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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  degree  at  this  the  thesis  in  University of  partial  fulfilment  of  of  department publication  this or  thesis for by  his  or  scholarly purposes may be her  representatives.  an advanced  Library shall make it  It  is  granted by the understood  that  of this thesis for financial gain shall not be allowed without  Department of  /"VU  ^Ctf^JZaJL  The University of British Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3  DE-6(3/81)  for  agree that permission for extensive  permission.  Date  requirements  British Columbia, I agree that the  freely available for reference and study. I further copying  the  €<*&^c&f  head of copying  my or  my written  ii  ABSTRACT  Valproic acid (VPA) is a broad spectrum antiepileptic widely  in the treatment  of absence and tonic-clonic  agent used  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, free  of two serious  teratogenicity.  side  effects  (E)-2-ene VPA appears to be  of VPA, namely  hepatotoxicity and  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 followed  of 80 /JL of plasma, urine or bile with ethyl  by derivatization  methyl-trifluoroacetamide).  with MTBSTFA  acetate  (N-tertiarybutyldimethylsilyl-N-  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 ii  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) either  bolus dose of  (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)-2ene 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  mL/min.kg and 7.4 + 1.1 mL/min.kg, respectively. The apparent  +  1.8  elimination  h a l f - l i f e 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 2 to 1 9 + 3  ° f 4-ene VPA from 13 ±  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  enterohepatic  parent  drug  recirculation  VPA, in the  (E)-2-ene rat  VPA and  4-ene  VPA showed  which produced secondary plasma  peaks in normal animals. Moreover, both (E)-2-ene VPA and 4-ene VPA showed  iv  a rapid but transient  choleretic effect  binding of 4-ene VPA was apparently  in the rat.  low (14-25%),  The plasma protein 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.  required to show a non-linear decline orders of  magnitude  higher  patients on VPA therapy.  It  than is,  4-ene  The plasma levels of 4-ene VPA (>200 /xg/mL) VPA levels  therefore,  in the rat (<1  are two  ng/ml) seen in  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 lasting pharmacologic effect than VPA.  (E)-2-ene  VPA may have a longer  V  TABLE OF CONTENTS CHAPTER  PAGE  Abstract  ii  Table of Contents  v  List of Tables  viii  List of Figures  x  List of Appendices  xi  Symbols and Abbreviations  xii  Acknowledgements  xv  A. INTRODUCTION A.l  VALPROIC ACID A.1.1. A.1.2. A.1.3. A.1.4. A.1.5. A.1.6. A.1.7.  A.2.  Pharmacologic Activity Pharmacokinetics Side Effects  12 13 15  4-ENE VALPROIC ACID A.3.1. A.3.2. A.3.3.  A.4.  1 2 3 4 8 9 10  (E)-2-ENE VALPROIC ACID A.2.1. A.2.2. A.2.3.  A.3.  Background Pharmacokinetics in Man Pharmacokinetics in Animals Metabolism Mechanism of Action Side Effects Hepatotoxicity  Toxicity Metabolism Pharmacokinetics  16 16 17  RATIONALE AND OBJECTIVES  19  vi B. EXPERIMENTAL B.l.  MATERIALS B.l.l. B.l.2.  Chemicals Instrumentation  22 23  Synthesis  24  B.2. METHODS B.2.1.  B.2.1.1. B.2.1.2. B.2.2.  B.2.3.  B.2.4.  (E)-2-ene VPA 4-ene VPA  Capillary GCMS Assay  28  B.2.2.1. B.2.2.2. B.2.2.3. B.2.2.4. B.2.2.5. B.2.2.6. B.2.2.7. B.2.2.8.  28 29 29 30 30 30 30 31  Standards Extraction and Derivatization Chromatography Optimum Derivatization Conditions Hydrolysis of Conjugate Calibration Curves Precision Extraction Efficiency  Rat Experiments  31  B.2.3.1. B.2.3.2. B.2.3.3. B.2.3.4. B.2.3.5. B.2.3.6.  31 31 32 34 35 35  Animal Handling Jugular Vein Cannulation Bile Duct Cannulation Pharmacokinetic Studies In Vitro Plasma Protein Binding Metabolism of (E)-2-ene VPA  Pharmacokinetic Analysis  C. C.l.  36  RESULTS  SYNTHESIS C.l.l. C.1.2.  C.2.  24 27  (E)-2-ene VPA 4-ene VPA  38 45  Chromatography Derivatization Kinetics Linearity and Reproducibility Hydrolysis of Conjugates  45 48 48 54  ASSAY C.2.1. C.2.2. C.2.3. C.2.4.  vi i C.2.5. C. 3.  Extraction Efficiency  54  PHARMACOKINETIC STUDIES C.3.1. C.3.2.. C.3.3. C.3.4. C.3.5. C. 3.6.  Pharmacokinetics in Normal Rats Pharmacokinetic Model Pharmacokinetics in Bile Exteriorized Rats Choleretic Effect In Vitro Protein Binding Metabolism of (E)-2-ene VPA  D.  54 66 71 84 84 88  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. D.3.1.2. D.3.1.3.  92 93 95  D.3.2.  Pharmacokinetics in Bile Exteriorized Rats D.3.2.1. D.3.2.2. D.3.2.3. D.3.2.4. D.3.2.5.  D.3.3.  Plasma Profile Pharmacokinetic Model 1ing Pharmacokinetic Parameters  Plasma Profile Biliary Elimination Conjugation Choleretic Effect Pharmacokinetic Parameters  In Vitro Protein Binding  96 96 97 98 99 100 101  SUMMARY AND CONCLUSIONS  106  REFERENCES  108  APPENDIX  128  vi i i LIST OF TABLES Table  Page  1.  Serum levels of VPA and its metabolites in patients on VPA monotherapy.  7  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 normal rats (Dose=20 mg/kg).  58  12.  Pharmacokinetic parameters of (E)-2-ene VPA in normal rats (Dose=100 mg/kg).  59  13.  Urinary excretion of unconjugated (E)-2-ene VPA in normal rats (Dose=20 mg/kg).  60  14.  Urinary excretion of unconjugated (E)-2-ene VPA in normal rats (Dose=100 mg/kg)  61  15.  Pharmacokinetic parameters of 4-ene VPA in normal rats (Dose=20 mg/kg).  62  16.  Pharmacokinetic parameters of 4-ene VPA in normal rats (Dose=100 mg/kg).  63  17.  Urinary excretion of unconjugated 4-ene VPA in normal rats (Dose=20 mg/kg).  64  18.  Urinary excretion of unconjugated 4-ene VPA in normal rats (Dose=100 mg/kg).  65  19.  Pharmacokinetic parameters of (E)-2-ene VPA in bileexteriorized rats (Dose=20 mg/kg).  72  ix Table  Page  20.  Pharmacokinetic parameters of (E)-2-ene VPA in bileexteriorized rats (Dose=100 mg/kg).  73  21.  Urinary excretion of unconjugated (E)-2-ene VPA in bile-exteriorized rats (Dose=20 mg/kg).  74  22.  Urinary excretion of unconjugated (E)-2-ene VPA in bile-exteriorized rats (Dose=100 mg/kg).  75  23.  Biliary elimination of unconjugated (E)-2-ene VPA in rats (Dose=20 mg/kg).  76  24.  Biliary elimination of unconjugated (E)-2-ene VPA in rats (Dose=100 mg/kg).  77  25.  Pharmacokinetic parameters of 4-ene VPA in bileexteriorized rats (Dose=20 mg/kg).  78  26.  Pharmacokinetic parameters of 4-ene VPA in bileexteriorized rats (Dose=100 mg/kg).  79  27.  Urinary excretion of unconjugated 4-ene VPA in bile-exteriorized rats (Dose=20 mg/kg).  80  28.  Urinary excretion of unconjugated 4-ene VPA in bile-exteriorized rats (Dose=100 mg/kg).  81  29.  Biliary elimination of unconjugated 4-ene VPA in rats (Dose=20 mg/kg).  82  30.  Biliary elimination of unconjugated 4-ene VPA in rats (Dose=100 mg/kg).  83  31.  Comparison of pharmacokinetic parameters of (E)-2-ene VPA in the rat.  85  32.  Comparison of pharmacokinetic parameters of 4-ene VPA in the rat.  86  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) 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.  43  6.  Total ion chromatogram of t-BDMS derivative (A) and El-mass spectrum (B) of 4-ene VPA.  44  7.  Selected-ion-chromatogram of t-BDMS derivatives of an extract from a spiked plasma sample.  46  8.  Selected ion chromatogram of blank plasma sample showing no interfering peaks at the lowest attenuation.  47  9.  Semi logarithmic plots of plasma concentrations of (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).  55  10.  Semilogarithmic plots of plasma concentrations of 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).  56  11  Semilogarithmic plots of plasma concentrations of (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).  67  12.  Semilogarthmic plots of plasma concentrations of 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).  68  13.  A typical choleretic effect and excretion rate plot of high dose of 4-ene VPA in the bile of a rat after high dose of 100 mg/kg.  69  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 chloride (Scheme 1) reaction.  129  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 (Dose=20 mg/kg).  133  6.  Plasma levels of (E)-2-ene VPA in normal rats, ng/ml (Dose=100 mg/kg).  134  7.  Plasma levels of 4-ene VPA in normal rats, ng/ml (Dose=20 mg/kg).  135  8.  Plasma levels of 4-ene VPA in normal rats, ng/ml (Dose=100 mg/kg).  136  9.  Plasma levels of (E)-2-ene VPA in bile-exteriorized rats, /ig/mL (Dose=20 mg/kg).  137  10.  Plasma levels of (E)-2-ene VPA in bile-exteriorized rats, /ig/mL (Dose=100 mg/kg).  138  11.  Plasma levels of 4-ene VPA in bile-exteriorized rats, /ig/mL (Dose=20 mg/kg).  139  12.  Plasma levels of 4-ene VPA in bile-exteriorized rats, /ig/mL (Dose=100 mg/kg).  140  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  Cl Cl  c o n  me1  j  -  apparent clearance due to the formation of conjugates apparent metabolic clearance  CIR  apparent renal clearance of unconjugated metabolite  Clj  apparent plasma clearance of unconjugated metabolite  cm  centimetre  Cone  concentration  C  plasma concentration of unconjugated metabolite at zero  Q  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  10  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  milliiitre  mm  millimetre  MTBSTFA  N-tertiarybutyldimethylsilyl-N-methyl  N  normal  na  not available  trifluoroacetamide  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  facilities,  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. this project  I am grateful to the following for their contributions to  in various ways:  Mr. Wayne Riggs, Mr. Stephen Clark, Mr.  Peter Phillips, Mr. Peter Martin, Ms. Sue Panesar, Ms. Sheila Tyner, Ms. Neil in Ratanshi, Mr. Ray deSouza and Mr. Dan Chan.  1 A.  A.l.  INTRODUCTION  VALPROIC ACID  A.1.1.  Background  Valproic acid (VPA) is a small, branched-chain fatty acid (Fig Its  synthesis was f i r s t  reported over a century ago (Burton  anticonvulsant properties were only discovered in 1963 1963;  Carraz  et  al.,  1964a; Carraz  et  1964b)  al.,  in  1882).  (Meunier  et  France.  1). Its al.,  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 VPA is  Valproic acid  a broad spectrum anticonvulsant  used in the  treatment of  generalized absence, myoclonic and tonic-clonic seizures (Gram and Bentsen 1985;  Dulac and Arthuis  1982). It 1986),  1984;  is also effective  partial  (Vajda et al.,  seizures  Feuerstein et  Covanis et  al.,  in controlling fever convulsions (Lee et  al.,  (Bruni  1983;  al.,  and Albright  1983),  status  1978) and photosensitive epilepsy (Harding et al.,  epilepticus 1978).  has been also employed to treat Lennox syndrome (Schobben et al., and epilepsies refractory  to  other  anticonvulsants  1980b)  (Redenbaugh et  1980). The drug is administered orally at a dose of 15-30 mg/kg daily. therapeutic  blood levels  Schweizer 1980; Gram et al.,  are  in the  1980).  range of  50-100 ^g/ml  It  (Klotz  al., Its and  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  Antonin 1977).  Its  by the  liver  (Perucca et  plasma elimination  half-life  1978a; Klotz and  al., 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 1978b; Gugler and von Unruh 1980). VPA follows exhibiting dosing  no change in  (Gugler  et  its  elimination  1977).  al.,  tj/2  linear  after  pharmacokinetics  single or  Plasma concentrations  al.,  of  multiple  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 apparent  volume of distribution  of 0.1-0.4  L/kg  (Gugler  has a small  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., VPA, between mother's milk  1983; Vajda et al.,  1-3% of the maternal (Nau et  al.,  1981). Only low levels of  serum levels,  1981a; Dickinson et  are secreted into al.,  the  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 et al.,  1979b). The elimination t ^  al.,  1981a; Dickinson  in neonates is 45-47 h, which is three  3 times longer than that in adults  (Nau et  1981a; Dickinson et  al.,  al.,  1979b).  A.1.3 Pharmacokinetics in Animals The disposition of VPA has been studied including the pig (Bonora et 1980),  the  dog (Loscher  (Ichimura et  al.,  1978;  in  many animal  species  1979), the monkey (Dickinson et  Loscher  and Esenwein 1978),  the  al., rabbit  1985), the rat (Loscher 1978) and the mouse (Loshcer and  al.,  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 drug is mainly distributed (Dickinson et  al.,  in the liver,  1979a). In the rabbit,  (Eymard et  al.,  kidneys and testes of the  1985).  rat  the highest levels of VPA are  found in kidneys, followed by liver, heart, GI tract and fat al.,  1971). The  (Ichimura et  4 Detailed disposition studies in the brain of the rat have shown that VPA  is  preferentially  (Mesdjian et al.,  distributed  in  the  1982; Hariton et al.,  cerebellum  and hippocampus  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  hypothalamus and medulla of the  dog after  a constant  accumulated in 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 VPA  produces  (Dickinson et al.,  a  190 min)  choleretic  effect  in  the  1982), and cat (Marshall et al.,  rat,  dog  and monkey  1984). Bile flow rate  increases by 2-3 times the basal flow, 30-60 min after a single dose of 4060 mg/kg to the rat,  cat and dog (Dickinson et  Klaassen 1981; Marshall et al., due to the osmotic activity  al.,  1982,  Watkins and  1984). VPA-induced choleresis is primarily  (Watkins and Klaassen 1981; Dickinson et  al.,  1982) of VPA conjugates excreted in bile. A.1.4 VPA is Kochen et  al.,  Loscher 1981;  Metabolism extensively  metabolized  1984; Schobben et Jakobs 1978;).  al.,  in man (Acheampong et  al.,  1983;  1980a; Gugler and von Unruh 1980;  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  CH =CH-CH  CH =CH-CH 2  2  2  2  CHCOOH  CHCOOH CH -CH -CH 3  CH -CH -CH 3  2  2  CH =CH-CH 2  2  4-Ene VPA  2  2  4,4'-Diene VPA  CHCOOGlu CHj-CHg-CH^ CH CH *CH  CH,=CH-CH  CH -CH=CH^ 3  -  VPA Glucuronide  3  2  CHCOOH I  2  CHCOOH  CH -CH -CH 3  OH •  CH CH *CH 2  2  T OH CH -CH-CH 3  CH -CH -CH^ 3  CH *CH -CH 3  2  CH -CH -CH 3  2  2  4-OH VPA  2  2(E),3'(E)-Diene VPA  1  3  3  2  'CHCOOH  2  CHCOOH CH -CH -CH 3  2  / 2  CHJ'CH^'CHJ  CH -CH -CH -CH-COOH 3  2-Ene VPA  CH -CH -CH  n CH -C-CH  3-OH VPA  2  COOH 2-Propylmalonic  4-Keto VPA  acid CH -CH -C^ 3  HOOC-CH, CHCOOH CH *CH -CH 3  2  C-COOH  CHj-CH-CH  OH  0  2-Propylglutaric acid  CH,-CH=CH 3 \  CH j"CHg"  I  / 2  I  C-COOH  2  2  C CHCOOH  2  2  CHCOOH  H00C-CH -CH  (E)-2,4-Diene VPA  I  CHCOOH CHj-CHg-CH^ 5-OH VPA  2  3-Ene VPA  VPA  _  2  2  C-COOH CH2*CH2*CH2  /  2  CHCOOH CH -CH -CH 3  2  2  2  2-Propylsuccinic a c i d  3-Keto 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 5hydroxy VPA, 4-hydroxy VPA, 3-ene VPA and 4-ene VPA, respectively, as their first  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 al.,  (Granneman et  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, VPAinduced  hepatic  damage has  been suggested to  metabolite, 4-ene VPA (Kesterson et al.,  1984).  be  caused by  a minor  7  TABLE 1. SERUM LEVELS OF VPA AND ITS METABOLITES IN PATIENTS ON VPA MONOTHERAPY 3  Metabolite  Mean Cone (/jg/mL)  VPA  % VPA Level  46.4  100  (E)-2-ene VPA  5.53  11.9  3-keto VPA  3.59  7.7  2.95  6.4  3-ene VPA  0.94  2.0  4-ene VPA  0.67  1.4  4-keto VPA  0.40  0.9  4-OH VPA  0.38  0.8  0.20  0.4  2-Propylglutaric acid^  0.20  0.4  (Z)-2-ene VPA  0.19  0.4  5-OH VPA9  0.18  0.4  2-Propylsuccinic acid^  0.04  0.1  b  C  C  (E)-2,3'-diene VPA ' c  d  d  e  f  f  (E)-2,4-diene VPA ' c  e  C  a, c, e, g,  (Abbott et a l . , 1986); b, parent drug; /8-oxidation; d, -y-dehydrogenation; 6-dehydrogenation; f, (w-1) hydroxylation; 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 potent  al.,  inhibitor  1975), an enzyme that breaks down GABA. VPA is also a 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 VPA administration glutamate  has been also reported that  raises GABA levels by increasing its  synthesis from  (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 i s , 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 1984).  The highest  GABA levels  are  found in  hypothalamus of the rat (Hariton et al., VPA  increases  the  threshold  the  olfactory  al.,  bulbs and  1984).  potential  for  excitation  of  nerve  membrane by blocking sodium and potassium conductance (VanDongen et  al.  1986).  of  VPA  has  also  been  reported  to  depress  the  firing  rate  spontaneously active cortical c e l l s , whose time course is parallel onset of anticonvulsant activity  in the rat  to the  (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  ability to raise GABA levels (Schumtz et al.,  its  1979; Kerwin and Olpe 1980).  9 Another ability  possible mechanism of  of  VPA is  ascribed to  its  to significantly decrease aspartate levels in the brain of mouse  (Schechter et al., 1983;  action  Patsalos  1978) and the rat Chapman et al., and  Loscelles  reduction in the cerebral  1981).  aspartate  The  time  1982; Chapman et course  of  al.,  VPA-induced  levels coincides with the period of  protection from audiogenic seizures in mice (Schechter et al., In actual practice, the antiepileptic its combined effect on neurotransmitters  1978).  activity of VPA may be due to  in the brain, and firing rate of  subcortical c e l l s .  A.1.6  Side Effects  VPA is considered a reasonably safe drug that shows mostly transient and mild side effects (Stefan et al., and Albright  1984; Feuerstein et al.,  1983). The most common adverse effects  1983; Bruni  are GI disturbances  including nausea, vomiting, abdominal cramps, diarrhea and indigestion in some patients  (Beran et al.,  noticed to cause stomatitis 1984; Murphy et al., et al.,  1980;  Simon and Penry 1975). VPA has been  (Russo 1981), pancreatitis  (Wyllie et  al.,  1981), change in appetite and weight gain (Feuerstein  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  alertness,  1984;  improved  Jeavons  and  behavior  Clark  1974)  and improvement  have of  reported  increased  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;  hypothyroidism (Salvatoni et al.,  Bruni and Wilder 1979;). VPA may produce 1983), in rare instances.  10 A serious toxic effect of VPA is teratogenicity (Lammer et al., Di Carlo et 1976)  1986). VPA is embryotoxic in the rabbit,  al.,  and mouse (Nau and Loscher  pronounced  effects  are  neural  abnormalities, embryolethality 1984; Kao et al.,  1984; tube  Brown et defects  rat  1987;  (Whittle  1980). The most  al.,  (exencephaly),  and reduced fetal weight  skeletal  (Nau and Loscher  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., have  further  teratogenic folinic  proposed effects.  acid  that  Trotz  zinc et  administration  al.  supplements (1987)  markedly  may  reduce VPA-induced  have recently  reduces  1983)  reported  that  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 Baillie 1985). In these patients, typical symptoms of anorexia, vomiting, lethargy, jaundice, hepatic therapy  failure  and terminal  (Zimmerman  and  coma developed within  Ishak  1982).  Autopsy  of  1-4 the  months of VPA 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 1982).  These  results  suggest  that  enzyme  inducers  may  metabolism to form a hepatotoxic metabolite (Granneman et al., Several  biochemical  abnormalities  have  been  al.,  enhance VPA 1984a).  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., al.,  1987).  VPA,  especially  when  co-administered  anticonvulsants, produces hyperammonemia (Ratnaike et al., al.,  with  1983).  VPA administration  causes impaired  fatty  other  1986; Zaccara et  1985). It inhibits gluconeogenesis in the rat (Turnbull et al.,  and in vitro in isolated rat hepatocytes (Rogiers et al., al.,  1984; Cotariu et  1983)  1985; Turnbull et acid metabolism  12  including reduced fatty acid synthesis, and decreased ^-oxidation (Becker and Harris reported  1 9 8 3 ;  Kesterson et  al.,  to develop hypocarnitinemia  liver cells (Sugimoto et al.,  Rats receiving VPA have been  1 9 8 4 ) .  and mitochondrial  swelling  These abnormalities are corrected by  1 9 8 7 ) .  administering supplements of L-carnitine  (Sugimoto et al.,  administration of VPA produces hyperglycinemia in the rat et al.,  Cherruau et al.,  1 9 8 5 ;  of the  1 9 8 7 ) .  Chronic  (Martin-Gallardo  It has been also reported to suppress  1 9 8 1 ) .  plasma levels of corticotropin (ACTH) in children (Kritzler et al.,  (E)-2-ENE  A.2.  A.2.1  VALPROIC ACID  Pharmacologic Activity  Ten metabolites of VPA have been tested for anticonvulsant in animals (Loscher and Nau of  1 9 8 3 ) .  the metabolites  electroshock  and/or  (Loscher and Nau  1 9 8 5 ;  1 9 8 5 ;  Loscher  significantly  raise  pentylenetetrazole Loscher  ene VPA and 4-ene VPA, are  1 9 8 1 ) .  8 0 - 9 0 %  1 9 8 1 ;  threshold  levels  (PTZ)-induced  Nine  1 9 8 0 ) .  for maximal  seizures  in mice  The two most active compounds,  (E)-2-  as active as VPA on a molar basis after  IP administration to mice (Loscher and Nau VPA is 6 6 mg/kg in rats  Schafer et al.,  activity  exhibiting  1 9 8 5 ) .  The  ED^Q  for (E)-2-ene  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  ratio of E D  50  for (E)-2-ene VPA/VPA is  0 . 8 1 ,  1 . 2 3  and  0 . 6 9  al.,  1 9 8 4 ) .  The  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  slightly  of ( E ) - 2 - e n e  VPA are  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 The  Pharmacokinetics pharmacokinetics  characterized. administered, monitored  In  some  and the  (Loscher  of  of  (E)-2-ene  the  plasma  or  and Nau 1982).  studies, tissue  VPA  have  the  parent  levels  On oral  of  not  been  well  drug  VPA was  (E)-2-ene  VPA were  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 0.10.5 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;  apparent elimination t ^  Nau and Loscher 1982). The  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 ^ an apparent volume of distribution of 1983) . A recent abstract (O'Connor et al.,  0.25  litres/kg  of 1.8 h, and has (Loscher and Nau  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  respectively (O'Connors et al.,  471  and  718  mL/kg  at  the  above  doses,  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 control  seizures  in  rats  and gerbils  effects  (Loscher et  at doses required to 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.,  ene VPA in mice is (Loscher et al.,  760 mg/kg, which is  1984).  1984). The L D  50  similar to 810 mg/kg for VPA  (E)-2-ene VPA is free of embryotoxic effects in  mice even at extremely high doses of 600 mg/kg (Loscher et al., whole embryo culture studies concentrations  for (E)-2-  (Lewandowski et  al.,  1986),  of 200 /xg/g show no abnormal development  1984). In  (E)-2-ene VPA 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., Kesterson et  a/.  1986). (1984)  have shown that  (E)-2-ene  VPA does not  produce hepatic steatosis in the rat. On its administration to rats, clinical  the  features such as serum urea nitrogen, SG0T, SGPT, ammonia levels  and ketone bodies are unaltered. These results strongly suggest that (E)-2ene 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 vitro studies  investigators  have reported the toxicity of 4-ene VPA. In  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. 4-ene VPA produces mitochondrial lesions  (1984) have reported that  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., the  fatty  acid  hydroxylation  1986; Rettenmeier et al.,  /J-oxidation  and epoxidation  complex, pathways  and  1985). It is metabolized by by  cytochrome  (Rettenmeier  et  P450-mediated  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, (Rettenmeier et  al.,  4,5-di-hydroxy VPA lactone,  AGA and PGA  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  metabolites were detected in the urine (Rettenmeier et a7., major metabolites  were 4-ene VPA glucuronide  (38.6%),  of 20  1986). Three  (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., a7.,  1986)  1984c). A recent article (Pollack et  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  exponential  with a terminal  elimination  curve in the monkey is bi-  half-life  of  2.3-3.6 h,  and a  18  CO,H HO.C  CO.H  CO.H OHC  CO,H MO> CO.H OH  CO,H  CO,H CO,H  CO,H  .OH  CO,H CO.H ^CHO OH  CO.H  CO,H  CO.H •*CO,H  Fig. 3. Proposed metabolic pathway for 4-ene VPA . (a, Rettenmeier et al., 1985) a  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.  antieplileptic  Following its  activity  administration, a delayed onset of  ranging from hours to  (Jeavons and Clark 1974; Rowan et al., withdrawn, a carry-over effect  weeks has  been  observed  1979). Similarly, after the drug is  both in animals and humans ranging from  weeks to months has been reported (Pellegrini et a / . , 1978; Harding et 1978;  its  Lockard and Levy 1976). A possible  al.,  explanation for these temporal  effects may be the formation of a pharmacologically active metabolite with longer h a l f - l i f e .  (E)-2-ene  VPA, a major mono-unsaturated metabolite  VPA, has been found to be as active as VPA in several  of  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  associated with VPA, especially been proposed (Nau et alternative  al.,  anticonvulsant.  hepatotoxicity  1984)  that  Since  it  and embryotoxicity. It has  (E)-2-ene is  not  side effects  VPA may be used as an  uncommon to  effective and safer drugs from drug metabolites,  develop more  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  initially  identified  as  'parent' drugs diazepam and phenacetin, respectively. of  (E)-2-ene  VPA have not been well  metabolites  of  the  The pharmacokinetics  characterized due to a lack of the  20 availability Abbott's  pure  laboratory  metabolites logical  of  of  compound  has  been  in a  VPA, including  to use those f a c i l i t i e s  sufficiently leader  (E)-2-ene  in  large  the  VPA. It  to synthesize  quantities.  synthesis was,  of  several  therefore,  (E)-2-ene  Dr.  most  VPA, develop a  sensitive assay for (E)-2-ene VPA and study its disposition in the rat.  II.  The metabolites  toxicities.  of a drug are often  Hepatotoxicities  responsible for drug-related  of acetaminophen and iproniazid,  their respective metabolites,  caused by  are classic examples of metabolite-induced  side effects (Mitchell et a7., 1973; Nelson et a7., 1978). Similarly, Reyelike  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., after  the  1983). Moreover, there is a latent period of 1-4 months,  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 i t 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)-2ene 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.  B.l.  EXPERIMENTAL  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  anhydrous sodium sulfate (granular), hydrochloric acid.  c.  Eastman Organic Chemicals (Rochester, N.Y.) Propionaldehyde, methansulfonyl  d.  chloride.  Fisher Scientific Company (Fair Lawn, N.J.) Quinoline, pyridine.  e.  Mallinkrodt Chemicals (St. Louis, MI) Para-toluenesulfonyl  f.  chloride.  Matheson Coleman and Bell Company. (Norwood, OH) Phosphorous tribromide, 1-bromopropane.  sulfuric  acid,  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 s i l i c a capillary column (25 metre x 0.32 bonded  phase  (0.25  (i  film  of  0V-1701)  was  mm I.D.) obtained  coated with a 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.  I.  (E)-2-ene VPA  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 d i s t i l l e d 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 atmosphere. n-Butyllithium (0.25 moles) was added dropwise to the 2  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 over  anhydrous  Na^O^,  and  the  solvent  removed  layer was dried  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 f i l t r a t e ,  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, were obtained  on the  and analyzed on GCMS. Several  chromatogram indicating that the  peaks  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 icewater, the mixture was extracted with f i r s t with dilute  H S0 , 2  4  then with  CHCI3.  NaHC0  3  The organic layer was washed  (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)  was placed in a 1-litre three-necked  in 750 mL of dry THF  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 d i s t i l l e d 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), were placed  in a 250 mL flask  bromine (0.15  mole) and PBr  equipped with  a water  attached to an all-glass gas absorption device,  3  reflux  (0.5 mole) condenser  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 and the reaction product HBr were 2  completely removed by distillation under reduced pressure of a water pump. The residue was d i s t i l l e d under high vacuum at 73°C at 0.01 mm to obtain 2bromo VPA (0.09 mole). 2-Bromo VPA (0.09 mole) and 100% ethanol  (0.26  for 48 h with 20 mL benzene and 1 mL cone. H S0 2  4  mole) were refluxed  in a round bottom flask  fitted with a Dean-Stark apparatus. The contents were washed with NaHC0  3  27 (5%), water, and dried over anhydrous Na S0 . Benzene and unreacted ethanol 2  were removed by d i s t i l l a t i o n ,  4  and 2-bromo VPA ethyl  was collected  by  d i s t i l l a t i o n 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 CaC^ guard tube.  head fitted  with an air condenser protected by a  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 2ene 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 unconjugated)  (E)-2-ene  (conjugated and  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 contained  standard 20,  respectively.  40,  solutions and  40  for  4-ene VPA in  /zg/mL of  For the estimation  plasma,  DNBA and HA each  of total  urine in  (conjugates and  and  bile  0.1N NaOH, 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 ZJL aliquot of  the plasma, urine or bile was added 80 fil of the  solution,  50 (ii of 2N HCI and 200 /JL of ethyl  internal  standard  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 Na S0 , and 60 /JL of dried organic extract 2  4  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 s i l i c a 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.  conditions were as follows: 260°C;  Other operating  injection port temperature, 240°C;  interface,  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: for  50°-100°C at 30°C/min; 100°-160°C at 8°C/min> hold  2 min; post run to 250°C at 30°C/min,  spectrometer was operated in a positive-ion  and hold for 2 min. The mass 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, extract,  10,  15 to  20 ill,  were added to 60 ill of the organic  and heated at 60°C for 1 h. A 1 ill aliquot was used for GCMS  analysis.  B.2.2.5. rat,  Hydrolysis of Conjugates : Urine samples from a  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  VPA or 4-ene VPA and the  internal  concentration of  VPA or 4-ene VPA in the  linear  (E)-2-ene  least-squares  regression  (E)-2-ene  standard DNBA were plotted  analysis  was  against  standard sample. A  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. different  Extraction Efficiency: Plasma samples (80 /il) of  concentrations  of  either  (E)-2-ene  acidified with HC1, and extracted with 200 ill  VPA or ethyl  4-ene  VPA were  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 longitudinal  with a nose cone containing an ether-soaked  swab. A  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 i r s t . A small incision was made, 3 mm below the f i r s t 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 l e d 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 bileexteriorization. The cannula was kept patent by flushing and r e f i l l i n g i t 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 l i v e r . A narrow glass rod was pushed under the bile duct between the two sutures. The free ends of the f i r s t suture were gently pulled towards the t a 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 i v e r . 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  peritoneum  on  the  hub had been  dorsal  side  of  removed, the  was  passed  abdominal  cavity  through and  the  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 30 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 t e r .  centrifuged  a fixed  in  angle  rotor  at  g for  1000  The units were  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. obtained  from  rats  Metabolism of (E)-2-ene VPA: Urine and bile samples  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, lactone,  215,  217, 329, 331 and 343 corresponding to 4-hydroxy ene VPA  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), hydroxy VPA (di derivative) were monitored.  3-hydroxy VPA, 3-keto VPA (di derivative), and allylglutaric acid (AGA),  3-  respectively,  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  10  + k ) Cj + k C * 12  dC /dt = kj Cj - k j C 2  2  2  2 1  2  2  where: Cj = concentration in compartment 1 at time t C  = concentration in compartment 2 at time t  2  * 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/ )  compartment  2  (Vj)  was calculated was  determined  as 0.693/K. from dose  The volume of (D)  divided  the central  by the  plasma  37  concentration at time zero (C ), which was obtained by extrapolation of the Q  log-linear regression line to zero time. The total body clearance C1 was T  calculated as dose divided by total  area under the plasma concentration-  time curve (AUC). Renal and biliary clearances, C1 and Clg, were obtained R  by multiplying the fractions of dose excreted unchanged (unconjugated) the  urine  and bile,  respectively,  formation of conjugates C l  c o n  by Cl j .  The clearance  due  to  in the  j was determined as the sum of the fractions  of dose recovered as conjugates in urine and bile collectively, multiplied by C1 . The metabolic clearance C l T  m e t  was obtained as the difference of C l j  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.  Cl.  SYNTHESIS  C.l.l. by  RESULTS  (E)-2-ene VPA: The synthesis of (E)-2-ene VPA was attempted  dehydration  of  chloride  (Scheme  analysis  of  the  the  3-hydroxy VPA ethyl  ester  1) and with toluenesulfonyl reaction  mixture,  from  with  chloride  the  methanesulfonyl (Scheme  2 ) . GCMS  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 1 0 , 11 and 12),  several  VPA ethyl  unidentified peaks,  and extremely small quantities  of 2-ene  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  gave 3-ene VPA. NMR (Appendix (3H,  CH=);  CH -CH ); 3  2  2.7-3.3,  1.1-1.6,  2)  of the product showed: 8  complex m  m (IH, CH-C=0),  and d i s t i l l a t i o n  (4H,  -CH -CH -); 2  amount of 2-ene VPA was present as indicated by ene VPA);  6.8-7.1,  • The desired  0.8-1.1,  1.6-2.0,  2  complex m  5.2-5.9,  under vacuum  (3H, CH3-  CH=CH). A small  (2H,  2.1-2.5,  d  triplet  m (CH -C= from 2  2-  t (trans CH=C from 2-ene VPA ). product (E)-2-ene VPA was successfully  synthesized  by  dehydrobromination, and then hydrolysis of 3-bromo VPA ethyl ester. After repeated  fractional  recrystal1izations  found to  be a mixture of 90% trans  at  -20°C,  and 10% cis  the  final  isomers  product was  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 . 4 9 min was identified to be the cis isomer of 2-ene VPA and the larger peak at 7 . 1 1 min was the trans isomer of  39  Scheme 1  CH -CH2-CH -CH -C00H 3  2  2  C H OH 2  5  CH -CH -CH -CH -COOC H 3  2  2  2  2  5  Li-CH CH -CH CH _  _  2  2  2  3  CH -CH -CHO 3  2  (CH ) CH-NH-CH(CH ) 3  2  3  CH -CH -CH -CH-C00C H 3  2  2  2  5  CH -CH -CHOH 3  2  C1-S0 -CH 2  (C H ) N 2  5  3  CH C12 2  CH -CH -CH -CH-COOC H 3  2  2  2  5  CH -CH -CH-0-S0 -CH 3  2  2  KH CH -CH -CH -C-COOC H 3  2  2  2  5  CH -CH -CH 3  2  2-Ene VPA ethyl ester  3  3  2  40  Scheme 2  CH -CH -CH -CH-C00C H 3  2  2  2  5  CH -CH -CHOH 3  2  p-Toluenesulfonyl chloride Pyridine  CH -CH -CH-COOC H 2  2  2  CH -CH=CH 3  5  CH -CH -CH -C-COOC H 3  2  2  CH -CH -CH 3  2  NaOH  CH -CH -CH-COOH ?  ?  I CH -CH=CH 3  3-Ene VPA  2  NaOH  CHo-CHo-CHo-C-COOH 3 Z Z „ CH -CH -CH 3  2  2-Ene VPA  5  41  Scheme 3  CH -CH -CH -CH -COOH 3  2  2  2  (CH ) C-NH-C(CH3)3 3 3  Li-CH -CH -CH CH _  2  2  2  CH CH -CH -Br _  3  2  CH -CH -CH -CH-COOH 3  2  2  CH CH CH _  -  3  2  2  Br? PBr,  CH -CH CH  Br  -  3  2  2  C H 0H 2  5  CH -CH -CH -C-COOC H 3  2  2  / VBr  CH3 CH CH _  -  2  2  5  2  Quinoline CH -CH -CH -C-C00C H 3  2  2  2  CH3 CH CH -  -  2  NaOH CHo-CHo-CHo-C-COOH  II CH3 CH -CH _  2  2-Ene VPA  5  2  3  42  Scheme 4  -CH2-CH2-CH2-COOH  Li-CH  2  -CH  2  -CH  2  -CH  3  (CH3)2CH-NH-CH(CH3) CH2=CH-CH -I 2  -CHo-CHp-CH-COOLi  I CH2=CH-CH2  HC1  -CHo-CHo-CH-COOH  I CH2=CH2-CH  4-Ene VPA  2  2  43  7.11 i2ee*-  leeee-  9866-  00  O  ?eeeij  3 f ec-M  6.43  lSeM-i  -4  it,' '  1  ',V '  ' i f  !  ' l  TIME (min)  199  B  1 1  I  ln .  •i ee  -  leeaee-j  KB  OO  o  r58  O -  oo  ieeeee-  MB  38 466&e-  •26  75  41  5 7  /  ,  155  \ I  M  125  I  211  1 4 1 ^  A^Ua.. M 0. i . . L _k _, 168 46 86 126 266  Id  281361 S  L  246  t  .  *  -—I—•—r——i—  286  326  366  375 /  !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  90688-  6 .68  70000^  CO  z o o.  50966-]  46090^  2&&00  1  10800-  5.6  6.0  7.0  8.0  9.0  I ' ' ' ' I  ie  .e  TIME (min)  6 .68  B  min. :  1 99  26006-  R00  24660-  ;-90  2266020000-  75  ^60  18600oo  16600-  o  1 4000"  t/0  i 16  -7e  -66  12000-  ^8  1 0606-  ^48  8086-  730  6600468099  200H  \  i is  129 \  E20 157 171  141  181  214  241  L  18  lib  40  80  160  126  206  240  m/z F i g . 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  butyldimethylsilyl characteristic  derivative  peak at  VPA. The mass of  (M-57)  +  (E)-2-ene mass  ion  spectrum of VPA showed  (m/z)  199.  an  the  tertiary  intense  and  NMR (Appendix 3)  analysis showed: 6 0.8-1.1, complex m (6H, 2CH ), 1.2-1.7, m (2H, CH -CH_ 3  3  2  CH -); 2.1-2.7, m (4H, CH_-CH= and -CH_ -C=); 6.8-7.1, t (IH, -CH=C, trans, 2  2  2  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 0.81.1,  t (3H, CH ); 1.1-1.7, complex m, (4H, -CH -CH ); 2.0-2.7, complex m 3  2  2  (3H, -CH-CH -CH=); 4.9-5.3, m (2H, CH =), 5.5-6.1, complex m (IH, -CH=). 2  C.2.  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  5  3  6  LU CO  z  o a.  m/z 1 9 7  CO LU CC  m/z 1 9 9 -  m/z 2 2 9  m/z 1 7 3  r  5  i  6  1  1  7  8 TIME  — i  9  (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.  1  10  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 including  experiments  2-ethylhexanoic  with  acid,  several  heptanoic  other and  internal  standards  1-methyl-1-cyclohexanoic  acids had shown an interference with cis-2-ene VPA, 4-ene VPA and trans-2ene 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, added to  50 /xL of  appreciable standards  change (Table 3).  ethyl  acetate extract  in the  peak areas  In the  subsequent  of  of  15 to 20 /xL of MTBSTFA, plasma sample,  4-ene VPA and the  analysis  of  samples,  showed no internal 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). for  all  regression lines.  The correlation coefficient,  Within-day coefficients  r, was > 0.997  of variation (CV) for  49  TABLE 2. EFFECT OF HEATING TIME ON DERIVATIZATION  3  Time (min)  4-ene VPA  DNBA  HA  0  4770  3340  5650  15  4760  3500  30  4200  60 120  (E)-2-ene VPA  DNBA  HA  7780  3440  5780  5540  8130  3610  5900  2830  5070  7530  3320  5670  4710  3380  5540  9690  3770  6280  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 PEAKAREAS  3  MTBSTFA (ML)  4-ene VPA  DNBA  HA  5  4200  2810  4520  10  4110  2780  15  4720  20  3680  a, peak area x 10  (E)-2-ene VPA  DNBA  HA  5790  2430  4820  5000  5610  2220  4660  3550  5400  6340  2470  4670  2460  4440  5490  2220  3880  50  TABLE 4. CALIBRATION CURVE DATA FOR (E)-2-ENE VPA IN RAT PLASMA (N=5) Added ng/ml  FoundMg/mL  CV  %  Found Mg/mL  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  0.40  1  2  CV  %  1=DNBA; 2=HA  TABLE 5. CALIBRATION CURVE DATA FOR (E)-2-ENE VPA IN RAT URINE (N=5) Added ng/ml  Found* Mg/mL  CV  %  Found /ig/mL  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  1  2  CV  %  2  2  51  TABLE 6. CALIBRATION CURVE DATA FOR (E)-2-ENE VPA IN RAT BILE (N=3) Added MO/mL  Found* jug/mL  CV  Found  %  CV  Hg/ml  %  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  1  2  2  52  TABLE 7. CALIBRATION CURVE DATA FOR 4-ENE VPA IN RAT PLASMA (N=6) Added Mg/mL  Found* Mg/mL  CV  0.50 2.0  %  Found /ig/mL  CV  0.6±0.08  13  0.6±0.05  8.9  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  2  2  %  1=DNBA; 2=HA  TABLE 8. CALIBRATION CURVE DATA FOR 4-ENE VPA IN RAT URINE (N=6) Added MQ/mL  Found* Mg/mL  CV  1  %  Found /jg/mL  2  CV  %  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  2  53  TABLE 9. CALIBRATION CURVE DATA FOR 4-ENE VPA IN RAT BILE (N=3) Added  Found*  CV  %  Found Mg/mL  CV  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  2.0  1  %  1=DNBA; 2=HA  TABLE 10. EFFECT OF HEATING TIME ON THE HYDROLYSIS OF CONJUGATES IN RAT URINE 3  (Time, h)  0.5  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  1  1.5  2  2  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  plasma concentrations recoveries recovery  concentrations  acetate were 99,  (E)-2-ene of  5,  were  VPA from 15  performed  at  three  different  20 and 40 ng/ml of 4-ene VPA. The average  of 5,  using ethyl of  studies  and  30  102 and 102%,  plasma jig/mL  was and  respectively. The  also was  100,  quantitative 99  and  at  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 f i r s t 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 Thereafter,  + 1.1  /ig/mL,  at  approximately 2 h after  the plasma levels of (E)-2-ene  the dose.  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 There was,  however,  a short i n i t i a l  period of  (Fig.9,10).  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 1.5  min  to  reach  trough  levels  of  26  +  11  1 / 2  and  of 28 + 6 and 18 + 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). total  There was no significant  plasma clearances  of  (E)-2-ene  change in the metabolic and  VPA, and in the  excreted as unconjugated, conjugated and total  fraction of dose  (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.  Parameter  PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg) la  2a  3a  4a  Mean  Weight  270  285  290  300 ,  286  Dose  5.4  5.7  5.8  6.0  5.7  90  84  98  66  85  300  390  250  300  310  23  18  28  23  23  »/  220  240  200  300  240  AUC^  4700  3300  7400  3100  4600  3  + t  l/2  6  e  % DOSE EXCRETED INURINE 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) C1  cl  R  met  C1  T  0.10  0.43  0.27  0.27  0.27  4.2  5.7  2.4  6.2  4.6  4.3  6.1  2.7  6.4  4.9  a, g; 6, mg/kg; c, ztg/mL; d, xlO"° m i n ; e, min; f, mL/kg; g, zig.min/mL -1  59  TABLE 12.  Parameter  PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg) lb  2b  3b  4b  Mean  Weight  260  260  310  320  288  Dose  26  26  31  32  29  410  460  480  390  430  320  260  190  260  260  22  27  36  27  28  v/  250  220  210  260  230  AUCS  29000  33000  35000  35000  33000  3  6  e  4.  % 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) C1 C1  R  met  ci  T  0.38  0.35  0.30  0.13  0.29  3.1  2.7  2.6  2.7  2.8  3.4  3.0  2.9  2.8  3.0  a, g; 6, mg/kg; c, m/ml; d,x l O f, mL/kg; g, /jg.min/mL  - 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)  la  0-2.5 2.5-8 8-24  2.68 2.44 6.60  2a  0-3 3-9 9-24  3.63 1.60 9.50  Cone (/jg/mL)  Amount (mg)  Time (h)  Cumulative Amt (mg)  Cumulative % of Dose  32 (400) 6.9 (89) 2.9 (3.3)  0,.09 (1.1) 0..02 (0.22) 0,.02 (0.02)  0-2.5 0-8 0-24  0.09 (1.1) 0.11 (1.3) 0.13 (1.3)  1.7 (20) 2.0 (24) 2.4 (25)  99 (360) 27 (190) - (5.0)  0,.36 (1.3) 0..04 (0.30) (0.05)  0-3 0-9 0-24  0.36 (1.3) 0.40 (1.6) 0.40 (1.6)  6.3 (23) 7.0 (28) 7.0 (29) cn o  3a  0-3.25 3.25-8 8-24  3.61 1.40 15.0  140 (480) 36 (140) - (")  0..52 (1.7) 0.,05 (0.19) ~ (-)  0-3.25 0-8 0-24  0.52 (1.7) 0.57 (1.9) 0.57 (1.9)  8.9 (30) 9.8 (33) 9.8 (33)  4a  0-8 8-24  10.1 15.2  22 (230) 2.2 (2.9)  0.,22 (2.3) 0.,03 (0.04)  0-8 0-24  0.22 (2.3) 0.25 (2.4)  3.7 (39) 4.2 (39)  Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA.  TABLE 14.  Rat  Time (h)  URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg) Volume (mL)  Cone (Mg/mL)  Amount (mg)  Time (h)  Cumulative Amt (mg)  Cumulative % of Dose  0-1.5 0-8.5 0-11.5 0-24  2.6 2.7 2.8 2.9  1.9 (15) 1.1 (1.7) 0.06 (0.06)  0-6.5 0-12.5 0-24  1.9 (15) 3.0 (17) 3.0 (17)  7.3 (57) 12 (64) 12 (64)  130 (1600) 960 (3500) 120 (190)  0.27 (3.4) 2.4 (8.7) 0.56 (0.91)  0-1 0-8.5 0-24  0.27 (3.4) 2.7 (12) 3.2 (13)  0.87 (11) 8.6 (39) 10 (42)  150 240 240 34  0.16 0.36 0.87 0.12  0-0.5 0-2 0-11.5 0-24  lb  0-1.5 1.5-8.5 8.5-11.5 11.5-24  3.0 2.28 2.00 4.65  860 64 29 13  2b  0-6.5 6.5-12.5 12.5-24  3.99 2.92 4.50  480 (3700) 360 (580) 14 (15)  3b  0-1 1-8.5 8.5-24  2.15 2.50 4.86  4b  0-0.5 0.5-2 2-11.5 11.5-24  1.05 1.50 3.61 3.56  (1900) (1600) (560) (79)  (480) (5300) (2100) (110)  2.6 0.14 0.06 0.06  (5.8) (3.6) (1.1) (0.37)  (0.50) (7.9) (7.4) (0.41)  0.16 0.52 1.4 1.5  (5.8) (9.4) (11) (11)  (0.50) (8.4) (16) (16)  Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA.  10 11 11 11  0.50 1.6 4.3 4.7  (22) (36) (40) (42)  (1.6) (24) (49) (51)  62  TABLE 15.  PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg)  lc  2c  3c  Weight  290  280  Dose  5.8  Parameter  4c  5c  6c  280  270  320  335  296 + 26  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  11  14  12  14  11  10  v/  210  180  240  270  190  170  210 + 40  AUC^  2200  2500  2200  2100  2400  2500  2300 + 160  3  6  4. t  l/2  e  Mean + SD  12 + 1.7  % 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) C1 cl  R  met  C1  T  0.85  0.26  0.32  0.51  0.51  0.35  8.3  7.8  8.8  8.8  7.9  7.7  8.2 + 0.5:  9.2  8.1  9.1  9.4  8.4  8.0  8.7 + 0.61  a, g; b, mg/kg; c, /zg/mL; d, xlO"^ min"*; e, min; f, mL/kg; g, zzg.min/mL  0.47 +  0.2:  63  TABLE 16.  PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg)  Id  2d  3d  4d  5d  6d  280  256  262  260  305  295  28  26  26  26  31  30  280  320  330  350  310  290  310 + 24  360  400  410  330  370  410  380 + 34  19  18  17  21  19  17  »/  350  320  300  290  320  350  AUC0  17000  15000  15000  19000i  19000  18000  Parameter Weight  3  Dose  6  kl'  t e l/2 l  5i  Mean + SD 276 + 20 28 + 2.1  18 + 1.5 320 + 25 17000 + 16i  DOSE EXCRETED 1 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  0.23 + o.i;  CLEARANCE (mL/min.kq) C1  0 1  R  met  C1  T  o .26  0 .32  0.43  0 .13  0.13  0 .14  .8  6 .2  6.1  5 .2  5.2  5 .6  5.7 + 0.4:  6 .0  6 .5  6.5  5 .3  5.3  5 .7  5.9 + 0.-5.  5  a. g; b, mg/kg; c, izg/mL; d, xlO"^ min"*; e, min; f, mL/kg; g, tig.min/mL  TABLE 17.  *at  Time (h)  Volume (mL)  URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN NORMAL RATS (DOSE=20 mg/kg) Cone (Mg/mL) 97 (230) 22 (110) 4.5 (8.6)  Amount (mg) 0.36 (0.86) 0.08 (0.39) 0.10 (0.20)  Time (h)  Cumulative Amt (mg)  Cumulativ % of Dose  0-3 0-7 0-24  0.36 (0.86) 0.43 (1.3) 0.54 (1.5)  6.2 (15) 7.5 (22) 9.3 (25)  0-2.5 0-5 0-8 0-24  0.18 0.18 0.18 0.18  (0.99) (1.2) (1.2) (1.2)  3.1 3.3 3.3 3.3  (18) (21) (22) (22)  0-0.5 0-4 0-8 0-24  0.12 0.17 0.18 0.20  (0.41) (0.98) (1.1) (1.1)  2.0 3.1 3.2 3.6  (7.2) (18) (19) [19)  lc  0-3 3-7 7-24  3.70 3.40 23.3  2c  0-2.5 2.5-5 5-8 8-24  2.23 1.24 1.35 8.00  78 6.9 -  (450) (142) (24) (-)  3c  0-0.5 0.5-4 4-8 8-24  1.47 2.59 1.90 8.50  78 22 4.7 2.1  (280) (220) (37) (2.2)  4c  0-2 2-8 8-24  3.00 7.40 8.55  61 (250) 15 (74) - (")  0.18 (0.75) 0.11 (0.54) - (")  0-2 0-8 0-24  0.18 (0.75) 0.30 (1.3) 0.30 (1.3)  3.4 [14) 5.5 [24) 5.5 [24)  5c  0-8 8-24  3.50 11.20  110 (440) - (3.1)  0.39 (1.6) - (0.035)  0-8 0-24  0.39 (1.6) 0.39 (1.6)  6.1 [24) 6.1 [25)  6c  0-8 8-24  2.95 15.50  90 (370) - (6.3)  0.27 (1.1) - (0.098)  0-8 0-24  0.27 (1.1) 0.27 (1.2)  4.0 [16) 4.0 ,18)  0.18 0.008 0.12 0.06 0.009 0.018  (0.99) (0.18) (0.033) (") (0.41) (0.58) (0.069) (0.019)  Numbers inside brackets indicate total (sum of conjugated and unconjugated) 4-ene VPA.  TABLE 18.  Volume (mL)  URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN NORMAL RATS (DOSE=100 mg/kg)  Rat  Time (h)  Cone (itg/mL)  Id  0-3 3-7.5 7.5-12 12-24  3.07 2.63 2.37 3.28  330 62 11 3.2  (1400) (0.85) (0.13) (0.018)  1.0 0.16 0.025 0.010  2d  0-1 1-6 6-10 10-24  1.85 1.29 2.14 3.40  260 530 25 14  (2600) (2700) (350) (21)  3d  0-1 1-2 2-8.5 8.5-11 11-13 13-24  0.35 2.35 2.27 1.59 1.62 4.52  230 610 85 5.5 11  (290) (2200) (700) (52) (68) - (9.3)  4d  0-1 1-2 2-10 10-12 12-24  0.51 1.50 1.29 1.30 3.54  110 250 130 9.1 8.9  5d  0-6 6-24  5.30 18.50  110 (1500) 8.5 (130)  0.57 (8.1) 0.16 (2.4)  0-6 0-24  0.57 (8.1) 0.73 (11)  1.9 (27) 2.4 (34)  6d  0-6 6-24  3.10 7.88  290 (1800) 9.9 (230)  0.91 (5.6) 0.078 (1.8)  0-6 0-24  0.91 (5.6) 0.99 (7.4)  3.1 (19) 3.3 (25)  (450) (1400) (2200) (160) (37)  Amount (mg)  Time (h)  Cumulative Amt (mg)  Cumulative % of Dose  (4.4) (2.2) (0.31) (0.06)  0-3 0-7.5 0-12 0-24  1.0 1.2 1.2 1.2  (4.4) (6.6) (6.9) (7.0)  3.6 4.2 4.3 4.3  (16) (24) (25) (25)  0.48 0.68 0.054 0.047  (4.9) (3.4) (0.75) (0.073)  0-1 0-6 0-10 0-24  0.48 1.2 1.2 1.3  (4.9) (8.3) (9.1) (9.2)  1.9 4.5 4.8 4.9  (19) (33) (36) (36)  0.08 1.4 0.19 0.009 0.018 -  (0.10) (5.2) (1.6) (0.083) (0.11) (0.042)  0-1 0-2 0-8.5 0-11 0-13 0-24  0.079 1.5 1.7 1.7 1.7 1.7  (0.10) (5.3) (6.9) (7.0) (7.1) (7.2)  0.3 5.8 6.6 6.6 6.6 6.6  (0.4) (21) (27) (27) (27) (28)  (0.23) (2.1) (2.9) (0.21) (0.13)  0-1 0-2 0-10 0-12 0-24  0.057 0.43 0.60 0.61 0.64  (0.23) (2.4) (5.2) (5.4) (5.6)  0.2 1.7 2.3 2.3 2.5  (0.9) (9.1) (20) (21) (21)  0.057 0.38 0.16 0.012 0.032  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  clearances  plasma,  renal  and  non-renal  (metabolic)  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 constants, and for  values  of  first-order transfer  kjQ, k^ and k j for (E)-2-ene VPA were 1.0, 2  2  4-ene VPA, 2.4,  generated curve (solid  0.89  line)  and 0.50  h" , 1  actual data points. At the trough levels,  0.69 and 1.3 h"*  respectively.  showed an acceptable however,  fit  rate  The model-  with most of the  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)  2  <D 100-  > c LU CN  0) > -J  NO SECONDARY PEAK 1T  (0  E  CO  Q.  0.1  3  Time, h Fig.  4  6  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 CD  <  Q_  10CN  c LU i  > CD  —I  i  10-  1^  CO  E  CO  Q_  0.1  f  NO SECONDARY PEAK  f 3  Time, h Fig.  5  6  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 elimination t high dose,  followed  an open one-compartment  model  with  of 20 + 3.4 and 13 + 1.8 min, respectively.  1 / 2  a brief  first-order  Following the  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  of 21+1.7 and  19 + 3.1 min, respectively.  1 / 2  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  eliminated  in  of the  65% of  the  low dose and 66% of  urine and bile,  collectively  the  high dose were  (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). recovered as conjugated  and total  In contrast,  the fraction of dose  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  Weight  320  285  280  310  299  Dose  6.4  5.7  5.6  6.2  6.0  100  79  100  81  91  390  400  280  340  350  18  17  25  20  20  200  250  200  240  220  2600  2100  3800  2500  2700  3  t l  l/2  vi  6  e  f  AUC^  % 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 Conj Total  -  12  8.4  6.3  5.9  8.1  39  29  29  22  29  51  37  34  28  38  CLEARANCE (mL/min.kq) R  0.13  0.33  0.22  0.44  0.28  B  0.93  0.81  0.33  0.47  0.64  4.2  5.2  2.8  4.2  4.1  7.8  9.7  5.3  8.0  7.7  C1  ci Cl  ci  conj T  a, g; b, mg/kg; c, /jg/mL; d, xlO" min" ; e, min; f, mL/kg; g, /jg.min/mL 5  1  73  TABLE 20.  Parameter  PHARMACOKINETIC PARAMETERS OF (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS (D0SE=100 mg/kg) If  2f  3f  4f  Mean  Weight  270  290  320  270  288  Dose  27  29  32  27  29  440  440  380  440  430  360  300  320  340  330  19  23  22  20  21  230  230  260  230  240  13000  20000  18000  16000  3  6  c. h t  c  -  d  l/2  h  f  AUC^  17000  % DOSE EXCRETED INURINE 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 INBILE Unconj  6.0  8.4  2.5  7.4  6.1  Conj  25  34  17  25  25  Total  31  42 .  19  32  31  CLEARANCE (mL/min • kq) C1  R  0.35  0.18  0.07  0.21  0.20  ci  B  0.45  0.43  0.13  0.45  0.37  5.2  3.0  3.0  2.8  3.5  7.5  5.0  5.4  6.1  6.0  Cl ^'conj ci T  a, g; 6, mg/kg; c, jug/mL; d, xlO"° min" ; e, min; f, mL/kg; g, /zg.min/mL 1  TABLE 21.  URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=20 mg/kg)  Rat  Time (h)  Volume (mL)  Cone (MOM)  Amount (mg)  Time (h)  Cumulative Amt (mg)  le  0-6 6-24  1.52 20.0  71 (730) - (-)  0.11 (1.1) - (-)  0-6 0-24  0.11 (1.1) 0.11 (1.1)  1.7 (17) 1.7 (17)  2e  0-6 6-24  3.4 21.5  47 (450) - (3.4)  0.16 (1.5) - (0.07)  0-6 0-24  0.16 (1.5) 0.16 (1.6)  2.8 (27) 2.8 (28)  3e  0-5.5 5.5-24  2.17 8.20  97 (720) 2.9 (3.0)  0.21 (1.6) 0.02 (0.02)  0-5.5 0-24  0.21 (1.6) 0.23 (1.6)  3.8 (28) 4.1 (29)  4e  0-5.5 5.5-24  4.90 4.80  69 (450) - (2.2)  0.34 (2.2) - (0.01)  0-5.5 0-24  0.34 (2.2) 0.34 (2.2)  5.5 (36) 5.5 (36)  Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA.  Cumulative % of Dose  TABLE 22.  Rat  Time (h)  URINARY EXCRETION OF UNCONJUGATED (E)-2-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=100 mg/kg) Volume (mL)  Cone (M9/mL)  Amount (mg)  Time (h)  Cumulative Amt (mg)  Cumulative % of Dose  3.20  400 (4100)  1.3 (13)  0-6.5  1.3 (13)  4.7 (49)  If  0-6.5 6.5-24  2f  0-6 6-24  1.93 11.80  58 (1700) 77 (430)  0.11 (3.3) 0.91 (5.1)  0-6 0-24  0.11 (3.3) 1.0 (8.4)  0.4 (12) 3.5 (29)  3f  0-6.5 6.5-24  11.33 5.60  35 (1100) 7.9 (45)  0.39 (12) 0.04 (0.25)  0-6.5 0-24  0.39 (12) 0.43 (13)  1.2 (38) 1.3 (39)  4f  0-6 6-24  2.25 7.80  190 (2000) 66 (230)  0.42 (4.5) 0.51 (1.8)  0-6 0-24  0.42 (4.5) 0.94 (6.3)  1.6 (17) 3.5 (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 (h)  Volume (mL)  Time (h)  Cumulative Amt (mg)  le  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  1.24 1.56 1.12 1.07 1.10 1.05  440 140 12 -  (1900) (520) (32) (1.3) (-) (-)  0.54 0.22 0.013 -  (2.4) (0.81) (0.035) (0.001) (-) (-)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  0.54 0.75 0.77 0.77 0.77 0.77  (2.4) (3.2) (3.2) (3.2) (3.2) (3.2)  8.4 12 12 12 12 12  (38) (50) (51) (51) (51) (51)  2e  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  1.15 1.53 1.10 0.91 0.86 0.74  330 65 2.6 -  (1400) (300) (8.0) (-) (-) (-)  0.38 0.10 0.003 -  (1.7) (0.45) (0.009) (-) (-) (-)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  0.38 0.48 0.48 0.48 0.48 0.48  (1.7) (2.1) (2.1) (2.1) (2.1) (2.1)  6.6 8.3 8.4 8.4 8.4 8.4  (29) (37) (37) (37) (37) (37)  3e  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  0.84 1.01 0.87 0.54 0.78 0.64  240 140 12 1.2 -  (1400) (750) (60) (2.8) (-) (-)  0.20 0.14 0.010 0.001 -  (1.2) (0.76) (0.015) (0.001) (-) (")  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  0.20 0.34 0.35 0.35 0.35 0.35  (1.2) (1.9) (1.9) (1.9) (1.9) (1.9)  3.5 6.1 6.1 6.3 6.3 6.3  (21) (34) (35) (35) (35) (35)  4e  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  1.05 1.21 1.00 0.94 0.86 0.81  220 100 5.1 -  (1200) (350) (23) (1.1) (-) (-)  0.23 0.13 0.005 -  (1.3) (0.43) (0.023) (0.001) (-) (-)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  0.23 0.36 0.36 0.36 0.36 0.36  (1.3) (1.7) (1.7) (1.7) (1.7) (1.7)  3.8 5.8 5.9 5.9 5.9 5.9  (21) (28) (28) (28) (28) (28)  Cone (/zg/mL)  Amount (mg)  Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA.  Cumulative % of Dose  --4  TABLE 24.  BILIARY ELIMINATION OF UNCONJUGATED (E)-Z-ENE VPA IN RATS (DOSE=100 mg/kg)  Time (h)  Volume (mL)  0-0.5 0.5-1.5 1.5-2.5 2.5-5.0 5.0-6.5  0.84 1.42 0.78 1.54 1.23  870 590 75 1.6  0-0.5 0.5-1.0 1.0-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-6.0  Cone (iig/mL)  Time (h)  Cumulative Amt (mg)  (3.7) (4.4) (0.23) (0.01) (-)  0-0.5 0-1.5 0-2.5 0-5 0-6.5  0.73 1.6 1.6 1.6 1.6  (3.7) (8.1) (8.3) (8.3) (8.3)  2.7 5.8 6.0 6.0 6.0  (14) (30) (31) (31) (31)  Amount (mg)  Cumulative % of Dose  0.73 0.84 0.058 0.002  ~  (4400) (3100) (290) (5.7) (-)  1.01 1.18 1.04 1.15 0.82 0.60 0.67  580 670 570 360 54 10 2.4  (2500) (3900) (3200) (1400) (180) (36) (8.7)  0.58 0.80 0.59 0.41 0.044 0.006 0.002  (2.5) (4.6) (3.4) (1.6) (0.15) (0.022) (0.006)  0-0.5 0-1.0 0-1.5 0-2.5 0-3.5 0-4.5 0-6.0  0.58 1.4 2.0 2.4 2.4 2.4 2.4  (2.5) (7.1) (11) (12) (12) (12) (12)  2.0 4.8 6.8 8.2 8.4 8.4 8.4  (8.7) (24) (36) (41) (42) (42) (42)  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-5.0 5.0-6.5  0.87 1.12 0.87 0.77 0.98 0.98  320 330 150 14 -  (2300) (2900) (1100) (46) (2.6) (1.2)  0.28 0.37 0.13 0.011 -  (2.0) (3.3) (0.93) (0.035) (0.002) (0.001)  0-0.5 0-1.5 0-2.5 0-3.5 0-5.0 0-6.5  0.28 0.64 0.77 0.79 0.79 0.79  (2.0) (3.3) (6.2) (6.2) (6.2) (6.2)  0.9 2.0 2.4 2.4 2.4 2.4  (6.2) (10) (19) (19) (19) (19)  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-6.0  0.93 1.32 1.07 0.84 0.83 1.09  730 620 400 87 5.4 -  (2500) (3300) (1700) (260) (17) ( - )  0.68 0.81 0.43 0.073 0.004 -  (2.3) (4.3) (1.8) (0.22) (0.14) ( ")  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-6.0  0.68 1.5 1.9 2.0 2.0 2.0  (2.3) (6.6) (8.4) (8.6) (8.8) (8.8)  2.5 5.5 7.1 7.4 7.4 7.4  (8.5) (24) (31) (32) (32) (32)  ~  Numbers inside brackets indicate total (sum of conjugated and unconjugated) (E)-2-ene VPA.  78  TABLE 25.  Parameter  PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=20 mg/kg) ig  2g  3g  Weight  270  285  Dose  5.4  C  Mean + SD  4g  5g  6g  290  310  265  305  288 + 18  5.7  5.8  6.2  5.3  6.1  5.8 + 0.4  85  88  100  96  74  100  91 + 11  560  550  620  420  560  590  12  13  11  16  12  12  v/  220  210  180  200  270  190  210 + 30  AUC^  1700  1700  1800  2400  1400  1800  1800 + 310  3  6  c  ^0  l  d  4. l  l/2  e  550 ± 70 13 + 1.8  % DOSE EXCRETED INURINE 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) C1 ci  R  B  Cl u 1  ci  conj T  0.08  0.04  0.15  0.11  0.36  0.33  0.18 + 0.1<  0.64  0.63  0.48  0.87  1.1  0.44  0.69 + 0.2'  6.3  4.5  4.8  2.8  4.6  4.2  4.5 + 1.1  12  12  11  8.5  14  11  a, g; o, mg/kg; c, ng/ml; d, xl0"° min" ; e, min; f, mL/kg; g, itg.min/mL 1  11 + 1.8  79  TABLE 26.  PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN BILE-EXTERIORIZED RATS (D0SE=100 mg/kg) lh  2h  3h  4h  5h  6h  Mean + SD  Weight  290  325  302  295  280  300  299 + 15  Dose  29  33  30  30  28  30  290  320  310  250  270  310  290 + 28  430  300  310  380  400  440  380 + 58  l/2  16  23  22  18  17  16  v,  340  320  320  400  370  320  350 + 35  15000  16000  16000  12000  12000  12000  14000 + 2100  Parameter 3  h t  6  d  f  AUC^  30 + 1.5  19 + 3.1  % DOSE EXCRETED INURINE 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  5.5 + 1.6  % DOSE EXCRETED IN BILE Unconj  4.7  8.4  3.8  3.9  6.1  4.5  Conj  15  18  16  12  16  15  15 + 2.0  Total  20  27  20  16  22  19  21 + 3.5  CLEARANCE (mL/min.kq) C1  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 conj  2.6  3.2  2.3  2.9  3.4  2.6  2.8 + 0.42  C1  6.8  6.4  6.1  8.0  8.7  8.4  7.4 + 1.1  u1  T  a, g; b, mg/kg; c, /ig/mL; d, xlO" min - - e f, mL/kg; g, /zg.min/mL 5  1  min;  TABLE 27.  URINARY EXCRETION OF UNCONJUGATED 4-ENE VPA IN BILE-EXTERIORIZED RATS (DOSE=20 mg/kg)  Rat  Time (h)  Volume (mL)  Cone (itg/mL)  Amount (mg)  ig  0-6 6-24  3.80 18.5  8.8 (270) ~ (-)  0.03 (1.0) ~ (-)  Time (h)  Cumulative Amt (mg)  Cumulative % of Dose  0-6 0-24  0.03 (1.0) 0.03 (1.0)  0.6 (19) 0.6 (19)  2g  0-6 6-24  3.00 4.70  6.9 (230) (-)  0.02 (0.68) (3.5)  0-6 0-24  0.02 (0.68) 0.02 (0.70)  0.4 (12) 0.4 (12)  3g  0-6 6-24  4.30 5.70  19 (300) ~ (3.8)  0.08 (1.3) ~ (0.02)  0-6 0-24  0.08 (1.3) 0.08 (1.3)  1.4 (23) 1.4 (23)  4g  0-6 6-24  2.80 3.84  28 (440) ~ (4.1)  0.08 (1.2) ~ (0.02)  0-6 0-24  0.08 (1.2) 0.08 (1.3)  1.3 (20) 1.3 (20)  5g  0-6 6-24  3.15 4.38  33 (220) 7.8 (21)  0.10 (0.69) 0.03 (0.09)  0-6 0-24  0.10 (0.69) 0.14 (0.78)  2.0 (13) 2.6 (15)  6g  0-6 6-24  3.75 5.14  35 (260) 9.2 (15)  0.13 (0.98) 0.05 (0.08)  0-6 0-24  0.13 (0.98) 0.18 (1.1)  2.2 (16) 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 (h)  Volume (mL)  Cone (Mg/mL)  Cumulative Amt (mg)  Cumulative % of Dose  lh  0-5 5-24  4.66 9.70  150 (1500) 4.8 (46)  0.70 (7.0) 0.047 (0.45)  0-5 0-24  0.70 (7.0) 0.75 (7.4)  2.4 (24) 2.6 (26)  2h  0-6 6-24  3.80 9.50  280 (2800) 6.2 (46)  1.1 (11) 0.059 (0.44)  0-6 0-24  1.1 (11) 1.1 (11)  3.3 (34) 3.5 (35)  3h  0-6 6-24  6.70 18.0  96 (1000) 5.7 (20)  0.64 (6.9) 0.10 (0.36)  0-6 0-24  0.64 (6.9) 0.74 (7.3)  2.1 (23) 2.5 (24)  4h  0-6 6-24  2.80 4.70  220 (2900) 39 (600)  0.62 (4.9) 0.18 (2.8)  0-6 0-24  0.62 (4.9) 0.81 (7.7)  2.1 (17) 2.8 (27)  5h  0-7 7-24  4.40 9.70  56 (1400) 2.4 (55)  0.25 (6.1) 0.023 (0.53)  0-7 0-24  0.25 (6.1) 0.27 (6.7)  0.9 (22) 1.0 (24)  6h  0-7 7-24  3.96 8.45  100 (1200) 2.9 (31)  0.40 (4.8) 0.025 (0.26)  0-7 0-24  0.40 (4.8) 0.42 (5.0)  1.3 (16) 1.4 (17)  Numbers inside brackets indicate total  Amount (mg)  Time (h)  (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  ig  2g  3g  4g  5g  6g  Time  Volume  Amount  Time  Cumulative  Cumulative  (mg)  (h)  Amt (mg)  X of Dose  200 (1500)  0.22 (1.7)  0-0.5  0.22 (1.7)  4.1 (31)  45 (280)  0.064 (0.41)  0-1.5  0.28 (2.1)  5.2 (38)  2.3 (4.9)  0.003 (0.01)  0-2.5  0.29 (2.1)  5.3 (38)  (-)  0-3.5  0.29 (2.1)  5.3 (38)  (-)  0-4.5  0.29 (2.1)  5.3 (38)  0.24 (1.5)  0-0.5  0.24 (1.5)  4.2 (26)  0.06 (0.34)  0-1.5  0.03 (1.8)  5.3 (32)  0.004 (0.01)  0-2.5  0.31 (1.8)  5.4 (32)  (-)  0-3.5  0.31 (1.8)  5.4 (32)  (-)  0-4.5  0.31 (1.8)  5.4 (32)  0.18 (1.2)  0-0.5  0.18 (1.2)  3.1 (20)  0.065 (0.31)  0-1.5  0.25 (1.5)  4.2 (26)  0-2.5  0.25 (1.5)  4.3 (26)  (-)  0-3.5  0.25 (1.5)  4.3 (26)  (-)  0-4.5  0.25 (1.5)  4.3 (26)  Cone  (h)  (mL)  (ug/mL)  0-0.5  1.10  0.5-1.5  1.43  1.5-2.5  1.19  2.5-3.5  1.15  -  (-)  -  3.5-4.5  1.00  -  (-)  -  0-0.5  1.10  220 (1300)  0.5-1.5  1.40  43 (240)  1.5-2.5  1.28  2.8  2.5-3.5  1.31  -  (-)  -  3.5-4.5  1.23  -  (-)  -  0-0.5  1.08  170 (1100)  0.5-1.5  1.60  41 (200)  1.5-2.5  1.38  2.2 (4.5)  0.003 (0.01)  2.5-3.5  1.17  -  (-)  -  3.5-4.5  1.08  -  (-)  -  (4.7)  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)  0.085 (0.11)  0-2.25  0.63 (1.5)  10 (24)  0.002 (0.002)  0-3.25  0.64 (1.5)  10 (24)  1.0-2.25  1.12  76 (97)  2.25-3.25  0.65  2.5 (3.0)  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)  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  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)  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)  0.002 (0.012)  (3.5)  1.5-2.5  1.11  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)  2.1 (11)  Numbers i n s i d e brackets indicate t o t a l (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 (h)  Volume (mL)  1h  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  0.70 1.24 1.07 0.88 0.80 0.85  600 490 290 25 3.8  0-0.5 0.5-1.0 1.0-1.5 1.5-2.0 2.0-3.5 3.5-5.0 5.0-6.0  1.15 1.30 1.10 1.24 1.34 1.57 0.87  610 630 450 260 250 4.8  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-6.0  0.71 1.38 1.23 0.89 0.75 1.08  480 260 300 70 7.6  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-6.0  0.76 1.23 0.75 0.55 0.49 1.03  500 420 290 42 6.3  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  1.05 2.04 1.46 1.29 1.05 0.97  500 520 75 5.7  0-0.5 0.5-1.5 1.5-2.5 2.5-3.5 3.5-4.5 4.5-5.5  0.95 2.00 1.38 1.12 1.04 0.91  480 350 110 15 8.3  2h  3h  4h  5h  6h  Cone (Mg/mL) (2000) (2600) (1000) (50) (6.6)  - (-) (2800) (2800) (680) (480) (270) (3.2)  - (-)  Amount (mg) 0.42 0.61 0.32 0.022 0.003  0.70 0.82 0.50 0.32 0.33 0.008  (1700) (2100) (930) (120) (12) - (2.0)  0.38 0.52 0.22 0.023 0.003  (2100) (1800) (240) (16) - (3.4)  - (-) (2000) (1600) (310) (160) (25)  - (-)  -  0.53 1.1 0.11 0.007  (1.4) [4.6) £5.7) C5.8) (5.8) C5.8)  1.5 3.6 4.6 4.7 4.7 4.7  (4.1) (16) (20) (20) (20) (20)  0-0.5 0-1.0 0-1.5 0-2.0 0-3.5 0-5.0 0-6.0  0.70 1.5 2.0 2.3 2.7 2.7 2.7  (3.2) (6.8) (7.6) (8.2) (8.5) (8.5) (8.5)  2.2 4.8 6.3 7.3 8.3 8.4 8.4  (10) (21) (24) (26) (27) (27) (27)  (1.7) (2.8) (1.4) (0.15) (0.011) (0.003)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-6.0  0.34 0.70 1.1 1.1 1.1 1.1  (1.7) (4.5) (5.9) (6.0) (6.1) (6.1)  1.1 2.3 3.6 3.8 3.8 3.8  (5.8) (15) (20) (20) (20) (20)  (1.3) (2.6) (0.70) (0.065) (0.006) (0.002)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-6.0  0.38 0.90 1.1 1.1 1.1 1.1  (1.3) (3.9) (4.6) (4.7) (4.7) (4.7)  1.3 3.1 3.8 3.9 3.9 3.9  (4.4) (14) (16) (16) (16) (16)  (2-2) (3.6) (0.35) (0.020) (0.003)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  0.53 1.6 1.7 1.7 1.7 1.7  (2.2) (5.8) (6.2) (6.2) (6.2) (6.2)  1.9 5.7 6.1 6.1 6.1 6.1  (8.0) (21) (22) (22) (22) (22)  (1.9) (3.2) (0.43) (0.18) (0.026)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  0.45 1.2 1.3 1.3 1.3 1.3  (1.9) (5.2) (5.6) (5.8) (5.8) (5.8)  1.5 3.8 4.4 4.4 4.5 4.5  (6.4) (17) (19) (19) (19) (19)  (3.2) (3.6) (0.75) (0.59) (0.36) (0.005)  - (-) 0.45 0.70 0.16 0.016 0.009  Cumulative % ofDose  0.42 1.0 1.4 1.4 1.4 1.4  - (-) 0.34 0.36 0.37 0.063 0.006  Cumu ati ve Amt Cmg)  0-0.5 0-1.5 0-2.5 0-3.5 0-4.5 0-5.5  - (-)  (2500) (2000) (1100) (170) (15) (3.0)  -  (1.4) (3.2) (1.1) (0.044) (0.005)  Time (h)  - (-)  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 4ene 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 PARAMETERS OF (E)-2-ENE VPA IN THE RAT NORMAL  Parameter k^ t  l/2  Vj  p-Value  310+60  260±50  NS  kl  NS  tl/2  230+20  NS  vi  1160±74  NS  AUC/D  0.27±0.13  0.29±0.11  NS  Clp  4.6+1.7  2.8±0.21  4.9+1.7  3.0+0.27  810+350  f  C1 9 R  met  100 mg/kg  a  240+40  e  AUC/D  C1  20 mg/kg  23+4.2  d  g  C1 ^ T  BILE EXTERIORIZED a  28+5.8  5.9+3.3  20 mg/kg  a  350±55  c  20±3.4  d  100 mg/kg  a  330+27 21±1.7  p-Value NS NS  220±29  240±17  NS  460±150  590+77  NS  9  0.28+0.13  0.20+0.11  NS  NS  C1 9  0.64+0.28  0.37+0.16  NS  NS  Cl ° ' c o n j-9  4.1±1.0  3.5+1.1  NS  C1 9  7.7+1.8  6.0+1.1  NS  % Dose Excreted in Urine Unconj  Parameter  e  f  B  T  9.4+3.2  NS  % Dose Excreted in Urine  Conj  26+6.4  40+11  NS  Unconj  3.5±1.6  3.3±1.4  NS  Total  32+6.3  50+11  NS  Conj  24±6.1  32+11  NS  Total  27±7.6  35+11  NS  a, Dose; b, p>0.05=Nonsignificant (NS);  % Dose Excreted in Bile  c, xlO" min" ; d, min; e, mL/kg; 5  1  f, /zg.min/mL.mg; g , mL/min.kg Each value is a mean of 4 observations + SD  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  Unconj  COMPARISON OF PHARMACOKINETIC PARAMETERS OF 4-ENE VPA IN THE RAT  TABLE 32.  BILE EXTERIORIZED  NORMAL Parameter kj t  c  e  AUC/D  f  a3 R  Cl  100 mg/kg  p-Value'  580±80  380±34  S  a  12+1.7  c f 1 / 2  Vj  20 mg/kg  g m e t  Clj?  a  18+1.5  S  ki t t  20 mg/kg 550±68  c  13±1.8  d  l/2  210±37  320+25  S  vi  390+27  620+53  S  AUC/D  0.47±0.21  0.23+0.12  S  ClR  8.2+0.51  5.7±0.43  S  ci  8.7±0.60  5.9+0.54  S  Cl ° ' c o n j-3  e  f  g  g B  C1 ^  % Dose Excreted in Urine Unconj  Parameter  100 mg/kg  p-Value'  380±60  S  a  19+3.1  S  210±31  350±35  s  310±37  460±60  s  0.18±0.14  0.16±0.06  NS  0.69±0.24  0.40±0.11  s  2.8±0.42  s  7. 4±1.1  s  4.5+1.1 11+1.8  T  a  % Dose Excreted in Urine  5.3+2.3  3.8+1.7  NS  Conj  17±2.1  24+5.7  S  Unconj  Total  22±3.1  28+5.7  S  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  a, Dose; 6, p>0.05=Nonsignificant (NS), % Dose Excreted in Bile o, p < 0.05 Significant (S); Unconj  6.2±2.4  5.5±1.6  NS  c, xlO" min" ; d, min; e, mL/kg; 5  1  Conj  23±6.5  15+2.0  S  Total  29±5.2  21+3.5  S  f, jLtg.min/mL.mg; g , mL/min.kg Each value is a mean of 6 observations + SD  87  TABLE 33. 4-Ene Cone {ng/ml)  PLASMA PROTEIN BINDING OF 4-ENE VPA  Ultrafiltrate Cone {ng/ml)  Bound Cone {ng/ml)  % Bound  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  metabolites (I and II)  urine  and bile  of  the  rat.  Only  two diene VPA  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.  D.l.  DISCUSSION  CHEMISTRY The 3-hydroxy VPA, VPA and 4-ene VPA were prepared by the treatment  of  an alkylcarboxylic  acid with  (LDA), to generate dianions derivative  such  as  alkyl  strong  base,  lithium diisopropylamide  which on alkylation with appropriate alkyl  halide  or aldehyde  aliphatic acid in good yield (Pfeffer et al.,  gave the  alpha-subsituted  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 d i s t i l l a t i o n . 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.,  D.2.  1977).  ASSAY The metabolites (E)-2-ene VPA and 4-ene VPA have been quantitated,  along with other VPA metabolites, al.,  1981b; Kochen et al.,  by GC (Loscher, 1981)  1983; Granneman, et al.,  and GCMS (Nau et  1984c) assay methods.  The major drawbacks of these methods are poor sensitivity  (Loscher, 1981),  long and tedious extraction procedures for sample preparation (Nau et 1981b; Kochen et al.,  1983) and long retention time (Kochen et al.,  al.,  1983).  A capillary GCMS procedure has been used to measure  4-ene VPA as  trimethylsilyl  (E)-2-ene VPA has  derivative  (Rettenmeier  et  al.,  1985).  also been determined by GCMS using chemical ionization (Granneman et 1984c; Schobben et  al.,  1980a). Abbott et  al.  (1986) have  its  al.,  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 i n i t i a l 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  (dimethylaminopyridine) were  of  a  added to  strongly  the  basic  reagent  catalyst,  solution.  DMAP  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 project.  Not long after  that,  of the metabolites  studied  in  this  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  and  extraneous  of  reducing the  substances.  chances  of  interference  by endogenous  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  hydrolysis. Dickinson et  al.  (1984) have reported that glucuronide ester  conjugates of VPA undergo rearrangement to resistant hydrolyze on medium,  incubation  however,  with  various  kinds  glucuronide and sulfate esters, Dickinson et al.,  the of  enzyme,  ensure  forms, which do not  /J-glucuronidase.  conjugate  complete  esters  of  In  alkaline  VPA, including  are hydrolyzed (Dickinson et  al.,  1984;  1979a).  The salient features of this assay method are that i t is an extremely simple  procedure  sample,  that  involves  a single  extraction  unlike the long procedure of Nau et  efficiency  is  virtually 100%.  There is  al.  of  the  biological  (1981). The extraction  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.,  D.3.  1987).  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, i n i t i a l l y , rapidly cleared from the plasma within the f i r s t 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.  showed a non-linear  Plasma Profile: Both (E)-2-ene VPA and 4-ene VPA  plasma decline  whereas VPA exhibited  at  concentrations  similar non-linear decline  lig/ml (Dickinson et al.,  above  200 /ig/mL,  at plasma levels > 100  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  therefore,  in every animal  and was,  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. was 23 min during the  first  The plasma t j /  hour following  the  2  of (E)-2-ene VPA  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 ^1/2' ^  r o m  1 2  t  o  1 8  min  of 1.5  fold in the  > 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)-2ene 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.,  extensively  1986)  are  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;  xenobiotic) systemic  is  Hill  and Drasar 1975). The unconjugated moiety  then  reabsorbed  circulation  (Klaassen  through  the  and Watkins  intestinal 1984).  wall  (drug or into  the  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. the  pharmacokinetic  compartment  models  Pharmacokinetic Modelling: In the early stages of  modelling were  (Harrison and Gibaldi  for  proposed  drugs to  undergoing  describe  their  EHC, simple plasma  1976; Chen and Gross 1979), especially  two  profiles  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,  compartments"  before  enters  entering  into 3-5 the  serially connected  blood  compartment.  "intestinal  Such  a model  essentially adds to the time required for the recirculating drug to reach the  systemic  circulation.  investigators (1982),  A  second  approach  was  including Veng Pedersen and Miller  Col burn  (1984),  and more recently  According to this modelling technique, introduced in the  solution  taken  by  other  (1980), Steimer et  by Shepard et  al.  (1985).  a continuous time-lag function  to the differential  provide the freedom to alter the time-lag  equations.  (tau),  al.  is  These models  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  a n 1 2  d ^21  w  e  r  clearance ( C l Vj.(kj  0  + k ) 12  e  u s e c  n e t  t  '  t  o  determine the effective  clearance (Cl ff) and net  ) as reported by Colburn (1984). C l o  e  e f f  may be calculated as  describe the intrinsic ability of liver to remove (E)-2-  95 ene VPA or 4-ene VPA from the  blood.  Cl  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 f f for (E)-2-ene VPA was 6.9 and for 4-ene e  VPA, 11 mL/min.kg. The C l f f e  normal rats,  values,  calculated from the plasma data of  are close to C l j in bile-exteriorized rats,  that the model-generated  values  for microconstants  which suggests  are f a i r l y 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. parameters different  of  (E)-2-ene  from those  Pharmacokinetic Parameters: The pharmacokinetic VPA in the  reported  rat determined  by O'Connor  et  al.  in  this  study  (1986)  in  are  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 C l j at the low doses are close to each other  (4.9  versus 4.0 mL/min.kg). However, the effect  dose is opposite explain  due  to  in these two studies. the  experimental design, blood collection.  lack  of  of increasing the  This discrepency is  information  in  their  not easy to  abstract  on  their  the schedule for blood sampling and the duration of  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;  substantial  Ogiso et  al.,  1986)  have shown that  VPA in fact  EHC in the rat. Therefore, the elimination half-life  undergoes (4.6 h)  and V (/S), 657 mL/kg for VPA as calculated by Loscher (1978) are different d  from the pharmacokinetic parameters such as Vj (143 mL/kg) of Ogiso et (1986).  Similarly O'Connor  et  al.  (1986)  may have  used  a  al.  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 C1 in the rat (4.9 mL/min.kg), T  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). C1 in the rat at the low dose of 4T  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 al.,  identical to that reported for the monkey (Rettenmeier  et  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 halfl i f e 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 (Dickinson et al., excretion  of  saturation  from 4 to  the  dose was raised  10 times  1979a). These observations suggest that as the biliary  VPA or one of  at  15%, when the  high  its  dose,  monounsaturated metabolites urinary  elimination  is  approaches  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.,  D.3.2.3. 4-ene  VPA seems to  1972a).  Conjugation: The conjugation of (E)-2-ene VPA and  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)  conjugates in the bile of the rat (Dickinson et al.,  was recovered as  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., active  1978), are transported into hepatocytes by carrier-mediated  transport  mechanisms.  monounsaturated metabolites difference  in the  hypothesis  is  also  A  reduced  transport  uptake  compared to VPA may be responsible  extent of  conjugation  supported by the  investigators. Rettenmeier et al.  amongst  experiments  of  for this  these compounds. carried  out  the  This  by other  (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 The  enzyme  UDP-glucuronyltransferase  substrate-specific  (UDPGT)  exists  in  affinity.  two  separate  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, in bile-exteriorized rats. This increase versus 79% of the dose) is significant slight  in the conjugation of VPA (62%  (Dickinson et al.,  (53% versus 57% of the dose) for  also increased  (E)-2-ene  1979a) and only  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. those observed  for VPA which has  been  These results are similar to  shown to  induce choleresis  primarily to osmotic activity in the bile (Dickinson et al., and Klaassen  1981).  The bile  flow  rate  increased  due  1982; Watkins  by approximately 19  /iL/piOle of 4-ene VPA excreted in bile. Since this value was greater than the  predicted  increase  pressure alone  in the  bile  flow  (7 /iL//xmole) due to  (Watkins and Klaassen 1981),  either  fluid  osmotic  absorption  is  inhibited in the ductular tract or an electrolyte  transport mechanism is  stimulated  these  by  unknown  mechanism.  In  addition,  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., VPA may also  contribute to the total  choleretic  1985) and (E)-2-ene  effect  seen after  the  administration of monounsaturated metabolites. After an i n i t i a l  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.  reported for VPA-induced choleresis  A similar observation was  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 ). The values of V for d  (E)-2-ene VPA and 4-ene VPA were, respectively,  d  -220 and 210 mL/kg, which  are almost half the V of 430 mL/kg for VPA in the rat receiving a dose of d  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 AUC  n o r m a l  .  AUCu  )  i  |  e  _  e  x  t  e  r  i  o  r  i  z  e  d  from the  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  1977), but is only 63% bound in the rat (Loscher 1978).  al.,  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 carryover 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 to  be metabolized.  It  metabolized to an active  is  possible (or toxic)  that  (E)-2-ene  metabolite.  expected  VPA may be further  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 VPA should be expected  even after  its  (E)-2-ene  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  (Loscher 1978)  4-ene VPA is  cleared  1.4  times as  fast  from the plasma of the rat (5.9 versus 4.2 mL/min.kg). The  plasma elimination of 4-ene VPA is dose-dependent levels required to show dose dependency  in the rat. The plasma  (>200 jug/mL) are two orders of  magnitude higher than 4-ene VPA levels (<lug/mL) seen in patients therapy.  It  as VPA  is,  therefore,  unlikely that  4-ene VPA is  on VPA  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 ene  VPA has  observed metabolite  in  been  documented  normal  rats  were abolished  in  circulation of (E)-2-ene VPA and 4the  following  rat. an  Secondary  IV bolus  in bile-exteriorized  plasma  dose  rats,  of  peaks either  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. 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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 CDC1 . 3  Appendix 4. NMR (80 MHz) spectrum of 4-ene VPA in CDC1  133  APPENDIX 5. PLASMA LEVELS OF (E) -2-ENE VPA IN NORMAL RATS, jig, (DOSE = 20 mg/kg) 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) -2-ENE VPA IN NORMAL RATS, (DOSE = 100 mg/kg) Time (min)  3b  4b  Mean  460  480  390  430  390  390  360  380  lb  2b  0*  410  5  370  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.  Time (min)  PLASMA LEVELS OF 4-ENE VPA IN NORMAL RATS, /zg/mL (DOSE = 20 mg/kg)  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  _  _  _  _  0.9  *  0.7  , extrapolated to time zero; na, plasma sample not available.  136  APPENDIX 8.  Time (min)  -ENE VPA IN NORMAL RATS, ng/ml PLASMA LEVELS OF 4(DOSE = 100 mg/kg) Mean + SD  Id  2d  3d  4d  5d  6d  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  16  na  270  na  na  na  na  22  na  na  220  na  na  na  30  180  190  -  230  200  210  35  na  na  160  na  na  na  57  na  na  75  na  na  na  60  71  47  -  68  96  75  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.  270 + 12  190 + 24  72 + 16  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) 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  Time (min)  , extrapolated to time zero.;  _  na  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  -  -  -  4.2  2.0  188  , extrapolated to time zero;  na, plasma sample not available.  

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