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Pharmacokinetics of two monounsaturated metabolites of valproic acid in the rat Singh, Kuldeep 1988

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Pharmacokinetics of Two Monounsaturated Metabolites Valproic Acid in The Rat by KULDEEP SINGH M. Pharm., Panjab University, 1979 M.Sc, Dalhousie University, 1982 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY in THE FACULTY OF GRADUATE STUDIES Faculty of Pharmaceutical Sciences (Division of Pharmaceutics) We accept this thesis as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA February 1988 ©Kuldeep Singh, 1988 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of /"VU ^Ctf^JZaJL €<*&^c&f The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 Date DE-6(3/81) i i ABSTRACT Valproic acid (VPA) is a broad spectrum antiepileptic agent used widely in the treatment of absence and tonic-clonic seizures. VPA is extensively metabolized and forms 17 metabolites in man. A monounsaturated metabolite, (E)-2-ene VPA, is at least as potent as the parent drug VPA in several animal models of epilepsy. Moreover, (E)-2-ene VPA appears to be free of two serious side effects of VPA, namely hepatotoxicity and teratogenicity. Another monounsaturated metabolite of VPA, 4-ene VPA, has been incriminated in the pathogenesis of fatal hepatic failure in children on VPA therapy. This thesis describes the synthesis of (E)-2-ene VPA and 4-ene VPA and the development of a simple and sensitive capillary gas chromatographic-mass spectrometric (GCMS) assay method for the estimation of (E)-2-ene VPA and 4-ene VPA in the biological fluids of the rat. This thesis also describes the pharmacokinetics of (E)-2-ene VPA and 4-ene VPA at two dose levels of 20 and 100 mg/kg in normal and bile exteriorized rats. A simple capillary GCMS assay method was developed that involves a single extraction of 80 /JL of plasma, urine or bile with ethyl acetate followed by derivatization with MTBSTFA (N-tertiarybutyldimethylsilyl-N-methyl-trifluoroacetamide). For an 80 /iL biological sample employed for extraction, the lowest detection limit for (E)-2-ene VPA was 60 ng/mL and for 4-ene VPA, 100 ng/mL. The calibration curves for (E)-2-ene VPA were linear over a fairly wide concentration range of 0.4-35 /xg/mL in plasma and 2-200 ng/ml in urine of the rat. Standard curves for 4-ene VPA were prepared in concentration ranges of 0.5-45 iig/mL in plasma and 2-80 ng/ml i i i in urine. 'The assay method is reliable, reproducible, and is able to separate the diene metabolites of (E)-2-ene VPA. For pharmacokinetic studies, a single intravenous (IV) bolus dose of either (E)-2-ene VPA or 4-ene VPA was administered to normal or bile-exteriorized rats. On increasing the dose from 20 to 100 mg/kg in normal rats, the apparent plasma clearance of (E)-2-ene VPA changed from 4.9 + 1.7 (SD) to 3.0 ± 0.3 mL/min.kg, and of 4-ene VPA decreased from 8.7 ± 0.6 to 5.9 + 0.5 mL/min.kg. A total (conjugates and unconjugates) of 32 + 6% of the low dose and 50 + 11% of the high dose of (E)-2-ene VPA was recovered in the urine of the rat. The second metabolite, 4-ene VPA, was eliminated in the urine to a relatively smaller extent (22 + 3% of the low dose and 28 + 6% of the high dose). In bile-duct cannulated rats, the apparent plasma clearance of (E)-2-ene VPA was 7.7 + 1.8 mL/min.kg at the low dose and 6.0 + 1.1 mL/min.kg at the high dose. The corresponding values for 4-ene VPA were l l + 1.8 mL/min.kg and 7.4 + 1.1 mL/min.kg, respectively. The apparent elimination half- l i fe of (E)-2-ene VPA remained unchanged at 20-21 min at the two dose levels, compared to a 1.5 fold increase in the t\/2 ° f 4-ene VPA from 13 ± 2 to 19+3 min. The fraction of the low dose (29 + 5%) eliminated in bile was significantly larger than at the high dose (21 + 4%), when calculated as the sum of conjugated and unconjugated 4-ene VPA. The biliary elimination of (E)-2-ene VPA showed a non-significant change from 38 + 10 to 31 + 9% on increasing the dose. Like the parent drug VPA, (E)-2-ene VPA and 4-ene VPA showed enterohepatic recirculation in the rat which produced secondary plasma peaks in normal animals. Moreover, both (E)-2-ene VPA and 4-ene VPA showed i v a rapid but transient choleretic effect in the rat. The plasma protein binding of 4-ene VPA was apparently low (14-25%), in the concentration range of 20-350 /jg/mL. The results indicate that 4-ene VPA is cleared much faster from the plasma than (E)-2-ene VPA in the rat. The plasma levels of 4-ene VPA required to show a non-linear decline (>200 /xg/mL) in the rat are two orders of magnitude higher than 4-ene VPA levels (<1 ng/ml) seen in patients on VPA therapy. It is , therefore, unlikely that 4-ene VPA is eliminated more slowly than VPA in man. On the other hand, the plasma elimination t ° f (E)-2-ene VPA in bile-exteriorized rats is longer than that reported for VPA, indicating that (E)-2-ene VPA may have a longer lasting pharmacologic effect than VPA. V TABLE OF CONTENTS CHAPTER PAGE Abstract i i Table of Contents v List of Tables v i i i List of Figures x List of Appendices xi Symbols and Abbreviations xii Acknowledgements xv A. INTRODUCTION A.l VALPROIC ACID A.1.1. Background 1 A.1.2. Pharmacokinetics in Man 2 A.1.3. Pharmacokinetics in Animals 3 A.1.4. Metabolism 4 A.1.5. Mechanism of Action 8 A.1.6. Side Effects 9 A.1.7. Hepatotoxicity 10 A.2. (E)-2-ENE VALPROIC ACID A.2.1. Pharmacologic Activity 12 A.2.2. Pharmacokinetics 13 A.2.3. Side Effects 15 A.3. 4-ENE VALPROIC ACID A.3.1. Toxicity 16 A.3.2. Metabolism 16 A.3.3. Pharmacokinetics 17 A.4. RATIONALE AND OBJECTIVES 19 vi B. EXPERIMENTAL B. l . MATERIALS B . l . l . Chemicals 22 B.l.2. Instrumentation 23 B.2. METHODS B.2.1. Synthesis 24 B.2.1.1. (E)-2-ene VPA 24 B.2.1.2. 4-ene VPA 27 B.2.2. Capillary GCMS Assay 28 B.2.2.1. Standards 28 B.2.2.2. Extraction and Derivatization 29 B.2.2.3. Chromatography 29 B.2.2.4. Optimum Derivatization Conditions 30 B.2.2.5. Hydrolysis of Conjugate 30 B.2.2.6. Calibration Curves 30 B.2.2.7. Precision 30 B.2.2.8. Extraction Efficiency 31 B.2.3. Rat Experiments 31 B.2.3.1. Animal Handling 31 B.2.3.2. Jugular Vein Cannulation 31 B.2.3.3. Bile Duct Cannulation 32 B.2.3.4. Pharmacokinetic Studies 34 B.2.3.5. In Vitro Plasma Protein Binding 35 B.2.3.6. Metabolism of (E)-2-ene VPA 35 B.2.4. Pharmacokinetic Analysis 36 C. RESULTS C . l . SYNTHESIS C . l . l . (E)-2-ene VPA 38 C.1.2. 4-ene VPA 45 C.2. ASSAY C.2.1. Chromatography 45 C.2.2. Derivatization Kinetics 48 C.2.3. Linearity and Reproducibility 48 C.2.4. Hydrolysis of Conjugates 54 vi i C.2.5. Extraction Efficiency 54 C. 3. PHARMACOKINETIC STUDIES C.3.1. Pharmacokinetics in Normal Rats 54 C.3.2.. Pharmacokinetic Model 66 C.3.3. Pharmacokinetics in Bile Exteriorized Rats 71 C.3.4. Choleretic Effect 84 C.3.5. In Vitro Protein Binding 84 C. 3.6. Metabolism of (E)-2-ene VPA 88 D. DISCUSSION D. l . CHEMISTRY 89 D.2. ASSAY 89 D.3. PHARMACOKINETICS 91 D. 3.1. Pharmacokinetics in Normal Rats 92 D.3.1.1. Plasma Profile 92 D.3.1.2. Pharmacokinetic Model 1ing 93 D.3.1.3. Pharmacokinetic Parameters 95 D.3.2. Pharmacokinetics in Bile Exteriorized Rats 96 D.3.2.1. Plasma Profile 96 D.3.2.2. Biliary Elimination 97 D.3.2.3. Conjugation 98 D.3.2.4. Choleretic Effect 99 D.3.2.5. Pharmacokinetic Parameters 100 D.3.3. In Vitro Protein Binding 101 SUMMARY AND CONCLUSIONS REFERENCES APPENDIX 106 108 128 vi i i LIST OF TABLES Table Page 1. Serum levels of VPA and its metabolites in patients 7 on VPA monotherapy. 2. Effect of heating time on derivatization. 49 3. Effect of MTBSTFA on the peak areas. 49 4. Calibration curve data for (E)-2-ene VPA in rat plasma. 50 5. Calibration curve data for (E)-2-ene VPA in rat urine. 50 6. Calibration curve data for (E)-2-ene VPA in rat bile. 51 7. Calibration curve data for 4-ene VPA in rat plasma. 52 8. Calibration curve data for 4-ene VPA in rat urine. 52 9. Calibration curve data for 4-ene VPA in rat bile. 53 10. Effect of heating time on the hydrolysis of conjugates. 53 11. Pharmacokinetic parameters of (E)-2-ene VPA in 58 normal rats (Dose=20 mg/kg). 12. Pharmacokinetic parameters of (E)-2-ene VPA in 59 normal rats (Dose=100 mg/kg). 13. Urinary excretion of unconjugated (E)-2-ene VPA in 60 normal rats (Dose=20 mg/kg). 14. Urinary excretion of unconjugated (E)-2-ene VPA in 61 normal rats (Dose=100 mg/kg) 15. Pharmacokinetic parameters of 4-ene VPA in normal 62 rats (Dose=20 mg/kg). 16. Pharmacokinetic parameters of 4-ene VPA in normal 63 rats (Dose=100 mg/kg). 17. Urinary excretion of unconjugated 4-ene VPA in normal 64 rats (Dose=20 mg/kg). 18. Urinary excretion of unconjugated 4-ene VPA in normal 65 rats (Dose=100 mg/kg). 19. Pharmacokinetic parameters of (E)-2-ene VPA in bile- 72 exteriorized rats (Dose=20 mg/kg). ix Table Page 20. Pharmacokinetic parameters of (E)-2-ene VPA in bile- 73 exteriorized rats (Dose=100 mg/kg). 21. Urinary excretion of unconjugated (E)-2-ene VPA in 74 bile-exteriorized rats (Dose=20 mg/kg). 22. Urinary excretion of unconjugated (E)-2-ene VPA in 75 bile-exteriorized rats (Dose=100 mg/kg). 23. Biliary elimination of unconjugated (E)-2-ene VPA in 76 rats (Dose=20 mg/kg). 24. Biliary elimination of unconjugated (E)-2-ene VPA in 77 rats (Dose=100 mg/kg). 25. Pharmacokinetic parameters of 4-ene VPA in bile- 78 exteriorized rats (Dose=20 mg/kg). 26. Pharmacokinetic parameters of 4-ene VPA in bile- 79 exteriorized rats (Dose=100 mg/kg). 27. Urinary excretion of unconjugated 4-ene VPA in 80 bile-exteriorized rats (Dose=20 mg/kg). 28. Urinary excretion of unconjugated 4-ene VPA in 81 bile-exteriorized rats (Dose=100 mg/kg). 29. Biliary elimination of unconjugated 4-ene VPA in 82 rats (Dose=20 mg/kg). 30. Biliary elimination of unconjugated 4-ene VPA in 83 rats (Dose=100 mg/kg). 31. Comparison of pharmacokinetic parameters of 85 (E)-2-ene VPA in the rat. 32. Comparison of pharmacokinetic parameters of 86 4-ene VPA in the rat. 33. Plasma protein binding of 4-ene VPA. 87 X LIST OF FIGURES Figure Page 1. Valproic acid. 1 2. Proposed metabolic pathway for VPA. 5 3. Proposed metabolic pathway for 4-ene VPA. 18 4. Time-lag pharmacokinetic model. 36 5. Total ion chromatogram of t-BDMS derivative (A) 43 of (Z)-2-ene VPA (6.49 min) and (E)-2-ene VPA (7.11 min), and El-mass spectrum (B) of (E)-2-ene VPA. 6. Total ion chromatogram of t-BDMS derivative (A) 44 and El-mass spectrum (B) of 4-ene VPA. 7. Selected-ion-chromatogram of t-BDMS derivatives 46 of an extract from a spiked plasma sample. 8. Selected ion chromatogram of blank plasma sample 47 showing no interfering peaks at the lowest attenuation. 9. Semi logarithmic plots of plasma concentrations of 55 (E)-2-ene VPA versus time following IV dose of 20 and 100 mg/kg in normal rats. Each point represents mean ± 95% confidence limits (N=4). 10. Semilogarithmic plots of plasma concentrations of 56 4-ene VPA versus time following IV dose of 20 and 100 mg/kg in normal rats. Each point represents mean ± 95% confidence limits (N=6). 11 Semilogarithmic plots of plasma concentrations of 67 (E)-2-ene VPA versus time following IV doses of 20 and 100 mg/kg in bile-exteriorized rats. Each point represents mean ± 95% confidence limits (N=4). 12. Semilogarthmic plots of plasma concentrations of 68 4-ene VPA versus time following IV dose of 20 and 100 mg/kg in bile-exteriorized rats. Each point represents mean ± 95% confidence limits (N=6). 13. A typical choleretic effect and excretion rate plot 69 of high dose of 4-ene VPA in the bile of a rat after high dose of 100 mg/kg. 14. A typical cumulative biliary excretion plot of 4-ene VPA versus time after 20 and 100 mg/kg. 70 xi APPENDIX Title Page 1. Total ion chromatogram of methanesulfonyl 129 chloride (Scheme 1) reaction. 2. NMR (80 MHz) spectrum of 3-ene VPA in CDCI3. 130 3. NMR (80 MHz) spectrum of 2-ene VPA in CDCI3. 131 4. NMR (80 MHz) spectrum of 4-ene VPA in CDCI3. 132 5. Plasma levels of (E)-2-ene VPA in normal rats, itg/mL 133 (Dose=20 mg/kg). 6. Plasma levels of (E)-2-ene VPA in normal rats, ng/ml 134 (Dose=100 mg/kg). 7. Plasma levels of 4-ene VPA in normal rats, ng/ml 135 (Dose=20 mg/kg). 8. Plasma levels of 4-ene VPA in normal rats, ng/ml 136 (Dose=100 mg/kg). 9. Plasma levels of (E)-2-ene VPA in bile-exteriorized 137 rats, /ig/mL (Dose=20 mg/kg). 10. Plasma levels of (E)-2-ene VPA in bile-exteriorized 138 rats, /ig/mL (Dose=100 mg/kg). 11. Plasma levels of 4-ene VPA in bile-exteriorized 139 rats, /ig/mL (Dose=20 mg/kg). 12. Plasma levels of 4-ene VPA in bile-exteriorized 140 rats, /ig/mL (Dose=100 mg/kg). xi i SYMBOLS AND ABBREVIATIONS AGA allylglutaric acid Amt amount AUC area under the plasma concentration-time curve from 0-» B biliary BBB blood brain barrier conj conjugated Clg apparent biliary clearance of unconjugated metabolite C l c o n j apparent clearance due to the formation of conjugates C l m e 1 - apparent metabolic clearance CIR apparent renal clearance of unconjugated metabolite Clj apparent plasma clearance of unconjugated metabolite cm centimetre Cone concentration CQ plasma concentration of unconjugated metabolite at zero time CSF cerebrospinal fluid CV coefficient of variation °C degree Celsius DNBA di-N-butylacetic acid (E) trans configuration (E)-2-ene VPA . trans 2-n-propyl-2-pentenoic acid EHC enterohepatic circulation EI electron impact 4-ene VPA 2-n-propyl-4-pentenoic acid eV electron volt xi i i GABA -y-aminobutyric acid GC gas chromatograph GCMS gas chromatograph-mass spectrometer GI gastrointestinal GIT gastrointestinal tract g gram HA hexanoic acid h hour I.D. internal diameter IP intraperitoneal IV intravenous kj apparent elimination rate constant in the log-linear phase between 0=2 hr k 1 0 apparent first-order elimination rate constant from compartment 1. kj2 apparent first-order transfer rate constant from compartment 1 to compartment 2. k2j apparent first-order transfer rate constant from compartment 2 to compartment 1. Lit. literature m/z mass/charge min minute mL mil l i i i t re mm millimetre MTBSTFA N-tertiarybutyldimethylsilyl-N-methyl trifluoroacetamide N normal na not available xiv polyethylene propylglutaric acid polytetrafluoroethylene standard deviation serum glutamic oxaloacetic transaminase serum glutamic pyruvic transaminase tau (time-lag) time tertiary-butyldimethylsilyl elimination half-life tetrahydrofuran millimetre of mercury pressure micron urinary microampere unconjugated (unchanged) uridine diphosphate microgram microlitre 2-n-propylpentanoic acid volume of the central compartment apparent volume of distribution cis configuration XV ACKNOWLEDGEMENTS I thank Dr. J.M. Orr for suggesting the work carried out in this project and for his supervision. I am deeply indebted to Prof. F.S. Abbott for providing all the necessary faci l i t ies, constant guidance, encouragement and friendship throughout my stay here. I am also thankful to the committee members, Prof. J .E . Axelson for the use of his instruments, Prof. D.V. Godin, Prof. J.G. Sinclair and Dr. K.M.J. McErlane for their suggestions and help. I gratefully acknowledge excellent technical assistance and valuable suggestions by Mr. Roland Burton. I am thankful to Dr. A. Acheampong and Mr. Ron Lee for donating compounds used for the identification of some of the metabolites. I am grateful to the following for their contributions to this project in various ways: Mr. Wayne Riggs, Mr. Stephen Clark, Mr. Peter Phill ips, Mr. Peter Martin, Ms. Sue Panesar, Ms. Sheila Tyner, Ms. Neil in Ratanshi, Mr. Ray deSouza and Mr. Dan Chan. 1 A. INTRODUCTION A . l . VALPROIC ACID A.1.1. Background Valproic acid (VPA) is a small, branched-chain fatty acid (Fig 1). Its synthesis was f irst reported over a century ago (Burton 1882). Its anticonvulsant properties were only discovered in 1963 (Meunier et al., 1963; Carraz et al., 1964a; Carraz et al., 1964b) in France. It was introduced as a drug (Depakene, Depakine, Epimyl or Ergenyl) in Europe in 1968 and in North America in 1978. CH^•CH2-CH2 \ CH-COOH / CH^-CH2-CH2 Fig.l Valproic acid VPA is a broad spectrum anticonvulsant used in the treatment of generalized absence, myoclonic and tonic-clonic seizures (Gram and Bentsen 1985; Dulac and Arthuis 1984; Feuerstein et al., 1983; Covanis et al., 1982). It is also effective in controlling fever convulsions (Lee et al., 1986), partial seizures (Bruni and Albright 1983), status epilepticus (Vajda et al., 1978) and photosensitive epilepsy (Harding et al., 1978). It has been also employed to treat Lennox syndrome (Schobben et al., 1980b) and epilepsies refractory to other anticonvulsants (Redenbaugh et al., 1980). The drug is administered orally at a dose of 15-30 mg/kg daily. Its therapeutic blood levels are in the range of 50-100 ^g/ml (Klotz and Schweizer 1980; Gram et al., 1980). 2 A.1.2 Pharmacokinetics in Man After oral administration, VPA is rapidly absorbed from the 61 tract. Peak plasma levels are reached within 1-2 h of its administration (Gugler and von Unruh 1980). VPA is completely bioavailable without any evidence of first-pass elimination by the liver (Perucca et al., 1978a; Klotz and Antonin 1977). Its plasma elimination half- l i fe is between 10-16 h in healthy volunteers (Perucca et a / . , 1978a; Gugler and von Unruh 1980), and 6-10 h in epileptic patients taking other anticonvulsants (Perucca et al., 1978b; Gugler and von Unruh 1980). VPA follows linear pharmacokinetics exhibiting no change in its elimination tj/2 after single or multiple dosing (Gugler et al., 1977). Plasma concentrations of VPA increase linearly when its dose is increased within the therapeutic range and above, up to 60 mg/kg (Nutt and Kupferberg 1979). The plasma clearance of VPA is in the range of 5-10 mL/min (Gugler and von Unruh 1980). It has a small apparent volume of distribution of 0.1-0.4 L/kg (Gugler and von Unruh 1980). VPA is highly bound (>90%) to plasma proteins, especially to albumin (Loscher 1978; Patel and Levy 1979). The free-fraction of VPA is the same in serum and in heparin-treated or EDTA-treated plasma (Cramer et al., 1983). At therapeutic doses, CSF and brain levels of VPA, 3-33 and 7-27 Mg/mL respectively, are directly proportional to free VPA concentration in the plasma (Rapeport et al., 1983; Vajda et al., 1981). Only low levels of VPA, between 1-3% of the maternal serum levels, are secreted into the mother's milk (Nau et al., 1981a; Dickinson et al., 1979b). The serum concentrations of VPA in newborn infants have been reported to be 1.4-1.7 times higher than the maternal serum levels (Nau et al., 1981a; Dickinson et al., 1979b). The elimination t ^ in neonates is 45-47 h, which is three 3 times longer than that in adults (Nau et al., 1981a; Dickinson et al., 1979b). A.1.3 Pharmacokinetics in Animals The disposition of VPA has been studied in many animal species including the pig (Bonora et al., 1979), the monkey (Dickinson et al., 1980), the dog (Loscher 1978; Loscher and Esenwein 1978), the rabbit (Ichimura et al., 1985), the rat (Loscher 1978) and the mouse (Loshcer and Esenwein 1978). The plasma elimination t j / 2 of VPA in the pig, dog, rabbit and mouse has been reported to be 87, 61-84, 75 and 50 min respectively. The shorter t j ^ of VPA in smaller animals has been attributed, in part, to lesser plasma protein binding of drug in such animals, thus providing a larger free-fraction for elimination. The free fraction (xlOO) of VPA in the dog, rat and mouse is 22, 37 and 88%, respectively (Loscher 1978). In an extensive study in the rat, Dickinson et al. (1979a) have reported that plasma elimination of VPA is linear at concentrations below 100 /ig/mL, and non-linear at higher concentrations. VPA undergoes enterohepatic circulation (EHC) in the rat, which produces secondary rises in its blood levels after single dose administration. In bile-exteriorized rats, VPA is eliminated with a t ^ of 11.3 min after the low dose of 15 mg/kg, and 16.7 after the high dose of 100 mg/kg. Approximately 60% of the administered dose is eliminated in the bile of the rat. After oral administration, VPA is rapidly distributed in the body of the rat (Eymard et al., 1971). The drug is mainly distributed in the l iver, kidneys and testes of the rat (Dickinson et al., 1979a). In the rabbit, the highest levels of VPA are found in kidneys, followed by l iver, heart, GI tract and fat (Ichimura et al., 1985). 4 Detailed disposition studies in the brain of the rat have shown that VPA is preferentially distributed in the cerebellum and hippocampus (Mesdjian et al., 1982; Hariton et al., 1984). In another study (Loscher and Nau 1983), the highest levels of VPA were found in substantia nigra of the rat after prolonged treatment. VPA is preferentially accumulated in hypothalamus and medulla of the dog after a constant infusion of VPA (Loscher and Nau 1983). The CSF levels of VPA are identical to its free levels in the plasma of dog (Frey and Loscher 1978). The authors have, however, shown that VPA is actively transported out of CSF, probably by the monocarboxylic acid transport system. The presence of an active transport system across BBB is also supported by experiments in cat (Hammond et al., 1981) , in which VPA is much more rapidly cleared from the brain ( t j / £ 41 min) than the plasma (tj/2 190 min) VPA produces a choleretic effect in the rat, dog and monkey (Dickinson et al., 1982), and cat (Marshall et al., 1984). Bile flow rate increases by 2-3 times the basal flow, 30-60 min after a single dose of 40-60 mg/kg to the rat, cat and dog (Dickinson et al., 1982, Watkins and Klaassen 1981; Marshall et al., 1984). VPA-induced choleresis is primarily due to the osmotic activity (Watkins and Klaassen 1981; Dickinson et al., 1982) of VPA conjugates excreted in bile. A.1.4 Metabolism VPA is extensively metabolized in man (Acheampong et al., 1983; Kochen et al., 1984; Schobben et al., 1980a; Gugler and von Unruh 1980; Loscher 1981; Jakobs 1978;). The proposed metabolic pathway for VPA is shown in Fig 2. The major routes of its metabolism are glucuronidation and /J-oxidation. Up to 20% of the administered dose is recovered as glucuronide in the urine (Granneman et al., 1984b; Bialer et al., 1985). /?-oxidation of 5 CH2=CH-CH2 CHCOOH CH3-CH2-CH2 CH2=CH-CH2 CHCOOH CH2=CH-CH2 CH3-CH2-CH2 CHj-CHg-CH^ CHCOOGlu VPA Glucuronide O H • CH 2 _CH 2*CH 2 CHj-CHg-CH^ 5-OH VPA H00C-CH2-CH2 C CH3-CH2-CH2/ CHCOOH HCOOH 2-Propylglutaric acid I CH3-CH2-CH2-CH-COOH COOH 2-Propylmalonic acid CH 3 -CH 2*CH 2 CHCOOH VPA I T OH CH3-CH-CH2 CH3*CH2-CH2 4-OH VPA I 0 n CH3-C-CH2 CHCOOH 'CHCOOH CH J 'CH^'CH J 4-Keto VPA HOOC-CH, CHCOOH CH3*CH2-CH2 2-Propylsuccinic acid 4-Ene VPA CH3-CH=CH^ I CH3-CH2-CH2/ 3-Ene VPA CHCOOH CH3-CH2-CH^ C-COOH CH j"CHg" 2-Ene VPA OH 1 CH3-CH2-CH CH3-CH2-CH2/ 3-OH VPA CHCOOH CH 3-CH 2-C^ CHCOOH CH3-CH2-CH2 3-Keto VPA 4,4'-Diene VPA CH,=CH-CH C-COOH CH2*CH2*CH2 (E)-2,4-Diene VPA CH,-CH=CH 3 \ C-COOH CHj-CH-CH 2(E),3'(E)-Diene VPA Fig. 2. Proposed metabolic pathway for VPA. 6 VPA produces cis and trans isomers of 2-ene VPA, and 3-keto VPA. Abbott et al. (1986) have reported that serum levels of trans 2-ene VPA and 3-keto VPA are 11.9 and 7.7%, respectively, of VPA serum levels at steady state in pediatric patients. The minor routes of VPA metabolism are u-hydroxylation, w-1 hydroxylation, -y-dehydrogenation and 6-dehydrogenation that produce 5-hydroxy VPA, 4-hydroxy VPA, 3-ene VPA and 4-ene VPA, respectively, as their f i rst metabolites. A hydroxamate metabolite of VPA has been recently identified in the urine of patients on VPA therapy (Libert et a7., 1986). The serum levels of 12 metabolites of VPA in patients on VPA monotherapy (Abbott et al., 1986) and their possible routes of formation are shown in Table 1. The metabolic fate of VPA has been extensively studied in several animal species including rat, rabbit, mouse, dog and monkey (Matsumoto et al., 1976; Jakobs and Loscher 1978; Ferrandes and Eymard 1977, Schobben et al., 1980a). VPA metabolism in animals, especially in the rat, is similar to that in man. A total of 48 compounds have been identified as metabolic products of VPA and its intermediate metabolites in the rat (Granneman et al., 1984c). The major interest in the metabolites of VPA emerges from their possible contribution to the pharmacologic and toxicologic effects of this drug. The /J-oxidation product, (E)-2-ene VPA, which is the major metabolite in the plasma of man, has been found to be as potent an anticonvulsant as VPA (Loscher et al., 1984). This metabolite has been considered to contribute significantly to the total VPA activity. On the other hand, VPA-induced hepatic damage has been suggested to be caused by a minor metabolite, 4-ene VPA (Kesterson et al., 1984). 7 TABLE 1. SERUM LEVELS OF VPA AND ITS METABOLITES IN PATIENTS ON VPA MONOTHERAPY3 Metabolite Mean Cone % VPA (/jg/mL) Level VPAb 46.4 100 (E)-2-ene VPAC 5.53 11.9 3-keto VPAC 3.59 7.7 (E)-2,3'-diene VPA c ' d 2.95 6.4 3-ene VPAd 0.94 2.0 4-ene VPAe 0.67 1.4 4-keto VPAf 0.40 0.9 4-OH VPAf 0.38 0.8 (E)-2,4-diene VPA c ' e 0.20 0.4 2-Propylglutaric acid^ 0.20 0.4 (Z)-2-ene VPAC 0.19 0.4 5-OH VPA9 0.18 0.4 2-Propylsuccinic acid^ 0.04 0.1 a, (Abbott et a l . , 1986); b, parent drug; c, /8-oxidation; d, -y-dehydrogenation; e, 6-dehydrogenation; f, (w-1) hydroxylation; g, w-hydroxylation. 8 A.1.5 Mechanism of Action VPA increases whole brain GABA content in the mouse (Godin et al., 1969; Nau and Loscher 1982) by inhibiting 7-aminobutyric acid transaminase (Fowler et al., 1975), an enzyme that breaks down GABA. VPA is also a potent inhibitor of succinic semialdehyde dehydrogenase (Harvey et al., 1975) and aldehyde reductase (Whittle and Turner 1978), enzymes present in the degradation pathway of the GABA shunt. It has been also reported that VPA administration raises GABA levels by increasing its synthesis from glutamate (Nau and Loscher 1982). When VPA is administered to mice, the brain GABA levels are significantly elevated, with a parallel increase in the enzymatic activity of glutamate decarboxylase, the enzyme responsible for GABA synthesis (Nau and Loscher 1982). The decline in GABA levels and glutamate decarboxylase activity is , however, much slower than the decline of VPA levels in the brain (Nau and Loscher 1982). The increase in GABA levels in different areas of the brain is not uniform (Hariton et al., 1984). The highest GABA levels are found in the olfactory bulbs and hypothalamus of the rat (Hariton et al., 1984). VPA increases the threshold potential for excitation of nerve membrane by blocking sodium and potassium conductance (VanDongen et al. 1986). VPA has also been reported to depress the firing rate of spontaneously active cortical cel ls, whose time course is parallel to the onset of anticonvulsant activity in the rat (McLean and Macdonald, 1986; Kerwin and Olpe 1980). In another study, the pharmacologic activity was evident as early as 1 min after IP administration of VPA to rats (Schmutz et al., 1979). Such a rapid onset of action was suggested to be due to the direct postsynaptic inhibitory effect of VPA that was independent of its ability to raise GABA levels (Schumtz et al., 1979; Kerwin and Olpe 1980). 9 Another possible mechanism of action of VPA is ascribed to its ability to significantly decrease aspartate levels in the brain of mouse (Schechter et al., 1978) and the rat Chapman et al., 1982; Chapman et al., 1983; Patsalos and Loscelles 1981). The time course of VPA-induced reduction in the cerebral aspartate levels coincides with the period of protection from audiogenic seizures in mice (Schechter et al., 1978). In actual practice, the antiepileptic activity of VPA may be due to its combined effect on neurotransmitters in the brain, and firing rate of subcortical cel ls. A.1.6 Side Effects VPA is considered a reasonably safe drug that shows mostly transient and mild side effects (Stefan et al., 1984; Feuerstein et al., 1983; Bruni and Albright 1983). The most common adverse effects are GI disturbances including nausea, vomiting, abdominal cramps, diarrhea and indigestion in some patients (Beran et al., 1980; Simon and Penry 1975). VPA has been noticed to cause stomatitis (Russo 1981), pancreatitis (Wyllie et al., 1984; Murphy et al., 1981), change in appetite and weight gain (Feuerstein et al., 1983; Bruni and Wilder 1979). VPA produces mild alopecia (Bruni and Albright 1983), and blood disorders such as thrombocytopenia (Winfield et al., 1976) and neutropenia (Symon and Russell 1983). Several authors (Dulac and Arthuis 1984; Jeavons and Clark 1974) have reported increased alertness, improved behavior and improvement of intellectual functions following VPA administration. A very few patients on VPA monotherapy have experienced drowsiness (Stefan et al., 1984) and reversible dementia (Zaret and Cohen 1986). VPA has been incriminated in causing resting and postural tremors (Coultar et a l . , 1980; Bruni and Wilder 1979;). VPA may produce hypothyroidism (Salvatoni et al., 1983), in rare instances. 10 A serious toxic effect of VPA is teratogenicity (Lammer et al., 1987; Di Carlo et al., 1986). VPA is embryotoxic in the rabbit, rat (Whittle 1976) and mouse (Nau and Loscher 1984; Brown et al., 1980). The most pronounced effects are neural tube defects (exencephaly), skeletal abnormalities, embryolethality and reduced fetal weight (Nau and Loscher 1984; Kao et al., 1981). Expectant mothers on VPA therapy have a high risk (1-2%) of bearing offspring with minor abnormalities (Koch et al., 1983) or major malformations including the neural tube defect spina bifida aperta (Lammer et al., 1987; Robert 1983; Bjerkedal et al., 1982; Jeavons 1984). Hurd and coworkers (1981, 1982) have reported that VPA binds to zinc, and lowers zinc and selenium levels in the plasma of animals and man. The authors have suggested that VPA-induced deficiency of these rare metals may be responsible for the teratogenic effects of VPA. They (Hurd et al., 1983) have further proposed that zinc supplements may reduce VPA-induced teratogenic effects. Trotz et al. (1987) have recently reported that folinic acid administration markedly reduces VPA-induced neural tube defects in the mouse. A.1.7 Hepatotoxicitv The most serious side effect of VPA is fatal hepatotoxicity in young children (Zafrani and Berthelot 1982). Over 80 cases of fatal hepatic failure, on VPA therapy, have been reported (Bjorge and Baill ie 1985). In these patients, typical symptoms of anorexia, vomiting, lethargy, jaundice, hepatic failure and terminal coma developed within 1-4 months of VPA therapy (Zimmerman and Ishak 1982). Autopsy of the liver showed microvesicular steatosis accompanied by cirrhosis or necrosis (Zimmerman and Ishak 1982). The hepatic injury and lesions caused by VPA (Keene et al., 1982) are similar to those of Jamaican Vomiting Syndrome (JVS) and 11 Reye-like syndrome (RS) produced by hypoglycin A and 4-pentenoic acid (Glasgow and Chase 1975). Since 4-ene VPA, a VPA metabolite is structurally similar to the hepatotoxin, 4-pentenoic acid, VPA-induced hepatotoxicity has been ascribed to its metabolite, 4-ene VPA (Zimmerman and Ishak 1982). Delay in the onset of illness until after 1 month of drug administration in 80% of cases also suggests that a metabolic idiosyncracy rather than hypersensitivity reaction is the cause of hepatic failure. This hypothesis is supported by the lack of hallmarks of hypersensitivity such as rash, itch and urticaria in patients. Moreover, co-administration of other anticonvulsants such as phenytoin and phenobarbital, which are known enzyme inducers, enhances the hepatotoxicity of VPA in patients. In young children of 2 years or less in age, the incidence of fatal hepatic failure of 1:7000 in individuals on VPA monotherapy is increased to 1:500 on VPA polytherapy (Dreifuss et al., 1987). Similarly, phenobarbital-treated rats show higher mortality and microvesicular steatosis with low doses of VPA compared to no deaths and no fatty liver in animals receiving VPA alone (Lewis et al., 1982). These results suggest that enzyme inducers may enhance VPA metabolism to form a hepatotoxic metabolite (Granneman et al., 1984a). Several biochemical abnormalities have been reported in the literature, that may or may not be related to overt hepatotoxicity. VPA administration produces a temporary elevation of the hepatic enzyme SGOT in man (Willmore et al., 1978) and rat (Cotariu et al., 1987), but a decrease in the serum levels of SGPT in the rat (Kesterson et al., 1984; Cotariu et al., 1987). VPA, especially when co-administered with other anticonvulsants, produces hyperammonemia (Ratnaike et al., 1986; Zaccara et al., 1985). It inhibits gluconeogenesis in the rat (Turnbull et al., 1983) and in vitro in isolated rat hepatocytes (Rogiers et al., 1985; Turnbull et al., 1983). VPA administration causes impaired fatty acid metabolism 1 2 including reduced fatty acid synthesis, and decreased ^-oxidation (Becker and Harris 1 9 8 3 ; Kesterson et al., 1 9 8 4 ) . Rats receiving VPA have been reported to develop hypocarnitinemia and mitochondrial swelling of the liver cells (Sugimoto et al., 1 9 8 7 ) . These abnormalities are corrected by administering supplements of L-carnitine (Sugimoto et al., 1 9 8 7 ) . Chronic administration of VPA produces hyperglycinemia in the rat (Martin-Gallardo et al., 1 9 8 5 ; Cherruau et al., 1 9 8 1 ) . It has been also reported to suppress plasma levels of corticotropin (ACTH) in children (Kritzler et al., 1 9 8 3 ) . A . 2 . (E)-2-ENE VALPROIC ACID A . 2 . 1 Pharmacologic Activity Ten metabolites of VPA have been tested for anticonvulsant activity in animals (Loscher and Nau 1 9 8 5 ; Loscher 1 9 8 1 ; Schafer et al., 1 9 8 0 ) . Nine of the metabolites significantly raise threshold levels for maximal electroshock and/or pentylenetetrazole (PTZ)-induced seizures in mice (Loscher and Nau 1 9 8 5 ; Loscher 1 9 8 1 ) . The two most active compounds, ( E ) - 2 -ene VPA and 4-ene VPA, are 8 0 - 9 0 % as active as VPA on a molar basis after IP administration to mice (Loscher and Nau 1 9 8 5 ) . The ED^Q for (E)-2-ene VPA is 6 6 mg/kg in rats exhibiting spontaneously occuring 'petit mal' seizures, 9 0 mg/kg in gerbils with generalized tonic-clonic seizures, and 2 2 5 mg/kg against PTZ-induced seizures in mice (Loscher et al., 1 9 8 4 ) . The ratio of ED 5 0 for (E)-2-ene VPA/VPA is 0 . 8 1 , 1 . 2 3 and 0 . 6 9 for the above seizure models in the rat, gerbil and mouse, respectively. Loscher and Nau ( 1 9 8 3 ) have reported that since the brain levels of (E)-2-ene VPA are slightly less than those of VPA after equimolar doses of the two, the relative pharmacologic activity of (E)-2-ene VPA is 1 . 3 times higher than that of VPA. After oral administration, (E)-2-ene VPA is approximately half 13 as potent as VPA in chemically induced seizure models in mice (Keane et a7., 1985). The low potency of (E)-2-ene VPA after oral administration may be due to its lesser absorption than VPA. Overall, (E)-2-ene VPA possesses a broad spectrum of anticonvulsant activity in a number of animal models. The mechanism of action of (E)-2-ene VPA is not known, except that it elevates GABA levels in the brain to the same extent as VPA (Keane et a7., 1985). A.2.2 Pharmacokinetics The pharmacokinetics of (E)-2-ene VPA have not been well characterized. In some of the studies, the parent drug VPA was administered, and the plasma or tissue levels of (E)-2-ene VPA were monitored (Loscher and Nau 1982). On oral administration of VPA via drinking water for 12 days, the plasma levels of (E)-2-ene VPA (0.7 ng/ml) were 20% of VPA plasma levels (3-4 ng/ml) in mice (Loscher and Nau 1982). After an IP dose of VPA to mice, the plasma elimination t\/2 ° f (E)-2-ene VPA was 130 min (Nau and Loscher 1982). On constant-rate administration of VPA for 7 days to mice, the elimination t j ^ of (E)-2-ene VPA was found to be 70 min (Nau and Zierer 1982). In man, the apparent elimination t j ^ of (E)-2-ene VPA has been reported to be 43 h (Pollack et a7., 1986). In human neonates, (E)-2-ene VPA is eliminated with a half-life of 47 h, which is identical to that for VPA (Nau et a7., 1981a). Tissue distribution studies have shown that hepatic concentration of (E)-2-ene VPA in mice was much lower than that for VPA (Nau and Loscher 1985). The liver-to-plasma concentration ratio for (E)-2-ene VPA was 0.1-0.5 and for VPA, 1.5-3 in mice (Nau and Loscher 1985). The brain levels of (E)-2-ene VPA are 3% of its total plasma levels at steady-state in mice (Nau and Zierer 1982). CSF levels of (E)-2-ene VPA are much lower than its 14 free concentration in the plasma of dog, but its brain levels are higher than its CSF levels (Loscher and Nau 1983). These results suggest that the metabolite is probably bound to the brain tissues. This hypothesis is supported by studies in rats showing that, on prolonged administration of VPA, a marked increase of (E)-2-ene VPA levels occurs in some regions of the brain, especially hippocampus, substantia nigra, superior and inferior colliculus and medulla (Loscher and Nau 1983). Moreover, after the withdrawl of VPA, (E)-2-ene VPA is cleared much more slowly than VPA from the brain of mice (Loscher and Nau 1982; Nau and Loscher 1982). The apparent elimination t ^ of (E)-2-ene VPA in the brain of mice is 240 min, which is 5 times longer than the 50 min half-life for VPA (Nau and Loscher 1982) . In a few studies, (E)-2-ene VPA was administered to animals and its pharmacokinetic parameters were determined (Nau and Zierer 1982). When equal oral doses of (E)-2-ene VPA and VPA are given individually to mice, the maximum plasma concentration of (E)-2-ene VPA is approximately 70% of that of VPA (Nau and Loscher 1985). After a constant-rate administration of (E)-2-ene VPA to mice, its plasma elimination is reported to be 71 min, and plasma clearance, 339 mL/h.kg (Nau and Zierer 1982). In the dog, (E)-2-ene VPA is eliminated from the plasma with a t ^ of 1.8 h, and has an apparent volume of distribution of 0.25 litres/kg (Loscher and Nau 1983) . A recent abstract (O'Connor et al., 1986) has reported that in rats, after IV bolus doses of 25, 75 and 225 mg/kg of (E)-2-ene VPA, the serum clearance changes biphasically from 3.95 to 3.76 mL/min.kg. The apparent volumes of distribution are 471 and 718 mL/kg at the above doses, respectively (O'Connors et al., 1986). (E)-2-ene VPA is more highly bound than VPA to plasma proteins. In 15 the plasma of mouse, dog and man, (E)-2-ene VPA is bound up to 97, 97 and 99.5% (Nau and Loscher 1985; Loscher and Nau 1983) respectively. Little information is available on the metabolism of (E)-2-ene VPA. Following oral administration of (E)-2-ene VPA to mice, three metabolites were detected in the plasma in the following decreasing order: 3-keto VPA, VPA, and 5-hydroxy VPA (Nau and Loscher 1985). A.2.3 Side Effects (E)-2-ene VPA shows no obvious side effects at doses required to control seizures in rats and gerbils (Loscher et al., 1984). It is, however, more sedating than VPA in mice as determined by rotarod (Keane et al., 1985) and chimney testing (Loscher et al., 1984). The LD 5 0 for (E)-2-ene VPA in mice is 760 mg/kg, which is similar to 810 mg/kg for VPA (Loscher et al., 1984). (E)-2-ene VPA is free of embryotoxic effects in mice even at extremely high doses of 600 mg/kg (Loscher et al., 1984). In whole embryo culture studies (Lewandowski et al., 1986), (E)-2-ene VPA concentrations of 200 /xg/g show no abnormal development of the embryo, whereas VPA levels of 40 /jg/g and above clearly induce teratogenic effects. Moreover, at equimolar concentrations of (E)-2-ene VPA and VPA in the culture medium, (E)-2-ene VPA levels in the embryo are less than those of VPA (Lewandowski et al., 1986). Kesterson et a/. (1984) have shown that (E)-2-ene VPA does not produce hepatic steatosis in the rat. On its administration to rats, the clinical features such as serum urea nitrogen, SG0T, SGPT, ammonia levels and ketone bodies are unaltered. These results strongly suggest that (E)-2-ene VPA may be free of hepatotoxicity seen with VPA. It may, therefore, be a suitable alternative antiepileptic agent free of serious side effects of VPA, namely embryotoxicity and hepatotoxicity. 16 A.3. 4-ENE VALPROIC ACID A.3.1 Toxicity Several investigators have reported the toxicity of 4-ene VPA. In vitro studies in rat hepatocytes have shown that 4-ene VPA significantly raises lactic dehydrogenase (LDH) index, a measure of hepatocyte toxicity (Kingsley et al., 1983). The metabolite, 4-ene VPA inhibits gluconeogenesis in isolated rat hepatocytes (Rogiers et al., 1985). It is also a strong inhibitor of /J-oxidation of medium chain fatty acid in homogenates of rat liver (Bjorge and Baillie 1985). Kesterson et al. (1984) have reported that 4-ene VPA produces mitochondrial lesions in hepatocytes, and inhibits 8-oxidation in the rat. It also causes severe microvesicular steatosis in the livers of animals. The authors (Kesterson et al., 1984) concluded that two different mechanisms may be responsible for ^-oxidation inhibition by VPA and 4-ene VPA in rats. LDgQ for 4-ene VPA in mice is 1000 mg/kg, on IV administration, compared to 630 mg/kg for VPA (Loscher and Nau 1985). A.3.2 Metabolism The metabolism of 4-ene VPA has been recently studied in animals (Rettenmeier et al., 1986; Rettenmeier et al., 1985). It is metabolized by the fatty acid /J-oxidation complex, and by cytochrome P450-mediated hydroxylation and epoxidation pathways (Rettenmeier et al., 1985). In isolated rat liver perfusion studies, eight metabolites of 4-ene VPA were identified namely 2,4-diene VPA, 3-hydroxy 4-ene VPA, 3'-oxo 4-ene VPA, 5'-hydroxy 4-ene VPA, 5-hydroxy, 4,5-di-hydroxy VPA lactone, AGA and PGA (Rettenmeier et al., 1985). The metabolism of 4-ene VPA, in the liver 17 perfusion studies, was affected by the length of the perfusion time. When the duration of the experiment was short (20 min), approximately 58% of the 4-ene VPA dose was recovered unchanged and 15% was converted to metabolites in the perfusate. Only 2% of the dose was excreted in bile. In a longer perfusion time study (60 min), 29% of the injected 4-ene VPA was unchanged, and 13% was collected as the sum of all the metabolites in the perfusate. A higher proportion of dose, 12%, was eliminated in the bile. Following 4-ene VPA administration to the monkey, a total of 20 metabolites were detected in the urine (Rettenmeier et a7., 1986). Three major metabolites were 4-ene VPA glucuronide (38.6%), (E)-2,4-diene VPA (8.9%) and 3'-oxo VPA (8.0%). Approximately 59% of the dose is recovered as unchanged 4-ene VPA and its metabolites, collectively, in the urine of the monkey. The proposed metabolic pathway for 4-ene VPA is shown in Fig 3. A.3.3 Pharmacokinetics Very l i t t l e information is available on the disposition of 4-ene VPA in animals or man. Since a very small fraction of the dose of VPA is converted to 4-ene VPA, the plasma levels of the latter are either too low to determine its pharmacokinetics or are undetected. In children on VPA monotherapy, the serum levels of 4-ene VPA are between 0.16-1.22 /ig/mL, which is only 1% of plasma VPA levels (Abbott et a7., 1986). Similarly in rats, only a small fraction (0.05%) of injected VPA is recovered as 4-ene VPA in the urine (Granneman et a7., 1984c). A recent article (Pollack et a7., 1986) has reported that the plasma elimination t ^ of 4-ene VPA in man, following VPA administration, is 50.7 h. In one study, 4-ene VPA was administered to the monkey (Rettenmeier et a7., 1986). The plasma concentration-time curve in the monkey is bi-exponential with a terminal elimination half-life of 2.3-3.6 h, and a 18 CO,H H O . C CO.H CO.H OH CO,H CO,H CO.H CO.H O H C CO,H CO.H OH CO,H CO,H MO> CO,H .OH CO,H ^CHO CO.H •*CO,H Fig. 3. Proposed metabolic pathway for 4-ene VPAa. (a, Rettenmeier et al., 1985) 19 clearance of 2.8-2.0 mL/min.kg. A large proportion of the dose (39%) is eliminated as glucuronide and 5% is excreted unchanged in the urine of the monkey (Rettenmeier et al., 1986). The plasma protein binding of 4-ene VPA in the monkey is 58-78%. A.4. RATIONALE AND OBJECTIVES I. The clinical effectiveness of valproic acid is not always related to its serum levels. Following its administration, a delayed onset of its antieplileptic activity ranging from hours to weeks has been observed (Jeavons and Clark 1974; Rowan et al., 1979). Similarly, after the drug is withdrawn, a carry-over effect both in animals and humans ranging from weeks to months has been reported (Pellegrini et a/ . , 1978; Harding et al., 1978; Lockard and Levy 1976). A possible explanation for these temporal effects may be the formation of a pharmacologically active metabolite with longer half-l ife. (E)-2-ene VPA, a major mono-unsaturated metabolite of VPA, has been found to be as active as VPA in several animal models of epilepsy. A knowledge of its disposition would be needed to estimate its contribution to the overall action of VPA. Moreover, recent studies have shown that (E)-2-ene VPA appears to be free of any serious side effects associated with VPA, especially hepatotoxicity and embryotoxicity. It has been proposed (Nau et al., 1984) that (E)-2-ene VPA may be used as an alternative anticonvulsant. Since it is not uncommon to develop more effective and safer drugs from drug metabolites, i t appears that (E)-2-ene VPA may also join the ranks of now commonly used drugs such as oxazepam and acetaminophen, which were init ial ly identified as metabolites of the 'parent' drugs diazepam and phenacetin, respectively. The pharmacokinetics of (E)-2-ene VPA have not been well characterized due to a lack of the 20 availability of pure compound in sufficiently large quantities. Dr. Abbott's laboratory has been a leader in the synthesis of several metabolites of VPA, including (E)-2-ene VPA. It was, therefore, most logical to use those facilities to synthesize (E)-2-ene VPA, develop a sensitive assay for (E)-2-ene VPA and study its disposition in the rat. II. The metabolites of a drug are often responsible for drug-related toxicities. Hepatotoxicities of acetaminophen and iproniazid, caused by their respective metabolites, are classic examples of metabolite-induced side effects (Mitchell et a7., 1973; Nelson et a7., 1978). Similarly, Reye-like syndrome produced by VPA in children has been attributed to its metabolite, 4-ene VPA (Rettenmeier et a7., 1986; Kesterson et a7., 1984). The concentrations of this toxic metabolite, 4-ene VPA, in serum and urine of patients or animals receiving VPA, are normally either undetectable or very low. When a patient develops symptoms of severe hepatic damage, the serum levels of 4-ene VPA are increased several fold over the normal values (Kochen et a7., 1983). Moreover, there is a latent period of 1-4 months, after the initiation of VPA therapy, before clinical symptoms of hepatotoxicity appear. These observations suggest that the toxic metabolite levels may gradually build up during the course of therapy, or it may be rather slowly eliminated from the system. A pharmacokinetic study of 4-ene VPA, including its biliary elimination and/or enterohepatic circulation, may explain to some extent its role in VPA-induced hepatotoxicity. 21 The major objectives of this project were: 1. To synthesize (E)-2-ene VPA and 4-ene VPA in sufficiently large quantities to carry out disposition studies. 2. To develop capillary GCMS assay methods for the estimation of (E)-2-ene VPA and 4-ene VPA in the biological fluids of the rat. 3. To determine the effect of dose, at two dose levels, on the pharmacokinetics of (E)-2-ene VPA and 4-ene VPA in the rat. 4. To study the biliary elimination of (E)-2-ene VPA and 4-ene VPA in bile-exteriorized rats. 5. To develop a pharmacokinetic model that may be applied to drugs showing enterohepatic circulation. 22 B. EXPERIMENTAL B . l . MATERIALS B. 1.1. Chemicals Chemicals and solvents were reagent grade, and were obtained from the following sources. a. Aldrich Chemical Company, Inc. (Milwaukee, WIS) Triethylamine, potassium hydride (35% oil dispersion), diisopropylamine, n-butyl1ithium (1.6 M in hexane), hexanoic acid, MTBSTFA reagent. b. BDH Chemicals (Toronto, Canada) Bromine, sodium hydroxide pellets, concentrated sulfuric acid, anhydrous sodium sulfate (granular), hydrochloric acid. c. Eastman Organic Chemicals (Rochester, N.Y.) Propionaldehyde, methansulfonyl chloride. d. Fisher Scientific Company (Fair Lawn, N.J.) Quinoline, pyridine. e. Mallinkrodt Chemicals (St. Louis, MI) Para-toluenesulfonyl chloride. f. Matheson Coleman and Bell Company. (Norwood, OH) Phosphorous tribromide, 1-bromopropane. 23 g. Sigma Chemical Company, (St. Louis MO) Valeric acid. h. Caledon Laboratories (Georgetown, Canada) Ethyl acetate (distilled-in-glass grade). B.1.2. Instrumentation Nuclear Magnetic Resonance Spectrometry Proton NMR spectra were recorded on a Bruker WP-80 spectrometer at the Department of Chemistry, UBC, using deuterated chloroform as solvent and tetramethylsilane as the internal standard. Gas Chromatography Mass Spectrometry A Hewlett Packard 5987A GCMS (5880A GC) equipped with a 2623A HP terminal and a 59824A scanning interface was used for GCMS analysis. A fused si l ica capillary column (25 metre x 0.32 mm I.D.) coated with a bonded phase (0.25 (i film of 0V-1701) was obtained from Quadrex Corporation, New Haven, Connecticut. For packed column chromatography, a Hewlett Packard 5700 GC interfaced to a Varian Mat 111 mass spectrometer with an on-line Varian 620L data analysis computer system was used. A glass column (2 metre x 2 mm I.D.) packed with 3% Desxil 300 (carborane/silicone) on 100/120 Supelcoport (Supelco Inc., Bellefonte, Pennsylvania) was used. 24 B.2. METHODS B.2.1. Synthesis B.2.1.1. (E)-2-ene VPA I. Valeric acid (1 mole), ethanol (3 moles), 1 mL cone, sulfuric acid and 400 mL benzene were refluxed overnight in a 1-litre round bottom flask fitted with a Dean-Stark apparatus. The contents were washed thoroughly with water, dried over anhydrous ^ S O ^ and disti l led to remove benzene at 66°C. The residue was distilled to collect ethyl valerate (0.78 moles) at 140°-143°C (Lit. 145°-146°C, The Merck Index, 1976a). A flame-dried 1-litre three-necked flask containing a magnetic stirring bar, and equipped with a dropping funnel with a septum inlet, a N 2 inlet, and an air condenser attached to a mercury bubbler was cooled in an ice-salt mixture. Diisopropylamine (0.25 moles, dried over calcium hydride, and distilled) and 200 mL tetrahydrofuran (THF) were placed in the flask under N 2 atmosphere. n-Butyllithium (0.25 moles) was added dropwise to the stirring mixture, followed by dropwise addition of ethyl valerate (0.25 moles) dissolved in 50 mL THF. The ice-salt mixture was replaced with a dry ice-acetone mixture. Propionaldehyde (0.25 moles) dissolved in 25 mL THF was added dropwise. The contents were stirred for 30 min, and the reaction quenched with 6N HCI (0.9 moles). The mixture was extracted with ether twice, the combined ether extracts washed with a weak solution of NaHC03 (5%) followed by three washings with water. The ethereal layer was dried over anhydrous Na^O^, and the solvent removed using a flash-film evaporator. Distillation of the residue gave 3-hydroxy VPA ethyl ester, at 92°-95°C at 4.5 mm (Lit. 105°C at 8 mm, Blaise and Bagard 1907) 3-hydroxy VPA ethyl ester (0.05 moles) dissolved in 40 mL methylene chloride (distilled in glass) was placed in a three-necked flask assembly 25 as described above, except that a drying tube was used in place of the mercury bubbler. A cold solution of triethylamine (0.075 moles) in 10 mL of methylene chloride was added gradually, followed by dropwise addition of methanesulfonyl chloride (0.052 moles) in 10 mL methylene chloride. The contents were stirred for 30 min, filtered and the precipitates were washed with ether. The washings were combined with the fi ltrate, and the solvent removed using a flash film evaporator. The residue was dissolved in 50 mL THF, and cooled to 0°C in an ice bath. Clean potassium hydride (KH, 0.1 mole), carefully cleaned with petroleum ether to remove mineral o i l , was added with the aid of THF. The flask was protected with a drying tube, and kept in ice for 2 h. The contents were stirred for 12 h at room temperature. Excess KH was decomposed by dropwise addition of cold glacial acetic acid (3 mL) in 5 mL THF, followed by gradual addition of water. The contents were extracted with ether, and analyzed on GCMS. Several peaks were obtained on the chromatogram indicating that the reaction did not proceed as expected. II. To a 250 mL 3-necked flask equipped with a drying tube, a dropping funnel and a magnetic stirring bar was added 3-hydroxy VPA ethyl ester (0.1 mole) in 20 mL dry pyridine. The flask was cooled in an ice-salt mixture. Toluenesulfonyl chloride (0.15 mole) in 40 mL pyridine was added dropwise, and the contents stirred for 2 days at room temperature. After adding ice-water, the mixture was extracted with C H C I 3 . The organic layer was washed first with dilute H 2 S 0 4 , then with NaHC0 3 (5%) and finally with water. The extract was dried with anhydrous ^ S O ^ the chloroform removed and the residue refluxed with IN NaOH for 4 h. After stirring the mixture for 2 days at room temperature, it was neutralized with 2N HC1, extracted with ether and worked up as described in I. Distillation of the residue gave a 26 fraction (Fl) at 110°-117°C/30 mm. This fraction was stirred overnight with 50 mL 4N NaOH, refluxed for 4 h, washed with ether, and acidified with 50% HCI. The acidified extract was re-extracted with ether and worked up as described in I. Distillation of the residue gave fraction (F2) at 125°C/4 mm. The product was identified to be 3-ene VPA (Lit. boiling point 116°C at 8 mm, Blaise and Bagard 1907). III. Diisopropylamine (0.8 mole, dried and distilled) in 750 mL of dry THF was placed in a 1-litre three-necked flask as described above in I. n-Butyllithium (0.8 mole) was added dropwise, followed by dropwise addition of valeric acid (0.4 mole) in 50 mL THF. The mixture was stirred for 20 min, and the ice bath was removed. Propyl bromide (0.44 moles, dried and distilled) in 40 mL THF was added over a period of 5 min, and contents stirred for 3 h at room temperature. The reaction was quenched with 400 mL 6N HCI, extracted with ether twice and worked up as before. The residue was disti l led at 123°-125°C at 14 mm (Lit 120°-121°C/14 mm, The Merck Index, 1976b) to obtain Valproic acid (0.22 mole). Valproic acid (0.14 mole), bromine (0.15 mole) and PBr3 (0.5 mole) were placed in a 250 mL flask equipped with a water reflux condenser attached to an all-glass gas absorption device, consisting of an inverted funnel dipped in water. The flask was heated in an oil bath at 70°C for 1 h, and then at 100°C for 3 h. Excess Br 2 and the reaction product HBr were completely removed by distillation under reduced pressure of a water pump. The residue was distil led under high vacuum at 73°C at 0.01 mm to obtain 2-bromo VPA (0.09 mole). 2-Bromo VPA (0.09 mole) and 100% ethanol (0.26 mole) were refluxed for 48 h with 20 mL benzene and 1 mL cone. H2S04 in a round bottom flask fitted with a Dean-Stark apparatus. The contents were washed with NaHC03 27 (5%), water, and dried over anhydrous Na2S04. Benzene and unreacted ethanol were removed by disti l lation, and 2-bromo VPA ethyl was collected by distil lation under vacuum at 106°-115°C/11 mm. 2-Bromo VPA ethyl ester (0.06 mole) and quinoline (0.18 mole, dried, and distilled) were stirred rapidly in a 100 mL round bottom flask equipped with a Claissen s t i l l head fitted with an air condenser protected by a CaC^ guard tube. The flask was rapidly heated on a heating mantle to obtain fractions Fl at 183°-186°C and F2 at 192°C at atmospheric pressure. Fl and F2 contained different proportions of cis and trans isomers of 2-ene VPA ethyl ester along with quinoline, a reactant. The fractions Fl and F2 were combined and the mixture was dissolved in ether. The organic layer was washed with 50 mL IN HC1 and then with water. The ethereal layer was dried over anhydrous Na^O^, and ether removed using a flash film evaporator to obtain 2-ene VPA ethyl ester, as verified by GCMS. The 2-ene VPA ethyl ester (0.045 mole) was refluxed for 6 h with 25 mL of 4N NaOH and 1 mL EtOH in a 100 mL flask. The contents were stirred overnight at room temperature, washed with ether twice, and acidified with 4N HC1. The mixture was extracted with ether and worked up as described earlier. Distillation of the residue at 112°-114°C at 2.4 mm (Lit 103°C/1 mm for (E)-2-ene VPA, Neuman and Holmes 1971) gave 2-ene VPA (0.029 mole). GCMS analysis showed that it was a 3:1 mixture of trans:cis isomers of 2-ene VPA. The product was dissolved in a small volume of chloroform, and stored at -20°C for several days to harvest crystals of (E)-2-ene VPA. B.2.1.2. 4-Ene VPA Di isopropyl amine (0.4 mole) and 300 mL dry THF were stirred under a N 2 atmosphere in a 1-litre 3-necked flask equipped as described above in B.2.1.1.I. n-Butyllithium (0.4 mole) was added dropwise, followed by 28 dropwise addition of valeric acid (0.2 mole) in 75 mL of THF. The ice-bath was removed, and allyl iodide (0.2 mole) in 25 mL THF was added gradually over a period of 10 min. The reaction was allowed to proceed for 3 h at room temperature, and quenched with 200 mL of ice-cold 20% HCI. The mixture was extracted with ether twice, and the organic layer worked up as before. Distillation of the residue gave 4-ene VPA (0.12 mole) at 103°-105°C at 6.5 mm (Lit 95°-100°C at 5 mm, Campos and Amaral 1965). B.2.2. Capillary GCMS Assay B.2.2.1. Standards: Plasma, urine or bile standards for (E)-2-ene VPA and 4-ene VPA were separately prepared by adding 50 /JL of appropriate stock solution in methanol to blank rat plasma, urine or bile, and the volume made up to 2 mL in volumetric tubes. Plasma, urine and bile standards of (E)-2-ene VPA were prepared in concentration ranges of 0.4-35, 2-200 and 1-150 ng/ml, respectively. The plasma standards for 4-ene VPA were prepared in the concentration range of 0.5-45 ng/ml, and urine and bile standards of 2-80 ng/ml. For the analysis of total (conjugated and unconjugated) (E)-2-ene VPA or 4-ene VPA in urine and bile, the same standards were used as described above. The internal standard solutions for (E)-2-ene VPA samples in plasma, urine and bile were prepared by dissolving both DNBA and HA in 0.1N NaOH to give final concentrations of 20, 80 and 80 ng/ml of each, respectively. The internal standard solutions for 4-ene VPA in plasma, urine and bile contained 20, 40, and 40 /zg/mL of DNBA and HA each in 0.1N NaOH, respectively. For the estimation of total (conjugates and unconjugates) (E)-2-ene VPA or 4-ene VPA in urine and bile, the internal standards were prepared by dissolving DNBA in 3N NaOH. 29 B.2.2.2. Extraction and Derivatization: To an 80 Z J L aliquot of the plasma, urine or bile was added 80 fil of the internal standard solution, 50 (ii of 2N HCI and 200 / J L of ethyl acetate in a 1 mL conical reaction vial with a PTFE-lined cap. The contents were mixed on a Fisher tumbler for 15 min, and centrifuged at 1000 g for 20 min. The top organic layer was dried over anhydrous Na2S04, and 60 / J L of dried organic extract was derivatized by heating at 60°C for 1 h with 20 /xL of MTBSTFA reagent. A 1 ztL sample was injected into the GCMS. To determine total (E)-2-ene VPA or 4-ene VPA in urine and bile, an 80 /xL sample was added to an 80 nl aliquot of internal standard solution in 3N NaOH, heated at 60°C for 1 h, acidified with 4N HCI and extracted with 200 /iL ethyl acetate as detailed above. B.2.2.3. Chromatography: A Hewlett-Packard GCMS, model 5987A, equipped with a fused si l ica capillary column, 25 m long and of 0.32 mm I.D., coated with a bonded stationary phase 0V-1701 of 0.25 n thickness (Quadrex Corporation, New Haven, Connecticut) was used. Other operating conditions were as follows: injection port temperature, 240°C; interface, 260°C; and ion source, 260°C. The injection mode was splitless. Helium was used as the carrier gas at a flow rate of 1 mL/min. GC oven temperature was programmed as follows: 50°-100°C at 30°C/min; 100°-160°C at 8°C/min> hold for 2 min; post run to 250°C at 30°C/min, and hold for 2 min. The mass spectrometer was operated in a positive-ion selected-ion-monitoring (SIM) mode and a source pressure of 3X10"^  Torr. Electron impact was the mode of ionization with an energy of 70 eV and emission current of 300 /xA. The intense mass ions, at (M-57)+, of tert-butyldimethylsilyl derivatives (t-BDMS) of (E)-2-ene VPA or 4-ene VPA, and the internal standard DNBA at 199 and 229, respectively, were monitored. 30 B.2.2.4. Optimum Derivatization Conditions: The effect of heating time on the derivatization of (E)-2-ene VPA or 4-ene VPA and the internal standards with MTBSTFA was studied as follows: Plasma aliquots of 80 fil, containing 200 /xg/mL of either (E)-2-ene VPA or 4-ene VPA, were extracted as described above. To 60 /il of the dried organic extract was added 15 fil of MTBSTFA, and heated at 60°C for 0, 15, 30, 60 and 120 min. A 1 /il aliquot was injected into GCMS. In another similar series of experiments, varying amounts of MTBSTFA, ranging from 5, 10, 15 to 20 ill, were added to 60 ill of the organic extract, and heated at 60°C for 1 h. A 1 ill aliquot was used for GCMS analysis. B.2.2.5. Hydrolysis of Conjugates : Urine samples from a rat, following 4-ene VPA administration, were heated at 60° for 0.5, 1, 1.5 and 2 h with internal standard prepared in 3N NaOH. The samples were extracted, derivatized and analyzed as described before. B.2.2.6. Calibration Curves: Peak area ratios of (E)-2-ene VPA or 4-ene VPA and the internal standard DNBA were plotted against concentration of (E)-2-ene VPA or 4-ene VPA in the standard sample. A linear least-squares regression analysis was performed to obtain a calibration curve. The concentration of the unknown sample was calculated from its peak area ratio and the regression equation of the calibration curve prepared on the same day. B.2.2.7. Precision: Within-day precision of the assay method was estimated by the analysis of six individually prepared samples, at the same standard concentration, on the same day. 31 B.2.2.8. Extraction Efficiency: Plasma samples (80 /il) of different concentrations of either (E)-2-ene VPA or 4-ene VPA were acidified with HC1, and extracted with 200 ill ethyl acetate. An 80 ill aliquot of the top organic layer was mixed with an equal volume of internal standard solution of DNBA prepared in ethyl acetate. The mixture was dried over anhydrous ^ S O ^ , an aliquot derivatized and analyzed. The peak area ratio of analyte/internal standard in these extracted samples was corrected for by multiplying by a factor of 2.5. This factor compensates for the 2.5 fold difference (200/80 ill) in the volumes of ethyl acetate used for the preparation of extracted samples compared to the unextracted samples. For the preparation of unextracted samples, solutions of (E)-2-ene VPA or 4-ene VPA were prepared in ethyl acetate at the same concentrations as the plasma samples. Aliquots (80 of these solutions were mixed with an equal volume of the internal standard in ethyl acetate and analyzed as above. The corrected peak area ratios of analyte/internal standard in extracted samples were compared to those in unextracted samples to obtain extraction efficiency of ethyl acetate. B.2.3. Rat Experiments B.2.3.1. Animal Handling: Male Sprague-Dawley rats weighing 250-350 g were obtained from the UBC Animal Care Centre. The animals were allowed to acclimatize to the surroundings for 2-3 days. They were kept in a 12 h day-and-night cycle (6 a.m. to 6 p.m.) at 21.5°C room temperature, and fed standard Purina rat chow and tap water ad libitum. B.2.3.3. Jugular Vein Cannulation: The rat was anesthetized with ether, and the hair removed from about 3-cm square of the skin on the ventral side of the neck. The animal was placed on its dorsal side, and its 32 limbs were taped to a surgical board with adhesive tape. The animal was kept anesthetized with a nose cone containing an ether-soaked swab. A longitudinal incision, 2-3 cm long, was made above the mid-point of the right collar bone. About 1 cm of the external jugular vein was exposed. A glass rod or a flat smooth piece of metal, 3 cm x 3 mm x 0.5 mm, was inserted under the vein. The vein was ligated anteriorly with a 4-0 silk suture, and another suture was placed approximately 7 mm below the f irst . A small incision was made, 3 mm below the first suture, in the wall of the vein by piercing it with a 23 gauge needle. A 25 cm long PE-50 tubing, with a smooth, rounded beveled edge, was inserted and pushed gently with a rotating action about 2.5 cm towards the heart. The second suture was tied around the vein and the cannula inside. The knot was made tight enough to secure the cannula properly but not too tight to collapse the tubing. The loose ends of the first suture were tied to another silk suture that was glued 2.7 cm above the beveled end of the cannula. The cannula was checked for its patency by aspiration, f i l led with heparin solution, and its dorsal free end was plugged with a pin. The free end of the cannula was exited dorsally between the scapulae. The ventral incision was closed with 3-0 catgut and 4-0 silk sutures. A wider-bore (1 cm) hard plastic tubing, whose one end was tied with a silk suture to the back of the animal, was used as a protective covering for the emerging cannula. The animals were kept individually in steel metabolic cages. The rats were allowed to recover for 1-2 days before dosing or bile-exteriorization. The cannula was kept patent by flushing and refi l l ing it daily with heparin solution (20 units/mL). B.2.3.3. Bile Duct Cannulation: The rat was anesthetized as described above, and shaved ventrally on the upper abdomen. The animal was 33 placed on its back and its limbs were taped on the surgical board. A midline abdominal incision, 3-cm long, was made posterior to the xiphoid cartilage. The duodenum and anterior segments of the small intestine were carefully pulled out to the left of the animal and placed on a pad, moistened with physiological saline, on the abdomen. The major lobes of the liver were pushed back towards the diaphragm or gently pulled out and placed on the chest wall and wrapped in a moist gauze. The bile duct above the pancreatic ducts was cautiously freed of the connective tissue with forceps. A 4-0 silk suture was tied tightly around the bile duct proximal to the pancreas. A second suture was kept loose at 4-5 mm in front of the first suture, towards the liver. A narrow glass rod was pushed under the bile duct between the two sutures. The free ends of the first suture were gently pulled towards the ta i l , and a 26 gauge needle was used to pierce into the bile duct, 2 mm in front of the suture knot. A 30 cm long PE-10 tubing with a beveled edge was pushed ~7 mm into the duct towards the l iver. The second ligature was tightened around the duct and the cannula inside. The bile flow was checked for even and continuous flow through the catheter. The free ends of the first silk suture were tied to a second 4-0 silk suture glued to the cannula. The viscera were then gently returned to the abdominal cavity in their respective positions. The cannula was slightly bent to make a wide loop inside the abdominal cavity. A large-bore needle, from which the hub had been removed, was passed through the peritoneum on the dorsal side of the abdominal cavity and pushed subcutaneously to exit at the tip only, between the scapulae on the back of the rat. The free end of the cannula was passed through the needle to bring it out at the back of the neck. The needle was then removed. The bile flow was checked before the abdominal incision was closed with 3-0 catgut and 3-0 silk sutures. A hard plastic tubing was used to protect the external 34 portion of the cannula as described above. The rats were kept in individual cages and allowed to recover for at least 2 h before dosing. B.2.3.4. Pharmacokinetic Studies: Two separate solutions of 20 and 100 mg/mL of each metabolite, (E)-2-ene VPA or 4-ene VPA, were prepared in water as sodium salts and the pH adjusted to 7.4 with HC1. The concentration of each solution refers to the free acid. A single IV bolus dose of 20 or 100 mg/kg was administered via the jugular vein cannula. The cannula was then flushed with 250 /iL of saline. Blood samples of about 0.2 mL were withdrawn typically at -10, 5, 15, 30, 60, 90, 120, 150, 180, 240, 300, 360, 420 and 480 min after the low dose; and at -10, 5, 15, 30, 60, 120, 180, 240, 300, 360, 435, 510, 585, 675 and 765 min following the high dose. In bile-exteriorized rats, blood samples were collected as described above for 6-8 h. After each blood sample, an equivalent volume of heparinized normal saline (20 units/mL) was administered into the cannula to prevent dehydration of the rat, and to prevent blood clotting in the cannula. The blood samples were immediately centrifuged in heparinized microhematocrit tubes (Caraway) and the plasma separated and stored at -20°C until analyzed. Total urine output samples were collected at various time intervals for 24 h. The volumes of the urine samples were measured, and aliquots stored in a freezer at -20°C. In bile-duct cannulated rats, 4 to 6 bile samples were collected usually at 0.5-1 h intervals during the first 6 h of the dose, and then at a longer interval of 6-24 h. The bile volume was measured, and samples stored as described for urine. 35 B.2.3.5. In Vitro Plasma Protein Binding: Aliquots of stock solutions of 4-ene VPA in methanol were diluted with pooled rat plasma to provide 4-ene VPA concentrations of 20, 50, 100, 150, 200, 250, 300 and 350 ng/ml. The final concentration of methanol in each spiked plasma sample was 2%. One mL of each plasma sample was added to the reservoir of an Amicon ultrafiltration unit equipped with a YMT (MPS-1) f i l ter . The units were centrifuged in a fixed angle rotor at 1000 g for 10 min to yield approximately 140 itL of ultrafiltrate. The filtrate was stored at -20°C prior to analysis. The concentration of bound 4-ene VPA was obtained as the difference between 4-ene VPA concentration in the plasma and 4-ene VPA concentration in the ultrafiltrate. Percent binding was calculated as the ratio of the bound over total 4-ene VPA concentration in the plasma multiplied by 100. B.2.3.6. Metabolism of (E)-2-ene VPA: Urine and bile samples obtained from rats receiving 100 mg/kg of (E)-2-ene VPA were pooled separately. The samples (500 /zL) were extracted without hydrolysis, and in another series after hydrolysis with 3N NaOH, as described above. The extracts were derivatized with MTBSTFA reagent. A 1 /zL aliquot was injected into the GCMS, and the mass ions at m/z 98, 112, 114, 197, 199, 195, 173, 201, 213, 215, 217, 329, 331 and 343 corresponding to 4-hydroxy ene VPA lactone, A^-3-heptanone, 3-heptanone, diene VPA, monounsaturated VPA, triene VPA, hexanoic acid, VPA, 3'-oxo-4-ene VPA, 3-keto VPA or 3-hydroxy ene VPA (mono derivative), 3-hydroxy VPA, 3-keto VPA (di derivative), 3-hydroxy VPA (di derivative) and allylglutaric acid (AGA), respectively, were monitored. 36 B.2.4. Pharmacokinetic Analysis: To describe the plasma profile of (E)-2-ene VPA or 4-ene VPA after the low dose administration to the rat, a time-lag pharmacokinetic model (Veng Pedersen and Miller 1980) was employed as shown in Fig. 4. 12 1 "* © 2 21 10 Fig. 4 Time-Lag Pharmacokinetic Model The differential equations for this model are: dCj/dt = - (k 1 0 + k 1 2 ) Cj + k 2 1 C 2 * dC2/dt = kj 2Cj - k 2 jC 2 where: Cj = concentration in compartment 1 at time t C 2 = concentration in compartment 2 at time t * C 2 = concentration in compartment 2 at time t - T t = time after dosing T (tau) = time-lag. The equations were solved numerically by MULTI(RUNGE), a non-linear least squares regression program (Yamaoka and Nakagawa 1983). The overall elimination rate constant, K, was calculated from the slope of the log-linear regression line of the plasma concentration-time curve. The apparent half-life ( tj / 2 ) was calculated as 0.693/K. The volume of the central compartment (Vj) was determined from dose (D) divided by the plasma 37 concentration at time zero (CQ), which was obtained by extrapolation of the log-linear regression line to zero time. The total body clearance C1T was calculated as dose divided by total area under the plasma concentration-time curve (AUC). Renal and biliary clearances, C1R and Clg, were obtained by multiplying the fractions of dose excreted unchanged (unconjugated) in the urine and bile, respectively, by Cl j . The clearance due to the formation of conjugates C l c o n j was determined as the sum of the fractions of dose recovered as conjugates in urine and bile collectively, multiplied by C1 T . The metabolic clearance C l m e t was obtained as the difference of Clj and Clp in normal rats. Various pharmacokinetic parameters obtained after the low dose were compared to those after the high dose, in normal or bile-exteriorized rats, using unpaired two-tailed Students' t-test. 38 C. RESULTS C l . SYNTHESIS C . l . l . (E)-2-ene VPA: The synthesis of (E)-2-ene VPA was attempted by dehydration of the 3-hydroxy VPA ethyl ester with methanesulfonyl chloride (Scheme 1) and with toluenesulfonyl chloride (Scheme 2 ) . GCMS analysis of the reaction mixture, from the methanesulfonyl chloride reaction, showed at least 12 peaks (Appendix 1) with large amounts of 3 -hydroxy VPA ethyl ester (peak 9 ) , 3 condensation products (peaks 10, 11 and 1 2 ) , several unidentified peaks, and extremely small quantities of 2-ene VPA ethyl ester (peak 3 ) . When the same reaction was carried out in the presence of toluenesulfonyl chloride, a larger proportion of 3-ene VPA than 2-ene VPA along with 4-hydroxy VPA and other products was obtained. Washing the ethereal layer with alkaline solution and distil lation under vacuum gave 3-ene VPA. NMR (Appendix 2) of the product showed: 8 0 . 8 - 1 . 1 , triplet (3H, CH 3-CH 2); 1 . 1 - 1 . 6 , complex m ( 4H, -CH 2-CH 2-); 1 . 6 - 2 . 0 , d ( 3H, CH3-CH=); 2 . 7 - 3 . 3 , m (IH, CH-C=0), 5 . 2 - 5 . 9 , complex m (2H, CH=CH). A small amount of 2-ene VPA was present as indicated by 2 . 1 - 2 . 5 , m (CH2-C= from 2-ene VPA); 6 . 8 - 7 . 1 , t (trans CH=C from 2-ene VPA ). • The desired product (E)-2-ene VPA was successfully synthesized by dehydrobromination, and then hydrolysis of 3-bromo VPA ethyl ester. After repeated fractional recrystal1izations at -20°C, the final product was found to be a mixture of 90% trans and 10% cis isomers of 2-ene VPA. Further attempts to increase the proportion of trans isomer of 2-ene VPA by recrystallization were unsuccessful. The purity of 2-ene VPA was confirmed by GCMS (Fig. 5 ) . The smaller peak at 6 .49 min was identified to be the cis isomer of 2-ene VPA and the larger peak at 7 .11 min was the trans isomer of 39 Scheme 1 CH3-CH2-CH2-CH2-C00H C2H5OH CH3-CH2-CH2-CH2-COOC2H5 Li-CH 2 _ CH 2 -CH 2 _ CH 3 CH3-CH2-CHO (CH3)2CH-NH-CH(CH3)2 CH 3-CH 2-CH 2-CH-C00C 2H 5 CH3-CH2-CHOH C 1 - S 0 2 - C H 3 (C 2H 5) 3N CH2C12 CH3-CH2-CH2-CH-COOC2H5 CH 3 -CH 2 -CH-0-S0 2 -CH 3 KH CH3-CH2-CH2-C-COOC2H5 CH3-CH2-CH 2-Ene VPA ethyl ester 40 Scheme 2 C H 3 - C H 2 - C H 2 - C H - C 0 0 C 2 H 5 CH 3-CH 2-CHOH p-Toluenesulfonyl chloride Pyridine C H 2 - C H 2 - C H - C O O C 2 H 5 CH 3-CH=CH NaOH CH ?-CH ?-CH-COOH I CH 3-CH=CH C H 3 - C H 2 - C H 2 - C - C O O C 2 H 5 C H 3 - C H 2 - C H NaOH CHo-CHo-CHo-C-COOH 3 Z Z „ CH 3 - C H 2 - C H 3-Ene VPA 2-Ene VPA 41 Scheme 3 CH3-CH2-CH2-CH2-COOH (CH3)3C-NH-C(CH3)3 Li-CH 2 -CH 2 -CH 2 _ CH 3 CH 3 _CH 2-CH 2-Br CH3-CH2-CH2-CH-COOH CH 3 _ CH 2 - CH 2 Br? PBr, CH 3-CH 2 -CH 2 Br C2H50H C H 3 - C H 2 - C H 2 - C - C O O C 2 H 5 / V C H 3 _CH 2 -CH 2 Br Quinoline CH 3-CH 2-CH 2-C - C 0 0 C 2H 5 CH3 -CH 2 -CH NaOH CHo-CHo-CHo-C-COOH II CH3 _CH 2-CH 2-Ene VPA 42 Scheme 4 - C H 2 - C H 2 - C H 2 - C O O H L i - C H 2 - C H 2 - C H 2 - C H 3 ( C H 3 ) 2 C H - N H - C H ( C H 3 ) 2 C H 2 = C H - C H 2 - I - C H o - C H p - C H - C O O L i I C H 2 = C H - C H 2 HC1 - C H o - C H o - C H - C O O H I C H 2 = C H 2 - C H 2 4-Ene VPA 43 00 O i2ee*-leeee-9866-?eeei-j 3 f ec-M lSeM-i 6.43 -4 7.11 it,' ' 1 ' , V ' ' i f ! ' l TIME (min) B OO o O -oo -leeaee-j ieeeee-466&e-41 1 9 9 I 1 1 7 5 5 7 / M L 1 2 5 , \ 155 I I 1 4 1 ^ A^Ua.. M 0. i . . L _k _, 211 Id 281361 S t . * 375 / l n . •i ee 46 86 126 1 6 8 266 246 - — I — • — r — — i — 286 326 366 K B r58 MB 38 •26 !8 m/z Fig. 5. Total ion chromatogram of t-BDMS derivative (A) of (Z)-2-ene VPA (6.49 min) and (E)-2-ene VPA (7.11 min), and El-mass spectrum (B) of (E)-2-ene VPA. 44 CO z o o . 90688-70000^ 50966-] 46090^ 2&&001 10800-5.6 6 .68 6.0 7 . 0 8.0 9.0 I ' ' ' ' I i e .e TIME (min) oo o t/0 B 26006-24660-22660-20000-18600-16600-1 4000" 12000-1 0606-8086-6600-4680-2 0 0 H 75 1 99 99 \ i i s 129 \ 141 157 l i b 171 1 8 1 6 .68 min. : i 16 R 0 0 ;-90 ^60 - 7 e -66 ^ 8 ^48 730 E20 L18 2 1 4 241 40 80 1 2 6 160 206 240 m/z Fig. 6. Total ion chromatogram of t-BDMS derivative (A) and El-mass spectrum (B) of 4-ene VPA. 45 2-ene VPA. From the relative sizes of these two peaks, the product was found to be 90% (E)-2-ene VPA. The mass spectrum of the tertiary butyldimethylsilyl derivative of (E)-2-ene VPA showed an intense and characteristic peak at (M-57)+ mass ion (m/z) 199. NMR (Appendix 3) analysis showed: 6 0.8-1.1, complex m (6H, 2CH3), 1.2-1.7, m (2H, CH3-CH_2-CH 2-); 2.1-2.7, m (4H, CH_2-CH= and -CH_2-C=); 6.8-7.1, t (IH, -CH=C, trans, strong). Trace amounts of cis isomer were shown by 5.9-6.2, t (IH, -CH=C, cis, weak). In all the pharmacokinetic studies, the amounts refer to the trans isomer of free acid of 2-ene VPA. C.1.2. 4-ene VPA: The synthesis of 4-ene VPA was carried out by the general procedure of Pfeffer et al. (1972). Valeric acid was treated with lithium diisopropylamide, and alkylated with allyl iodide to give 4-ene VPA. The purity of the compound was established by capillary GCMS analysis (Fig. 6A) that showed a single peak. The mass spectrum of the t-BDMS derivative of 4-ene VPA (Fig. 6B) showed an intense characteristic peak at mass ion (m/z) 199 obtained by the loss of 57 a.m.u. (atomic mass units) from the t-BDMS derivative. NMR (Appendix 4) analysis showed: sigma 0.8-1.1, t (3H, CH 3); 1.1-1.7, complex m, (4H, -CH 2-CH 2); 2.0-2.7, complex m (3H, -CH-CH2-CH=); 4.9-5.3, m (2H, CH2=), 5.5-6.1, complex m (IH, -CH=). C.2. ASSAY C.2.1. Chromatography: A typical selected ion chromatogram obtained from an extract of spiked plasma sample is shown in Fig. 7. The retention times for (E)-2-ene VPA, 4-ene VPA, HA, DNBA, diene VPA I and diene VPA II were 7.50, 6.74, 5.72, 8.94, 7.90 and 8.44 min, respectively. The chromatographic peaks were sharp, symmetrical, well separated from each other and resolved at the base 2 3 5 6 LU CO z o a. CO LU CC m/z 173 m/z 199-m/z 197 m/z 2 2 9 r i 1 1 — i 1 5 6 7 8 9 10 TIME (min) Fig. 7. Selected-ion-chromatogram of t-BDMS derivatives of an extract from a spiked plasma sample. Peaks 1, 2, 3, 4, 5, and 6 correspond to hexanoic acid, 4-ene VPA, (E)-2-ene VPA, (E)-2,4-diene VPA, (E)-2,3'-diene VPA and di-N-butylacetic acid, respectively. 47 TIME (min) 8. Selected-ion-chromatogram of a blank plasma sample showing no interfering peaks at the lowest attenuation. 48 line. No interfering peaks were seen in the blank plasma (Fig. 8), urine and bile extracts, except for an extremely small endogenous component that contributed approximately 1% to the peak area of HA. The error in the estimation of peak area of HA was minimal. The chromatographic run was complete within 12 min. Preliminary experiments with several other internal standards including 2-ethylhexanoic acid, heptanoic and 1-methyl-1-cyclohexanoic acids had shown an interference with cis-2-ene VPA, 4-ene VPA and trans-2-ene VPA, respectively. HA and DNBA were selected for further experiments. C.2.2. Derivatization Kinetics The effect of heating for 0, 15, 30, 60 and 120 min at 60°C showed that derivatization with MTBSTFA was extremely rapid. Even without heating, silylation was almost instantaneous (Table 2). To ensure complete derivatization, heating for 1 h was chosen for all subsequent studies. The effect of varying amounts from 5, 10, 15 to 20 /xL of MTBSTFA, added to 50 /xL of ethyl acetate extract of plasma sample, showed no appreciable change in the peak areas of 4-ene VPA and the internal standards (Table 3). In the subsequent analysis of samples, 15 /xL of MTBSTFA was added to the organic extracts. C.2.3. Linearity and Reproducibility Calibration curves of (E)-2-ene VPA were linear in the concentration range of 0.4-35 /xg/mL in plasma, 2-200 /xg/mL in urine and 1-150 ng/ml in bile (Tables 4-6). Similarly, the standard curves for 4-ene VPA were prepared at concentrations of 0.5-45 /xg/mL in plasma, and 2-80 /xg/mL in urine and bile (Tables 7-9). The correlation coefficient, r, was > 0.997 for all regression lines. Within-day coefficients of variation (CV) for 49 TABLE 2. EFFECT OF HEATING TIME ON DERIVATIZATION3 Time (min) 4-ene VPA DNBA HA (E)-2-ene VPA DNBA HA 0 4770 3340 5650 7780 3440 5780 15 4760 3500 5540 8130 3610 5900 30 4200 2830 5070 7530 3320 5670 60 4710 3380 5540 9690 3770 6280 120 4880 3520 5790 9140 3480 5950 a, peak area x 10 . Each reading is a mean of 3 samples. TABLE 3. EFFECT OF MTBSTFA ON THE PEAK AREAS3 MTBSTFA (ML) 4-ene VPA DNBA HA (E)-2-ene VPA DNBA HA 5 4200 2810 4520 5790 2430 4820 10 4110 2780 5000 5610 2220 4660 15 4720 3550 5400 6340 2470 4670 20 3680 2460 4440 5490 2220 3880 a, peak area x 10 50 TABLE 4. CALIBRATION CURVE DATA FOR (E)-2-ENE VPA IN RAT PLASMA (N=5) Added ng/ml Found-Mg/mL CV1 % Found2 Mg/mL CV2 % 0.40 0.41±0.02 4.9 0.40±0.03 7.5 2.0 1.9±0.04 2.1 2.0±0.2 7.7 5.0 4.8+0.1 2.7 4.9±0.2 4.1 10 9.8±0.3 2.9 10±0.4 4.0 15 16±0.5 3.2 15±0.6 3.9 25 25+0.5 2.0 25±0.5 2.1 35 35±0.5 1.5 35+0.6 1.7 1=DNBA; 2=HA TABLE 5. CALIBRATION IN CURVE DATA FOR (E)-2-ENE RAT URINE (N=5) VPA Added ng/ml Found* Mg/mL CV1 % Found2 /ig/mL CV2 % 2.0 2.0±0.1 5.0 2.1±0.2 7.8 5.0 5.2±0.1 1.9 5.0±0.4 7.3 30 29+0.7 2.3 31±1.2 4.0 60 59±1.8 3.1 61+1.9 3.1 100 97±6.0 6.1 102±4.3 4.2 150 154+3.4 2.2 150±2.5 1.7 200 199±2.9 1.4 199±3.9 2.0 1=DNBA; 2=HA 51 TABLE 6. CALIBRATION IN CURVE DATA FOR (E)-2-ENE RAT BILE (N=3) VPA Added MO/mL Found* jug/mL CV1 % Found2 Hg/ml CV2 % 1.0 0.9±0.2 23 0.9±0.4 51 10 9.3±0.4 4 8.9±1.0 11 25 27±1.6 6 26±2.2 9 60 58±2.4 4 61+2.9 5 100 100±0.9 1 100±2.3 2 150 150+0.7 0.5 150±1.7 1 1=DNBA; 2=HA 52 TABLE 7. CALIBRATION CURVE DATA FOR 4-ENE VPA IN RAT PLASMA (N=6) Added Mg/mL Found* Mg/mL CV1 % Found2 /ig/mL CV2 % 0.50 0.6±0.08 13 0.6±0.05 8.9 2.0 2.1±0.1 6.8 2.0+0.1 6.4 10 10+0.6 5.7 9.9±0.6 5.9 20 20+1.1 5.5 20±0.8 4.0 30 30±2.2 7.1 30±1.0 3.3 45 45±1.4 3.1 45+0.7 1.6 1=DNBA; 2=HA TABLE 8. CALIBRATION CURVE DATA FOR 4-ENE VPA IN RAT URINE (N=6) Added Found* CV1 Found2 CV2 MQ/mL Mg/mL % /jg/mL % 2.0 1.9+0.08 4.2 1.9±0.08 4.2 10 10±0.5 4.8 10±1.0 9.8 20 19±0.9 4.9 20±0.5 2.6 40 40+2.4 6.0 40±1.7 4.2 60 59±2.0 3.5 60+1.2 2.0 80 81±1.3 1.7 80±0.4 0.5 1=DNBA; 2=HA 53 TABLE 9. CALIBRATION CURVE DATA FOR 4-ENE VPA IN RAT BILE (N=3) Added Found* CV1 Found CV2 % Mg/mL % 2.0 2.5±0.3 14 2.8±1.4 51 10 10+0.2 2 10±0.2 2 20 19±1.3 7 19±2.1 11 40 39±1.0 3 39±1.5 4 60 60±1.3 2 62±2.9 5 80 81+0.6 0.7 80±1.0 1 1=DNBA; 2=HA TABLE 10. EFFECT OF HEATING TIME ON THE HYDROLYSIS OF CONJUGATES IN RAT URINE3 (Time, h) 0.5 1 1.5 2 4-ENE VPA 5880 6110 5920 6320 DNBA 2500 2390 2390 2690 Area Ratio 2.35 2.55 2.48 2.35 a, peak area x 100 Temperature = 60°C 54 (E)-2-ene VPA in plasma standards varied from 2-4% with DNBA as internal standard, and 2-6% when HA was used as internal standard. The corresponding values of within-day CV for 4-ene VPA plasma standards were 4-7% and 2-3%. The t-BDMS derivatives of the analyte and the internal standards showed no significant change in their peak area ratios when stored at -20°C for 2 weeks. C.2.4. Hydrolysis of Conjugates Table 10 shows that the maximum hydrolysis of the conjugates of 4-ene VPA, excreted in the urine of the rat, was achieved by heating with 3N NaOH at 60°C for 0.5 h. Further heating up to 2 h did not alter the peak area of 4-ene VPA or its peak area ratio with the internal standard. C.2.5. Extraction Efficiency Extraction efficiency studies were performed at three different plasma concentrations of 5, 20 and 40 ng/ml of 4-ene VPA. The average recoveries using ethyl acetate were 99, 102 and 102%, respectively. The recovery of (E)-2-ene VPA from plasma was also quantitative at concentrations of 5, 15 and 30 jig/mL and was 100, 99 and 100%, respectively. C.3. PHARMACOKINETIC STUDIES C.3.1. Pharmacokinetics in Normal Rats Figures 9 and 10 represent the semi logarithmic plots of mean plasma concentrations of (E)-2-ene VPA and 4-ene VPA versus time following the low and high doses to normal rats. Following the low dose of 20 mg/kg, the plasma level of (E)-2-ene VPA and 4-ene VPA declined rapidly with a t ^ 55 Fig. 9. Semilogarthimic plots of plasma concentrations of (E)-2-ene VPA versus time following IV dose of 20 (•) and 100 ( o ) mg/kg in normal rats. Each point represents mean + 95% confidence limits (N=4). Solid line represents model-generated curve. 56 _j 1000q E Time, h Fig. 10. Semilogarithmic plots of plasma concentrations of 4-ene VPA versus time following IV dose of 20 (• ) and 100 (o) mg/kg in normal rats. Each point represents mean ± 95% confidence limits (N=6). Solid line represents model-generated curve. 57 of 23 + 4 and 12 + 2 min, respectively, during the first h. The plasma concentration of (E)-2-ene VPA reached trough levels of 5.7 + 5.6 jug/mL and for 4-ene VPA, 1.6 + 1.1 /ig/mL, at approximately 2 h after the dose. Thereafter, the plasma levels of (E)-2-ene VPA and 4-ene VPA started to rise, showing a secondary peak at 4 h. This secondary peak was attributed to enterohepatic circulation of both the metabolites in the rat. After 4 h, (E)-2-ene VPA and 4-ene VPA were eliminated slowly from the plasma with apparent population t j / 2 of 55 and 73 min., respectively. The plasma profile of (E)-2-ene VPA and 4-ene VPA, after the high dose of 100 mg/kg each, was similar to that of the low dose (Fig.9,10). There was, however, a short initial period of an apparently non-linear plasma decline of (E)-2-ene VPA and 4-ene VPA at concentrations above 200 /zg/mL. In the log-linear phase, between 30 to 120 min, the plasma level of (E)-2-ene VPA and 4-ene VPA declined with apparent t 1 / 2 of 28 + 6 and 18 + 1.5 min to reach trough levels of 26 + 11 and 6.9 + 2.3 fig/ml, respectively. The plasma levels then started to rise indicating EHC of the administered metabolite. Following the secondary plasma peaks, (E)-2-ene VPA and 4-ene VPA were more slowly eliminated from plasma with apparent population t j / 2 of 99 and 73 min respectively. The plasma levels of (E)-2-ene VPA and 4-ene VPA in individual normal rats are tabulated in Appendices 5-8. Tables 11 and 12 summarize the pharmacokinetic parameters calculated from individual animals describing the above data. For (E)-2-ene VPA, the apparent volume of the central compartment was unaltered at the two dose levels (Table 31). There was no significant change in the metabolic and total plasma clearances of (E)-2-ene VPA, and in the fraction of dose excreted as unconjugated, conjugated and total (E)-2-ene VPA in the urine of the rat when the dose was increased by 5 fold (Tables 31, 13, 14). There 58 TABLE 11. PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg) Parameter la 2a 3a 4a Mean Weight3 270 285 290 300 , 286 Dose6 5.4 5.7 5.8 6.0 5.7 90 84 98 66 85 300 390 250 300 310 + e t l / 2 23 18 28 23 23 »/ 220 240 200 300 240 AUC^ 4700 3300 7400 3100 4600 % DOSE EXCRETED IN URINE Unconj 2.4 7.0 9.8 4.2 5.9 Conj 22 22 23 35 26 Total 25 29 33 39 32 CLEARANCE (mL/min. •kq) C 1 R 0.10 0.43 0.27 0.27 0.27 c lmet 4.2 5.7 2.4 6.2 4.6 C 1 T 4.3 6.1 2.7 6.4 4.9 a, g; 6, mg/kg; c, ztg/mL; d, xlO"° min - 1 ; e, min; f, mL/kg; g, zig.min/mL 59 TABLE 12. PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg) Parameter lb 2b 3b 4b Mean Weight3 260 260 310 320 288 Dose6 26 26 31 32 29 410 460 480 390 430 320 260 190 260 260 4. e 22 27 36 27 28 v / 250 220 210 260 230 AUCS 29000 33000 35000 35000 33000 % DOSE EXCRETED IN URINE Unconj 11 12 10 4.7 9.4 Conj 31 52 32 46 40 Total 42 64 42 51 50 CLEARANCE (mL/min. kq) C1R 0.38 0.35 0.30 0.13 0.29 C1met 3.1 2.7 2.6 2.7 2.8 c i T 3.4 3.0 2.9 2.8 3.0 a, g; 6, f, mL/kg; mg/kg; c, m/ml; d, g, /jg.min/mL x l O - 5 min "1; e, min; TABLE 13. URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg) Rat Time (h) Volume (mL) Cone (/jg/mL) Amount (mg) Time (h) Cumulative Amt (mg) Cumulative % of Dose la 0-2.5 2.68 32 (400) 0, .09 (1.1) 0-2.5 0.09 (1.1) 1.7 (20) 2.5-8 2.44 6.9 (89) 0. .02 (0.22) 0-8 0.11 (1.3) 2.0 (24) 8-24 6.60 2.9 (3.3) 0, .02 (0.02) 0-24 0.13 (1.3) 2.4 (25) 2a 0-3 3.63 99 (360) 0, .36 (1.3) 0-3 0.36 (1.3) 6.3 (23) 3-9 1.60 27 (190) 0. .04 (0.30) 0-9 0.40 (1.6) 7.0 (28) 9-24 9.50 - (5.0) (0.05) 0-24 0.40 (1.6) 7.0 (29) 3a 0-3.25 3.61 140 (480) 0. .52 (1.7) 0-3.25 0.52 (1.7) 8.9 (30) 3.25-8 1.40 36 (140) 0. ,05 (0.19) 0-8 0.57 (1.9) 9.8 (33) 8-24 15.0 - (") ~ (-) 0-24 0.57 (1.9) 9.8 (33) 4a 0-8 10.1 22 (230) 0. ,22 (2.3) 0-8 0.22 (2.3) 3.7 (39) 8-24 15.2 2.2 (2.9) 0. ,03 (0.04) 0-24 0.25 (2.4) 4.2 (39) cn o Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA. TABLE 14. URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (Mg/mL) (mg) (h) Amt (mg) % of Dose lb 0-1.5 3.0 860 (1900) 2.6 (5.8) 0-1.5 2.6 (5.8) 10 (22) 1.5-8.5 2.28 64 (1600) 0.14 (3.6) 0-8.5 2.7 (9.4) 11 (36) 8.5-11.5 2.00 29 (560) 0.06 (1.1) 0-11.5 2.8 (11) 11 (40) 11.5-24 4.65 13 (79) 0.06 (0.37) 0-24 2.9 (11) 11 (42) 2b 0-6.5 3.99 480 (3700) 1.9 (15) 0-6.5 1.9 (15) 7.3 (57) 6.5-12.5 2.92 360 (580) 1.1 (1.7) 0-12.5 3.0 (17) 12 (64) 12.5-24 4.50 14 (15) 0.06 (0.06) 0-24 3.0 (17) 12 (64) 3b 0-1 2.15 130 (1600) 0.27 (3.4) 0-1 0.27 (3.4) 0.87 (11) 1-8.5 2.50 960 (3500) 2.4 (8.7) 0-8.5 2.7 (12) 8.6 (39) 8.5-24 4.86 120 (190) 0.56 (0.91) 0-24 3.2 (13) 10 (42) 4b 0-0.5 1.05 150 (480) 0.16 (0.50) 0-0.5 0.16 (0.50) 0.50 (1.6) 0.5-2 1.50 240 (5300) 0.36 (7.9) 0-2 0.52 (8.4) 1.6 (24) 2-11.5 3.61 240 (2100) 0.87 (7.4) 0-11.5 1.4 (16) 4.3 (49) 11.5-24 3.56 34 (110) 0.12 (0.41) 0-24 1.5 (16) 4.7 (51) Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA. 62 TABLE 15. PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg) Parameter lc 2c 3c 4c 5c 6c Mean + SD Weight3 290 280 280 270 320 335 296 + 26 Dose6 5.8 5.6 5.6 5.4 6.4 6.7 5.9 + 0.5 94 110 83 75 100 120 98 + 17 610 490 590 490 620 680 580 + 80 4. e t l / 2 11 14 12 14 11 10 12 + 1.7 v / 210 180 240 270 190 170 210 + 40 AUC^ 2200 2500 2200 2100 2400 2500 2300 + 160 % DOSE EXCRETED IN URINE Unconj 9.3 3.3 3.6 5.5 6.1 4.0 5.3 + 2.3 Conj 16 18 16 18 19 14 17 + 2.1 Total 25 22 19 24 25 18 22 + 3.1 CLEARANCE (mL/min.kq) C 1 R 0.85 0.26 0.32 0.51 0.51 0.35 0.47 + 0.2: c lmet 8.3 7.8 8.8 8.8 7.9 7.7 8.2 + 0.5: C 1 T 9.2 8.1 9.1 9.4 8.4 8.0 8.7 + 0.61 a, g; b, f, mL/kg; mg/kg; c, /zg/mL; d, g, zzg.min/mL xlO"^ min"*; e, min; 63 TABLE 16. PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg) Parameter Id 2d 3d 4d 5d 6d Mean + SD Weight3 280 256 262 260 305 295 276 + 20 Dose6 28 26 26 26 31 30 28 + 2.1 280 320 330 350 310 290 310 + 24 k l ' 360 400 410 330 370 410 380 + 34 t e l l / 2 19 18 17 21 19 17 18 + 1.5 »/ 350 320 300 290 320 350 320 + 25 AUC0 17000 15000 15000 19000 i 19000 18000 17000 + 16i 5 i DOSE 1 EXCRETED IN URINE Unconj 4.3 4.9 6.6 2.5 2.4 3.3 3.8 + 1.7 Conj 21 31 21 19 32 22 24 + 5.7 Total 25 36 28 21 34 25 28 + 5.7 CLEARANCE (mL/min.kq) C 1 R o .26 0 .32 0.43 0 .13 0.13 0 .14 0.23 + o.i; 0 1 met 5 .8 6 .2 6.1 5 .2 5.2 5 .6 5.7 + 0.4: C1T 6 .0 6 .5 6.5 5 .3 5.3 5 .7 5.9 + 0.-5. a. g; b, mg/kg; c, izg/mL; d, xlO"^ min"*; e, min; f, mL/kg; g, tig.min/mL TABLE 17. URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg) *at Time Volume Cone Amount Time Cumulative Cumulativ (h) (mL) (Mg/mL) (mg) (h) Amt (mg) % of Dose lc 0-3 3.70 97 (230) 0.36 (0.86) 0-3 0.36 (0.86) 6.2 (15) 3-7 3.40 22 (110) 0.08 (0.39) 0-7 0.43 (1.3) 7.5 (22) 7-24 23.3 4.5 (8.6) 0.10 (0.20) 0-24 0.54 (1.5) 9.3 (25) 2c 0-2.5 2.23 78 (450) 0.18 (0.99) 0-2.5 0.18 (0.99) 3.1 (18) 2.5-5 1.24 6.9 (142) 0.008 (0.18) 0-5 0.18 (1.2) 3.3 (21) 5-8 1.35 - (24) - (0.033) 0-8 0.18 (1.2) 3.3 (22) 8-24 8.00 - (-) - (") 0-24 0.18 (1.2) 3.3 (22) 3c 0-0.5 1.47 78 (280) 0.12 (0.41) 0-0.5 0.12 (0.41) 2.0 (7.2) 0.5-4 2.59 22 (220) 0.06 (0.58) 0-4 0.17 (0.98) 3.1 (18) 4-8 1.90 4.7 (37) 0.009 (0.069) 0-8 0.18 (1.1) 3.2 (19) 8-24 8.50 2.1 (2.2) 0.018 (0.019) 0-24 0.20 (1.1) 3.6 [19) 4c 0-2 3.00 61 (250) 0.18 (0.75) 0-2 0.18 (0.75) 3.4 [14) 2-8 7.40 15 (74) 0.11 (0.54) 0-8 0.30 (1.3) 5.5 [24) 8-24 8.55 - (") - (") 0-24 0.30 (1.3) 5.5 [24) 5c 0-8 3.50 110 (440) 0.39 (1.6) 0-8 0.39 (1.6) 6.1 [24) 8-24 11.20 - (3.1) - (0.035) 0-24 0.39 (1.6) 6.1 [25) 6c 0-8 2.95 90 (370) 0.27 (1.1) 0-8 0.27 (1.1) 4.0 [16) 8-24 15.50 - (6.3) - (0.098) 0-24 0.27 (1.2) 4.0 ,18) Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA. TABLE 18. URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (itg/mL) (mg) (h) Amt (mg) % of Dose Id 0-3 3.07 330 (1400) 1.0 (4.4) 0-3 1.0 (4.4) 3.6 (16) 3-7.5 2.63 62 (0.85) 0.16 (2.2) 0-7.5 1.2 (6.6) 4.2 (24) 7.5-12 2.37 11 (0.13) 0.025 (0.31) 0-12 1.2 (6.9) 4.3 (25) 12-24 3.28 3.2 (0.018) 0.010 (0.06) 0-24 1.2 (7.0) 4.3 (25) 2d 0-1 1.85 260 (2600) 0.48 (4.9) 0-1 0.48 (4.9) 1.9 (19) 1-6 1.29 530 (2700) 0.68 (3.4) 0-6 1.2 (8.3) 4.5 (33) 6-10 2.14 25 (350) 0.054 (0.75) 0-10 1.2 (9.1) 4.8 (36) 10-24 3.40 14 (21) 0.047 (0.073) 0-24 1.3 (9.2) 4.9 (36) 3d 0-1 0.35 230 (290) 0.08 (0.10) 0-1 0.079 (0.10) 0.3 (0.4) 1-2 2.35 610 (2200) 1.4 (5.2) 0-2 1.5 (5.3) 5.8 (21) 2-8.5 2.27 85 (700) 0.19 (1.6) 0-8.5 1.7 (6.9) 6.6 (27) 8.5-11 1.59 5.5 (52) 0.009 (0.083) 0-11 1.7 (7.0) 6.6 (27) 11-13 1.62 11 (68) 0.018 (0.11) 0-13 1.7 (7.1) 6.6 (27) 13-24 4.52 - (9.3) - (0.042) 0-24 1.7 (7.2) 6.6 (28) 4d 0-1 0.51 110 (450) 0.057 (0.23) 0-1 0.057 (0.23) 0.2 (0.9) 1-2 1.50 250 (1400) 0.38 (2.1) 0-2 0.43 (2.4) 1.7 (9.1) 2-10 1.29 130 (2200) 0.16 (2.9) 0-10 0.60 (5.2) 2.3 (20) 10-12 1.30 9.1 (160) 0.012 (0.21) 0-12 0.61 (5.4) 2.3 (21) 12-24 3.54 8.9 (37) 0.032 (0.13) 0-24 0.64 (5.6) 2.5 (21) 5d 0-6 5.30 110 (1500) 0.57 (8.1) 0-6 0.57 (8.1) 1.9 (27) 6-24 18.50 8.5 (130) 0.16 (2.4) 0-24 0.73 (11) 2.4 (34) 6d 0-6 3.10 290 (1800) 0.91 (5.6) 0-6 0.91 (5.6) 3.1 (19) 6-24 7.88 9.9 (230) 0.078 (1.8) 0-24 0.99 (7.4) 3.3 (25) Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA. 66 was, however, a trend towards smaller metabolic and total clearance values at the high dose than at the low dose. For 4-ene VPA, the apparent volume of the central compartment increased 1.5 times with a five fold increase in the dose (Tables 15,16). The fraction of dose excreted as conjugates and total 4-ene VPA in urine increased significantly with the dose (Table 32). However, the apparent total plasma, renal and non-renal (metabolic) clearances decreased significantly with an increase in the dose of 4-ene VPA (Table 32). More than 95% of 4-ene VPA excreted in urine was recovered within 12 h of the dose (Tables 17,18). C.3.2. Pharmacokinetic Model The plasma data of (E)-2-ene VPA and 4-ene VPA obtained from normal rats receiving the low dose (Fig 9,10) were fitted to the proposed time-lag pharmacokinetic model using an optimal tau (T) value of 1.75 for (E)-2-ene VPA and 4-ene VPA. The calculated values of first-order transfer rate constants, kjQ, k^2 and k 2 j for (E)-2-ene VPA were 1.0, 0.69 and 1.3 h"* and for 4-ene VPA, 2.4, 0.89 and 0.50 h" 1, respectively. The model-generated curve (solid line) showed an acceptable f i t with most of the actual data points. At the trough levels, however, the predicted values were lower than the observed plasma levels. The residual sum of squares was found to be the least, 63 and 12 Mg/mL, for (E)-2-ene VPA and 4-ene VPA, respectively, at a time-lag value of 1.75 h. Since the proposed model assumes first-order transfer processes between the compartments, it was not applied to the plasma profile after the high dose since that showed a short non-linear phase at concentrations > 200 /zg/mL. 67 1000: O) 0) > - J (0 E CO Q. 100-2 > c LU CN 1T 0.1 <D NO SECONDARY PEAK 3 Time, h 4 6 Fig. 11. Semilogarthmic plots of plasma concentrations of (E)-2-ene VPA versus time following IV dose of 20 (• ) and 100 ( o ) mg/kg in bile-exteriorized rats. Each point represents mean ± 95% confidence limits (N=4). 68 1000^. .E < 10CN Q_ c LU i > CD —I CO E CO Q_ 10-1^  0.1 CD i f f NO SECONDARY PEAK 3 Time, h 5 6 Fig. 12. Semilogarthmic plots of plasma concentrations of 4-ene VPA versus time following IV dose of 20 (•) and 100 ( o ) mg/kg in bile exteriorized rats. Each point represents mean ± 95% confidence limits (N=6). 69 Fig. 13. A typical choleretic effect, and excretion rate plot of 4-ene VPA in the bile of a rat after high dose of 100 mg/kg. Bile flow rate ( • ) , and conjugated 4-ene VPA ( ® ) and unconjugated 4-ene VPA ( A ) in bile. 70 Fig. 14. A typical cumulative biliary excretion plot of 4-ene VPA versus time after 20 (•) and 100 ( o ) mg/kg. 71 C.3.3. Pharmacokinetics in Bile-Exteriorized Rats The semilogarithmic plots of average plasma levels of (E)-2-ene VPA and 4-ene VPA versus time in bile exteriorized rats are shown in Fig. 11 and 12, respectively. After the 20 mg/kg dose, (E)-2-ene VPA and 4-ene VPA plasma profile followed an open one-compartment model with first-order elimination t 1 / 2 of 20 + 3.4 and 13 + 1.8 min, respectively. Following the high dose, a brief period of non-linear plasma decline was observed at concentrations above 200 H9/ml for both the metabolites. Thereafter, (E)-2-ene VPA and 4-ene VPA were eliminated with an apparent t 1 / 2 of 21+1.7 and 19 + 3.1 min, respectively. No secondary plasma peaks were seen in these bile-exteriorized rats. The plasma levels of (E)-2-ene VPA and 4-ene VPA in individual bile-exteriorized rats are shown in Appendices 9-12. Various pharmacokinetic parameters of (E)-2-ene VPA at the low and the high dose in bile-duct cannulated rats are shown in Tables 19 and 20. The fraction of (E)-2-ene VPA dose, measured as a sum of unconjugated and conjugated (E)-2-ene VPA, in the urine showed a slight but non-significant increase with the dose. The percentage of (E)-2-ene VPA dose eliminated in bile showed a non-significant decrease with an increase in the dose (Table 31). A total of 65% of the low dose and 66% of the high dose were eliminated in the urine and bile, collectively (Tables 21-24). Plasma clearance of (E)-2-ene VPA decreased by 22%, when the dose was increased by 5 fold. In the case of 4-ene VPA, there was a significant increase in the apparent volume of the central compartment, and in the fraction of dose excreted as conjugated and total 4-ene VPA in urine with an increase in the administered dose (Tables 32, 25, 26). In contrast, the fraction of dose recovered as conjugated and total 4-ene VPA in bile, the total plasma clearance and the metabolic clearance decreased by approximately 40% with a 72 TABLE 19. PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN BILE-EXTERIORIZED (DOSE=20 mg/kg) Parameter le 2e 3e 4e Mean Weight3 320 285 280 310 299 Dose6 6.4 5.7 5.6 6.2 6.0 100 79 100 81 91 390 400 280 340 350 t e l l / 2 18 17 25 20 20 v i f 200 250 200 240 220 AUC^ 2600 2100 3800 2500 2700 % DOSE EXCRETED IN URINE Unconj 1.7 2.8 4.1 5.5 3.5 Conj 16 25 24 30 24 Total 17 28 29 36 27 % DOSE EXCRETED IN BILE Unconj 12 8.4 6.3 5.9 8.1 Conj - 39 29 29 22 29 Total 51 37 CLEARANCE 34 (mL/min.kq) 28 38 C1R 0.13 0.33 0.22 0.44 0.28 ci B 0.93 0.81 0.33 0.47 0.64 Cl conj 4.2 5.2 2.8 4.2 4.1 c i T 7.8 9.7 5.3 8.0 7.7 a, g; b, f, mL/kg; mg/kg; c, /jg/mL; d, g, /jg.min/mL xlO"5 min"1; e, min; 73 TABLE 20. PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS (D0SE=100 mg/kg) Parameter If 2f 3f 4f Mean Weight3 270 290 320 270 288 Dose6 27 29 32 27 29 c. c - 440 440 380 440 430 h d 360 300 320 340 330 t l / 2 19 23 22 20 21 h f 230 230 260 230 240 AUC^ 13000 20000 18000 16000 17000 % DOSE EXCRETED IN URINE Unconj 4.7 3.5 1.3 3.5 3.3 Conj 44 26 38 20 32 Total 49 29 39 24 35 % DOSE EXCRETED IN BILE Unconj 6.0 8.4 2.5 7.4 6.1 Conj 25 34 17 25 25 Total 31 42 . CLEARANCE 19 (mL/min 32 • kq) 31 C 1 R 0.35 0.18 0.07 0.21 0.20 c i B 0.45 0.43 0.13 0.45 0.37 Cl ^'conj 5.2 3.0 3.0 2.8 3.5 c i T 7.5 5.0 5.4 6.1 6.0 a, g; 6, mg/kg; c, jug/mL; d, xlO"° min"1; e, min; f, mL/kg; g, /zg.min/mL TABLE 21. URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=20 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (MOM) (mg) (h) Amt (mg) % of Dose le 0-6 1.52 71 (730) 0.11 (1.1) 0-6 0.11 (1.1) 1.7 (17) 6-24 20.0 - (-) - (-) 0-24 0.11 (1.1) 1.7 (17) 2e 0-6 3.4 47 (450) 0.16 (1.5) 0-6 0.16 (1.5) 2.8 (27) 6-24 21.5 - (3.4) - (0.07) 0-24 0.16 (1.6) 2.8 (28) 3e 0-5.5 2.17 97 (720) 0.21 (1.6) 0-5.5 0.21 (1.6) 3.8 (28) 5.5-24 8.20 2.9 (3.0) 0.02 (0.02) 0-24 0.23 (1.6) 4.1 (29) 4e 0-5.5 4.90 69 (450) 0.34 (2.2) 0-5.5 0.34 (2.2) 5.5 (36) 5.5-24 4.80 - (2.2) - (0.01) 0-24 0.34 (2.2) 5.5 (36) Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA. TABLE 22. URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=100 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (M9/mL) (mg) (h) Amt (mg) % of Dose If 0-6.5 6.5-24 3.20 400 (4100) 1.3 (13) 0-6.5 1.3 (13) 4.7 (49) 2f 0-6 6-24 1.93 11.80 58 77 (1700) (430) 0.11 0.91 (3.3) (5.1) 0-6 0-24 0.11 1.0 (3.3) (8.4) 0.4 3.5 (12) (29) 3f 0-6.5 6.5-24 11.33 5.60 35 7.9 (1100) (45) 0.39 0.04 (12) (0.25) 0-6.5 0-24 0.39 0.43 (12) (13) 1.2 1.3 (38) (39) 4f 0-6 6-24 2.25 7.80 190 66 (2000) (230) 0.42 0.51 (4.5) (1.8) 0-6 0-24 0.42 0.94 (4.5) (6.3) 1.6 3.5 (17) (24) Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA. *, accidentally lost. TABLE 23. BILIARY ELIMINATION OF UNCONJUGATED (E)-2-ENE VPA IN RATS (DOSE=20 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (/zg/mL) (mg) (h) Amt (mg) % of Dose le 0-0.5 1.24 440 (1900) 0.54 (2.4) 0-0.5 0.54 (2.4) 8.4 (38) 0.5-1.5 1.56 140 (520) 0.22 (0.81) 0-1.5 0.75 (3.2) 12 (50) 1.5-2.5 1.12 12 (32) 0.013 (0.035) 0-2.5 0.77 (3.2) 12 (51) 2.5-3.5 1.07 - (1.3) - (0.001) 0-3.5 0.77 (3.2) 12 (51) 3.5-4.5 1.10 - (-) - (-) 0-4.5 0.77 (3.2) 12 (51) 4.5-5.5 1.05 - (-) - (-) 0-5.5 0.77 (3.2) 12 (51) 2e 0-0.5 1.15 330 (1400) 0.38 (1.7) 0-0.5 0.38 (1.7) 6.6 (29) 0.5-1.5 1.53 65 (300) 0.10 (0.45) 0-1.5 0.48 (2.1) 8.3 (37) 1.5-2.5 1.10 2.6 (8.0) 0.003 (0.009) 0-2.5 0.48 (2.1) 8.4 (37) 2.5-3.5 0.91 - (-) - (-) 0-3.5 0.48 (2.1) 8.4 (37) 3.5-4.5 0.86 - (-) - (-) 0-4.5 0.48 (2.1) 8.4 (37) 4.5-5.5 0.74 - (-) - (-) 0-5.5 0.48 (2.1) 8.4 (37) 3e 0-0.5 0.84 240 (1400) 0.20 (1.2) 0-0.5 0.20 (1.2) 3.5 (21) 0.5-1.5 1.01 140 (750) 0.14 (0.76) 0-1.5 0.34 (1.9) 6.1 (34) 1.5-2.5 0.87 12 (60) 0.010 (0.015) 0-2.5 0.35 (1.9) 6.1 (35) 2.5-3.5 0.54 1.2 (2.8) 0.001 (0.001) 0-3.5 0.35 (1.9) 6.3 (35) 3.5-4.5 0.78 - (-) - (-) 0-4.5 0.35 (1.9) 6.3 (35) 4.5-5.5 0.64 - (-) - (") 0-5.5 0.35 (1.9) 6.3 (35) 4e 0-0.5 1.05 220 (1200) 0.23 (1.3) 0-0.5 0.23 (1.3) 3.8 (21) 0.5-1.5 1.21 100 (350) 0.13 (0.43) 0-1.5 0.36 (1.7) 5.8 (28) 1.5-2.5 1.00 5.1 (23) 0.005 (0.023) 0-2.5 0.36 (1.7) 5.9 (28) 2.5-3.5 0.94 - (1.1) - (0.001) 0-3.5 0.36 (1.7) 5.9 (28) 3.5-4.5 0.86 - (-) - (-) 0-4.5 0.36 (1.7) 5.9 (28) 4.5-5.5 0.81 - (-) - (-) 0-5.5 0.36 (1.7) 5.9 (28) --4 Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA. TABLE 24. BILIARY ELIMINATION OF UNCONJUGATED (E)-Z-ENE VPA IN RATS (DOSE=100 mg/kg) Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (iig/mL) (mg) (h) Amt (mg) % of Dose 0-0.5 0.84 870 (4400) 0.73 (3.7) 0-0.5 0.73 (3.7) 2.7 (14) 0.5-1.5 1.42 590 (3100) 0.84 (4.4) 0-1.5 1.6 (8.1) 5.8 (30) 1.5-2.5 0.78 75 (290) 0.058 (0.23) 0-2.5 1.6 (8.3) 6.0 (31) 2.5-5.0 1.54 1.6 (5.7) 0.002 (0.01) 0-5 1.6 (8.3) 6.0 (31) 5.0-6.5 1.23 ~ (-) ~ (-) 0-6.5 1.6 (8.3) 6.0 (31) 0-0.5 1.01 580 (2500) 0.58 (2.5) 0-0.5 0.58 (2.5) 2.0 (8.7) 0.5-1.0 1.18 670 (3900) 0.80 (4.6) 0-1.0 1.4 (7.1) 4.8 (24) 1.0-1.5 1.04 570 (3200) 0.59 (3.4) 0-1.5 2.0 (11) 6.8 (36) 1.5-2.5 1.15 360 (1400) 0.41 (1.6) 0-2.5 2.4 (12) 8.2 (41) 2.5-3.5 0.82 54 (180) 0.044 (0.15) 0-3.5 2.4 (12) 8.4 (42) 3.5-4.5 0.60 10 (36) 0.006 (0.022) 0-4.5 2.4 (12) 8.4 (42) 4.5-6.0 0.67 2.4 (8.7) 0.002 (0.006) 0-6.0 2.4 (12) 8.4 (42) 0-0.5 0.87 320 (2300) 0.28 (2.0) 0-0.5 0.28 (2.0) 0.9 (6.2) 0.5-1.5 1.12 330 (2900) 0.37 (3.3) 0-1.5 0.64 (3.3) 2.0 (10) 1.5-2.5 0.87 150 (1100) 0.13 (0.93) 0-2.5 0.77 (6.2) 2.4 (19) 2.5-3.5 0.77 14 (46) 0.011 (0.035) 0-3.5 0.79 (6.2) 2.4 (19) 3.5-5.0 0.98 - (2.6) - (0.002) 0-5.0 0.79 (6.2) 2.4 (19) 5.0-6.5 0.98 - (1.2) (0.001) 0-6.5 0.79 (6.2) 2.4 (19) 0-0.5 0.93 730 (2500) 0.68 (2.3) 0-0.5 0.68 (2.3) 2.5 (8.5) 0.5-1.5 1.32 620 (3300) 0.81 (4.3) 0-1.5 1.5 (6.6) 5.5 (24) 1.5-2.5 1.07 400 (1700) 0.43 (1.8) 0-2.5 1.9 (8.4) 7.1 (31) 2.5-3.5 0.84 87 (260) 0.073 (0.22) 0-3.5 2.0 (8.6) 7.4 (32) 3.5-4.5 0.83 5.4 (17) 0.004 (0.14) 0-4.5 2.0 (8.8) 7.4 (32) 4.5-6.0 1.09 - ( - ) - ( " ) 0-6.0 2.0 (8.8) 7.4 (32) Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA. 78 TABLE 25. PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=20 mg/kg) Parameter ig 2g 3g 4g 5g 6g Mean + SD Weight3 270 285 290 310 265 305 288 + 18 Dose6 5.4 5.7 5.8 6.2 5.3 6.1 5.8 + 0.4 C c ^0 85 88 100 96 74 100 91 + 11 ld 560 550 620 420 560 590 550 ± 70 4. e l l / 2 12 13 11 16 12 12 13 + 1.8 v / 220 210 180 200 270 190 210 + 30 AUC^ 1700 1700 1800 2400 1400 1800 1800 + 310 % DOSE EXCRETED IN URINE Unconj 0.62 0.36 1.4 1.3 2.6 2.9 1.5 + 1.0 Conj 19 12 22 19 12 14 16 + 4.0 Total 19 12 23 20 15 17 18 + 3.9 % DOSE EXCRETED IN BILE Unconj 5.3 5.4 4.3 10 7.8 4.0 6.2 + 2.4 Conj 33 27 22 14 21 23 23 + 6.5 Total 38 32 26 24 29 27 29 + 5.2 CLEARANCE Iml/m i n. kq) C1R 0.08 0.04 0.15 0.11 0.36 0.33 0.18 + 0.1< c i B 0.64 0.63 0.48 0.87 1.1 0.44 0.69 + 0.2' Cl u 1 conj 6.3 4.5 4.8 2.8 4.6 4.2 4.5 + 1.1 c i T 12 12 11 8.5 14 11 11 + 1.8 a, g; o, mg/kg; c, ng/ml; d, xl0"° min"1; e, min; f, mL/kg; g, itg.min/mL 79 TABLE 26. PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN BILE-EXTERIORIZED RATS (D0SE=100 mg/kg) Parameter lh 2h 3h 4h 5h 6h Mean + SD Weight3 290 325 302 295 280 300 299 + 15 Dose6 29 33 30 30 28 30 30 + 1.5 290 320 310 250 270 310 290 + 28 h d 430 300 310 380 400 440 380 + 58 t l / 2 16 23 22 18 17 16 19 + 3.1 v, f 340 320 320 400 370 320 350 + 35 AUC^ 15000 16000 16000 12000 12000 12000 14000 + 2100 % DOSE EXCRETED IN URINE Unconj 2.6 3.5 2.5 2.8 0.96 1.4 2.3 + 0.94-Conj 23 31 22 24 23 15 23 + 5.1 Total 26 35 24 27 24 17 25 + 5.8 % DOSE EXCRETED IN BILE Unconj 4.7 8.4 3.8 3.9 6.1 4.5 5.5 + 1.6 Conj 15 18 16 12 16 15 15 + 2.0 Total 20 27 20 16 22 19 21 + 3.5 CLEARANCE (mL/min.kq) C 1 R 0.18 0.23 0.15 0.22 0.08 0.19 0.16 + 0.06 ci B 0.32 0.53 0.30 0.32 0.53 0.38 0.40 + 0.11 Cl u 1 conj 2.6 3.2 2.3 2.9 3.4 2.6 2.8 + 0.42 C 1 T 6.8 6.4 6.1 8.0 8.7 8.4 7.4 + 1.1 a, g; b, f, mL/kg; mg/kg; c, /ig/mL; d, g, /zg.min/mL xlO"5 min - 1 - e min; TABLE 27. URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=20 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (itg/mL) (mg) (h) Amt (mg) % of Dose ig 0-6 3.80 8.8 (270) 0.03 (1.0) 0-6 0.03 (1.0) 0.6 (19) 6-24 18.5 ~ (-) ~ (-) 0-24 0.03 (1.0) 0.6 (19) 2g 0-6 3.00 6.9 (230) 0.02 (0.68) 0-6 0.02 (0.68) 0.4 (12) 6-24 4.70 (-) (3.5) 0-24 0.02 (0.70) 0.4 (12) 3g 0-6 4.30 19 (300) 0.08 (1.3) 0-6 0.08 (1.3) 1.4 (23) 6-24 5.70 ~ (3.8) ~ (0.02) 0-24 0.08 (1.3) 1.4 (23) 4g 0-6 2.80 28 (440) 0.08 (1.2) 0-6 0.08 (1.2) 1.3 (20) 6-24 3.84 ~ (4.1) ~ (0.02) 0-24 0.08 (1.3) 1.3 (20) 5g 0-6 3.15 33 (220) 0.10 (0.69) 0-6 0.10 (0.69) 2.0 (13) 6-24 4.38 7.8 (21) 0.03 (0.09) 0-24 0.14 (0.78) 2.6 (15) 6g 0-6 3.75 35 (260) 0.13 (0.98) 0-6 0.13 (0.98) 2.2 (16) 6-24 5.14 9.2 (15) 0.05 (0.08) 0-24 0.18 (1.1) 2.9 (17) Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA. TABLE 28. URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=100 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (Mg/mL) (mg) (h) Amt (mg) % of Dose lh 0-5 4.66 150 (1500) 0.70 (7.0) 0-5 0.70 (7.0) 2.4 (24) 5-24 9.70 4.8 (46) 0.047 (0.45) 0-24 0.75 (7.4) 2.6 (26) 2h 0-6 3.80 280 (2800) 1.1 (11) 0-6 1.1 (11) 3.3 (34) 6-24 9.50 6.2 (46) 0.059 (0.44) 0-24 1.1 (11) 3.5 (35) 3h 0-6 6.70 96 (1000) 0.64 (6.9) 0-6 0.64 (6.9) 2.1 (23) 6-24 18.0 5.7 (20) 0.10 (0.36) 0-24 0.74 (7.3) 2.5 (24) 4h 0-6 2.80 220 (2900) 0.62 (4.9) 0-6 0.62 (4.9) 2.1 (17) 6-24 4.70 39 (600) 0.18 (2.8) 0-24 0.81 (7.7) 2.8 (27) 5h 0-7 4.40 56 (1400) 0.25 (6.1) 0-7 0.25 (6.1) 0.9 (22) 7-24 9.70 2.4 (55) 0.023 (0.53) 0-24 0.27 (6.7) 1.0 (24) 6h 0-7 3.96 100 (1200) 0.40 (4.8) 0-7 0.40 (4.8) 1.3 (16) 7-24 8.45 2.9 (31) 0.025 (0.26) 0-24 0.42 (5.0) 1.4 (17) Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA. 82 TABLE 29. BILIARY ELIMINATION OF UNCONJUGATED 4-ENE VPA IN RATS (0OSE=20 mg/kg) Rat Time Volume Cone Amount Time Cumulative Cumulative (h) (mL) (ug/mL) (mg) (h) Amt (mg) X of Dose ig 0-0.5 1.10 200 (1500) 0.22 (1.7) 0-0.5 0.22 (1.7) 4.1 (31) 0.5-1.5 1.43 45 (280) 0.064 (0.41) 0-1.5 0.28 (2.1) 5.2 (38) 1.5-2.5 1.19 2.3 (4.9) 0.003 (0.01) 0-2.5 0.29 (2.1) 5.3 (38) 2.5-3.5 1.15 - (-) - (-) 0-3.5 0.29 (2.1) 5.3 (38) 3.5-4.5 1.00 - (-) - (-) 0-4.5 0.29 (2.1) 5.3 (38) 2g 0-0.5 1.10 220 (1300) 0.24 (1.5) 0-0.5 0.24 (1.5) 4.2 (26) 0.5-1.5 1.40 43 (240) 0.06 (0.34) 0-1.5 0.03 (1.8) 5.3 (32) 1.5-2.5 1.28 2.8 (4.7) 0.004 (0.01) 0-2.5 0.31 (1.8) 5.4 (32) 2.5-3.5 1.31 - (-) - (-) 0-3.5 0.31 (1.8) 5.4 (32) 3.5-4.5 1.23 - (-) - (-) 0-4.5 0.31 (1.8) 5.4 (32) 3g 0-0.5 1.08 170 (1100) 0.18 (1.2) 0-0.5 0.18 (1.2) 3.1 (20) 0.5-1.5 1.60 41 (200) 0.065 (0.31) 0-1.5 0.25 (1.5) 4.2 (26) 1.5-2.5 1.38 2.2 (4.5) 0.003 (0.01) 0-2.5 0.25 (1.5) 4.3 (26) 2.5-3.5 1.17 - (-) - (-) 0-3.5 0.25 (1.5) 4.3 (26) 3.5-4.5 1.08 - (-) - (-) 0-4.5 0.25 (1.5) 4.3 (26) 4g 0-0.5 0.79 410 (990) 0.32 (0.78) 0-0.5 0.32 (0.78) 5.2 (13) 0.5-1.0 0.62 360 (930) 0.22 (0.58) 0-1.0 0.55 (1.4) 8.8 (22) 1.0-2.25 1.12 76 (97) 0.085 (0.11) 0-2.25 0.63 (1.5) 10 (24) 2.25-3.25 0.65 2.5 (3.0) 0.002 (0.002) 0-3.25 0.64 (1.5) 10 (24) 3,25-4.75 0.99 - (-) - (-) 0-4.75 0.64 (1.5) 10 (24) 4.75-6.0 0.85 - (-) - (-) 0-6.0 0.64 (1.5) 10 (24) 5g 0-0.5 1.06 290 (1100) 0.31 (1.2) 0-0.5 0.31 (1.2) 5.7 (23) 0.5-1.5 1.47 73 (220) 0.11 (0.33) 0-1.5 0.41 (1.5) 7.8 (29) 1.5-2.5 1.22 2.4 (3.5) 0.003 (0.004) 0-2.5 0.42 (1.5) 7.8 (29) 2.5-3.5 1.00 - (-) - (-) 0-3.5 0.42 (1.5) 7.8 (29) 3.5-4.5 0.91 - (-) - (-) 0-4.5 0.42 (1.5) 7.8 (29) 6g 0-0.5 0.95 190 (1300) 0.18 (1-2) 0-0.5 0.18 (1.2) 2.9 (20) 0.5-1.5 1.25 51 (360) 0.064 (0.45) 0-1.5 0.24 (1.7) 4.0 (27) 1.5-2.5 1.11 2.1 (11) 0.002 (0.012) 0-2.5 0.25 (1.7) 4.0 (27) 2.5-3.5 1.02 - (-) - (-) 0-3.5 0.25 (1.7) 4.0 (27) 3.5-4.5 0.78 - (-) - (-) 0-4.5 0.25 (1.7) 4.0 (27) Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA. 83 TABLE 30. BILIARY ELIMINATION OF UNCONJUGATED 4-ENE VPA IN RATS (DOSE=100 mg/kg) Rat Time Volume Cone Amount Time Cumu ati ve Cumulative (h) (mL) (Mg/mL) (mg) (h) Amt Cmg) % of Dose 1h 0-0.5 0.70 600 (2000) 0.42 (1.4) 0-0.5 0.42 (1.4) 1.5 (4.1) 0.5-1.5 1.24 490 (2600) 0.61 (3.2) 0-1.5 1.0 [4.6) 3.6 (16) 1.5-2.5 1.07 290 (1000) 0.32 (1.1) 0-2.5 1.4 £5.7) 4.6 (20) 2.5-3.5 0.88 25 (50) 0.022 (0.044) 0-3.5 1.4 C5.8) 4.7 (20) 3.5-4.5 0.80 3.8 (6.6) 0.003 (0.005) 0-4.5 1.4 (5.8) 4.7 (20) 4.5-5.5 0.85 - (-) - (-) 0-5.5 1.4 C5.8) 4.7 (20) 2h 0-0.5 1.15 610 (2800) 0.70 (3.2) 0-0.5 0.70 (3.2) 2.2 (10) 0.5-1.0 1.30 630 (2800) 0.82 (3.6) 0-1.0 1.5 (6.8) 4.8 (21) 1.0-1.5 1.10 450 (680) 0.50 (0.75) 0-1.5 2.0 (7.6) 6.3 (24) 1.5-2.0 1.24 260 (480) 0.32 (0.59) 0-2.0 2.3 (8.2) 7.3 (26) 2.0-3.5 1.34 250 (270) 0.33 (0.36) 0-3.5 2.7 (8.5) 8.3 (27) 3.5-5.0 1.57 4.8 (3.2) 0.008 (0.005) 0-5.0 2.7 (8.5) 8.4 (27) 5.0-6.0 0.87 - (-) - (-) 0-6.0 2.7 (8.5) 8.4 (27) 3h 0-0.5 0.71 480 (2500) 0.34 (1.7) 0-0.5 0.34 (1.7) 1.1 (5.8) 0.5-1.5 1.38 260 (2000) 0.36 (2.8) 0-1.5 0.70 (4.5) 2.3 (15) 1.5-2.5 1.23 300 (1100) 0.37 (1.4) 0-2.5 1.1 (5.9) 3.6 (20) 2.5-3.5 0.89 70 (170) 0.063 (0.15) 0-3.5 1.1 (6.0) 3.8 (20) 3.5-4.5 0.75 7.6 (15) 0.006 (0.011) 0-4.5 1.1 (6.1) 3.8 (20) 4.5-6.0 1.08 - (3.0) - (0.003) 0-6.0 1.1 (6.1) 3.8 (20) 4h 0-0.5 0.76 500 (1700) 0.38 (1.3) 0-0.5 0.38 (1.3) 1.3 (4.4) 0.5-1.5 1.23 420 (2100) 0.52 (2.6) 0-1.5 0.90 (3.9) 3.1 (14) 1.5-2.5 0.75 290 (930) 0.22 (0.70) 0-2.5 1.1 (4.6) 3.8 (16) 2.5-3.5 0.55 42 (120) 0.023 (0.065) 0-3.5 1.1 (4.7) 3.9 (16) 3.5-4.5 0.49 6.3 (12) 0.003 (0.006) 0-4.5 1.1 (4.7) 3.9 (16) 4.5-6.0 1.03 - (2.0) - (0.002) 0-6.0 1.1 (4.7) 3.9 (16) 5h 0-0.5 1.05 500 (2100) 0.53 (2-2) 0-0.5 0.53 (2.2) 1.9 (8.0) 0.5-1.5 2.04 520 (1800) 1.1 (3.6) 0-1.5 1.6 (5.8) 5.7 (21) 1.5-2.5 1.46 75 (240) 0.11 (0.35) 0-2.5 1.7 (6.2) 6.1 (22) 2.5-3.5 1.29 5.7 (16) 0.007 (0.020) 0-3.5 1.7 (6.2) 6.1 (22) 3.5-4.5 1.05 - (3.4) - (0.003) 0-4.5 1.7 (6.2) 6.1 (22) 4.5-5.5 0.97 - (-) - (-) 0-5.5 1.7 (6.2) 6.1 (22) 6h 0-0.5 0.95 480 (2000) 0.45 (1.9) 0-0.5 0.45 (1.9) 1.5 (6.4) 0.5-1.5 2.00 350 (1600) 0.70 (3.2) 0-1.5 1.2 (5.2) 3.8 (17) 1.5-2.5 1.38 110 (310) 0.16 (0.43) 0-2.5 1.3 (5.6) 4.4 (19) 2.5-3.5 1.12 15 (160) 0.016 (0.18) 0-3.5 1.3 (5.8) 4.4 (19) 3.5-4.5 1.04 8.3 (25) 0.009 (0.026) 0-4.5 1.3 (5.8) 4.5 (19) 4.5-5.5 0.91 - (-) - (-) 0-5.5 1.3 (5.8) 4.5 (19) Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA. 84 5-fold increase in the dose. For both the low and the high dose, a total of 47-46% was excreted in the urine and bile collectively (Tables 27-30). The fraction of dose excreted as unconjugated 4-ene VPA in urine and bile was not altered significantly (Table 32). C.3.4. Choleretic Effect A typical choleretic effect of the high dose of 4-ene VPA in the rat is shown in Fig. 13. The normal bile flow rate of 0.82 mL/h increased rapidly to 1.4 mL/h within the first 0.5 h of 4-ene VPA injection. The bile flow declined slowly for the next hour and rapidly thereafter to normal values within 4 h. The excretion rate of 4-ene VPA in bile samples plotted against the mid-points of the time intervals for sample collection is also shown in the Fig. 13. The maximal excretion rate of conjugated 4-ene VPA (3.48 mg/h) in bile was observed within the first half-hour of the dose. The excretion rate of conjugated 4-ene VPA in bile then declined almost parallel to the bile flow rate. Approximately 95% of the biliary elimination of 4-ene VPA was complete within 3 h of the dose (Fig. 14). Only low levels of unconjugated 4-ene VPA were found in the bile. A similar choleretic effect was observed after the administration of low dose of 4-ene VPA (Table 29). The duration of maximal bile flow rate after the low dose was, however, shorter than after the high dose. A similar choleretic effect of (E)-2-ene VPA on the bile flow rate, and its elimination in the bile of the rat was observed (Tables 23, 24) as described above for 4-ene VPA. C.3.5. In Vitro Protein Binding: The plasma protein binding of 4-ene VPA was apparently low (14-25%), at various concentrations ranging from 20 to 350 /zg/mL (Table 33). TABLE 31. COMPARISON OF PHARMACOKINETIC NORMAL Parameter 20 mg/kga 100 mg/kga p-Value k^ 310+60 260±50 NS t l / 2 d 23+4.2 28+5.8 NS V j e 240+40 230+20 NS AUC/Df 810+350 1160±74 NS C1R9 0.27±0.13 0.29±0.11 NS C 1met g 4.6+1.7 2.8±0.21 NS C1T^ 4.9+1.7 3.0+0.27 NS % Dose Excreted in Urine Unconj 5.9+3.3 9.4+3.2 NS Conj 26+6.4 40+11 NS Total 32+6.3 50+11 NS a, Dose; b, p>0.05=Nonsignificant (NS); c, xlO"5 min"1; d, min; e, mL/kg; f, /zg.min/mL.mg; g , mL/min.kg Each value is a mean of 4 observations + SD PARAMETERS OF (E)-2-ENE VPA IN THE RAT BILE EXTERIORIZED Parameter 20 mg/kga 100 mg/kga p-Value k l c 350±55 330+27 NS t l / 2 d 20±3.4 21±1.7 NS v i e 220±29 240±17 NS AUC/Df 460±150 590+77 NS Clp 9 0.28+0.13 0.20+0.11 NS C1B9 0.64+0.28 0.37+0.16 NS Cl -9 ° 'conj 4 .1±1.0 3.5+1.1 NS C1T9 % 7.7+1.8 Dose Excreted 6.0+1.1 in Urine NS Unconj 3 .5±1.6 3.3±1.4 NS Conj 24±6.1 32+11 NS Total % 27±7.6 Dose Excreted 35+11 in Bile NS Unconj 8.1+2.8 6.1+2.6 NS Conj 29±6.8 25+6.8 NS Total 38+9.5 31+9.3 NS TABLE 32. COMPARISON OF PHARMACOKINETIC NORMAL Parameter 20 mg/kga 100 mg/kga p-Value' k j c 580±80 380±34 S t 1 / 2 c f 12+1.7 18+1.5 S V j e 210±37 320+25 S AUC/Df 390+27 620+53 S aR3 0.47±0.21 0.23+0.12 S C l m e t g 8.2+0.51 5.7±0.43 S Clj? 8.7±0.60 5.9+0.54 S % Dose Excreted in Urine Unconj 5.3+2.3 3.8+1.7 NS Conj 17±2.1 24+5.7 S Total 22±3.1 28+5.7 S a, Dose; 6, p>0.05=Nonsignificant (NS), o, p < 0.05 Significant (S); c, xlO"5 min"1; d, min; e, mL/kg; f, jLtg.min/mL.mg; g , mL/min.kg Each value is a mean of 6 observations + SD PARAMETERS OF 4-ENE VPA IN THE RAT BILE EXTERIORIZED Parameter 20 mg/kga 100 mg/kga p-Value' k i c 550±68 380±60 S t d  tl/2 13±1 .8 19+3.1 S v i e 210±31 350±35 s AUC/Df 310±37 460±60 s ClR g 0 . 1 8 ± 0 . 1 4 0 . 1 6 ± 0 . 0 6 NS c i B g 0 . 6 9 ± 0 . 2 4 0 . 4 0 ± 0 . 1 1 s Cl -3 ° 'conj 4.5+1.1 2 . 8 ± 0 . 4 2 s C1T^ 11+1.8 7. 4±1.1 s % Dose Excreted in Urine Unconj 1.5+1.0 2.3+0.94 NS Conj 16±4 .0 23±5.1 S Total 18+3.9 25+5.8 S % Dose Excreted in Bile Unconj 6 . 2 ± 2 . 4 5 . 5 ± 1 . 6 NS Conj 2 3 ± 6 . 5 15+2.0 S Total 2 9 ± 5 . 2 21+3.5 S 87 TABLE 33. PLASMA PROTEIN BINDING OF 4-ENE VPA 4-Ene Cone Ultrafiltrate Bound % Bound {ng/ml) Cone {ng/ml) Cone {ng/ml) 20 15 + 1.7 5.1 + 1.7 26 + 8.3 50 38 + 2.2 12 + 2.2 24 + 4.4 100 82 + 4.9 18 + 4.9 18 + 4.9 150 120 + 1.3 30 + 1.3 20 + 0.8 200 170 + 5.9 31 + 5.9 16 + 3.0 250 210 + 1.5 36 + 1.5 14 + 0.6 300 240 + 8.3 58 + 8.3 19 + 2.8 350 300 + 2.6 53 + 2.6 16 + 1.2 Each value is a mean + SD of 3 observations. 88 C.3.6. Metabolism of (E)-2-ene in the rat The presence of several potential metabolites of (E)-2-ene VPA was investigated in the urine and bile of the rat. Only two diene VPA metabolites (I and II) were found in conjugated form in the urine and bile of the rat. Diene VPA I may be either 2(E),4-diene VPA or 2(E),3'(Z)-diene VPA or a mixture of the two, since both isomers have identical retention times. Diene VPA II was tentatively assigned the structure of (E)-2,3'-diene VPA, based on the same retention time as of an authentic synthetic sample. No conclusive evidence was found for the reverse conversion of (E)-2-ene VPA to VPA. Nau and Loscher (1985) have reported that the major metabolites of (E)-2-ene VPA in the plasma of mice are 3-keto VPA, VPA and 5-hydroxy VPA, but no diene VPA metabolites were mentioned. These results may be explained by the difference in the metabolic pathways of (E)-2-ene VPA in different species. 89 D. DISCUSSION D. l . CHEMISTRY The 3-hydroxy VPA, VPA and 4-ene VPA were prepared by the treatment of an alkylcarboxylic acid with strong base, lithium diisopropylamide (LDA), to generate dianions which on alkylation with appropriate alkyl derivative such as alkyl halide or aldehyde gave the alpha-subsituted aliphatic acid in good yield (Pfeffer et al., 1972). Dehydration of 3-hydroxy VPA gave a mixture of 3-ene VPA and 2-ene VPA as reported by Blaise and Bagard (1907). 3-Ene VPA was separated from 2-ene VPA by double reverse extractions, and vacuum disti l lation. 2-Ene VPA was obtained by dehydrobromination of 2-bromo-2-propylvaleric acid ethyl ester with quinoline, followed by saponification and acidification (Thallandier et al., 1977). D.2. ASSAY The metabolites (E)-2-ene VPA and 4-ene VPA have been quantitated, along with other VPA metabolites, by GC (Loscher, 1981) and GCMS (Nau et al., 1981b; Kochen et al., 1983; Granneman, et al., 1984c) assay methods. The major drawbacks of these methods are poor sensitivity (Loscher, 1981), long and tedious extraction procedures for sample preparation (Nau et al., 1981b; Kochen et al., 1983) and long retention time (Kochen et al., 1983). A capillary GCMS procedure has been used to measure 4-ene VPA as its trimethylsilyl derivative (Rettenmeier et al., 1985). (E)-2-ene VPA has also been determined by GCMS using chemical ionization (Granneman et al., 1984c; Schobben et al., 1980a). Abbott et al. (1986) have successfully employed silylation, with t-BDMS derivatizing reagents, for the estimation of VPA and its metabolites in human saliva, serum and urine. The t-BDMS 90 derivatives are very stable (de Jong et al., 1980), and provide higher sensitivity than TMS derivatives for the estimation of VPA metabolites. In the init ial stages of the development of this assay method, t-BDMS chloride was employed to study optimum conditions for derivatization of VPA, since the drug was available in large quantities to carry out experimentation. The reagent was dissolved in dry pyridine at a concentration of 50% w/v. The reagent solution derivatized the analyte very slowly, and took 6 h of heating at 60°C for complete esterification. To increase the speed of this reaction, various concentrations of a strongly basic catalyst, DMAP (dimethylaminopyridine) were added to the reagent solution. At a DMAP concentration of 20%, the reaction rate was enhanced several fold and was complete within 30 min. Chromatographic analysis showed peaks of the reagent which did not interfere with the peak of VPA, and had different retention time than that of either of the metabolites studied in this project. Not long after that, MTBSTFA was introduced to prepare t-BDMS derivatives of alcohols and carboxylic acids (Mawhinney and Madson 1982). With MTBSTFA, derivatization was instantaneous on its addition to a carboxylic acid. Moreover, no extraneous peaks were seen since the reagent peak emerges very early along with the solvent peak under the present GC conditions. This reagent also offers the convenience of being sold in a ready to use form. Addition of a large excess of MTBSTFA (20 nl or greater) gradually decreased the peak areas on repeated injections into GCMS. The t-BDMS derivatives provide a large (M-57)+ mass ion peak on electron impact MS. Thus, selected ion monitoring of m/z 199, corresponding to (E)-2-ene VPA and 4-ene VPA, and 229 for DNBA were carried out to estimate the amount of the analyte. Monitoring a higher mass offers the advantage of reducing the chances of interference by endogenous and extraneous substances. 91 Conjugates of (E)-2-ene VPA and 4-ene VPA, in urine and bile, were hydrolyzed at pH > 12 by heating with 3N NaOH to ensure complete hydrolysis. Dickinson et al. (1984) have reported that glucuronide ester conjugates of VPA undergo rearrangement to resistant forms, which do not hydrolyze on incubation with the enzyme, /J-glucuronidase. In alkaline medium, however, various kinds of conjugate esters of VPA, including glucuronide and sulfate esters, are hydrolyzed (Dickinson et al., 1984; Dickinson et al., 1979a). The salient features of this assay method are that it is an extremely simple procedure that involves a single extraction of the biological sample, unlike the long procedure of Nau et al. (1981). The extraction efficiency is virtually 100%. There is no concentration step involved. Using an 80 /zL plasma sample, concentrations as low as 60 and 100 ng/mL of (E)-2-ene VPA and 4-ene VPA, respectively, were detected with a signal-to-noise ratio of 4. The assay method provide higher sensitivity than the GC method of Loscher (1981). Calibration curves are linear over a wide range of concentrations in the plasma, urine and bile of the rat. The assay method is able to separate the diene VPA metabolites of (E)-2-ene VPA and 4-ene VPA without any interference. The assay method is sensitive, specific and requires only 12 min for elution of the metabolites and the internal standard after an injection into the GCMS (Singh et al., 1987). D.3. PHARMACOKINETICS The pharmacokinetics of (E)-2-ene VPA and 4-ene VPA in the rat were similar to those reported for VPA (Dickinson et al., 1979a). Both (E)-2-ene VPA and 4-ene VPA were, init ial ly , rapidly cleared from the plasma within the first 1-2 h after the dose, followed by recirculation into plasma due to enterohepatic circulation. Thus, the plasma levels were maintained for 92 several hours after a single injection. Like the parent drug VPA, (E)-2-ene VPA and 4-ene VPA were mainly eliminated as conjugates in urine and bile. D.3.1. Pharmacokinetics in Normal Rats D.3.1.1. Plasma Profile: Both (E)-2-ene VPA and 4-ene VPA showed a non-linear plasma decline at concentrations above 200 /ig/mL, whereas VPA exhibited similar non-linear decline at plasma levels > 100 lig/ml (Dickinson et al., 1979a). The much higher concentration of (E)-2-ene VPA and 4-ene VPA in the collected plasma samples compared to the highest plasma standard in the calibration curve may contribute to the non-linear plasma profile of these metabolites. However, this non-linear behavior was observed in every animal and was, therefore, assumed to be due to the saturation of one or more of the metabolic pathways and/or an excretory process(s) at the above concentrations. The plasma t j / 2 of (E)-2-ene VPA was 23 min during the first hour following the 20 mg/kg dose and it increased by 1.2 times to 28 min following the 100 mg/kg dose. The plasma profile of 4-ene VPA also showed a similar increase of 1.5 fold in the ^1/2' ^ r o m 1 2 t o 1 8 m i n > during the first h following the two doses. These results are similar to those reported for VPA, which showed a 3.6-fold longer t j ^ , from 12 to 41 min, when the dose was increased from 15 to 150 mg/kg (Dickinson et al., 1979a). There was a time lag of at least 1 h, before plasma levels of (E)-2-ene VPA, 4-ene VPA and the parent drug VPA (Ogiso et al., 1986; Dickinson et al., 1979a) started to rise due to EHC in the rat. After the appearance of a secondary peak at 3-4 h, the metabolites were slowly eliminated from the plasma. This terminal, slow elimination of (E)-2-ene VPA and 4-ene VPA from plasma cannot be called a /3-phase, as has been pointed out by Dickinson et al. (1979a), since reabsorption of the xenobiotic would s t i l l be occuring from the GIT of the rat. Experiments in 93 bile-exteriorized rats in the present study have confirmed that i f (E)-2-ene VPA or 4-ene VPA is prevented from entering the GIT of the rat via the bile, secondary plasma peaks and the subsequent slow elimination phase are abolished. Several drugs including acetaminophen (Watari et al., 1983), VPA (Dickinson et al., 1979a) and xenobiotics such as 4-ene VPA (Rettenmeier et al., 1986) are extensively conjugated with glucuronic acid before elimination. Glucuronide conjugates are secreted into bile which flows into the intestine via the duodenum. Inside the lumen of the GIT of the rat, as well as other animals, glucuronic acid esters are hydrolyzed by B-glucuronidase, an enzyme produced mainly by resident microorganisms (Clark et al., 1969; Hill and Drasar 1975). The unconjugated moiety (drug or xenobiotic) is then reabsorbed through the intestinal wall into the systemic circulation (Klaassen and Watkins 1984). The ^-glucuronidase enzyme activity is negligible in the duodenum and ileum, and is maximal in the cecum of the rat (Marsh et al., 1952). The lack of glucuronidase activity in the proximal part of the intestine explains, in part, the time lag associated with the appearance of a secondary peak of (E)-2-ene VPA and 4-ene VPA in the plasma of the rat. Moreover, the processes of deconjugation and absorption of unconjugated molecules through the GI wall may also add to the delay in the circulation. Ogiso and coworkers (1986) have also shown that deconjugation of VPA is the rate limiting step during its enterohepatic circulation in the rat. D.3.1.2. Pharmacokinetic Modelling: In the early stages of the pharmacokinetic modelling for drugs undergoing EHC, simple two compartment models were proposed to describe their plasma profiles (Harrison and Gibaldi 1976; Chen and Gross 1979), especially in the rat 94 which does not have a gall bladder and therefore secretes bile more or less continously into the GIT. However, such models are not suitable for the plasma data of (E)-2-ene VPA, 4-ene VPA or VPA in the rat due to the delay in the transfer of the molecules from bile to blood as described above. To solve this problem, Dahlstrom and Paalzow (1978) successfully applied a multi-segment-loop intestine model to describe the EHC of morphine in the rat. The drug, in bile, enters into 3-5 serially connected "intestinal compartments" before entering the blood compartment. Such a model essentially adds to the time required for the recirculating drug to reach the systemic circulation. A second approach was taken by other investigators including Veng Pedersen and Miller (1980), Steimer et al. (1982), Col burn (1984), and more recently by Shepard et al. (1985). According to this modelling technique, a continuous time-lag function is introduced in the solution to the differential equations. These models provide the freedom to alter the time-lag (tau), and also to study the effect of such a change on the overall disposition of a drug undergoing EHC. In addition, more than one time-lag function can be chosen to describe repetitive recycling (Colburn, 1984). The plasma data, after the low dose administration of (E)-2-ene VPA and 4-ene VPA to the rat, were fitted to a relatively simple version of the model proposed by Veng Pedersen and Miller (1980) and Colburn (1984). The differential equations were solved by MULTI(RUNGE), a non-linear least squares regression program (Yamaoka and Nakagawa 1983). The time-lag value was chosen to provide a best f i t of the model-predicted curve to actual plasma data points, as measured by the least sum of residual squares. The calculated values of microconstants k 1 0 , k 1 2 a n d 2^1 w e r e u s e c ' t o determine the effective clearance (Cl eff) and net clearance ( C l n e t ) as reported by Colburn (1984). C l e f f may be calculated as Vj . (k j 0 + k 1 2 ) t o describe the intrinsic ability of liver to remove (E)-2-95 ene VPA or 4-ene VPA from the blood. C l n e t is obtained by Vj.kjQ to estimate the permanent elimination of the metabolite from the body of the rat. The calculated values of C l e f f for (E)-2-ene VPA was 6.9 and for 4-ene VPA, 11 mL/min.kg. The C l e f f values, calculated from the plasma data of normal rats, are close to Clj in bile-exteriorized rats, which suggests that the model-generated values for microconstants are fairly accurate. This pharmacokinetic model, however, does not explain all the plasma data points, especially at the trough levels approximately 2 h after the dose. Therefore, its applicability is partially limited. D.3.1.3. Pharmacokinetic Parameters: The pharmacokinetic parameters of (E)-2-ene VPA in the rat determined in this study are different from those reported by O'Connor et al. (1986) in several respects. The total plasma clearance of (E)-2-ene VPA decreased from 4.9 to 3.0 mL/min.kg on increasing the dose from 20 to 100 mg/kg. In contrast, O'Connor et al. (1986) have reported that the serum clearance of (E)-2-ene VPA increased from 4.0 to 6.1 mL/min.kg when the dose was raised from 25 to 75 mg/kg in the rat. The value of Clj at the low doses are close to each other (4.9 versus 4.0 mL/min.kg). However, the effect of increasing the dose is opposite in these two studies. This discrepency is not easy to explain due to the lack of information in their abstract on their experimental design, the schedule for blood sampling and the duration of blood collection. It may be speculated that the difference in the strains of the rat used could produce different results. Secondly, a short duration of blood collection, up to 2 h or less, could miss the recirculation of (E)-2-ene VPA in the blood, and erroneously give a smaller value of AUC and a larger C l j . On the other hand, i f the blood sampling is infrequent, a 96 recycled drug may appear to exhibit a two-compartment model profile (Colburn 1984). As an example, in a pharmacokinetic study of VPA in the rat in which blood samples were collected infrequently, the plasma data were fitted to a two compartment model by Loscher (1978). The pharmacokinetic experiments in which blood samples were withdrawn frequently (Dickinson et al., 1979; Ogiso et al., 1986) have shown that VPA in fact undergoes substantial EHC in the rat. Therefore, the elimination half-life (4.6 h) and Vd(/S), 657 mL/kg for VPA as calculated by Loscher (1978) are different from the pharmacokinetic parameters such as Vj (143 mL/kg) of Ogiso et al. (1986). Similarly O'Connor et al. (1986) may have used a different experimental protocol or different modelling techniques from ours to arrive at their results. A comparison of the total apparent plasma clearance of (E)-2-ene VPA in different animals species shows that the C1T in the rat (4.9 mL/min.kg), following the low dose, is similar to that in the mouse (5.7 mL/min.kg) (Nau and Zierer 1982). 4-ene VPA is cleared much faster from the plasma of the rat than in the monkey (Rettenmeier et al., 1986). C1T in the rat at the low dose of 4-ene VPA (8.7 mL/min.kg) was on an average 3.7 times faster than that in the monkey, probably due to the higher metabolic rate in smaller animals. The urinary elimination of 4-ene VPA in the unconjugated form was 5% in the rat, which is identical to that reported for the monkey (Rettenmeier et al., 1986). The rat, however, excreted less than half as much 4-ene VPA in conjugated form in the urine as does the monkey. D.3.2. Pharmacokinetics in Bile-Exteriorized Rats D.3.2.1. Plasma Profile: In bile-exteriorized rats, (E)-2-97 ene VPA and 4-ene VPA were eliminated with respective plasma t j / 2 of 20 and 13 min at the low dose, 21 and and 19 min at the high dose. The elimination half-life remained unaltered for (E)-2-ene VPA and increased 1.5 times for 4-ene VPA with a five fold increase in their respective doses. A similar increase of 1.5 times, from 11 to 17 min, was reported for VPA plasma half-l i fe in bile-exteriorized rats given doses of 15 and 150 mg/kg (Dickinson et al., 1979a). No secondary plasma peaks were observed in these rats. D.3.2.2. Biliary Elimination: The biliary elimination of (E)-2-ene VPA decreased from 38 to 31% of the administered dose, and 29 to 21% for 4-ene VPA, with a 5 times increase in the dose. The urinary excretion, however, increased from 27 to 35% of (E)-2-ene VPA dose, and 18 to 25% of 4-ene VPA dose with a five fold increase in their respective doses. These results are similar to those of VPA which showed an enhanced urinary elimination from 4 to 15%, when the dose was raised 10 times (Dickinson et al., 1979a). These observations suggest that as the biliary excretion of VPA or one of its monounsaturated metabolites approaches saturation at the high dose, urinary elimination is enhanced as a complementary excretory pathway. The marked differences in the extent of biliary excretion of these chemically similar compounds of almost identical molecular weights, may be due to the subtle structural changes introduced by the presence and position of a double bond in the molecule. A molecular weight threshold of approximately 200 Dal tons for quaternary ammonium compounds and 300-325 Daltons for aromatic anions is essential for the elimination of a molecule in the bile of the rat (Welling 1986; Hirom et al., 1972b). However, a large molecule or size is not the sole factor in determining the extent of elimination in the bile. Molecules of similar size, but of slightly 98 different structures, have been reported to be eliminated to significantly different extents in the bile of the rat (Hirom et al., 1972a). D.3.2.3. Conjugation: The conjugation of (E)-2-ene VPA and 4-ene VPA seems to be the major route of metabolism and subsequent elimination in the rat. For the administered low dose (20 mg/kg), 29% of (E)-2-ene VPA and 23% of 4-ene VPA were eliminated as conjugates in the bile. For the high dose (100 mg/kg), 25% of (E)-2-ene VPA and 15% of 4-ene VPA were eliminated as conjugates in the bile. In contrast, a much larger percentage (58-61%) of VPA doses (15 and 150 mg/kg) was recovered as conjugates in the bile of the rat (Dickinson et al., 1979a). Approximately 53% of the low dose and 57% of the high dose of (E)-2-ene VPA was recovered in conjugated form collectively in the bile and urine. The corresponding values for 4-ene VPA were 39% and 38% , and for VPA, 62% and 76% in the rat (Dickinson et al., 1979a). Thus, conjugation, which is the most prominent route of elimination in the rat decreases in the order: VPA > (E)-2-ene VPA > 4-ene VPA. The difference in the extent of conjugation may be attributed to one of the following factors: The organic anions, including fatty acids (Renaud et al., 1978), are transported into hepatocytes by carrier-mediated active transport mechanisms. A reduced transport uptake of the monounsaturated metabolites compared to VPA may be responsible for this difference in the extent of conjugation amongst these compounds. This hypothesis is also supported by the experiments carried out by other investigators. Rettenmeier et al. (1985) have reported that in isolated rat liver perfusion studies, approximately 4 times larger amounts of conjugated 4-ene VPA were recovered in the bile when the length of the perfusion time was increased from 20 to 60 min. These results suggest that 4-ene VPA is transferred to hepatocytes in a time-dependent fashion, probably mediated 99 by a transport system. Similarly, Nau and Loscher (1985) have reported that (E)-2-ene VPA appears to enter the liver by an active transport mechanism in the mouse. The second factor that may be partly responsible for varying extents of conjugation is the nature of the enzyme, especially substrate affinity. The enzyme UDP-glucuronyltransferase (UDPGT) exists in two separate substrate-specific and inducer-selective forms (Watkins and Klaassen 1982; Dutton and Burchell 1977). A higher affinity of UDPGT enzyme for VPA than (E)-2-ene VPA, which in turn is greater than the affinity for 4-ene VPA, may contribute to this variance in the degree of conjugation of these compounds. An interesting observation is that, with an increase in the dose of VPA (Dickinson et al., 1979a) and (E)-2-ene VPA, the fraction of the dose excreted as conjugates in the urine and bile, collectively, also increased in bile-exteriorized rats. This increase in the conjugation of VPA (62% versus 79% of the dose) is significant (Dickinson et al., 1979a) and only slight (53% versus 57% of the dose) for (E)-2-ene VPA. Practically no change was observed in the fraction of 4-ene VPA dose (39% versus 38%) eliminated as conjugates between the low and high dose. These results indicate that metabolic pathways other than conjugation and/or excretory routes for the elimination of VPA and (E)-2-ene VPA may be approaching saturation at the high dose. Thus, a larger fraction of the high dose may be available for conjugation. D.3.2.4. Choleretic Effect: The choleretic effect of (E)-2-ene VPA and 4-ene VPA may be partly due to increased osmotic pressure in the bile canaliculi, created by large quantities of conjugated moieties. This hypothesis is supported by the observation that the bile flow rate was 100 directly proportional to the excretion rate of 4-ene VPA conjugates in the bile (Fig 13). Moreover, the duration of maximal flow rate was dose-dependent, being shorter (0.5 h) after the low dose and longer (>2 h) after the high dose of either of the metabolites. These results are similar to those observed for VPA which has been shown to induce choleresis due primarily to osmotic activity in the bile (Dickinson et al., 1982; Watkins and Klaassen 1981). The bile flow rate increased by approximately 19 /iL/piOle of 4-ene VPA excreted in bile. Since this value was greater than the predicted increase in the bile flow (7 /iL//xmole) due to osmotic pressure alone (Watkins and Klaassen 1981), either fluid absorption is inhibited in the ductular tract or an electrolyte transport mechanism is stimulated by unknown mechanism. In addition, these monounsaturated metabolites of VPA may have a direct effect on a hormone such as secretin, which regulates canalicular secretion of bile. Moreover, (E)-2-ene VPA and 4-ene VPA are partially metabolized into dienes, which are largely excreted as conjugates in the bile. The presence in the bile of dienes and other possible metabolites of 4-ene VPA (Rettenmeier et al., 1985) and (E)-2-ene VPA may also contribute to the total choleretic effect seen after the administration of monounsaturated metabolites. After an initial choleresis produced by (E)-2-ene VPA or 4-ene VPA, the bile flow rate decreased steadily over the period of study. The flow rate invariably decreased to values slightly smaller than those observed before the administration of the metabolite. A similar observation was reported for VPA-induced choleresis in the rat (Dickinson et al., 1982). Depletion of the bile acid pool may be responsible for reduction in the bile formation, and the consequent reduced bile flow rate in the rat. D.3.2.5. Pharmacokinetic Parameters: After the low dose, the 101 plasma elimination of both the metabolites follows an open one-compartment model: thus, the apparent volume of distribution of the central compartment (Vj) is the same as the volume of distribution (V d). The values of V d for (E)-2-ene VPA and 4-ene VPA were, respectively, -220 and 210 mL/kg, which are almost half the V d of 430 mL/kg for VPA in the rat receiving a dose of 15 mg/kg (Dickinson et al., 1979a). The smaller volume of distribution of (E)-2-ene VPA may be due to higher plasma protein binding than VPA in the rat (Loscher and Nau 1983). A simple calculation of blood volume of -15 mL and a total body water volume of -200 mL (Altman and Dittmer) in a 300 g rat suggests that both the metabolites may not be restricted to the body fluids, but they appear to penetrate and perhaps bind to some degree to the tissues of the rat. The AUC of (E)-2-ene VPA or 4-ene VPA, due to the EHC phase alone, may be estimated by subtracting the average A U C u ) i | e _ e x t e r i o r i z e d from the A U C n o r m a l . The contribution of EHC to the total AUC of (E)-2-ene VPA was 40% at the low dose and 49% at the high dose. The corresponding values for 4-ene VPA were 23% and 24%. These results suggest that the extent of recirculation for (E)-2-ene VPA was twice as much as that of 4-ene VPA, an observation which agrees with the greater biliary elimination of (E)-2-ene VPA than 4-ene VPA, at the low dose, in the rat. D.3.3. In Vitro Protein Binding: 4-ene VPA is bound in vitro to a very small extent (14-25%) to the plasma proteins of the rat. Rettenmeier et al. (1986) have reported that 4-ene VPA binding is 58-78% in the monkey. These results suggest that the binding of 4-ene VPA decreases with the size of the animal. A similar observation was made with VPA, which is highly bound (90%) to the plasma proteins of the man and monkey (Levy et al., 1977), but is only 63% bound in the rat (Loscher 1978). 102 The plasma protein binding of VPA has been studied by equilibrium dialysis, ultrafiltration and ultracentrifugation (Barre et al., 1985). The results obtained with ultrafiltration were similar to those obtained with equilibrium dialysis (Barre et al., 1985). Thus, it is assumed that the use of ultrafiltration would yield reasonably accurate results with 4-ene VPA. 103 The plasma levels of a drug are often correlated to its pharmacologic response in man and animals. Occasionally, the correlation is poor due to several factors such as the presence of an active metabolite, tolerance to drug, delayed response because of the time required for equilibration of drug in plasma to that at the site of action, and time delays due to indirect pharmacologic activity. The poor correlation in the plasma level of VPA and its anticonvulsant activity has been speculated to be due to the formation of an active metabolite, (E)-2-ene VPA. The present results obtained in the rat show that (E)-2-ene VPA is cleared more slowly than VPA (Loscher 1978) from the body of the animal (3.0 versus 4.2 mL/min.kg). These results suggest that (E)-2-ene VPA may be responsible for the carry-over effect seen after withdrawl of VPA. Moreover, the plasma, urine and biliary profiles of (E)-2-ene VPA in the rat indicate that the metabolite exhibits dose-dependent pharmacokinetics, probably due to saturation of its metabolism at the high dose. Only 10% or less of the administered dose is recovered unchanged in the urine. Thus, 90% of the metabolite is expected to be metabolized. It is possible that (E)-2-ene VPA may be further metabolized to an active (or toxic) metabolite. The presence of (E)-2,3 diene VPA, an active diene metabolite of (E)-2-ene VPA, in the plasma of the rat further substantiates the contribution of (E)-2-ene VPA to the pharmacologic activity of the parent drug VPA. In addition, (E)-2-ene VPA undergoes extensive enterohepatic circulation in the rat, which delays its elimination from the body of the animal. Recirculated (E)-2-ene VPA is redistributed to the site of action, the brain, which prolongs its pharmacologic activity in the animal. This was apparent during experimentation where rats were sedated within 5-10 min of the dose and remained sedated for 10 h after the high dose. The duration and intensity of sedation was less at the low dose. 104 Paradoxical as it may be, the plasma levels of (E)-2-ene VPA may not be easily correlated with pharmacologic activity due to the same factors that were applied to VPA above. While this work was in progress, Loscher and Nau (1983) have shown that on repeated administration, (E)-2-ene VPA is gradually accumulated at the site of action, the brain of the rat. Moreover, (E)-2-ene VPA is eliminated much more slowly than VPA from the brain of animals. Therefore, continued pharmacologic activity of (E)-2-ene VPA should be expected even after its blood levels have declined below analytical limits. It is suggested that to evaluate the pharmacologic activity of VPA, it is imperative to monitor the parent drug, and its active metabolites, especially (E)-2-ene VPA and (E)-2,3-diene VPA. Unfortunately, the site of action, the brain in this case, is not accessible for the measurement of a drug/metabolite; instead blood and urine samples are the only biological fluids that can be collected without causing bodily harm. Thus, when the plasma levels of (E)-2-ene VPA have declined below the limits of the assay method, urinary recovery of the metabolites may indicate whether or not the metabolite is s t i l l present in the body. The delayed hepatotoxic reaction of VPA has been speculated to be caused by another metabolite, 4-ene VPA. The delay in the onset of toxicity symptoms may be due to slow elimination of the toxicant. The present work, however, suggests that 4-ene VPA is cleared 1.4 times as fast as VPA (Loscher 1978) from the plasma of the rat (5.9 versus 4.2 mL/min.kg). The plasma elimination of 4-ene VPA is dose-dependent in the rat. The plasma levels required to show dose dependency (>200 jug/mL) are two orders of magnitude higher than 4-ene VPA levels (<lug/mL) seen in patients on VPA therapy. It is, therefore, unlikely that 4-ene VPA is eliminated more 105 slowly than VPA in man. Like its parent drug, 4-ene VPA produces a choleretic effect in the rat, and undergoes enterohepatic circulation. The role of these effects in the hepatic damage suspected of 4-ene VPA is uncertain. Of the administered dose, only 5% is excreted unchanged in the urine and the rest of it is probably metabolized. At the most 47-46% of the low and the high dose was recovered as a total of conjugated and unconjugated 4-ene VPA in the urine and bile, collectively. Thus, more than 50% of the dose was either metabolized to other compounds and/or excreted in the feces or stored in the liver (Rettenmeier et al., 1985). Our animal studies do not rule out the possibility of irreversible binding of 4-ene VPA or one of its metabolic toxic species to the liver. 106 SUMMARY AND CONCLUSIONS 1. A simple, sensitive and selective capillary GCMS assay method was developed that could detect concentrations as low as 60 ng/mL of (E)-2-ene VPA and 100 ng/mL of 4-ene VPA in the biological fluids of the rat. 2. (E)-2-ene and 4-ene VPA apparently follow linear pharmacokinetics at the low dose of 20 mg/kg. Non-linear plasma decline was observed at plasma levels greater than -200 tzg/mU after the high dose of 100 mg/kg. 3. The plasma protein binding of 4-ene VPA was apparently low (14-25%) in the concentration range of 20-350 /jg/mL, indicating that binding may not be the cause of dose-dependent elimination of 4-ene VPA in the rat. 4. For the first time, enterohepatic circulation of (E)-2-ene VPA and 4-ene VPA has been documented in the rat. Secondary plasma peaks observed in normal rats following an IV bolus dose of either metabolite were abolished in bile-exteriorized rats, confirming the existence of EHC. 5. Following the low dose of (E)-2-ene and 4-ene VPA, the plasma profile including the appearance of secondary plasma peaks can be described by a time-lag pharmacokinetic model. 6. Conjugation of (E)-2-ene and 4-ene VPA, followed by excretion in the urine and bile was the major mode of elimination for both the metabolites in the rat. 7. Less than 10% of the administered dose of (E)-2-ene or 4-ene VPA was excreted unchanged in the urine. 107 8. A marked but transient choleretic effect was observed within the first hour of the administration of (E)-2-ene or 4-ene VPA in the rat. The choleretic effect was thought to be partly due to an osmotic pressure effect similar to that seen for the parent drug, VPA. 9. 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Sodium valproate in the induction of unusual hepatotoxicity. Hepatology 2: 648-649, 1982. Zaret B.S. and Cohen R.A. Reversible valproic acid-induced dementia: A case report. Epilepsia 27: 234-240, 1986. 127 Zimmerman H.J. and Ishak K.G. Valproate induced hepatic injury: Analysis of 23 fatal cases. Hepatology 2: 591-597, 1982. 128 129 oo o CL. F 2 3 - o u — i 1000 250 500 750 T I M E (sec) Appendix 1. Total ion chromatogram of ether layer of methanesulfonyl chloride (Scheme 1) reaction. Glass column (2 metre x 2 mm I.D.) was packed with 3% Dexsil 300 on 100/120 Supelcoport. The temperature was programmed to hold for 2 min at 50°C and was then raised to 270°C at a rate of 16°C/min. Peak 3 = 2-ene VPA ethyl ester, peak 9 = 3-0H VPA ethyl ester, peaks 10-12 were condensation products. Appendix 2. NMR (80 MHz) spectrum of 3-ene VPA in CDC1 131 Appendix 3. NMR (80 MHz) spectrum of 2-ene VPA in CDC13. Appendix 4. NMR (80 MHz) spectrum of 4-ene VPA in CDC1 133 APPENDIX 5. PLASMA LEVELS OF (E) (DOSE = -2-ENE VPA 20 mg/kg) IN NORMAL RATS, jig, Time (min) la 2a 3a 4a Mean 0* 90 84 98 66 85 5 79 70 90 na 77 6.5 na na na 56 15 53 46 64 42 51 30 39 26 48 26 35 60 14 7.9 30 11 16 90 7.5 2.9 17 4.9 8.0 120 5.0 2.3 14 1.7 5.7 150 4.9 2.5 20 1.1 7.1 180 3.8 2.7 22 5.0 8.4 240 11 8.0 8.2 5.3 8.2 300 1.6 3.1 6.9 1.0 3.1 360 0.4 1.0 2.9 - 1.4 420 - 0.6 1.2 -480 _ _ 0.5 _ *, extrapolated to time zero; na, plasma sample not available. 134 >PENDIX 6. PLASMA LEVELS OF (E) (DOSE = -2-ENE VPA 100 mg/kg) IN NORMAL RATS, Time (min) lb 2b 3b 4b Mean 0* 410 460 480 390 430 5 370 390 390 360 380 15 na- 280 260 320 280 30 na 260 230 240 240 39 180 na na na 60 82 120 120 120 110 120 13 25 39 25 26 180 14 na 45 43 34 240 33 47 46 56 45 300 39 43 38 33 38 360 28 na 42 21 30 435 15 20 30 40 26 510 20 14 23 15 18 585 17 6.1 16 16 14 675 12 0.7 2.8 4.0 4.8 765 6.8 _ 1.5 2.1 3.5 *, extrapolated to time zero; na, plasma sample not available. 135 APPENDIX 7. PLASMA LEVELS OF 4-ENE VPA IN NORMAL RATS, /zg/mL (DOSE = 20 mg/kg) Time (min) lc 2c 3c 4c 5c 6c Mean + SD * 0 94 110 83 75 104 120 98 + 17 5 64 82 59 66 76 86 72 + 11 15 43 54 39 37 43 46 44 + 6.0 30 17 29 15 14 15 14 17 + 5.7 60 2.8 5.7 2.7 4.5 2.6 2.1 3.4 + 1.4 90 1.4 na 1.7 4.9 1.4 0.9 2.1 + 1.6 120 1.3 0.6 1.2 3.7 1.3 1.4 1.6 + 1.1 150 2.4 1.4 na 2.8 1.7 2.0 2.1 + 0.6 156 na na 1.5 na na na 180 2.9 - 2.8 1.9 2.0 2.4 2.4 + 0.5 240 2.7 - 4.2 0.8 2.5 2.2 2.5 + 1.2 300 1.4 - na 0.6 1.5 1.3 1.2 + 0.4 360 _ _ 0.7 _ _ 0.9 , extrapolated to time zero; na, plasma sample not available. 136 APPENDIX 8. PLASMA LEVELS OF 4-(DOSE -ENE VPA IN NORMAL = 100 mg/kg) RATS, ng/ml Time (min) Id 2d 3d 4d 5d 6d Mean + SD 0* 280 320 330 350 310 290 310 + 24 5 280 na 300 320 290 280 300 + 16 8 na 290 na na na na 15 270 na na 280 250 270 270 + 12 16 na 270 na na na na 22 na na 220 na na na 30 180 190 - 230 200 210 190 + 24 35 na na 160 na na na 57 na na 75 na na na 60 71 47 - 68 96 75 72 + 16 120 7.1 5.1 5.2 11 7.8 5.4 6.9 + 2.3 180 14 15 6.4 14 5.6 13 11 + 4.3 240 11 8.5 11 11 7.1 9.6 9.6 + 1.5 300 5.4 4.4 4.5 9.2 18 8.8 9.8 + 6.3 360 3.9 5.4 4.3 9.3 15 5.8 9.7 + 6.6 435 4.1 2.8 2.5 5.5 4.2 5.1 5.4 + 4.1 510 2.1 - - 1.5 3.2 3.6 2.6 + 0.9 600 0.9 _ _ 0.9 1.0 1.1 1.0 + 0.1 , extrapolated to time zero; na, plasma sample not available. 137 APPENDIX 9. PLASMA LEVELS OF (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS, ng/ml (DOSE = 20 mg/kg) Time (min) lc 2c 3c 4c Mean 0* 101 79 102 81 91 5 na 62 85 62 73 8 76 na na na na 15 na 42 65 48 54 17 54 na na na na 30 29 25 44 31 32 60 10 8.2 21 12 13 90 3.1 2.2 9.2 4.1 4.7 120 0.8 0.6 3.3 1.2 1.5 150 _ _ 1.5 0.5 180 , extrapolated to time zero; na, plasma sample not available. 138 APPENDIX 10. PLASMA LEVELS OF (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS, /ig/mL (DOSE = 100 mg/kg) Time (min) Id 2d 3d 4d Mear 0* 440 440 380 440 430 5 370 410 na 390 390 8 na na 360 na 15 260 340 340 310 310 30 170 260 na 220 210 39 na na 210 na 60 61 108 99 76 86 90 13 48 34 27 31 120 7.5 18 11 8.5 11 150 na na 5.4 2.4 180 na 2.9 na 1.5 187 na . na 1.7 na 240 - - na -262 na na _ na , extrapolated to time zero.; na, plasma sample not available. 139 APPENDIX 11. PLASMA LEVELS OF 4-ENE VPA IN BILE-EXTERIORIZED RATS, /zg/mL (DOSE = 20 mg/kg) Time (min) ig 2g 3g 4g 5g 6g Mean + SD 0* 85 88 103 96 74 103 91 + 11 5 77 73 70 80 67 80 74 + 5.5 15 38 36 42 49 33 45 40 + 5.9 30 13 16 18 28 12 15 17 + 5.7 60 2.1 3.1 2.4 6.4 2.0 1.1 2.8 + 1.8 90 0.7 0.6 - 2.3 0.6 0.5 1.0 + 0.8 120 0.4 150 , extrapolated to time zero; na, plasma sample not available. 140 APPENDIX 12. PLASMA LEVELS OF 4-ENE VPA IN BILE-EXTERIORIZED RATS, /xg/mL (DOSE = 100 mg/kg) Time (min) lh 2h 3h 4h 5h 6h Mean + SD 0* 290 320 310 250 270 310 290 + 28 5 290 290 300 240 260 290 280 + 25 15 280 240 280 220 230 250 250 + 24 30 180 215 230 200 160 160 190 + 29 60 90 97 95 65 59 58 77 + 19 90 38 37 34 19 13 8.0 25 + 13 120 9.5 20 19 10 4.9 2.3 11 + 7.2 150 3.9 6.1 4.3 1.8 0.8 1.1 3.0 + 2.1 180 0.7 na na - - -188 4.2 2.0 , extrapolated to time zero; na, plasma sample not available. 

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