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Metabolism and pharmacokinetic studies of valproic acid using stable isotope techniques Zheng, Jiaojiao 1993

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METABOLISM AND PHARMACOKINETIC STUDIES OF VALPROIC ACID USING STABLE ISOTOPE TECHNIQUES by JIAOJIAO ZHENG B.Sc. (Chem.) Zhejiang Normal University, 1983 M.Sc. (Chem.) Shanghai Institute of Materia Medica, 1986  A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE in THE FACULTY OF GRADUATE STUDIES Faculty of Pharmaceutical Sciences (Division of Pharmaceutical Chemistry) We accept this thesis as conforming to the required standard  THE UNIVERSITY OF BRITISH COLUMBIA January 1993 © JIAOJIAO ZHENG, 1993  In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission.  (Signature)  Department of  phad-rri.  The University of British Columbia Vancouver, Canada  Date  ^  DE-6 (2/88)  24 ,  g3  ii  ABSTRACT  Valproic acid (VPA) is an anticonvulsant agent widely used in the treatment of several types of epileptic seizures. The drug is unique within its therapeutic class, in terms of its mechanism of action, its chemical structure, as well as its extensive biotransformation into at least 16 different metabolites. The interest in VPA metabolites has been stimulated by the potential of VPA to produce severe hepatotoxicity. Metabolites 4-ene VPA and 2,4-diene VPA are thought to be responsible for the rare but fatal hepatotoxicity associated with VPA. Thus, methodology to study the pharmacokinetics of VPA metabolites is important to an evaluation of the role that metabolites may play during VPA therapy.  Stable isotope techniques and gas chromatography mass spectrometry (GCMS) have been used in several areas of research on VPA. For example the application of a stable isotope labelled analog as a "pulse dose" in antiepileptic drug studies allows the elimination kinetics of the drug to be determined without discontinuing therapy and risking the exacerbation of seizures. In the present study, [21-10PA and [13C4]VPA were evaluated as to their applicability to pharmacokinetic studies of VPA. Pharmacokinetic parameters of VPA, [21-161VPA were measured in a healthy human volunteer. Potential isotope effects of [2H6JVPA and [13C4]VPA were studied based on the urine recovery ratio or AUC ratio of VPA and its metabolites to their isotope labelled analogs. No apparent isotope effect was found in the metabolism of [13C4]VPA, which makes [13C4]VPA qualified for use in a "pulse dose" manner. Upon [2H6JVPA  iii  administration, a large isotope effect was observed in the metabolic formation of [ 2 4]5-0H VPA and [ 2 H3]2-PGA, and a small isotope effect was apparent for the formation of [ 2 H6](E)-2,4-diene VPA. Based on the latter observation it was proposed that the formation of 2,4-diene VPA might occur partly from 3-ene VPA. (E)- and (Z)-3-ene VPA were synthesized to test this proposal.  The use of stable isotope labelled analogs as internal standards can minimize the variance arising from extraction of VPA metabolites due to a slight pH change or incompleteness of derivatization due to time and temperature. In order to obtain optimal analytical results, eight deuterium labelled VPA metabolites were synthesized as internal standards, which included [ 2 H7]VPA, [ 2 H7]2-ene VPA, [ 2 H7]3-keto VPA, [ 2 H7]3-0H VPA, [ 2 H7]4-ene VPA, [ 2 H7]4-0H VPA, [ 2 H7]4-keto VPA, and [ 2 H7]5-0H VPA. These internal standards were applied to the analysis of VPA, [ 13 C4]VPA and their metabolites in serum and urine samples collected from two nonpregnant sheep following single dose administration of VPA:[ 13 C4]VPA (50:50).^The elimination half-life of VPA in the sheep was estimated to be approximately 2.5-5 hours.  GCMS conditions for both electron ionization (EI) and negative chemical ionization (NCI) were optimized to obtain the best resolution and sensitivity for VPA metabolites. A single temperature program with a run time of 47 min was established for NCI analysis of PFB derivatives of VPA, [ 2 H6]VPA and their metabolites. Two temperature programs were investigated for the EI analysis of t-BDMS derivatives. One run time of  iv  35 min was used for VPA unsaturated metabolites, while a run time of 20 min was used for the more polar metabolites of VPA.  All the urine and serum samples were analyzed with both El and NCI techniques. PFB derivatives of VPA metabolites analyzed by the NCI technique gave higher sensitivity and better resolution than t-BDMS derivatives of VPA metabolites analyzed by El methods. All urine samples were hydrolyzed with glucuronidase and with NaOH solution. No difference was observed between the results obtained with the different hydrolysis methods, which indicated that there was little or no flglucuronidase-resistant conjugate present in the urine samples of this human volunteer after urine samples were kept at -20 °C for about two months. The conjugated fractions were measured, more than 90% of VPA and its unsaturated metabolites were excreted into urine in the form of their glucuronic conjugates. Metabolites 3-0H, 4-0H and 5-0H VPA were excreted partly as glucuronides, while 3-keto, 4-keto VPA, 2-PSA and 2PGA were excreted mostly as the free metabolites.  The present investigation reaffirmed the importance of GCMS assay techniques to studies of VPA pharmacokinetics and disposition. Stable isotope labelled internal standards improved the accuracy and precision of VPA metabolite analysis by GCMS. [ 13 C4JVPA was ideal for "pulse dose" VPA studies of pharmacokinetics, in which techniques it was demonstrated that the pharmacokinetic parameters of VPA metabolites will be obtained for the first time in pediatric patients.  v  TABLE OF CONTENTS  ABSTRACT^  ii  LIST OF TABLES^  ix  LIST OF FIGURES^  xii  LIST OF SCHEMES^  xiv  LIST OF ABBREVIATIONS ^ ACKNOWLEDGEMENT^  xv xviii  1.  Introduction  1  1.1  Overview of VPA  1  1.2  Mechanism of action  2  1.2.1  GABAergic hypothesis of VPA  2  1.2.2  VPA potentiates the postsynaptic response to GABA  4  1.2.3  VPA action on neuronal membrane  4  1.3  Metabolism  5  1.3.1  Conjugated metabolites of VPA  6  1.3.2  fl-Oxidation pathway of VPA administration  9  1.3.3  w-Oxidation and (w-1) oxidation  10  1.4  Pharmacokinetics and pharmacodynamics  11  1.5  Toxicity  14  1.6  Chemical^derivatization and analysis  17  1.7  Stable isotope techniques  18  1.8  Specific objectives  24  2.  Experimental  26  2.1  Chemicals and instrumentation  26  vi  2.1.1  Chemicals and reagents  26  2.1.2  VPA metabolites and internal^standards  27  2.1.3  Nuclear magnetic resonance spectrometry  27  2.1.4  Centrifuges  28  2.1.5  Packed column gas chromatography - mass spectrometry  28  2.1.6  Capillary column gas chromatography - mass spectrometry  29  2.1.7  Mass selective detector (MSD)  30  2.2  Chemistry  30  2.2.1  Synthesis of [ 2 H7]VPA  30  2.2.2  Synthesis of [ 2 H7]4-ene VPA  31  2.2.3  Esterification of [ 2 H7]4-ene VPA  32  2.2.4  Synthesis of [ 2 H7]4-keto VPA  33  2.2.5  Synthesis of [ 2 H7]4-0H VPA  34  2.2.6  Synthesis of [ 2 H7]5-0H VPA  35  2.2.7  Synthesis of [ 2 H7]3-keto VPA  36  2.2.7a  Synthesis of ethyl 3-keto pentanoate  36  2.2.7b  Alkylation of ethyl 3-keto-pentanoate with [ 2 H7]bromopropane  37  2.2.8  Synthesis of [ 2 H7]3-0H VPA  38  2.2.9  Synthesis of [ 2 H7]2-ene VPA  39  2.2.10  Synthesis of (E)-3-ene VPA  40  2.2.11  Synthesis of (Z)-3-ene VPA  41  2.3  Pharmacokinetic studies  42  2.3.1  Pharmacokinetic study with [ 2 H6]VPA  42  2.3.2  Pharmacokinetic study with [ 13 C4]VPA  43  2.3.2a  Human study  43  2.3.2b  Animal^study  43  2.4  Metabolic studies of (E)- and^(Z)-3-ene VPA  44  vi i  2.4.1^Study design^  44  2.4.2^Metabolism of (Z)- and (E)-3-ene VPA^  44  2.5^Calibration curves^  44  2.6^Extraction and derivatization ^  48  2.7^Calculation and data evaluation ^  52  2.7.1^Pharmacokinetic parameters^  52  2.7.2^Isotope effects^  52  2.7.3^Conjugated fraction of VPA and its metabolites in urine samples^  52  2.7.4^Evaluation of data^  53  3.^Results and discussion^  54  3.1^Synthesis of deuterium labelled internal standards ^54 3.1.1^Synthesis of [2H7]VPA^  54  3.1.2^Synthesis of [21-17]4-ene VPA^  59  3.1.3^Synthesis of [ 2 H7]4-keto VPA^  63  3.1.4^Synthesis of [2H7]4-0H VPA^  66  3.1.5^Synthesis of [ 2 H7]5-0H VPA^  69  3.1.6^Synthesis of [ 2 H7]3-keto VPA^  72  3.1.6a Synthesis of ethyl 3-keto pentanoate 72 3.1.6b Alkylation of ethyl 3-keto pentanoate with [2H7]bromopropane 75 3.1.7  Synthesis  of  [2H7]3-0H  VPA  79  3.1.8^Synthesis of (E)-[ 2 H7]2-ene VPA^  82  3.1.9^Stereoselective syntheses of (E)- and (Z)-3-ene VPA ^86 3.2^Optimizing GCMS conditions for the analysis of VPA metabolites in El (t-BDMS derivatives) and NCI (PFB derivatives) modes ^92  viii  3.3  Pharmacokinetics of [ 2 H6]VPA and its metabolites in a healthy volunteer  97  3.4  Isotope effects of [ 2 H6]VPA metabolism  106  3.5  Isotope effects with respect to [ 13 C4]VPA metabolism  111  3.6  Urinary recoveries of VPA and its metabolites  114  3.7  Conjugated fraction of VPA and its metabolites in urine samples  119  3.8  Comparison of analyzing and hydrolyzing methods  123  3.9  A pharmacokinetic study of VPA in sheep using [ 13 C4]VPA  125  3.10  Metabolic studies of (Z)-and (E)-3-ene VPA  132  4.  Summary and conclusions  135  5.  References  138  ix  LIST OF TABLES  Table 1: Stock solution concentrations (ug/mL) used fer the preparation of calibration curves for VPA, [916]VPA and their metabolites. ^  45  Table 2: Stock solution concentrations (ug/mL) used f9K the preparation of calibration curves for VPA, [1C4] VPA and their metabolites. ^  47  Table 3: Mass to charge (m/z) for the internal standards (*), VPA, and VPA metabolites that were used for ion monitoring in the NCI (PFB derivatives) and El (t-BDMS derivatives) mode.^  51  Table 4: List of the negative ions monitored and retention times for the PFB derivatives of VPA, [LHOPA, their metabolites and internal standards (I.S.) for the NCI analysis mode. ^  93  Table 5: Positive ions monitored and the retention times of the t-BDMS derivatives of VPA, [LHOPA, their unsaturated metabolites, and the internal standards (I.S.) in the El analysis mode.^  95  Table 6: Positive ions monitored and the retention times of the t-BDMS derivatives of VPA, [LHOPA, their keto and hydroxyl metabolites and internal standards in the El analysis mode.^  96  Table 7: Linearity of calibration curves for quantitative assays of VPA, VPA metabolites and their [LH7]-labelled analogues which were isolated from urine saples of a human volunteer administered with 700 mg of VPA:[917PPA (50:50) every 12 hours for two and half days.^ 98 Table 8: Linearity of calibration curves for quantitative assays of VPA, VPA metabolites and their [917]-labelled analogues in serum total, serum free and saliva samples of a human volunteer administered with 700 mg of VPA:rH7PPA (50:50) every 12 hours for two and half days. ^  99  Table 9: Pharmacokinetic parameters of VPA(I) and [2H6]VPA(II) measured by NCI technique in serum and saliva samples of one subject under steady state conditions (5 oral doses of 700 mg of VPA : [9161VPA).^  103  Table 10: Pharmacokinetic Parameters of deuterium labelled and unlabeled metabolites of VPA measured in a healthy volunteer under steady state conditions; all data were based on NCI results. ^  105  x  Table 11: Area under curve (AUC) ratios of VPA, VPA metabolites to their deuterium labelled analogs over 12 hours after the final dose in the serum samples of a,healthy volunteer administered 5 doses of 700 mg VPA:[ 62 H ]VPA (50:50); all values were based on NCI results.  108  Table 12: Steady state urinary recovery molar ratio of VPA aqd its metabolites to their deuterium labelled analogs, rHOPA and metabolites in a healthy human volunteer administered 5 doses of 700 mg of VPA:[ H6]VPA (50:50), based on 12 hour urine collected following the final dose*. ^109 Table 13: Metabolic equivalence of [ 13 C4]VPA and VPA based 911 mean TIC peak area ratio of VPA metabolites to their [ 1° C4]labelled analogs (13 serum samples and urine sample collected 3-9 hr after the dose from a healthy human volunteer administered a single dose of 700 mg of VPA: ["C4]VPA were analyzed by NCI techniques).^113 Table 14: Steady state urinary recoveries of VPA, [ 2 H6]VPA and metabolites (free plus conjugated) in the urine collected 12 hr following the final dose , hydrolyzed with NaOH solution, and analyzed by NCI GCMS.^  115  Table 15: Steady state urinary recoveries of VPA, [ 2 H6]VPA and metabolites (free plus conjugated) in a urine sample collected for 12 hours following the final dose, hydrolyzed with glucuronidase and analyzed by NCI GCMS. ^116 Table 16: Steady state urinary recoveries of VPA, [ 2 H6]VPA and metabolites (free plus conjugated, in the urine collected 12 hours following the final dose , hydrolyzed with NaOH solution, and analyzed by EI GCMS.^  117  Table 17: Steady state urinary recoveries of VPA, [ 2 4]VPA and metabolites (free plus conjugated) in the urine collected 12 hours following the final dose*, hydrolyzed with glucuronidase, and analyzed by EI GCMS. ^  118  xi Table 18: Conjugated fraction (%) of VPA and its metabolites in urine samples collected for 12 hours after final dose measured by different hydrolysis and assay methods.  121  Table 19: P-values of paired t-test over different hydrolysis and analysis methods.^  122  Table 20: Correlation coefficients (r 2 , n=11) between concentrations of VPA urine metabolites measured by different hydrolysis (base or enzyme) and analysis (NCI or EI) methods. Urine samples were collected from a human volunteer in the multiple dose study (700 mg of VPA : [ H6]VPA (50:50) every 12 hr for 2.5 days). ^ 124 Table 21: The retention time and m/z values of the (M-57) 4" diagnostic ions of VPA and its metabolites isolated from serum sample ,of sheep dosed with single i.v. dose of T' 1 g of VPACOPA (50:50).^  127  Table 22: Linearity of calibration curves for the quantitative analysis of VPA, VPA metabolites and their C-13 labelled analogues isolated from urine samples 9f sheep dosed i.v. with a single dose of 1000 mg of VPA:[ C4]VPA (50:50). ^130 Table 23: Pharmacokinetic parameter,for VPA(I), [ 13 C4]VPA(II), (E)-2-ene VPA (III) and ["C4](E)-2-ene VPA (IV) measured in two sheep dosed i.v. with a single 1000 mg dose of VPA:[ i3 C4]VPA (50:50); based on serum samples which were analyzed by EI GCMS.^  131  Table 24: Retention time and peak area of monitored ions m/z 199 and 197 which represent parent drug 3-ene VPA and its diene metabolites. 134  xii  LIST OF FIGURES Fig. 1.^Proposed metabolic pathways of VPA in humans. The broken line indicates a likely metabolic route not yet confirmed. The compounds in brackets have also not been confirmed (Kassahun et al., 1989).^  7  Fig. 2:^The structure of 1-0-acyl-P-linked VPA glucuronide. ^8 Fig. 3:^GCMS mass spectra of the methyl esters of [2H7]/PA (top) and VPA (bottom). ^  57  Fig. 4:^1 H NMR spectrum of [2H7]VPA. ^  58  Fig. 5:^GCMS mass spectra of the methyl esters of [2H7]4-ene VPA (top) and 4-ene VPA (bottom).^  61  Fig. 6:^IH NMR spectrum of [ 2 H7]4-ene VPA.^  62  Fig. 7:^GCMS mass spectra of ethyl esters of [2H7]4-keto VPA (top) and 4-keto VPA (bottom).^  65  Fig. 8:^GCMS mass spectra of ethyl esters of [2H7]4-0H VPA (top) and 4-0H VPA (bottom).^  68  Fig. 9:^GCMS mass spectra of the ethyl esters of [2H7]5-0H VPA (top) and 5-0H VPA (bottom).^  71  Fig. 10: GCMS mass spectra of ethyl 3-keto-pentanoate. ^73 Fig. 11: 1 H NMR spectrum of ethyl 3-keto-pentanoate. ^74 Fig. 12: GCMS mass spectra of the ethyl esters of [2H7]3-keto VPA (top) and 3-keto VPA (bottom).^  77  Fig. 13: 111 NMR spectrum of ethyl ester of [2H7]3-keto VPA.^78 Fig. 14: GCMS mass spectra of the ethyl esters of [2H7]3-0H VPA (top) and 3-0H VPA (bottom).^  81  Fig. 15: GCMS mass spectra of the ethyl esters of (E)-[2H7]2-ene VPA (top) and (E)-2-ene VPA (bottom).^  84  Fig. 16: IH NMR spectrum of the ethyl ester of [ 2 H7]2-ene VPA.^85 Fig. 17: GCMS mass spectra of the methyl esters of (E)- (top) and (Z)-3-ene VPA (bottom). ^  89  Fig. 18: 1 H NMR spectrum of (E)-3-ene VPA. ^  90  Fig. 19: IH NMR spectrum of (Z)-3-ene VPA. ^  91  Fig. 20: Elimination curves of VPA and [21-16]VPA in serum total (top), serum free (middle) and saliva (bottom) which were measured with NCI techniques. 101 Fig. 21: Time course (12 hr) of labelled and unlabeled a-oxidation metabolites of VPA.^  104  Fig. 22: SIM chromatograms of,[21-17PPA (top, internal standard), VPA (middle), and [-"COPA (bottom).^  128  x iv  LIST OF SCHEMES Scheme 1: Sample handling procedure for serum (total and free) and saliva samples.^  49  Scheme 2: Sample handling procedure for urine samples. ^50 Scheme 3: Synthesis of [ 2 H7]VPA.^  56  Scheme 4: Synthesis of [ 2 H7]4-ene VPA.^  60  Scheme 5: Synthesis of [ 2 H7]4-keto VPA.^  64  Scheme 6: Synthesis of [ 2 H7]4-0H VPA.^  67  Scheme 7: Synthesis of [ 2 H7]5-0H VPA.^  70  Scheme 8: Synthesis of [ 2 H7]3-keto VPA.^  76  Scheme 9: Synthesis of [ 2 H7]3-0H VPA.^  80  Scheme 10: Synthesis of (E)-[ 2 H7]2-ene VPA.^  83  Scheme 11: Synthesis of (E)-3-ene VPA. ^  88  XV  LIST OF ABBREVIATIONS  AUG^Area Under the Curve BDZ^Benzodiazepines bp^Boiling Point n-BuLi^n-Butyllithium CL^Clearance cm^Centimeter CNS^Central Nervous System CSF^Cerebral Spinal Fluid d^Doublet DBU^1,8-Diazabicyclo[5,4,0]undec-7-ene E^Trans El^Electron Impact Et0H^Ethanol eV^Electron Volt GABA^Gamma Aminobutyric Acid GABA-T^Gamma Aminobutyric Acid Transaminase GAD^Glutaric Acid Decarboxylase GCMS^Gas Chromatography Mass Spectrometry h^Hour HMPA^Hexamethylphosphoramide Hz^Hertz i.d.^Internal Diameter IDMS^Isotope Dilution Mass Spectrometry I.S.^Internal Standard i.v.^Intravenous  xvi  J^Coupling Constant in Hertz KE^Elimination Rate Constant kg^Kilogram L^Litre LiICA^Lithium n-Isopropylcyclohexylamine LDA^Lithium Diisopropylamide m^Multiplet  M^Molarity M +^Molecular Ion Me0H^Methanol mg^Milligram 2-MGA^2-Methylglutaric Acid MHz^Megahertz MIDAS^Michigan Interactive Data analysis System min^Minute mL^Milliliter mmoles^Millimoles MSD^Mass Spectrometry Detector MW^Molecular Weight m/z^Mass/charge NaOH^Sodium hydroxide NCI^Negative Chemical Ionization NMR^Nuclear Magnetic Resonance PFB^Pentafluorobenzyl 2-PGA^2-Propylglutaric Acid 2-PSA^2-Propylsuccinic Acid  q^Quartet  xv ii  SIM^Selective Ion Monitoring SSA-DH^Succinic Semialdehyde Dehydrogenase t^Triplet ti/2^Half Life t-BDMS^tertiary-Butyldimethylsilyl TMS^Trimethylsilyl THF^Tetrahydrofuran TIC^Total Ion Chromatogram U^Unit ug^Microgram uL^Microlitre Vd^Volume of Distribution w^Wide Z^Cis  xix  DEDICATION  To Xudong and my parents  xviii  ACKNOWLEDGEMENT  I sincerely thank Dr. Frank S. Abbott for his generous support and excellent supervision throughout this program. I am grateful to the committee members Dr. James Orr, Dr. Stelvio Bandiera, Dr. James Axelson, and Dr. Kathleen MacLeod for their effort and helpful suggestions. Special thanks go to Mr. R. Burton for his valuable assistance in GCMS and computer work; Mr. D. Yu for his kind help in GCMS analysis of sheep samples. I very much appreciate the assistance from my lab mates, Dr. R. Lee, Dr. K. Kassahun, Mr. A. Borel, Ms. S. Panesar, Mr. J. Palaty and Ms. S. Gopaul.  1  1. Introduction  1.1 Overview of VPA  Valproic acid (VPA) is a relatively new antiepileptic compound whose pharmacological properties were discovered in 1963 (Meunier et  al., 1963). Since its first clinical use in France in 1964 (Carraz, 1964), valproate or VPA has rapidly established itself worldwide as a major antiepileptic drug against several types of seizures. In 1983, VPA was marketed as a syrup and gelatin capsule under the trade name Depakene R . It was soon recognized as a highly effective first line drug against the primary generalized tonic-clonic, and myoclonic seizures. The drug is unique within its therapeutic class, in terms of both its mechanism of action and its chemical structure, and as a consequence, it has been the focus of much basic and applied research (Gram et a7., 1985; Chapman et al., 1982).  In recent years, interest in VPA metabolites has been stimulated by the potential of VPA to produce severe hepatotoxicity (Bohan et al., 1987; Dickinson et al., 1985; Kuhara et al., 1985; Zimmerman et al., 1982). The risk of fatal hepatic dysfunction has been assessed at 1 in 37000 in patients receiving VPA as monotherapy, and as high as 1 in 500 in children younger than 2 years of age who are receiving VPA in combination with other anticonvulsants (Dreifuss et al., 1987).  Although several studies of the pharmacokinetics of VPA and its metabolites in adults have been completed, no pharmacokinetic study of  2  VPA metabolites has been reported for children, the age group most susceptible to severe hepatotoxicity. Developing methodology that could be applied to a study of the pharmacokinetics of VPA metabolites in pediatric patients is one of the objectives of the present study.  1.2 Mechanism of Action  The mechanism of action of VPA is not clear. ^There are three proposed mechanisms of action: ^1) VPA increases brain 7-aminobutyric acid (GABA) (Feriello  et a7., 1983); 2) VPA potentiates the  postsynaptic response to GABA (Macdonald and Bergey, 1979) and 3) VPA exerts a direct membrane effect (Slater and Johnson, 1978). A brief description for each hypothesis is presented as follows.  1.2.1 GABAergic hypothesis of VPA VPA is able to antagonize seizures induced by GABA antagonists, bicuculline and picrotoxin (Frey and Loscher, 1976; Worms and Lloyd, 1981) and seizures induced by inhibitors of GABA synthesis, 3mercaptopropionic acid, isoniazid and allyl-l-glycine (Dren  et al.,  1979). Research indicated that GABA levels in the whole brain of rodents are elevated within 15-60 min of administration of VPA (Schechter  et al., 1978; Perry and Hansen, 1978) and these remain  elevated for 3-8 hours (Nau and Loscher, 1982). Godin (1969) reported that VPA inhibits  in vitro GABA - transaminase (GABA-T), the enzyme in  the first step of GABA degradation. Harry  et 0.(1975) also presented  their finding that VPA is a more potent inhibitor of  in vitro succinic  3  semialdehyde dehydrogenase (SSA-DH), the next enzyme in the GABA degradative pathway. Conversely, the activity of regional (Phillips and Fowler, 1982) and whole brain (Loscher, 1981) glutamic acid decarboxylase (GAD), the GABA synthesizing enzyme, is increased after VPA administration. The inhibited activities of GABA degradation enzymes and the increased GAD activity all result in increased GABA levels.  NH2-CH2-CH2-CH2-COOH  Structure of GABA  In the neuron, GABA is contained in the synaptosomes of nerve terminals as well as in the neuronal metabolic pool of soma and glial cells.^However, only the synaptosomal fraction is involved in neurotransmission.^Therefore, unless a clear distinction is made between the two pools, it is impossible to establish the effectiveness of increased levels of whole brain GABA in increasing GABA-mediated inhibition (Balazs et al., 1970).  Drugs which increase GABA function and elevate convulsant thresholds act as anticonvulsants. The fact that GABA levels in the brain increase after VPA administration has been the basis for the above hypothesis concerning the mechanism of action of this drug. However, the available evidence is still insufficient to make a final assessment of the validity of this hypothesis.  4  1.2.2 VPA potentiates the postsynaptic response to GABA  VPA has been shown to potentiate GABA-mediated postsynaptic inhibition^in^vitro,^which^is^similar^to^the^anticonvulsant benzodiazepines (BDZ) and barbiturates (MacDonald, 1986). The potentiation of GABA by VPA was also observed in the rat cortical neurons in the substantia nigra  (Kerwin et al.,  1980). However, the  concentration of VPA initially used to potentiate GABA response was higher than that seen  in vivo  (Harrison and Simmonds, 1982) and when the  concentration was reduced to reflect serum levels, the results of potentiation could not be repeated. On the basis of  in vitro  binding  studies, Ticku and Davis (1981) suggested that VPA action may be exerted at the picrotoxin binding site of the GABA receptor-chloride ionophore complex in the postsynaptic membrane. However, upon further investigation using a tritiated analogue, no evidence of binding to brain membranes was found (Morre  et al.,  1984).  1.2.3 VPA action on Neuronal Membrane  When VPA is at concentrations 15 to 50 times higher than clinical levels, an increase in membrane conductance to e has been observed in the Aplysia neuron, a powerful hyperpolarizing mechanism (Slater and Johnson, 1978). Valproate at "therapeutic" cerebral spinal fluid (CSF) levels limits the depolarization-induced sustained repetitive firing to a few action potentials (McClean and MacDonald, 1986) through a blockage of voltage-sensitive Na+ influx. Similarly, in studying hippocampal slices, Franceschetti  et al  (1986) found that VPA markedly depressed  5  frequency potentiation and paired pulse facilitation. ^VPA also suppressed spontaneous epileptiform activity and prolonged the after discharge elicited by antidromic stimulation (Franceschetti et al., 1986).  From the discussion above, VPA was shown to possess direct membrane effects at clinically obtainable levels. However, the relationship between these direct membrane effects in these test systems and VPA's anticonvulsant effect, although plausible, remain unknown.  Although there is considerable evidence to support each hypothesis, for the mechanism of action of VPA, none of the hypotheses can satisfactorily explain all of its anticonvulsant activity. It is therefore, probable that VPA acts through more than one mechanism in providing its broad anticonvulsant effects.  1.3 Metabolism  The structure of VPA (1) - a branched chain fatty acid, differs dramatically from the substituted heterocyclic ring structure which is common to other anticonvulsants.  CH3-CH2-CH2 CH-COON CH3 - CH2 - CH2  (1)  6  Despite it structural simplicity, the metabolic fate of VPA is complex because of its branched chain structure. This short-chain fatty acid is metabolized in the body by a combination of mitochondrial, microsomal and cytosolic enzymes to produce at least sixteen known metabolites (Gugler et al., 1980; Acheampong et al., 1983, 1985; Kassahun et al., 1989; Rettie et al., 1987). In mammals, the fate of VPA is mainly hepatic metabolism since only 1-3% of the dose is excreted unchanged in the urine (Gugler et al., 1980; Bailer et al., 1985). The major metabolic pathways of VPA include conjugation of VPA with glucuronic acid, a-oxidation, w-oxidation and (w-1)-oxidation. A series of unsaturated, hydroxyl and glucuronic acid conjugated metabolites are formed. Figure 1 summarizes the metabolic pathways of VPA in human (Kassahun et al., 1989).  1.3.1 Conjugated metabolites of VPA Direct conjugation^of valproate with glucuronic ^acid^is quantitatively the most important route of valproate biotransformation. Metabolism of valproate is dose-dependent, and, at least in humans and rats, glucuronidation accounts for progressively more of a dose as the dose or blood concentration increases (Dickinson et al., 1979; Granneman et al., 1984). In humans, glucuronidation of VPA varies between 20-70% of recovered dose (Abbott et al., 1986; Chapman et al., 1982). Dickinson et al. (1989) reported VPA glucuronide conjugation accounts for 59.3 + 25.6 % of VPA dose, based on a study in 24 epileptic patients  7  4,4'-diene VPA  ^  4-ene VPA  ^  VPA^3-keto-4-ene VPA  00H  00H  00-OW  z  VPA  VPA glucuronide  VPA^  3-ene  VPA  (E,E)-2,3'-diene  CIOH^ 00H  5-0H  (E)-2.4-diene  4-OH VPA  N  1  //  00H  51:7\  (E)-2-ene  VPA  VPA^(Z)-2-ene  VPA  4'-keto-2-ene  VPA  1 00H  00H  HOOC OH  2-PGA  4-keto VPA  3-0H  VPA  3-keto  VPA  1 /...\/COOH  COON 2- PMA  HOOC  ZN -I 2-PSA  Fig. 1: Proposed metabolic pathways of VPA in humans. The broken line indicates a likely metabolic route not yet confirmed. The compounds in brackets have also not been confirmed (Kassahun et al., 1989).  8  under steady state conditions. The corresponding conjugate, 1-0-acyl-fllinked ester glucuronide (Figure 2), is excreted into urine. This conjugate is also present at high concentrations in the bile of rats given VPA (Dickinson et al., 1979), and is consistent with the finding that VPA undergoes enterohepatic recycling in rats (Dickinson et a7., 1985a).  CH3-CH2-CH2 CH-CO CH3-CH2-CH2  Fig. 2: The structure of 1-0-acyl-8-linked VPA glucuronide.  It should be noted that glucuronidation also represents an important pathway of biotransformation for primary metabolites of VPA that have been formed by initial oxidative processes (Rettenmeier et al., 1985; 1986a; 1986b). In the case of hydroxylated VPA metabolites, both ether and ester glucuronides can result.  Other minor conjugation routes of VPA metabolism exist. Carnitine conjugates (Bohan et e., 1984) were found in the urine of pediatric patients receiving prolonged administration of the drug. VPA glycine conjugates have been identified in rat urine, together with somewhat greater quantities of glycine conjugates of unsaturated VPA metabolites (Granneman et al., 1984). The existence of a VPA conjugate with  9  coenzyme A as a metabolic intermediate in liver tissue was proposed by the evidence from animal studies (Thurston  et al.,  1983; 1985), even  though rigorous structural characterization of this conjugate has not been reported. A novel metabolite of VPA, 5-(N-acetylcystein-S-y1)3-ene  VPA identified in rat and human urine has been reported by Kassahun  al  et  (1991). In their studies, 5-(glutathion-S-y1)-3-ene VPA was detected  in rat bile following the administration of either (E)-2,4-diene or 4ene VPA, and it was assumed that GSH reacted  in vivo  with an activated  form of the diene, namely with the CoA ester.  1.3.2 B Oxidation pathway of VPA administration -  The second major route of VPA metabolism is 0-oxidation. Metabolites generated keto VPA (Granneman  via  this pathway are 2-ene VPA, 3-0H VPA and 3-  et al.,  1984). It is noteworthy that the structures  of these three metabolites are formally analogous to the sequential intermediates of fatty acid 10-oxidation (Prickett and Baillie, 1984). The available evidence indicates that VPA and endogenous lipids compete for the enzymes of 0-oxidation (Bjorge and Baillie, 1985). In fact, VPA and its metabolites are thought to serve as competitive inhibitors of fatty acid ig-oxidation, which causes the rare but fatal hepatotoxicity.  Recently, stable isotope labelling techniques have been employed to investigate the fl-oxidation pathway of VPA metabolism in the rat. The findings indicated that have demonstrated that 3-0H VPA is not as was originally suspected an exclusive product of 0-oxidation, but has a dual origin  in vivo  being derived largely by direct, cytochrome P450-  10  dependent hydroxylation of the parent drug (Rettenmeier  et al., 1987).  This study also confirmed an earlier observation (Nau and Zierer, 1982) that 3-keto-VPA appeared to be formed mainly by oxidation of 2-ene VPA rather than derived from 3-0H VPA.  Recent studies on mitochondrial metabolism of VPA (Li  et al., 1991,  Bjorge and Baillie, 1991) identified 3-keto VPA together with three  viz. (E)-2-ene VPA, 3-ene VPA and (E,E)-2,3'  unsaturated metabolites,  diene VPA after incubating VPA with freshly isolated rat liver mitochondria. The 3-ene VPA and (E,E)-2,3'-diene VPA were subsequently shown to be metabolites of 2-ene VPA. All three unsaturated metabolites were shown to serve as precursors of 3-keto VPA when incubated with mitochondrial preparations (Bjorge and Baillie, 1991). Metabolite 3-0H VPA was not detected as a metabolite of VPA in this  in vitro system.  However, trace amounts of 3-0H-VPA CoA were detected by HPLC. It was concluded that crotonase catalyzes the hydration of 2-ene-VPA CoA to 3OH-VPA CoA, and that oxidation of the latter species to the corresponding 3-keto metabolite is mediated by a novel NAD + -dependent 3hydroxyacyl-CoA dehydrogenase (Li  et al., 1991).  1.3.3 w Oxidation and (w 1) Oxidation -  -  Products of w-oxidation of VPA are 5-0H VPA, 2-propylglutaric acid (2-PGA) and 2-propylmalonic acid (2-PMA), while (w-1) oxidation results in 4-0H VPA, 4-keto VPA and 2-propylsuccinic acid (2-PSA) (Granneman  al., 1984).  et  11  As in the case of its endogenous counterparts, VPA undergoes hydroxylation at the 4 and 5 position by the action of cytochrome P-450 enzymes (Prickett and Baillie, 1984), mainly in liver tissue but also in other organs. A possible mechanism to account for the formation of 4-0H and 5-0H VPA was proposed. A carbon-centered VPA free radical was formed  via  hydrogen atom abstraction from position 4 or 5 by the  perferryl oxygen of the heme prosthetic group, then followed by recombination of carbon radical-perferric hydroxide radical pairs to yield the isomeric alcohols (Rettie from 4-0H VPA (Granneman  et al.,  et al.,  1987). 4-Keto VPA is formed  1984), and was first identified as a  human metabolite of VPA in a stable isotope "pulse dose" experiment (Acheampong  et al.,  1983).  2-PGA and 2-PSA are believed to arise from further oxidation of 5OH and 4-0H VPA, respectively (Granneman  et al.,  1984).  1.4 Pharmacokinetics and Pharmacodynamics  VPA can be administered by intravenous (I.V.), oral and rectal routes. Among them, the oral route is by far the most widely used. Despite differences in the population (healthy volunteer versus epileptic patients) and formulation (oral solution, immediate release tablet, enteric-coated tablet), the absolute bioavailability of sodium valproate was consistently found to be close to unity (Levy and Shen, 1989). The various forms of oral VPA essentially differ only in the rate of absorption. The peak plasma levels are usually attained within  12  0.5-2 hours after administration of an oral solution or coated tablet of  VPA.  VPA is highly bound (90%) to human plasma albumin at therapeutic concentrations (Levy and Lai, 1982). This property tends to keep most of the drug within the vascular compartment. A value of 0.1-0.4 L/Kg of Vd (volume of distribution) is an indication that the distribution of  VPA is limited to the circulation and rapidly exchangeable extracellular water (Gugler and Von Unruh, 1980).  The clearance of VPA is independent of liver blood flow but is highly dependent on the free fraction. It has been recognized that the blood level-dose relationship for VPA is highly variable between patients. VPA is eliminated almost exclusively by hepatic metabolism (>96% of administered dose, Levy and Shen, 1989). The reported plasma (or metabolic) clearance in healthy volunteers is in the range of 6-11 mL/Kg/h (Levy and Shen, 1989). Children younger than five, 5 to 10 and 10 to 15 years of age have been reported to have mean VPA clearances of 48.3, 39.1 and 24.8 mL/Kg/h respectively (Dodson and Tasch, 1981). The clearance of VPA was found to increase (14.4-16.5 ml/Kg/h) in adults on polytherapy, and is thought to be the result of hepatic enzyme induction (Schappel et al., 1980; Hoffman et al., 1981).  The elimination half-life of VPA in plasma ranges from 8-16 hours in adult epileptics (Gugler and Von Unruh, 1980; Bowdle et al., 1980) and 3-12 hr in children (Cloyd et al., 1983).  13  Besides aging and coadministration of other drugs, pregnancy also affects the clearance of VPA. It was reported (Plasse et al., 1979) that the blood level-to-dose ratio began to decline in the latter part of the second trimester in a pregnant woman and continued through the early part of the third trimester, finally reaching nadir within 3 weeks of delivery. Following parturition, VPA levels rose rapidly and regained pre-pregnancy values within 2-3 weeks. A similar experience in five pregnant patients was cited by Philbert and Dam (1982). Part of the reason for the apparent increase in clearance during late gestation is a decrease in maternal serum protein binding of VPA as a result of elevated nonesterified fatty acids and hypoalbuminemia (Nau and Krauer, 1986).  VPA exhibits several distinct pharmacodynamic characteristics as compared with traditional antiepileptic drugs. The anticonvulsant effect of VPA has been shown repeatedly to correlate poorly with the steady-state serum VPA concentration (Chadwick, 1984; Minns et al., 1982). Also, a striking dissociation between serum VPA concentration and the time course of antiepileptic response has been demonstrated in patients (Rowan, 1979a; 1979b;) and in several experimental models of epilepsy (Lockard and Levy, 1976; Pellegrini et al., 1978; Walter et  al., 1980).  Notably, maximal anticonvulsant effect is usually not observed during initial drug therapy until sometime after the attainment of steady-state serum VPA concentrations. In addition, following discontinuation of VPA administration, seizure control persists long  14  after the intact drug has been cleared from the systemic circulation (Lockard and Levy, 1976; Harding et al., 1978). Since VPA is extensively metabolized by the liver, one explanation for the above phenomena is that one or more of its metabolites may contribute significantly to the antiepileptic action of the drug and that these metabolites are eliminated more slowly than the parent drug.  In fact, the unsaturated metabolites 2-ene VPA, 3-ene VPA, 4-ene VPA (Loscher, 1981; Loscher et al., 1985) and 2,3'-diene (Abbott et al., 1988) were found to have significant anticonvulsant activity in rodent models.  1.5 Toxicity  Adverse reactions to VPA may be divided into physiological or doserelated side effects, metabolic effects, and unusual or rare drug reactions. Idiosyncratic reactions and effects other than those due to the pharmacology of the drug may be mediated by the formation of unusual or novel metabolites. Altered target organ responses may also result from genetic abnormalities. Finally, teratogenicity represents an adverse drug reaction which, in the case of valproate, appears to be dose-related (Jaeger-Roman et al., 1986; Diliberti, et al., 1984).  Among the idiosyncratic side effects of VPA, hepatotoxicity draws the most attention. A comprehensive, retrospective analysis of the cases reported in the United States from 1978 to 1984 has provided definitive information on the primary risk for fatal liver failure with  15  valproate treatment (Dreifuss  et al.,  1987). According to this study,  patients at risk are children two years old or younger who receive valproic acid as part of anticonvulsant polypharmacy and who also have other medical problems besides severe epilepsy, e.g., mental retardation, developmental delay, and metabolic disorder. The incidence of hepatotoxicity found is as high as 1 in 500 in pediatric patients with multi- therapy. Outside of this group, the overall risk of fatal hepatic dysfunction with valproate (1 in 12,000 with polytherapy versus 1 in 45,000 with monotherapy) is still higher among patients receiving multiple anticonvulsants. In addition, no cases of hepatic failure were identified among those persons over the age of 10 who were administered valproate monotherapy (Dreifuss  et al.,  1987).  The most common histopathological feature of VPA-induced liver injury is microvesicular steatosis, similar to that produced by the toxic metabolites of hypoglycin A and by 4-pentenoic acid (Rettie  al.,  et  1988) which is a potent inhibitor of mitochondrial fatty acid  metabolism (Corredor et al., 1967). Evidence has been obtained from studies  al.,  in vivo  1984) and  Thurston  (Mortensen, 1980; Mortensen  in vitro  et al.,  (Thurston  et al.,  et al.,  1980; Kesterson  et  1983; Bjorge & Baillie, 1985;  1985) that VPA inhibits the fi-oxidation of endogenous  fatty acids.  Studies in animals have indicated that several metabolites contribute to the toxic effects of VPA. The 4-ene VPA and 2,4-diene VPA metabolites are hepatotoxic in rats (Kesterson  et al.,  1984) and are  thought to be responsible for the rare but fatal hepatotoxicity  16  associated with VPA. These metabolites may cause damage to liver mitochondria, inhibit fatty acid a-oxidation activity and cause accumulation of hepatic lipids (Zimmerman  et al., 1982; Rettenmeier et  al., 1986a). Additional studies with 4-ene VPA served to reinforce the view that this terminal olefin, like 4-pentenoic acid, acts as a mechanism-based irreversible inhibitor of enzymes of the fatty acid /3oxidation complex, and then induces the microvesicular steatosis  in vivo  (Rettenmeier et al., 1985, 1986a). In fact, 4-ene VPA was shown to be the most toxic metabolite of VPA in the rat (Kingsley  et al., 1983).  Since the formation of 4-ene VPA is mediated by cytochrome P450, the prevalence of 4-ene VPA may be increased by the presence of enzyme inducers (Rettie et al., 1987). This may in part be the explanation why patients on polytherapy are at considerably greater risk of developing fatal hepatic dysfunction than patients receiving valproate monotherapy.  Kassahun  et al. (1990) reported the discovery of GSH 3-ene VPA in  rat bile following the administration of either (E)-2,4-diene VPA or 4ene VPA. Since 2,4-diene is considered to be a mitochondrial oxidation product of 4-ene VPA CoA (Rettenmeier  et al., 1985), it is conceivable  then that, by virtue of its electrophilic nature, (E)-2,4-diene VPA CoA may bind to a nucleophilic site of a mitochondrial enzyme. This would account for the potent inhibition of mitochondrial a-oxidation of fatty acids observed for (E)-2,4-diene VPA in rats (Kesterson  et al., 1984).  An alternate mechanism for the hepatotoxicity of the reactive (E)-2,4diene VPA CoA ester, according to Kassahun's study could be the localized depletion of GSH in mitochondria.  17  Since fl-oxidation of VPA would require that the drug be in the form of its CoA thioester (Bjorge and Baillie, 1991; Li et al., 1991), it appears likely that the effects of VPA on the hepatic lipid metabolism are mediated in part by the ability of the drug to sequester limited pools of CoASH, the obligatory cofactor for fl-oxidation.  1.6 Chemical Derivatization and Analysis  Gas chromatography mass spectrometry (GCMS) is widely used for the analysis of VPA and its metabolites. The reported GCMS methods include electron impact (El) of the t-butyldimethylsilyl (t-BDMS) and trimethylsilyl (TMS) derivatives (Abbott, et al., 1986; Nau et al., 1981; Rettenmeier et al., 1986b; 1989; Tatsuhara et al., 1987) and negative chemical ionization (NCI) of pentafluorobenzyl (PFB) derivatives (Abbott et al., 1987; Kassahun et al., 1989). A complete GCMS assay using El techniques for VPA and its metabolites was reported by Abbott et al. (1986). With the El method, t-BDMS derivatives of VPA and its metabolites have advantages over TMS derivatives except for that of 3-0H VPA. When derivatized with t-BDMS, VPA and its metabolites have increased sensitivity in El mode because of the intense (M-57)4fragments formed in contrast to the less intense (M-15)1" fragment from TMS derivatives. While very good sensitivity was obtained for the tBDMS derivatized VPA unsaturated metabolites, problems arise in the analysis of the keto and hydroxy metabolites which can yield either mono- or di- derivatives depending upon the derivatization conditions used. The 3-0H VPA does not derivatize readily under the conditions suitable for derivatizing other metabolites with t-BDMS.  18  NCI GCMS is a very sensitive and specific assay method for the analysis of valproic acid metabolites. The PFB derivatives produce abundant [M-181]  -  ions except for 3-keto VPA which gave an [M-181-0O2]  -  ion. However, when a combination of PFB and TMS (for hydroxy and 3-keto moiety) derivatization was used, 3-keto VPA formed an [M-181]  -  ion which  was practically the only ion present in the mass spectrum. Hydroxy metabolites derivatized by this method gave significant improvement in both peak shape and sensitivity of detection (Kassahun et al., 1989, 1990). The PFB derivatives (NCI mode) proved to be 30-50 times more sensitive than the t-BDMS derivatives under the El mode (Kassahun et al., 1989).  1.7 Stable Isotope Techniques  Stable isotope techniques have been used in different areas of VPA studies including steady-state kinetics (Acheampong et al., 1984), bioavailability studies (Strong et al., 1975), drug interaction studies (Von Unruh et al., 1980), drug metabolism studies (Acheampong et al., 1983), and mechanistic studies of drug metabolism (Rettie et al., 1988; Rettenmeier et al., 1987).  The identification of VPA metabolites is hindered by the chemical lability of some of the metabolites as well as the structural similarity of VPA to endogenous compounds. The use of stable-isotope labelled drugs has been widely applied to facilitate identification of drug metabolites (McMahon et al., 1973; Pohl et al., 1975). Usually an  19  equimolar mixture of unlabeled and stable isotope-labelled drug is administered and biological fluids are examined by mass spectrometry for characteristic ion-doublets indicative of drug-derived metabolic products.  A stable isotope technique was used in this lab (Acheampong et al., 1983) to search for new metabolites of VPA in human serum and urine. GCMS proved useful to clarify the identity of compounds described either as endogenous compounds or as VPA metabolites. In this study, a pulse dose of di-[(3,3,3-2H3)-propy1]-acetic acid ( [2H6}VPA, (2) ) was administered to a human volunteer at steady state serum concentrations of unlabeled VPA. The location of the deuterium on the terminal carbons provided ion doublets in the mass spectra of GC metabolite peaks with mass differences of either three or six amu.  C2H3-CH2-CH2 \  CH-COOH  r2u ru ru 1 ‘- H3-112-'-'12  (2)  Stable isotope technique can also determine changes in the kinetic parameters of a drug in patients on multiple dose therapy (Sullivan et  al., 1975; Kapetanovic et al., 1980). To determine kinetic parameters of an anticonvulsant in patients on multiple dosing or multi-therapy, a 'pulse' dose of stable isotope-labelled drug offers a convenient  20  technique. Application of this method to antiepileptic drug studies allows the elimination kinetics of the drug to be determined without discontinuing therapy and risking the exacerbation of seizures. The labelled drug elimination phase can be followed for 3-4 half-lives during subsequent uninterrupted multiple dosing of unlabeled drug. This technique was applied first by Von Unruh  et al.  (1980) using di-[(2,3'-  2 H2) propyl]-acetic acid to study the elimination kinetics of VPA under steady state conditions in patients on combined antiepileptic drug therapy. Similar methodology was applied by Acheampong  et al.  in which  a human volunteer was given a single dose of di-[(3,3,3- 2 H3)-propy1]acetic acid (Acheampong  et al.,  1984). In the latter case, an isotope  effect was observed for metabolites in the co-oxidation pathway.  A stable isotope-labelled drug which shows a significant biological isotope effect is not appropriate for use in pharmacokinetic studies, particularly when metabolites are to be measured. Thus the position of the label in the drug should be optimal for minimizing isotope effects on metabolic reactions. In the present study, potential isotope effects in the  in vivo  metabolism of [ 13 C4]VPA and [ 2 H6]VPA will be  investigated. If no significant isotope effect is found in [ 13 C4]VPA as expected, [ 13 C4]VPA will be used in pharmacokinetic studies of VPA and its metabolites in pediatric patients. Hence, pharmacokinetic parameters for VPA metabolites will be determined for the first time in children.  Stable isotope methodology, besides providing the identification of new metabolites and determining elimination kinetics under steady state  21  conditions, also represents a powerful technique for studies on the origins of drug metabolites and for the elucidation of complex metabolic inter-relationships in vivo.  VPA or its metabolites are thought to serve as competitive inhibitors of fatty acid fl-oxidation, which causes the rare but fatal hepatotoxicity. It is known that VPA is metabolized to three products (2-ene VPA, 3-0H VPA and 3-keto VPA) whose structures are formally analogous to the sequential intermediates of fatty acid fl-oxidation (Prickett & Baillie, 1984). Rettenmeier and coworkers (1987) attempted to determine whether these three metabolites share a common metabolic origin in vivo and represent products of fl-oxidation activity using stable isotope techniques. In that study, the metabolism of 2-[2H1]VPA (3) and 3,3-[ 2 H2]VPA (4) was compared to unlabeled VPA. When 2-[2H1]VPA was administered to rats, the metabolite 3-0H VPA exhibited partial labeling (60%). This result demonstrated clearly that the 3-0H VPA formed in that experiment was not produced exclusively via fl-oxidation. When [3,3- 2 H2]VPA was given to rats, the deuterium content of 3-keto-VPA was very close to that of 2-ene VPA, which suggested that 3-keto-VPA was formed solely via 2-ene VPA.  CH3 CH2 C2N\  CH3CH2CN\ C2H-COOH CH3C H2CH 2  ^  2 [21.11]VPA (3) -  CH-COOH  ^  CH3 C H2CH2  3-[2H2]VPA (4)  22  Since kinetic isotope effects can be associated with the formation of some metabolites, stable isotope techniques are useful in mechanistic studies of drug metabolism. Rettie and his coworkers (1988) used this technique in studying the formation of 4-ene VPA. The metabolite 4-ene VPA was shown to be the most toxic metabolite of VPA in rat hepatocytes in culture (Kingsley et al., 1983) and to be considerably more potent as an inducer of steatosis in young rats than was the parent drug (Kesterson et al., 1984).  Based on the supposition that a carbon-centered free radical, localized at either C-4 or C-5 is formed before eliminating hydrogen to yield 4-ene VPA (Rettie et al., 1987), the mechanism of the denaturation reaction to form 4-ene was studied by inter- and intra- molecular deuterium isotope effect experiments using deuterium substitutions of VPA at either C-4 or C-5 (Compounds 5, 6, 7, 2, Rettie et al., 1988).  CH3C2H2CH2  CH3C 2 H2CH2 CH-COON ;  CH-COON ru r2u ru /  CH3CH2CH2  %-113k, "2L12  4-[ 2 H2]VPA (5)  C 2 H3CH2CH \  4,4'-[ 2 H4]VPA (6)  C 2 H3CH2CH \ CH-COON ;  CH-COON  CH3CH2CH2  C2H"3'-"2'-"2 CH 2 ru CH I 3 ru  5-[ 2 H3]VPA (7)  5,5'-[ 2 H6]VPA (2)  23  A large primary kinetic isotope effect was obtained for the formation of 4-0H VPA from 4-[ 2 H2]VPA and for the formation of 5-0H VPA from 542H3]VPA. Conversely, some fl-secondary kinetic isotope effects could be observed for the formation of 4-OH-VPA from 5-[2H3]VPA and for the formation of 5-0H VPA from 4-[2H2]VPA. After calculating the isotope effects of 4-ene VPA, 4-0H VPA and 5-0H VPA, the authors found that the formation of 4-ene VPA and 4-0H from hexadeuterated VPA 2 showed a small or no isotope effect, while high isotope effects were observed for 4-ene VPA and 4-0H VPA formed from compound 6. It was then concluded that removal of a hydrogen atom from the subterminal C-4 position of VPA to form a carbon-centered free radical at C-4 is rate limiting in the formation of both 4-ene and 4-0H, and the influence which the second hydrogen/deuterium exerts to form 4-ene on the overall kinetics of the reaction is small. Therefore, it is a carbon-centered free radical, localized at C-4 instead of C-5 that is formed before eliminating hydrogen to yield 4-ene VPA (Rettie et al., 1988).  Another application of stable isotopes is also called isotope dilution mass spectrometry (IDMS). IDMS methods for organic analytes involve spiking a sample with a labelled version of the analyte as an internal standard, processing the sample, and then measuring the ratio of unlabeled to labelled analyte by using GCMS. IDMS has been the technique of choice for definitive methods at the National Institute of Standards and Technology, since it does not depend on sample recovery, shows high precision, and can be tested for bias and unknown interferences.  24  When stable-isotope labelled VPA and its metabolites were used as internal standards, calibration curves with high correlation coefficients and good reproducibilities were obtained (Abbott et al., 1986; Kassahun et al., 1990; Von Unruh et al., 1980). The variance in extraction of VPA metabolites due to a slight pH change or incompleteness of derivatization due to time and temperature can be minimized by using stable isotope-labelled analogs as internal standards.^In GCMS analysis, especially under CI conditions, the spectra depend on source pressure and temperature.^More accurate measurements can be made with a stable isotope-labelled internal standard since the physicochemical properties of the internal standard closely approximate that of the analyte.  1.8 Specific objectives  1.  To investigate potential isotope effects of the metabolism of  [ 2 H6]VPA and [ 13 C4]VPA in a healthy human volunteer.  2.  To determine the pharmacokinetics of [ 2 H6]VPA, and its  metabolites in a healthy volunteer, and compare the pharmacokinetic behavior of [ 2 H6]VPA and its metabolites with those of unlabeled analogs.  3.^The applicability of [ 13 C4]VPA for pulse dose studies of the pharmacokinetics of VPA and metabolites requires the development of a  25  precise and sensitive assay technique. ^This project will focus on evaluating and improving GCMS methods for the assay of labelled and unlabeled VPA metabolites.  4.  As part of the assay development, urine, serum and saliva  samples are to be analyzed using different ionization methods (NCI and El) to compare the sensitivities and reproducibilities of these two GCMS methods. The conjugated fraction of VPA and its metabolites in urine are to be measured by hydrolyzing conjugates with alkali and with glucuronidase, in order to examine if any glucuronidase resistant conjugates exist.  5.  To synthesize stable isotope-labelled internal standards in  order to facilitate the quantitative analysis of VPA, [ 13 C4]VPA and their metabolites by GCMS. The labelled metabolites to be synthesized are: [2117]VPA;^ [2117]2-ene VPA; [2H713-keto VPA;^[2H7]3-OH VPA; [2117]4-ene VPA;^[21-17]5-0H VPA; [21-17]4-01-1 VPA;^[2H7]4-keto VPA  6. To perform preliminary study of the use of stable isotope labelled internal standards in a pharmacokinetics assay of [13C4] VPA and its metabolites in nonpregnant sheep.  26  2. Experimental  2.1 Chemicals and Instrumentation  2.1.1 Chemicals and Reagents  Chemicals were reagent grade and were obtained from the following sources:  Aldrich Chemical Co. (Milwaukee, WI): Butyllithium (1.6 M in hexane), 1,8-diazabicyclo[5,4,0] undec-7-ene (DBU), DiazaldR, diisopropylamine, diisopropylethylamine, hexamethylphosphoramide, methanesulfonyl chloride, triethylamine, (E)-2pentenoic acid, bromopropane, lithium aluminum hydride, propionyl chloride, 4-pentenoic acid, tetrahydrofuran, isopropylcyclohexylamine.  MSD Isotopes (Montreal, Canada): 1,2,3,1'-[ 13 C4]-2-propylpentanoic acid, [ 2 H7] propyl bromide.  BDH Chemicals (Toronto, Ontario): Benzene, chloroform, ether, hydrochloric acid, magnesium sulfate, sodium hydroxide, sodium sulfate, sulfuric acid, hexane.  Caledon Laboratories Ltd. (Georgetown, Ontario): Dichloromethane, ethanol, ethyl acetate.  ICN Pharmaceuticals Inc. (Plainview, NY):  27  Di-n-propylacetic acid (Valproic acid)  2.1.2 VPA metabolites and internal standards  The synthesis of the following VPA metabolites for use as analytical standards has been described elsewhere: 3-ene VPA (stereochemistry undetermined), 4-ene VPA, 3-0H VPA, 4-0H VPA, 5-0H VPA, 3-keto VPA, 4-keto VPA, 2-PSA and 2-PGA (Acheampong et al., 1983); (E,E)-2,3'-diene VPA (Acheampong et al., 1985); (E)-2,4-diene VPA (Lee et al., 1989).  The internal standards used for the assay of VPA, [2H6]VPA and their metabolites were [2H3]2-ene VPA, synthesized by Abbott et al. (1986); [2H3]3-keto VPA, a kind gift from Dr. T.A. Baillie (University of Washington, School of Pharmacy, Seattle, WA); di-n-butylacetic acid; and 2-methylglutaric acid (2-MGA). In both the El and NCI modes, di-nbutylacetic acid was used as the internal standard for VPA and [2H6]VPA; [2H3]3-keto VPA for 3-keto VPA and [24]3-keto VPA; 2-MGA for 2-PSA, 2PGA and their deuterated analogs; [ 2 H3]2-ene VPA for all other metabolites.  2.1.3 Nuclear Magnetic Resonance Spectrometry  High field 1H NMR spectra were obtained on the Bruker WH-400 and Varian XL-300 spectrometers in the Department of Chemistry, University of British Columbia NMR facility. Spectra were acquired in CD3C1 with tetramethylsilane as an internal standard. Carbon-13 NMR spectra were  28  obtained on the Varian XL-300 spectrometer in the Department of Chemistry.  2.1.4 Centrifuges  Unbound drugs were separated from serum samples by the use of ultrafiltration. Protein becomes selectively partitioned into a fraction of the sample volume (retentate), while free ligand passes essentially unhindered through the membrane along with solvent into the ultrafiltrate.^The physical separation does not change free ligand volume or concentration.^Centrifree TM micropartition system (Amicon, W.R. Grace & Co, Danvers, MA) and Beckman centrifuge, model J2-21, equipped with a 45° rotor were used to achieve this purpose. Serum samples were centrifuged at a speed of 3500 rpm for 30 minutes.  2.1.5 Packed Column Gas Chromatography  -  Mass Spectrometry  GCMS analysis of synthesized compounds was performed on a Hewlett Packard 5700A gas chromatograph interfaced to a Varian MAT-111 mass spectrometer using a variable slit separator. Electron impact (EI) data were recorded in scanning mode with a range of 15 - 750 mass units every 5 seconds, and the data were processed using a Packard Bell computer (IBM AT clone) and a program developed in our laboratory. Total ion current (TIC) plots were based on scanning range of m/z 50 - 500.  The instrument was operated with an emission current of 300 uA, ionization of 70 eV, and source pressure of 5 x 10 -6 Torr. The column  29  (1.8 m x 2 mm i.d.) was packed with 3% Dexsil 300 on 100 - 120 mesh Supelcoport. The oven temperature program was 50 - 300 °C at 16 °C/min. Injection port temperature was 250 °C and the separator temperature set at 250 °C. The carrier gas, with a flow of 25 ml/min, was helium.  2.1.6 Capillary Column Gas Chromatography - Mass Spectrometry  The quantitative and qualitative analyses of VPA and its metabolites were performed on a Hewlett Packard 5987A GCMS having an open-split interface. Data recording and processing were managed with a HP-1000 on-line computer. El techniques were used to analyze t-BDMS derivatives with an electron ionization energy of 70 eV. Operating conditions were: source temperature 200 °C, transfer line temperature 240 °C, injection port temperature 240 °C and helium flow-rate 1 ml/min. The capillary column was an OV-1701 bonded phase, 25 m x 0.32 mm i.d., with a film thickness of 0.25 um (Quadrex Scientific, New Haven, CT, U.S.A.). Two temperature programs were utilized for the GCMS, EI analysis. The temperature program used for VPA and its unsaturated metabolites was initiated at 50 °C, programmed at 30 °C/min to 110 °C, held for 18 min before being increased to 260 °C at rate of 8 °C/min. The temperature program used for the remaining polar metabolites of VPA was initiated at 50 °C, programmed to 100 °C at 30 °C/min, then increased to 250 °C at rate of 8 °C/min.  In the negative ion chemical ionization (NICI) mode, ultra-high purity methane was used as the reagent gas. The capillary column used was DB-1 (25m x 0.25mm, with a film thickness of 0.25 um, J&W  30  Scientific, Rancho Cordora, CA, U.S.A.). The source pressure was 0.8 1.2 Torr, ionization energy 200 eV, and the emission current 250 uA. The instrument was programmed for SIM as well. The initial oven temperature was 50 °C, programmed to 140 °C, held at 140 °C for 20 min before being increased to 260 °C at 5 °C/min.  2.1.7 Mass Selective Detector (MSD)  All samples obtained from the VPA study in sheep were extracted, derivatized with t-BDMS and analyzed with a HP 5890 GC interfaced to a HP 5971A MSD using EI mode. Data recording and processing were managed with a HP VECTRA R 486 data system. Operation conditions were: injection temperature 250 °C, ion source temperature 180 °C, GCMS interface temperature 280 °C, and helium flow-rate 1 ml/min. The capillary column used was DB-1701 (30m x 0.25mm, with a film thickness of 0.25 um, J&W Scientific, Rancho Cordora, CA, U.S.A.). The initial oven temperature was 80 °C, programmed to 100 °C at rate of 10 °C/min, then to 130 °C at 2 °C/min, and finally to 260 °C at 30 °C/min and held for 8 minutes.  2.2 Chemistry:  2.2.1 Synthesis of ( 2H71VPA  In a dry 250 ml 3-neck flask equipped with a mechanical stirrer, drying tube, dropping funnel, and reflux condenser, n-butyllithium in hexane (30 mL of 1.6 M, 48 mmol) was added dropwise to a solution of diisopropylamine (6.8 mL, 48 mmol distilled over CaH2) in 40 mL of THE  31  (distilled over L1A1H4) at 0 °C under N2 atmosphere and stirred for another 30 minutes. Distilled valeric acid (2.45 mL, 23 mmol) in 10 mL of THF was added dropwise so that the temperature of the reaction never exceeded 5 °C. The solution at first became milky, but when 5 mL of HMPA was added the solution became clear. The reaction was allowed to proceed for 30 min before [2H7] bromopropane (18 mmol) was added to the mixture. The ice/water bath was then removed and the mixture stirred at room temperature for 3 h. The reaction was again cooled in ice and quenched with 15% HC1 (cooled) until a pH of 1 was attained. The mixture was extracted with diethyl ether (50 mL x 3), the ethereal layer washed with 10% HC1 and water, and then dried over anhydrous Na2SO4. The solvent was removed by flash evaporation and the residue was fractionally distilled (110-113 °C/10 mm Hg) to afford 1.2 g of pure [2H7] VPA (yield 44%). A small portion of the sample was derivatized with diazomethane (Levitt, 1973) for GCMS analysis. GCMS mass spectrum of the methyl ester of [ 2 H7]VPA, m/z(%): 89(100), 123(30), 117(28), 61(23), 134(5), 106(5).  1H NMR of [2H7]VPA (CDC13): 6 0.93 (t, 3H, CH3), 1.37 (m, 2H, CH3CH2), 1.46-1.62 (m, 2H, CH2CH), 2.39 (dd, 1H, CHCOOH), 8.0 (w, 1H, COOH).  2.2.2 Synthesis of [2H7]4-ene VPA  In a dry 500 mL 3-necked flask equipped with a dropping funnel, condenser, mechanical stirrer, and drying tube, 90 mL of n-butyllithium (0.144 mol) in hexane was added dropwise to a solution of  32  diisopropylamine (20.2 mL, 0.144 mol) in 120 mL of THE (distilled over LiA1H4) in an ice/water bath under N2 atmosphere. ^The mixture was stirred for 30 min.^4-Pentenoic acid (6.75 mL, 0.066 mol) was added dropwise over a period of 15 min, followed by 15 mL of HMPA (0.066 mol). The reaction was then allowed to proceed for 30 min. [ 2 H7] Bromopropane (6.2 g, 0.06 mol) was added and the ice/water bath removed. The mixture was stirred at room temperature for 2.5 hours before being quenched with 15% HC1 solution. The aqueous layer was extracted 3 times with ether and the combined ether extracts dried over anhydrous Na2SO4. The solvent was removed by flash evaporation. A yield of 5.4 gram (77%) of [ 2 H7]4-ene VPA was obtained by fractional distillation (110-120 °C/10 mm Hg). A small portion of the sample was derivatized with diazomethane (Levitt, 1973) for GCMS analysis.  GCMS mass spectrum of the methyl ester of 4-ene [ 2 H7]VPA, m/z(%): 113(100), 59(67), 104(58), 115(37), 132(11), 163(M + , 2).  1 H NMR of 4-ene [ 2 H7]VPA (CDC13): (5 2.28 (m, 1H, CH2CH), 2.38-2.43 (m, 2H, CH2CH), 5.03 (dd, 1H, CH2=CHCH2, J=9 Hz, cis), 5.08 (dd, 1H, CH2=CHCH2, J=15 Hz, trans), 5.78 (m, 1H, CH2=CH), 8.5 (w, 1H, COOH).  2.2.3 Esterification of ( 2H7]4-ene VPA  In a 100 mL round-bottomed flask equipped with Dean-Stark apparatus, drying tube, and a reflux condenser, [ 2 H7]4-ene VPA (3.7 g, 0.027 mol), ethanol (6 mL), concentrated sulfuric acid (0.5 mL), and 12 mL of benzene were refluxed at 90 °C for 46 hours. The organic layer  33  was consecutively washed with saturated NaHCO3 solution and water until the wash was neutral, then dried over anhydrous Na2SO4. A yield of 3.75 g of ethyl [2H7]4-ene VPA was obtained by fractional distillation (80 °C/10 mm Hg, 85% yield).  GCMS mass spectrum of the ethyl ester of [2H7]4-ene VPA, m/z(%): 104(100), 127(88), 59(86), 99(73), 143(17), 189(e, 1).  2.2.4 Synthesis of [2H734-keto VPA  Benzoquinone (1.32 g) and PdC12 ( 0.033 g) were added to a mixture of 38.5 mL of dimethyformamide and 5.5 mL of water in a 50 mL roundbottomed flask equipped with a reflux condenser. To this stirred mixture, ethyl [2H7]4-ene VPA (1.87 g, 0.013 mol) was then added dropwise with a syringe over a period of 10 min and the reaction was allowed to proceed at room temperature for 22 h. The product was then poured into 20 mL of cold 10% HC1 solution and extracted 3 times with ether (80 mL x 3). The combined organic layer was washed three times with 20 mL of 10% NaOH solution, once with saturated NaC1 solution, and was then dried over anhydrous Na2SO4. The solvent was removed by flash evaporation and the residue was fractionally distilled to afford 1.18 g of pure ethyl [ 2 H7]4-keto VPA ( 110-115 °C/10mm Hg, 60% yield).  To 0.54 mL of ethyl [2H7]4-keto VPA in a 25 ml round-bottomed flask, 1.55 g of NaOH in 6 mL of H20 and 5 mL of CH3OH was added and heated at 50 °C for 4 h. ^Using an oil bath at 120°C, methanol and ethanol were distilled off at atmospheric pressure.^The remaining  34  aqueous solution was washed with ether (3 x 20 mL) to extract any unhydrolyzed ethyl ester. The remaining aqueous solution was then acidified to pH 2 with diluted HC1 and extracted with ether three times (3 x 40 mL).^The combined ethereal layers were dried over anhydrous Na2SO4.^The ether was evaporated under flash evaporation and the residue was further dried under vacuum (0.5 mm Hg) for 3 hours. [ 2 H7]4keto VPA (0.43 g) was obtained (93% yield). A small portion of the sample was derivatized with diazomethane (Levitt, 1973) for GCMS analysis.  GCMS mass spectrum of the methyl ester of [ 2 H714-keto VPA, m/z(%): 43(100), 122(88), 87(40), 89(33) 75(28), 103(20), 148(17), 164(2), 179(M + ,1).  2.2.5 Synthesis of 2H7]4-0H VPA [  To a well stirred mixture of 170 mg of NaBH4 (4.47 mmol) in 8 mL of ethanol, 0.64 g (3.3 mmol) of ethyl [ 2 H7]4-keto VPA was added dropwise over a period of 10 min. The reaction was allowed to proceed for 60 min at room temperature, and then quenched with saturated NH4C1 solution. The aqueous solution was extracted 3 times (3 x 20 mL) with ether and the combined ethereal layers were dried over anhydrous Na2SO4.^The solvent was removed under flash evaporation.^The residue was fractionalized by distillation (115-118 °C / 8 mm Hg ) and 170 mg (yield 26%) of ethyl [ 2 H7]4-0H VPA was then obtained.  35  GCMS mass spectrum of ethyl [2H7]4-0H VPA, m/z(%): 101(100), 43(45), 134(8), 116(5), 151(2).  Ethyl [2H7]4-0H VPA (170 mg), 0.75 mL methanol and 0.3 g of NaOH in 3 mL of H20 were heated at 50 °C for 2 hours. After being cooled, the solution was washed with ether to remove any unhydrolyzed ester.  2.2.6 Synthesis of [2H7] 5-0H VPA To 0.75 g of ethyl [2H7]4-ene VPA (4.1 mmol) in dry THF in a 50 mL three neck round-bottomed flask cooled to 4 °C with ice. Under N2, 1.5 mL of borane - THE was added dropwise. After the mixture was stirred at room temperature for 30 min, the flask was immersed into an ice/water bath and 100 gl of water was added to destroy excess hydride. To this was added after 5 min, 4 mL of 1.5 N NaOH followed with 0.5 mL of 30 % H202. The reaction temperature was maintained at 40-50 °C for 1 h. The reaction was then quenched by pouring into 20 mL of ice/water.^The solution was extracted 3 times with 30 mL of ether. ^The combined ethereal layer was washed with water followed by saturated NaC1 solution and then dried over anhydrous Na2SO4.^The solvent was removed under flash evaporation.^The residue was purified by flash column chromatography (Rettenmeier  et al., 1985) using a mobile phase of 5%  methanol in ethyl acetate. A yield of 150 mg (19%) of ethyl [2H7]5-0H VPA was obtained.  GCMS mass spectrum of the ethyl ester of [2H7]5-0H VPA, m/z(%): 101(100), 57(28), 115(12), 150(7), 196(M+, 1).  36  2  Ethyl [ H7]5-0H VPA (150 mg) was hydrolyzed by being heated with 1 mL of methanol and 0.35 g of NaOH in 4 mL of H2O at 60  °C  for 4 hours.  After cooled, the solution was washed with ether to remove any unhydrolyzed ester.  2.2.7 Synthesis of [2H733-keto VPA  2.2.7a. Synthesis of ethyl 3-keto pentanoate  In a 1000 mL 3-necked flask equipped with a dropping funnel and mechanical stirrer, isopropylcyclohexylamine (40 mL) and THE (250 mL) under N2 atmosphere were cooled to 0  °C  with ice. N-butyllithium (150  mL, 0.24 mol, 1.6 M in Hexane) was added dropwise over a period of 10 min. The mixture was stirred for another 20 min before immersing the flask into a dry ice/acetone bath (-78 °C). Ethyl acetate (11 mL, 0.12 mol) dried over Na2SO4 was then added dropwise over a period of 10 min followed by propionyl chloride (9.3 mL). The reaction was allowed to proceed for an additional 15 min before being quenched by adding 4 N HC1 slowly to a pH of 2. The product was extracted 3 times (100 mL x 3) with ether. The ethereal layer was washed with NaHCO3 solution and then dried over anhydrous Na2SO4. The solvent was removed under vacuum using flash evaporation. The residue was fractionally distilled to afford 9.5 g of ethyl 3-keto pentanoate (70 °C/5 mm Hg, yield 55%).  GCMS mass spectrum of ethyl 3-keto pentanoate, m/z(%): 57(100),  +  29(95), 43(39), 98(14), 115(12.5), 144(1%1 ,12).  37  300 MHz 1H-NMR of ethyl 3-keto pentanoate (CDC13): 6 1.1(t, 3H, CH3-CH2), 1.3(t, 3H, CH3-CH20), 2.6(q, 2H, CH3CH2C0), 3.45(s, 2H, COCH2CO2), 4.2(q, 2H, CH20).  2.2.76 Alkylation of ethyl 3 keto pentanoate with [2H7] bromopropane -  -  Sodium metal (1.4 g, 0.06 mol) cut into small pieces was placed in a 50 mL 3-necked flask equipped with a dropping funnel filled with ethanol (20 mL) and protected from moisture with drying tubes. A wet towel was kept in readiness to control the vigor of the subsequent reaction. Absolute ethanol (10 mL) dried over Mg was added to the sodium producing a vigorous reaction. As the reaction subsides, more alcohol was introduced to maintain vigorous, but controllable refluxing. In this manner, most of the Na reacted rapidly. Finally the remainder of ethanol was added and the mixture refluxed by a heating mantle until the Na had reacted completely. Ethyl 3-keto pentanoate (8.6 g, 0.06 mol) was added dropwise over a period of 10 min, and the mixture was stirred and refluxed for another 20 min. [ 2 H7] Bromopropane (7 g, 0.054 mol) was then introduced dropwise, and the reaction allowed to reflux for 7 hours. The solution was filtered to remove the sodium bromide after cooling. The filtered NaBr was washed with ethanol. The ethanol solutions were combined and the ethanol was evaporated by flash evaporation. Water was added to the residue and extracted with ether 4 times (4 x 50 mL). The combined organic layer was dried over anhydrous Na2504 over night. The ether was removed under flash evaporation and  38  the residue fractionally distilled to yield 8.2 gram of ethyl [ 2 H7]3keto VPA (75-80 °C /3 mm Hg, 78% yield).  GCMS of ethyl [ 2 H7]3-keto VPA, m/z(%): 57(100), 103(42), 137(18), 145(9), 164(3), 193(M + ,1).  1 11 NMR of ethyl [ 2 H7]3-keto VPA (CDC13): 6 1.15(t, 3H, CH3CH2), 1.26(t, 3H, CH3CH20), 2.55(m, 2H, CH3CH2), 3.42(d, 1H, COCHCO2), 4.18(q, 2H, CH3CH2O).  Ethyl [ 2 H7]3-keto VPA (60 mg) was hydrolyzed by stirring with 1 mL of 3N NaOH and 0.4 mL of methanol at room temperature for two hours. Unhydrolyzed ester was extracted with hexane.  2.2.8 Synthesis of [2H7] 3-0H VPA In a dry 100 mL flask, ethyl [ 2 H7]3-keto VPA (5 g, 0.026 mol) was added dropwise to the well mixed NaBH4 (1.425 g 0.0375 mol) in 60 mL of ethanol. The reaction was then allowed to proceed with stirring for 1 hour before being quenched with saturated NH4C1. The solution was extracted 5 times (5 x 50 mL) with ether, and the ethereal layer dried over anhydrous Na2SO4. The solvent was removed under flash evaporation and the residue further dried under vacuum (0.5 mm Hg). The reaction was found to be quantitative with 4.75 g (94% yield) of ethyl [ 2 H7]3-0H VPA being obtained.  39  GCMS of the ethyl ester of [2H7] 3-0H VPA, m/z(%): 103(100), 75(52), 120(37),137(30), 166(25), 150(17), 196(M+, 1).  Ethyl [2H7]3-0H VPA (45 mg) was hydrolyzed by refluxing with lmL 3N NaOH and 0.6 mL methanol at 50 °C for two hours. Unreacted ester was extracted with 3 mL of hexane. The aqueous solution was then acidified with 4 N HC1 solution to pH of 2 and extracted with ether (5mL x 3). The ethereal solution was then evaporated under flash evaporation to afford [2H7]3-0H VPA.  2.2.9 Synthesis of [2H7] 2-ene VPA In a 50 mL flask equipped with a mechanical stirrer, ethyl [2H7]3OH VPA (1.5g, 7.7 mmol), triethylamine (1.34 mL, 9.5 mmol) and dichloromethane (15 mL) were cooled to 0 °C in an ice bath. Methanesulfonyl chloride (0.83 mL, 9.9 mmol) in dichloromethane was cooled to 0 °C and added dropwise to the stirred mixture. The mixture was stirred for a further 60 min at room temperature. A few mL of ether were added to the mixture to precipitate the salt. The mixture was then filtered and the solvents removed by flash evaporation. The residue was reconstituted in 20 mL of dry THE, a solution of DBU (1,8-diazabicyclo [5.4.0] undec-7-ene, 1.43 mL, 9.5 mmol) was added and the contents gently refluxed for 3 hours. The reaction was quenched with water and the aqueous layer extracted 3 times (3 x 30 mL) with ether. The organic layer was washed first with 1M HC1, then with 1M NaOH solution and finally dried over anhydrous Na2SO4.  40  The product was purified by flash column chromatography (Rettenmeier et al., 1985) using a mobile phase of 2-5% ethyl acetate in hexane. A yield of 260 mg of ethyl [ 2 H7]2-ene VPA was obtained (19% yield). The product was confirmed with I H NMR to be mostly E isomer with a small portion of Z isomer.  GCMS of ethyl [ 2 H7]2-ene VPA, m/z(%): 115(100), 57(95), 132(87), 177(M + , 82), 97(42), 104(40), 148(37).  1 11 NMR of ethyl [ 2 H7]2-ene VPA (CDC13): 1.06(t, 3H, CH3CH2), 1.3(t, 3H, CH3CH2O), 2.2(m, 2H, CH3CH2), 4.2(q, 2H, CH3CH20), 6.73(t, 1H, CH=C).  Ethyl [ 2 H7]2-ene VPA (50 mg) was hydrolyzed by heating with 1 mL of 3N NaOH and 0.3 mL of methanol at 80 °C for 20 hours.  2.2.10 Synthesis of (E)- 3-ene VPA  A flame-dried 250 mL three-necked flask equipped with a graduated separatory funnel and mechanical stirrer was flushed with N2 and immersed in an ice-water bath. Diisopropylamine (5.6 mL, 0.04 mol) and 40 mL of THE were placed in the flask, 25 mL of n-butyllithium (1.6 M in hexane, 0.04 mol) was added dropwise from the funnel. The mixture was allowed to stir for 20 min. The flask was then immersed in a dry ice/acetone bath (-78 °C), HMPA (7.9 mL, 0.044 mol) was added and the mixture was stirred for 30 min. Ethyl (Z)-2-pentenoate (5.7 mL, 0.039 mol) was added to the mixture dropwise over a period of 15 min, followed  41  by dropwise addition of bromopropane (4.4 mL, 0.048 mol). ^After stirring for 30 min, the reaction was quenched with 15% HC1 solution to a pH of 2. The aqueous layer was extracted twice with 20 mL of ether. The combined organic layer was washed with saturated NaHCO3 solution, with water, and then dried over anhydrous Na2SO4. The ester was then hydrolyzed by heating with 8 mL of 3N NaOH solution and 2 mL of methanol at 50 °C for 2 hours. Unhydrolyzed ethyl ester was extracted with hexane, and ethanol and methanol were distilled under atmospheric pressure. The aqueous solution was then acidified with 4N HC1 solution to a pH of 1-2, and extracted three times (3 x 50 ml) with ether. Ether was removed under flash evaporation. The residue was fractionally distilled to afford 1 g of pure (E)-3-ene VPA (85 °C/1 mm Hg, yield 18%).  GCMS of the methyl ester of (E)-3-ene VPA, m/z(%): 55(100), 97(25), 113(18), 127(7), 156(M+,2).  1H NMR of (E)-3-ene VPA (CDC13): 6 0.9(t, 3H, CH3CH2), 1.34(m, 2H, CH3CH2), 1.53(m, 1H, CH2CH), 1.75(m, 1H, CH2CH), 1.70(d, 3H, CH3C=), 2.98(dd, 1H, CHC00), 5.44(dd, 1H, J=15Hz, =CHCH), 5.6(dt, 1H, J=15Hz, CH3CH=).  2.2.11 Synthesis of (Z) 3 ene VPA -  -  (Z)-3-ene VPA was synthesized by the same method as described for the synthesis of (E)-3-ene VPA, but with the starting material being ethyl (E)-2-pentenoate.  42  GCMS of the methyl ester of (Z)-3-ene VPA, m/z(%): 55(100), 97(25), 113(18), 127(7), 156(M + ,3).  1 H NMR of (Z)-3-ene VPA (CD30D): S 0.93(t, 3H, CH3CH2), 1.34(m, 2H, CH3CH2), 1.48(m, 1H, CH2CH), 1.72(m, 1H, CH2CH), 1.66(d, 3H, CH3C=), 3.35(dd, 1H, CHC00), 5.36(dd, 1H, J=11Hz, =CHCH), 5.6(dt, 1H, J=11Hz, CH3CH=).  2.3 Pharmacokinetic studies  2.3.1 Pharmacokinetic study with [2H6]VPA A healthy male volunteer (my research supervisor) participated in a multiple-dose study of the pharmacokinetics of [ 2 H6]VPA. A 700 mg dose consisting of VPA:[ 2 N6]VRA (50:50) was given orally to the volunteer every 12 hours for a period of two and half days. Following the fifth and final dose, blood samples were withdrawn at 0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 9, 12, 24, 48, 96, 168, 240 and 336 h. Blood samples were allowed to clot and then centrifuged to provide serum samples, which were transferred to sterile vacutainers and stored at -20°C until analyzed. Saliva samples were taken at selected times convenient with the taking of blood samples. Saliva production was stimulated with a 5% citric acid solution rinse of the mouth. Following the first dose, urine samples were collected in 12 hour blocks for 2 days and then in 2 or 3 day blocks for another 8 days. Total urine volume and pH values were recorded and homogeneous aliquots were saved. Saliva and urine  43  samples were also stored at -20°C. ^All samples were analyzed quantitatively by GCMS (HP 5987A) using El analysis of the t-BDMS derivatives and NCI analysis of the PFB derivatives. Dibutylacetic acid, 2-methylglutaric acid and the stable isotope labelled metabolites, [2H3]2-ene VPA and [2H3]3-keto VPA were used as internal standards.  2.3.2 Pharmacokinetic study with [13C4]VPA 2.3.2a Human study: A healthy human volunteer ( my research supervisor, 70 Kg) was given a single oral dose consisting of 700 mg of VPA:[13C4]VPA (50:50). Blood samples were collected 0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 9, 12, 24, 48 and 72 h after the single dose and allowed to clot. The samples were centrifuged to obtain serum samples. Urine samples were collected at convenient blocks for three days after the dose. Total urine volume and pH values were recorded and homogeneous aliquots were saved. All samples were stored at -20°C until analyzed by GCMS using NCI techniques.  2.3.2b Animal study: Two nonpregnant sheep (60 and 73.6 Kg respectively) were each administered i.v. a single dose of 1000 mg of VPA : [  13 C4]VPA (50:50).  Blood^samples^were collected^at^5 min^before^injection,  2, 6,  10, 15,  20,^30,^45,^60 min,^1.5,^2,^2.5,^3,^3.5,^4,^5,^6,^8,^10, 12, 24, 36, 48, 72 h after the single dose. All the samples were allowed to clot before centrifuging to collect serum samples. Urine samples were collected every half an hour, then in 1 hour, 2 hour, 12 hour, or 24 hour blocks.  44  Bile samples were taken at selected time 0-30 min, 60-90 min, 2.5-3.5 h, 5-5.5 h, 7-7.5 h, 11-11.5 h, 24-24.5 h, 36-36.5 h, 48-48.5 h, and 7272.5 h after administration. The volume of urine and bile samples were recorded, and the pH values of urine samples were measured. All samples were stored at -20  ° C until the time of analysis.  2.4 Metabolic studies of (E)- and (Z)-3-ene VPA  2.4.1 Study design The metabolic studies of (Z)-3-ene VPA and (E)-3-ene VPA were performed in rats. The animals were kept in a restraint cage equipped with a funnel and a glass container to collect the urine.  2.4.2 Metabolism of (Z)  -  and (E) 3 ene VPA -  -  (Z)- and (E)-3-ene VPA (150 mg/kg dose) were separately administrated i.p. to two rats (adult male Wistar rats weighing 270 and 308 g).^A blood sample from each rat was taken 2 h after the dose, allowed to clot, and centrifuged to obtain a serum sample. ^Urine samples were collected every 24 hours for two days. Samples were stored at -20  ° C until derivatized and analyzed by GCMS using EI techniques.  2.5 Calibration Curves  2  To analyze VPA, [ H6]VPA and their metabolites in the steady-state study, calibration curves having different concentration ranges were  45  prepared for urine, serum total, and serum free (saliva) samples respectively. Table 1 summarizes the concentrations of stock solutions of VPA and VPA metabolites used in the preparation of the different calibration curves.  Table 1: Stock solution concentrations (ug/mL) used for the preparation of calibration curves for VPA, rHOVPA and their metabolites.  COMPOUNDS  URINE  SERUM TOTAL  SERUM FREE (SALIVA)  VPA  509.2  124.4  12.4  4-ene VPA  1.5  1.5  0.15  (E+Z)-3-ene VPA  0.985  1.97  0.197  (Z)-2-ene VPA  4.188  (E)-2-ene VPA  103.6  20.43  2.043  (E)-2,4-diene VPA  23  1.02  0.102  (E,E)-2,3'-diene VPA  49.2  5.03  0.503  3-keto VPA  300  10  1  4-keto VPA  40.8  1.04  0.104  3-0H VPA  28.3  0.98  0.098  4-01-1 VPA  52.8  1.908  0.1908  5-0H VPA  53.4  0.95  0.095  2-PSA  19.7  0.124  0.0124  2-PGA  99.6  1.004  0.1004  46  Calibration curves for VPA, [ 2 H6]VPA and their metabolites were made from samples prepared by the following dilution of the stock solution described in Table 1.  Amount of stock solution(%) *  * **  Control or water ** (%)  100  0  80  20  60  40  40  60  20  80  0  100  total amount: 1 mL for EI, 0.25 mL for NCI. water was used as a control for serum free.  A second set of calibration curves was prepared to analyze the samples from the pharmacokinetic study in sheep which were administered a single dose of VPA:[ 13 C4]VPA (50:50). Table 2 summarizes the concentrations of stock solutions used for the dilution of VPA and VPA metabolites for the calibration standard samples.  47  Table 2: Stock solution concentc4tions (ug/mL) used for the preparation of calibration curves for VPA, ["C4] VPA and their metabolites.  COMPOUNDS^  Concentration (ug/mL)  VPA^  20  4-ene VPA^  2  (E+Z)-3-ene VPA^  4  (Z)-2-ene VPA^  20  (E)-2-ene VPA^  20  (E)-2,4-diene VPA^  4  (E,E)-2,3'-diene VPA ^  8  3-keto VPA^  4  4-keto VPA^  2  3-0H VPA^  4  4-011 VPA^  4  5-011 VPA^  2  2-PSA^  2  2-PGA^  2  48  Calibration curves for VPA, [ 13 C4]VPA and their metabolites were made from samples prepared by the following dilution of the stock solution described in Table 2.  Amount of stock solution(%) *  Control or water ** (%)  100  0  50  50  25  75  12.5  87.5  6.25  93.75  1.56  98.44  49  2.5 Extraction and Derivatization  Extraction and derivatization procedures for the biological samples are summarized in Schemes 1 and 2.  Serum (total or free) or Saliva sample (0.25 mL for NCI, 1 mL for El) + internal standards / Adjust to pH=2 with 4N HC1 i Extract twice with lmL ethyl acetate (centrifuge to optimize separation) i Dry with Na2SO4, reduce volume to 200 ul with N2  1  I  / Add 10 ul PFBB (30% in ethyl acetate)^50 ul of MTBSTFA and 10 ul diisopropylethylamine ,,^i^ 60 'C for 1 h^  I 60 °C for 1 h  inject 1 ul^ (NCI assay)^  inject 1 ul (El assay)  Scheme 1:^Sample handling procedure for serum (total and -free) and saliva samples.  50  Urine sample (0.25 mL for NCI, 1 mL for EI) + internal standards i Hydrolyzing conjugates  *  Adjust to pH=2 with 4N HC1  Extract with 1 mL ethyl acetate  1  Dry with Na2SO4, reduce volume to 200 ul with N2  I^ I Add 10 ul PFBB (30% in ethyl acetate) ^50 ul of MTBSTFA and 10 ul diisopropylethylamine „ i 60 uC for 1 h^  inject 1 ul (NCI assay)  60  °C  for 1 h  i inject 1 ul (EI assay)  Scheme 2: Sample handling procedure for urine samples. *  The glucuronide conjugates in urine were hydrolyzed using two techniques:^(i). enzymatic hydrolysis by fl-glucuronidase at 37uC for 24 h (ii). alkaline (NaOH solution, pH=12) hydrolysis at 60 C for 1 h.  °  All samples were run in selected ion monitoring (SIM) mode. Table 3 summarizes the ions of derivatized VPA and metabolites that were monitored for the EI or NCI ionization techniques.  51  Table 3: Mass to charge (m/z) for the internal standards (*), VPA, and VPA metabolites that were used for ion monitoring in the NCI (PFB derivatives) and El (t-BDMS derivatives) mode.  COMPOUNDS  ^  NCI^El (M-181)-^(M-57)+  Dibutyl acetic acid*^171^229 VPA^  143^201  (E)-[2H3] 2-ene VPA*^144^202 4-ene VPA^141^199 (E+Z)-3-ene VPA^141^199 (Z)-2-ene VPA^141^199 (E)-2-ene VPA^141^199 (E)-2,4-diene VPA^139^197 (E,E)-2,3'-diene VPA^139^197 4-keto VPA^157^215 3-0H VPA^ 231^217 4-0H VPA^ 231^217 5-0H VPA^ 231 (monoderiv)^331 (dideriv) {2H3}3-keto VPA*^232 (dideriv)^332 (dideriv) 160 (monoderiv)^218 (monoderiv) 3-keto VPA^229 (dideriv)^329 (dideriv) 157 (monoderiv)^215 (monoderiv) 2-MGA*^  325^317  2-PSA^  339^331  2-PGA^  353^345  52  2.7 Calculation and Data Evaluation  2.7.1 Pharmacokinetic parameters  The pharmacokinetic parameters, area under the curve (AUC), halflife (t1/2), elimination rate constant (KE), distribution volume (Vd), and clearance (CL) of [ 2 H6]VPA and some of its metabolites were calculated using the equations of Gibaldi and Perrier (1982) and compared with those of VPA and its metabolites.  2.7.2 Isotope effects Isotope effects of [ 2 H6]VPA were measured from a) serum data based on the AUC ratio of [ 2 H0]VPA and its metabolites to [ 2 H6]VPA and its deuterated metabolites, and b) urine data determined from the recovery ratio of [ 2 H0]VPA and its metabolites to [ 2 H6]VPA and its metabolites in urine samples collected for 12 hours after the final dose.  Isotope effects of [ 13 C4]VPA was measured from serum data based on the concentration ratio of VPA and its metabolites to [  13 C4]VPA  and its  metabolites.  2.7.3 Conjugated fraction of VPA and its metabolites in urine samples  The glucuronide conjugates in urine were hydrolyzed using two techniques, enzymatic hydrolysis by fl-glucuronidase at 37 ° C for 24 hours and alkaline (NaOH solution, pH=12) hydrolysis at 60 ° C for 1 hour. The  53  efficacy of the two hydrolyzing methods and whether any fl-glucuronidaseresistant conjugates exist or not were determined by comparing the results of the two hydrolysis methods. The conjugated fractions were determined from the concentration differences of free drug and metabolites in hydrolyzed and unhydrolyzed urine samples respectively.  2.7.4 Evaluation of Data  A MIDAS based computer program containing methodological statistics was used to evaluate the two analytical methods, NCI and El, and to compare the efficiencies of the two hydrolysis methods, fl-glucuronidase and treatment with alkali (NaOH solution, pH=12).  54  3. Results and Discussion  3.1 Synthesis of deuterium labelled internal standards  Stable isotope-labelled analogs provide optimal internal standards for GCMS assays. By using stable isotope-labelled analogs as internal standards, the lowest variance factors due to sample manipulation and instrumental errors was produced (Claeys  et al., 1977). Ideally the  labelled internal standard should have a mass difference of at least 3 mass units from the analyte to minimize interference from natural isotopes and thus avoid correction for mass overlap. While [2H6]VPA is suitable for VPA analysis ( Au = 6), it is not a good internal standard for [13C4]VPA ( Au = 2). Based on the above principle, [2H7]VPA should provide an ideal internal standard for both [13C4]VPA and VPA analysis by GCMS. The previously reported assays (Abbott Acheampong  et al., 1986;  et al., 1983; Kassahun et al., 1989) for VPA metabolites from  this laboratory could also be improved by the availability of adequate stable isotope labelled internal standards of the metabolites. Thus, syntheses were carried out to produce several deuterium labelled VPA metabolites.  3.1.1 Synthesis of [2H7]VPA [2H7]VPA was synthesized by a method for the alkylation of metalated aliphatic acids (Pfeffer  a-  et al., 1972). The procedure is  outlined in scheme 3. Distilled pentanoic acid was converted into the a-anion by means of two equivalents of lithium diisopropylamide in tetrahydrofuran (THF)-hexane solution. Hexamethylphosphoramide (HMPA)  55  was added to the mixture to make the reaction more specific and efficient, due to its chemical and physical properties. First, the metalated straight-chain acids are insoluble in THF, but readily dissolve in the highly dipolar solvent, HMPA; second, because organolithium compounds are known to associate to higher molecular weight aggregates, the degree of polymerization (n=2-6) varying with solvent and structure (Mallan and Bebb, 1969), the aggregation state is disrupted by complexation with polar solvents in the formation of a solvent-separated ion pair of higher reactivity than the original aggregate. A strong dipolar solvent like HMPA should accordingly be more efficient in association with metalated carboxylates than THF by forming a complex of higher reactivity, presumably a solvent-separated ion pair (Pfeffer et al., 1972). Pfeffer et al. (1972) also illustrated by comparative experiments that HMPA, in addition to solubilising dianions, also accelerates the alkylation rates, and quantitative conversion was observed in HMPA solution when 1.5 equivalents of alkyl bromide was added.  This method was first used to synthesize VPA in this lab by Acheampong (1985). The same method was applied in synthesizing [ 2 H7]VPA, the only difference being the use of [ 2 H7]propyl bromide. Since [ 2 H7]propyl bromide is very expensive, we could not afford to redistill it before use, hence, the yield is less than the reference (91%, Lee, 1987). The final product was confirmed to be [ 2 H7]VPA by comparing its GCMS mass spectrum and NMR spectrum with unlabeled VPA. The mass spectrum of the methyl ester derivative of [ 2 H7]VPA which was prepared by derivatizing with diazomethane (Levitt, 1973) revealed  56  fragment ions at m/z 136(M-C2H5)+, 123(M-C3H6)+, 117(M-C32H6)+, and 89(C2H2CH2COOCH3)+, corresponding to VPA fragments at m/z 129(M-C2H5)+, 116(M-C3H6)+, and 87(CH2CH2COOCH3)+ respectively (Figure 3). The 1H NMR of the synthesized product also confirmed the [2H7]VPA structure (Figure 4).  CH3-CH2-CH2-CH2-COOH  ^  valeric acid  / LDA/THF  CH3-CH2-CH2-CH--000-^  dianion  [2H7] propyl bromide  CH3-CH2-CH2  CH-COOH 9 /  C2H3-C2H2-C412  Scheme 3: Synthesis of [2H7]VPA.  [2H7]VPA  57  69 (C 2 H2CH2COOCH3 +  CH3 CH2 CH2 -  -  CH -COOCH3  C 2H3-C 2H2 -C 2H2 (M-C32[16 )4- ^(M-C3H6) 117 123  61  18  1117,,^I, 111 1 1 1, . .111 5  I,  106  I ti a^ s^a,  134  Al  87 (CH2CH2C0OCH3  C H3 -C H 2 - CH 2  CH -COOCH 3 C H3 - C H2 - CH2  ( M - C3 H 6 ) 57 18  116  43 29  (M - C2H5 ) 469  99  129  M+ 159  IMIIMIN■111111■•■1111  Fig. 3.^GCMS mass spectra of the methyl esters of [ 2 H7]VPA (top) and VPA (bottom).  CH3-CH2-CH2 CH-COOH C2H3-C 2 H2-C2 H2  CE3-CH2  i  CH3CH2-CH CH-COOH  // CH3-CH2  CH3CH2-C1  A 'L ^J 8.5  2.5  2.8^6.5  6.8^5.5  Fig. 4:^111 NMR spectrum of [2H7]VPA.  5.0^4.5 999  4.0^3.5  3.9^2.5  2.8^1.5^1.2  59  3.1.2 Synthesis of [2H7]4-ene VPA  4-Ene VPA can usually be prepared via its ethyl ester, by reaction of ethyl valerate and allyl bromide according to the general method for the alkylation of esters at the a-carbon atom (Cregge, et al., 1973). However, this method was not suitable for introducing the [2H7] deuterium labelled side chain. It turns out the a-metalation method was also applied by Pfeffer (1972) to olefinic acids. For example, 10undecenoic and (Z)-9-octadecenoic acids, were converted to their a anions and then alkylated in good yields to the a-branched chain unsaturated acids. The isolated double bonds in these acids were found to be unaltered positionally or geometrically in the formation of their dianions by lithium diisopropylamide. An attempt to synthesize [2H7]4ene VPA using this method was performed by starting first with the synthesis of unlabeled 4-ene VPA. Good yields were obtained for 4-ene VPA, and thus [2H7] 4-ene VPA was synthesized in this study starting from 4-pentenoic acid (Scheme 4) by Pfeffer's method.  [2H7]4-ene^VPA^was^successfully^purified^by^fractional distillation. The GCMS mass spectrum of the methyl ester of [2H7]4-ene VPA was compared with that of the methyl ester of 4-ene VPA (Figure 5). Fragments of [2117]4-ene VPA occurred at m/z 163 (e), 129 (M-C22H5)+, 113(M-C32H7)+, and 104(M-COOCH3)+ and correspond to those of 4-ene VPA at m/z 156 (Mt), 127 (M-C2H5)+, 113(M-C3H7)+, and 97(M-COOCH3)+. 1H-NMR (Figure 6) confirmed the presence of the terminal double bond: 8 5.78 (m, 1H, CH2=CH), 5.12 (d, 1H, J=17 Hz, proton at C-5 trans to proton at C-4), 5.08 (d, 1H, J=11 Hz, proton at C-5 cis to proton at C-4).  60  CH2=CH-CH2-CH2-000H  4-pentenoic acid  LDA/THF  CH2=CH-CH2-CH - -000 -  dianion  [ 2 H7] propyl bromide  CH2=CH-CH2  /  CH-COON  C 2 H3-C 2 H2-C 2 H2  Scheme 4: Synthesis of [ 2 H7]4-ene VPA.  [ 2 H7]4-ene VPA  61  Fig. 5: GCMS mass spectra of the methyl esters of 4-ene [ 2 H7PPA (top) and 4-ene VPA (bottom).  CH 2=CH  Fig. 6:^1H NMR spectrum of [2H7]4-ene VPA.  63  3.1.3 Synthesis of [2H7]4-keto VPA  Ethyl [2H7]4-ene VPA was oxidized by benzoquinone to ethyl [2H714keto VPA using palladium chloride as catalyst. This procedure is wellknown as the Wacker process, and is one of the most important industrial processes employing transition metal catalysts (Smidt et al., 1959). There are several methods which can selectively oxidize terminal double bonds to methyl ketones; of them, the palladium(II) chloride-catalyzed oxidation of terminal olefins seems to be one of the best (Tsuji, 1984). Since the oxidation proceeds under mild conditions, various functional groups, such as esters, carboxylic acids, aldehydes, are unchanged during the reaction (Tsuji, 1984). The conversion of ethyl [2H714-ene VPA to ethyl [2H7]4-keto VPA was quantitatively completed in 22 hours. Scheme 5 summarizes the synthesis procedure of [2H7]4-keto VPA.  The GCMS mass spectrum of ethyl [2H7]4-keto VPA (Figure 7) revealed a molecular ion (m/z 193) and other fragments, m/z 148 (M-OCH2CH3)+, 136 (M-CH3COCH2)+, and 108 (C2H3C2H2C2H2CH2C00)+, which correspond to ethyl 4-keto VPA fragments, m/z 186 (M+), 141 (M-OCH2CH3)+, 129 (M-CH3COCH2)+, and 101 (CH3CH2CH2CH2C00)+.  64  CH2=CH-CH2  \ 9  CH-COON  [2H7]4-ene VPA  //  C 2 H3 - C 2 H2 - C4 2  EtOH, H2SO4 Benzene  I  CH2=CH-CH2  \  ,/  CH -CO0C2H5  Ethyl [ 2 H7]4-ene VPA  C 2 H3-C 2 H2 -C 42  Benzoquinone PdC12  CH3-C O-CH2  \  CH -CO0C2H5  Ethyl [ 2 H7]4-keto VPA  c2H r c2H 2 _cr_9 l_1 /2  Na0H/Me0H  CH3 -CO -CH2  \  CH-COOH^  9 /2 c2H 3 _c2H 2 _u_H  Scheme 5: Synthesis of [ 2 H7]4-keto VPA.  [2H7]4-keto VPA  65  Fig. 7: GCMS mass spectra of ethyl esters of (2H7]4-keto VPA (top) and 4-keto VPA (bottom).  66  3.1.4 Synthesis of 1-2H7,14-0H VPA The synthesis of [ 2 H7]4-0H VPA was accomplished via its ethyl ester by reducing the ethyl ester of [ 2 H7]4-keto VPA with NaBH4. The reaction was quantitatively completed within 60 min at room temperature, when excess NaBH4 was used. The steps in the synthesis are summarized in Scheme 6. The GCMS mass spectrum of ethyl [ 2 H7]4-0H VPA was compared with that of unlabeled ethyl 4-0H VPA (Figure 8). The fragments of ethyl [ 2 H7]4-0H VPA at m/z 150 (M-C2HSO), 134, 116 and 101 correspond to ethyl 4-0H VPA at m/z 143, 127, 113 and 110.  67  CH3 -CO -CH2 CH -CO0C2H5  ^  Ethyl [ 2 H7]4-keto VPA  C 2 H3-C 2 H2-C 2 H2  NaBH4/EtOH  OH CH3-CH-CH 2  CH -CO0C2H5  two isomers of ethyl [ 2 H7]4-0H VPA  C 2 H3-C 2 H2-C 2 H2  NaOH/MeOH  OH CH3 -CH-CH2  CH-COOH C 2 H3-C 2 H2-C 2 H2  ^  ^  Scheme 6: Synthesis of [ 2 H7]4-0H VPA.  two isomers of [ 2 H7]4-0H VPA  68  Fig. 8: GCMS mass spectra of ethyl esters of [2H7]4-0H VPA (top) and 4OH VPA (bottom).  69  3.1.5 Synthesis of [2H7] 5-0H VPA The ethyl ester of [ 2 H7]5-0H VPA was prepared from ethyl [ 2 H7]4 -ene VPA by the same method that Rettenmeier  et  a/.(1985) used to synthesize  5-0H VPA. Scheme 7 summarizes the synthesis procedure. Ethyl [ 2 H7]4ene VPA was hydroborated into the corresponding trialkylborane, which was then oxidized with alkaline hydrogen peroxide to provide ethyl [ 2 H7}5-0H VPA. The GCMS mass spectrum (Figure 9) of ethyl [ 2 H7]5-0H VPA indicates the molecular ion at m/z 195 and other fragments at m/z 164 (M-CH3OH) + , 150 (M-C2H5OH) + , 115, and 101, corresponding to fragments of ethyl 5-0H VPA at m/z: 157 (M-CH3OH) + , 143 (M-C2H5OH) + , 113, and 100.  70  CH2=CH-CH2  \  ethyl [ 2 H7]4-ene VPA  CH-COOC2H5  9 /2 c2H 3 _c2H 2 _c41  H3 B- BH3  -CH2-CH2-CH2  C 2 H3-C 2 H2 -C 2 H2  \ CH-COOCH2CH3 /  3  trialkylborane  /  H202/NaOH  HOCH2 CH2 CH2 -  -  C 2 H3-C 2 H2-C 2 H 2  \ CH-COOCH2CH3^ethyl [ 2 H7]5-OH VPA /  NaOH/MeOH  HOCH2-CH2-CH2  C 2 H3-C 2H2-C 2 H 2  \ CH-COON^ /  Scheme 7: Synthesis of [ 2 H7]5-0H VPA.  [2H7]5-0H VPA  71  Fig. 9: GCMS mass spectra of the ethyl esters of [2H7]5-0H VPA (top) and 5-0H VPA (bottom).  72  3.1.6 Synthesis of [ 2H7]3-keto VPA  Ethyl 3-keto VPA is usually synthesized by alkylating the enolate ester of valeric acid with propionyl chloride, a very well established method for the synthesis 3-keto VPA (Acheampong, 1982). However, [ 2 H7]pentanoic acid would have to be prepared first if this method were applied in the synthesis of [ 2 H7]3-keto VPA. This adds to increased costs and a lower yield based on the availability of [ 2 H7]bromopropane. It is optimal to introduce the [ 2 H7]propyl side chain in the final step of the synthesis for considerations of expense and efficacy. Accordingly, we designed a synthesis of ethyl 3-keto pentanoate, which could then be alkylated with [ 2 H7]bromopropane to produce the desired product.  3.1.6a Synthesis of ethyl 3-keto pentanoate:  Ethyl 3-keto pentanoate was synthesized according to the method published by Micheal  et a/.(1971) for the preparation of fl-keto esters.  Scheme 8 summarizes the procedure. Ethyl acetate was converted into the corresponding lithium enolate ester by reaction with lithium  N-  isopropylcyclohexylamide (LiICA). The enolate ester was then condensed with propionyl chloride to form the desired fl-keto ester without significant self-condensation. However, attack of the enolate ester at the ketone carbonyl of the product could lead to decreased yields. This possibility was minimized by using an extra equivalent of the generating base, LiICA, thereby converting the product into the relatively inert fiketo enolate ester. The product gave only one peak in the TIC upon GCMS  73  analysis and proved to be ethyl 3-keto pentanoate from the GCMS mass spectrum and from IH NMR, after purification by fractional distillation. GCMS mass spectrum (Figure 10) of the product shows a molecular ion of ethyl 3-keto-pentanoate at m/z 144, and other fragments m/z 115(MC2H5)+, 98(M-C2H5OH)+, 87(M-CH3CH2C0)+, 69(M-CH3CH2COOH)+, 57(CH3CH2C0)+. 111 NMR (Figure 11) also confirmed the structure of the product to be ethyl 3-keto-pentanoate.  57  29  (CH3CH2C0 )4-  CH3 -02-C-CH2-COOC 2H5  43  1 ^+ (M-C21-150H)+  69 18  1.,  (M-CH3CH2CO)  (M-C2F15)  r^.11,^ir F 98  Fig. 10: GCMS mass spectra of ethyl 3-keto-pentanoate.  m+ .144  ^lit  CH3CH2CO  CH3CH2O  0 II  CH3 -C H2-C-CH2 COOC2H5 -  j I  1  9  I  1  TtitioillitijilfilliiiTi  l  1  11^til—IM-1—VilFITI1^111—$11-1111111111  l l I  1^•^8^7^6^5^M^3^  Fig. 11: 1 H NMR spectrum of ethyl 3-keto-pentanoate.  11 l  2  1  11 i  11■1111111  75  3.1.66 Alkylation of ethyl 3-keto pentanoate with [2H7]bromopropane  Ethyl 3-keto-pentanoate was converted into the corresponding sodium fl-keto enolate ester with sodium ethoxide which was prepared by adding absolute alcohol to freshly cut Na metal. The enolate was then alkylated with [2H7]bromopropane to afford a high yield of ethyl [2H7]3keto VPA (Scheme 8). [2H7]3-keto VPA was purified by fractional distillation. Figure 12 shows the mass spectra of ethyl [2H7]3-keto VPA and ethyl 3-keto VPA. The molecular ion (m/z 193) and other fragments of [2H7]ethyl 3-keto VPA at m/z 164 (M-C2H5)+, 145 (M-C32H6)+, 137 (MCH2CH2C0)+, 103 (C2H2CH2C00C2H5)+, correspond to those of ethyl 3-keto VPA at m/z 186 (e), 157 (M-C2H5)+, 144 (M-C3H6)+, 130 (M-CH2CH2C0)+, 101 (CH2CH2C00C2H5)+. IH NMR (Figure 13) of ethyl [2H7]3-keto VPA confirmed the structure and purity of product.  76  CH3-CO0C2H5  ethyl acetate  LiICA/THF CH3CH2C0C1  CH3-CH2-CO-CH2-CO0C2H5  ethyl 3-keto pentanoate  EtONa [ 2 H7]propyl bromide  CH3 -CH2 -CO  \  C 2 H3 C 2 H2 C 2 H2 -  /  CH -CO0C2H5  ethyl [ 2 H7]3-keto VPA  -  NaOH/MeOH  CH3-CH2-00  C 2 H3-C 2 H2-C 2 H2  \ /  CH-COOH^  Scheme 8: Synthesis of [ 2 H7]3-keto VPA.  [2H7]3-keto VPA  77  57  CH 3-CH2 -CO  C2H3-C2H2 - C42  /  CH -COOC2H5  (C2H2CH2C00C2H5)-1-  29  103  (H-CH2CH2C0)-1137 75  I  116^Ii  Si  45  18  (M-C324)+ (M-C2H5)-1-  m÷  145^  57  164  (CH2CH2C00C2H5)-1-  29  193  CH3 -CH2 -C O  Mil  CH-CO0C2H5 CH3-C H2-CH2 (M-CH2CH2 C0 ) -1130  7  (M-C3H6)-1144  43  83  115  (M-C2H5)4157 187  Fig. 12: GCMS mass spectra of the ethyl esters of [2H7]3-keto VPA (top) and 3-keto VPA (bottom).  CH3 CH 0 CH3CH2C0  CH3-CH2-00 CH-CO0C2H5 C2H3-C2H2-C2H2  CH20  ^I) G.5^G0  5. 5  1^,^,  5.0^4.5  4.0^3.5 PPM  3.0  2. 5  Fig. 13: 1 H NMR spectrum of ethyl ester of [ 2 H7]3-keto VPA.  2.^5^.^.5^0.0  79  3.1.7 Synthesis of 1 -2H733-0H VPA The ethyl ester of [ 2 H7]3-0H VPA was synthesized by reducing the ethyl ester of [ 2 H7]3-keto VPA with NaBH4, the same method that was used in the synthesis of [ 2 H7]4-0H VPA. Scheme 9 presents the synthetic procedure.  The product was confirmed to be ethyl [ 2 H7]3-0H VPA by comparison of its GCMS mass spectrum with that of ethyl 3-0H VPA (Figure 14). The fragments of ethyl [ 2 H7]3-0H VPA at m/z: 166 (M-C2H5) + , 150 (M-0C2H5) + , 137 (M-CH3CH2CHO) + , 120 (CH3CH2COCHC3 2 H7) + , 103 (C 2 H2CH2CO2C2H5) + , correspond to the fragments of ethyl 3-0H VPA at m/z: 159 (M-C2H5) + , 143 (M-0C2H5) + , 130 (M-CH3CH2CHO) + , 113 (CH3CH2COCHC3H7) + , and 101 (CH2CH2CO2C2H5) 1".  ^  80  CH3-CH2-CO  \  CH-CO0C2H5  ^ ethyl CH713-keto VPA  c2H3_c2H2_c2H2 /  NaBH4/Et0H  OH  1  CH3-CH2-CH  Two isomers of \^ CH -CO0C2H5^ ethyl [ 2 H7P-OH VPA /  ^C2H3-C2H2-C2H2  Na0H/Me0H  OH  1  CH3-CH2-CH  \  Two isomers of CH-COOH  c2H3_c2H2 -C 2H2/  Scheme 9: Synthesis of [2H7]3-0H VPA  [2H7]3-OH VPA  81  Fig. 14: GCMS mass spectra of the ethyl esters of [2H7}3-OH VPA (top) and 3-0H VPA (bottom).  82  3.1.8 Synthesis of (E)-[ 2H7]2-ene VPA Ethyl (E)-[ 2 H7]2-ene VPA was synthesized from ethyl [ 2 H7]3-0H VPA according to the procedure in scheme 10, and based on the method of Lee et al., (1989) to synthesize (E)-2-ene VPA. The formation of the mesyl ester of [ 2 H7]3-0H VPA was completed within one hour after methanesulfonyl chloride was added. Nucleophilic elimination of the mesyl group by 1,8-diazabicyclo [5.4.0]undec-7-ene (DBU) afforded ethyl [ 2 H7]2-ene VPA.^The separation and purification of product was performed by column chromatography.^The main impurity was unreacted ethyl [ 2 H7]3-0H VPA, which combined with the stationary phase tightly because of the hydroxyl group. The impurity eluted only when using a highly polar solvent, therefore, it was easy to separate the relatively non-polar ethyl [ 2 H7]2-ene VPA from it. Moreover, being an a,$unsaturated ester, ethyl [ 2 H7]2-ene VPA was visible under UV light, which facilitated monitoring the separation. The solvent was removed under flash evaporation from the fraction shown to contain the 2-ene  VPA, and the residue was dried under vacuum. The 1 H NMR (Figure 16) confirmed the product to be ethyl [ 2 H7](E) 2-ene VPA, with a small portion of (Z) isomer, which agrees with Lee's result (1989). Figure 15 shows the mass spectra of labelled and unlabeled ethyl (E)-2-ene VPA. The fragments of ethyl [ 2 H7]2-ene VPA at m/z 177 (e), 148 (M-C2H5) + , 132 (M-0C2H5) + , 115 (CH2=C(C 2 H2)C00C2H5) + , and 97 (CH3CH=CHC(C0)=C 2 H2) + , correspond to those of ethyl 2-ene VPA at m/z 170 (e), 141 (M-C2H5) + , 125(M-0C2H5) + , 113(CH2=C(CH2)C00C2H5) + , and 95(CH3CH=CHC(C0)=CH2) + .  83  OH CH3-CH2-CH  ethyl [ 2 H7D-OH VPA  CH -CO0C2H5 C2H 3-C2H2-C2H2  MsC1 Et3N, CH2C12  OSO2C1 mesyl ester of ethyl  CH3-CH2-CH^ C-CO0C2H5^  [2H7D-OH VPA  C2H3-C2H2-C2H2  DBU/THF  CH3-CH2-CH C-COOC2H5  ethyl (E)-[2H7]2-ene VPA  9 /  C2H3-C2H2-CLH2  Na0H/Me0H  CH3-CH2-CH C-COOH 9 / c2H3_c2H2_cm2  Scheme 10: Synthesis of (E)-[ 2 H7]2-ene VPA.  (E)-[2H7]2-ene VPA  84  Fig. 15: GCMS mass spectra of the ethyl esters of (E)-42H7]2-ene VPA (top) and (E)-2-ene VPA (bottom).  Fig. 16: IH NMR spectrum of the ethyl ester of [2H7]2-ene VPA.  86  3.1.9 Stereoselective syntheses of (E)- and (Z)-3-ene VPA (E)-3-ene VPA was synthesized as outlined in scheme 11, by a method based on general procedures described by Herrmann et al. (1973). According to the research of Kende and Toder (1982), alkylation of ethyl (Z)-2-alkenoate yields stereospecifically ethyl (E)-3-alkenoate. The a,fl-unsaturated ester ethyl (Z)-2-pentenoate was converted into the lithium enolate by the non-nucleophilic form of lithium diisopropylamide (LDA) which is a 1:1 complex of LDA and HMPA. Besides being employed as base, LDA might also act as a nucleophile and conjugatively add to the unsaturated ester at a rate competitive with proton abstraction. After applying HMPA to modify LDA, no Micheal addition to (Z)-2-pentenoate was observed. The lithium enolate was then alkylated by bromopropane to afford ethyl (E)-3-ene VPA. Ethyl (E)-3-ene VPA was hydrolyzed with base and the free acid purified by fractional distillation. The product was confirmed by GCMS and 1 H-NMR to be pure (E)-3-ene VPA. GCMS mass spectrum (Figure 17) of 3-ene VPA after methylation affords ions at m/z 156, 127, 113, 97, 55, corresponding to the molecular ion (M + ) and fragments (M-C2H5) + , (M-C3H7) + , (M-COOCH3) + , and (CH3CH=CHCH2) + respectively.  The NMR spectrum (Figure 18) confirmed the stereoselectivity of this reaction. The proton at C-4 has a chemical shift value at 5.6 ppm, and is split into a 16 peak multiplet with coupling constants of 15 Hz (with the proton at C-3), 6.5 Hz (with CH3), and 1 Hz (with the proton at C-2); the proton at C-3 has a chemical shift value of 5.43 ppm, and  87  is split into a 16 peak multiplet with coupling constants of 15 Hz (with the proton at C-4), 8.6 Hz (with the proton at C-2) and 2 Hz (with CH3).  (Z)-3-ene VPA was synthesized by the same method as (E)-3-ene VPA except the starting material was ethyl (E)-2-pentenoate. The GCMS mass spectrum (Figure 17) of the methyl ester of (Z)-3-ene VPA was identical with that of the methyl ester of (E)-3-ene VPA. However, 1 H NMR can distinguish between these two isomers. Figure 19 shows the NMR spectrum for (Z)-3-ene VPA. The signal for the vinylic proton at C-4 appears at 5.6 ppm, and occurs as a 16 peak multiplet with coupling constants of 11 Hz (with the proton at C-3), 6.5 Hz (with CH3), and 1 Hz (with the proton at C-2); the vinylic proton at C-3 shows a signal at 5.36 ppm, and is split into a 16 peak multiplet with coupling constants of 11 Hz (with the proton at C-4), 9.5 Hz (with the proton at C-2), and 2 Hz (with CH3 adjacent to the C-4).  The purity of both (E)- and (Z)-3-ene VPA were checked by GCMS and by NMR, before they were used in the metabolism studies.  ^ ^  88  (Z)-ethyl 2-pentenoate CH3-CH2^CO0C2H5  / LDA/HMPA, THF  ^CH3^H \ / C=C / \ H^CHCO0C2H5 I Li  1^  bromopropane  CH3^H \ / C=C / \ H^CHCO0C2H5 / CH3CH2CH2  I  ^  ethyl (E)-3-ene VPA  Na0H/Me0H  CH3^H \ / C=C / \ H^CHCOOH CH3CH2CH2  lithium enol ate  /  Scheme 11: Synthesis of (E)-3-ene VPA.  (E)-3-ene VPA  89  Fig. 17: GCMS mass spectra of the methyl esters of (E)- (top) and (Z)3-ene VPA (bottom).  ^  CH3CH2  ^CH3  ^H / C=C / H^CHCOOH CH3CH2CH2  CH3CH= r-CH_CHCOOH  2.5^7.5  5.5^G. fi  Fig. 18: IH NMR spectrum of (E)-3-ene VPA.  L. 5.8^•^5.6^ 5.4^5.2  -CHCHCOOH  till  111111T 1114^  4  Fig. 19: 1H NMR spectrum of (Z)-3-ene VPA.  1-1-1  ir  ^ITT-  92  3.2 Optimizing GCMS conditions for the analysis of VPA metabolites in EI (t-BDMS derivatives) and NCI (PFB derivatives) modes  When a mixture of VPA and stable isotope labelled VPA is administered to a subject, the number of metabolite peaks to be quantitated by GCMS will double.^Adequate resolution of metabolite peaks becomes even more critical.^Under a constant carrier gas flow rate (1 ml/min), oven temperature is certainly the most important factor for good resolution when using either EI or NCI techniques.  By performing several experiments with different oven temperature programs, we obtained relatively satisfying results for both the EI and NCI methods. Table 4 lists the retention times of the PFB derivatives of VPA, [ 2 H6]VPA and their metabolites analyzed by NCI technique, with a GC oven temperature initiated at 50 °C, programmed to 140 °C at 30°C/min, held for 20 min, and then increased to 260 °C at 8 °C/min.  The PFB derivatives of VPA, [ 2 H6]VPA and their metabolites have good chromatographic properties and give sharp and symmetric peaks even during a relatively long GC run time. All of the derivatized metabolites gave an abundant ion at [M-181] - corresponding to the loss of the PFB moiety. These ions were monitored in NCI SIM mode for the quantitation of metabolites.  93  Table^4:^List^of the^negative, ions monitored^and^retention^times^for the^PFB^derivatives ^of^VPA,^[9-16]VPA,^their^metabolites^and^internal standards^(I.S.)^for the NCI analysis mode. COMPOUNDS  NEGATIVE ION MONITORED (m/z)  Dibutyl^acetic acid^(I.S.) [2H3](Z)-2-ene VPA^(I.S.) [ 2 H3](E)-2-ene VPA^(I.S.) [2H3]3-keto VPA (I.S.) 2-MGA^(I.S.) VPA [ 2 H6]VPA 4-ene VPA [24]4-ene VPA 3-ene VPA [ 2 H03-ene VPA (Z)-2-ene VPA [24](Z)-2-ene VPA (E)-2-ene VPA [ 2 H6](E)-2-ene VPA 2,4-diene VPA [ 2 H5]2,4-diene VPA (E,Z)-2,3'-diene VPA [24](E,Z)-2,3'-diene VPA (E,E)-2,3'-diene VPA [241(E,E)-2,3'-diene VPA 4-keto VPA [ 2 H04-keto VPA 3-0H VPA [24]3-0H VPA 4-0H VPA [ 2 H614-0H VPA 5-0H VPA [ 2 H5]5-0H VPA 3-keto VPA [ 2 H6]3-keto VPA 2-PSA [2H3]2-PSA 2-PGA [2H3]2-PGA * . isomers;  **  di-derivatives  171 144 144 232 325** 143 149 141 146 141 147 141 147 141 147 139 144 139 145 139 145 157 160 231 237 231 237 231 236 229 235 339** 342** 353** 356**  tR (min) 27.0 15.35 18.50 32.31 43.23 14.67 14.46 14.34 14.14 14.92 14.71 15.44 15.21 18.62 18.33 19.93 19.21 18.93 18.60 22.43 22.03 25.03 24.87 29.69 29.53 29.30, 30.36* 29.14, 30.21* 33.28 33.19 32.36 32.26 41.76 41.71 43.43 43.38  94  Good resolution of all peaks was obtained by using the GC condition described above. The 4-ene VPA peak which is frequently overlapped by high concentrations of VPA was well separated from VPA with retention time difference of about 0.34 min. (E,Z)-2,3'-diene VPA, which is usually missed by EI analysis of the t-BDMS derivative, was detected and well separated from (E)-2,4-diene VPA with a retention time difference of up to 1 minute.  Two different temperature programs were investigated for t-BDMS derivatives of VPA and metabolites when analyzed by GCMS in EI mode. Table 5 summarizes retention times of the t-BDMS derivatives of the unsaturated metabolites of VPA and [ 2 H6]VPA with the GC temperature initiated at 50 °C, programmed at 30 °C/min to 110 °C, held for 18 min and then increased to 260 °C at a rate of 10 °C/min. All unsaturated metabolites were eluted by 26 min and gave good resolution and peak shapes by this temperature program. However, the run time was too long for the keto and hydroxyl metabolites and these did not produce sharp and symmetric peak shapes because of their high polarity. Thus, another temperature program was set to analyze polar metabolites. Table 6 shows retention times of t-BDMS derivatives of VPA, [ 2 H6]VPA and their keto and hydroxyl metabolites with the GC temperature initiated at 50 °C, programmed to 100 °C at 30 °C/min, then increased to 250 °C at a rate of 8 °C/min. Total run time was less than 20 min.  95  Table 5: Positive ion monitored and the retention times of the t-BDMS derivatives of VPA, [915]VPA, their unsaturated metabolites, and the internal standards (I.S.) in the El analysis mode.  COMPOUNDS  ION MONITORED (m/z)  tR (min)  [2H3](Z)-2-ene VPA ^(I.S.)  202  16.79  [2H3](E)-2-ene VPA (I.S.)  202  20.41  VPA  201  15.71  [2H6]VPA  207  15.66  4-ene VPA  199  15.68  [24]4-ene VPA  204  15.53  3-ene VPA  199  15.88  [2H5]3-ene VPA  205  15.62  (Z)-2-ene VPA  199  16.86  [2H5](Z)-2-ene VPA  205  16.61  (E)-2-ene VPA  199  20.59  [2H6](E)-2-ene VPA  205  20.19  2,4-diene VPA  197  22.88  [2H5]2,4-diene VPA  202  22.71  (E,E)-2,3'-diene VPA  197  24.91  [24](E,E)-2,3'-diene VPA  203  24.71  96  Table 6:^Positive ion^monitored and the retention times of the t-BDMS derivatives^of VPA,^[ H6]VPA,^their^keto^and^hydroxyl^metabolites^and internal^standards in the EI analysis mode.  COMPOUNDS  ION MONITORED (m/z)  tR (min)  Dibutyl^acetic acid^(I.S.)  229  11.02  [ 2 H3](E)-2-ene VPA^(I.S.)  202  9.45  [ 2 H3]3-keto VPA (I.S.)  218  11.33  VPA  201  8.55  [ 2 H6JVPA  207  8.48  4-keto VPA  215  11.96  [ 2 H6]4-keto VPA  221  11.89  3-OH VPA  217  11.50, 11.83 *  [ 2 4]3-0H VPA  223  11.43, 11.77 *  4-OH VPA  217  11.37, 11.94 *  [ 2 4]4-0H VPA  223  11.30, 11.90 *  5-OH VPA  331**  16.25  [ 2 4]5-0H VPA  336**  16.21  3-keto VPA  332**  15.85  [ 2 4]3-keto VPA  335**  15.83  2-MGA^(I.S.)  317**  16.16  2-PSA  331**  16.39  [ 2 H3]2-PSA  334**  16.36  2-PGA  345**  17.70  [ 2 H3]2-PGA  348**  17.67  * isomers,  ^**  di-derivatives  97  3.3^Pharmacokinetics of [2H6]VPA and its metabolites in a healthy volunteer  A 700 mg dose consisting of VPA:[24]VPA (50:50) was given orally to a healthy human volunteer every 12 hours for a period of two and half days to perform a multiple-dose study of the pharmacokinetics of VPA and [2H6]VPA. Following the final dose, blood samples were withdrawn at certain times and serum samples were then obtained by centrifugation. Saliva samples were taken at selected times convenient with the taking of blood samples after stimulation with a 5% citric acid solution rinse of the mouth. Following the first dose, urine samples were collected in 12 hour blocks for 2 days and then in 2 or 3 day blocks for another 8 days.  All samples were analyzed quantitatively by GCMS (HP 5987A) using El analysis of the t-BDMS derivatives and NCI analysis of the PFB derivatives with the GC condition we discussed in section 3.2.. Urine or serum samples (0.25 mL) were extracted and derivatized with PFB and then TMS to be made ready for NCI analysis, while 1 mL of urine or serum was required to be derivatized with t-BDMS for El analysis. Dibutylacetic acid was used as an internal standard to analyze VPA and [2H6JVPA, 2-methylglutaric acid for 2-PSA and 2-PGA, [2H3]3-keto VPA for 3-keto VPA and [2H6]3-keto VPA, and [2H312-ene VPA for the rest of VPA metabolites and their deuterium labeled analogs. Calibration curves with different standard concentrations were made for urine, serum total, serum free and saliva respectively, and run for both PFB and t-BDMS  98  derivatives. Table 7 and 8 summarize the coefficients of determination for calibration curves of VPA metabolites.  Good linearity of calibration curves was obtained for most of the unsaturated VPA metabolites and 3-keto VPA, since [ 2 H3]2-ene VPA and [ 2 H3]3-keto VPA were used as internal standards. However, the linearity could be improved for 4-keto and hydroxyl metabolites, if adequate stable isotope labelled internal standards were used for those metabolites.  Table 7: Linearity of calibratiorl curves for quantitative assays of VPA, VPA metabolites and their [ H7] labelled analogues which were isolated frpm urine samples of a human volunteer administered with 700 mg of VPA:[917]VPA (50:50) every 12 hours for two and half days.  r2 Metabolites  NCI  EI  VPA  0.986  0.984  4-ene  0.999  0.997  3-ene (Z)-2-ene (E)-2-ene  0.997 0.999 1.000  0.997 0.999 0.996  (E)-2,4-diene  0.998  0.992  (E,Z)-2,3'-diene (E,E)-2,3'-diene  0.971 0.993  N.D. 0.980  4-keto 3-keto  0.987 0.998  0.987 0.998  3-0H 4-0H  0.996 0.984  0.987 0.983  5-0H  0.990  0.978  2-PSA  0.990  0.999  2-PGA  0.997  0.998  99  Table 8: Linearity (r2) of calibratipn curves for quantitative assays of VPA, VPA metabolites and their [917] labelled analogues in serum total, serum free and saliva samples of a human volunteer administered with 700 mg of VPA:VH7NPA (50:50) every 12 hours for two and half days.  Serum Total  Serum Free & Saliva  Metabolites  NCI  El  NCI  El  VPA  0.999  0.997  0.998  0.999  4-ene  0.995  0.997  0.992  0.993  3-ene  0.996  N.D.  0.992  0.991  (Z)-2-ene  0.999  1.000  1.000  0.998  (E)-2-ene  1.000  1.000  1.000  0.999  (E)-2,4-diene  0.991  0.999  0.984  0.993  E,Z 2,3'-diene  0.994  N.D.  0.992  N.D.  E,E 2,3'-diene  0.994  0.998  0.992  0.994  4-keto  0.997  0.953  0.961  0.994  3-keto  0.998  0.999  0.998  0.999  3-0H  0.995  0.960  0.992  N.D.  4-0H  0.962  0.906  N.D.  N.D.  5-0H  0.971  N.D.  0.995  N.D.  2-PSA  N.D.  0.998  N.D.  N.D.  2-PGA  0.994  0.996  0.876  0.985  100  Pharmacokinetic parameters of VPA and its metabolites were obtained based on the serum and saliva data. The elimination rate constants (KE) were obtained from the slopes of the log serum or saliva concentration  vs time plot using statistical linear regression program. Total body clearance (CL) and volume of distribution (Vd) were obtained from the following equation where area under the curve (AUC) over 12 hours after the final dose was calculated using a computer program. Dose CL = KE x Vd AUC  Figure 20 shows elimination curves of VPA and [ 2 H6]VPA in serum total, serum free and saliva which were measured with NCI techniques.  101  50  ^  100  ^  150^200  ^  250  ^  300  ^  350  HOURS 10.000  (t) 4,  1.000 (C1/7  E J'o's^0.100 o_  0.010  50  ^  100  ^  150^200  ^  250  ^  300  ^  350  HOURS  o^1.000  o c  0100 0.050  0^10^20^30  ^  40  ^  50  HOURS  Fig. 20: Elimination Curves of VPA (A ) and [2H6]VPA (I ) in serum total (top), serum free (middle) and saliva (bottom) which were measured with NCI techniques.  102  From the elimination curves shown in figure 20, [ 2 H6]VPA shows very similar pharmacokinetic behavior to VPA. Table 9 presents the pharmacokinetic parameters of both VPA and [ 2 H6]VPA measured in this volunteer under steady state conditions.  These [ 2 H6]VPA (Table 9) parameters are in agreement with the results that Acheampong  et al.  obtained for a pulse dose of [ 2 H6]VPA  (1984). The pharmacokinetic equivalency of the labelled and unlabeled VPA might indicate that the two main metabolic pathways, glucuronidation and 0-oxidation, cannot be affected by primary deuterium isotope effects in the metabolism of [ 2 H6]VPA.  To help demonstrate the equivalency of VPA and [ 2 H6]VPA with respect to metabolism  via  the fl-oxidation metabolic pathway, the time  course (12 hours) of both labelled and unlabeled 0-oxidative metabolites of VPA are illustrated in Figure 21.  Table 10 lists the terminal elimination half-life and elimination constant values of the 0-oxidation and other metabolites of VPA and [ 2 H6]VPA. Deuterium labelled 2-ene, 3-keto and 3-0H VPA have very similar elimination behaviors as their unlabeled analogues.  103  Table 9: Pharmacokinetic Parameters of VPA(I) and [2N6]VPA(II) measured by NCI technique in seruT and saliva samples of one subject after 5 oral doses of 700 mg of VPA:[ NOPA (50:50).  CL  AUC  VD  (mg.h/L)  (L/Kg)  t1/2  KE  (h)  (h-1)  (L/h/Kg)  I  19.8  0.035  0.0079  636  0.226  II  20.4  0.034  0.0078  642  0.229  I  15.1  0.046  0.134  37.22  2.92  II  14.7  0.047  0.154  32.38  3.29  I  13.6  0.051  II  13.6  0.052  Serum Total  Serum Free  Saliva  104  O ^  ^  A_A  [2HOVPA  • -• [2H6]3-keto VPA 3-0H VPA  V 2-ene VPA  v  3- keto VPA  VPA  [2H6]2-ene VPA  •  • [21i]3-0H VPA  100. 0 00  10.000 -  s  A^A  Li  t^  1.000-  • 0.10°0.050 ^ ^ ^ 0^2 6^8 10^12 HOURS  Fig. 21:^Time courses (12 hours after last dose) of labelled and unlabeled fl-oxidation m4abolites of VPA in the subject administered 5 doses of 700 mg of VPA:[H7]VPA (50:50).  105  Table 10:^Pharmacokinetic Parameters of some deuterium labelled and unlabeled metabolites of VPA measured in a healthy volunteer under steady state conditions, all data were based on NCI results.  Metabolites of VPA  Apparent t1/2^(h)  Apparent KE^(h -1 )  4-ene  15.0  0.046  [ 2 H6]4-ene  14.7  0.047  3-ene  80.4  0.0086  [ 2 4]3-ene  75.2  0.0092  (E)-2-ene  31.2  0.0222  (E)-[ 2 H6]2-ene  29.7  0.0237  2,4-diene  50.1  0.0139  [ 2 H5]2,4-diene  41.0  0.0169  (E,E)-2,3'-diene  27  0.0259  [ 2 H6](E,E)-2,3'-diene  26  0.0269  3-keto  28.5  0.024  [ 2 H6]3-keto  32.1  0.0216  4-keto  32.7  0.0212  [ 2 H6]4-keto  34.8  0.0199  3-0H  35.7  0.0194  [ 2 4]3-0H  32.8  0.0211  106  The half-life values of 3-keto and 2-ene VPA determined in this study are very close to the reported values of Pollack et al.'s studies (1986).^Basically, elimination of the metabolites of VPA were slow compared with the parent drug. ^If one or more of these metabolites exerted a significant anticonvulsant action, their presence might explain the general clinical observation of a slow onset of maximal anticonvulsant effect and a prolonged duration of action of VPA. As discussed in the introduction, the unsaturated metabolites 2-ene VPA, 3ene VPA, 4-ene VPA (Loscher, 1981; Loscher et al., 1985) and 2,3'-diene (Abbott et al., 1988) were found to have significant anticonvulsant activity in rodent models.  3.4 Isotope Effects of [ 2 H6]VPA Metabolism.  When used as a 'pulse' dose to investigate the pharmacokinetic parameters of a drug, the stable isotope labelled analogue should not show isotope effects with respect to metabolic reactions and should have the same pharmacokinetic behavior as the unlabeled analogue. There are two main reasons for these requirements. First, the pharmacokinetics of the labelled analogue must represent those of the unlabeled drug if the pulse dose method is to prove successful. Secondly and most important, the labelled drug should not be expected to switch metabolic pathways as a result of introducing a stable isotope, otherwise, severe toxicity may be caused. Because isotope effects are possible, these should be evaluated based on the labelling position of stable isotopes and mechanism of the drug metabolism, and confirmed with experiments before use of the labelled drug in patients.  107  Since [2H6]VPA has been labelled with deuterium on the terminal carbons, the two main metabolic pathways, a-oxidation and glucuronidation should not be affected by any deuterium isotope effect. However, for w-oxidation which occurs at terminal protons, isotope effects were expected for this metabolic pathway.  Potential isotope effects of [ 2 H6]VPA were investigated in the healthy volunteer from a) serum data based on the AUC ratio of [2H0]VPA and its metabolites to [ 2 H6]VPA and its deuterated metabolites, and b) urine data based on the recovery ratio of [2H0]VPA and its metabolites to their stable isotope labelled counterparts during the 12 hours following the last dose. All urine samples were measured by both El and NCI. Tables 11 and 12 show data of the serum AUC ratios and urine recovery ratios of VPA and its metabolites to their deuterium analogues. Several conclusions can be drawn from the results.  108  Table 11: Area under curve (AUC) ratios of VPA and VPA metabolites to their deuterium labelled analogs over 12 hours after the final dose in serum samples of a healthy volunteer administered 5 doses of 700 mg VPA:rHOVPA (50:50). All values were based on NCI results.  AUC^(mg.h/l)  AUC Ratio  Metabolites  2H0  2u H6  2H0/24  VPA  636.26  493.8  1.28  4-ene  1.675  1.294  1.29  3-ene  10.08  6.768  1.49*  (Z)-2-ene  6.021  4.827  1.25  (E)-2-ene  31.45  26.26  1.20  2,4-diene  1.538  1.03  1.49*  (E,E)-2,3'-diene  7.365  6.663  1.10  4-keto  3.424  1.78  1.93*  3-keto  46.84  33.11  1.41  3-0H  3.063  2.363  1.29  4-0H  3.402  2.663  1.28  5-0H  1.075  0.028  38.4*  2-PGA  1.268  N.D.  N.D.  *: Potential isotope effects based on AUC ratio.  109  Table 12:^Steady state urinary recovery molar ratio of ,VPA and its metabolites to their deuterium labelled analogs, ^[ 2H 6]VPA and metabolites in a healthy human volunteer administered 5 doses of 700 mg of VPA:[9-16]VPA (50:50), based on 12 hour urine collected following the final dose*.  Metabolites  Ratio 2 H0/ 2 H6 NCI + Base l  NCI + Enzyme 2^EI + Base 3  EI + Enzyme 4  VPA  1.12  1.21  1.10  1.09  4-ene  1.08  1.25  N.D.  N.D.  3-ene  1.29  1.20  1.34  1.22  (Z)-2-ene  0.97  1.28  1.27  1.23  (E)-2-ene  0.97  1.13  1.30  1.27  (E)-2,4-diene  1.36  1.42  1.59  1.63  (E,Z)-2,3'-diene  0.87  1.03  N.D.  N.D.  (E,E)-2,3'-diene  1.00  1.04  1.26  1.23  4-keto  0.90  1.20  0.90  1.13  3-keto  1.14  1.45  3.23  3.12  3-0H  1.10  1.15  1.27  1.67  4-0H  1.00  1.22  2.33  2.98  5-0H  6.50  6.38  6.04  5.89  2-PSA  1.11  1.00  1.20  1.20  2-PGA  15.5  16.3  14.6  15.2  N-Acetyl^Cysteine Conjugate of (E)-2,4-diene (NCI)  1.54  * Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [ 2 H6]VPA. 1. Urine samples were hydrolyzed by alkali and analyzed by NCI. 2. Urine samples were hydrolyzed by glucuronidase and analyzed by NCI. 3. Urine samples were hydrolyzed by alkali and analyzed by EI. 4. Urine samples were hydrolyzed by glucuronidase and analyzed by EI.  110  From serum data, [21-105-01-1 VPA shows prominent isotope effects, while deuterated 3-ene, 2,4-diene and 4-keto also show slight isotope effects based on this AUC ratios.  According to the urine data, a large isotope effect was observed in the metabolic formation of [ 2 1-105-OH VPA and [ 2 1-13}2-PGA, which agrees with the finding of Acheampong et al. (1984). This result was expected because 5-0H VPA and 2-PGA are the products of metabolic w-oxidation by mixed function oxidative enzymes. As mentioned in the introduction, 5OH VPA is formed via abstraction of a hydrogen from position 5 to form a carbon-centered free radical(Rettie et al., 1987). An isotope effect is predicted if the extraction of hydrogen is the rate limiting step.  Since 5-0H VPA and 2-PGA only account for a small portion of the total metabolites, the decrease in the formation of [ 2 H05-01-I VPA and [21-3]2-PGA did not markedly affect the elimination kinetics of [2H6]VPA.  No major isotope effects were observed in the other metabolic pathways, based on urine data, including the formation of 4-ene VPA, where one deuterium is lost to form the product. This result supports a recently reported mechanism for the cytochrome P-450 desaturation metabolism (Rettie et al., 1988) of VPA in which a carbon centered radical at C-4 serves as an intermediate, and this step is rate limiting in the formation of 4-ene VPA.  Surprisingly, a small isotope effect was apparent for the formation of [ 2 H6](E)-2,4-diene VPA, from both serum and urine data. ^It is  111  believed that 2,4-diene VPA is formed  via 2-ene and 4-ene VPA (Porubek  et al., 1988). The formation of [ 2 H6](E)-2,4-diene VPA should not show any isotope effect since the formation of [ 2 H6]4-ene VPA does not. It was also found by my colleague Dr. Kassahun that the ratio of the Nacetylcysteine conjugate of (E)-2,4-diene VPA to its deuterium counterpart was as high as 1.54 (Table 10), when the same urine samples were analyzed. Based on this information and our findings of a small isotope effect for 2,4-diene VPA formation from [ 2 H5]VPA, we therefore proposed that the formation of 2,4-diene VPA might occur partly from 3ene VPA. Removal of a deuterium atom from the C-5 position in [ 2 H03ene VPA may be the first step in the formation of 2,4-diene VPA. Consequently, an isotope effect is expected for this pathway, and thus might explain the observed result. An experiment was designed to test this proposal, and the results of that experiment will be discussed later.  3.5 Isotope effects with respect to [ 13 C4]VPA metabolism:  A healthy human volunteer participated in this study. He was given a single oral dose consisting of 700 mg of VPA:0 3 C4]VPA (50:50). The structure of [ 13 C4]VPA (8) is illustrated below.  CH3-CH2- 13 CN 3 CH- 13 COOH  1  ru^ 13ru 2 CH3-2 -^ (8)  112  Thirteen blood samples were collected at 0, 0.5, 1, 1.5, 2, 2.5, 3, 5, 7, 9, 12, 24, 48 and 72 h after the single dose and allowed to clot. The blood samples were centrifuged to obtain serum samples. A few urine samples were collected at convenient time blocks. All urine and serum samples were analyzed using NCI techniques, and the potential isotope effects of [13C4JVPA were studied based on the concentration ratio of VPA and its metabolites to [13C4]VPA and its metabolites in urine or serum samples. The results are presented in Table 13. No apparent isotope effects were observed, thus qualifying [ 13 C4]VPA to be used in pharmacokinetic studies of VPA metabolites in pediatric patients. The ratios of [13C0/13C4]VPA for 4-0H VPA and 5-0H VPA wre different from unity, but these differences were accounted for by a high back ground for the unlabeled VPA metabolites.  113  Table 13: Metabolic equivalence of [ 13 C4]VPAAnd VPA based on mean TIC peak area ratio of VPA metabolites to their ["C4]-labelled analogs (13 serum samples and a urine sample collected 3-9 hr after the dose from a healtbx^human^volunteer^administered^a^single^dose^of^700^mg^of VPA:["C4]VPA (50:50) were analyzed by NCI techniques).  Metabolites  • (^Co/^C4) 13^13^. Ratio Urine Serum  4-ene VPA  0.954  0.974  3-ene VPA  0.982  1.154  (Z)-2-ene VPA  0.780  0.884  (E)-2-ene VPA  1.018  1.110  VPA  1.006  0.987  2,4-diene VPA  1.020  1.112  (E,E)-2,3'-diene VPA  1.041  1.104  4-keto VPA  1.041  1.045  3-0H VPA  1.038  1.122  4-011 VPA  1.413*  1.335*  3-keto VPA  0.891  0.956  2-PGA  0.934  0.894  5-0H VPA  6.331*  1.675*  * Area ratio of 4-OH and 5-011 VPA are over 1 due to high background at the same retention time as 4-OH and 5-011 VPA when monitoring ion m/z 231.  114  3.6 Urinary recoveries of VPA and its metabolites  The urinary recoveries of VPA, [21-16]VPA and their metabolites in the study of multiple doses (one dose every 12 hr for 2 and half days) were measured in urine samples collected for 12 hours after the final dose.  This study found that VPA glucuronide and 3-keto VPA are the predominant urinary metabolites, which is consistent with previous studies (Abbott  et al.,  1986; Pollack  et al.,  1986; Dickinson  et al.,  1989) on VPA metabolites in humans. About 50% of the VPA dose is recovered as conjugated metabolites, while 25% is recovered  via  the /3-  oxidation pathway. The remaining oxidative metabolites in urine, the unsaturated and hydroxyl metabolites, together account for only a small fraction of the recovered VPA-derived products in urine.  Tables 14 to 17 present the metabolite concentrations measured by El or NCI after hydrolysis with either glucuronidase or sodium hydroxide solution. The conjugated fraction will be discussed in section 3.7.  115  Table 14: Steady state urinary recoveries of VPA, [ 2 H6]VPA and metabolites (free plus * conjugated) in the urine collected 12 hr following the final dose , hydrolyzed with NaOH solution, and analyzed by NCI GCMS.  Metabolites  VPA 4-ene 3-ene (Z)-2-ene (E)-2-ene (E)-2,4-diene (E,Z)-2,3'-diene (E,E)-2,3'-diene 4-keto 3-keto 3-0H 4-0H 5-0H 2-PSA 2-PGA  Recoveries(% of dose) ** 2 Ho  2H6  59.9 0.056 0.036 0.037 0.89 0.99 1.66 1.26 1.64 26.6 2.03 2.83 1.24*** 0.30 3.10***  53.3 0.052 0.028 0.038 0.92 0.73 1.90 1.26 1.83 23.4 1.84 2.84 0.19 0.27 0.20  * Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [ 2 H6]VPA. ** Recoveries are calculated on a molar basis. *** Qualify as an isotope effect.  116  Table 15: Steady state urinary recoveries of VPA, [2H6]VPA and metabolites (free plus ionjugated) in urine sample collected 12 hours following the final dose , hydrolyzed with glucuronidase and analyzed by NCI GCMS.  Metabolites  Recoveries(% of dose)** 2 Ho^2 H6 48.3 0.045  39.8 0.036  0.024 0.041  0.020 0.032  0.87 0.94  0.77 0.66 2.02  (E,E)-2,3'-diene  2.09 1.11  4-keto 3-keto  2.01 16.4  1.07 1.67 11.3  3-0H  2.09  1.81  4-0H 5-0H 2-PSA  4.40 1.34*** 0.29  3.60 0.21 0.29  2-PGA  3.26***  0.20  VPA 4-ene 3-ene (Z)-2-ene (E)-2-ene (E)-2,4-diene (E,Z)-2,3'-diene  * Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [2H ]VPA. ** Recoveries are calculated on a molar basis. *** Qualify as an isotope effect.  117  Table 16: Steady state urinary recoveries of VPA, [2H6]VPA and Metabolites (free plus ionjugated) in the urine collected 12 hours following the final dose , hydrolyzed with NaOH solution, and analyzed by El GCMS.  Metabolites^  Recoveries(% of dose)** 2 Ho  2u n6  VPA  45.6  41.6  4-ene  0.065  3-ene  0.055 0.047  N.D. 0.041  (Z)-2-ene (E)-2-ene  0.037  1.13  0.86  (E)-2,4-diene  0.54  0.34  (E,E)-2,3'-diene  1.45  1.15  4-keto 3-keto  2.25 29.8  2.50 9.22  3-0H  2.70 3.39  2.12  4-0H  1.45  5-0H 2-PSA  5.80***  0.96  0.24  0.20  2-PGA  2.92***  0.20  * Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [2H6]VPA. ** Recoveries are calculated on a molar basis. *** Qualify as an isotope effect.  118  Table 17:^Steady state urinary recoveries of VPA, [ 2 H6]VPA and metabolites (free plus conjugated) in the urine collected 12 hours following^the^final^dose*, hydrolyzed with glucuronidase,^and^analyzed by EI GCMS.  Recoveries(% of dose) **  Metabolites 2 Ho  24  VPA  42.1  39.2  4-ene  0.055  N.A.  3-ene  0.028  0.023  (Z)-2-ene (E)-2-ene (E)-2,4-diene  0.042 1.00  0.034 0.79  0.49  0.30  (E,E)-2,3'-diene  1.15  0.93  4-keto 3-keto 3-0H  1.31 18.7  1.16 5.98  2.11 3.70  1.26 1.24  1.59*** 0.30 3.80***  0.27 0.25 0.25  4-0H 5-011 2-PSA 2-PGA  * Steady state dose consisted of 2.43 mmol VPA and 2.33 mmol [ 2 H6]VPA. ** Recoveries are calculated on a molar basis. *** Qualify as isotope effect.  119  3.7 Conjugated Fraction of VPA and Its Metabolites in Urine Samples  VPA and most of its metabolites undergo phase II conjugation prior to excretion. The most important conjugate in urine is the glucuronic acid conjugate, which is susceptible to hydrolysis with fl-glucuronidase, and with alkali or strong acid (Dickinson  Studies by Dickinson  et al., 1985a).  et al. (1979; 1982) of the disposition of VPA  in the rat, monkey and dog revealed that, in bile and urine samples, a proportion of the total conjugated VPA, as determined by alkaline hydrolysis, was resistant to cleavage by fl-glucuronidase, suggesting that nonglucuronide conjugates were present. Further studies by Dickinson  et al. (1984) indicated that acid- and base- catalyzed  intramolecular acyl migration of the valproate moiety in valproate glucuronides away from the C-1 position could occur. In the subsequent process of ring-opening mutarotation and lactonization, six structural isomers and lactones could be formed which were not substrates for flglucuronidase, but could be hydrolyzed in strong alkaline media. The disposition of these rearranged glucuronides of the primary isomer (Dickinson  in vivo differs from that  et al., 1985a; 1985b; 1986), and it is  of some interest that abnormally high concentrations of VPA conjugates, consisting largely of the rearranged glucuronide isomers, were detected in the plasma of a patient diagnosed with VPA-associated hepatobiliary and renal dysfunction (Dickinson  et al., 1985b).  In the present study, we used both enzyme fl-glucuronidase and alkali NaOH solution to hydrolyze VPA and its metabolite conjugates.  120  Whether any glucuronidase-resistant conjugates exist or not were determined based on results of these two hydrolysis methods. The conjugated fractions were obtained from the concentration differences of recovered drug and metabolites in hydrolyzed (total) and unhydrolyzed (free) urines. All urine samples were analyzed with both the EI and NCI methods. Table 18 shows the conjugated fractions of VPA and its metabolites obtained with the different hydrolysis and assay methods.  About 95% of VPA in urine was in the form of the glucuronide conjugate and all unsaturated metabolites were excreted mainly as their glucuronide conjugates. However, other metabolites of VPA particularly polar metabolites show relatively low or no conjugation. These results are in agreement with the findings of Kassahun et al. (1989) for the excretion of VPA and metabolites in the urine of pediatric patients.  121  Table 18: Conjugated fraction (%) of VPA and its metabolites in urine samples collected for 12 hours after final dose measured by different hydrolysis and assay methods.  Metabolites^ Conjugated Fraction (%) Enzyme + NCI *^Base + NCI *^Enzyme + EI * VPA^96^97 4-ene^96^97 3-ene^79^86 (Z)-2-ene^99^99 (E)-2-ene^99^99 (E)-2,4-diene^98^98 (E,Z)-2,3'-diene^96^95 (E,E)-2,3'-diene^96^97 4-keto^34^19 3-keto^0^0 3-0H^47^45 4-0H^22^0 5-0H^22^15 2-PSA^0^0 2-PGA^7^0  * Base:^Hydrolyzed with NaOH solution, Enzyme: Hydrolyzed with glucuronidase, NCI:^Analyzed by NCI GCMS, EI:^Analyzed by EI GCMS.  93 89 61 92 89 84 N.D. 81 0 0 44 65 0 20 24  Base + EI * 93 91 80 92 90 86 N.D. 85 35 0 56 62 49 0 0  122  No apparent differences were observed between the fl-glucuronidase and alkaline catalyzed hydrolysis (Table 18), as measured by a paired ttest. Table 19 presents the t-test results. No significant difference was indicated between glucuronidase and alkaline hydrolysis (p-values: 0.4069, 0.1141), with samples analyzed with either NCI or El.^We can therefore,^assume that there was little glucuronidase-resistant conjugate present in the urine samples of this subject after being stored at -20 °C for about 2 months. A greater number of subjects would be required to give this finding statistical significance.  Table 19:^P-values of paired t-test over different hydrolysis and analysis methods.  ^ *Enzyme + NCI *Base + El ^ *Enzyme + El^0.7776 0.1141 ^ ^ * Base + NCI 0.4069 0.8115  * Base:^Hydrolyzed with NaOH solution, Enzyme: Hydrolyzed with glucuronidase, NCI:^Analyzed by NCI GCMS, El:^Analyzed by El GCMS.  123  3.8 Comparison of analysis and hydrolysis methods  As discussion above, all urine samples from the multiple dose study were hydrolyzed with both NaOH solution and glucuronidase, and analyzed by both EI and NCI techniques. The data from different method combinations were compared to see if the same results were obtained. Table 19 presents the t-test results of comparison of four groups of urine data measured by different method (hydrolysis and analysis) combination. No significant difference (p-value >> 0.05) among those four groups of data was indicated by this statistic results.  Further comparison of these four groups of data were performed on a MIDAS based computer program containing methodological statistics. Table 20 lists the correlation coefficients r 2 between those four groups of data.  Alkaline and glucuronidase hydrolysis gave very good consistency, when samples were analyzed by the NCI technique. The results from the NCI and EI methods also gave good consistency except for 5-0H VPA, when urine samples were hydrolyzed with enzyme. The poor agreement for 5-0H VPA could be due to EI analysis, since very good agreement was obtained between the two hydrolysis methods when 5-0H VPA was analyzed by NCI. Comparing these four groups of data of total urine VPA and metabolites, we can say that the combined method of base hydrolysis and EI analysis is not as good as the other three.  124  Table 20: Coefficients of determination (r2, n=11) between concentrations of VPA urine metabolites measured by different hydrolysis (base or enzyme) and analysis (NCI or El) methods. Urine samples were collected from a human,volunteer participated in the multiple doses study (700 mg of VPA : rHOVPA (50:50) every 12 hr for 2.5 days).  Metabolites  Correlation Coefficients NCI*  El*  Base^Enzyme  Unhydrolyzed*  3-ene  0.98968 0.97400  0.99668 0.97742  0.98718 0.96690  0.97977 0.95566  0.67333** 0.79158**  2-ene^(Z)-  0.96690  0.99339  0.97621  0.98726  0.35921**  2-ene^(E)VPA  0.99391  0.99846  0.98287  0.99561  0.84806**  0.97912  0.98785  0.98547  0.96284  0.81675**  2,4-diene 2,3'-diene  0.99594 0.97460  0.99777 0.99190  0.98856 0.94098  0.99397 0.94878  0.54398** 0.29505**  4-keto 3-0H 4-0H  0.95869 0.98686  0.89630 0.89371  0.95808 0.95017  0.94769 0.96591  0.97706 0.91851  3-keto  0.95681 0.96737  0.59668** 0.91728 0.99358 0.99383  0.95648 0.96563  0.87108 0.99271  5-0H  0.99854  0.52438** 0.61801** 0.62679** 0.95798  2-PSA  0.95541  0.99230  0.95195  0.95161  0.98570  2-PGA  0.99005  0.99584  0.99193  0.99827  0.98440  4-ene  NCI: Correlation between alkaline and glucuronidase hydrolysis results, when measured by NCI GCMS. El:^Correlation between alkaline and glucuronidase hydrolysis, when measured by El GCMS. Base: Correlation between NCI and El analysis methods when hydrolyzed with NaOH solution. Enzyme: Correlation between NCI and El analysis methods when hydrolyzed with glucuronidase. Unhydrolyzed: Correlation between NCI and El analysis results of **^non-conjugated components in urine samples. Poor correlation  125  For unhydrolyzed (free) urine samples, two analysis methods gave different correlation values for different metabolites. VPA and its unsaturated metabolites which exist mainly as conjugates in urine show poor agreement between the two analysis methods. Other metabolites which are not conjugated or have low levels of conjugates in urine show very good consistency when analyzed by the two different methods. Since strong acidic solution can dissociate the conjugates, it could be possible that some conjugates were hydrolyzed when urine samples were adjusted to pH=2 during extraction. The partial hydrolysis results in the variation of free fraction of VPA and its unsaturated metabolites that are excreted into urine mostly as their conjugated forms.  3.9 A Pharmacokinetic study of VPA in sheep using [ 13 C4]VPA  The pharmacokinetics of VPA in sheep were studied by giving a single dose (I.V.) of 1000 mg of VPA:[ 13 C4]VPA (50:50) to each of two sheep.^Blood samples were collected and allowed to clot before centrifuging to collect serum samples. ^All serum samples were then analyzed by EI GCMS quantitatively, using the [ 2 H7]VPA and its metabolites that were synthesized as internal standards. When stable isotope-labelled analogs are applied as internal standards, SIM mode is the ideal method for quantitative analysis. Figure 21 illustrates the SIM chromatograms of VPA [ 13 C4]VPA and [ 2 H7]VPA. These three analogs show very sharp peaks and can be readily differentiated based on their ion masses. As mentioned in the introduction, there should be no natural isotope interference since the mass difference is equal or higher than 3 mass units. The separation and quantitation of VPA, VPA  126  metabolites and their C-13 labelled analogs were completed in a single chromatographic run of 29.5 minutes in length, C-13 labelled metabolites had very similar retention time as their unlabeled analogs, with about 0.01 min difference. Table 21 presents the retention times and m/z values of the (M-57)1- diagnostic ions for VPA and its metabolites.  127  Table 21: The retention time and m/z values of the (M-57)+ diagnostic ions of VPA and its metabolites iso14ed from serum samples of sheep dosed with single dose of 1 g of VPAT'COVPA (50:50).  Metabolites*  Retention time  Ion monitored  (min)  (m/z)  VPA  13.64  201  4-ene VPA  13.88  199  (E)-3-ene VPA  14.05  199  (Z)-3-ene VPA  14.29  199  (Z)-2-ene VPA  16.58  199  (E)-2-ene VPA  14.64  199  (Z)-2,4-ene VPA  17.05  197  (E)-2,4-diene VPA  17.78  197  (E,Z)-2,3'-diene VPA  17.80  197  (E,E)-2,3'-diene VPA  18.71  197  3-keto VPA  21.79  329b  4-keto VPA  20.03  215  3-0H VPA  19.66, 19•93a  217  4-0H VPA  12.21, 12•41a  100c  5-0H VPA  22.14  331b  2-PSA  22.18  331b  2-PGA  22.50  345b  Two isomers a Diderivatives b 7-Lactone c *^ 13 [ C4]VPA and its metabolites were monitored by SIM with diagnostic ions which were four mass units higher than those of their unlabeled analogs.  128  Abundance  Ion 208.00: 1001010.D  150000  100000  50000  0 Time -> Abundance  11.00  12.00  15.00 13.00 14.00 Ion 201.00: 1001010.D  16.00  17.00  11.00  12.00  15.00 13.00 14.00 Ion 205.00: 1001010.D  16.00  17.00  150000  100000  50000  Time -> Abundance 150000-  100000-  50000-:  Time ->^11.00^12.00^13.00^14.00^15.00^16.00^17.00  Fig. 22:^SIM Oromatograms of [ 2 H7]VPA (top, internal standard), VPA (middle), and [ IJ C4]VPA (bottom).  129  Excellent standard curves were obtained for most of the metabolites. The coefficients of determination obtained for calibration curves of metabolites isolated from standard urine samples are presented in Table 22.  As Table 22 shows, the calibration curves have very good linearity. The coefficients of determination r2 for all metabolites, were greater than 0.994, except for 3-0H and 3-keto VPA. Calibration curves for 3-0H and 3-keto VPA could be expected to improve had [2H7]3-0H VPA and [2H7]3-keto VPA been used as internal standards. These two internal standards were not synthesized until after this analysis had been completed. Our results thus support the contention that optimal calibration curves are obtained by using stable isotope-labelled analogues as internal standards when samples are analyzed with GCMS. Unfortunately, [2H7]4-ene VPA could not be used in this assay because of interference from [13C4]VPA with a mass difference of only I mass units. Retention times were very close and the sample contained a high concentration of [ 13 C4]VPA. Thus to apply [2H7]4-ene VPA as an internal standard, some modification in the GC conditions would be necessary to adequately separate VPA and the 4-ene VPA peaks. The NCI method should be suitable for application of [2H7]4-ene VPA as an internal standard because the 4-ene precedes VPA in retention time.  130  Table 22: Linearity of calibration curves for the quantitative assays of VPA, VPA metabolites and their C-13 labelled analogues isolated from urine,,samples of sheep dosed I.V. with a single dose of 1000 mg of VPA:["C4]VPA (50:50).  Metabolites *^r 2 VPA^  ^  Internal Standards  1.000^[2H7]VPA  4-ene VPA^ 0.999^[2H7]VPA 3-ene VPA^ 0.999^[2H7]VPA (Z)-2-ene VPA^0.999^[2H7]VPA (E)-2-ene VPA^0.999^[2H7]VPA (E)-2,4-diene VPA^0.999^[2H7]VPA (E,Z)-2,3'-diene VPA^0.999^[2H7]VPA (E,E)-2,3'-diene VPA^0.996^[2H7]VPA 3-keto VPA^0.975^[2H7]4-keto VPA 4-keto VPA^0.999^[2H7]4-keto VPA 3-0H VPA^ 0.988^[2H7]VPA 4-0H VPA^ 0.996^[2H7]VPA 4-0H VPA isomer^0.990^[2H7]VPA 5-0H VPA^ 0.994^[2H7]5-0H VPA 2-PGA^  0.997^[2H7]VPA  *^[ 13 C4]VPA and its metabolites were analyzed using the calibration curves for the unlabeled analogs.  131  Pharmacokinetic parameters for VPA, [13C4]VPA and the major serum metabolites, 2-ene VPA and [13C4]2-ene VPA were determined for the two sheep that were studied. The results are presented in table 23.  Table 23: Pharmacoktnetic Parameters for VPA(I), [13C4]VPA(II), (E)-2ene VPA (III) and ["C4](E)-2-ene VPA (IV) meamred in two sheep dosed I.V. with a single dose of 1000 mg of VPAT'CWPA (50:50). Serum samples were analyzed by El GCMS.  t1/2  KE  CL  AUC  VD  (h)  (h-1)  (L/h/Kg)  (mg.h/L)  (L/Kg)  I  5.09  0.136  0.122  55.9  0.897  II  4.98  0.139  0.127  53.42  0.913  III  5.63  0.123  IV  5.63  0.123  I  2.42  0.286  0.159  52.5  0.556  II  2.41  0.287  0.160  52.0  0.557  III  4.33  0.160  IV  3.89  0.178  Sheep 411  Sheep #2  132  No apparent isotope effect of [ 13 C4]VPA metabolism was observed in these two sheep based on AUC ratio of VPA to [ 13 C4]VPA, which is in agreement with the result we obtained from the study in a healthy human volunteer.  Since few studies of VPA pharmacokinetics have been carried out in sheep, very little information is available on the metabolic fate of valproic acid in this species. Nevertheless, this initial study of [ 13 C4]VPA indicates that this compound should prove ideal for the study of placental transfer and the determination of drug pharmacokinetics in both mother and fetus.  3.10 Metabolic studies of (Z)-and (E)-3-ene VPA  As discussed in the section 3.4., it was suggested that, 2,4-ene VPA may be partially formed from 3-ene VPA. To further investigate this hypothesis, we synthesized (Z)-3-ene and (E)-3-ene VPA for metabolism studies in rats.  The syntheses of (E)- and (Z)-3-ene VPA were discussed in section 3.1.9. Both (E)-and (Z)-3-ene VPA were analyzed by GCMS and by NMR and determined to be of very high purity before they were used in metabolism studies.  (Z)- and (E)-3-ene VPA (150 mg/kg dose) were separately administrated i.p. to two rats (adult male Wistar rats weighing 270 and 308 g). A blood sample (about 200 ul) from each rat was taken 2 h after  133  the dose, allowed to clot, and centrifuged to obtain a serum sample. Urine samples were collected every 24 hours for two days. Samples were stored at -20 °C until derivatized and analyzed by GCMS using El techniques.  In the SIM mode used, m/z 197 and 199 were monitored to see the diene VPA metabolites and the parent drugs (E)- and (Z)-3-ene VPA. From the assay results, two diene VPA metabolites were present in urine samples of both sheep, one metabolite was confirmed to be (E,E)-2,3'diene VPA. The other diene VPA could be either (E,Z)-2,3'-diene VPA or 2,4-diene VPA since they had very close retention time under El analysis conditions. It was likely that (E,E)-2,3'-diene VPA was the main diene metabolite of (E)-3-ene VPA, while (E,Z)-2,3'-diene or 2,4-diene VPA accounts for most of the diene metabolites of (Z)-3-ene VPA. Table 24 shows the peak area of all the ions monitored in urine samples. Since (E)-2,4-diene VPA and (E,Z)-2,3'-diene VPA were not differentiated well under that condition, it is not apparent that (E)-2,4-diene VPA is one of metabolites of 3-ene VPA. Further analysis by using NCI mode has to be done before making a final conclusion.  The t-BDMS derivative of (Z)-3-ene VPA had a slightly longer retention time than that of (E)-3-ene VPA, when run on an OV-1701 column.^It is usually the (E)-isomer of unsaturated fatty acids that has the longer retention time with this stationary phase. ^The reason for this reversal of elution order by the (E)- and (Z)- isomers of 3-ene VPA is not readily apparent.  134  Table 24:^Retention time and peak area of monitored^ions m/z^199 and 197 which represent parent drug 3-ene VPA and diene metabolites respectively isolated from urine of rats dosed with either (Z)- or (E)3-ene VPA (150 mg/kg).  Metabolites^(tR)  JZ)-3-ene VPA 0-24h^24-48h  jE)-3-ene VPA 0-24h^24-48h  (Z)-3-ene VPA (8.53 min)  178442944  21974892  N.D.  N.D.  (E)-3-ene VPA (8.38 min)  N.D.  N.D.  243174752  9520370  * (E,Z)-2,3'-diene VPA (10.82 min)  66036848  3494949  4874712  118144  (E,E)-2,3'-diene VPA (12.83 min)  21874212  1503089  17302344  548160  *  or (E)-2,4-diene VPA  135  4. Summary and Conclusions  4.1 GCMS conditions for both El and NCI were optimized to obtain optimal resolution and sensitivity for VPA metabolites and their stable isotope-labelled analogs. A single temperature program with a run time of 47 min was established for NCI analysis of PFB derivatives of VPA, VPA metabolites and their [24] labelled analogs. Two temperature programs were investigated for t-BDMS derivatives of VPA and VPA metabolites and their [24] labelled analogs, one with a run time of 35 min was used for VPA unsaturated metabolites, the other with a run time of 20 min was for more polar metabolites of VPA.  4.2 In the 12h period immediately following the dose, t112 and AUC values for VPA and [2H6]VPA in the human volunteer were not different. AUC values and urinary recoveries of unlabeled and labelled metabolites over the same time period were equivalent except for 5-0H VPA and 2propylglutaric acid (2-PGA). Significant isotope effects were observed for 5-0H VPA and 2-PGA. No apparent isotope effect was observed for 4ene VPA, which supported the mechanism that a carbon centered radical at C-4 of VPA serves as an intermediate, and this step is rate limiting in the formation of 4-ene VPA.  A small isotope effect was observed for 2,4-diene VPA. It was then proposed that the formation of 2,4-diene VPA might occur partly from 3ene VPA. An experiment was designed to test this proposal.  136  4.3 (E)- and (Z)- 3-ene VPA were synthesized. Both were used in metabolic studies in rats. The metabolites diene VPA and parent drug 3ene VPA were monitored in urine samples of rats dosed with either (E)or (Z)-3-ene VPA by SIM EI mode. Both (E)- and (Z)- isomers of 3-ene VPA were shown to have two diene VPA metabolites. One is confirmed to be (E,E)-2,3'-diene VPA, the other could be either 2,4-diene VPA or (E,Z)-2,3'-diene VPA since they had the same retention time. The (E)-3ene VPA had a higher level of metabolite (E,E)-2,3'-diene VPA, while (Z)-3-ene VPA was metabolized into more (E,Z)-2,3'-diene VPA (or 2,4diene VPA).  4.4 More than 90% of VPA and its unsaturated metabolites (2-ene, 3-ene, 4-ene, 2,4-diene and 2,3'-diene-VPA) were in the form of their glucuronic acid conjugates when excreted into urine. Metabolites 3-0H, 4-0H and 5-0H VPA were excreted partly as glucuronides, while, 3-keto, 4-keto VPA, 2-PSA and 2-PGA were excreted mostly as free metabolites.  4.5 No difference was observed between the concentrations of total VPA and its metabolites when urine samples were hydrolyzed with glucuronidase and alkali NaOH solution. Therefore, it seems that there was no glucuronidase-resistant conjugate present in the urine samples of this healthy human volunteer after urine samples were kept at -20 °C for about two months.  NCI and EI analyzing techniques show very good agreements for most metabolites in urine samples.^PFB derivatives of VPA metabolites  ^  137  analyzed by NCI technique give higher sensitivity and better resolution than t-BDMS derivatives of VPA metabolites analyzed by El methods.  4.6 No apparent isotope effect was observed in the metabolism of [13C4]VPA based on concentration ratios of VPA and its metabolites to their [13C4] labelled analogs in serum and urine samples of a healthy human volunteer. Thus it would appear that [ 13 C4]VPA is suitable for the pharmacokinetic studies of VPA metabolites in pediatric patients when given as a "pulse" dose.  4.7^Eight deuterium labelled compounds that included [2H7]VPA, [2H7]4-ene , [2H7]4-keto, [2H7]4-0H, [2H7]5-0H, [2H7]3-keto, [2H7]3-0H, and [2H7]2-ene VPA were synthesized and used as internal standards in a GCMS assay of VPA, [ 13 C4]VPA and their metabolites for the pharmacokinetic studies in sheep.  4.8^Pharmacokinetics of VPA was studied in sheep dosed with VPA:[13C4]VPA (50:50). ^No isotope effect was observed for the [13C4JVPA.^The elimination half-life of VPA in these two sheep was estimated to be approximately 5.0 and 2.4 hr respectively.  138  5. References Abbott, FS., and Acheampong, AA., (1988) Quantitative structureanticonvulsant activity relationships of valproic acid, related carboxylic acids and tetrazoles, Neuropharmacology, 27, 287. 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