA 140Significance of 4-ene VPA formation to the mechanism ofVPA hepatotoxicity 141Formation of the hydroxy metabolites of ‘[PA 143Effect of anti-rat cytochrome P-450b and P-450h antibodies on(E)-2-ene VPA metabolite profiles from rat livermicrosomes 146A comparison of the effects of PB, CBZ and CBZE induction onthe in vitro metabolism of (E)-2-ene VPA in rat livermicrosomes 147Effect of CFB treatment on the metabolism of VPA and (E)-2-ene ‘[PA 151Clinical relevance 151Summary and conclusions 153References 155viiiLIST OF TABLESTable 1. Summary comparing the nomenclature of Ryan and Levin(1990) and Nelson et al. (1993) for isozymes of cytochrome P450 purified from rat liver microsomes 29Table 2. Summary of total hepatic cytochrome P-450 content(nmollmg protein, mean ± s.d.) and change in cytochrome P450 relative to the untreated group or to the respectivevehicle control group for the PB, CFB, CBZ and CBZEtreatment groups (n=4) 57Table 3. Summary of PROD (nmol resorufinlminlmg protein) andchanges in PROD relative to the untreated group or to therespective vehicle control group for the PB, CFB, CBZ andCBZE treatment groups (n=4) 68Table 4. Comparison of mean PROD activities of CBZE 3, 7, 10 and14 day treated groups as a percent of the PROD activities ofthe PB and CBZ 3, 7, 10 and 14 day treatment groups 70Table 5. Cytochrome P-450b (pmolJ5 pmol of spectrally determinedcytochrome P-450 or as percent of total hepatic cytochromeP-450) in microsomes from rats treated with either PB, CBZfor 3, 7, 10 or 14 days or CBZE for 3, 7, 10 or 14 days 73Table 6. A comparison of the metabolism of VPA to 3-OH VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats,relative to the untreated group or to the respective vehiclecontrol group (n=4) 79Table 7. A comparison of the metabolism of VPA to 4-OH VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats,relative to the untreated group or to the respective vehiclecontrol group (n=4) 83Table 8. A comparison of the metabolism of VPA to 5-OH VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats,relative to the untreated group or to the respective vehiclecontrol group (n=4) 86Table 9. A comparison of the metabolism of VPA to 4-ene VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats,relative to the untreated group or to the respective vehiclecontrol group (n=4) 90ixList of TablesTable 10. A comparison of the metabolism of VPA to 4-keto VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats,relative to the untreated group or to the respective vehiclecontrol group (n=4) 94Table 11. A comparison of the metabolism of (E)-2-ene VPA to (E,E)2,3’-diene VPA by microsomes from PB, CFB, CBZ andCBZE treated rats, relative to the untreated group or to therespective vehicle control group (n=4) 98Table 12. A comparison of the metabolism of (E)-2-ene VPA to (E)-2,4-diene by microsomes from PB, CFB, CBZ and CBZE treatedrats, relative to the untreated group or to the respectivevehicle control group (n=4) 102Table 13. Total 12 h urinary recoveries of CBZ, CBZE and CBZD (jig)from rats treated with CBZ 100 mg/kg every 12 h 115Table 14. Urinary recoveries (12 h) of CBZ, CBZE and CBZD aspercent of dose administered from rats treated with CBZ 100mg/kgeveryl2h 117Table 15. Total 12 h urinary recoveries of CBZE and CBZD (jig) fromrats treated with CBZE 50 mg/kg every 12 h 118Table 16. Urinary recoveries (12 h) of CBZE and CBZD as percent ofdose from rats treated with CBZE 50 mg/kg every 12 h 119xLIST OF FIGURESFigure 1. Summary of vaiproic acid metabolism .7Figure 2. Summary of carbamazepine metabolism 23Figure 3. Summary of the extraction procedure for vaiproic acid andmetabolites 42Figure 4. Summary scheme of the extraction of carbamazepine andmetabolites from urine of rats. 45Figure 5. Carbon monoxide sodium dithionite-reduced differencespectrum of hepatic microsomal cytochrome P-450 53Figure 6. Cytochrome P450 content (nmol of spectrally determinedcytochrome P-4501mg protein, mean ± s.d.) of microsomesfrom control, PB, NS, CFB and CO treated rats (n4) 54Figure 7. Cytochrome P-450 content (nmol of spectrally determinedcytochrome P-450/mg protein, mean ± s.d.) of microsomesfrom CBZ, CBZE and PG 3, 7, 10 and 14 day treated rats(n=4). 55Figure 8. SDS-PAGE gel of rat liver microsomal fractions from varioustreatment groups 60Figure 9. SDS-PAGE gel of rat liver microsomal fractions from varioustreatment groups 61Figure 10. Immunoblot of rat liver microsomal proteins probed withanti-rat cytochrome P-450b antibody 62Figure 11. Immunoblot of rat liver microsomal proteins probed withanti-rat cytochrome P-450b antibody 63Figure 12. Microsomal 0-dealkylation of pentoxyresorufin andethoxyresorufin (mriol resorufinlminlmg protein, mean ±s.d.) by microsomes from control, PB, NS, CFB and COtreated rats (n=4) 65Figure 13. Microsomal 0-dealkylation of pentoxyresorufin (nmolresorufinlminlmg protein, mean ± s.d.) by microsomes fromCBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4) 66xiList ofFiguresFigure 14. Microsomal 0-dealkylation of ethoxyresorufin (nmolresorufmnlminlmg protein, mean ± s.d.) by microsomes fromCBZ, CBZE and P03, 7, 10 and 14 day treated rats (n=4)..... 67Figure 15. Formation of 3-OH VPA (jig, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from untreated, PB, NS, CFBand CO treated rats (n=4) 77Figure 16. Formation of 3-OH VPA (jig, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from CBZ, CBZE and PG 3, 7,10 and 14 day treated rats (n=4) 78Figure 17. Formation of 4-OH VPA (jig, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from untreated, PB, NS, CFBand CO treated rats (n=4) 80Figure 18. Formation of 4-OH VPA (jig, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from CBZ, CBZE and PG 3, 7,10 and 14 day treated rats (n=4) 81Figure 19. Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes-(2 nmol of spectrallydetermined cytochrome P-450) from untreated, PB, NS, CFBand CO treated rats (n=4) 84Figure 20. Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from CBZ, CBZE and PG 3, 7,10 and 14 day treated rats (n=4) 85Figure 21. Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from untreated, PB, NS, CFBand CO treated rats (n=4) 88Figure 22. Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitrometabolism of ‘[PA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from CBZ, CBZE and PG 3, 7,10 and 14 day treated rats (n=4) 89xiiList ofFiguresFigure 23. Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from untreated, PB, NS, CFBand CO treated rats (n=4) 92Figure 24. Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from CBZ, CBZE and PG 3, 7,10 and 14 day treated rats (n=4) 93Figure 25. Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from thein vitro metabolism of (E)-2-ene VPA by microsomes (2 nmolof spectrally determined cytochrome P-450) from untreated,PB, NS, CFB and CO treated rats (n=4) 96Figure 26. Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from thein vitro metabolism of (E)-2-ene VPA by microsomes (2 nmolof spectrally determined cytochrome P-450) from CBZ, CBZEand PG 3, 7, 10 and 14 day treated rats (n=4) 97Figure 27. Formation of (E)-2,4-diene VPA (jig, mean ± s.d.) from the invitro metabolism of (E)-2-ene VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from untreated,PB, NS, CFB and CO treated rats (n=4) 100Figure 28. Formation of (E)-2,4-diene VPA (jig, mean ± s.d.) from the invitro metabolism of (E)-2-ene VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from CBZ, CBZEand PG 3, 7, 10 and 14 day treated rats (n=4) 101Figure 29. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 3-OH VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3day treated rats 104Figure 30. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 4-OH VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3day treated rats 106Figure 31. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 5-OH V.PA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3day treated rats 107Figure 32. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 4-ene VPA by microsomes (2 nmol ofxliiList ofFiguresspectrally determined cytochrome P-450) from PB and CBZ 3day treated rats 108Figure 33. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 4-keto VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3day treated rats 109Figure 34. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of (E)-2-ene VPA to (E,E)-2,3’-diene VPA bymicrosomes (2 nmol of spectrally determined cytochrome P450) from PB and CBZ 3 day treated rats 110Figure 35. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of (E)-2-ene VPA to (E)-2,4-diene VPA bymicrosomes (2 nmol of spectrally determined cytochrome P450) from PB and CBZ 3 day treated rats 111Figure 36. HPLC chromatograms of a) standards of CBZ, CBZE, CBZDand MCBZ, b) extracted blank rat urine sample, c) extractedspiked rat urine sample and d) extracted rat urine sample.Peak 1, CBZD, peak 2, CBZE, peak 3, CBZ and peak 4,MCBZ 114Figure 37. Cytochrome P-450 catalyzed metabolism of VPA to 3-OHVPA, 4-OH VPA, 5-OH VPA, 3-ene VPA and 4-ene VPA 138Figure 38. Structural similarity amongst hypoglycin, 4-pentenoic acidand 4-ene VPA 142Figure 39. The f-oxidation pathway of VPA metabolism inmitochondria 149xivLIST 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 acid2-ene VPA 2-propyl-2-pentenoic acid2-PGA 2-propyiglutaric acid2-PSA 2-propylsuccinic acid3-ene VPA 2-propyl-3-pentenoic acid3-keto VPA 2-propyl-3-oxopentanoic acid3-OH VPA 2-propyl-3-hydroxypentanoic acid4-ene‘4TPA 2-propyl-.4-pentenoic acid4-keto VPA 2-propyl-4-oxopentanoic acid4-OH VPA 2-propyl-4-hydroxypentanoic acid5-OH VPA 2-propyl-5-hydroxypentanoic acidAUC area under the serum concentration versus time curveBis N,N’-methylene-bis-acrylamideBSA bovine serum albuminCBZ carbamazepineCBZD trans-lO, 1 1-dihydroxy-lO, 1 1-dihydrocarbamazepine(carbamazepine diol)CBZE carbamazepine-10,1 1-epoxideCFB clofibrateCO corn oilCoA coenzyme AE transEDTA ethylenediaminetetraacetic acidg gram(s)xvList of abbreviationsGC gas chromatographyGCMS gas chromatography-mass spectrometryGSH glutathioneh hour(s)HEPES N- [2-Hydroxyl] piperazine-N- [2-ethanesulfonic acicHPLC high performance liquid chromatographyi.d. internal diameteri .p. intraperitonealIgG immunoglobulin, antibodyJVS Jamaican Vomiting Sicknessk thousandkg kilogram(s)micron(s), micrometer(s)MCBZ lO-methoxycarbamazepineMCPA methylenecyclopropylacetic acidMES maximal electroseizurespg microgram(s)mg milligram(s)2-MGA 2-methylgiutaric acidmm minute(s)microlitremM millimolarMSD mass selective detectorMTBSTFA N-tert-butyldimethylsilyl-N-methyltrifluoroacetamideNAC N-acetylcysteineNADH nicotinamide adenine dinucleotide, reducedNADPH nicotinamide adenine dinucleotide phosphate, reducedxviList of abbreviationsND not detectedNS normal salinePA 4-pentenoic acidPB phenobarbitalPBS phosphate buffered salinePG propylene glycolPROD pentoxyresorufin 0-dealkylationPTZ pentylenetetrazoleRS Reye’s syndromes.d. standard deviationSDS sodium dodecyl sulphateSDS-PAGE sodium dodecyl suiphate-polyacrylamide gelelectrophoresisSGOT serum glutamic oxaloacetic transaminaseaspartate aminotransferaseSGPT serum glutamic pyruvic transaminasealanine aminotransferaseSIM selected ion monitoringtBDMS tertiary-butyldimethylsilylTEMED N,N,N1,1V’-tetramethylethylenediamineVd volume of distributionVPA vaiproic acid (2-propylpentanoic acid)xviiDEDICATIONTo my parents for their support throughout the years.xviiiACKNOWLEDGEMENTSI would like to acknowledge my supervisor Dr. Frank Abbott for hisguidance 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 andexpertise. My appreciation also to Drs. David Seccombe and James Orr for theirconstructive and helpful comments. Thanks also to my able chairperson, Dr.James Axelson.I would also like to acknowledge the following people for their help withthe animal studies beyond the call of duty and friendship: Anthony Borel, GraceChan and Michael Gentleman. My extreme gratefulness to Dr. Dianchen Yu forhis help with the GCMS analyses.The figure of the summary of vaiproic acid metabolism in Introductionwas generously provided by John Gordon. The metabolism figures in Discussionwere 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.xixINTRODUCTIONEpilepsy affects approximately 20 to 40 million people throughout theworld (Rail and Schleifer, 1990). The incidence of epilepsy is higher in childrenthan in adults, with approximately 8 in 1000 children under the age of 7 yearsdemonstrating epilepsy. Epilepsy is characterized by abnormal phenomena ofmotor, sensory, autonomic or psychic origin. It can be designated as primary(idiopathic) or secondary (symptomatic) epilepsy. Primary epilepsy has noknown identifiable cause while secondary epilepsy may be caused by variousfactors including trauma, neoplasms, infection or cerebrovascular disease. TheInternational Classification of Epileptic Seizures describes in detail the varioustypes of epilepsy (Dreifuss, 1990).A number of anticonvulsant agents are available for the treatment ofepilepsy. Approximately 80% of patients can be effectively treated with a singleanticonvulsant 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 oftenused in combination therapy in efforts to optimize seizure control (Cloyd et al.,1985).Because treatment of epilepsy is a long term endeavour, the opportunitiesfor undesirable interactions with other xenobiotics are immense. An interactionmay result in the formation of a metabolite(s) which not only adversely affectsseizure control but also proceeds to a resultant toxic reaction. Although the sideeffects associated with VPA usage are generally of the mild gastrointestinaltype, incidents of teratogenicity, pancreatitis (Isom, 1984) and hepatotoxicity(Jager-Roman et al., 1986) have also been reported. Hepatotoxicity in mostcases has involved young children on multiple anticonvulsant therapy (Dreifuss1Introductionet 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 similarin structure to 2 known hepatotoxic terminal olefins, 4-pentenoic acid andmethylenecyclopropylacetic acid. Metabolism of 4-ene VPA via mitochondrial -oxidation may result in the formation of chemically reactive intermediateswhich can alklyate cellular macromolecules. Thus, it is postulated thatcoadministration of other anticonvulsant drugs, a number of which are known tobe enzyme inducing agents, will enhance the formation of the potentially toxicmetabolite(s).Because of the frequency of the combination of VPA and CBZ and thepotential risks of toxicity that might result from induction of VPA metabolism, itis important to characterize this interaction completely. This study will allowus 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. Sincethis thesis focuses on characterizing the effect of CBZ on the in vitro metabolismof VPA, a brief discussion on the metabolism of VPA and the toxicity associatedwith its usage will be presented. The major serum metabolite of VPA, 2-eneVPA appears to be devoid of the severe toxicities associated with ‘[PA and thushas been considered as a potential alternative anticonvulsant agent (Loscher,1992). Therefore, it was deemed important to also investigate the effect of CBZinduction on 2-ene VPA metabolism. A brief review on 2-ene VPA as well as abrief summary of the literature on CBZ and the interaction between VPA andCBZ will also be presented. In addition, a brief review on induction ofcytochrome P-450 by phenobarbital is also presented.2IntroductionVALPROIC ACIDOriginally synthesized for use as a solvent over a century ago (Burton,1881), VPA has only been available for therapeutic use in North America since1978 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 seizuretypes 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 seizuretypes, VPA is widely used either as a single agent (monotherapy) or incombination with other anticonvulsants (polytherapy). Other uses for VPAinclude prophylactic treatment of febrile convulsions (Lee et at., 1986), post-trauma epilepsy, status epilepticus, acute mania and alcohol withdrawal(Rimmer and Richens, 1985).MetabolismSince VPA shares in common the metabolic pathways of fatty acids, abrief review on fatty acid metabolism is presented.Fatty acid metabolismFatty acids consist of a terminal carboxyl group and an alkyl side chainand are of the basic formula, CH3-(CH2)-COOH (Devlin, 1986). Fatty acidsare mainly metabolized via n-oxidation as their coenzyme A (CoA) esters. In 3-oxidation, 2 carbon fragments are sequentially removed from the carboxylterminal after dehydrogenation, hydration, oxidation and thiolysis (Devlin,1986; Stryer, 1981).Mitochondrial f3-oxidation offatty acidsThe first step in the f3-oxidation cycle is the activation of the fatty acid toa fatty acyl CoA which occurs either in the endoplasmic reticulum or in the3Introductionouter mitochondrial membrane (Devlin, 1986; Montgomery et al., 1990). Theactivation 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 chainenzyme is specific for acetate and propionate, a medium chain enzyme specificfor 4 to 10 carbon fatty acids, a long chain enzyme specific for fatty acids longerthan 12 carbons and a separate enzyme for arachidonic acid. The 2 enzymesspecific for short and medium chain fatty acids are mitochondrial in locationwhilst the other 2 enzymes are located in the endoplasmic reticulum.Because the mitochondrial membrane is impermeable to CoA and itsderivatives, a carrier is necessary for transportation of the fatty acid across themembrane (Devlin, 1986). This function is performed by carnitine which isrequired for the transport of activated fatty acids of chain length 12 to 18carbons across the mitochondrial membrane. The acyl group is transferred bycarnitine palmitoyl transferase I to the hydroxyl group on the carnitinemolecule from the sulphur atom of CoA on the outer surface of the membrane.At the inner mitochondrial membrane, the acyl group is transferred fromcarnitine back to CoA by carnitine palmitoyl transferase II. However, shortchain fatty acids can directly diffuse across the membrane and become activatedto the CoA derivatives in the matrix compartment of the mitochondrion, i.e. theoxidation of short chain fatty acids is independent of carnitine.Inside the mitochondrion, the CoA derivatives are oxidized by one of agroup of acyl CoA dehydrogenases (Devlin, 1986). These enzymes are specificfor a certain chain length; palmitoyl CoA dehydrogenase for medium and longchain fatty acids, while the other 3 enzymes, octanoyl CoA and 2 butyryldehydrogenases, are specific for shorter chain fatty acids. The function of thesedehydrogenases is to remove 2 hydrogen atoms to form an enoyl CoA with atrans double bond between the second and the third carbon atoms. The 24Introductionhydrogen atoms are accepted by flavin adenine dinucleotide (FAD) andultimately, 2 electrons are channelled into the electron transport system.The x,3-unsaturated acyl CoA accepts a molecule of water, a reactioncatalyzed by enoyl CoA hydrase to form L-3-hydroxyacyl CoA (Devlin, 1986). L3-hydroxyacyl CoA is oxidized by 3-hydroxyacy1 CoA dehydrogenase to [3-ketoacyl CoA which is further oxidized in the [3-position by f3-ketothiolase. CoAis inserted and cleavage occurs at the [3-carbon to yield acetyl CoA and asaturated acyl CoA with 2 fewer carbons than the original substrate.The steps described above are repeated until a 4 carbon butyryl CoAremains as the intermediate. Butyryl CoA is [3-oxidized to yield acetoacetyl CoAand subsequently 2 molecules of acetyl CoA.Feroxisomal [3-oxidation offatty acidsThe [3-oxidation of fatty acids also occurs in peroxisomes, subcellularorganelles 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 13-oxidation in 3 ways. First, carnitine is not required for fatty acid entry into theperoxisome (Lazarow, 1987). Second, the initial dehydrogenation step inperoxisomes is catalyzed by a cyanide-insensitive oxidase leading to theformation of hydrogen peroxide which is eventually eliminated. Third, theenzymes involved in the cycle differ slightly, in that 3 proteins perform the 4reactions of peroxisomal [3-oxidation whereas in mitochondrial [3-oxidation, 4proteins are involved. It appears that the role of peroxisomes is to shorten thechain length of relatively long fatty acids for [3-oxidation in the mitochondria,since peroxisomal [3-oxidation is unable to proceed beyond 8 carbons in theshortening of long chain fatty acids.5IntroductionOmega and omega-i oxidation offatty acidsMetabolism of fatty acids via o- and 0)-i oxidation represent minorbiotransformation pathways (Devlin, 1986). Primarily, medium chain lengthfatty acids undergo metabolism via these oxidative pathways which occur in theendoplasmic reticulum of many tissues. Omega oxidation involves hydroxylationat the methyl carbon on the opposite end from the carboxyl group while o-1oxidation occurs at the penultimate carbOn atom next to the terminal methylgroup. After hydroxylation, the fatty acid may be further oxidized to adicarboxylic acid at which stage f3-oxidation can occur from either end of themolecule. The co- and 0)-i oxidations are cytochrome P-450 mediated events(Montgomery et at., 1990).Metabolism of valproic acidVPA, an 8 carbon, branched chain fatty acid, possesses an uniquestructure amongst the wide array of anticonvulsant agents. Unlike otheranticonvulsant agents, VPA lacks a nitrogen moiety. Despite its simplestructure, VPA undergoes extensive biotransformation (figure 1) via severalpathways: glucuronidation, 13-oxidation and (0- and co-i oxidation (Loscher,1981a; Granneman et at., 1984a). Very small quantities (3 to 7%) of theunchanged drug are recovered in the urine (Schobben et at., 1975; Dickinson etat., 1989). Glucuronidation and f3-oxidation are the 2 major metabolic pathwaysin both man and rat (Granneman et at., 1984a).The glucuronide conjugate accounts for approximately 11 to 68% ofurinary recovery in man (Granneman et at., 1984a; Dickinson et at., 1989). Withincreasing doses of VPA, glucuronidation, which occurs in the endoplasmicreticulum, increases at the expense of 13-oxidation (Granneman et at., 1984a;Granneman et at., 1984b). Conjugation with glycine occurs but is a minormetabolic pathway.6COOHOOH2-eneVPA(B)-and(Z)-IsomersCOOH3-OHVPAVPA COOH4-eneVPAil.. COOH24-dIeneVPA(E)- and(Z)-isomersOHCOOH4-OHVPACOO-GIuCOOHHOO•COOHCOOHHOOC2-PSACOOH/\/c/\3-eneVPA(B)-and(Z)-Isomers./NVPA.glucuronide23-dieneVPA(E,E)-and(EZ)-isomers5-OHVPACOOHHOOC2-PGA4-ketoVPA/3-ketoVPAIFigure1.Summaryofvaiproicacidmetabolism(Yuetal.).IntroductionMitochondrial 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 themitochondria, VPA is activated to VPA-CoA, then dehydrogenated to 2-ene VPACoA by acyl-CoA dehydrogenase, then hydrated by enoyl-CoA hydrase to 3-OHVPA-CoA and finally dehydrogenated to 3-keto VPA-CoA by 3-hydroxyacyl-CoAdehydrogenase. The 3-keto VPA metabolite is the terminal product of thispathway due to prevention of thiolytic cleavage by the enzyme 3-ketoacyl CoAthiolase (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 enhancedexcretion of 3-keto VPA was observed in rats after pretreatment with clofibrate,a known peroxisomal inducer (Heinemeyer et at., 1985). VPA is alsohydroxylated to 3-OH VPA microsomally via co-2 oxidation (Prickett and Baillie,1984). Mean urinary recoveries of the 13-oxidation pathway metabolites accountfor 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 VPAand 2-propylglutaric acid (2-PGA) (Granneman et at., 1984a). The metabolite, 2-PGA, is the end product of co-oxidation and does not undergo further metabolismvia 13-oxidation (Kuhara and Matsumoto, 1974). Oxidation of VPA at the co-iposition leads to 4-OH VPA, 4-keto VPA and 2-propylsuccinic acid (2-PSA). Thehydroxylated metabolites are not further metabolized to the unsaturatedmetabolites (Granneman et at., 1984a). The co- and co-i oxidation pathwaysaccount 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-eneVPA (Granneman et at., 1984a). 4-Ene VPA represents less than 1% of therecovered dose in man (Abbott et at., 1986; Dickinson et at., 1989). The8Introductionmetabolism 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 resultin the formation of diunsaturated metabolites. For example, 4-ene VPA and 3-ene 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 beenidentified as 2,3’-diene VPA (Acheampong and Abbott, 1985). (E)-2-ene VPA isdehydrogenated to both diunsaturated metabolites and is 13-oxidized to 3-ketoVPA (Granneman et at., 1984a; Loscher et at., 1992).Metabolism of 4-ene VPAThe 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 bileand 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 gammalactone, 2-PGA and the parent compound, 4-ene VPA. Most metabolites werederived via either 13-oxidation or cytochrome P-450 mediated reactions. Sixmetabolic pathways for the biotransformation of 4-ene VPA were assigned: 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 dicarboxylicacid (2-PGA), reduction followed by oxidation to the diacid, epoxidation to thegamma butyrolactone derivative and hydroxylation at the C-3 position to form3-OH-4-ene VPA. Twenty metabolites of 4-ene VPA were identified in the urineof 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,9Introductionother metabolites identified included (E)-3-ene VPA, 4-OH VPA, 4-OH VPAlactone, 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 theglycine conjugate of (E)-2,4-diene VPA were also identified. Again,glucuronidation and 13-oxidation were identified as the major metabolicpathways and - and (0-1 oxidation as minor biotransformation routes.Metabolite activityThe metabolites (E)-2-ene ‘[PA, 4-ene VPA (Loscher, 1981b), 3-ene VPA(Kochen and Scheffner, 1980) and (E,E)-2,3’-diene VPA (Abbott andAcheampong, 1988) possess anticonvulsant activity. (E,E)-2,3-diene VPA is asactive as (E)-2-ene VPA in mice (Abbott and Acheampong, 1988). (E)-2-ene VPAand 4-ene ‘[PA are the most active of the metabolites, displaying approximately60 to 90% of the potency of ‘[PA although they are more sedating than ‘[PA inmice (Nau and Loscher, 1984; Loscher and Nau, 1985). 2-Ene VPA wasdetectable in mouse plasma and brain 2 days after discontinuation of VPA andmay be responsible for the elevated seizure threshold in the absence ofdetectable VPA brain levels (Loscher and Nau, 1984).Adverse effectsSide effectsThe 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, weightgain, hyperkinesia, fine postural tremor, drowsiness and transienthallucinations (Dulac et al., 1986). Tremor, weight gain, transient hair loss andlimb 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 limiting10Introductionneutropenia and thrombocytopenia have also been observed with VPA (Barr etal., 1982) as well as reduced platelet adhesiveness and prolonged bleeding time(Smith and Bleck, 1991). Dementia has also been observed in patients on VPAtherapy, although there was prompt remission after withdrawal of the drug(Zaret and Cohen, 1986).Biochemical effectsBiochemical effects of VPA include hyperammonemia, inhibition of f3-oxidation (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 dicarboxylicacids. 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 etal., 1980). In a 6 year old male with Reye’s syndrome, increased amounts oflactic and adipic acids as well as increased quantities of 2-PGA, the end productof 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, 5-OH VPA and 2-PSA produced a concentration-dependent inhibition ofgluconeogenesis from lactate (Rogiers et al., 1985). The extent of toxicity indecreasing order was VPA and 4-ene VPA, 5-OH VPA, 4-OH VPA, (E)-2-eneVPA and 2-PGA.VPA interferes with the folate-dependent one carbon enzyme responsiblefor glycine cleavage, resulting in hyperglycinemia in patients and animalstreated with VPA (Carl, 1986). Hyperglycinemia and hyperglycinuria areobserved in rats administered VPA chronically for several weeks at dosesranging from 0.3 to 1.2 mmoLlkg (Cherruau et al., 1981). Chronic11Introductionadministration of 1% VPA to young rats resulted in significant increases inblood, liver and brain glycine levels (Martin-Gallardo et at., 1985).Hyperammonemia and hyperbilirubinemia are also associated with VPAtherapy (Matsuda et at., 1986; Ratnaike et at., 19.86). \TPA inhibits ureasynthesis in rat hepatocytes (Coude, 1983; Turnbull et at., 1983).Coadministration of other anticonvulsants with VPA, particularly phenobarbitalor 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 whichthemselves are not toxic but cause an increased metabolic need for carnitine toexcrete the more toxic metabolites. Patients on VPA therapy display decreasedplasma carnitine levels accompanied by elevated blood ammonia levels (Ohtaniet at., 1982). Oral administration of carnitine 50 mg/kg/day for 4 weeks wassuccessful in correcting the VPA induced carnitine deficiency andhyperammonemia. Carnitine deficiency may be the end result ofhyperammonemia. In mice, VPA exerts an immediate but transient effect oncarnitine metabolism (Rozas et at., 1990). Single doses of VPA in thetherapeutic range for man decreased hepatic levels of free CoA, acetyl CoA andfree 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 furthermetabolites in the matrix of the hepatic mitochondria (Turnbull et at., 1983) orby altering the integrity of the inner mitochondrial membrane or by actions onthe 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 may12Introductionbe due to sequestration of CoA as valproyl-CoA (Veitch and Van Hoof, 1990).Increased activity of the peroxisomal n-oxidation enzymes in rat liver wasobserved after chronic administration of VPA (Hone and Suga, 1985; Ponchautet at., 1991). Decreased cytochrome P-450 levels were observed in ratsadministered VPA (172.8 to 259.2 mg/kg/day) (Cotariu et at., 1985).PancreatitisPancreatitis 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 physiciansrevealed 15 additional cases of VPA induced pancreatitis (Asconape et at., 1993).A further 6 cases of pancreatitis in addition to hepatic failure with VPA usagehave also been reported in the literature (Binek et at., 1991). Most of the casesof VPA implicated pancreatitis (77%) involved patients under the age of 20 yearsand 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 VPApolytherapy. VPA pancreatitis manifests itself initially as abdominal pain andvomiting (Parker et at., 1981; Wyllie et at., 1984; Rosenberg et at., 1987).Increased serum amylase and lipase levels have also been observed in somecases (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 mostcases the reaction is mild with rapid resolution once VPA is discontinued(Asconape et at., 1993). VPA induced pancreatitis appears to be an idiosyncraticreaction, unrelated to VPA dosage or serum concentrations.Embryotoxicity / teratogenicityVPA possesses the potential for teratogenicity in all species thus farinvestigated, including man, monkey and rodent (Cotariu and Zaidman, 1991).13IntroductionAlthough the exact mechanism by which VPA exerts its teratogenic effects hasnot been established, possible mechanisms include alteration of fetalglutathione status, alteration of fetal lipid metabolism, effects on folate or zinclevels 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 forteratogenic activity include a free carboxyl group attached to a carbon atomwhich is substituted with only 2 alkyl chains (Nau and Siemes, 1992). The -Hatom is quite important for teratogenic activity; substitution abolishesteratogenic 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 isobserved if the alkyl chain length is shortened or lengthened (Nau and Scott,1987; Nau and Siemes, 1992). Teratogenic activity may be expressed throughchiral interactions of the branched chain carboxylic acids with variousembryonic 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 andhyperconvex nails (Yerby et al., 1992). A consistent facial phenotype (epicanthalfolds, flat nasal bridge etc.) was observed in 7 children who had been exposed toVPA in utero (DiLiberti et al., 1984).A 1 to 2% incidence in humans of VPA induced neural defects isassociated 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 implicated14Introductionto 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 bifidaaperta, but incidents of myelomeningocele have been reported (Nau and Siemes,1992).The intensity of the teratogenic response as measured by neural tubedefects in mice, was dependent on the concentrations of VPA achieved in themother and fetus (Nau, 1985). In pregnant mice, both the dose of VPA and areaunder the serum concentration versus time curve (AUC) correlate withembryolethality and fetal weight retardation (Nau, 1985).Although therapeutic doses of VPA in pregnant women do not affect fetalgrowth, the risk for fetal perinatal distress increases with higher doses of VPA(Jager-Roman et al., 1986). VPA administration to rhesus monkeys at a humantherapeutic dose of 20 mg/kg/day during organogenesi did not yield any adverseeffects (Mast et al., 1986). However, a dose of 200 mg/kg/day caused low birthweights, craniofacial and skeletal defects and a dose of 300 mg/kg/day wasembryolethal (Mast et al., 1986). A later study in rhesus monkeys (20 to 600mg/kg) also resulted in a dose dependent developmental toxicity whichmanifested itself as increased embryo/fetal mortality, intrauterine growthretardation and craniofacial and skeletal defects (Hendrickx et al., 1988).In whole rat embryos, VPA at doses greater than 40 mg/kg/day causedabnormal development in 30% of embryos (Lewandowski et aL, 1986). In ratsadministered VPA 300 mg/kg daily on embryonic days 7 to 18, a decrease inmaternal bodyweight and fetal weight and an increase in malformations wereobserved (Vorhees et at., 1991).HepatotoxicityAlthough side effects associated with VPA use are generally mild, anumber of cases of fatal hepatotoxicity have been reported (Kochen et at., 1984;15IntroductionJager-Roman et al., 1986). Hepatotoxicity associated with VPA usage is of 2types, either dose-related or idiosyncratic (Dreifuss et al., 1987). Dose-relatedVPA hepatotoxicity resolves with a decrease in dosage or discontinuation of thedrug. Up to 44% of patients on VPA therapy demonstrate dose related increasesin liver enzyme levels (Sussman and McLain, 1979) but these increases are notpredictive of hepatotoxicity since less than 0.01% of patients develop fatalhepatotoxicity (Dreifuss et al., 1987). The fatal but rare idiosyncratichepatotoxicity is irreversible and dose-independent.A recent survey of reported cases of fatal hepatotoxicity associated withVPA usage in the United States between 1978 and 1984 concluded that age andpolytherapy were the major determinants (Dreifuss et al., 1987). The incidenceof VPA induced hepatotoxicity decreased with increasing age. The incidencewas highest in those children under 2 years of age on polytherapy (1/500)compared to the same age group on monotherapy (1/7000). The overallincidence of VPA hepatotoxicity was 1/10,000. Many of the patients whodeveloped hêpatotoxicity suffered from other medical conditions includingmental retardation, developmental delay, congenital abnormalities andmetabolic disorders which conspired to place them at higher risk. A follow-upstudy for the period 1985 to 1986 demonstrated a decline in the incidence ofhepatic fatalities related to VPA from 1/10,000 to 1/49,000. This occurreddespite an overall increased usage of VPA but as a single agent rather than inpolytherapy (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 Jamaican16IntroductionVomiting Sickness (JVS), Rey&s Syndrome (RS) and 4-pentenoic acid (PA)toxicity (Lewis et al., 1982; Nau and Loscher, 1984). Clinically, VPAhepatotoxicity shares similar manifestations with JVS, RS and PA toxicity.Reye’s syndrome was first described in 21 children (Reye et al., 1963) andthe etiology of RS remains unknown. RS involves encephalopathy and fattydegeneration of the liver, kidney and occasionally other organs. Other clinicalfeatures include fever, convulsions, vomiting, hypoglycemia and increasedserum glutamic oxaloacetic transaminase (SGOT) and serum glutamic pyruvictransaminase (SGPT) levels.Hypoglycin A (hypoglycin, L-x-amino-2-methylenecyclopropylpropionicacid), an amino acid found in unripe akee fruit and in the seeds of severalvarieties of maple trees, is responsible for JVS (Tanaka et al., 1976). Hypoglycinis metabolized to methylenecyclopropylacetic acid (MCPA) which forms an enoylCoA 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 andcoenzyine A by MCPA (Bressler et al., 1969). Hypoglycin causeshyperammonemia in rats (Glasgow, 1983) and hypoglycemia is common inpatients with JVS (Jelliffe and Stuart, 1954). In addition, encephalopathy andfatty degeneration of the viscera are common features. The incidence of JVSdecreases after the age of 10 (Reye et al., 1963).4-Pentenoic acid is a structural analogue of hypoglycin. In rats, PAproduces similar features to JVS and RS including hypoglycemia, fattydegeneration 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 3-keto-4-pentenoyl-CoA (Schulz, 1983) through inhibition of the 13-oxidation17Introductionenzyme 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 moreeffective inhibitor of the enzyme (Schulz, 1983). The inhibition andmorphological manifestations caused by PA can be partially reversed by Lcarnitine (Billington et at., 1978; Sugimoto et at., 1990).The formation of toxic metabolites is thought to be responsible for VPAhepatotoxicity. Because of the similarities between VPA hepatotoxicity, JVS,RS and PA toxicity, a metabolite of VPA similar in structure to PA and MCPAmay be responsible for VPA associated toxicity. The hepatotoxicity of VPA isthought to be caused by its mono- and/or diunsaturated metabolites (Kochen etat., 1984). An increased formation of the diunsaturated metabolites appears tobe characteristic in fatal hepatic failure. 4-Ene VPA was detected in the urineof 6 patients and 4,4’-diene in 3 of the patients who died of VPA related hepaticfailure (Scheffner et at., 1988). The metabolite, 4-ene VPA, is structurallysimilar to 4-pentenoic acid and MCPA and may play an important role in VPAassociated hepatotoxicity. One 7 year old patient on phenobarbital and VPAtherapy who died from hepatic failure resembling Reye’s syndrome (Kochen etat., 1983) demonstrated plasma and urine concentrations 4 to 5 times thenormal levels of several unsaturated metabolites including 4-ene VPA. The 4,4’-diene VPA was also present. The f3-oxidation pathway appeared to be inhibitedas 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-eneVPA and 2,4-diene VPA caused hepatic steatosis and inhibition of 13-oxidation(Granneman et al., 1984c; Kesterson et at., 1984). These unsaturatedmetabolites may be further metabolized to the chemically reactive 3-keto-4-eneVPA which could then alkylate mitochondrial proteins including enzymesinvolved in 13-oxidation (Rettenmeier et at., 1986b). This mechanism is based on18Introductionobservations with 4-pentenoic acid which is transformed to a reactiveintermediate that alkylates and destroys the terminal enzyme of the 13-oxidationpathway (Schulz, 1983; Fong and Schulz, 1983).In rat liver homogenates, VPA and 4-ene VPA caused inhibition ofdecanoic acid f3-oxidation (Bjorge and Baillie, 1985). VPA depletes free CoApoois (Fears, 1985) and may cause a transient and mild inhibition of the f3-oxidation pathway by sequestration of CoA (Kesterson et aL, 1984). 4-Ene VPAproduces a more potent and prolonged inhibition by forming CoA esters whichdirectly inhibit enzymes in the 13-oxidation pathway. VPA inhibitsmitochondrial 13-oxidation by forming vaiproyl CoA which is a weak inhibitorbut acts at a different site than 4-pentenoic acid and hypoglycin (Sherratt andVeitch, 1984). VPA, 4-ene VPA and 2,4-diene VPA trap intramitochondrial freeCoA (Ponchaut et at., 1992).The glutathione conjugate of (E)-2,4-diene VPA was identified in the bileof 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 of4-ene VPA, in vivo activation to a CoA ester which readily interacts withglutathione may explain the high urinary recovery. In patients with VPAassociated hepatic failure, the levels of (E)-2,4-diene VPA recovered in the urineas the N-acetylcysteine conjugate were 3 to 4 times the levels observed inhealthy patients (Kassahun et at., 1991).(E)-2-ENE VPAThe metabolite (E)-2-ene VPA has been touted as a potentialanticonvulsant agent due to its lack of teratogenicity (Nau et at., 1984; Nau andLoscher, 1986; Nau, 1986), lack of embryotoxicity (Loscher et at., 1984;19IntroductionLewandowski et al., 1986; Nau, 1986) and apparent lack of hepatotoxicity(Kesterson et al., 1984; Schafer and Luhrs, 1984; Loscher, 1992) in experimentalanimals.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 aseffective 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 clonicthreshold of pentylenetetrazole (PTZ) induced seizures (Semmes and Shen,1991).The spectrum of activity of (E)-2-ene VPA is similar to that of VPAwithout the potential for embryotoxicity even at doses of 600 mg/kg (Loscher etal., 1984). In 4 different models of anticonvulsant activity, (E)-2-ene VPAactivity was similar to that of VPA. (E)-2-ene VPA was more potent in generaltonic clonic seizures in gerbils and in petit mal recurrent seizures in rats. In themaximal electroseizures (MES) and PTZ tests in mice, doses of 200 to 300 mg/kgof (E)-2-ene VPA were more sedating than VPA. However, sedation was notobserved in rats or gerbils at these doses. The anticonvulsant activity of (E)-2-ene VPA is of shorter duration (2 h compared to 5 h) than VPA in mice afterdoses of 4 mmollkg (Keane et al., 1985).Teratogenicity(E)-2-ene VPA possesses very little teratogenic potential (Nau, 1986). Atsimilar 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 folatemetabolism at doses of 500 mg/kg i.p. (Wegner and Nau, 1992). In ratsadministered (E)-2-ene VPA at doses of 300 mg/kg daily on embryonic days 7 to20Introduction18, no increases in the percentage of resorptions or malformations wereobserved (Vorhees et al., 1991). Increasing the dose to 400 mg/kg decreasedfetal bodyweight by approximately 8% but with no change in resorptions ormalformations. Abnormal embryo development or retardation of growth wasnot observed after administration of 2 doses of 400 mg/kg on day 10 ofpregnancy in rats (Klug et aL, 1990). In whole rat embryos, (E)-2-ene VPA didnot produce any adverse effects at doses up to 200 mg/kg/day (Lewandowski etal., 1986).MetabolismTo 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 the13-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 likelyresulting from isomerization (Vorhees et al., 1991). In addition, in urine up to2% of the administered dose was recovered as V.PA (Granneman et al., 1984a).CARBAMAZEPINECarbamazepine (CBZ, 5-.carbamoyl-5H-dibenz [b,f] azepine, carbamoyliminostilbene), an iminostilbene derivative (Eadie and Tyrer, 1989), isstructurally similar to the tricyclic antidepressants (Kutt, 1989). CBZ iseffective 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 erraticabsorption from the gastrointestinal tract (Rail and Schleifer, 1990). Peakplasma levels are attained in 2 to 8 h. Therapeutic plasma levels of CBZ are inthe range of 3 to 14 ig/mL. CBZ has a relatively long half-life ranging from 8 to72 h. The drug is highly plasma protein bound (75 to 90%).21IntroductionCarbamazepine undergoes extensive metabolism (figure 2) via the livermicrosomal enzyme system, with less than 1% of the parent drug excretedunchanged in the urine (Bertilsson and Tomson, 1986). The major route ofmetabolism is the epoxide-diol pathway where CBZ is transformed tocarbamazepine-10,11-epoxide (CBZE) which is further hydrolyzed to trans10,1 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 CBZDaccounts for 30 to 60% of the administered dose. Other metabolic pathwaysinclude hydroxylation in the 2, 3 and 9 positions and account for approximately15% of the dose.Carbamazepine induces its own metabolism as well as the metabolism ofother drugs via induction of the hepatic microsomal enzyme system (Bertilssonand 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 (Neuvonenet 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 ofthe epoxide-diol pathway (Eichelbaum et at., 1984). CBZ autoinduction in manhas been reported to occur within 2 or 3 days (Pynnonen, 1979) althoughmaximal CBZ autoinduction may require 3 to 4 weeks (Bleck, 1990). In a groupof 77 patients, autoinduction of CBZ metabolism appeared to be complete withinone 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 antipyrine22COHH2 (CBZ)CarbamazepineI/CONHCOHHI/•1/2.OH.CBZCBZ-1O11poxdo+/ /epoxidehydrolase//C H2OHCONHCONH3.OH.CBZCONH29-OH-CBZS.trans-CBZ.dIoI0 0Figure2.Summaryofcarbamazepinemetabolism.Introductionclearance in healthy volunteers (Rapeport et al., 1983).In rats, chronic administration of CBZ (25 mg/kg i.p. every 12 h for 7days) 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 andinduced cytochrome P-450, NADPH cytochrome P-450 reductase, aminopyrineN-demethylase and UDP-glucuronyltransferase (Wagner and Schmid, 1987).Regnaud et al. (1988) attempted to determine the dose dependency of CBZinduction in rats. CBZ was administered intraperitoneally at 60, 120 and 200mg/kg/day for 4 days. Epoxide hydrolase activity appeared to increase withincreasing dose as did aminopyrine N-demethylase activity. Cytochrome P-450and aniline hydroxylase activity did not increase at doses higher than 120mg/kg/day.Carbamazepine-lO,11 -epoxideCarbamazepine-10,11-epoxide is an active metabolite and serum levelsare usually 15 to 55% and 5 to 81% of the parent compound in adults andchildren, respectively (Bertilsson and Tomson, 1984). CBZE possessesanticonvulsant activity similar to CBZ in various animal models (Tomson andBertilsson, 1991). In 7 adult patients, no change was observed in seizure controlwhen CBZE was substituted for CBZ (Tomson et al., 1990). CBZE wasequipotent to CBZ in controlling pain due to trigeminal neuralgia (Tomson andBertilsson, 1984). CBZE has a shorter half-life than CBZ (approximately 6 h) inman and is metabolized almost completely to CBZD, with 67 to 100% of theepoxide dose recovered as CBZD (Tomson et al., 1983; Spina et al., 1988).When CBZE was administered to rats intraperitoneally at a dose of 100mg/kg daily for 3 and 7 days, maximal induction of epoxide hydrolase andglutathione transferase appeared to be achieved in 3 days (Jung et al., 1980).24IntroductionHowever, CBZE treatment did not alter total hepatic cytochrome P-450 levels.INTERACTION BETWEEN VPA AND CBZBecause VPA undergoes such extensive biotransformation in the body,coadministration of metabolic inducing agents may result in the increasedformation of potentially toxic metabolites. Interactions of VPA with otheranticonvulsant drugs have been studied most extensively. These interactionsmay be pharmaceutical, pharmacokinetic or pharmacodynamic (Smith andBleck, 1991).In polytherapy with other anticonvulsants, (CBZ, phenytoin andphenobarbital) shorter serum VPA half-lives are observed compared tomonotherapy (Kutt, 1984). In addition, the ratio of VPA steady-state plasmalevels 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 inducingVPA metabolism (Kutt, 1984). CBZ affects VPA disposition in both pediatricand adult patients (Kutt, 1984; Baciewicz, 1986). Lower serum VPAconcentrations, despite higher VPA doses, have been observed in both adult(Reunanen et al., 1980) and pediatric patients (Abbott et al., 1986) on VPA andCBZ compared to VPA alone. VPA serum half-life decreased and plasmaclearance (Clv) increased when VPA was coadministered with CBZ in adultepileptic patients (Hoffman et al., 1986).Bowdie and coworkers (1979) demonstrated that administration of CBZ ata dose of 200 mg daily in healthy volunteers resulted in increased VPAclearance and decreased VPA steady state levels after 2 weeks. A change in theelimination rate constant, Ke, was not observed.25IntroductionIn 5 adult volunteers, the plasma clearance of VPA was significantlyincreased following CBZ coadministration (Panesar et al., 1989). The volume ofdistribution (Vd) of VPA remained unchanged after CBZ, thus providing supportfor 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 inthe Vd was not expected. 4-Ene VPA serum levels were unchanged after CBZadministration but urinary recovery of this metabolite was increased after CBZ.The serum data suggested induction of the 0)- and 0)-i oxidative pathways byCBZ. The major unsaturated metabolite, (E)-2-ene VPA, was significantlyreduced in serum and urine suggesting at first that induction of 13-oxidation viaperoxisomes had occurred. The 13-oxidation end product, 3-keto VPA, however.was not increased suggesting that (E)-2-ene VPA metabolism was shunted intoalternate pathways. Furthermore, urinary recoveries based on the assay of VPAand metabolites could not confirm an increase in VPA metabolism. Thus, itwould appear that either VPA metabolite elimination occurred via non-renalroutes or the assay failed to detect a significant proportion of the VPAmetabolites. The urinary recovery of VPA and metabolites representingapproximately 65% of the dose was consistent with other investigators (Pollacket al., 1986).In a study performed in our laboratory (unpublished data), significantdifferences were observed between the VPA metabolite profiles of 16 pediatricpatients on VPA and CBZ polytherapy and the profiles of 37 pediatric patientson VPA monotherapy. VPA and unsaturated metabolites were significantlyreduced in the serum of the VPA and CBZ group. The pediatric patient profilesdid not show an increase in urinary 4-ene VPA elimination yet decreased serum4-ene VPA concentrations were observed with CBZ coadministration. Inductionof 4-ene VPA metabolism may have occurred, resulting in the formation of26Introductionsecondary f3-oxidation metabolites such as 3’-keto-4-ene VPA and the postulatedreactive 3-keto-4-ene VPA which our assay did not detect. Urinary profiles weregenerally a reflection of the serum data. Thus, there was apparent induction ofVPA metabolism via the 0)-, 0)-i and 13-oxidation pathways by CBZ. Again, thenet recovery of VPA and metabolites in the urine of the VPA and CBZ group didnot account for the increased VPA metabolism. An apparent induction of the (0-and e-1 oxidation pathways of VPA metabolism was also observed in anotherpediatric group on combined VPA and CBZ therapy (Kassahun et at., 1990).CYTOCHROME P-450The hepatic microsomal mixed-function oxidase systems consists ofcytochrome P-450 (a hemoprotein), NADPH-cytochrome P-450 reductase andphospholipids (Lu and Levin, 1974.) Cytochrome P-450 is a fairly ubiquitousenzyme, present in virtually every tissue including lung, small intestine, liver,adrenals, testis, kidney and duodenum (Okey, 1990) with the exception oferythrocytes and striated muscle (Guengerich, 1991). Cytochrome P-450 is alsopresent in the mitochondria and has been shown to be involved in themetabolism of carcinogens including aflatoxin Bi, benzo[a]pyrene anddimethylnitrosoamine (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 whichare responsible for the biotransformation of hydrophobic molecules to morehydrophilic molecules which can undergo further metabolism by phase IIenzymes prior to their excretion in either urine or bile (Leroux et at., 1989).However, situations arise where metabolism by cytochrome P-450 results in theformation of a reactive metabolite which may ultimately cause hepatotoxicity asin the case of cocaine (Boelsterli et at., 1992) and VPA (Rettie et at., 1987).27IntroductionThe expression of the constitutive forms of cytochrome P-450 is dependenton 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, thephysiological status of the animal, starvation and hypertension. As an example,nutritional deficiences generally result in decreased rates of xenobioticmetabolism in rat liver microsomal fractions (Yang et al., 1992).Xenobiotics capable of inducing cytochrome P-450 can be grouped into 6categories: 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 withaliphatic 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 transcriptionalactivation, mRNA stabilization, or by protein stabilization.The nomenclature of Levin and co-workers (Ryan and Levin, 1990) will beused in this thesis when referring to the isozymes of cytochrome P-450. Asummary comparing their nomenclature to that of Nelson et al., (1993) isprovided in table 1. In humans, orthologues have been identified for ratcytochromes P-450j and P-450p (Soucek and Gut, 1992).Induction of cytochrome P-450 by phenobarbitalPhenobarbital (PB) is an effective inducer of a number of isozymes ofcytochrome P-450 in rats and other laboratory animals (Waxman and Azaroff1992). In addition to induction of cytochrome P-450, PB also induces a numberof other enzymes which are involved in the metabolism of xenobiotics includingaldehyde dehydrogenase, epoxide hydrolase, NADPH-dependent:cytochrome P450 reductase, UDP-glucuronyltransferase and glutathione transferases.Induction is invoked by the parent compound itself as opposed to its major28IntroductionTable 1. Summary comparing the nomenclature of Ryan and Levin (1990)and Nelson et al. (1993) for isozymes of cytochrome P-450 purifiedfrom rat liver microsomes.Ryan and Levin Nelson et al.cytochrome P-450a CYP2A1cytochrome P-450b CYP2B1cytochrome P-450e CYP2B2cytochrome P-450f CYP2C7cytochrome P-450g CYP2C13cytochrome P-450h CYP2C11cytochrome P-450j CYP2E1cytochrome P-450k CYP2C6cytochrome P-450p CYP3A1Adapted from Nelson et al. (1993), Ryan and Levin (1990) and Soucek and Gut(1992).29Introductionmetabolite, p-hydroxyphenobarbital (Cresteil et al., 1980).Phenobarbital induction results in proliferation of smooth endoplasmicreticulum 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, arapid increase in the cytochrome P-450 levels in rough endoplasmic reticulum isobserved within 3 h and maximal levels are achieved within 6 h (Ernster andOrrenius, 1965). Six hours after the administration of PB, the levels ofcytochrome P-450 in the smooth endoplasmic reticulum slowly start to increaseand after 12 h surpass the levels achieved in the rough endoplasmic reticulum.Maximal induction with phenobarbital is achieved in 3 days compared to24 h with 3-methylcholanthrene treatment (Greim et aL, 1981). With thebarbiturates, the extent of induction in rats is directly related to plasma half-life, i.e. compounds possessing longer plasma half-lives are more effectiveinducers of cytochrome P-450 (Toannides and Parke, 1975). Total hepaticcytochrome P-450 levels in rat liver returned to baseline levels within 5 daysafter discontinuation of PB injections (Ernster and Orrenius, 1965). The halflives of cytochromes P-450b and P-450e are approximately 37 h (Parkinson etal., 1983).Some isozymes of cytochrome P-450 in rats are modestly induced after PBtreatment while others are more dramatically affected (Waxman and Azaroff,1992). For example in adult rats, PB treatment induces a 2 to 4 fold increase incytochromes P-450a (Thomas et al., 1981) and P-450k (Waxman et at., 1985) andmodestly induces cytochrome P-450p (Heuman et at., 1982). Conversely, up to40 fold increases in cytochromes P-450e and P-450b have been reported (Thomaset at., 1981; Thomas et at., 1987). In addition to PB, phenothiazine, SKF-525A,Ardor 1254, isosafrole, trans-stilbene oxide (Thomas et at., 1981), acetone and30Introductionethanol (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 sequencehomology (Fujii-Kuriyama et at., 1982).Induction of cytochrome P-450 by PB in rat liver is due primarily to newlysynthesized cytochrome P-450b/e protein that results from increased steadystate levels of cytochrome P-450b/e mRNA (Phillips et at., 1981; Waxman andAzaroff, 1992). A 20 fold induction in mRNA was observed after PB induction inrats (Phillips et at., 1981). Stabilization of mRNA or proteins is not believed tobe involved in the mechanism of PB induction.Microheterogeneity of cytochromes P-450b and P-450e also exists in somestrains of rats (Vlasuk et at., 1982; Wilson et at., 1987; Oertle et at., 1991). Inmale Sprague-Dawley rats, PB induction resulted in the identification of 6members of the cytochrome P-450b/e family by monoclonal antibodies andpartial sequence analysis of tryptic peptides (Oertle et al., 1991). Three of the 6proteins belonged to the cytochrome P-450b family whilst the other 3 wereidentified as members of the cytochrome P-450e family.Cytochrome P-450b metabolizes a wide spectrum of lipophilic drugs inaddition to steroids including androgens and androstenedione (Waxman andAzaroff, 1992). Cytochrome P-450e has a similar substrate profile but is not asactive as cytochrome P-450b. The differences in activity between these 2isozymes may arise due to several factors. The cytochrome P-450e isozyme maybe more susceptible to denaturation during the purification process or requiresthe presence of as yet unknown specific phospholipids upon reconstitution(Christou et at., 1987). Alternatively, it is possible that activation of cytochromeP-450b occurs during the purification process from a membrane environment toa reconstituted system.31IntroductionCLINICAL IMPLICATIONSEnzyme induction is “the process which increases the rate of synthesis ofan enzyme relative to its normal rate of synthesis in the uninduced organism”(Gelboin and Wiebel, 1971). Induction of the liver enzymes results in anenhancement in metabolic rate and thus directly affects duration and intensityof drug actions in man and animals. Induction can alter the steady stateconcentrations of the parent compound and its metabolites in addition to theirelimination (Gillette, 1979) and possibly result in the formation of potentiallytoxic metabolites.Such is the case for VPA where induction of metabolism appears to play amajor role in the production of toxicity. In a retrospective study of fatalhepatotoxicity associated with VPA usage, young children on polytherapy weremore susceptible to development of toxicity than those on VPA monotherapy(Dreifuss et at., 1987).In young rats, PB pretreatment was necessary for VPA to demonstrateliver 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 withPB (Lewis et at., 1982). Glucuronidation and 0)- and 0)-i oxidation of VPA werereported to increase following PB induction (Watkins et at., 1982; Heinemeyer etat., 1985).The potential hepatotoxin, 4-ene ‘[PA, which was produced via thecytochrome P-450 oxidation of VPA, was observed only with microsomes fromphenobarbital treated rats (Rettie et at., 1987). At low concentrations of 4-eneVPA (10 ig/mL), significant alterations to the membrane permeability of guineapig hepatocytes were not observed unless phenytoin or phenobarbital was alsopresent (Yu et at., 1991).Clofibrate (CFB) pretreatment in rats resulted in enhanced 13-oxidation of32IntroductionVPA (Heinemeyer et al., 1985). Clofibrate is a known peroxisomal proliferator(Lazarow, 1987) and these observations suggested that peroxisomal 13-oxidationof VPA had occurred. There was also some microsomal induction since excretionof 4-OH VPA in rats was reported to increase following CFB treatment(Heinemeyer et al., 1985). In rats, 3-methyicholanthrene pretreatment alsoinduced 3-OH VPA formation.Increased production of 4-ene VPA by microsomal metabolism whichmight then be readily converted by 13-oxidation to a reactive metabolite couldhave serious consequences regarding the risk potential of the VPA and CBZdrug combination. CBZ is not likely to be a peroxisomal inducer because it doesnot contain a carboxyl group, like other known peroxisome proliferators(Lundgren et al., 1987). However, the major metabolite of CBZ is an epoxidewhich in turn is metabolized by epoxide hydrolase to CBZD (Tybring et al.,1981; Eichelbaum et al., 1985). Increased epoxide hydrolase activity withperoxisome proliferation has been reported (Oesch and Schladt, 1987; Moodyand Hammock, 1987). Since CBZ induces its own metabolism via induction ofthe enzymes of the epoxide-diol pathway (Eichelbaum et al., 1985), enhanced f3-oxidation may result.Our goal, then, is to detail the VPA and CBZ interaction based on thechanges in VPA metabolism resulting from CBZ induction. The rat will be usedas the model. Although CBZ is known to be an enzyme inducer, neither thetime course of induction nor the extent of induction has previously beendetermined. The effects of CBZE on microsomal enzymes will also beinvestigated. VPA and (E)-2-ene VPA metabolite profiles will be compared inanimals pretreated with CBZ, CBZE, PB and CFB. The hypothesis to be testedis that CBZ enhances VPA toxicity through enhanced production of toxic ‘[PAmetabolites.33SPECIFIC OBJECTiVES1. To determine the time course and the extent of induction of hepaticmicrosomal enzymes in the rat by CBZ at a given dose. The effectsproduced by CBZ will be compared to the commonly used inducing agentPB and to CFB, a known peroxisomal inducer.2. To determine the time course and the extent of induction of hepaticmicrosomal enzymes in the rat by CBZE at a given dose. These resultswill be compared to those obtained for CBZ, PB and CFB.3. To determine the contribution by CBZE to the overall induction producedby CBZ.4. To identify the isozyme(s) of cytochrome P-450 induced by CBZ and CZBEand determine if the same isozyme(s) are induced by PB.5. To identify the metabolites from the in vitro metabolism of VPA usingmicrosomal fractions from CBZ, CBZE, PB and CFB treated rats.6. To identify the products resulting from the in vitro metabolism of (E)-2-ene VPA using microsomal fractions from CBZ, CBZE, PB and CFBtreated rats.34EXPERIMENTALREAGENTS AWD MATERIALSVaiproic acid and metabolitesVaiproic acid (di-n-propylacetic acid) was obtained from K and K FineChemicals, ICN Biochemicals Inc. (Plainview, NY). The metabolites, (E)-2-eneVPA, 3-ene VPA, 4-ene VPA, 3-OH VPA, 4-OH VPA, 5-OH VPA, 3-keto VPA, 4-keto VPA, 2-propylgiutaric acid (2-PGA) and 2-propylsuccinic acid (2-PSA) usedfor the preparation of the calibration curves and for in vitro incubations weresynthesized 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 standardsThe deuterated internal standards,[2H7]E-2-ene VPA,[2H7]4-ene VPA,[2H7]3-OH VPA,[2H7]5-OH VPA,[2H713-keto VPA,[2H7]4-keto VPA and[2H7]VPA were synthesized in our laboratory (Zheng, M.Sc. thesis, 1993). 2-Methylgiutaric acid (2-MGA) was obtained from Aldrich Chemical Company.Carbamazepine and metabolitesCarbamazepine, carbamazepine-10,1 1-epoxide, carbamazepine- 10,1 1-dioland lO-methoxycarbamazepine for use as standards in the HPLC analyses weregenerously 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 fromSigma Chemical Company (St. Louis, MO, U.S.A.).35ExperimentalReagentsChemicals, solvents and reagents were obtained from the followingsources:BDH CHEMICALS (Vancouver, B.C., Canada).Acetonitrile OmniSolve® grade, ammonium acetate, calcium chloride,citric acid anhydrous, di-potassium hydrogen orthophosphate, di-sodiumhydrogen orthophosphate, dichloromethane OmniSolve® grade,ethylenediaminetetraacetic acid (EDTA), hydrochloric acid, magnesiumchloride, methanol OmniSolve® grade, potassium dihydrogen orthophosphate,potasssium chloride, sodium chloride, sodium dihydrogen orthophosphate,sodium hydroxide, sodium sulphate anhydrous, sulphuric acid andtrichioroacetic acid.BlO-RAD LABORATORIES (Richmond, California, U.S..A).Acrylamide 99.9%, ammonium persuiphate 98%, bis (IVN’-methylenebisacrylamide), 2-mercaptoethanol, SDS-PAGE 10 to lOOK molecular weightstandards, SDS-PAGE 40 to 250K molecular weight standards, sodium dodecylsulphate (SDS) and TEMED (N,N,N’,N’-tetramethyethylenediamine).BOEHRINGER MANNHEIM CANADA LTD. (Laval, Quebec, Canada).Bovine serum albumin, fraction V (BSA), bovine serum albumin, fattyacid free, fraction V, nicotinamide adenine dinucleotide, reduced (NADH) andnicotinamide adenine dinucleotide phosphate, reduced (NADPH).CALEDON (Georgetown, Ontario, Canada).Ethyl acetate distilled-in-glass grade.36ExperimentalFISHER SCIENTIFIC LTD. (Vancouver, B.C., Canada).Creatinine.INTER MEDICO (Markham, Ontario, Canada)Goat F(ab’)2 anti-rabbit IgG (G+L) horseradish peroxidase conjugatedIgG, 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 antibodiesThe 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), were37Experimentalprepared and generously provided by Dr. Stelvio Bandiera’s group. Theantibodies were raised in female New Zealand rabbits immunized with theelectrophoretically homogeneous proteins. IgG was purified from a pooi of heat-inactivated high-titer antisera obtained from multiple bleedings from severalrabbits using a combination of caprylic acid precipitation followed byammonium sulphate precipation and a final cleanup on a DEAE-Sephacelcolumn (Bandiera and Dworschak, 1992). Antibody concentration wasdetermined spectrophotometrically at 280 nm, E1 cm = 13 for a 1% solution.Each antibody was extensively immunoabsorbed in a manner analogous to thatdeveloped for anti-rat cytochromes P-450f and P-450g to generate monospecificantibody (anti-rat cytochromes P-450f, P-450g and P-450h). The anti-ratcytochrome P-450b antibody was polyspecific.The specificity of each antibody was assessed using Ouchterlony doublediffusion analysis, noncompetitive ELISA and immunoblots. The anti-ratcytochrome P-450f, P-450g and P-450h antibodies only reacted with the antigenof immunization and did not react with any other purified cytochrome P-450. Inthe case of the anti-rat cytochrome P-450b antibody, it reacted with cytochromeP-450e and also with a third, noninducible member of the cytochrome P-450 2Bfamily.METHODSInduction studiesAnimalsAdult male Long Evans rats (190 to 225 g, Charles River, Montreal) wereused for the experiments. After arrival, rats were allowed to recover for 3 to 5days prior to commencing the studies. Rats were fed standard rat chow (Purina38Experimental5001®) ad libitum and allowed drinking water ad libitum. The rats werehoused on corn cob bedding (Anderson’s®) in a room with controlled light (14 h)and dark (10 h) cycles.Treatment of solvents and compoundsNormal saline (NS), corn oil (CO) and propylene glycol (PG) were filteredvia either a 0.2 or 0.45 t filter (Gelman FP-Vericel) prior to use as vehicles forphenobarbital (PB), clofibrate (CFB), carbamazepine (CBZ) and carbamazepine10,11-epoxide (CBZE). Compounds were dissolved or suspended such that thevolume 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 theappropriate vehicle.Treatment of animals with carbamazepineAdult male Long Evans rats (4 per group) were treated i.p. with CBZsuspended 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 thelast dose of CBZ, each rat was placed in a separate metabolic cage and urinecollected for the 12 h period until sacrifice.Treatment of animals with carbamazepine-10,11 -epoxideRats (4 per group) were treated i.p. with CBZE suspended in PG at a doseof 50 mg/kg every 12 h for 3, 7, 10 and 14 days. Again, control rats received anequivalent volume of PG for each dose studied. After administration of the lastdose of CBZE, rats were placed in metabolic cages and urine collected for the 12h period until sacrifice.Treatment of animals with phenobarbitalRats were administered PB dissolved in normal saline (NS) 75 mg/kg i.p.39Experimentaldaily for 4 days. Vehicle control animals received an equivalent volume of NS.Treatment of animals with clofibrateAnimals were administered CFB diluted in corn oil (CO) at a dose of 350mg/kg i.p. daily for 7 days. Vehicle control animals received injections of CO.Treatment of animals with vaiproic acidVPA 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 metabolitesStock solutions of internal standards for GCMSTo decrease pipetting errors, the required internal standards were mixedsuch that the amounts required for each sample could be added simultaneously.A 200 tL aliquot contained the following amounts of each internal standard:[H71E-2-ene VPA 200 ng,[2H7]4-ene VPA 100 ng,[2H7]3-OHVPA 400 ng,[2H7]5-OH VPA 400 ng,[2H7]4-keto VPA 100 ng,[2H7]3-keto VPA 200 ng,[2H7]VPA 100 ng and 2-MGA 50 ng. Stock solutions were kept frozen at - 20 °Cuntil needed.Preparation ofstandard curves in phosphate bufferA bulk stock solution (hereafter referred to as standard 5) was preparedin 0.2 M phosphate buffer, pH 7.4. For the calibration curve 200, 400, 600, 800and 1,000 !IL of standard 5 were made up to a final volume of 1 mL withphosphate buffer. One mL of the phosphate buffer served as the blank for thecalibration curve. The stock solution was stored at - 20 °C until required.40ExperimentalStandard curve for VPA and metabolitesThe 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-ketoVPA, 2-PSA, 2-PGA, (E,E)-2,3’-diene VPA, (E)-2,4-diene VPA and (E)-2-ene VPA0, 20, 40, 60, 80 and 100 ng/mL; 5-OH VPA 0, 40, 80, 120, 160 and 200 ng/mL; 3-OH 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 metabolitesThe 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 4p,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 peakarea of metabolite or VPA to that of the respective internal standard versus theconcentration of VPA or the particular metabolite. The deuterated compoundsserved as the internal standards for their respective undeuterated counterparts.Deuterated (E)-2-ene VPA served as the internal standard for 3-ene VPA andthe diene metabolites. Deuterated VPA served as the internal standard for 4-OH VPA while 2-MGA was used as the internal standard for the 2 dicarboxylicacid metabolites. Standard curves were prepared and injected into the GCMSwith each batch of samples.Extraction of VPA and metabolites from standard samples and incubatesThe extraction procedure for VPA and metabolites is shown in figure 3.Two hundred iiL of internal standard mixture were added to each tubecontaining the sample for analysis or standard sample (1 mL). The pH of thestandard curve and incubates was adjusted to between 1.5 to 2.0 The final41ExperimentalMicrosomal Incubate or Standard SamplelmLInternal Standard Mixture (200 E.LL)2-MGA (50 ng)[H7]VPA (100 ng)21E-2-ene VPA (200 ng)[H7]4-ene VPA (100 ng)[2H713-OH VPA (400 ng)[2H7]5-OH VPA (400 ng)]3-keto VPA (200 ng)[2H7]4-keto VPA (100 ng)$Adjust pH to 1.5 - 2Adjust final volume to 3 mL with H201Extract with 3 mL ethyl acetate x 30 mm x 2Organic layerDry over anhydrous sodium sulphateConcentrate to 200 .tL4Derivatize with MTBSTFA 100 iLHeat for 1 h at 60 °CInject 1 jiL into GCMSFigure 3. Summary of the extraction procedure for valproic acid andmetabolites.42Experimentalvolume was adjusted to 3 mL with distilled water. Each sample was extractedtwice 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 overanhydrous sodium sulphate by vortexing for 1 mm, waiting 10 mm andcentrifuging for 10 mm at 3,000 rpm. The dried organic layer was transferred toanother test tube, concentrated to approximately 200 iL, transferred to a 1 mLconical vial and derivatized with 100 j.tL MTBSTFA for 60 mm at 60 °C. Thederivatized samples were transferred to autosampler vials and 1 tL injectedinto the GCMS.Carbamazepine and metabolitesPreparation of stock solutions for HPLCStock solutions of CBZ, CBZE and CBZD at a concentration of 1 mg/mL inmethanol were prepared. A stock solution of lO-methoxycarbamazepine (MCBZ,internal standard) also at a concentration of 1 mg/mL was prepared in methanoland 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 ofstandard curve for CBZ and metabolitesA bulk standard stock solution was prepared in rat urine from PG treatedrats. Standard curves were prepared from 2, 4, 6, 8, 12, 16 and 20 pgImL ofCBZ, CBZE and CBZD. Unspiked urine served as the blank for the standardcurve. Peak area ratios of CBZ, CBZE or CBZD to internal standard wereplotted versus concentration to prepare calibration curves.Extraction of CBZ, CBZE and CBZD from urine samplesCBZ and its 2 metabolites, CBZE and CBZD were extracted from urineusing a procedure based on literature methods of Elyas et al. (1982) and Kumps43Experimentalet 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 internalstandard (MCBZ) and 500 ilL of 300 mM phosphate buffer, pH 6.7. The samplewas extracted with 2.5 mL of ethyl acetate by gentle rotation for 10 mm andcentrifuged at 2,500 rpm for 10 mm. The top organic layer was transferred to asecond tube and evaporated under a gentle nitrogen stream in a water bath at40 °C, reconstituted with 200 jiL of acetonitrile, evaporated and reconstitutedagain with 200 tL of acetonitrile. The samples were transferred to autosamplervials and for each sample 20 jiL were injected into the liquid chromatograph.InstrumentationVaiproic acid and metabolitesThe analyses were performed on a Hewlett-Packard 5890 Series II gaschromatograph interfaced with a Hewlett-Packard 5971A Mass SelectiveDetector and equipped with a 7673 autosampler. A Hewlett-Packard Vectra®25T 486 computer, Hewlett-Packard Video Graphics Colour Display and aHewlett-Packard Laserjet Series II printer accompanied the MSD. Operatingconditions for tBDMS derivatives were source and injection port temperatures of240 °C and an interface temperature of 270 °C. Helium (carrier gas) flow was 1mL/min and the operating electron ionization energy for the mass spectrometerwas 70 eV. Source temperature was 180 °C. Injection mode was splitless andcolumn 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. Temperatureprogramming 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 run44ExperimentalUrine (400 j.tL)4 jig (100 jIL) MCBZ500 jiL 300 mM phosphate buffer, pH 6.7IExtract with 2.5 mL ethyl acetate x 10 mmCentrifuge at 2,500 rpm for 10 mmIOrganic layerEvaporate to dryness under N2• IReconstitute with 200 jiL acetonitrile x 2Evaporate to dryness under N2 x 2Reconstitute with 200 p.L acetonitrileInject 20 jiL into HPLCFigure 4. Summary scheme of the extraction of carbamazepine andmetabolites from urine of rats. -45Experimentaltime was approximately 29 mm.Selected ion monitoring mode was used for the analyses. The ionsscanned were mlz 100 (4-OH ‘[PA lactone), mlz 197 (dienes), mlz 199 (enes), mlz201 (‘[PA), mlz 206 ([27]E-2-ene VPA and [2H7]4-ene VPA), mlz 208([2H7]VPA), (mlz 213 (3-keto-4’-ene VPA, monoderivative), mlz 215 (3- and 4-keto VPA, monoderivative), rnlz 217 (3-OH VPA), mlz 222 ([H7]4-keto VPA),mlz 224([H7J3-OH VPA), mlz 317 (2-methyiglutaric acid), mlz 327 (3-keto-4’-ene VPA, diderivative), m/z 329 (3-keto VPA, diderivative), mlz 331 (5-OH ‘[PAand 2-PSA), mlz 336([2H7]3-keto ‘[PA), mlz 338([2H715-OH VPA) and mlz 345(2-PGA).Carbamazepine and metabolitesA Hewlett-Packard Series II 1090 Liquid Chromatograph equipped with aHP3396A integrator with a Beckman Ultrasphere® ODS column, particle size 5urn, 250 mm length, I.D. 4.6 mm was used for the analysis. Detectionwavelength 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 and85% A and 15% B at 30 mm. Stop time was 50 mm. Ten minutes were allowedbetween each run for a total run time of 60 mm.Preparation of subcellular fractions from rat liversInitially, assays were to be performed using mitochondrial, peroxisomaland microsomal fractions and thus differential centrifugation of rat liverhomogenates according to the method of Cook et al., (1986) was used. The liverswere removed, washed in 0.9% NaC1 and homogenized in 20 mL of 0.25 Msucrose/0.1% ethanoll5 mM Tris/1.15% KC1 solution. The homogenate wascentrifuged at 600 g for 10 mm (4,200 rpm, J-20 rotor), the supernatant filtered46Experimentalthrough 4 layers of cheesecloth and centrifuged at 7,500 g for 10 mm (8,000rpm, J-20 rotor) to obtain the mitochondrial fraction. The 7,500 g supernatantwas centrifuged at 17,000 g for 10 mm (12,000 rpm, J-20 rotor) to obtain theperoxisomal fraction. The 17,000 g supernatant was centrifuged at 100,000 gfor 60 mm (33,500 rpm, 50.2 Ti rotor) to obtain the microsomal fraction. Themitochondrial and peroxisomal fractions were each washed once with the abovebuffer and resuspended in either 0.25 M sucrose for the mitochondrial fractionor 0.25 M sucrose with 0.1% ethanol for the peroxisomal fraction. Themicrosomal pellet was washed once in buffer containing 1.15% KC1 and 10 mMEDTA buffer and then resuspended in an equivalent volume of 0.25 M sucrose.The samples were stored in cryovials at - 65 °C. Eventually, only themicrosomal fractions were used.Determination of protein content of various subcellular fractionsThe protein content of the various subcellular fractions was determinedaccording to the method of Lowry et al. (1951). The analyses were performed ona Hewlett-Packard 8452A diode array spectrophotometer in triplicate. BSA wasused as the protein standard.Determination of cytoclirome P-450 content in hepatic microsomesCytochrome P-450 content in the microsomal fraction from varioustreated groups was determined on a Hewlett-Packard 8452A diode arrayspectrophotometer using the method of Omura and Sato (1964). Microsomalprotein was diluted 1:25 in a buffer containing 0.1 M sodium phosphate, pH 7.4,20% glycerol and 0.1 M EDTA. Carbon monoxide was bubbled through onecuvette (sample) for 1 mm and a small amount of sodium dithionite was addedto each cuvette and mixed thoroughly. After one mm, the sample was scannedover the range of 325 to 625 nm. The amount of cytochrome P-450 in the sample47Experimentalwas calculated using the millimolar extinction coefficient of 91 cm’ mM1- forcytochrome P.450 (Omura and Sato, 1964). The analyses were performed induplicate.Gel electrophoresis of microsomal proteinSDS-polyacrylamide gel electrophoresis was performed according to themethod of Laemmli (1970) using a Hoeffer vertical slab gel unit. The separatinggel was 7.5% acrylamide-bis, 0.375 M Tris-HC1, 0.1% SDS, 0.042% ammoniumpersulphate and 0.03% TEMED. The stacking gel was 3% acrylamide-bis, 0.125M 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 2mm 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 forproteins to travel through stacking gel) and then the current was increased to25 mA per gel for approximately 2 h until the dye front reached the bottom ofthe 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 tobe quantitated, 5 pmol of spectrally determined microsomal cytochrome P-450were loaded per lane.ImmunoblotImmunoblotting was performed according to the method of Towbin et al.(1979) using a Hoeffer TE 52 Transphor® unit equipped with a power lid. Theproteins were resolved using SDS-PAGE and electrophoretically transferredonto a sheet of 0.20 ji nitrocellulose transfer membrane (BA-S 83, Schleicher &48ExperimentalSchuell, Keene, NH). The transfer was performed at 0.4 mA for 2 h in a coldcabinet using a precooled buffer containing 20% methanol, 0.02 M Tris, 0.154 Mglycine and 0.008% SDS at pH 8.3. After completion of the transfer, thenitrocellulose sheet was placed in an utility box containing 50 mL of blockingbuffer (1% BSA and 3% skim milk powder (Carnation®) in phosphate bufferedsaline (PBS)) and stored at 4 °C overnight or until development.For development of the blot, the blocking buffer was discarded and theprimary 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 hat 37 °C in a shaking water bath (Haake). The primary antibody was discardedand the nitrocellulose sheet washed 3 times for 10 mm with wash buffer (0.05%Tween in PBS). The secondary antibody, goat anti-rabbit peroxidase conjugatedantibody, was diluted (1:3,000 dilution) in antibody dilution buffer andincubated for 2 h at 37 °C in a shaking water bath. The nitrocellulose sheet waswashed 3 times at 10 mm each. The reaction was then visualized using asubstrate solution containing 3 mL of 0.018% 4-chloro-1-naphthol in methanol,30 jiL of 30% H20 and 47 mL of PBS. The reaction was allowed to proceeduntil visually satisfactory and terminated by submersing the nitrocellulosesheet in a tray of distilled water.Quantitation of cytochrome P-450b in microsomal protein from PB,CBZ and CBZE treated ratsA 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. Microsomalprotein equivalent to 5 pmol of spectrally determined cytochrome P-450 wasloaded per lane. The conditions for gel electrophoresis and immunoblottingwere as noted above. Rabbit anti-rat cytochrome P-450b antibody at aconcentration of 5 jig/mL was used to probe the blot. The staining intensity of49Experimentalthe bands from these experiments was quantitated using the VISAGE® 110 BioImage Analyzer (Bio Image, Ann Arbor, MI) using whole band analysis andoptical density.In vitro microsomal metabolism of VPA and (E)-2-ene VPAIn vitro metabolism of VPA and (E)-2-ene VPA was performed accordingto the method of Rettie et at. (1987). Five hundred jiL 0.2 M phosphate buffer,pH 7.4, 10 j.tL 300 mM MgC12, 2 nmol of spectrally determined cytochrome P450 (as microsomal protein), 10 .tL 0.01 M NADPH and 10 .tL 0.01 M NADHadjusted with water to a final volume of 1,000 jiL were combined in glass screwcapped test tubes and preincubated for 10 mm at 37 °C. The reaction wasinitiated with either 10 j.tL of 0.04 M VPA or 40 jiL of 0.01 M (E)-2-ene VPA andallowed to proceed for 40 mm at 37 °C. The reaction was terminated with 1 mLof 10% HC1 and the samples extracted as outlined above for GCMS analysis.In vitro microsomal metabolism of YPA and (E)-2-ene YPA in thepresence of anti-rat cytochrome P-450b or anti-rat cytochrome P-450hantibodyThese incubations were performed as above, except that increasingamounts of one of anti-rat cytochrome P-450b antibody, anti-rat cytochrome P450h antibody or control rabbit IgG was preincubated with the microsomalprotein (2 nmol of spectrally determined cytochrome P-450) for 10 mm prior toinitiation 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, 2and 2.5 mg IgG/nmol of cytochrome P-450.50ExperimentalIn vitro microsomal metabolism of VPA and (E)-2-ene VPA in thepresence of both anti-rat cytochrome P-450b and anti-rat cytochromeP-450h antibodies.These incubations were performed as above, except that the microsomalprotein was preincubated with both anti-rat cytochrome P-450b and anti-ratcytochrome P-450h antibodies for 10 mm prior to initiation of the reaction bythe addition of VPA or (E)-2-ene VPA. The antibodies were each used at aconcentration of 2 mg IgG/nmol of cytochrome P-450.Microsomal 0-dealkylation of ethoxyresorufin and pentoxyresorufinMicrosomal 0-dealkylation rates were determined according to themethod of Burke et al. (1985) using either pentoxyresorufin or ethoxyresorufinas the substrate. The assays were performed on a RF-540 Shimadzuspectrofluorometer equipped with a Shimadzu DR-3 Data Recorder. Excitationwavelength was 530 nm and emission wavelength was 582 nm with a slit widthof 5 nm. The reaction mixture contained 1.93 mL of 0.1 M Hepes/5 mM MgC12,pH 7.8, 10 tL of either 1 mM pentoxyresorufin or 1 mM ethoxyresorufin inDMSO and 50 iL of microsomal protein diluted in sucrose to 2 mg/mL. Thereaction was initiated with the addition of 10 iiL of 50 mM NADPH diluted inthe above mentioned buffer. The fluorescence reading was recorded everyminute for 10 mm. Enzyme activity was expressed as either nmol resorufinformedlminlmg protein or nmol resorufin formedlminlnmol cytochrome P-450.Statistical analysisStatistical analysis was performed using one way ANOVA (NewmanKeuls test). The level of statistical significance chosen was p 0.05.51RESULTSThe goals of this project were to identify the isozyme(s) of cytochrome P450 induced by carbamazepine (CBZ) and its major metabolite, carbamazepine10,11-epoxide (CBZE) and to determine the effect of these inducers on the invitro metabolism of vaiproic acid (VPA) and its major metabolite, (E)-2-ene VPA.These effects of CBZ and CBZE on cytochrome P-450 were then to be comparedto those of the classic inducer, phenobarbital (PB) and to clofibrate (CFB) whichhad been reported to induce VPA metabolism in rats (Heinemeyer et aL, 1985).QUANTITATIONAND IDENTIFICATION OF CYTOCHROMES P.450 INHEPATIC MICROSOMESQuantitation of total hepatic microsomal cytochrome P-450 contentPB (Remmer and Merker, 1963), CBZ (Wagner and Schmid, 1987;Regnaud et al., 1988) and CFB (Gibson, 1992) have been reported to inducecytochrome P-450. The effectiveness of these compounds to induce cytochromeP-450 was compared. Total cytochrome P-450 content in rat hepatic microsomesisolated from the various treatment groups was measured according to themethod of Omura and Sato (1964). The terms “untreated animals”, “uninducedanimals” and “control animals” are used interchangeably to describe animalswhich did not receive any compound or vehicle. A representative carbonmonoxide sodium dithionite-reduced cytochrome P-450 difference spectrum isshown in figure 5.A graphic representation of the total hepatic cytochrome P-450 content ofmicrosomes (mean ± s.d.) from untreated, PB, NS, CFB and CO treated rats isshown in figure 6 and is similarly detailed for microsomes from CBZ, CBZE andPG treated rats in figure 7. The changes in total hepatic cytochrome P-45052ResultsFigure 5. Carbon monoxide sodium dithionite-reduced difference spectrum ofhepatic microsomal cytochrome P-450.0.200000.140000.08000LJ02C0.02000—.01000400 S00 600U V EL E N G TH53Resultst2J0SCDtoiiiS0C)0C)0S3.02.52.01.51.00.50.0Figure 6.control PB NS CFB COCytochrome P-450 content (nmol of spectrally determinedcytochrome P-450/mg protein, mean ± s.d.) of microsomes fromcontrol, PB, NS, CFB and CO treated rats (n=4). a significantigreater than microsomes from untreated animals (p 0.05),significantly greater than microsomes from appropriate vehiclecontrol animals. Cytochrome P-450 was determined as outlined inthe Experimental section.54Results3.02.5ScD 2.01.5S0.c: 10C.)0C.)—0.50S0.0Figure 7. Cytochrome P-450 content (nmol of spectrally determinedcytochrome P-450/mg protein, mean ± s.d.) of microsomes fromCBZ, CBZE and PG 3, 7, 10 and 14 day treated rats (n=4). asignificantly greater than microsomes from untreate animals (0.6± 0.04 nmol cytochrome P-4501mg protein, p 0.05), significantlygreater than microsomes from appropriate vehicle control animalstreated over the same time period, C significantly greater thanmicrosomes from CBZE treated animals over the same time period.Cytochrome P-450 was determined as outlined in the Experimentalsection.CBZ CBZE PG55Resultscontent relative to the control group and to the respective vehicle groups for thePB, CFB, CBZ and CBZE treatment groups are summarized in table 2. Themean cytochrome P-450 content of microsomes prepared from PB (1.5 ± 0.2 nmolcytochrome P-450/mg protein, 2.3 fold increase), CFB (1.2 ± 0.2, 1.9 foldincrease), CBZ 3 day (1.3 ± 0.2, 2.1 fold increase), CBZ 7 day (1.5 ± 0.4, 2.3 foldincrease), CBZ 10 day (1.5 ± 0.3, 2.3 fold increase) and CBZ 14 day (1.2 ± 0.2, 1.8fold increase) treated rats were all significantly greater when compared to themean cytochrome P-450 content of microsomes from untreated animals (0.6 ±0.04 nmol cytochrome P-4501mg protein). No significant differences wereobserved when either the NS, CO, CBZE 3, 7, 10 and 14 day or PG 3, 7, 10 and14 day treated groups were compared to the control group.When PB treated animals were compared to the NS (vehicle) treatedgroup, the mean cytochrome P450 content was significantly higher (1.5 ± 0.2versus 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 ofmicrosomes 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, 7and 10 day treated rats was significantly greater than the PG (vehicle) treatedrats for the corresponding time period (1.5 to 1.8 fold increase, table 2). TheCBZ 14 day treated group was an exception where a significant difference wasnot observed for the amount of total cytochrome P-450 found when compared tothe corresponding PG vehicle treated group.Hepatic microsomes from the CBZE treatment groups did not display anysignificant increases in the amount of total cytochrome P-450 when compared totheir corresponding PG vehicle controls (figure 7, table 2).56ResultsTable 2. Summary of total hepatic cytochrome P.450 content (nmollmgprotein, mean ± s.d.) and change in cytochrome P-450 relative tothe untreated group or to the respective vehicle control group forthe PB, CFB, CBZ and CBZE treatment groups (n=4). CytochromeP-450 was determined as outlined in the Experimental section.Cytochrome P-450 (nmollmg protein)fold foldTreatment increase increaserelative to relative tonmollmg protein untreated vehiclemean ± s.d. group groupUntreated 0.6 ± 0.04PB 1.5 ± 02ab 2.3 1.9NS 0.8 ± 0.05 1.3CFB 1.2 ± 02a 1.9 1.2CO 1.0±0.2 1.7CBZ 3 day 1.3 ± 02ab 2.1 1.8CBZ 7 day 1.5 ± 0.4 2.3 1.7CBZ 10 day 1.5 ± 03abc 2.3 1.5CBZ 14 day 1.2 ± 02a 1.8 1.4CBZE 3 day 1.1 ± 0.2 1.7 1.5CBZE 7 day 0.9 ± 0.2 1.4 1.0CBZE1Oday 0.9±0.1 1.5 1.0CBZE 14 day 0.9 ± 0.1 1.4 1.1PG 3 day 0.7 ± 0.2 1.2PG7day 0.9±0.2 1.5PGlOday 1.0±0.2 1.7PG 14 day 0.8 ± 0.2 1.3(p 0.05)a significantly greater than microsomes from untreated animalsb significantly greater than microsomes from appropriate vehicle controlanimals treated over the same time periodc significantly greater than microsomes from CBZE treated animals over thesame time period57ResultsComparable amounts of hepatic microsomal cytochrome P-450 were foundfor all 4 CBZ treatment groups as well as for the PB and CFB treated rats, withno statistical differences observed. Increases ranged from 1.8 to 2.3 fold overthe untreated group (table 2).Cytochrome P-450 levels were found to be consistent over the time courseof treatment in both the CBZ and CBZE treated groups (table 2). However, thetotal microsomal cytochrome P-450 levels of the CBZ 7 and CBZ 10 day groupswere found to be higher than the corresponding CBZE treated group.Identification of the cytochrome P-450 isozymes induced by CBZ andCBZE using SDS-PAGE and Western blot techniquesPrevious literature has suggested that CBZ may induce the sameisozymes of cytochrome P-450 as PB (Wagner and Schmid, 1987). Based on thisinformation, studies were performed to determine if cytochrome P-450b (Ryan etat., 1982) (cytochrome P-4502B1, Nelson et at., 1993), an isozyme known to beinduced by PB, was also induced by either CBZ or CBZE. CFB served as acontrol since it is known to induce cytochrome P-452 (CYP4A1, lauric acidhydroxylase) (Leroux et at., 1989).The antibodies against various isozymes of cytochrome P-450 wereprepared by Dr. Bandier&s group. The polyspecific antibody against ratcytochrome P-450b also reacted with cytochrome P-450e, another isozymeinduced by PB.The nomenclature of Levin and co-workers will be used throughout thisthesis 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) isprovided in table 1 in the introduction.Representative SDS-PAGE gels of microsomal protein isolated from thelivers of rats treated with either CBZ or CBZE over the time course of treatment58Resultsare shown in figures 8 and 9. In both gels, lanes 2, 9 and 18 are the molecularweight standards while lane 3 is the purified cytochrome P-450b standard.Lane 5 contains microsomal protein from the PB treatment group while lanes10, 12, 14 and 16 contain microsomal protein from either the CBZ 3, 7, 10 and14 day treatment groups, respectively in figure 8 or from the CBZE 3, 7, 10 and14 day treatment groups, respectively in figure 9. Lanes 11, 13, 15 and 17contain microsomal protein from PG 3, 7, 10 and 14 day treated groups,respectively. After the separated proteins were electrophoretically transferredto a nitrocellulose membrane and probed with the antibody directed against ratcytochrome P-450b, microsomal protein from both CBZ and CBZE treated ratsreacted positively, indicating that cytochrome P-450b was present (figures 10and 11). Cytochrome P-450b could be detected in the microsomes after 3 days oftreatment with either CBZ or CBZE. A second band due to reaction of theantibody with cytochrome P-450e was also observed just above the cytochromeP-450b band. Neither cytochrome P-450b nor cytochrome P-450e was detectedin microsomes from untreated, NS, CO and PG treated animals. Surprisingly, apositive reaction was also observed with microsomal protein from the CFBtreated group suggestive of the presence of cytochromes P-450b and P-450e.Microsomal protein isolated from VPA treated rats did not react with theanti-rat cytochrome P-450b antibody, indicating that VPA does not influencethis particular isozyme of cytochrome P-450 (data not shown).Microsomal protein from none of the treatment groups reacted to anyappreciable degree with anti-rat cytochrome P-450f or anti-cytochrome P-450g(data not shown).59ResultsMwt(kDa)97.4-- —-—.—— — — —— . —66.2— — —-ian_aii42.7— -31 —21.5 . .--- — .•Lane 2 3 4 5 6 7 8 9 10 1112 131415161718Figure 8. SDS-PAGE gel of rat liver microsomal fractions from varioustreatment 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; lane10, CBZ 3 day; lane 11, PG 3 day; lane 12, CBZ 7 day; lane 13, PG7 day; lane 14, CBZ 10 day; lane 15, PG 10 day; lane 16, CBZ 14day; lane 17, PG 14 day and lane 18, molecular weight standards.—-—-- _____-ø_, —-- — -60ResultsMwt(kDa)97.4 -—--———66.2--‘ —, w42.7-31 —21.5—Lane 2 34 5 6 7 8 9 1011 12131415161718Figure 9. SDS-PAGE gel of rat liver microsomal fractions from varioustreatment 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; lane10, 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 weightstandards.61ResultsLane 3 4 5 6 7 8 9 10 11 12 13 14 15 16Figure 10. Immunoblot of rat liver microsomal proteins probed with anti-ratcytochrome 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, CBZ14 day.62Results3 4 5 6 7 8 9 10111213 1415 16Figure 11. Immunoblot of rat liver microsomal proteins probed with anti-ratcytochrome 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; lane13, PG 7 day; lane 14, CBZE 10 day; lane 15, PG 10 day; lane 16,CBZE 14 day.63ResultsIn vitro 0-dealkylation of pentoxyresorufin and ethoxyresorufincatalyzed by hepatic microsomal protein from the various treatmentgroupsTo the best of our knowledge, the isozyme(s) of cytochrome P-450 inducedby CBZ and CBZE have not previously been identified. Pentoxyresorufin andethoxyresorufin were used as substrates for the microsomal 0-dealkylationreactions to confirm the identification of cytochrome P-450b isozyme ofcytochrome P.450 induced by CBZ and CBZE. Pentoxyresorufin is a preferredsubstrate for cytochrome P-450b (PB inducible) while ethoxyresorufin is apreferred substrate for cytochrome P-450c (3-methylcholanthrene inducible)(Burke et al., 1985).The results from the 0-dealkylation of ethoxyresorufln andpentoxyresorufin by microsomes from untreated, PB, NS, CFB and CO treatedrats are shown in figure 12. In the untreated, CFB, or CO treated rats,ethoxyresorufin was not utilized as a substrate while 0-dealkylation ofethoxyresorufin occurred only to a minor degree in the PB and NS groups. Onthe other hand it was readily apparent that pentoxyresorufin was utilized as asubstrate to varying degrees by microsomes from all 5 treatment groups.The microsomal O-dealkylation of pentoxyresorufin and ethoxyresorufinby microsomes from CBZ, CBZE and PG treated animals over the time course oftreatment is illustrated in figures 13 and 14, respectively. Pentoxyresorufin,without question, was the preferred substrate by microsomes from all of thetreatment groups. Induction of ethoxyresorufin 0-dealkylation activity wasobserved 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 thevehicle control groups are summarized in table 3.64Resultsci)0bO0‘i2ci)-l0121086420Figure 12.control PB NS CFB COMicrosomal 0-dealkylation of pentoxyresorufin andethoxyresorufln (nmol resoruflnlminlmg protein, mean ± s.d.) bymicrosomes from control, PB, NS, CFB and CO treated rats (n=4).a siguiflntly greater than microsomes from untreated animals (p0.05), U significantly greater than microsomes from appropriatevehicle control animals treated over the same time period.Microsomal 0-dealkylation was determined as outlined in theExperimental section.65Results.,-100c14.z0U)a)0121086420Figure 13.CBZ CBZE PGMicrosomal 0-dealkylation of pentoxyresorufin (nmolresorufinlminlmg protein, mean ± s.d.) by microsomes from CBZCBZE and PG 3, 7, 10 and 14 day treated rats (n=4).significantly greater than microsomes from untreate4 animals (0.1± 0.01 nmol resorufinlminlmg protein, p 0.05), ‘ significantlygreater than microsomes from appropriate vehicle control animalstreated over the same time period, C significantly greater thanmicrosomes from CBZE treated animals over the same time period.Microsomal 0-dealkylation was determined as outlined in theExperimental section.66Results1.0substrate: ethoxyresorufin3 day0.8- 7day__lOdayiIIIIIII[ 14 day-‘• 04‘ ::___.______CBZ CBZE PGFigure 14. Microsomal 0-dealkylation of ethoxyresorufin (nmolresorufinlminlmg protein, mean ± s.d.) by microsomes from CBZ,CBZE and PG 3, 7, 10 and 14 day treated rats (nz4). Microsomal0-dealkylation was determined as outlined in the Experimentalsection.67ResultsTable 3. Summary of PROD (nmol resorufinlminlmg protein) and changesin PROD relative to the untreated group or to the respectivevehicle control group for the PB, CFB, CBZ and CBZE treatmentgroups (n=4). PROD was determined as outlined in theExperimental section.Pentoxyresorufin O-dealkylation Activity (PROD)fold foldTreatment increase increaserelative to relative tonmol resorufin formedlminlmg protein untreated vehiclemean ± s.d. group groupUntreated 0.1 ± 0.01PB 6.6 ±0•5ab 66 12NS 0.6±0.1 6CFB 0.4±0.1 4 3CO 0.1±0.1 1CBZ 3 day 8.9 ±1•3abc 89 34CBZ7 day ±i.2abc 98 38CBZlOday 8.0±i.4abc 80 40CBZ 14 day 69±i.5abc 69 53CBZE 3 day 3.9 ± 10ab 39 15CBZE7day 3.2±2.0 32 12CBZE 10 day 2.6 ± 05ab 25 13CBZE14day 5078.b 25 19PG3day 0.3±0.1 3PG7day 0.3±0.2 3PGlOday 0.2±0.1 2PGl4day 0.1±0.1 1(p 0.05)a significantly greater than microsomes from untreated animalsb significantly greater than microsomes from appropriate vehicle controlanimals treated over the same time periodC significantly greater than microsomes from CBZE treated animals over thesame time period68ResultsThere was no statistical difference in the 0-dealkylation ofpentoxyresorufin by microsomes from the CFB treated animals when comparedto the CO treated group, although the rate for the CFB induced microsomes was3 fold greater than that of the CO induced microsomes (figure 12).The rates of pentoxyresorufln O-dealkylation by microsomes preparedfrom PB, CBZ and CBZE treated rats were significantly greater when comparedto the microsomes from the untreated animals, with the increases ranging froma low of 25 fold for the CBZE 10 and 14 day groups to a high of 98 fold for theCBZ 7 day group (table 3).Similarly, the rates of pentoxyresorufin 0-dealkylation by microsomesfrom PB, CBZ or CBZE treated rats were significantly greater when comparedto the O-dealkylation rates of their respective vehicle control groups with theincreases ranging from 12 fold for the PB group to 53 fold for the CBZ 14 daygroup (table 3).When the inducing agents were compared, the rates of pentoxyresorufin0-dealkylation by microsomes from CBZ 3 and 7 day treated animals weresignificantly greater than the mean pentoxyresorufin 0-dealkylation rate formicrosomes from PB treated rats. The higher pentoxyresorufin 0-dealkylationrates compared to the PB treated group were only true for the CBZ 3 and 7 daytreated groups as the rates for the CBZ 10 and 14 day treated groups appearedto decline (table 3).Pentoxyresorufin O-dealkylation rates for microsomes from CBZE 3, 7, 10and 14 day treated rats were not statistically different from each other over thecourse of treatment. When CBZE pentoxyresorufin 0-dealkylation rates overthe time course were compared to those of the corresponding CBZ treated groupor to the PB treated group, CBZE rates were approximately 32 to 43% of theappropriate CBZ treated group and 38 to 58% of the PB treated group (table 4).69ResultsTable 4. Comparison of mean PROD activities of CBZE 3, 7, 10 and 14 daytreated groups as a percent of the PROD activities of the PB andCBZ 3, 7, 10 and 14 day treatment groups. PROD was determinedas outlined in the Experimental section.PROD (nmol resorufin formed/mm/mg protein)CBZE CBZE CBZE CBZE3 day 7 day 10 day 14 day(%) (%) (%) (%)PB 58 49 39 38CBZ3day 43 - - -CBZ7day - 33 - -CBZlOday - - 32 -CBZ 14 day - - - 3670ResultsThe 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 CBZ14 day treated animals was significantly lower than the rates for microsomesfrom CBZ 3, 7 and 10 day treated rats.Results similar to those discussed above for rates normalized to proteincontent were obtained when the rate of either pentoxyresorufin orethoxyresorufin 0-dealkylation was normalized to total cytochrome P-450 (datanot 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 weresignificantly increased when compared to the untreated group (17 to 42 fold) orto their respective vehicle control group (6 to 69 fold). The rates ofpentoxyresorufin 0-dealkylation of the CBZ treated groups were comparable tothe PB treated group with the exception of the CBZ 3 day treated group whichwas significantly higher (6.8 ± 0.4 versus 4.5 ± 0.4 nmol resorufinlmin/nmolcytochrome. P-450). CBZE pentoxyresorufin 0-dealkylation rates over the timecourse were approximately 61 to 80% of the PB treated group and 48 to 64% ofthe appropriate CBZ treated groups (data not shown).Quantitation of cytochrome P-450b in microsomes from CBZ, CBZE andPB treated rats by SDS-PAGE and Western blot techniquesWhile PB is known to induce cytochrome P-450b (Waxman and Azaroff,1992), induction of cytochrome P-450b by CBZ or CBZE has not been reportedpreviously. Since it had been verified in the previous sections that cytochromeP-450b was inducible by CBZ, this particular isozyme was quantitated inmicrosomes from CBZ 3, 7, 10 and 14 day, CBZE 3, 7, 10 and 14 day and PBtreated rats to determine if the inducing abilities of CBZ and CBZE werequantitatively similar to that of PB. Cytochrome P-450b content was71Resultsquantitated from Western blots based on the intensity of the bands using theVisage Bio-Image Analyser.The amount of cytochrome P-450b quantitated in microsomes from ratstreated with PB, CBZ and CBZE over the time course is shown in table 5 aspmol cytochrome P-450b/5 pmol total cytochrome P-450 (loaded per lane) and asa percentage of total hepatic cytochrome P-450. The percent of cytochrome P450b 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 treatedmicrosomes when compared to PB induced microsomes (table 5). Themicrosomes from the PB treated group contained 3.3 ± 0.1 pmol of cytochromeP-450b/5 pmol of spectrally determined cytochrome P-450, representing 65% oftotal hepatic cytochrome P-450 content. The microsomes from the CBZ 3 daytreated group contained 3.3 ± 0.8 pmol cytochrome P-450b15 pmol of spectrallydetermined cytochrome P-450 representing 66% of total hepatic cytochrome P450. Although mean quantities of the isozyme in the CBZE induced microsomes(mean 31 to 53%, over the time course) appeared lower than in microsomes fromboth the PB (65%) and CBZ induced microsomes (mean 39 to 66% over the timecourse), no statistical differences were observed between the 3 treatmentgroups.IN VITRO METABOLISM OF VPA AND (E)-2-ENE VPAEnzyme induction plays an important role in the formation of toxicmetabolites of VPA. Enzyme induction due to polytherapy is associated with ahigh incidence of VPA induced hepatotoxicity (Dreifuss et al., 1987). The effectsof PB and CBZ on the in vitro metabolism of VPA have been briefly investigatedwhilst the effects of CBZE induction have not been yet investigated. In thepresent work, the effects of induàtion by PB, CBZ, CBZE and CFB on the in72ResultsTable 5. Cytochrome P-450b (pmolI5 pmol of spectrally determinedcytochrome P-450 or as percent of total hepatic cytochrome P-450)in microsomes from rats treated with either PB, CBZ for 3, 7, 10 or14 days or CBZE for 3, 7, 10 or 14 days. Microsomal protein (5pmol of spectrally determined cytochrome P-45Oflane) wasseparated by SDS-PAGE and probed using an anti-rat cytochromeP-450b antibody as outlined in the Experimental section. (n=3,mean ± s.d.).Treatment cytochrome P-450b cytochrome P-450bpmoIJ5 pmol P-450 (% of total cytochrome P-450)PB 3.3±0.1 65±2CBZ 3 day 3.3 ± 0.8 66 ± 16CBZ7day 2.7±0.8 54±16CBZ 10 day 2.7 ± 0.5 55 ± 11CBZ 14 day 1.9 ± 0.4 39±8CBZE3day 1.8±0.8 35± 17CBZE 7 day 2.7 ± 1.4 53±29CBZE 10 day 1.6 ± 0.7 33 ± 14CBZE 14 day 1.6 ± 1.1 31 ±2373Resultsvitro metabolism of VPA were investigated.Analysis of VPA and metabolites by GCMSThe GCMS assay previously developed in our laboratory (Abbott et al.,1986) was used for the analysis of reaction products extracted from the in vitromicrosomal metabolism of VPA and (E)-2-ene VPA. It was possible to separateand detect 16 metabolites of VPA. The deuterated compounds,[2H7]E-2-eneVPA,[2H7]4-ene VPA,[2H7]3-OH VPA,[2H7]5-OH VPA,[2H7]3-keto VPA,[2H7]4-keto VPA and[2H7]VPA recently synthesized in our laboratory (Zheng,M.Sc. thesis, 1993) were used as internal standards.In vitro metabolism conditions for VPA and (E)-2-ene VPAThe in vitro metabolism of VPA and (E)-2-ene VPA by microsomes fromthe various treatment groups was investigated to determine the formation ofcytochrome P-450 mediated metabolites and the effect of induction on theformation of these metabolites. The method used in. this work was based on aprocedure from the literature (Rettie et al., 1987) and is outlined in theExperimental section. The amount of VPA used in the incubations was chosensuch that the VPA peak in the GCMS chromatogram did not overlap extensivelywith 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 atan equivalent molar dose to VPA to allow a direct comparison. Cytochrome P450 was varied over the concentration range of 1 to 6 nmol of spectrallydetermined cytochrome P-450 per incubation and it was determined that 2 nmolof spectrally determined microsomal cytochrome P-450 per in vitro incubationwere adequate to obtain quantifiable amounts of VPA or (E)-2-ene VPAmetabolites as measured by our GCMS method. Over this range of cytochromeP-450 investigated, a linear relationship was not observed between the product74Resultsand the amount of protein. The use of 2 nmol of spectrally determinedcytochrome P-450 per incubation avoided the use of excessive amounts ofmicrosomal protein since the amount of product recovered did not increase. Anoptimal incubation time was investigated in order to allow the reaction toproceed to completion. Incubation times of 20, 30, 40, 50 or 60 mm wereexamined. It was determined that the amount of metabolites formed did notchange considerably but to be on the safe side an incubation time of 40 mm wasselected.Not all 16 metabolites of VPA that could be quantitated in the GCMSassay were monitored. Firstly, not all of the metabolites are cytochrome P-450generated 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 were3-OH VPA, 4-OH VPA, 5-OH VPA, 4-ene VPA and 4-keto VPA. Only 2metabolites, (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 forincluded the formation of (E)-2-ene VPA from VPA and the formation of 3-eneVPA 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 factproducts of cytochrome P-450 mediated metabolism, the incubations wereperformed using boiled microsomes, or in the absence of cofactors, or in theabsence of substrate to eliminate artifacts. Under these conditions, none of theexpected metabolites of either VPA or (E)-2-ene VPA were detected (data notshown).75ResultsIn vitro metabolism of YPAFormation of 3-OH VPA from VPAThe amount of 3-OH VPA formed from VPA (0.4 jimol) by microsomesfrom control, PB, NS, CFB and CO treated animals is shown in figure 15 whilethat by microsomes from CBZ, CBZE and PG treated animals is illustrated infigure 16. Very small quantities of 3-OH VPA were detected for the PG andCBZE 14 day treated groups. The changes in the formation of 3-OH ‘[PA by thePB, CFB, CBZ and CBZE treated groups relative to the untreated group and totheir respective vehicle control group are summarized in table 6.The formation of 3-OH VPA from VPA by microsomes from PB treatedanimals (0.40 ± 0.09 jig) was significantly increased 7 fold when compared tountreated microsomes ( 0.05 ± 0.02 jig) while 3 to 4 fold increases were observedfor 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 PBtreated group when compared to the NS treated group (0.40 ± 0.09 jig versus0.04 ±0.01 jig) (figure 15, table 6). Increases of 4 to 20 fold were observed whenthe 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 PBtreated rats was significantly greater when compared to all other treatmentgroups.Formation of 4-OH VPA from VPAThe 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 bymicrosomes 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, CBZ76Results0.5ab0.40.2C’Dcontrol PB NS CFB COFigure 15. Formation of 3-OH VPA (gig, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from untreated, PB, NS, CFB and CO treatedrats (n=4). a significntly greater than microsomes from untreatedanimals (p 0.05), U significantly greater than microsomes fromappropriate vehicle control animals treated over the same timeperiod. Microsomal incubations were performed as outlined in theExperimental section.77ResultsFormation of 3-OH VPA (.tg, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 daytreated rats (n=4). a significantly greater tian microsomes fromuntreated animals (0.05 ± 0.02 j.tg, p 0.05), significantly greaterthan microsomes from appropriate vehicle control animals treatedover the same time period, C significantly greater than microsomesfrom CBZE treated animals over the same time period.Microsomal incubations were performed as outlined in theExperimental section.0.50.4C)0.3X 0.20.1.0.0Figure 16.CBZ CBZE PG78ResultsTable 6. A comparison of the metabolism of VPA to 3-OH VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats, relative tothe untreated group or to the respective vehicle control group(n=4). Microsomal incubations and quantitation of 3-OH VPA wereperformed as outlined in the Experimental section.Formation of 3-OH VPAfold foldTreatment increase increaseamount formed relative to relative tomean ± s.d. untreated vehicle(hg) group groupUntreated 0.05 ± 0.02PB 0.40 ± 0.09 7 9NS 0.04 ± 0.01 --CFB 0.10±0.01 2 2CO 0.04 ± 0.01 --CBZ 3 day 0.20 ± 0.02 4 20CBZ 7’day 0.10 ± 0.05 3 6CBZ 10 day 0.10 ± 0.04 3 6CBZ 14 day ***-- 4CBZE 3 day 0.10 ± 0.02 2 9CBZE 7 day 0.10 ± 0.05 2 4CBZE 10 day 0.10 ± 0.01 -- 2CBZE 14 day *** -- --PG3day *** --PG 7 day 0.03 ± 0.01 --PG 10 day 0.02 ±0.02 --PG 14 day---- increase less than 2 foldtrace quantities detected79ResultsbJC)042.01.51.00.50.0control PB NS CFB COFigure 17. Formation of 4-OH VPA (.tg, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from untreated, PB, NS, CFB and CO treatedrats (n=4). a signific.ntly greater than microsomes from untreatedanimals (p 0.05), KJ significantly greater than microsomes fromappropriate vehicle control animals treated over the same timeperiod. Microsomal incubations were performed as outlined in theExperimental section.80ResultsC-)>04Figure 18. Formation of 4-OH VPA (rig, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 daytreated rats (n=4). a significantly greater tan microsomes fromuntreated animals (0.09 ± 0.01 rig, p 0.05), significantly greaterthan microsomes from appropriate vehicle control animals treatedover the same time period, c significantly greater than microsomesfrom CBZE treated animals over the same time period.Microsomal incubations were performed as outlined in theExperimental section.2.01.51.00.50.0CBZ CBZE PG81Resultsand CBZE relative to the untreated group and to the appropriate vehicle controlgroups 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 wasincreased 15 fold (1.30 ± 0.08 jig) and was significantly greater than all othertreatment groups. Increases for the CBZ treated groups ranged from 7 to 12fold when compared to the untreated group (table 7).Of particular note was the comparison of amounts of 4-OH VPA producedby microsomes from the CBZ and CBZE treated groups when compared to theirappropriate PG control groups (figure 18). The increases ranged from a low of 9fold for the CBZE 10 and 14 day treatment groups to a high of 52 fold for theCBZ 10 day treatment group (table 7) because very low quantities of 4-OH VPAwere detected for the PG treated groups.The CBZ treated group over the time course yielded significantly higheramounts of 4-OH VPA when compared to the appropriate CBZE treated group(figure 18).Formation of 5-OH VPA from VPAThe amount of 5-OH VPA produced by microsomes from PB, NS, CFB,CO and untreated animals is depicted in figure 19 while that by microsomesfrom CBZ, CBZE and PG treated animals are shown in figure 20. A summary ofthe changes in the formation of 5-OH VPA by induced microsomes is given intable 8.Compared to the untreated group (16 ± 3 ng), the amount of 5-OH VPAproduced 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 forthe CFB, CBZ and CBZE 3 and 14 day treated groups when compared to theuntreated group (table 8).82ResultsTable 7. A comparison of the metabolism of VPA to 4-OH VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats, relative tothe untreated group or to the respective vehicle control group(n=4). Microsomal incubations and quantitation of 4-OH VPA wereperformed as outlined in the Experimental section.Formation of 4-OH VPAfold foldTreatment increase increaseamount formed relative to relative tomean ± s.d. untreated vehicle(jig) group groupUntreated 0.09 ± 0.01PB 1.30 ± 0.08 15 14NS 0.10±0.01--CFB 0.20 ± 0.04 2 2CO 0.10±0.01--CBZ 3 day 0.80 ± 0.10 10 40CBZ 7 day 0.60 ± 0.30 7 30CBZ 10 day 1.10 ± 0.50 12 52CBZ 14 day 0.60 ± 0.04 7 29CBZE 3 day 0.30 ± 0.10 3 12CBZE 7 day 0.20 ± 0.10 3 11CBZE 10 day 0.20 ± 0.10 2 9CBZE 14 day 0.20 ± 0.04 2 9PG3 day 0.02±0.02--PG7 day 0.01±0.00--PG 10 day 0.01 ± 0.00--PG 14 day 0.01 ± 0.00---- increase less than 2 fold83Results160140120100806040200Figure 19. Formation of 5-OH VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from untreated, PB, NS, CFB and CO treatedrats (n=4). a significgntly greater than microsomes from untreatedanimals (p 0.05), ‘ significantly greater than microsomes fromappropriate vehicle control animals treated over the same timeperiod. Microsomal incubations were performed as outlined in theExperimental section.control PB NS CFB CO84ResultsFormation of 5-OH VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P.450) from CBZ, CBZE and PG 3, 7, 10 and 14 daytreated rats (n=4). a significantly greqer than microsomes fromuntreated animals (16 ±3 ng, p 0.05), u significantly greater thanmicrosomes from appropriate vehicle control animals treated overthe same time period, c significantly greater than microsomes fromCBZE treated animals over the same time period. Microsomalincubations were performed as outlined in the Experimentalsection.160140120100>6040200Figure 20.CBZ CBZE PG85ResultsTable 8. A comparison of the metabolism of VPA to 5-OH VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats, relative tothe untreated group or to the respective vehicle control group(n=4). Microsomal incubations and quantitation of 5-OH VPA wereperformed as outlined in the Experimental section.Formation of 5-OH VPAfold foldTreatment increase increaseamount formed relative to relative tomean ± s.d. untreated vehicle(ng) group groupUntreated 16 ± 3PB 87±7 5 6NS 15±3--CFB 43±10 3 2CO 26±11--CBZ3day 126±8 8 3CBZ7day 43±18 3 4CBZlOday 44±13 3 5CBZl4day 86±6 5 2CBZE3day 87±13 5 2CBZE7day 27±9 2 3CBZE1Oday 21±6-- 2CBZE 14 day 45 ± 7 3--PG3day 38±10 2PG7day 10±2--POlOday 10±3--PGl4day 39±8 2-- increase less than 2 fold86ResultsThe formation of 5-OH VPA from VPA by microsomes from the PBtreated rats was significantly increased 6 fold when compared to the NS group(table 8). When the CBZ treated groups were compared to the appropiate PGtreated group over the time course, significant increases ranging from 2 to 5 foldwere observed. A significant difference was not observed between the CBZEtreated 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-OHVPA was significantly higher when the CBZ treated groups were compared tothe corresponding CBZE treated group. The amount of 5-OH VPA formed fromVPA by CBZ 3 day treated microsomes was significantly greater when comparedto PB microsomes and all other treatment groups (table 8).Formation of 4-ene VPA from VPAThe 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 summarizesthe increases in 4-ene VPA formation by the inducing agents when compared tothe untreated and the vehicle control groups.PB, CBZ and CBZE were capable of enhancing 4-ene VPA formationrelative 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 3day treatment also yielded a 2 fold increase in 4-ene formation. Very smallquantities of 4-ene VPA were detected for the CBZE 7, 10 and 14 day and thePG treatment groups.The metabolism of VPA to 4-ene VPA was induced 5 fold by PB treatmentwhen compared to the NS group (17 ± 2 ng versus 3 ± 0.6 ng) while theformation of 4-ene VPA by the CBZ treated groups was increased compared to87Results25201050Figure 21. Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from untreated, PB, NS, CFB and CO treatedrats (n=4). a signific.ntly greater than microsomes from untreatedanimals (p 0.05), U significantly greater than microsomes fromappropriate vehicle control animals treated over the same timeperiod. Microsomal incubations were performed as outlined in theExperimental section.control PB NS CFB CO88Results25 3day7day10 day20 14 day-abcabc abc15 abc1oabI50 -___-_____________________CBZ CBZE PGFigure 22. Formation of 4-ene VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 daytreated rats (n=4). a significantly greter than microsomes fromuntreated animals (3 ± 1 ng, p 0.05), u significantly greater thanmicrosomes from appropriate vehicle control animals treated overthe same time period, C significantly greater than microsomes fromCBZE treated animals over the same time period. Microsomalincubations were performed as outlined in the Experimentalsection.89ResultsTable 9. A comparison of the metabolism of VPA to 4ene VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats, relative tothe untreated group or to the respective vehicle control group(n=4). Microsomal incubations and quantitation of 4-ene VPA wereperformed as outlined in the Experimental section.Formation of 4-ene VPAfold foldTreatment increase increaseamount formed relative to relative tomean ± s.d. untreated vehicle(ng) group groupUntreated 3 ± 1PB 17±2 6 5NS 3±1 --CFB 5±1 2CO 4±1 --CBZ3day 13±2 5CBZ7day 13±3 4CBZlOday 13±2 4CBZl4day 12±2 4CBZE3day 7±1 2CBZE 7 day ***CBZE 10 day ***CBZE 14 dayPG3 day ***PG7 day ***PG 10 day ***PG 14 day ***-- increase less than 2 foldtrace quantities detected90Resultsthe PG treated groups (table 9). The production of 4-ene VPA by the CBZtreatment groups was significantly higher when compared to the CBZE and PGtreated groups over the time course. Only the formation of 4-ene VPA by theCBZE 3 day treated group was significantly greater when compared to the PG 3day treated group.Formation of4-keto VPA from VPAThe mean quantities of 4-keto VPA produced from VPA by microsomesfrom the various treatment groups are displayed in figures 23 and 24. Theeffects of induction by PB, CFB, CBZ and CBZE on the formation of 4-keto VPArelative to the untreated and vehicle control groups are summarized in table 10.The formation of 4-keto VPA was significantly enhanced by CBZ over thetime course. Very large increases relative to the untreated group wereobserved. However, this increase could not be determined because only tracequantities of 4-keto VPA were formed by microsomes from the untreated group.When compared to the vehicle controls, significant increases of 3 to 7 foldwere observed for the CBZ treatment groups, plus a 4 fold increase for theCBZE 3 day treatment group (table 10). Despite much higher mean quantitiesof 4-keto VPA for the PB treated group, it was not significantly greater whencompared to the NS treated group (figure 23). The formation of 4-keto VPA bythe CBZ 3, 7 and 10 day treated groups was significantly greater whencompared to the PB treated group and the corresponding CBZE treated group.In vitro metabolism of (E)-2-ene VPASince (E)-2-ene VPA is the major metabolite of VPA in the serum andpossesses anticonvulsant activity, it was important to study the effects ofvarious inducing agents on its in vitro metabolism. The PB induced in vitrometabolism of (E)-2-ene VPA has only recently been reported (Kassahun and91ResultsbiJ002520151050Figure 23.control PB NS CFB COFormation of 4-keto VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from untreated, PB, NS, CFB and CO treatedrats (n=4). a significantly greater than microsomes from untreatedanimals (p 0.05). Microsomal incubations were performed asoutlined in the Experimental section.92Results>06050403020100CBZ CBZE PGFigure 24. Formation of 4-keto VPA (ng, mean ± s.d.) from the in vitrometabolism of VPA by microsomes (2 nmol of spectrally determinedcytochrome P-450) from CBZ, CBZE and PG 3, 7, 10 and 14 daytreated rats (n=4). a significantly greater tian microsomes fromuntreated animals (0.1 ± 0.01 ng, p 0.05), significantly greaterthan microsomes from appropriate vehicle control animals treatedover the same time period, C significantly greater than microsomesfrom CBZE treated animals over the same time period.Microsomal incubations were performed as outlined in theExperimental section.93ResultsTable 10. A comparison of the metabolism of VPA to 4-keto VPA bymicrosomes from PB, CFB, CBZ and CBZE treated rats, relative tothe untreated group or to the respective vehicle control group(n=4). Microsomal incubations and quantitation of 4-keto VPAwere performed as outlined in the Experimental section.Formation of 4-keto VPAfold foldTreatment increase increaseamount formed relative to relative tomean ± s.d. untreated vehicle(ng) group groupUntreatedPB 16±4 * 4NS 4±2 *CFB 7±5 * 2CO 4±1 *CBZ3day 37±14 * 7CBZ7day 33±8 * 7CBZlOday 44±7 * 3CBZl4day 25±15 * 4CBZE3day 20±12 * 4CBZE7day 1±3 * 2CBZE1Oday 18±5 * --CBZE14day 8±2 * --PG3day 5±1 *PG7day *** *PGlOday 18±7 *PGl4day 6±3 *- increase less than 2 foldtrace quantities detected* increase> 20 fold94ResultsBaillie, 1993). If(E)-2-ene VPA is to be utilized as an anticonvulsant agent, it islikely to be used in combination with CBZ, thus necessitating investigation intothe effects of induction on its metabolism. (E)-2-ene VPA is metabolized toseveral diunsaturated metabolites, one ((E,E)-2,3’-diene VPA) of whichpossesses anticonvulsant activity (Abbott and Acheampong, 1988) whileanother, ((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 VPAThe production of (E,E)-2,3’-diene VPA from (E)-2-ene VPA bymicrosomes from control, PB, NS, CFB and CO treated rats is shown in figure25 while that by microsomes from CBZ, CBZE and PG treated rats is illustratedin figure 26. The changes in the formation of (E,E)-2,3’-diene VPA relative tothe untreated and vehicle groups are summarized in table 11.The amount of (E,E)-2,3’-diene VPA formed from (E)-2-ene VPAsignificantly increased 9 fold and 2 fold, respectively for the PB and CFB treatedgroups when compared to the untreated group (table 11). Significant increasesof 2 to 8 fold were observed for the CBZ treated groups when compared to theuntreated group (table 11). The formation of (E,E)-2,3’-diene VPA bymicrosomes from rats treated for 3, 7 and 10 days with CBZE increasedsignificantly 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 microsomesfrom 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-eneVPA by microsomes from CBZ treated animals over the time course weresignificantly greater when compared to microsomes from PG treated animalsover 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 treated95Results.LJ>ci)ci-4302520151050Figure 25. Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from the invitro metabolism of (E)-2-ene VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from untreated, PB, NS,CFB and Co treated rats (n=4). a significant1y greater thanmicrosomes from untreated animals (p 0.05), U significantlygreater than microsomes from appropriate vehicle control animalstreated over the same time period. Microsomal incubations wereperformed as outlined in the Experimental section.control PB NS CFB Co96ResultsbOG)cjjC\2302520151050CBZ CBZE PGFigure 26. Formation of (E,E)-2,3’-diene VPA (ng, mean ± s.d.) from the invitro metabolism of (E)-2-ene VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from CBZ, CBZE and PG3, 7, 10 and 14 day treated rats (n=4). a significantly greater thamicrosomes from untreated animals (2 ± 1 ng, p 0.05),significantly greater than microsomes from appropriate vehiclecontrol animals treated over the same time period, significantlygreater than microsomes from CBZE treated animals over thesame time period. Microsomal incubations were performed asoutlined in the Experimental section.97ResultsTable 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 treatedrats, relative to the untreated group or to the respective vehiclecontrol group (n=4). Microsomal incubations and quantitation of(E,E)-2,3’-diene VPA were performed as outlined in theExperimental section.Formation of (E,E)-2,3’-diene VPAfold foldTreatment increase increaseamount formed relative to relative tomean ± s.d. untreated vehicle(ng) group groupUntreated 2 ± 1PB 18±4 9 7NS 3±1 --CFB 4±1 2 2CO 2±1 --CBZ3day 17±2 8 3CBZ7day 14±2 7 4CBZlOday 11±3 5 5CBZl4day 4±1 2 9CBZE3day 10±1 5 2CBZE7day 9±1 5 3CBZE1Oday 6±1 2 2CBZE14day 2±1 -- 3PG3day 6±1 3PG7day 5±1 3PG 10 day ***PG 14 day ***-- increase less than 2 fold>‘ trace quantities detected98Resultsgroups significantly increased 2 to 3 fold when compared to the PG treatedgroups (table 11, figure 26).In addition, (E,E)-2,3’-diene VPA values for the CBZ 3, 7, 10 and 14 daytreated groups were significantly higher than the CBZE treated group over thesame time period (figure 26). The formation of (E,E)-2,3’-diene VPA from (E)-2-ene VPA by PB (18 ± 4 ng) was significantly greater when compared to all othergroups, 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 VPAThe 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 infigures 27 and 28. The changes in the formation of (E)-2,4-diene VPA bymicrosomes from the PB, CFB, CBZ and CBZE treated groups relative to theuntreated and vehicle control groups are summarized in table 12.When compared to microsomes isolated from untreated control rats, theproduction of (E)-2,4-diene VPA was significantly increased 13 fold for the PBtreated group, 6 fold for the CBZ 3 day treated group, 13 fold for the CBZ 7 daytreated group, 11 fold for the CBZ 10 day treated group, 5 fold by the CBZ 14day treated group and 9 fold for the CBZE 7 day treated group (table 12). Nosignificant differences were observed in the formation of (E)-2,4-diene VPAwhen the other treatment groups were compared to the control group.The formation of (E).-2,4-diene VPA by microsomes from PB treatedanimals 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 wassignificantly increased for the CBZ treated groups when compared to the PGtreated groups over the same time course with increases of 6 to 12 fold (figure28, table 12). Of the CBZE treated groups, only the CBZE 7 day treated groupwas significantly enhanced when compared to the PG 7 day treated group (4 fold99Results0a)a).-44.03.53.02.52.01.51.00.50.0control PB NS CFB COFigure 27. Formation of (E)-2,4-diene VPA (tg, mean ± s.d.) from the in vitrometabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrallydetermined cytochrome P450) from untreated, PB, NS, CFB andCO treated rats a significanty greater than microsomesfrom untreated animals (p 0.05), significantly greater thanmicrosomes from appropriate vehicle control animals treated overthe same time period. Microsomal incubations were performed asoutlined in the Experimental section.100Resultsr I 3day7 dayabc10 daygi abc l4day2.5ab2.04 1.5 abc] abc—‘ 1.0::_______________CBZ CBZE PGFigure 28. Formation of (E)-2,4-diene VPA (jig, mean ± s.d.) from the in vitrometabolism of (E)-2-ene VPA by microsomes (2 nmol of spectrallydetermined cytochrome P-450) from CBZ, CBZE and PG 3, 7, 10and 14 day treated rats (n=4). a significantly greater thamicrosomes from untreated animals (0.20 ± 0.02 jig, p 0.05),significantly greater than microsomes from appropriate vehiclecontrol animals treated over the same time period, significantlygreater than microsomes from CBZE treated animals over thesame time period. Microsomal incubations were performed asoutlined in the Experimental section.101ResultsTable 12. A comparison of the metabolism of (E)-2-ene VPA to (E)-2,4-dieneby microsomes from PB, CFB, CBZ and CBZE treated rats, relativeto the untreated group or to the respective vehicle control group(n=4). Microsomal incubations and quantitation of (E)-2,4-dieneVPA were performed as outlined in the Experimental section.Formation of(E)-2,4-diene VPAfold foldTreatment increase increaseamount formed relative to relative tomean ±s.d. untreated vehicle(‘g) group groupUntreated 0.20 ± 0.02PB 2.5 ± 0.60 13 12NS 0.20±0.10 --CFB 0.40 ± 0.10 -- --CO 0.20 ± 0.10 --CBZ 3 day 1.2 ± 0.2 6 12CBZ 7 day 2.5 ± 0.6 13 6CBZlOday 2.1±1.1 11 6CBZ 14 day 1.0±0.2 5 6CBZE3day 0.5 ±0.2 2 5CBZE7day 1.6±0.6 9 4CBZE1Oday 0.4±0.04 2 3CBZE14day 0.3±1.0 2 2PG3day 0.1±0.04 --PG 7 day 0.5 ± 0.02 2PG 10 day 0.1 ± 0.10 --PG 14 day 0.2± 0.05 ---- increase less than 2 fold102Resultsincrease). 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 7day and CBZ 10 day treated rats. The amount of (E)-2,4-diene VPA formed bythe CBZ treated groups were significantly higher than the corresponding CBZEtreated groups.Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA and (E)-2-ene VPA by inicrosomes from PB and CBZtreated ratsCytochrome P-450b has been implicated in the formation of 4-ene \TPAfrom VPA (Rettie et al., 1988). Since cytochrome P-450b had been identified asan isozyme capable of being induced by CBZ and PB, the effect of anti-ratcytochrome P-450b antibody on the in vitro metabolism of VPA and (E)-2-eneVPA was investigated. Thus, the extent of involvement of cytochrome P-450b inthe microsomal metabolism of VPA and (E)-2-ene VPA could be determined.Microsomal protein was incubated with either the anti-rat cytochrome P-450bantibody or control IgO for 10 mm prior to initiation of the reaction by additionof 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 VPAThe effects of anti-rat cytochrome P-450b on the in vitro metabolism ofVPA to 3-OH VPA, 4-OH VPA, 5-OH VPA, 4-ene VPA and 4-keto VPA bymicrosomes from rats treated with either CBZ for 3 days or with PB isillustrated in figures 29 to 33.The metabolism of \TPA to 3-OH VPA by microsomes prepared from PBand CBZ 3 day treated rats was inhibited 63% and 52%, respectively at thehighest antibody concentration of 2.5 mg of IgG/nmol of cytochrome P-450(figure 29).103Results10090W807000,600g5040>30c201000.0 1.0 2.0 3.0mg IgG/nmol cyt. P—450Figure 29. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 3-OH VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3 daytreated rats. Microsomes were prepared from 4 pooled livers.Microsomal incubations were performed as outlined in theExperimental section.o PB• CBZ3day0.5I I1.5 2.5104ResultsThe formation of 4-OH VPA from VPA by microsomes prepared from PBand CBZ treated rats microsomes was inhibited 81% and 88%, respectively atthe highest IgG concentration investigated (figure 30).The biotransformation of VPA to 5-OH VPA using microsomes from PBand CBZ 3 day induced rats was inhibited 60% and 45%, respectively at thehighest antibody concentration investigated (figure 31).The metabolism of VPA to 4-ene VPA by microsomes from PB and CBZ 3day treated rats was completely inhibited in each case at an antibodyconcentration 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 CBZ3 day induced rats was blocked 86% and 75%, respectively in the presence of theantibody at the highest concentration examined (figure 33).Effect ofanti-rat cytochrome P-450b antibody on the in vitro metabolism of(E)-2-ene VPAThe 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 fromrats 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 fromCBZ treated rats was inhibited approximately 18% at the highest antibodyconcentration (2.5 mg of IgG/nmol of cytochrome P-450) examined while it wasinhibited 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 microsomesfrom PB and CBZ treated rats, respectively at the highest antibodyconcentration investigated (figure 35).105Results0-4-)0C-)0-c10Figure 30. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 4-OH VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3 daytreated rats. Microsomes were prepared from 4 pooled livers.Microsomal incubations were performed as outlined in theExperimental section.a PB• CBZ3day10090807060504030201000.0 0.5 1.0 1 .5 2.0 2.5mg IgG/nmol cyt. P—4503.0106Results0s-I0C)0cFigure 31. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 5-OH VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3 daytreated rats. Microsomes were prepared from 4 pooled livers.Microsomal incubations were performed as outlined in theExperimental section.0 PB• CBZ3day1 2011010090807060504030201000.0I I0.5 1.0I I1 .5 2.0 2.5mg IgG/nmol cyt. P—4503.0107Results1 20110100090o 80C)t4j 7006050>. 40101.0 3.0mg lgG/nmol cyt. P—450Figure 32. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 4-ene VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3 daytreated rats. Microsomes were prepared from 4 pooled livers.Microsomal incubations were performed as outlined in theExperimental section.0 PB. CBZ3dayI I1.50.5 2.0 2.5I I108Results1 2011004) 900C)o 70— 60124 50o 40U.) 30‘ 201000.0 1.0 2.0mg IgG/nmol cyt. P—450Figure 33. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of VPA to 4-keto VPA by microsomes (2 nmol ofspectrally determined cytochrome P-450) from PB and CBZ 3 daytreated rats. Microsomes were prepared from 4 pooled livers.Microsomal incubations were performed as outlined in theExperimental section.0 PBCBZ3dayI I I0.5 1 .5 2.5 3.0109Results00C)0G)a)C)c’•Figure 34. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of (E)-2-ene VPA to (E,E)-2,3’-diene VPA bymicrosomes (2 nmol of spectrally determined cytochrome P-450)from PB and CBZ 3 day treated rats. Microsomes were preparedfrom 4 pooled livers. Microsomal incubations were performed asoutlined in the Experimental section.1201101009080706050403020100—0.50 PB0.0I I I• CBZ3day0.5 1.0 1.5 2.0 2.5mg IgG/nmol cyt. P—4503.0110Results— 1100- 1000 90I.o 80‘— 7060504020Figure 35. Effect of anti-rat cytochrome P-450b antibody on the in vitrometabolism of (E)-2-ene VPA to (E)-2,4-diene VPA by microsomes(2 nmol of spectrally determined cytochrome P-450) from PB andCBZ 3 day treated rats. Microsomes were prepared from 4 pooledlivers. Microsomal incubations were performed as outlined in theExperimental section.1 20300—0.5 0.0 0.5 1.0 1.5 2.0 2.5 3.0mg IgG/nmol cyt. P—450111ResultsEffect of anti-rat cytochrome P-450h on the in vitro metabolism of VPAand (E)-2-ene VPA by untreated microsomesCytochrome P-450h, a male specific isozyme, catalyzes a number ofmetabolic reactions including progesterone and testosterone 2c- and 16a-hydroxylation (Ryan and Levin, 1990). The effect of an antibody specific forcytochrome P-450h was investigated to determine if this isozyme was alsoinvolved in the metabolism of VPA or (E)-2-ene VPA, particularily to themetabolites whose formation was not completely inhibited in the presence of theanti-rat cytochrome P-450b antibody.Anti-rat cytochrome P-450h antibody did not exhibit any significanteffects on the metabolism of VPA by control microsomes (data not shown). Inorder to examine the possibility of an additive effect on VPA metabolism, theanti-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 ofIgG/nmol of cytochrome P-450) to microsomes prepared from CBZ 3 day or PBtreated rats. No differences were observed in the inhibition profiles of 3-OHVPA, 4-OH VPA and 5-OH VPA (data not shown). The results obtained weresimilar 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 theantibody against rat cytochrome P-450h on the in vitro metabolism of (E)-2-eneVPA to (E,E)-2,3’-diene VPA and (E)-2,4-diene VPA by microsomes preparedfrom untreated animals was investigated. As in the case of VPA, the antibodydid 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 (2mg/nmol cytochrome P-450) and anti-rat cytochrome P-450b antibody (2112Resultsmg/nmol cytochrome P-450) were examined in either CBZ 3 day or PBmicrosomes, no significant inhibitory actions were observed.QUANTITATION OF CBZ, CBZE AND CBZDCBZ is known to induce its own metabolism via the epoxide-diol pathway(Eichelbaum et aL, 1985). The metabolites of CBZ and CBZE were quantitatedin 12 h rat urine collections over the 14 day time course to confirm if thispathway was induced with chronic administration.Analysis of CBZ and metabolites in rat urine by HPLCThe methods of Elyas et al. (1982) and Kumps et al. (1985) were combinedto provide the methodology for CBZ and metabolite analysis by HPLC. Ethylacetate was found to be a more efficient extraction solvent thandichloromethane for CBZD. The isocratic method of Elyas et al. (1982) wasfound to be incapable of adequately separating CBZD from the endogenouscompounds present in rat urine so a gradient method was developed.Representative chromatograms of CBZ and metabolite standards, withcomparison to CBZ and metabolites extracted from a rat urine sample and aspiked urine sample are shown in figure 36. The assay was linear over theconcentration range examined.Urinary recoveries of CBZ, CBZE and CBZD after dosing with CBZThe 12 h urinary recoveries of CBZ and metabolites after dosing rats withCBZ at 100 mg/kg are shown in table 13. Very small quantites of CBZ wererecovered in all 12 h collections, ranging from mean quantities of 17.3 jig for theCBZ 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 magnitudehigher than the CBZ levels.113Resultsd3 42L/1Figure 36. HPLC chromatograms of a) standards of CBZ, CBZE, CBZD andMCBZ, b) extracted blank rat urine sample, c) extracted spiked raturine sample and d) extracted rat urine sample. Peak 1, CBZD,peak 2, CBZE, peak 3, CBZ and peak 4, MCBZ. Chromatographyconditions were as described in the Experimental section.a b21a,43C4114ResultsTable 13. Total 12 h urinary recoveries of CBZ, CBZE and CBZD (jig) fromrats treated with CBZ 100 mg/kg every 12 h. (n=4, mean ± s.d.).CBZ, CBZE and CBZD were quantitated as described in theExperimental section.Treatment CBZ CBZE CBZD(jig) (jig) (jig)CBZ3 day 17.3± 15.5 317±256 602 ±461CBZ 7 day 44.4 ± 25.3 270 ± 138 337 ± 152CBZ 10 day 35.9 ± 19.7 267 ± 94 551 ± 279CBZ 14 day 38.4 ± 8.3 339 ± 195 365 ± 110115ResultsThe recoveries of CBZ, CBZE and CBZD expressed as a percent of thedose of CBZ administered are shown in table 14. Only 0.1 to 0.2% of the doseadministered was recovered as CBZ. The recoveries of CBZE and CBZDrepresented 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 hfollowing administration of the last dose.No significant differences were observed in the urinary recoveries overthe 14 day treatment period investigated.Urinary recoveries of CBZE and CBZD after dosing with CBZEThe 12 h urinary recoveries of CBZE and CBZD after administration ofCI3ZE at a dose of 50 mg/kg are summarized in table 15. The mean 12 hrecovery of CBZE ranged from 121 to 504 jig while the mean recovery of CBZDranged from 177 to 523 jig over the same time period.The recoveries of CBZE and CBZD expressed as a percent of the dose ofCBZE administered are shown in table 16. The mean recovery of CBZE as apercent of the dose administered ranged from 0.4 to 2.0% while the recovery ofCBZD ranged from 0.6 to 2.0% over the same time period. The total doserecovered as CBZE and CBZD ranged from 1.5 to 4.0% of the dose administered.As with the CBZ treatment groups, no significant differences wereobserved over the 2 week period of CBZE administration.116ResultsTable 14. Urinary recoveries (12 h) of CBZ, CBZE and CBZD as percent ofdose administered from rats treated with CBZ 100 mg/kg every 12h. (n=4, mean ± s.d.). CBZ, CBZE and CBZD were quantitated asdescribed in the Experimental section.Treatment CBZ CBZE CBZD total(% of dose) (% of dose) (% of dose) (% of dose)CBZ 3 day 0.1 ± 0.1 1.6 ± 1.3 3.0 ± 2.4 4.6 ± 3.6CBZ7day 0.2±0.1 1.2±0.6 1.5±0.7 2.9±1.1CBZ 10 day 0.2 ± 0.1 1.1 ± 0.4 2.3 ± 1.1 3.5 ± 1.5CBZ 14 day 0.2 ± 0.1 1.4 ± 0.8 1.5 ± 0.5 3.0 ± 1.3117ResultsTable 15. Total 12 h urinary recoveries of CBZE and CBZD (jig) from ratstreated with CBZE 50 mg/kg every 12 h. (n=4, mean ± s.d.). CBZEand CBZD were quantitated as described in the Experimentalsection.Treatment CBZE CBZD(jig) (jig)CBZE3day 247 ±83 447 ±494CBZE7day 504± 174 523± 139CBZE1Oday 121±83 319±256CBZE14day 249±134 177±83118ResultsTable 16. Urinary recoveries (12 h) of CBZE and CBZD as percent of dosefrom rats treated with CBZE 50 mg/kg every 12 h. (n=4, mean ±s.d.). CBZE and CBZD were quantitated as described in theExperimental section.Treatment CBZE CBZD total(% of dose) (% of dose) (% of dose)CBZE 3 day 1.1 ± 0.4 2.0 ± 2.2 3.1 ± 2.6CBZE 7 day 2.0 ± 0.6 2.0 ± 0.5 4.0 ± 1.1CBZE1Oday 0.4±0.3 1.1±0.9 1.6±1.1CBZE 14 day 0.9 ± 0.5 0.6 ± 0.3 1.5 ± 0.5119DISCUSSIONCHOICE OF EXPERIMENTAL CONDITIONSAnimal modelPrevious work suggested the rat should provide a reasonably good modelto study the interaction between VPA and CBZ since the metabolism of bothCBZ (Faigle and Feldmann, 1982) and VPA (Granneman et al., 1984a) in the ratare quite similar to that of man. CBZ undergoes biotransformation in rat andhuman by the same major metabolic pathways: epoxidation of the 10,11 doublebond, hydroxylation of the 6-membered aromatic rings, N-glucuronidation of thecarbamoyl side chain and substitution on the 6 membered rings by sulphurcontaining groups (Faigle and Feldmann, 1982; Faigle and Feldmann, 1989). Inboth rat and man, the 2 major metabolic pathways of VPA metabolism areglucuronidation and 3-oxidation (Granneman et al., 1984a).The male Long Evans rats used in this study weighed from 195 to 230 gand were approximately 7 to 9 weeks of age and, thus, are considered to beadults. In male, untreated Wistar rats, total hepatic cytochrome P-450 contentremained unchanged from 1 week to 24 months of age and low levels ofcytochromes P-450b and P-450e were detectable (Imaoka et aL, 1991).Choice of vehicle for CBZ and CBZECBZ 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, isreadily soluble in organic solvents (Kerr and Levy, 1989). Some organic solventssuch as acetone and ethanol are known to induce cytochrome P-450 in rats120Discussion(Soucek and Gut, 1992). Thus, propylene glycol was chosen as the suspendingvehicle for CBZ and CBZE because in humans it did not produce changes in theantipyrine test at an oral dose of 55 mL (Nelson et al., 1987). Although therehas 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 twicedaily) were much lower than in the 1989 study (8 mLfkg 3 times daily viagastric intubation).GCMS analysis of VPA and metabolitesA modified version of the gas chromatograph-mass spectrometric assaypreviously developed in our laboratory was employed for the analysis of VPAand its metabolites in the microsomal incubates (Abbott et al., 1986). Thesamples were analyzed on a gas chromatograph equipped with a mass selectivedetector. With this assay, it was possible to simultaneously quantitate VPA and16 metabolites using the selected ion monitoring mode. Briefly, the methodemployed a capillary column and the extracted samples were derivatized withMTBSTFA to yield the tBDMS derivatives. The [M-57] ion which was formedfrom the loss of the t-butyl group was monitored. The modified assay utilized 2-MGA as the internal standard for the 2 diacid metabolites of VPA, 2-PSA and 2-PGA. In addition, heptadeuterated analogues of VPA and some of the VPAmetabolites,[2H7]4-ene VPA,[2H7]2-ene VPA (E and Z),[2H713-keto VPA,[2H7]4-keto VPA,[2H7]3-OH VPA and[2H7]5-OH VPA, were used as internalstandards. Correlation coefficients of the calibration curves were 0.990 orbetter. The assay has been described at a recent conference (Yu et al., 1992) anda manuscript is currently in preparation (Yu et at.).121DiscussionChoice of metabolites monitored from the in vitro microsomalmetabolism of VPA and (E)-2-ene VPAInitially upon injection of the derivatized extracts of the microsomalincubates, 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 bedistinguished from background and subsequently only 3-OH VPA, 4-OH VPA, 5-OH VPA, 4-keto VPA and 4-ene VPA were monitored and quantitated. Inextracts of incubates where (E)-2-ene VPA was employed as the substrate,neither 3-ene VPA nor VPA peaks were detectable above the background noisealthough these metabolites have been reported as products of 2-ene VPAmetabolism 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-eneVPA, that arise from mitochondrial [3-oxidation were not detected from the invitro microsomal metabolism of (E)-2-ene VPA. Consequently, only the (E)-2,4-diene VPA and (E,E)-2,3’-diene VPA metabolites were monitored as the productsof rat hepatic microsomal metabolism of (E)-2-ene ‘[PA.HPLC analysis of CBZ and metabolitesThe HPLC assay used for the analysis of CBZ, CBZE and CBZD from raturine samples was a combination of 2 procedures from the literature (Elyas etat., 1983; Kumps et at., 1985). Kumps and co-workers (1985) were able toseparate a number of anticonvulsants and their metabolites (16 compounds intotal) in serum. The extraction of CBZD was greatly enhanced by changing theextraction solvent from dichloromethane to ethyl acetate. Many attempts toseparate the CBZD peak from endogenous compounds extracted from the urine,including varying the composition of the mobile phase, changing columns andemploying various gradient systems did not yield the extent of separation122Discussionclaimed in the literature (Kumps et al., 1985). Urine may contain moreendogenous compounds compared to serum, leading to a more complicatedchromatographic separation.A COMPARISON OF THE INDUCTION OF RAT HEPATIC MICROSOMALCYTOCHROME P-450 CONTENT BY PB, CBZ, CBZE AJTD OTHERINDUCING AGENTSCytochrome P-450 content in hepatic microsomes from untreated ratsCytochromes P-450b and P-450e were not detected on the immunoblots ofhepatic microsomal protein from untreated rats. This was not surprising sincethese isozymes are present in very low quantities in untreated rat liver(Waxmann and Azaroff 1992). In untreated rats, cytochrome P-450b content isreported to be highest in the lung in comparison to liver, kidney, adrenal orsmall intestine while cytochrome P-450e content is highest in the liver (Christouet at., 1987). With PB induction, the highest levels of both cytochromes P-450band P-450e are found in the liver. Sex differences in rats have been observed inthe induction of these 2 isozymes (Yamazoe et at., 1987).Cytochromes P-450b and P-450e are minor constituents of the cytochromeP-450 pool, representing 5% or less of the total cytochrome P-450 content ofhepatic microsomes from untreated male or female Long Evans rats (Thomas etat., 1981). In uninduced Long Evans, male immature and adult rats,cytochrome P-450b content was 4% and 2%, respectively, of the total hepaticcytochrome P-450 content (Thomas et at., 1981). The expression of cytochromeP-450b in uninduced male rats from 5 different strains (Sprague-Dawley, LongEvans, Wistar, Brown Norway and Fischer F344) varied considerably (< 2 to 9pmoLfmg protein) (Wilson et at., 1987). However, the expression of cytochromeP-450e exhibited very little interstrain variability (17 ± 5 pmoll mg protein) with123Discussionthe exception of the Brown Norway strain (8.5 pmollmg protein).Effect of VPA on cytochrome P-450 contentVPA is generally not considered to be an enzyme inducing agent althoughthere are conflicting reports in the literature. For instance, VPA did not affecttotal rat hepatic microsomal cytochrome P-450 content when administered atdoses 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 100mg/kg or 200 mg/kg i.p. daily for 30 days, a dose dependent increase inmitochondrial carnitine acetyltransferase activity occurred but no changes wereobserved in the total hepatic cytochrome P-450 content (Singh et at., 1987).Cotariu et at. (1985) observed a decrease in cytochrome P450 content when VPAwas administered to male rats whereas Rogiers et at. (1988) observed asignificant increase in total cytochrome P-450 content when VPA wasadministered to rats at a dose of 100 mg/kg i.p. daily for 10 days. In the currentstudy, VPA administration did not result in increased total hepatic cytochromeP-450 content. Furthermore, anti-rat cytochrome P-450b antibody did not reactwith microsomal protein isolated from the livers of rats treated with VPA. Theeffects of VPA on cytochrome P-450, thus, remain unclear. Perhaps a longertreatment with VPA is necessary to elicit changes in cytochrome P-450. Wetreated 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 cytochromeP-450 content with administration of VPA over 10 days at a lower dose.Effect of CFB on hepatic cytochrome P-450 contentCorn oil is commonly used as a vehicle for CFB and other compounds notreadily soluble in aqueous solutions and when administered as such it has notbeen reported to affect hepatic cytochrome P-450 (Thomas et at., 1981). In this124Discussionstudy, corn oil itself, appeared to cause an increase in the total hepaticcytochrome P-450 content. Increased levels of cytochromes P-450d, P-450e, P450j and P-450p have been observed when CO (20%) was administered as adietary source (Yoo et at., 1992).CFB treatment appeared to induce total hepatic cytochrome P-450content when compared to the untreated group. However, when compared tothe CO treated group, the increase was not statistically significant. A higherdose 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. dailyfrom 500 mg/kg i.p. daily. Based on reports in the literature, a wide range ofCFB doses have been utilized for induction experiments in rats. Bachmann andco-workers (1988) used doses of 200 mg/kg of CFB i.p. daily for 3 days andHeinemeyer 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 insignificantly increased (33.6%) cytochrome P-450 levels when the CFB treatedgroup was compared to a peanut oil control group (Sharma et at., 1988a). In thecurrent study, only a 22% increase was observed when the CFB treated groupwas compared to the CO control group. The assumption then, is that either thedose of CFB that we employed andlor the time interval of CFB treatment wasinadequate to achieve maximal induction of total hepatic cytochrome P-450content.Cytochrome P-452 (lauric acid hydroxylase, cytochrome P-450 4A1), is theisozyme 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 ofcytochrome P-450 induced by PB or by polycyclic hydrocarbons (Tamburini etat., 1984). Thus, the reaction of anti-rat cytochrome P-450b antibody withmicrosomal protein from CFB treated rats on some Western blots was somewhat125Discussionsurprising (figures 10 and 11). This reaction was not observed on all Westernblots and may be due to carryover.Cytochrome P-452 constituted approximately 6% of the total cytochromeP-450 pool in untreated male Long Evans rats and after CFB administrationincreased to 11% (Chinje and Gibson, 1991). Although specific cytochrome P452 was not quantitated in this study, it has been reported to increase in a dosedependent manner when CFB (50 to 250 mg/kg daily) was administered to ratsfor 3 days via gastric intubation (Sharma et al., 1988b).Ethoxyresorufin, the preferred substrate for cytochrome P-450c, which isinduced by 3-methylcholanthrene, was not utilized as a substrate by microsomesfrom CBZ treated rats (Burke et al., 1985). Although the microsomal proteinfrom the CFB treated rats appeared to cross react with cytochrome P-450b,pentoxyresorufin was utilized as a substrate only to the same extent asmicrosomal protein from untreated rats (figure 12) further demonstrating thedifferences 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 hepaticcytochrome P-450 content in ratsTo the best of our knowledge, there are no reports in the literature wherethe time dependency of cytochrome P-450 inductiôn by CBZ in animal modelshas been examined. Thus, the time period tested for the induction ofcytochrome P-450 by CBZ was based on the assumption that 2 weeks ofinduction in rats should be comparable to the reported autoinduction of CBZmetabolism 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 3or 7 days did not demonstrate any differences in total hepatic cytochrome P-450content between the 2 time periods of treatment (Jung et al., 1980). Therefore,126Discussionrats were treated for 3, 7, 10 or 14 days in order to determine the time course forinduction by CBZ and CBZE.When the CBZ or CBZE treated groups were compared over the 14 daytreatment period, statistical differences were not observed in total cytochromeP-450 content. This would indicate that 3 days of treatment with CBZ or CBZEwere sufficient to yield maximal induction of cytochrome P-450 at the dosesemployed in this study.The dose of CBZ employed in the current study was based on reports thatan oral dose of 400 mg/kg in rats yielded serum levels of CBZ equivalent to the 8to 10 ig/mL levels observed in man (Morselli et al., 1971) and that the oralabsorption of CBZ is reported to range from 58 to 86% in monkeys (Morselli andFrigerio, 1975). Carbamazepine-10,11-epoxide was dosed at half the CBZ dosebased on reported serum CBZE concentrations after dosing with CBZ to beusually 10 to 50% those of the parent drug in humans (Bertilsson and Tomson,1986). Carbamazepine and carbarnazepine-10,11-epoxide were suspended inpropylene glycol at a concentration of 100 mg/mL and 50 mg/mL, respectively.While not specifically investigated in this work, the induction propertiesof CBZ and presumably also of CBZE, suggest dose dependency in humans.CBZ invoked dose dependent induction of antipyrine clearance which returnedto control values within 2 weeks after discontinuation of CBZ therapy in adultvolunteers (Rapeport et al., 1983). At higher doses of CBZ in patients, increasesin dosage resulted in disproportionately low elevations of CBZ plasmaconcentrations as a result of dose dependent induction (Tomson et al., 1989). Inhumans, CBZ demonstrated dose dependent induction as evidenced by acurvilinear relationship between dose and steady state concentrations(Kudriokova et al., 1992). In rats, however, a statistical difference was notobserved between a 60 mg/kg and a 100 mg/kg twice daily dose when the127Discussioninduction of various enzyme activities was examined (Regnaud et al., 1988).The observed increase in hepatic microsomal cytochrome P-450 content inthe PB and CBZ treated groups when compared to the untreated control groupwere not unexpected since 2 to 3 fold increases in cytochrome P-450 contenthave been observed after PB treatment in rats (Conney, 1967; Phillips et al.,1981). Regnaud and co-workers (1988) observed significant increases in totalhepatic cytochrome P-450 content when CBZ treated rats were compared tocontrol rats. A 48% increase in total hepatic cytochrome P-450 content by CBZtreatment over 4 days in rats compared to the control group was reported byWagner and Schmid (1987).The total hepatic cytochrome P-450 content of microsomes from each ofthe CBZ 3, 7 and 10 day treated groups was significantly increased whencompared to their corresponding PG treated group while there was no differencein the total hepatic cytochrome P-450 content of the CBZ 14 and PG 14 daytreated groups (figure 7). A plateau may have been reached whereby thecontinued presence of CBZ did not result in a further-increase in cytochrome P450 content; i.e. tolerance to induction by CBZ had developed. Anotherconsideration is that since the inducer must be present in high concentrations toproduce induction (Conney, 1967) and because CBZ metabolism isautoinducible, lower concentrations of CBZ can be expected at the site of actionwhen studied at the longest time point. In order to invoke induction, theinducing agent must be able to maintain adequate intracellular concentrationsafter repeated administration (Conney, 1967). In one case of PB induction inrats, a plateau in cytochrome P.450 levels was achieved after 5 days possibly asthe result of the formation of a repressor as opposed to any increase inmetabolism of PB (Ernster and Orrenius, 1965; Orrenius et al., 1965).In order to investigate the possibility of autoinduction of CBZ and CBZE128Discussionmetabolism over the time course, rat blood samples were collected at sacrificeafter 3, 7, 10 and 14 days of treatment. Unfortunately, the concentrations ofCBZ and metabolites that were present 12 to 13 h after administration of thelast dose were extremely low and thus unquantifiable. However, CBZ andmetabolites were quantitated in urine collected for 12 h following the last doseof either CBZ or CBZE. The urinary recoveries of CBZ, CBZE and CBZDrepresenting the expoxide-diol metabolic pathway did not increase over the 2week time period and ranged from 2.9 to 4.6% of the administered dose. In apreviously reported rat study, the recovery of compounds in the epoxide-diolpathway (CBZ, CBZE and CBZD) in a 24 h urine collection accounted for 7 to10% of the dose of CBZ administered (Regnaud et al., 1988). Our recoveries aretherefore significantly lower and may be due to differences in our assay and/orthe strain of rat used. The low recoveries observed for metabolites in theepoxide-diol pathway make it difficult to use such data to conclude ifautoinduction had in fact occurred. It is possible that other metabolites of CBZwere formed that we did not or could not measure. A possible clue to this is thestudy by Regnaud and co-workers (1988) where 50% of the CBZ dose wasrecovered in the urine in the form of thioethers. These thioethers were notspecifically identified but suggest an increased formation of mercapturatemetabolites after repeated administration of CBZ to rats. We were not able topursue the identification and quantitation of such metabolites in thisinvestigation.The lack of statistical differences apparent between the observed totalhepatic cytochrome P-450 values for the CBZE and PG treated groups may bedue to the lower dose of CBZE used compared to CBZ. Previously, CBZEadministration to Sprague-Dawley rats at a dose of 100 mg/kg daily i.p. foreither 3 or 7 days did not affect total hepatic cytochrome P-450 when compared129Discussionto the control group (Jung et at., 1980). Assuming that CBZE induction is a dosedependent phenomenon as reported for CBZ, the CBZE even at lower serumconcentration could play an important role in the induction of cytochrome P-450.If plasma half-life plays an important role in achieving maximal induction forthe barbiturates (loannides and Parke, 1975), perhaps this is also true for CBZand CBZE. Repeated administration of CBZ enhances its own elimination andthis may also be true for CBZE (Faigle and Feldmann, 1982). The mean plasmahalf-life of CBZ after a single dose was 35.6 ± 15.3 h and decreased to 20.9 ± 5.0h after chronic administration in patients (Eichelbaum et at., 1985). The plasmahalf-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). Theshorter plasma half-life of CBZE could also be responsible for limiting itseffectiveness at inducing total hepatic cytochrome P-450.The mean recoveries of CBZE and CBZD in the 12 h urine followingadministration of CBZE was only 1.5 to 4% of the administered CBZE dose. Bycomparison, it was reported that 15% and 1% of the dose was recovered asCBZE and CBZD, respectively, in urine collected for 5 days from SpragueDawley rats dosed with 4 mg of CBZE i.v. (Kerr and Levy, 1989). In man, aftersingle doses of CBZE, the urinary recovery of CBZD varies from 67 to 90% of thedose 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 speciesdifference in the metabolism of CBZE to CBZD with rats being much lessefficient than humans in producing the diol metabolite.A comparison of the effects of PB, CBZ and CBZE on the induction ofcytochrome P-450bPrevious reports in the literature speculated that CBZ should induce thesame isozyme(s) of cytochrome P-450 as does PB (Faigle and Feldmann, 1982;130DiscussionWagner and Schmid, 1987). The results of our study would appear to supportthat speculation. After treatment for just 3 days with either CBZ or CBZE,cytochrome P-450b was clearly detectable as was cytochrome P-450e. The ratanti-cytochrome P-450b antibody used in the present study reacts withcytochrome P-450e, also inducible by PB (Dutton and Parkinson, 1989).Cytochromes P-450b and P-450e are 2 of the major PB inducible isozymes andmay 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-450bband and has a slightly higher apparent molecular weight of 52,500 daltonsversus 52,000 daltons for cytochrome P-450b (Ryan and Levin, 1990). Rat livercytochromes P-450b and P-450e share greater than 97% amino acid sequencehomology as determined from amino acid and cDNA analysis (Fujii-Kuriyama etal., 1982). Cytochromes P-450b and P-450e are not immunochemicallyseparable 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 hepaticcytochrome P-450 isolated from the PB treated group of rats (table 5). Over the14 day time course, microsomal cytochrome P-450b content varied from 39 to66% of the total cytochrome P-450 for the CBZ treated groups and 31 to 53% forthe CBZE treated groups. Mean quantities of cytochrome P-450b in the CBZEtreated groups appeared to be lower than both the PB and CBZ induced groupsbut did not test statistically different. The specific content of cytochrome P-450bin the cytochrome P-450 protein of the PB and CBZ 3 day treatment groups wasvery similar to literature values. After PB treatment of male Long Evans rats,microsomal cytochrome P-450b content ranged from 43 to 57% of the totalhepatic cytochrome P-450 content (Thomas et al., 1981). In PB induced adult,male Sprague-Dawley rats, cytochrome P-450b represented 51% of the total131Discussionhepatic cytochrome P-450 (Wilson et at., 1987). Thus, the values for cytochromeP-450b obtained after PB induction in our study are very close to the reportedliterature values. Furthermore, our results suggest that at the doses used inthis 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 providedcomparable induction of cytochrome P-450b.Although the induction of cytochrome P-450b in the PB, CBZ and CBZEtreatment groups was confirmed by the use of antibodies, quantitation of themonooxygenase enzyme activities by fluorimetric assays can provide furthercorroboration of the identity of the isozymes induced. Fluorometric assaysinclude 0.-dealkylation of coumarins (umbelliferones), phenoxazones(resorufins), fluorescein (Mayer et at., 1989) and quinolones (Mayer et at., 1990).Of these, pentoxyresorufin and ethoxyresorufin have been demonstrated to bespecifically metabolized by the isozymes of cytochrome P-450 induced by PB and3-methyicholanthrene, respectively (Mayer et at., 1989). Although we did notstudy the effects of antibodies on microsomal dealkylation, an antibody againstcytochrome P-450b inhibited pentoxyresorufin dealkylation more than 90% inhepatic microsomes from PB induced rats (Dutton and Parkinson, 1989).In the present study, pentoxyresorufin was found to be the preferentialsubstrate for the 0-dealkylation of reactions catalyzed by cytochromes P-450induced not only by PB but by CBZ and CBZE as well (figures 12 to 14). Therates of pentoxyresorufin 0-dealkylation on a per mg of protein basis bymicrosomes from the PB treated group were increased 12 fold over the vehiclecontrol group (table 3). Induction with PB is reported to result in anenhancement of pentoxyresorufin 0-dealkylation activity over a wide range of20 to 283 fold in rats (Burke et at., 1985; Dutton and Parkinson, 1989; Lubet etat., 1990; Mayer et at., 1990). The values obtained in this study for the PB132Discussiontreated group were slightly lower when compared to the literature values.Pentoxyresorufin 0-dealkylation rates for microsomal protein from the CBZ (34to 53 fold increase) and the CBZE (12 to 19 fold) treatment groups were also inthe 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, cytochromeP-450b content and enhancement of pentoxyresorufin 0-dealkylation rates thatare very similar to that obtained with typical PB induction in rats. Although wedid not examine the following enzyme activities, CBZ administration to rats isreported to result in increased activities of UDP-glucuronyltransferase,NADPH-cytochrome P-450 reductase (Faigle and Feldmann, 1982; Wagner andSchmid, 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 forCBZ induction, albeit of lower magnitude, were similar to those observed withPB administration.The rates for pentoxyresorufin 0-dealkylation for the CBZE treated ratsranged from 32 to 43% that of the CBZ treatment groups and appear to reflectthe decreased dose of CBZE that was tested. Furthermore, these rates alsoreflect the lower levels of cytochrome P-450b quantitated in microsomal proteinfrom the CBZE treatment groups, with the exception of the CBZE 7 daytreatment group. An example in the literature that was similar to thesefindings is the reported induction of microsomal ethoxycoumarin 0-dealkylationby 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 wereobserved when CBZE was administered for an additional 4 days.133DiscussionEffect of CBZ, CBZE and PB treatment on cytochromes P-450f and P450gThe microsomal protein isolated from the CBZ, CBZE and PB treatmentgroups did not react to any appreciable degree with anti-rat cytochrome P-450for anti-rat cytochrome P-450g antibody. These results are consistent with whatis known about these particular isozymes of cytochrome P-450. Cytochrome P450f is a constitutive isozyme present at higher concentrations in females thanin males and is regulated by age (Leroux et al., 1989). Cytochrome P-450g is aconstitutive, male specific isozyme which is regulated by both sex and age(Soucek and Gut, 1992). These isozymes are normally present in smallquantities in untreated rat hepatic microsomes. Cytochromes P-450f and P450g represent approximately 1.4% and 0.8%, respectively of the total hepatic P450 content of hepatic microsomes from untreated immature male Long Evansrats (Bandiera et al., 1986). In adult rats, the amounts of cytochromes P-450fand P-450g increase to approximately 7% and 17% of total hepatic cytochromeP-450, respectively (Bandiera et al., 1986). Furthermore, cytochromes P-450fand P-450g are relatively refractory to induction by all common classes of P-450inducers (Bandiera et al., 1986).In summary, hepatic cytochrome P-450b in rats was significantly inducedby 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 wasfound to be sufficient to yield induction comparable to the usual PB dose of 75mg/kg i.p. for 4 days. CBZE at a dose of 50 mg/kg i.p. twice daily wasapproximately 50% as effective as CBZ for both the induction of cytochrome P—450b and the enhancement of pentoxyresorufin 0-dealkylation and is attributedto the lower dose used. The induction of cytochrome P-450b andpentoxyresorufin 0-dealkylation by CBZ and CBZE has not previously been134Discussionreported. We attempted to confirm this induction by examination of themetabolic profile of CBZ and CBZE in rats, but the urinary recoveries ofmetabolites did not provide substantial evidence for the induction of theepoxide-diol metabolic pathway.IN VITRO METABOLISM STUDIES OF VPA AND (E)-2-ENE VPAIEFFECTSOF INDUCING AGENTSIn order to detail the effects of CBZ induction on VPA metabolism that isapparent from patient interaction studies and from in vivo studies in rats, itwas important to investigate in vitro effects of CBZ induction on VPAmetabolism and one of its major metabolites, (E)-2-ene VPA. Induction by CBZand CBZE was compared to the classic inducer, PB and to CFB. Since maximalinduction of total cytochrome P-450 content, cytochrome P-450b and microsomalpentoxyresorufin 0-dealkylation appeared to be achieved after 3 days of CBZtreatment, only the group treated with CBZ for 3 days will be compared to thePB, CFB and CBZE 3 day treatment groups.Interaction between YPA and CBZAs the number of medications that a patient is prescribed increases, sodoes the possibility of adverse drug interactions (Mclnnes and Brodie, 1988).CBZ induces the metabolism of VPA in healthy subjects and in epilepticpatients (Levy and Pitlick, 1982). In epileptic patients on VPA and CBZ, steadystate VPA levels were 37 to 64% lower than predicted from single dose studies ofVPA (Levy and Pitlick, 1982). In one volunteer study, CBZ caused increasedclearance of VPA accompanied by decreased steady state plasma concentrations(Bowdie et al., 1979). The effect of CBZ on VPA clearance does not result fromcompetition for protein binding sites because CBZ plasma levels are lowcompared to the amount of albumin present and thus, CBZ should not act as a135Discussiondisplacing agent (Levy and Pitlick, 1982).In a volunteer study conducted in this laboratory, the volume ofdistribution of VPA did not change upon CBZ administration suggesting thatenzyme induction and not a competition for plasma protein binding sites wasresponsible for the increased clearance of VPA and the resulting decreasedserum levels, AUC and half-life values (Panesar et at., 1989). The majorunsaturated metabolite, (E)-2-ene VPA, was significantly decreased in serumand urine, suggesting that it too was being cleared as a result of inducedmetabolism. The serum levels of 4-ene VPA were unchanged after CBZadministration but urinary recoveries, mainly as the glucuronide conjugate,were increased after CBZ. When the urinary recoveries of VPA and itsmetabolites were compared before and after CBZ administration, an increase inmetabolism elicited by CBZ could not be confirmed. Either VPA metaboliteswere eliminated via non-renal routes or the GCMS assay failed to detect asignificant proportion of the VPA metabolites. Thus, in order to verifSr thisapparent in vivo induction of VPA metabolism, it was deemed important toexamine 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 YPAmetabolite profiles from rat liver microsomesAnti-rat cytochrome P-450b and P-450h antibodies were employed todetermine the extent of involvement of cytochromes P-450b and P-450h in the invitro microsomal metabolism of VPA and (E)-2-ene VPA. These antibodies,prepared and made available to us by Dr. Bandiera’s research group, were usedin a concentration range of 0.5 to 2.5 mg of IgG/nmol of spectrally determinedcytochrome P-450.The metabolism of VPA to 4-ene VPA by microsomal protein from PB andCBZ 3 day treated rats was completely inhibited in the presence of the anti-rat136Discussioncytochrome P-450b antibody while the formation of 3-OH VPA and 5-OH VPAwas only partially inhibited. The formation of 4-OH VPA and 4-keto VPA wasinhibited by 75% or greater. These results suggest that cytochrome P-450b isthe major isozyme of cytochrome P-450 involved in the metabolism of VPA to 4-ene VPA, 4-OH VPA and 4-keto VPA while other isozymes of cytochrome P-450participate in the formation of 3-OH VPA and 5-OH VPA.The results obtained for the microsomal metabolism of VPA to 4-ene VPAdemonstrate that formation of this putative hepatotoxin occurs directly fromVPA and not via dehydration of either 4-OH VPA or 5-OH VPA (Kochen andScheffner, 1980; Granneman et at., 1984a). Metabolic studies of stable isotopeanalogues of VPA had indicated that the origin of 4-ene VPA was different fromthat of 3-ene VPA and (E)-2-ene VPA (Rettenmeier et at., 1987).Thus, induction of a particular isozyme of cytochrome P-450, namelycytochrome P-450b, may play an important role in VPA induced hepatotoxicity.The formation of 4-ene VPA occurs via cytochrome P-450b catalyzed oxidation ofa nonactivated alkyl substituent to the corresponding olefin (Baillie, 1988). Thiscytochrome P-450 isozyme functions as a desaturase (figure 37) oxidizing certainalkanes to olefins without an intermediate alcohol due to partitioning betweenhydroxylation and desaturation reactions (Guengerich, 1990). Initialabstraction of a hydrogen atom generates a transient free-radical intermediatethat partitions between recombination (alcohol formation) and elimination(olefin production). Based on the observed metabolism of deuterium labeledVPA, 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.450mediated formation of 17f3-hydroxy-4,6-androstadien-3-one from testosterone viadual hydrogen abstraction (Nagata et at., 1986). The 63-hydrogen is abstractedforming a transient radical, followed by abstraction of the 73-hydrogen to form137DiscussionCOOH (FoOHJ2 (Fej3 COGHh: k-+ -— ..1[FeOH]2 A H20 (3-Ene VPA)-,.- I‘I_, I(FeOHI3÷ -000H [FeOH]3 [Fe]3 GOGH(I) OH[FeOH][FeO]3(3-Hiiroxy VPA)COGH [FeOH] [FeO]3 GOGH (VPA)(Ill) [FeO][FeOH]3[FeJ3 [FeOGOGH GOGH [FeOH]3 [Fe] HO GOGHHO (II)[FeOHJ (4-Hytiroxy VPA)(5-Hydroxy VPA)[FeOH]2GOGH [FeOH]2 [Fe COOHH20 (4-Ene VPA)Figure 37. Cytochrome P-450 catalyzed metabolism of VPA to 3-OH VPA, 4-OH VPA, 5-OH VPA, 3-ene VPA and 4-ene VPA. (Based on Rettieet at., 1988).138Discussionthe resultant double bond. The free radical intermediate generated solely bycytochrome P-450b which partitions between alcohol and olefin formationexplains our results in that the antibody to cytochrome P-450b not onlycompletely blocked 4-ene VPA formation but also the formation of 4-OH VPAand the derived 4-keto VPA (figures 30, 32 and 33).In order to further affirm the specificity of cytochrome P-450b in themetabolism of VPA to 4-ene VPA and 4-OH VPA, one other antibody tocytochrome P-450 was tested. Cytochrome P-450h is a male specific isozyme ofcytochrome P-450 which catalyzes a number of metabolic reactions (Ryan andLevin, 1990). It is non-detectable in newborn rats but rapidly increases at 4 to 6weeks of age and then plateaus (Waxman et at., 1985). The anti-rat cytochromeP-450h antibody did not exhibit any significant effects on the metabolism ofVPA by microsomes from untreated, PB or CBZ 3 day treated groups, evidencethat VPA or (E)-2-ene VPA are not substrates for cytochrome P-450h.Since the antibody directed against cytochrome P-450b was unable tocompletely inhibit the metabolism of VPA to 3-OH VPA and 5-OH VPA, otherisozymes of cytochrome P-450 may be involved. Another isozyme of cytochromeP-450 inducible by PB is cytochrome P-450p (Soucek and Gut, 1992). Inductionof cytochrome P-450p significantly increased the biotransformation of CBZ toCBZE in mouse hepatic microsomes and this reaction was inhibited bygestodene, a cytochrome P-450p inhibitor (Pirmohamed et at., 1992). CBZ andCBZE may also induce cytochrome P-450p. To determine whether cytochromeP-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-450pantibody are required.139DiscussionA comparison of the effects of PB, CBZ and CBZE induction on the invitro metabolism of VPAPhenobarbital pretreatment resulted in enhanced metabolism of VPA to3-OH VPA, 4-OH VPA, 5-OH VPA, 4-ene VPA and 4-keto VPA. Similar resultswere obtained with the CBZ treated groups.Formation of4-ene VPAGranneman and co-workers (1984a) were one of the first groups to reportthat PB induction increased 4-ene VPA formation in VPA treated rats. Ourwork confirms that CBZ and CBZE are capable of this action as well. Rettie andco-workers (1987) were the first to report the in vitro formation of 4-ene VPAfrom VPA as a result of a cytochrome P-450 mediated reaction. The 4-ene VPAmetabolite could only be observed in the presence of PB induced rat livermicrosomes. Similar results were obtained for liver microsomes from PBinduced rabbits, mice and humans with 2.5 to 8.4 fold increases in the formationof 4-ene VPA, depending upon the species (Rettie et al., 1988). A comparativestudy using microsomes from CBZ, phenytoin and PB induced rats (80 mg/kg for4 days) indicated that PB was the more effective inducer of this desaturationpathway leading to 4-ene VPA (Rettie et al., 1987). In our study, the formationof 4-ene VPA was increased 6 fold and 5 fold respectively, by the PB and CBZ 3day treated groups when compared to the untreated group. Only a 2 foldincrease was observed with the CBZE 3 day treated group.The 4-ene VPA metabolite is not easily detected because of the low serumlevels. For example, in a survey of 49 patients, 4-ene VPA was only detected inthe 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 andCBZ group plus the induction effects of CBZ in forming increased 4-ene VPA.Extremely sensitive assays are required in order to reliably quantifr significant140Discussionchanges in 4-ene VPA serum concentrations. Using a GCMS assay, theformation clearance of 4-ene VPA was determined to increase 2 fold in patientswho were on combined therapy with VPA and either CBZ or phenytoin (Levy etal., 1990).Significance of4-ene VPA formation to the mechanism of VPA hepatotoxicityThere are currently 2 major hypotheses regarding the mechanism(s) ofVPA induced hepatotoxicity. The first proposed mechanism for VPA toxicityentails the depletion of free CoA due to sequestration by VPA to form valproylCoA and derivatives (Becker and Harris, 1983; Thurston and Hauhart, 1992).The reduced concentrations of free CoA result in increased levels of fatty acidsand decreased 13-oxidation. A second proposed mechanism that our laboratoryfavours, is that hepatotoxicity of VPA is mediated through the formation of atoxic metabolite and 4-ene VPA has been implicated as the most likelycandidate (Zimmerman and Ishak, 1982; Rettenmeier et al., 1985; Rettenmeieret al., 1986b). The 4-ene VPA metabolite is similar in structure to thehepatotoxin 4-pentenoic acid (PA) and to MCPA, the metabolite of hypoglycin(figure 38). VPA hepatotoxicity shares many similar manifestations withJamaican Vomiting Sickness (JVS), Reye’s Syndrome (RS) and PA toxicity(Lewis et al., 1982; Nau and Loscher, 1984). PA is bioactivated to the reactiveelectrophilic species, 3-keto-4-pentenoic acid, which alkylates 3-ketoacyl CoAthiolase, the terminal enzyme of mitochondrial 13-oxidation (Schulz, 1983; Fongand Schulz, 1983). It has been postulated that analogous to PA, 4-ene VPAundergoes 13-oxidation to (E)-2,4-diene VPA which is ultimately bioactivated tothe electrophilic metabolite, 3-keto-4-ene VPA (Rettenmeier et al., 1985). Thismetabolite has just recently been identified in rats although it is present in verytrace quantities and specialized techniques were required for its identification(Kassahun et al., 1993).141DiscussionH2CCH—CHr—CH2COOH(4-Pentenoic add)NH2H2C=C— CH—CH2—CH COOH(Hypoglycin)H2C=C— CH—CH2—COOH(Methylene-cydopropytacetic add)C3H7H2C=CH— CH— CH—COOH(4-EneVPA)Figure 38. Structural similarity amongst hypoglycin, 4-pentenoic acid and 4-ene VPA.142DiscussionIt has been established that VPA and 4-ene VPA undergo metabolicactivation in rat liver, both in vivo and in vitro, to electrophilic intermediatesthat bind covalently to cellular macromolecules (Porubek et at., 1989). Insupport of a reactive metabolite mechanism, Kassahun et at. (1991) found thatin rats administered 4-ene ‘[PA, the major metabolite recovered in bile was thegluthathione (GSH) conjugate of (E)-2,4-diene ‘[PA. GSH is a tripeptide whichreacts with a variety of electrophilic compounds aiding in the minimization ofcellular injury (Reed, 1990; DeLeve and Kaplowitz, 1991). Urinary Nacetylcysteine (NAC) conjugates are the end products of GSH conjugation. TheNAC conjugate of (E)-2,4-diene ‘[PA was a prominent urinary metabolite in ratsthat 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 bedue to localized mitochondrial depletion of GSH, leading to oxidative stress andsubsequent cellular injury (Kassahun et at., 1991). Thus, patients who aresubjected to increased serum levels of 4-ene VPA andlor have deficiencies intheir glutathione defense mechanisms could be at an increased risk to VPAinduced hepatotoxicity. In the retrospective study by Dreifuss and co-workers(1987), ‘[PA polytherapy, especially when in combination with CBZ, wasimplicated as a major factor in the increased incidence of VPA hepatotoxicity.Formation of the hydroxy metabolites of VPAThe formation of the hydroxy metabolites was significantly enhanced byCBZ and PB induction while smaller increases were observed with CBZEtreatment. The hydroxy metabolites of VPA (3-OH ‘[PA, 4-OH ‘[PA and 5-OHVPA) have been demonstrated in human embryo homogenates of liver, lung,brain and adrenals (Rettie et at., 1986) and rat liver microsomes (Prickett andBaillie, 1984). The formation of 3-OH VPA from VPA was thought to bemediated primarily via the 13-oxidation pathway (Bjorge and Baillie, 1991; Li et143Discussionat., 1991) but evidence suggests that it may partly arise via cytochrome P-450mediated o-2 oxidation (Prickett and Baillie, 1984). The o-2 oxidation pathway,inducible by PB, is a minor metabolic route for the rat hepatic microsomalmetabolism of simple alkanes, such as n-hexane (Frommer et at., 1972) and nheptane (Frommer et at., 1974).Phenobarbital treatment in rats was reported to primarily enhance the o1 oxidation pathway of VPA metabolism with the urinary recoveries of 4-OHVPA, 4-keto VPA and 2-PSA being increased by a factor of 3.8 (Granneman etat., 1984a). The urinary excretion of 5-OH VPA was also increased. In onepediatric patient who died from hepatic failure associated with VPA and PBpolytherapy, increased quantities of 2-PGA, the endproduct of the (0-oxidation ofVPA, 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-OHVPA) hydroxylation while CBZ was more efficient for the induction of (0-oxidation (5-OH VPA). This could, therefore, have implications regarding theincidence of VPA induced hepatic failure depending on the associated drugs andthe metabolic pathway responsible for the ultimate hepatotoxic response.Despite the presence of 3-OH VPA, 3-ene VPA was not detected in themicrosomal incubates from the metabolism of VPA. In rats, 3-ene VPA was notobserved as a metabolite of 3-OH VPA (Granneman et at., 1984a). These resultsindicate that 3-ene VPA probably arises through a different mechanism thanthe 4-ene VPAI4-OH VPA pathway described in figure 37. Alternatively, thedegree of 3-ene VPA formation may have been below the level of detection ofour assay.The oxidation of fatty acids involving hydroxylation at the o, o-1 or o-2positions occurs microsomally and can be succeeded by furtherbiotransformation via alcohol or aldehyde dehydrogenases to the dicarboxylic144Discussionacids or keto acids (Bjorkhem, 1972a). Dehydrogenation reactions can occur ineither the soluble or microsomal fraction, although the microsomaldehydrogenase activity is not as efficient as that of the soluble fraction(Bjorkhem, 1972b). This may explain why 4-keto VPA was detected in the invitro microsomal metabolism of VPA although it was not expected, since theformation of 4-keto VPA is the second step in the o-1 oxidation pathway. Basedon the theory that partitioning occurs between desaturation and hydroxylationas illustrated in figure 37, if larger quantities of the alcohol are formed (i.e. 4-OH VPA) then a larger concentration of substrate is present for alcoholdehydrogenases to form 4-keto VPA. CBZ was a more effective inducer of theformation of 4-keto VPA than was PB which may also relate to the lowerquantities of 4-ene VPA observed with the CBZ treated group.In a discussion of the effects of induction by CBZ and CBZE on themetabolism 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 ofVPA to some metabolites. There is some precedence in the literature for thisinhibition by PG from studies in mice. Administration of PG (4 mLfkg i.p.) tomice within 3 h of receiving acetaminophen, provided protection againstacetaminophen induced liver toxicity (Hughes et al., 1991). This protection byPG was perhaps afforded by inhibition of the formation of the toxic metaboliteN-acetyl-p-benzoquinoneimine by cytochrome P-450, a mechanism by whichethanol 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 3days), aminopyrine demethylase activity was observed to decrease. Thisdepressant effect of PG on aminopyrine demethylase activity was abolished145Discussionwhen PB was co-administered. We cannot be certain that induction by CBZ andCBZE produced the same effects as PB. Alternate vehicles which lack theinhibitory effects may need to be considered for further studies of CBZ andCBZE induction.Effect of anti-rat cytochrome P-450b and P-450h antibodies on (E)-2-eneVPA metabolite profiles from rat liver microsomesThe major serum metabolite of VPA, (E)-2-ene VPA, has been touted as apossible anticonvulsant agent (Nau et at., 1984). (E)-2-ene VPA possessesanticonvulsant activity similar to VPA without exhibiting the toxicities(teratogenicity, embryotoxicity and hepatotoxicity) associated with the parentcompound (Honack et at., 1992). In rats, (E)-2-ene VPA is metabolized toseveral diunsaturated metabolites, (E,E)-2,3’-diene VPA and 2,4-diene VPA, aswell as 3-keto VPA (Loscher et at., 1992). (E,E)-2,3’-diene VPA, the majordiunsaturated metabolite in the serum also possesses anticonvulsant activity(Acheampong and Abbott, 1985). However, the second diunsaturatedmetabolite, (E)-2,4-diene VPA, produces hepatic steatosis in rats (Granneman etat., 1984c; Kesterson et at., 1984).The effect of the anti-rat cytochrome P-450b antibody on the metabolismof (E)-2-ene VPA was investigated to determine the extent of this isozyme’sinvolvement. The metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA inmicrosomes 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 thepresence of the anti-rat cytochrome P-450b antibody. The anti-rat cytochromeP-450h antibody did not exert any inhibitory effects on the metabolism of (E)-2-ene VPA. These results clearly indicate that cytochrome P-450b plays animportant role in the metabolism of (E)-2-ene VPA to (E)-2,4-diene VPAanalogous to the formation of 4-ene VPA from VPA. However, other cytochrome146DiscussionP-450 isozymes appear to be responsible for the formation of (E,E)-2,3’-dieneVPA. Further details of the in vitro metabolism of (E)-2-ene VPA and thesignificance of our findings with respect to the prospective use of this compoundas an anticonvulsant agent are described below.A comparison of the effects of PB, CBZ and CBZE induction on the invitro metabolism of (E)-2-ene YPA in rat liver microsomesThe production of (E,E)-2,3’-diene VPA by microsomes isolated from thePB, CBZ 3 day and CBZE 3 day treatment microsomes was significantlyenhanced over controls as was the formation of (E)-2,4-diene VPA by the PB andCBZ 3 day treatment groups. The extent of induction by CBZ and PB asmeasured 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 VPAmetabolite was not detected. We were also unable to distinguish the peaks forVPA or 3-ene VPA from the background in the SIM chromatograms of theextracted incubates. VPA, albeit in small quantities, has been detected in ratsafter administration of (E)-2-ene VPA (Granneman et at., 1984a; Loscher et at.,1992). The metabolite 3-ene VPA was detected in rat plasma afteradministration 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 enzymesare not responsible for the reduction of (E)-2-ene VPA to VPA nor for theisomerization 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 the13-oxidation product 3-keto VPA (Granneman et at., 1984a).The detection of (E)-2,3’-diene VPA as a microsomal metabolite of (E)-2-ene VPA in our study was unusual because its formation has been attributed to147Discussionf3-oxidation mechanisms in mitochondria (Bjorge and Baillie, 1991) that aresummarized in figure 39. The (E)-2-ene VPA metabolite reversibly isomerizes to3-ene VPA, that in turn is metabolized by the 3-oxidation pathway to (E,E)-2,3-diene VPA (Bjorge and Baillie, 1991). However, our in vitro results indicatethat 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 CBZ3 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 isas effective as PB for the induction of this desaturation pathway. A significantincrease in the formation of (E)-2,4-diene VPA occurred only in the CBZE 7 daytreatment group which also corresponded to the maximal increase observed forcytochrome P-450b by CBZE. Similar results have recently been reported forthe effects of PB on the induction of (E)-2-ene VPA metabolism to (E)-2,4-dieneVPA in vitro (Kassahun and Baillie, 1993). The (E)-2,4-diene VPA was a majormetabolite 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 toinvoke 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 6cases of fatal VPA hepatotoxicity (Kochen et at., 1984). In patients with VPAassociated hepatic failure, the urinary recovery of (E)-2,4-diene VPA as the NACconjugate was increased 3 to 4 fold (Kassahun et at., 1991). Therefore, thesignificant enhancement of (E)-2,4-diene VPA formation by CBZ and PB on themetabolism of (E)-2-ene VPA raises important questions regarding the potential148DiscussionCOOHCoraffon-VPA gIucunde(VPA)p-oddalionCOOL-f COOH COOH H000_+OH(3HroVPA) ((E)—2-Ene VPA) ((E)-3-Erie VPA) ((Z)-3--Ene VPA)ICOOHCOOH0(3-Keto VPA) ÷((E,E)-2, 3-Oiene ((E,Z)-2,3-DieneVPA) VPA)Figure 39. The 13-oxidation pathway of VPA metabolism in mitochondria.149Discussionsafety of this drug. This is especially true should (E)-2-ene VPA be used insituations of polytherapy.Consideration should also be given to the relative amounts of (E)-2,4-diene VPA that is formed from either VPA or (E)-2-ene VPA administration. Acomparison 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)-2,4-diene VPA greatly exceeded that of 4-ene VPA. For example, with the PBtreatment group, 17 ng of 4-ene VPA were formed compared to 2.5 pg of (E)-2,4-diene VPA. For the CBZ treatment groups over the 2 week treatment period, 11to 13 ng of 4-ene VPA were produced in comparison to 1 to 2.5 tg of the (E)-2,4-diene VPA. Thus, the desaturation catalyzed by cytochrome P-450b in (E)-2-eneVPA metabolism appears to be considerably more efficient than for VPA. Onepossible explanation is that because of the conjugated structure of the (E)-2,4-diene VPA product, radical intermediates are being stabilized by the doublebond 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 thatthe relative lack of toxicity reported for (E)-2-ene VPA should be perceived withcaution. 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 hepatotoxicthan VPA. One rationale for such a hypothesis may be related to the organellein which the (E)-2,4-diene VPA is formed. With (E)-2-ene VPA, the desaturationto (E)-2,4-diene VPA occurs microsomally in comparison to mitochondrialformation 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-dieneVPA produced in microsomes can react with the much larger pooi of cytosolic150DiscussionOSH (Reed, 1990).Effect of CFB treatment on the metabolism of VPA and (E)-2-ene VPACFB treatment did not appear to have any significant effects on themetabolism of either VPA or (E)-2-ene VPA although the CO vehicle may haveexerted an effect which overshadowed or minimized any CFB invoked effects.When olive oil, was used as a vehicle for CFB, an increased urinary excretion of4-OH VPA, 2-PGA and 3-keto VPA after VPA administration to CFB treatedrats was observed (Heinemeyer et al., 1985).The cytochrome P-450 isozymes inducible by CFB possess the uniqueability to -oxidize fatty acids (Bains et al., 1985). The mechanism by whichCFB affects fatty acid metabolism is thought to be as follows. Administration ofCFB elicits an increase in cytochrome P-452, leading to increased ohydroxylation of fatty acids. 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 ofshorter 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 andthe f3-oxidation metabolite 3-keto VPA (Heinemeyer et at., 1985.) However, in amore recent study, CFB pretreatment in rats did not significantly affect themetabolism of VPA, suggesting a minimal role for peroxisomal mediated 13-oxidation (Bachmann et at., 1988). Based on the results of our study, neitherVPA nor (E)-2-ene VPA were substrates for cytochrome P-452.CLINICAL RELEVANCECytochrome P-450b was the major isozyme in rats induced by PB, CBZand to a lesser extent by CBZE. The in vitro microsomal formation of 4-ene VPAfrom VPA and (E)-2,4-diene VPA from (E)-2-ene VPA was enhanced by151Discussionpretreatment with all 3 drugs and was inhibited completely in the presence ofthe anti-rat cytochrome P-450b antibody. Thus, both of these metabolitesappear to be formed by a common desaturation mechanism. Both of thesemetabolites 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-eneVPA have been observed in cases of VPA associated hepatotoxicity (Kochen etat., 1983; Scheffner et at., 1988). The demonstrated inducibility of (E)-2-eneVPA to (E)-2,4-diene VPA suggests that the ‘toxicity free’ nature of (E)-2-eneVPA needs to be re-evaluated, particularly when the drug is administered incombination with inducing agents such as CBZ. Alternatively, if (E)-2-ene VPAproves to be free of hepatotoxicity, the proposed role of (E)-2,4-diene VPA in thehepatotoxicity of VPA will need reassessment.Future studies in rats should examine the induction of cytochrome P-450by an equivalent molar dose of CBZE to that of CBZ and thus establish theapparent dose dependent properties of CBZE. In previous studies of VPAmetabolism in patients, CBZ significantly reduced serum (E)-2-ene VPAconcentrations but not those of (E)-2,4-diene VPA. The results from this studysuggest that enhanced metabolism of (E)-2-ene VPA to (E)-2,4-diene VPA orinhibition of 3-oxidation occurs. Any further studies must include mitochondrialand peroxisomal effects of CBZ and CBZE, in light of the evidence thatmitochondrjal cytochromes P.450 are involved in the activation of aflatoxin B1(Niranjan et at., 1984; Shayiq and Avadhani, 1989) and may also play a role inVPA metabolism. In addition, the effects of induction on phase II metabolism ofVPA and metabolites, including glutathione conjugates should be investigated.152SUMMARY AND CONCLUSIONSCBZ was compared to PB with respect to induction of total hepaticcytochrome P-450, cytochrome P-450b and the catalysis ofpentoxyresorufin 0-dealkylation in rats. For maximal induction of theseparameters, an i.p. dose of 100 mg/kg twice daily for 3 days of CBZappeared 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 similarinduction profile to that of CBZ. Cytochrome P-450b andpentoxyresorufin 0-dealkylation values for the CBZE treatment groupswere approximately 50% of the CBZ treatment groups.3. Evidence for autoinduction of CBZ metabolism was sought by examiningthe urinary recoveries of metabolites that constitute the epoxide-diolpathway. No differences in metabolite recoveries were found afterchronic administration of CBZ.4. Studies with anti-rat cytochrome P-450b antibody indicated thatcytochrome P-450b was the primary isozyme that catalyzes thebiotransformation of VPA to 4-ene VPA, 4-OH VPA and 4-keto ‘[PA. Inaddition, cytochrome P-450e may also be involved. Results from theinhibition experiments indicated that all 3 metabolites probably arisefrom a common intermediate.5. Inhibition studies with anti-rat cytochrome P-450b antibody on theformation of (E)-2,4-diene VPA from (E)-2-ene VPA strongly suggest thatcytochrome P-450b is the primary isozyme responsible for thisbiotransformation.153Summary and Conclusions6. The amount of (E)-2,4-diene VPA formed from (E)-2-ene VPA per nmol ofcytochrome P-450 was approximately 100 fold greater than the conversionof VPA to 4-ene VPA as measured in microsomes from either CBZ 3 dayor PB treated rats. The enhanced production of (E)-2,4-diene VPA, ametabolite known to be hepatotoxic in rats, could have severeconsequences regarding the relative safety of (E)-2-ene VPA if usedtherapeutically and in combination with other drugs.7. Anti-rat cytochrome P-450h antibody did not inhibit the metabolism ofVPA 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 themetabolism of both VPA and (E)-2-ene VPA to potentially hepatotoxicmetabolites.8. A major metabolite of VPA, (E,E)-2,3’-diene VPA, recently shown to be amitochondrial metabolite of 3-ene VPA, was identified in this study as amicrosomal metabolite of (E)-2-ene VPA. Inhibition studies indicatedcytochrome P-450b to be only partially responsible for the conversion of(E)-2-ene VPA to (E,E)-2,3’-thene VPA.9. 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