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Pharmacokinetics, tissue distribution and serum protein binding of (E,Z)-2,3’-diene VPA in rats Moshenko, Janice Lynn 1996

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P H A R M A C O K I N E T I C S , TISSUE DISTRIBUTION A N D S E R U M P R O T E I N BINDING O F (E,Z)-2,3'-DIENE V P A IN R A T S by JANICE LYNN MOSHENKO B.Sc. (Pharmacy), University of British Columbia, 1991 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 September, 1996 © Janice Lynn Moshenko, 1996 In presenting this thesis in partial fulfilment of the requirements for an advanced degree at the University of British Columbia, I agree that the Library shall make it freely available for reference and study. I further agree that permission for extensive copying of this thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. It is understood that copying or publication of this thesis for financial gain shall not be allowed without my written permission. Department of CVVa^c^£ce-oAi.egA ^3>cX<ar\r The University of British Columbia Vancouver, Canada Date - S ^ p - ^ • ^ l ^ j b DE-6 (2/88) 11 ABSTRACT Valproic acid (VPA) is a broad spectrum anticonvulsant drug presently used for the management of various seizure disorders. Despite its reputation for being a relatively safe antiepileptic, it has been characterized by two potentially fatal side effects, namely hepatotoxicity and teratogenicity. Hence, the development of analogues of VPA lacking the toxic side effects but possessing the desired attributes of conventional anticonvulsants would be of great benefit therapeutically. One interesting observation with VPA therapy is a delayed anticonvulsant effect that occurs after discontinuation of VPA and after VPA has been cleared from the systemic circulation. Since VPA is extensively metabolized and its metabolites have been shown to possess anticonvulsant activity, the possibility exists that one or more of VPA's active metabolites may be contributing to the prolongedpharmacological activity of VPA. One such metabolite, (E,Z)-2,3'-diene VPA, has been of interest in our laboratory. This diene was preferentially found in rat brain after administration of VPA. Preliminary investigations with this compound demonstrated that it possessed equivalent anticonvulsant activity to VPA using the PTZ seizure test in rats and mice, and minimal muscle spasticity and sedative effects when compared to VPA. These studies suggested that this diene may have potential as an alternative medication to VPA. Therefore, investigations into the pharmacokinetics and disposition of (E,Z)-2,3'-diene VPA in rats were conducted in order to assess the diene's contributions to VPA activity and its potential as an alternative medication to VPA. Ul After successful synthesis of (E,Z)-2,3'-diene VPA, pharmacokinetic, tissue distribution, and serum protein binding experiments were initiated. Serum protein binding of (E,Z)-2,3'-diene VPA was determined and was found to be concentration-dependent. Saturation of serum proteins both in vitro and ex vivo occurred at concentrations in excess of 65 [ig/rnL. In order to investigate the dose-related pharmacokinetics of (E,Z)-2,3'-diene VPA, the compound was administered i.p. at three different dose levels to rats and the serum elimination profile determined. Enterohepatic circulation was apparent for all three dose levels. Dose-dependent kinetics was observed among the pharmacokinetic parameters, and was attributed to serum protein binding, saturable metabolism, and/or changes due to enterohepatic circulation. The excretion of conjugated and parent (E,Z)-2,3'-diene VPA was determined in the urine and bile of rats. Rats were administered 75, 150 and 300 mg/kg of the compound and urine collected. The fraction of the dose excreted in urine as parent drug remained relatively unchanged as dose increased, yet the fraction of the dose excreted as glucuronides increased with increasing dose. Glucuronide excretion in urine remained relatively unchanged as dose increased from 150 to 300 mg/kg, suggesting saturable elimination via the glucuronide pathway. Bile samples were collected from rats administered 150 mg/kg i.p. of (E,Z)-2,3'-diene VPA over 6 h. The drug was excreted as glucuronides in bile, although to a lesser extent than that of VPA, and induced a choleretic effect that appeared to be related to the amount of glucuronides produced. iv After single dose administration of (E,Z)-2,3'-diene VPA to rats, the elimination profiles in serum, liver, whole remaining brain and eleven brain regions were determined. The regional distribution of (E,Z)-2,3'-diene VPA in the brain was relatively homogenous. At low brain concentrations, the diene persisted in 10 of 11 brain regions. Braimserum concentration ratios remained below unity, suggesting that an active fatty-acid transport mechanism may play a role in diene transport and that transport of the drug out of the brain exceeds transport into the brain. Based on the AUC values, a low liver distribution was observed for the diene, which was approximately 1/2 that found in serum. Liver: serum concentration ratios were found to decrease over time. Liver AUC values were markedly lower than that reported for VPA at equivalent doses, suggesting that (E,Z)-2,3'-diene VPA may be potentially less hepatotoxic than VPA. The relationship between serum (E,Z)-2,3 '-diene VPA concentrations and brain and liver tissue concentrations was investigated. A nonlinear distribution pattern was observed for brain and liver tissues when compared to total serum, while the diene concentrations in brain and liver increased linearly with corresponding free serum levels. Concentration-dependent serum protein binding of the diene may play a significant role in the brain and liver distribution of the diene in rats and may be responsible for the nonlinear distribution patterns observed in these tissues. The results from these studies indicated that (E,Z)-2,3'-diene VPA reflects certain desired attributes of an antiepileptic drug which further strengthens our contention that the diene may be a suitable candidate for development as an anticonvulsant drug. v T A B L E O F C O N T E N T S Page A B S T R A C T ii T A B L E O F C O N T E N T S v L I S T O F T A B L E S x L I S T O F F I G U R E S xi L I S T O F S C H E M E S xiv L I S T O F A B B R E V I A T I O N S xv A C K N O W L E D G M E N T S xviii 1. INTRODUCTION 1 1.1 Overview 1 1.2 Mechanism of Action of V P A • 2 1.3 Metabolism and Toxicity of V P A 4 1.4 Chemical Derivatization and Analysis of V P A Using G C M S 9 1.5 Pharmacokinetics and Pharmacodynamics of V P A 10 1.6 The Potential Role of (E)-2-ene V P A , (E,E)-2,3'-diene V P A and 14 (E,Z)-2,3'-diene V P A as Alternative Therapy to V P A 1.7 Objectives 20 1.7.1 Specific Obj ectives 20 vi 2. M A T E R I A L S AND M E T H O D S 22 2.1 Supplies 22 2.1.1 Chemicals and Materials 22 2.1.2 (E,Z)-2,3'-diene VPA and Internal Standard Solutions 23 2.1.3 Animals 23 2.2 Instrumentation 24 2.2.1 Centrifuges 24 2.2.2 Gas Chromatography-Mass Selective Detection 24 2.3 Animal Experiments 25 2.3.1 Dose-Dependent Pharmacokinetics and Metabolic Study of 25 (E,Z)-2,3'-diene VPA 2.3.1.1 Preparation of Drug Solutions for Injection 25 2.3.1.2 Serum Collection 25 2.3.1.3 Urine Collection 26 2.3.1.4 Bile Collection 26 2.3.2 Pharmacokinetics and Tissue Distribution in Brain, Serum and Liver 27 2.3.2.1 Preparation of Drug Solution for Injection 27 2.3.2.2 Tissue Collection 27 2.3.2.3 Brain Dissection Procedure 27 2.4 Analytical Procedures 29 2.4.1 Preparation of Brain and Liver Samples for Drug Assay 29 2.4.2 Protein Binding of (E,Z)-2,3'-diene VPA 30 2.4.2.1 Ex Vivo Protein Binding 30 / vii 2.4.2.2 In Vitro Protein Binding 30 2.4.2.3 Binding of (E,Z)-2,3'-diene VPA to the YMT Membrane 31 2.4.3 Calibration Curves 31 2.4.4 Extraction and Derivatization 33 2.4.4.1 Bile and Urine Samples 33 2.4.4.2 Brain and Liver Samples 34 2.4.4.3 Serum and Ultrafiltrate Samples 34 2.4.5 Extraction Efficiency 36 2.5 Data Analysis 37 2.5.1 Calculation of Pharmacokinetic Parameters 37 2.5.2 Computer Modelling 37 2.5.3 Statistics 40 3. R E S U L T S 41 3.1 Analysis of (E,Z)-2,3'-diene VPA by EI-GCMS 41 3.1.1 Chromatography and Method of Detection of (E,Z)-2,3'-diene VPA 41 3.1.2 Calibration Curves, Inter-Assay Variation and Analytical Recoveries 43 3.2 Serum Protein Binding of (E,Z)-2,3'-diene VPA 45 3.2.1 Ex Vivo Protein Binding 45 3.2.2 In Vitro Protein Binding 45 3.3.3 Binding of (E,Z)-2,3'-diene VPA to the Ultrafiltration Apparatus 46 and YMT Membrane 3.3 Dose-Dependent Pharmacokinetics and Metabolic Study of 49 (E,Z)-2,3'-diene VPA viii 3.3.1 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Serum 49 3.3.2 Excretion of (E,Z)-2,3'-diene VPA and Conjugated 53 (E,Z)-2,3'-diene VPA in Urine 3.3.3 Excretion of (E,Z)-2,3'-diene VPA and Conjugated 53 (E,Z)-2,3'-diene VPA in Bile 3.4 Pharmacokinetics and Tissue Distribution of (E,Z)-2,3'-diene VPA in 57 Serum, Brain and Liver 3.4.1 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Serum 57 3.4.2 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Whole Remaining 61 Brain and Selected Brain Regions 3.4.3 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Liver 62 3.4.4 BraimSerum Concentration Ratios and the Relationship Between 65 Brain, Free Serum and Total Serum Concentrations 3.4.5 Relationship Between Liver, Free Serum and Total Serum 70 Concentrations 4. DISCUSSION 7.3 4.1 Analysis of (E,Z)-2,3'-diene VPAbyEI-GCMS 74 4.2 Serum Protein Binding of (E,Z)-2,3'-diene VPA 76 4.3 Dose-Dependent Pharmacokinetics and Metabolic Study of 79 (E,Z)-2,3'-diene VPA 4.3.1 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Serum 79 4.3.2 Excretion of (E,Z)-2,3'-diene VPA and Conjugated 85 (E,Z)-2,3'-diene VPA in Urine 4.3.3 Excretion of (E,Z)-2,3'-diene VPA and Conjugated 86 (E,Z)-2,3'-diene VPA in Bile 4.4 Pharmacokinetics and Tissue Distribution of (E,Z)-2,3'-diene VPA in 88 Serum, Brain and Liver ix 4.4.1 Profile of (E,Z)-2,3'-diene VPA in Serum 90 4.4.2 Profile of (E,Z)-2,3'-diene VPA in Whole Remaining Brain and 92 Selected Brain Regions 4.4.3 Profile of (E,Z)-2,3'-diene VPA in Liver 99 5 . C O N C L U S I O N S 103 6. R E F E R E N C E S 106 7. A P P E N D I C E S 122 X LIST OF TABLES Table Page 1 The mean effective doses against PTZ-induced seizures and the slopes 19 of the log dose-response plots for each compound tested in male Sprague-Dawley rats. 2 An example of the dilution protocol used for the preparation of a high 32 range calibration curve for analysis of (E,Z)-2,3'-diene VPA content in liver tissue. 3 Inter-assay variation based on the slopes of the calibration curves over 44 a 12 month period for (E,Z)-2,3'-diene VPA. 4 Ex vivo serum protein binding of (E,Z)-2,3'-diene VPA by ultrafiltration at 47 25 °C. 5 In vitro serum protein binding of (E,Z)-2,3'-diene VPA by ultrafiltration at 47 25 °C. 6 Apparent pharmacokinetic parameters in serum for three dose levels of 52 (E,Z)-2,3'-diene VPA in rats. 7 Calculated pharmacokinetic parameters in serum for three dose levels of 52 (E,Z)-2,3'-diene VPA in rats. 8 Apparent pharmacokinetic parameters in serum, whole remaining brain and 60 liver following i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene VPA in rats. 9 Apparent pharmacokinetic parameters in whole remaining brain and 11 brain 64 regions following i.p. administration of 150 mg/kg (E,Z)-2,3'-diene VPA in rats. 10 The time (tm ax) to maximum peak concentration (Cmax) and area under the 99 curve (AUCo-ioh) in brain tissues following i.p administration of equivalent doses of 150 mg/kg of either VPA, (E)-2-ene VPA, or (E,E)-2,3'-diene VPA to Sprague-Dawley rats (n=8). XI LIST OF FIGURES Figure Page Chemical structures of VPA and three of its metabolites that are under 18 investigation as possible alternatives to VPA therapy. Section lines used for dissection of rat brain; dorsal view on left, ventral 28 on right. The location of the representative lines courtesy of Zeman and Innes (1963). Time-lag two compartment pharmacokinetic model of (E,Z)-2,3'-diene VPA 38 in rats after an i.p. dose of 75, 150, or 300 mg/kg of (E,Z)-2,3'-diene VPA. Time-lag four compartment pharmacokinetic model of (E,Z)-2,3'-diene VPA 39 in rats after an i.p. dose of 150 mg/kg of (E,Z)-2,3'-diene VPA. Electron-ionization mass spectrum of a f-BDMS derivative of 42 (E,Z)-2,3'-diene VPA. Sample electron-ionization mass chromatograms of: 42 (A) (E,Z)-2,3'-diene VPA (m/z 197) (B) [2H7]-2-ene VPA (m/z 206). A representative calibration curve used for the quantitation of high levels 44 of (E,Z)-2,3'-diene VPA in rat serum. 8 Relationship between free serum and total serum concentrations of 48 (E,Z)-2,3'-diene VPA in vitro and ex vivo. xii 9 Concentration-time plot of (E,Z)-2,3'-diene VPA in serum following the i.p. 51 administration of 75, 150 and 300 mg/kg of (E,Z)-2,3'-diene VPA to rats. 10 Amount of (E,Z)-2,3'-diene VPA excreted in urine as unchanged and 55 conjugated drug following i.p. injection of 75, 150 or 300 mg/kg of (E,Z)-2,3'-diene VPA. 11 (E,Z)-2,3'-Diene VPA conjugate excretion and bile flow rate following the i.p. 56 administration of 150 mg/kg of (E,Z)-2,3'-diene VPA. 12 Concentration-time curve of (E,Z)-2,3'-diene VPA in rat serum, liver and brain 59 following the i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene VPA. 13 Concentration-time curves of (E,Z)-2,3'-diene VPA in rat serum (SER), 63 cerebellum (CER), medulla (MED), pons (PONS), corpus striatum (CS), hippocampus (HIP), olfactory bulb (OB), corpus callosum (CC), frontal cortex (FC), inferior colliculus (IC), superior colliculus (SC), whole remaining brain (WRB), and substantia nigra (SN) following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene VPA. 14 Braimtotal serum ratios of (E,Z)-2,3'-diene VPA in cerebellum (CER), 66 medulla (MED), pons (PONS), corpus striatum (CS), hippocampus (HIP), olfactory bulb (OB), corpus callosum (CC), frontal cortex (FC), inferior colliculus (IC), superior colliculus (SC), whole remaining brain (WRB), and substantia nigra (SN) of rats at tmax and t]2h following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene VPA. 15 Tissue concentrations relative to total and free serum concentrations of 67 (E,Z)-2,3'-diene VPA in whole remaining brain (WRB) and liver (LIV) at tmax (ranging from 7.5 to 22.5 min) and tL2h following 150 mg/kg i.p. administration of the compound. 16 Rat whole remaining brain concentrations versus free serum concentrations of 68 (E,Z)-2,3'-diene VPA following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene VPA to rats. 17 Rat whole remaining brain concentration versus total serum concentration of 69 (E,Z)-2,3'-diene VPA following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene VPA to rats. 18 Rat liver concentrations versus free serum concentrations of (E,Z)-2,3'-diene VPA following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene VPA to rats. 71 Rat liver concentrations versus total serum concentrations of (E,Z)-2,3'-diene VPA following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene VPA to rats. xiv LIST OF SCHEMES Scheme p a g e 1 Proposed metabolic pathways for VPA. 8 2 General extraction and derivatization scheme for the analysis of unconjugated 35 drug in biological fluids and liver or brain homogenate. XV LIST OF ABBREVIATIONS AUC area under the concentration-time curve P elimination rate constant °C degrees Celsius CC corpus callosum CER cerebellum Cl clearance C m a x peak drug concentration CNS central nervous system CS corpus striatum CV coefficient of variation E trans EHC enterohepatic recirculation EI electron ionization EV electron volt FC frontal cortex GABA gamma aminobutyric acid GC gas chromatography h hour HC1 hydrochloric acid HIP hippocampus IC inferior colliculus I.D. internal diameter i.p. intraperitoneal i.v. intravenous kg kilogram LOQ limit of quantitation M + molecular ion MCA monocarboxylic acid MED medulla |lg microgram mg milligram min minute mL millilitre MSD mass selective detection MTBSTFA N-methyl-N-(rer?-butyldimethylsilyl)-trifluoroacetamide m/z mass/charge NaOH sodium hydroxide OB olfactory bulbs PONS pons psi pounds per square inch PTZ pentylenetetrazole r2 coefficient of determination SC superior colliculus SD standard deviation SIM selected ion monitoring SN substantia nigra t|/2 half life tmax time to reach peak drug concentration r-BDMS terf-butyl dimethyl silyl V c volume of the central compartment VPA valproic acid WRB whole remaining brain Z cis 2,3'-diene VPA 2-(l'-propenyl)-2-pentenoic acid 2-ene VPA 2-n-propyl-2-pentenoic acid A C K N O W L E D G M E N T S I would like to express my gratitude to my supervisor, Dr. Frank Abbott, for his excellent support, guidance, and encouragement throughout these studies. I am sincerely grateful to the committee members Dr. Stelvio Bandiera, Dr. Kathleen MacLeod, Dr. Keith McErlane and Dr. Wayne Riggs for their time and helpful contributions. A special thanks go to Mr. Roland Burton for his assistance with the GCMSD, Dr. Mark Klitenick at the Department of Neurology at U.B.C. for the assistance and demonstrations with rat brain dissections, our summer student, Mr. Steven Ma, for his contributions with the serum protein binding experiments, and to Mr. Sanjeev Kumar, Mr. Harvey Wong and Mr. John Kim for their help with computer modelling and the GCMSD. My sincere thanks to my colleagues Dr2. Wei Tang, Dr. Jan Palaty, Dr. Anthony Borel, Mr. Ali Tabatabaei, and Ms. Sashi Gopaul for their assistance, constant support and helpful advice. I would also like to thank my family and friends who heard my numerous complaints about rats and late night experiments but were always there for support. Financial support was provided by PMAC-Health Research Foundation, Berlex Biosciences, and The Faculty of Pharmaceutical Sciences and is gratefully acknowledged. 1 1. INTRODUCTION 1.1 OVERVIEW Valproic acid (VPA) is a broad-spectrum antiepileptic drug that was fortuitously discovered by Meunier et al. in 1963 to possess anticonvulsant properties. It is a unique anticonvulsant in that its simple branched-chain fatty acid structure differs dramatically from the structure of conventional anticonvulsant medications. Marketed in 1978 under the trade name Depakene®, VPA was quickly recognized as a highly effective medication as sole or adjunctive therapy for the management of generalized (absence, tonic-clonic, myoclonic) partial (simple, complex, secondary generalized) and compound/combination seizures (Davis etal, 1994). VPA is generally well tolerated with minimal adverse effects, including dyspepsia, heartburn, nausea, vomiting, anorexia and weight gain (Davis et al, 1994). However, the use of VPA has been limited due to the infrequent incidence of a potentially fatal hepatotoxicity, idiosyncratic in nature, with greatest susceptibility in pediatric patients and patients receiving polytherapy (Dreifuss et al, 1987; 1989; Scheffner et al, 1988; Kondo et al, 1990; Bryant and Dreifuss, 1996). In addition, the use of VPA is further compromised due to its teratogenic potential (Omzigt etal, 1992). Since the properties of VPA are believed to be related to its branched-chain fatty acid-like structure, various analogues of VPA have been developed and investigated for their potential as alternative anticonvulsants to VPA without the harmful side effects. 2 Preferentially, an analogue that exhibits similar attributes as VPA, namely equivalent efficacy, low neurotoxicity and low risk of hepatotoxic and teratogenic effects would be of great benefit therapeutically. One of the minor metabolites of VPA, (E,Z)-2,3'-diene VPA, is of interest in our laboratory and is the topic of this thesis. 1.2 MECHANISM OF ACTION OF VPA The mechanism of action of VPA in the treatment of epilepsy has not been fully elucidated, although several investigations have demonstrated the involvement of brain synaptosomal y-aminobutyric acid (GABA), one of the inhibitory neurotransmitters in the CNS. GABA functions by binding to the GABA A receptor after its release from presynaptic neurons, resulting in an influx of chloride ions and subsequent hyperpolarization of the post-synaptic membrane (McNamara, 1996). One theory that has been postulated in an effort to explain the mechanism of action of VPA is a reduction in GABA-transaminase (GABA-T), the first enzyme in the GABA degradation pathway, which subsequently results in an elevation of GABA levels. An initial study by Godin et al. (1969) demonstrated a pronounced in vitro inhibition of GABA-T by VPA. Additional research with mouse synaptosomes reported increased GABA levels due to a competitive inhibition of GABA-T, although the reduction in activity of this synaptosomal enzyme was not very large (Loscher, 1981; Loscher, 1993). 3 Studies have also suggested that VPA is a potent inhibitor of the second enzyme in the GABA metabolic pathway, namely succinic semialdehyde dehydrogenase (SSA-DH). Van der Laan et al. (1979) reported that VPA exhibits an inhibitory effect on this enzyme in vitro. The results suggested that increased levels of GABA levels in the brain result from inhibition of SSA-DH, which may result from two mechanisms: 1) increased levels of succinic semialdehyde (the first metabolic product of GABA metabolism) initiates the reverse reaction of GABA-T, subsequently increasing GABA levels, or, 2) the increased levels of succinic semialdehyde inhibit GABA degradation, thus indirectly inhibiting the forward reaction (van der Laan, 1979). Another possibility that has been proposed suggests that GABA levels are increased by VPA through activation of glutamic acid decarboxylase (GAD), the GABA synthesizing enzyme. Following VPA administration to mice (Loscher, 1981; Loscher, 1989) and rats (Phillips and Fowler, 1982) increased levels of GAD were noted. Phillips and Fowler (1982) noted enhanced GAD activity in discrete brain regions, namely the medulla, pons, cerebellum and midbrain regions. In addition, Loscher et al. (1993) reported increased activity of GAD both in vitro and in vivo, and also demonstrated increased GABA turnover in discrete brain regions. The studies demonstrated, that increased GAD levels may be important in the mechanism responsible for increased GABA levels. Other biochemical mechanisms of VPA's anticonvulsant action have also been investigated. For example, studies performed by Chapman et al. (1982) demonstrated a reduced level of excitatory amino acids such as aspartic acid, glutamic acid, and y-hydroxybutyric acid (GHB) by VPA. Voltage dependent blocking of sodium channels, 4 resulting in the inhibition of sustained repetitive firing, has also been demonstrated (McLean and McDonald, 1986). Meshki Baf et al. (1994) also demonstrated alterations in monoamine (namely norepinephrine, dopamine, and serotonin) levels in various brain regions of the rat. Clearly, there are many postulated mechanisms of action of VPA in the treatment of epilepsy. With VPA having a broad spectrum of anticonvulsant activity, it would seem feasible to assume that more than one of the aforementioned mechanisms of action are responsible for the diverse pharmacological activity observed with VPA therapy. 1.3 METABOLISM AND TOXICITY OF VPA During the past 15 years, there has been great interest in the metabolism of VPA due to studies which suggested that metabolites of VPA may contribute to the anticonvulsant effects as well as the hepatotoxic and teratogenic effects of VPA (Nau and Loscher, 1984; Nau and Loscher, 1986; Baillie, 1988). Many VPA metabolites have been synthesized and have been shown to be pharmacologically active (Loscher et al., 1985; Abbott and Acheampong, 1988; Elmazar et al, 1993; Palaty and Abbott, 1995), teratogenic (Klug et al, 1990), or hepatotoxic (Zimmerman and Ishak, 1982; Jeavons and Clark, 1984; Kesterson et al, 1984; Granneman et al, 1984; Baillie, 1992; Fisher et al, 1992). Nevertheless, the metabolic fate of VPA is highly complex and warrants a full investigation. VPA undergoes extensive hepatic metabolism by a number of pathways, the two most prominent being glucuronidation and (3-oxidation, followed by P450 mediated pathways that 5 include co- and co-1-oxidation (Granneman et al, 1984; Rettenmeier et al, 1987; Zaccara et al, 1988) (scheme 1). Renal excretion is a minor route of elimination with only 1-3% of the dose excreted unchanged (Gugler and von Unruh, 1980; Bialer et al, 1985). The major conjugated metabolite of VPA excreted into the urine in humans and most animals is the glucuronic acid conjugate (Dickinson et al, 1979, 1989; Granneman et al, 1984). This conjugate has also been detected at high concentrations in the bile and urine of rats that were administered VPA (Dickinson et al, 1979). Several studies have reported extensive conjugation to VPA-glucuronide in humans, ranging.from 20-60% of the dose administered (Gugler et al, 1977; Granneman et al, 1984; Bialer et al, 1985; Pollack et al, 1986; Dickinson et al, 1989). A characteristic of glucuronide formation in rats is enterohepatic cycling. Glucuronide conjugates that are excreted into the bile are subsequently secreted into the intestine where hydrolysis by the bacterial enzyme -^glucuronidase may occur, thus liberating VPA from its conjugated state. The free VPA can then be reabsorbed from the intestine and reenter the systemic circulation (Dickinson et al, 1979, 1985). Several studies examining the pharmacokinetics of VPA have reported that VPA undergoes enterohepatic recycling in rats (Dickinson et al, 1979; Ogiso et al, 1986; Liu et al, 1990). A secondary serum concentration peak attributed to hepatobiliary cycling has also been observed for several VPA analogues (Singh et al, 1988; 1990; Liu et al, 1993). Another major route of metabolism for VPA is (3-oxidation. The first product of this metabolic pathway is the unsaturated compound 2-ene VPA (both (E)- and (Z)-isomers exist), which can subsequently be metabolized to 3-hydroxy VPA and 3-keto VPA. The (E,E)- and 6 (E,Z)-2,3'-diene VPA isomers result from p-oxidation of 3-ene VPA, formed through isomerization of 2-ene VPA (Bjorge and Baillie, 1991). Other routes of metabolism include co- and co-1 oxidation, which lead to the formation of 4-ene VPA, 3-hydroxy VPA, 4-hydroxy VPA, 5-hydroxy VPA, 4-keto VPA, 2-propylglutaric acid (2-PGA), 2-propylsuccinic acid (2-PSA) and 2-propylmalonic acid (2-PMA) (scheme 1) (Granneman et al, 1984). The di-unsaturated metabolite, (E)-2,4-diene VPA, is formed through p-oxidation of 4-ene VPA (Baillie and Rettenmeier, 1989). Several investigators have proposed that two unsaturated metabolites, 4-ene VPA and 2,4-diene VPA, may play a critical role in VPA-induced hepatotoxicity (Kesterson et al, 1984; Rettenmeier et al, 1985). Kesterson et al. (1984) demonstrated that these two metabolites induced microvesicular steatosis in the rat, a disorder characterized by lipid droplets in the mitochondria with the pathophysiology being similar to that produced by Reye's syndrome and Jamaican vomiting sickness (Zimmerman and Ishak, 1982). In vitro experiments demonstrated that 4-ene VPA inhibits the mitochondrial fatty acid p-oxidation pathway (Bjorge and Baillie, 1985). The unsaturated metabolite (E)-2,4-diene VPA was suggested to deplete mitochondrial glutathione or to generate 3-keto-4-ene VPA CoA, a compound which destroys the terminal enzyme in the 8-oxidation pathway, namely p-ketothiolase, thus preventing mitochondrial function (Rettenmeier et al., 1985; Baillie, 1988; Kassahun et al, 1991). It has been proposed that inhibition of this enzyme subsequently prevents fatty acid metabolism, leading to hepatotoxicity (Schulz, 1983). In addition, studies have indicated an increased potential for hepatotoxicity with pediatric patients on polytherapy. Specifically, phenobarbital, a known inducer of liver 7 cytochrome P450 (Perucca et al, 1984), has been administered to animals and humans in conjunction with VPA. Results indicated an increase in the metabolic conversion of VPA to 4-ene VPA (Kondo et al, 1990, 1992). Although the exact mechanism of VPA-induced hepatotoxicity has yet to be fully elucidated, evidence to date favors the involvement of 4-ene VPA and (E)-2,4-diene VPA (Tang et al, 1995). Both VPA and its metabolites have been implicated as potential teratogens. Clinical studies with VPA have revealed an increased risk of production of various congenital malformations, the most significant risk occurring during the first trimester of pregnancy. Teratogenic effects include facial anomalies, congenital heart defects, and neural tube defects such as spina bifida (Kaneko et al, 1988; Omtzigt et al, 1992). In animal studies, it was revealed that exposure to VPA during pregnancy may result in skeletal malformations and neural tube defects. Several metabolites of VPA have been demonstrated to be embryotoxic in mice and rats (Nau and Loscher, 1984; Klug et al, 1990). The 4-ene VPA metabolite appears to be the most potent teratogen with toxic effects similar to VPA (Nau and Loscher, 1984; Klug et al, 1990; Gofflot et al, 1996). Interestingly, the S-enantiomer of 4-ene VPA has been found to be more teratogenic than its R-enantiomer (Hauck and Nau, 1992). However, other investigators have reported that VPA itself and not its metabolites may be the potent teratogen in vivo (Dreifuss, 1989). 9 1.4 CHEMICAL DERIVATIZATION AND ANALYSIS OF VPA USING GCMS Of particular interest to many investigators is the use of gas chromatography-mass spectrometry for the detection and quantitation of VPA and its metabolites (Acheampong et al, 1983; Abbott et al., 1986; Kassahun et al, 1989; Rettenmeier et al, 1989; Fisher et al, 1992; Darius and Meyer, 1994; Yu et al, 1995). Among the methods employed using GCMS are negative-ion chemical ionization (NICI) and electron ionization (EI). NICI involves soft ionization whereupon an electron beam ionizes a reagent gas, which in turn reacts with the sample molecules. Because the electron beam does not interact directly with the molecules of interest, energy transfer is low (approximately 5 eV) resulting in less fragmentation of molecules and detection of a molecular or pseudomolecular ion peak. NICI employs pentafluorobenzyl bromide (PFB) derivatives that produce an abundant [M-181]" ion for VPA and its metabolites. This analytical method is highly sensitive for the detection of valproic acid metabolites and has been demonstrated to be up to 50 times more sensitive than the f-BDMS derivatives detected under EI mode (Kassahun et al, 1989). EI involves high energy (70-80 eV) bombardment of the sample molecules by an electron beam, resulting in the production of molecular ions. Most organic molecules require 7-13 eV to ionize; therefore, the parent molecules will often possess excess energy. This energy results in extensive fragmentation into daughter ions of lesser molecular weight. Various GCMS methods have used EI of the r-butyldimethylsilyl (f-BDMS) and trimethylsilyl (TMS) derivatives (Abbott et al, 1986; Fisher et al, 1992; Darius and Meyer, 1994; Yu et al, 1995). The EI method and ?-BDMS derivatives offer an advantage over the TMS 10 derivatives in that a highly intense [M-57]+ ion, characteristic of a fragmented r-BDMS derivative under EI mode, has provided enhanced sensitivity when compared with the less intense [M-15]+ ion, characteristic of TMS derivatives (Darius and Meyer, 1994). Thus, analysis of f-BDMS derivatives under EI mode has become a popular method of analysis of VPA and its analogues. The applicability of this method has been demonstrated recently by Yu et al. (1995) who developed a sensitive and reproducible method for the determination of VPA and its metabolites in sheep biological fluids using gas chromatography with mass-selective detection (operated under EI mode) along with selected ion monitoring. 1.5 PHARMACOKINETICS AND PHARMACODYNAMICS OF VPA The pharmacokinetics and tissue distribution of VPA have been studied extensively in humans and animals (Dickinson et al, 1979; Aly and Abdel-Latif, 1980; Gugler and von Unruh, 1980; Ogiso et al, 1986; Bialer et al, 1985; Yu et al, 1987). In humans, VPA is administered in several dosage forms as either the free acid or sodium salt and is well absorbed with peak plasma levels occurring in less than 2 h. Absolute bioavailability of VPA has been reported to be close to unity (Klotz and Antonin, 1977; Perucca et al, 1978). Administration of VPA together with a meal may delay the absorption but not the extent of absorption of the drug (Levy et al, 1980; Royer-Morrot et al, 1993). The therapeutic plasma concentration of VPA in adults has been suggested to range from 50 to 100 |Xg/mL, although plasma concentrations as high as 120 (ig/mL have been 11 reported without any significant neurotoxic effects (Davis et al, 1994). In children aged 9 months to 18 years, the therapeutic range has been reported to be slightly lower at 30-80 p.g/mL (Farrell et al, 1986). Plasma half-life values for VPA in adult humans range from 12 to 16 h, which may be shorter in patients on polytherapy (Perucca et al, 1977; Bowdle et al, 1980; Gugler and von-Unruh, 1980). Shorter half-life values ranging from approximately 2 to 15 h have been reported for children (Cloyd et al, 1983; Hall et al, 1985). In humans, the apparent volume of distribution of VPA has been reported to range from 0.1 to 0.4 L/kg, indicating that VPA is confined to the circulation and extracellular fluids (Klotz and Antonin, 1977; Perucca et al, 1978; von Unruh et al, 1980). The low volume of distribution for VPA reflects its high degree of serum or plasma protein binding. Volume of distribution for VPA is believed to be increased with increasing drug concentrations as a result of saturable protein binding (Liu and Pollack, 1993; Yu and Shen, 1996). Higher volume of distribution values have been reported for patients on polytherapy (Klotz and Antonin, 1977; Perucca et al, 1978; Levy et al, 1980). The volume of distribution for rats has been reported to be similar to that of humans (0.14-0.43 L/kg) (Dickinson etal, 1979; Ogiso etal, 1986). Plasma clearance of VPA in humans ranged from 6-8 mL/kg-h and was independent of liver blood flow (Klotz and Antonin, 1977; Perucca et al, 1978). The free fraction of VPA in plasma is larger than the hepatic extraction ratio (0.02), thus VPA clearance is restrictive and only unbound VPA is cleared (Klotz and Antonin, 1977). Epileptic patients on polytherapy have exhibited up to a 2-fold higher clearance values, attributed to induction of liver metabolizing enzymes caused by drugs such as phenytoin, carbamazepine and phenobarbital 12 (Perucca et al, 1978; Hoffman et al, 1981; May and Rambeck, 1985). Clearance values for children aged 2 to 10 years was reported higher than adult clearance values (Hall et al., 1985). In humans, more than 85% of VPA is bound to plasma proteins, predominantly to the albumin fraction (Klotz and Antonin, 1977; Loscher, 1978). VPA can displace other drugs from albumin binding sites (Levy and Koch, 1982). The binding of VPA is concentration-dependent with saturation of binding occurring at concentrations greater than 80-85 |lg/rnL (Cramer and Mattson, 1979; Cramer et al, 1986) although one study reported saturation at plasma concentrations greater than 50 (Ig/mL (Bowdle et al, 1980). This concentration-dependence in the plasma or serum protein binding of VPA may play a role in nonlinear kinetics that has been associated with VPA. An increase in serum protein binding of VPA, resulting in an increase in free fraction of the drug, has resulted in nonlinear elimination of VPA (Bowdle et al, 1980; May and Rambeck, 1985; Gomez Bellver et al, 1993). Nonlinear binding has also been observed in rats in studies performed both ex vivo (Brouwer et al, 1993) and in vitro (Semmes and Shen, 1990; Haberer and Pollack, 1994). The plasma elimination profiles of VPA in humans have been demonstrated to be first-order in nature and to decline biexponentially after i.v. and oral dosing; therefore, a two-compartment model has been applied to describe the kinetics of VPA in humans (Klotz and Antonin, 1977; Gugler et al, 1977). In rats, a limited distribution phase has been observed followed by first-order elimination after i.v. administration of a low dose of VPA, whereas at higher doses, the initial elimination phase was nonlinear (Dickinson et al, 1979; Lawyer et al, 1980). In addition, a secondary drug concentration peak has been observed in rats, attributed to hepatobiliary cycling since VPA is conjugated extensively to glucuronides (Dickinson et al, 13 1979; Ogiso et al, 1986). Hence, the elimination kinetics of VPA in rats is complex and follows first-order elimination only at low doses, and is further complicated by the presence of enterohepatic cycling. Tissue distribution studies for VPA have been limited to animals, with the exception of a few human brain distribution studies in which cortical brain samples were obtained from patients undergoing neurosurgery (Vajda et al, 1981; Weiser et al, 1991; Shen et al, 1992; Adkison et al, 1995). All studies reported low brain concentrations and low brain:serum concentrations of VPA, in contrast to other antiepileptic medications such as phenytoin which exhibited a brain:serum ratio greater than unity (Friel et al, 1989). VPA exhibits several unique pharmacodynamic properties unlike other conventional anticonvulsant medications. Studies in both humans (Jeavons and Clark, 1974; Rowan et al, 1979) and animals (Loscher and Nau, 1982) have revealed a weak correlation between anticonvulsant activity and plasma concentrations of the drug, in that protection against seizures is not maximal until steady-state serum or brain concentrations of VPA are attained. The involvement of one or more of VPA's metabolites in the activity was hypothesized, and this stimulated efforts to elucidate the mechanism of this phenomenon. 14 1.6 T H E P O T E N T I A L R O L E O F (E)-2-ENE VPA, (E,E)-2,3-DIENE V P A A N D (E,Z)-2,3'-DIENE V P A AS A L T E R N A T I V E T H E R A P Y T O V P A An interesting observation with respect to the pharmacology of VPA is the observed delay in anticonvulsant activity (Lockard and Levy, 1976; Nau and Loscher, 1982; Liu and Pollack, 1994) and a persistence of anticonvulsant activity after VPA has been cleared from the systemic circulation (Nau and Loscher, 1982; Pollack et al, 1986). The mechanisms responsible for these effects are uncertain, although several investigations have suggested that one or more VPA metabolites may possess anticonvulsant activity and may contribute to the prolonged anticonvulsant effect observed with VPA (Loscher, 1981; Loscher and Nau, 1985). In animal seizure models, analogues of VPA have demonstrated varying degrees of anticonvulsant activity when compared to VPA (Loscher et al, 1985; Abbott and Acheampong, 1988; Elmazar et al, 1993; Palaty and Abbott, 1995); therefore, it is feasible that accumulation of one or more of these metabolites may contribute to the anticonvulsant effects of VPA. This possibility has led to extensive testing of various VPA analogues. Several investigations have focussed on the major serum metabolite, (E)-2-ene VPA, because this monoene was initially reported to be the only metabolite detected in mouse brain three days after discontinuation of VPA therapy, while VPA itself was undetected (Nau and Loscher, 1982). In addition, detection of this monoene was reported in the substantia nigra, superior and inferior colliculi, hippocampus and medulla of rat brain after discontinuation of VPA treatment (Loscher and Nau, 1983). Nau and Loscher (1982) also reported that the half-life of (E)-2-ene VPA in brain tissue was markedly longer than that of VPA. Because 15 (E)-2-ene VPA was detected in rat brain over time, and because its elimination from brain and plasma was slower than that of VPA, it was postulated that (E)-2-ene VPA may accumulate in r brain regions and may contribute to the pharmacological activity of VPA. Thus, further investigations were conducted in an effort to assess the anticonvulsant potential of (E)-2-ene VPA. Additional studies with (E)-2-ene VPA demonstrated that this compound may have several advantages over VPA. Firstly, (E)-2-ene VPA has been shown to be more potent or equipotent in suppressing seizures in animals when compared to VPA (Loscher et al, 1984; Semmes and Shen, 1991; Loscher et al, 1991). High plasma protein binding (greater than 90%) has been observed with the monoene, that would potentially reduce drug transfer into the liver and may serve to be more advantageous than VPA with respect to hepatotoxicity (Nau and Loscher, 1985; Semmes and Shen, 1990). In addition, a relative lack of hepatotoxic (Kesterson et al, 1984; Schafer and Liihrs, 1984; Loscher et al, 1992; Loscher et al, 1993) and teratogenic (Loscher et al, 1984; Nau and Loscher, 1984; Nau, 1986; Vorhees et al, 1991) effects have also been reported by several investigators. In light of the favorable anticonvulsant activity, high plasma protein binding, and a relative lack of hepatotoxic and teratogenic effects, (E)-2-ene VPA was proposed to be a potential alternative to VPA therapy. However, other investigators have reported conflicting results with respect to the potential for using (E)-2-ene VPA as a substitute for VPA. Keane et al. (1985) reported the monoene to be half as potent as VPA in mice seizure models. Loscher et al. (1988) reported a weak relationship between the anticonvulsant activity and brain levels of (E)-2-ene VPA 16 after administration of (E)-2-ene VPA in rats. Various neurotoxicity experiments (such as rotarod and loss of righting reflex) in animals have indicated that (E)-2-ene VPA may be up to twice as neurotoxic as VPA (Keane et al, 1985; Loscher et al, 1991). Lee (1991) reported the monoene displayed a shorter residence time in rat brain, greater neurotoxicity and a lack of accumulation in any brain region as compared to the parent drug. Furthermore, one investigation reported to have found high levels of (E)-2,4-diene VPA after treatment with (E)-2-ene VPA, which suggests the potential for hepatotoxicity (Lin et al, 1991). As investigations continued into (E)-2-ene VPA's undetermined role as a future antiepileptic agent, investigators focussed on other VPA metabolites as potential replacement therapy. One such metabolite, (E,E)-2,3'-diene VPA, has been of interest in our laboratory (figure 1). This diene was found to be as prevalent as (E)-2-ene VPA in human serum (Kassahun et al, 1990; Fisher et al, 1992) and was also found to possess equivalent potency to (E)-2-ene VPA when tested for anticonvulsant activity in mice (Abbott and Acheampong, 1988). Interestingly, Loscher et al. (1989) did not detect accumulation of this diene in brain tissue after chronic administration of VPA to rats. Similarly, Lee (1991) reported (E,E)-2,3'-diene VPA undetectable in rat brain tissue following i.p. administration of 150 mg/kg VPA. However, pharmacokinetic studies performed with (E,E)-2,3'-diene VPA revealed localization and apparent accumulation of the diene in specific brain regions, but only at lower plasma drug concentrations (Lee, 1991). Lee (1991) also reported several attributes of (E,E)-2,3'-diene VPA, including rapid absorption, initial prolonged concentrations in plasma and brain, and low liver concentrations. However, negative attributes for (E,E)-2,3'-diene VPA, such as 17 a lower potency (table 1) and greater neurotoxicity when compared to VPA, subsequently eliminated the diene's potential as a likely alternative to VPA. Although the (E,E)-2,3'-diene VPA was undetected in rat brain tissue after administration of VPA, Lee (1991) observed an isomer of (E,E)-2,3'-diene VPA, namely (E,Z)-2,3'-diene VPA, in detectable quantities in rat brain tissue after administration of 150 mg/kg of VPA. The difference in detection for the two isomers was most unexpected, and the rationale has yet to be fully elucidated. This observation led to preliminary investigations with (E,Z)-2,3'-diene VPA. After successful synthesis of the (E,Z)-2,3'-diene VPA (Lee et al, 1989) the compound was subjected to anticonvulsant testing using the PTZ test in rats. Most surprisingly, tests revealed that (E,Z)-2,3'-diene VPA was 1.6 times more potent than its (E,E)-isomer, and equivalent in potency to VPA (Lee, 199.1) (table 1). Furthermore, Lee (1991) observed less muscle spasticity and sedative effects after (E,Z)-2,3'-diene VPA when compared to the (E,E)-isomer. The low neurotoxicity and equal anticonvulsant potency results obtained from preliminary studies with (E,Z)-2,3'-diene VPA make it an attractive investigational compound and a potential candidate as an alternative to VPA therapy. 18 C O O H (E)-2-ene VPA (E,E)-2,3'-cliene VPA C O O H (E,Z)-2,3'-cliene VPA Figure 1. Chemical structures of V P A and three of its metabolites that are under investigation as possible alternatives to V P A therapy. 19 Table 1. The mean effective dose against PTZ-induced seizures and the mean slope of the log dose-response plots for each compound tested in male Sprague-Dawley rats (n=8)1. Compounds were administered as solutions of their sodium salts and corrections made for the small isomer content of the unsaturated compounds. PTZ (70 mg/kg) was administered s.c. at the time of apparent peak maximum in brain for the compound being tested. C O M P O U N D E D 5 0 , mg/kg S L O P E VPA 158 (144-187) 1.2 (1.1-1.3) (E)-2-ENEVPA 185 (156-199) 1.2 (1.0-1.3) (E,E)-2,3'-DIENE VPA 2 266 (199-350) 1.7 (1.1-2.5) (E,Z)-2,3'-DIENE VPA 168 (154-196) 1.2 (0.8-1.8) () - 95% confidence limits 1 -Adapted from Lee (1991) 2 - Significantly different from VPA (p<0.05) 20 1.7 OBJECTIVES The favorable anticonvulsant potency and low neurotoxicity results obtained in the preliminary study with (E,Z)-2,3'-diene VPA suggest that (E,Z)-2,3'-diene VPA could potentially be used as an alternative to VPA therapy. To test this hypothesis, a number of investigations were conducted into the pharmacokinetics and tissue distribution of the compound in an effort to determine whether the diene reflects certain desirable qualities displayed by conventional anticonvulsants, namely rapid absorption and distribution into the brain as well as low liver distribution. Results from these studies were compared with known literature values for VPA and its analogues, and the information obtained was used to assess the potential for (E,Z)-2,3'-diene VPA as a new anticonvulsant medication. 1.7.1 Specific Objectives 1. To develop and employ an effective method for the analysis of (E,Z)-2,3'-diene VPA in rat tissue samples using EI-GCMSD. 2. To determine the serum protein binding characteristics of (E,Z)-2,3'-diene VPA in the rat. 3. To investigate the dose-dependent pharmacokinetics of (E,Z)-2,3'-diene VPA after administration of three different doses of the compound to rats. 21 4. To investigate the excretion of conjugated and unconjugated (E,Z)-2,3'-diene VPA in rat urine and bile. 5. To determine the elimination kinetics and tissue distribution of (E,Z)-2,3'-diene VPA in rat serum, brain, and liver tissues. 22 2. MATERIALS AND METHODS 2.1 SUPPLIES 2.1.1 Chemicals and Materials All chemicals were reagent grade. Chemicals and supplies were obtained from the following companies: The Centrifree™ micropartition MPS-1 ultrafiltration system with YMT membranes was purchased from Amicon Corporation (Danvers, MA). Anhydrous sodium sulfate was obtained from BDH Inc. (Vancouver, BC). Ethyl acetate was purchased from Caledon Laboratories Ltd. (Edmonton, AB). Polyethylene tubing PE-10 was obtained from Clay Adams (Parsippany, NJ). Hypodermic BD 23 Gl needles and tuberculin lcc syringes were purchased from Canlab (Mississauga, ON). Borosilicate glass autosampler vials, screw-top vials, Vacutainer® blood collection tubes, and Alkacid Wide-Range pH strips were purchased from Fisher Scientific (Vancouver, BC). Cryovials® w ere obtained from Ingram and Bell (Richmond, BC). DB-1701 fused-silica capillary columns (30 m x 0.25 mm ID., 0.25 um film thickness) were purchased from J & W Scientific (Folsom, CA). N-methyl-N-(tert-butyldimethylsilyl)-trifluoroacetamide (MTBSTFA) and Tuf-Bond Discs were obtained from Pierce Chemical Co. (Rockford, IL). 23 Urethane was purchased from Sigma Chemical Co. (St. Louis, MO). Micro-centrifuge tubes, inserts for autosampler vials, and Pellet-Pestle Disposable Mixers were from VWR Scientific (Edmonton, AB). 2.1.2 (E,Z)-2,3 '-diene VPA and Internal Standard Solutions (E,Z)-2,3'-Diene VPA (2-(Z)-(l'-propenyl)-(E)-2-pentenoic acid) was synthesized by Dr. J. Palaty (Palaty and Abbott, 1995) according to the procedure of Lee et al. (1989). Aqueous solutions of (E,Z)-2,3'-diene VPA for i.p. administration and for analytical standards were prepared by dissolving the free acid in an equivalent amount of 3M NaOH. The pH of the solution was adjusted to 7.4 with 3N HC1. All concentrations used for (E,Z)-2,3'-diene VPA were corrected for the 5% (E,E)-2,3'-diene. VPA content. Solutions of (E,Z)-2,3'-diene VPA for injection were prepared fresh daily. The internal standard, [2H7]-2-ene VPA ([2H7]-2-n-propyl-(E)-2-pentenoic acid), was synthesized in our laboratory by J. J. Zheng (1993) using the procedures as described by Acheampong et al. (1985). [2H7]-2-Ene VPA was also prepared from the free acid as described above and prepared fresh daily in distilled water at a concentration of 10 ug/mL. 2.1.3 Animals Male Sprague-Dawley rats weighing 201-319 g were obtained from the Animal Care Facility at the University of British Columbia. The animals were housed in plastic cages with free access to food and water and were maintained on a 12 h light/12 h dark cycle. Room temperature was maintained at 22 °C. 24 2.2 INSTRUMENTATION 2.2.1 Centrifuges A Beckman GP centrifuge equipped with a GH-3.7 rotor (Palo Alto, CA) was used during the pharmacokinetic and tissue distribution study to separate serum from blood samples, and during the drug extraction procedure to separate the organic and aqueous layers. A Fischer Scientific Micro Centrifuge Model 235C (13,600 g - fixed) was used during the dose-dependent pharmacokinetic study to separate serum from blood samples. Free (unbound) drug was separated from serum samples by ultrafiltration using a Beckman J2-21 centrifuge equipped with a JA-17 rotor (Palo Alto, CA). 2.2.2 Gas Chromatography-Mass Selective Detection The assay was performed on a Hewlett-Packard 5890 Series II gas chromatograph (GC) which was coupled to a Hewlett-Packard 5971A mass selective detector (MSD) (Hewlett-Packard, Avondale, PA). A DB-1701 fused-silica column (30 m x 0.25 mm ID., 0.25 pm thickness) was used. GC conditions were as follows: injector temp: 250 °C; detector: 270 °C; oven: programmed from 80-100 °C (10 °C/min), 100-130 °C (2 °C/min) and 130-260 °C (30 °C/min); helium head pressure: 10 psi. The MSD operated under electron ionization conditions (70 eV) at a source temperature of 275 °C. Emission current was 300 uA. Ions for the parent drug and the internal standard (m/z 197 and 206, respectively) were monitored using selected-ion-monitoring (SIM). A 1 u,L aliquot of sample was injected using a Hewlett Packard 7673A autosampler. Total run time was 30.6 min. 25 The MSD data system, a Hewlett-Packard ChemStation Version A.02.01 on a Hewlett-Packard Vectra 486 with a Hewlett-Packard 7360 controller, was used to control the entire process including data collection. 2.3 A N I M A L E X P E R I M E N T S 2.3.1 Dose-Dependent Pharmacokinetics and Metabolic Study of (E,Z)-2,3 '-diene VPA 2.3.1.1 Preparation of Drug Solutions for Injection Aqueous solutions of (E,Z)-2,3'-diene VPA were prepared fresh daily at concentrations of 39.5, 79, and 158 mg/mL (corrected for the 5% (E,E)-isomer content) for administration of 75, 150 and 300 mg/kg of the drug, respectively. 2.3.1.2 Serum Collection A group of five rats weighing 239-319 g was studied at each dose of (E,Z)-2,3'-diene VPA. Following administration of an i.p. injection of the selected dose of the drug, samples of blood (0.2 mL) were withdrawn from the tail artery into microcentrifuge tubes at specified time intervals (-15, 7.5, 15, 22.5, 30, 45, 60, 120, 240, 360, 540, and 720 min). Blood samples were placed on ice, allowed to clot, and centrifuged at 3000 rpm for 20 min. The serum was then promptly removed, transferred to a Cryovial® and frozen at -20° C until derivatized and analyzed by GCMS. 26 2.3.1.3 Urine Collection A group of three rats weighing 252-290 g were studied at each dose of (E,Z)-2,3-diene VPA. Animals were injected i.p. with 75, 150 or 300 mg/kg of (E,Z)-2,3'-diene VPA and were immediately housed in metabolic cages equipped with a funnel and glass container to collect the urine. The animals were allowed free access to food and, water throughout the experiment. Total urine was collected for 24 h. The urine produced was removed at 12 and 24 h after the administered dose and was frozen at -20° C until derivatized and analyzed by GCMS. 2.3.1.4 Bile Collection Prior to surgery, the animals were anesthetized with urethane (1.2 g per kg, i.p.). The common bile duct was isolated and cannulated using a 30 cm strand of PE-10 tubing and was secured and allowed to externalize through the muscle wall. The bile drained from the tubing into a Cryovial® situated 15 cm below the animal. A 30-min bile sample was collected prior to administration of (E,Z)-2,3'-diene VPA to permit calculation of control bile flow rate. With the rat bile duct cannulated, three rats weighing 226-241 g were administered an i.p. injection of 150 mg/kg of (E,Z)-2,3'-diene VPA. Bile was then collected for 6 h at specified time intervals (0-1, 1-2, 2-3, 3-4, 4-5, and 5-6 h). Bile samples were immediately frozen at -20° C until derivatized and analyzed by GCMS. 27 2.3.2 Pharmacokinetics and Tissue Distribution in Brain, Serum and Liver 2.3.2.1 Preparation of Drug Solution for Injection An aqueous solution of 79 mg/mL of (E,Z)-2,3'-diene VPA was prepared fresh daily (corrected for the 5% (E,E)-isomer content) for administration of 150 mg/kg of the drug. 2.3.2.2 Tissue Collection The elimination kinetics of (E,Z)-2,3'-diene VPA were determined using a dose of 150 mg/kg of (E,Z)-2,3'-diene VPA and eight male Sprague Dawley rats weighing 201-264 g for each time point (-15, 7.5, 15, 22.5, 30, 45, 60, 120, 240, 360, 540, and 720 minutes). At the appropriate time interval, the animals were sacrificed with carbon dioxide gas for 30 sec, decapitated using a Harvard small animal decapitator, and blood, liver, and brain tissues collected. Blood was collected into Vacutainers® and placed on ice, allowed to clot, and centrifuged at 3000 rpm for 20 min. The resultant serum was removed and stored in Cryovials® at -78° C until derivatized and analyzed by GCMS. The liver was weighed and homogenized as outlined in section 2.4.1. Each rat brain was quickly removed from the cranium, placed onto a cold plate (chilled at -20° C) within 30 sec after decapitation, and dissected into 11 brain regions within 6 min after decapitation. 2.3.2.3 Brain Dissection Procedure Brain regions were identified (Thompson, 1978; Paxinos and Watson, 1982) and the details of the dissection technique as follows: the olfactory bulbs which remained in the ethmoid fossae were removed with forceps. Further dissection of the brain was completed 28 Figure 2. Section lines used for dissection of rat brain; dorsal view on left, ventral on right. The location of the respective lines courtesy of Zeman and Innes (1963). using a "blunt dissection" technique whereupon each brain region was uncovered, identified, and removed in the order that the regions became visible. The cerebellum was carefully removed from the hindbrain and separated from the pons by a horizontal incision at the level of the fourth ventricle. After a dorso-sagittal incision was made to facilitate division of the cerebral cortices, the cortices were carefully separated and the inner brain exposed. Proceeding in a caudo-rostral direction, the corpus callosum was quickly collected followed by the inferior colliculi, superior colliculi and hippocampi. The corpus striata was removed at 'B' as shown in figure 2 and the frontal cortex could then be resected at 'A'. The remaining 29 cortex was then removed and stored as "whole remaining brain". The remaining portion of the brain was then placed on dry ice in a ventral position. A tissue slice was then made at the levels of ' C and 'D', the substantia nigra was identified and then quickly removed. Finally, the medulla oblongata was dissected by a transverse incision at 'E'. The remaining portion of the brain was stored with whole remaining brain. Upon the removal of each brain region, each sample was pooled from the eight animals from each time point and immediately frozen on dry ice. The whole remaining brain was treated individually. 2.4 ANALYTICAL PROCEDURES 2.4.1 Preparation of Brain and Liver Samples for Drug Assay Brain and liver samples were made to a specified volume or weight using 0.1 M phosphate buffer (pH 7.4). The liver samples were made to a final volume of 40 mL. In order to minimize error, brain tissues were made to a specified weight such that the resulting concentration was 500 mg brain tissue per 1 g brain homogenate. The whole remaining brain, liver, and pooled brain regions exceeding 0.2 g were homogenized over ice by three passes using a Potter-Elvehjem tissue grinder equipped with a Teflon pestle. Pooled brain regions weighing less than 0.2 g were homogenized over ice using a Pellet-Pestle Disposable Mixer. All homogenized samples were subsequently stored at -78° C until analyzed. 30 2.4.2 Protein Binding of (E,Z)-2,3 '-diene VPA 2.4.2.1 Ex Vivo Protein Binding The serum protein binding of (E,Z)-2,3'-diene VPA was determined from the rat serum samples collected during the pharmacokinetic and tissue distribution study. A 500 uX aliquot of each rat serum sample was individually pipetted into an Amicon micropartition apparatus fitted with a YMT ultrafiltration membrane, and samples were centrifuged at 25 °C for 20 minutes at 1650 g to separate free drug from serum proteins. A 20 |lL aliquot of the ultrafiltrate or serum was then assayed for determination of free or total drug concentration, respectively. 2.4.2.2 In Vitro Protein Binding Control rat serum was collected, pooled, and spiked with (E,Z)-2,3'-diene VPA. The concentrations of (E,Z)-2,3'-diene VPA investigated were 0, 15, 30, 45, 60, 120, 240, 360, and 720 ug/mL. Samples were prepared as follows: 50 uL aliquots of (E,Z)-2,3'-diene VPA (in distilled water) were pipetted into separate test tubes and made to a volume of 2 mL with control rat serum to obtain the required concentrations. The samples were vortex mixed and incubated for 2 h in a 37° C water bath. For determination of free serum levels in each sample, a 500 (iL aliquot of each serum sample was pipetted into an individual Amicon® micropartition apparatus. Samples were centrifuged at 25° C for 20 min at 1650 g to separate free drug from serum proteins. A 20 U.L aliquot of ultrafiltrate or serum was then assayed for determination of free and total drug concentration, respectively. 31 2.4.2.3 Binding of (E,Z)-2,3'-diene VPA to the YMT Membrane For the determination of the amount of the drug that binds to the YMT membrane, the following procedure was used. Samples of (E,Z)-2,3'-diene VPA in water were prepared at concentrations of 1, 30, 100 and 600 pg/mL. A 500 uL aliquot of each sample was pipetted into the Amicon® micropartition apparatus. The samples were centrifuged as previously mentioned. A 20 u\L aliquot of ultrafiltrate or drug solution was then assayed for determination of free and total drug concentration, respectively. 2.4.3 Calibration Curves Samples prepared for calibration curves were treated in the same manner as test samples. Standard (E,Z)-2,3'-diene VPA and [2H7]-2-ene VPA solutions were prepared fresh daily. Both high and low concentration ranges were used for the preparation of calibration curves for analysis of (E,Z)-2,3'-diene VPA in tissues and biological fluids. For the preparation of calibration curves for biological fluids, a 20 pL aliquot of either diluted control bile, urine, serum or ultrafiltrate was spiked with 20 pL of standard drug solution prepared at several concentrations, and diluted with 160 pL distilled water. Control standards at 0 pg/mL contained either 20 pL of diluted control bile, distilled water, control serum or ultrafiltrate (for bile, urine, serum or ultrafiltrate calibration curves, respectively), and 180 uL distilled water. For preparation of high calibration curves for liver homogenate, a stock solution of (E,Z)-2,3'-diene VPA in water was prepared at a concentration of 2.5 mg/mL. A 150 p i aliquot of this solution was diluted in control tissue homogenate to yield a 150 p:g/mL 32 standard tissue solution, which was then diluted with control tissue homogenate as outlined in table 2 to yield the concentrations used for the calibration curves. Low range calibration curves for liver homogenate were prepared in a similar manner using a 0.1 jig/mL standard tissue solution of (E,Z)-2,3'-diene VPA. Calibration curves for brain tissues were prepared in a similar manner as liver tissues using 100 |ig/mL and 0.1 u.g/mL standard tissue solutions for the preparation of high and low range calibration curves, respectively. The entire whole brain was homogenized and used as control brain tissue for the analysis of whole remaining brain and the 11 brain regions. Table 2. An example of the dilution protocol used for the preparation of a high range calibration curve for analysis of (E,Z)-2,3'-diene V P A content in liver tissue. Sample Standard Tissues . Solution (\x.L) Control Tissue Homogenate (\x.L) Final Concentration (Hg/mL) 1 0 100 0 2 10 90 15 3 20 80 30 4 40 60 60 5 50 50 75 6 60 40 90 7 80 20 120 8 100 0 150 33 2.4.4 Extraction and Derivatization The assays used were modified procedures developed by Abbott et al. (1986). All samples were analyzed in duplicate. General extraction and derivatization procedures for the biological samples are summarized in scheme 2. 2.4.4.1 Bile and Urine Samples Bile and urine were analyzed for both conjugated and unconjugated drug. For the analysis of unconjugated (E,Z)-2,3'-diene VPA, an amount equivalent to 20 pL of bile or urine was transferred to a clean vial. Bile samples were diluted with distilled H 2 0, vortex mixed, and an amount equivalent to 20 pL of bile was transferred from each sample to a clean vial. To each bile or urine sample was added 180 pL of distilled water, 100 pL of internal standard solution, 30 pL of 2M HC1 and 2 mL of ethyl acetate. The samples were mechanically rotated for 20 min, and then centrifuged for 10 min to break the emulsion formed. The organic layer was transferred to a clean vial and dried over anhydrous Na2SC>4. The samples were again vortex mixed, mechanically rotated, and centrifuged for 10 min. Following transfer of the organic layer to a clean vial, the volume was reduced to 50-100 pJL under nitrogen. The contents were then transferred to a 1 mL screwcap vial, derivatized with 50 pL of MTBSTFA, and heated at 60° C for 60 min. Each sample was then transferred to a 1 mL autosampler vial. A 1 pL aliquot was injected into the GCMSD via the autosampler. For analysis of conjugates of (E,Z)-2,3'-diene VPA in bile or urine, a 100 pL aliquot of bile or 500 pL aliquot of urine was treated with 3M NaOH to make the pH 12-13, followed by incubation for 60 min at 60° C to hydrolyze conjugates. Upon cooling to room 34 temperature, 3M HC1 were added to neutralize each sample. Samples were then left at room temperature for 15 min. An amount equivalent to 20 pL of bile or urine was transferred to a clean vile. Bile samples were then diluted with distilled H 20, vortex mixed, and an amount equivalent to 20 p i of bile was transferred from each sample to a clean vial. To each bile or urine sample was added 180 p i of distilled H 20, 100 p i of internal standard, 30 p i of 2M HC1 and 2 mL of ethyl acetate. The samples were then extracted and derivatized as described above. 2.4.4.2 Brain and Liver Samples To a 100 pll aliquot of brain or liver homogenate was added 100 pi of internal standard solution, 100 pi of distilled water, 25 pL (brain) or 27 pi (liver) of 2M HC1 and 2 mL of ethyl acetate. The samples were then subjected to identical extraction and assay procedures as previously mentioned. 2.4.4.3 Serum and Ultrafiltrate Samples To a 20 pi aliquot of ultrafiltrate or serum was added 180 pi of distilled water, 100 pi of internal standard solution, 30 p i of 2M HC1 and 2 mL of ethyl acetate. The samples were then extracted and assayed according to the aforementioned procedure. 35 Bile, urine, serum or liver or brain homogenate I 2M HCL 100 |ll internal standard distilled water I Extracted with 2 mL ethyl acetate Centrifuge 3000 rpm x 10 min I Discard aqueous fraction Dry over anhydrous Na2SC>4 1 Centrifuge 3000 rpm x 10 min I Transfer organic layer to Reacti-Vial® Reduce volume to 50-100 |il under N 2 I Add 50 |ll MTBSTFA Heat 60 min at 60°C I Inject 1 |ll via autosampler Scheme 2. General extraction and derivatization scheme for the analysis of unconjugated drug in biological fluids and liver or brain homogenate. 36 2.4.5 Extraction Efficiency For the determination of the extraction efficiency of (E,Z)-2,3'-diene VPA from brain and liver tissue homogenate, the following procedure was used. Solutions of (E,Z)-2,3'-diene VPA were prepared in control brain or liver homogenate at concentrations of 0.5, 1.0, 10.0 and 50.0 |lg/mL. For the calibration curve, solutions of (E,Z)-2,3'-diene VPA in ethyl acetate were prepared at the same concentrations. A 100 uX aliquot of each spiked brain or liver homogenate was extracted using the procedure outlined in section 2.4.4. To the extracted samples, the internal standard in ethyl acetate was added. Samples were derivatized and assayed as outlined previously in section 2.4.4. A 100 |lL aliquot of each solution of (E,Z)-2,3'-diene VPA in ethyl acetate was also derivatized and assayed in a similar manner. Calibration curves were prepared using peak area ratios from chromatograms obtained from the samples of (E,Z)-2,3'-diene VPA in ethyl acetate. From the calibration curves, the concentration of (E,Z)-2,3'-diene VPA extracted from brain and liver tissue homogenate could be determined. Extraction efficiency was then determined from the ratio of the concentration of drug extracted from brain or liver tissues to the theoretical concentration of drug in ethyl acetate. 37 2.5 DATA ANALYSIS 2.5.1 Calculation of Pharmacokinetic Parameters Apparent pharmacokinetic parameters were calculated using standard compartmental methods (Gibaldi and Perrier, 1982). The terminal elimination constant, (3, was calculated from the linear portion of the log serum concentration-time plot. The apparent ti/2 was determined from 0.693/(3. Area under the concentration-time curves were obtained by the trapezoidal rule and determined from 0 to 12 h (AUC0-i2h). Clearance was determined as Dose/AUC0-i2h unless otherwise indicated. Bioavailability (F) was not determined in this study and therefore clearance was expressed as Cl0-i2h/F. 2.5.2 Computer Modelling A two-compartment time-lag model (figure 3) was used to describe the elimination profile of (E,Z)-2,3'-diene VPA in rats for the dose-dependent pharmacokinetic study. A nonlinear least-squares regression program MULTI(RUNGE) (Yamaoka and Nakagawa, 1983) was used to fit serum concentration-time data to appropriate compartmental models and to calculate pharmacokinetic parameters (volume of the central compartment (Vc) and first-order microconstants (ka, k,2, k 2i, ki0)). The mass balance differential equations for the two-compartment model were: dCi/dt = -(k,o + k l2) x C, + k21 x A 2 * / V c + ka x DOSE x e"kat / V c dA2/dt =-k2, x A 2 * + k,2 x C i x V c 38 ka • kio Figure 3. Time-lag two compartment pharmacokinetic model of (E,Z)-2,3'-diene V P A in rats after an i.p. dose of 75, 150, or 300 mg/kg (E,Z)-2,3'-diene VPA. Compartment 1, sampled blood compartment; compartment 2, gut lumen; T, time lag; k ] 0 , k J 2 , k 2 i and ka, first-order rate constants. where C\ is the drug concentration in compartment 1 at time t, A 2 is the amount of drug in compartment 2 after time T (where T is the time lag), k [ 2 and k2, are first-order rate constants for the transfer of drug between compartment 1 and 2, k i 0 is the first-order rate constant for the elimination of drug from compartment 1, ka is the first-order absorption rate constant for compartment 1, and V c is the volume of the central compartment. The time-lag (T) was chosen at the apparent onset of enterohepatic cycling for each set of data which corresponded to 132 ± 7, 157 ±,8, and 345 ± 7 min for dose levels of 75, 150 and 300 mg/kg, respectively. For the pharmacokinetic and tissue distribution study, a four-compartment time-lag model (figure 4) was used to describe the elimination profile of (E,Z)-2,3'-diene VPA in rat serum, brain and liver tissues. A nonlinear least-squares regression program ADAPTII was Compartment 1 k l 2 k2 i Compartment 2 39 used to fit tissue concentration-time data to appropriate compartmental models and to calculate pharmacokinetic parameters (volume of the central compartment (Vc) and first-order microconstants (ka, k12, k2i, k10, ki3, k 3 i , kM, k,4)). The mass balance differential equations for the four-compartment model are located in Appendix 1. Compartment 3 kn ka k 3i k ] 4 Compartment 1 kin Compartment 4 ki2 k21 Compartment 2 Figure 4. Time-lag four compartment pharmacokinetic model of (E,Z)-2,3'-diene V P A in rats after an i.p. dose of 150 mg/kg (E,Z)-2,3'-diene VPA. Compartment 1, sampled blood compartment; compartment 2, gut lumen; compartment 3, brain; compartment 4, liver;T, time lag; k ] 0 , k i 2 , k 2 i , k ] 3 , k 3 J , k ] 4 , k 4 i , and k a, first-order rate constants. 40 2.5.3 Statistics All data are expressed as mean + standard deviation. The statistical differences among pharmacokinetic parameters for the dose-dependent study were determined using one-way ANOVA. All statistical tests applied the p value of less than 0.05 to assess significance of data. Linear regression was used to calculate the regression equation coefficients, correlation coefficient r2, and function results for each regression curve analyzed. 41 3. RESULTS 3.1 ANALYSIS OF (E,Z)-2,3'-DIENE VPA BY EI-GCMS 3.1.1 Chromatography and Method of Detection of (E,Z)-2,3'-diene VPA Electron ionization-gas chromatography mass spectrometry using £-BDMS derivatives was an effective method to obtain optimal resolution and sensitivity of (E,Z)-2,3'-diene VPA for all biological samples. The EI mode involves high energy (70 eV) bombardment of the parent molecules by an electron beam, resulting in the extensive fragmentation into daughter ions of less molecular weight. Figure 5 represents the EI-GCMSD spectrum of (E,Z)-2,3-diene VPA. The intense m/z [M-57]+ ion, corresponding to the loss of the tert-butyl fragment of the f-BDMS derivatives, was used to quantitate (E,Z)-2,3'-diene VPA. Ions for the parent drug and internal standard (m/z 197 and 206, respectively) were monitored using selected ion monitoring. A sample chromatogram is illustrated in figure 6. The chromatographic conditions used in the assay provided the conditions necessary for adequate resolution of the r-BDMS derivatives of (E,Z)-2,3'-diene VPA and [2H7]-2-ene VPA and resulted in sharp and symmetrical peaks. 4 2 0 O CC TJ c < 5000000 4500000 4000000 3500000^  3000000 2500000-j 2000000 1500000 1000000 500000^  0 75 55 . 95 12 50 100 39 197 [M - 57] + o 254 150 CH3: CH3 Si— i-| | -C—C CH3 j CH3 [ M - 57 ]+ 200 250 300 350 m/z Figure 5. Electron-ionization mass spectrum of a f-BDMS derivative of (E,Z)-2,3'-diene V P A . Fragmentation patterns as illustrated in the inset diagram are described in the text. CD O c CO T3 .Q < 400000 200000 16.01 T i m e CD o c CO T3 c _Q < 400000J 200000 T i m e o -> 14.65 i 0 i A 17.77 15.00 16.00 17.00 18.00 19.00 B 14.59 17.18 18.85 15.00 16.00 17.00 18.00 19.00 Figure 6. Sample electron-ionization mass chromatograms of: (A) (E,Z)-2,3'-diene V P A (m/z 197) (B) [2H7]-2-ene V P A (m/z 206) 43 3.1.2 Calibration Curves, Inter-Assay Variation and Analytical Recoveries Calibration curves of the (E,Z)-2,3'-diene VPA / [2H7]-2 -ene VPA peak ion area ratios vs. known concentrations of (E,Z)-2,3'-diene VPA were prepared using peak area ratios from chromatograms of the injected standards. The calibration curves obtained for all biological samples showed linearity within the concentration ranges used, with coefficients of determination ranging from 0.994 to 0.999. A sample calibration curve for rat serum is illustrated in figure 7. The reproducibility and reliability of the assay method was checked over a period of 12 months. The relative standard deviation based on the slopes of the calibration curves obtained during this time period ranged between 5.6 and 10.4%, indicating adequate reproducibility (table 3). The calibration standards for bile provided the best overall assay precision with a 5.6% relative standard deviation. The limit of quantitation for the diene was the concentration of the lowest calibration standard, 0.01 |lg/rnL, with a coefficient of variation of less than 17% for the tissues studied. A signal to noise ratio greater than 3:1 for m/z 197 was used as a reference. Extraction efficiencies for the diene, measured at concentrations ranging from 1 to 50 |i.g/mL, ranged between 63.5 to 73.5% for brain and 56.0 to 62.7% for liver. 44 10 n 0 60 120 180 240 300 360 420 480 (E,Z)-2,3'-Diene VPA Concentration (|Lig/mL) Figure 7. A representative calibration curve used for the quantitation of high levels of (E,Z)-2,3'-diene V P A in rat serum. Table 3. Inter-assay variation based on the slopes of the calibration curves over a 12 month period for (E,Z)-2,3'-diene VPA. (n represents the number of replicate analyses performed with each biological sample). Brain (n = 12) Liver (n = 6) Serum' (n=9) Ultrafiltrate Serum2 (n = 13) Urine (n=4) Bile (n = 6) 6.8 % 9.5 % 9.4% 10.4 % 9.2 % 9.9 % 5.6% 1 determined from calibration curves prepared for the pharmacokinetic and tissue distribution study 2 determined from calibration curves prepared for the dose-dependent pharmacokinetic study 45 3.2 SERUM PROTEIN BINDING OF (E,Z)-2,3'-DIENE VPA The serum protein binding characteristics of (E,Z)-2,3 '-diene VPA were investigated in order to determine whether the diene possesses similar binding characteristics as VPA and its analogues. Both ex vivo and in vitro studies were performed. 3.2.1 Ex Vivo Protein Binding Serum samples collected from the pharmacokinetic and tissue distribution study over a 12 h period were used for the ex vivo determination of free and total serum concentrations of (E,Z)-2,3'-diene VPA. The extent of serum protein binding could then be calculated over a broad concentration range (table 4). Serum protein binding ranged from 90-93% bound at serum concentrations less than 40 pg/mL to 54% bound at serum concentrations of 355.5 p, g/mL. A plot of free serum concentration vs. total serum concentration is displayed in figure 8. As the total concentration of (E,Z)-2,3'-diene VPA in rat serum increased from 14.3 to 35.2 p,g/mL, the free serum concentration increased only slightly. However, a dramatic increase in free serum concentration that appeared almost linear was observed when the total serum concentration was increased from 167.0 to 355.5 p\g/mL. 3.2.2 In Vitro Protein Binding The in vitro plot of free fraction of (E,Z)-2,3'-diene VPA vs. total serum concentration of (E,Z)-2,3'-diene VPA is displayed in figure 8 and the results are displayed in table 5. The data demonstrates a progressive decrease in the percentage drug bound with increasing 46 concentration, similar to that observed with the ex vivo study. In addition, similar trends in protein binding were observed with the in vitro study when compared to the ex vivo data in that the free serum concentrations of (E,Z)-2,3'-diene VPA increased only slightly when serum concentrations increased from 15.5 to 64.9 pg/mL, yet free levels increased dramatically when serum concentrations exceeded 123.5 pg/mL. 3.2.3 Binding of (E,Z)-2,3'-diene VPA to the Ultrafiltration Apparatus and YMT Membrane The binding of (E,Z)-2,3'-diene VPA to the ultrafiltration apparatus and the YMT membrane was investigated and was found to decrease with increasing concentration of (E,Z)-2,3'-diene VPA in water. The degree of binding was determined to be 3.46%, 0.17%, 0.02%, and 0.005% at diene concentrations of 1, 30, 100 and 600 p.g/mL, respectively. Free serum concentrations were corrected for drug binding as follows: first, an average of the degree of binding from 1-30 pg/mL, 30-100 p.g/mL, and 100-600 pg/mL was determined and the results were 1.82, 0.09, and 0.01%, respectively. Secondly, the observed in vitro and ex vivo serum concentrations values were classified into the three ranges (1-30 pg/mL, 30-100 pg/mL, and 100-600 pg/mL) and corrected accordingly. 47 Table 4. Ex vivo serum protein binding of (E,Z)-2,3'-diene V P A by ultrafiltration at 25 °C. Each value is expressed as the mean concentration from 5 animals ± SD. Total Concentration Free Concentration* .. (ug/mL) .. Percent Bound "... .,..:YyM^^E-i:-:...'l 355.5 ± 28.3 164.2 ± 22.7 54.0 ± 4.0 311.8 ± 36.2 139.0 ± 28.4 55.6 ± 8.0 236.9 ± 13.9 102.0 ± 5.9 57.0 ± 1.2 167.0 ± 29.8 52.8 ± 10.4 68.4 ± 3.0 53.7 ± 5.4 8.7 ± 2.0 84.2 ± 2.4 35.2 ± 2.7 2.4 + 0.5 93.2 ± 1.3 14.3 + 1.3 1.3 ± 0.1 90.4 ± 2.4 * values are corrected for drug binding to the Amicon ultrafiltration apparatus with YMT membrane. Table 5. In vitro serum protein binding of (E,Z)-2,3-diene V P A by ultrafiltration at 25 °C. Each value is expressed as the mean concentration from duplicate samples ± SD. Total Concentration (fig/mL) Free Concentration* (ug/mL) Percent Bound (%) 583.6 351.2 39.8 284.7 136.9 51.9 251.9 96.4 61.8 123.5 30.5 75.3 64.9 5.8 91.3 38.5 1.9 96.2 15.5 0.7 95.4 * values are corrected for drug binding to the Amicon® ultrafiltration apparatus with YMT membrane. 48 360 -i 300 240 a 180 120 H 60 0 e x v i v o • i n v i t r o o o cv • o o o 0 " < > 0 ^ , — 0 100 200 300 400 500 600 T o t a l S e r u m C o n c e n t r a t i o n ( ( i g / m L ) Figure 8. Relationship between free serum and total serum concentrations of (E,Z)-2,3'-diene V P A in vitro and ex vivo. Each ex vivo data point represents the mean concentration obtained from 5 animals. Each in vitro data point represents the mean concentration obtained from duplicate samples. Free serum values were corrected for drug binding to the Amicon® ultrafiltration apparatus with YMT membrane. Error bars for the ex vivo data have been omitted for clarity. 49 3.3 DOSE-DEPENDENT PHARMACOKINETICS AND METABOLIC STUDY OF (E,Z)-2,3-DIENE VPA The dose-dependent pharmacokinetics of (E,Z)-2,3'-diene VPA were investigated to assess whether the pharmacokinetic parameters change upon administration of increasing dose levels of the diene to rats. In addition, the excretion of parent and conjugated drug in urine and bile was determined. 3.3.1 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Serum The pharmacokinetics of (E,Z)-2,3'-diene VPA after administration of 75, 150 and 300 mg/kg of the drug was investigated to establish whether the elimination or reabsorption characteristics of the drug are dose-related. The concentration-time plots of (E,Z)-2,3'-diene VPA in serum over a 12-h period after i.p. injection of three different doses of the drug are illustrated in figure 9. All three doses exhibited either a secondary rise in concentration or a plateau in the serum profile, followed by a slower elimination phase. After the 75 and 150 mg/kg doses, initial elimination patterns were similar followed by a secondary increase in drug concentration or a plateau in the serum profile occurring at approximately the same time (120 min). After the 300 mg/kg dose, the initial rate of decline was less than that of the lower doses. In addition, the secondary rise in concentration occurred later (300 min) and was more apparent than that of the lower doses. The apparent pharmacokinetic parameters of (E,Z)-2,3'-diene VPA are illustrated in table 6. The drug was rapidly absorbed (tmax ranged between 15 and 22.5 min) and attained 50 peak serum concentrations of 232.4, 387.1, and 730.8 pg/mL for doses of 75, 150 and 300 mg/kg, respectively. The AUC0-i2h values tended to increase with increasing dose although there was no statistical difference between the low and intermediate doses. When the dose was increased from 150 to 300 mg/kg, AUC0-i2h increased three-fold. The (3 and ti/2 values remained relatively unchanged when the dose was increased from 75 to 150 mg/kg; however, when the dose was again increased to 300 mg/kg, p was reduced by 50% and the ti / 2 doubled. The calculated pharmacokinetic parameters of (E,Z)-2,3'-diene VPA are shown in table 7. The V c increased 1.4 fold as the dose increased from 75 to 300 mg/kg. The calculated values of ki 0, k [ 2 and k2i were used to determine effective clearance (Cleff) and net clearance (Clnet) (Colburn, 1984). Similar Cleff and Clnet values were calculated for the 75 and 150 mg/kg doses, whereas a 1.4-1.6 fold decrease in both Cl values were calculated for the 300 mg/kg dose. 51 -|—I—l—l—|—l—l—l—|—l—l—l—j—l—l—l—|—l—l—l—|—l—l—l—|—l—l—I—|—I—I—I—|—I—I—1—|—I—I—I—|—I—I—I—|—l—l—l—|—I—I 0 60 120 180 240 300 360 420 480 540 600 660 720 Time (min) Figure 9. Concentration-time plots of (E,Z)-2,3' -diene V P A in serum following the i.p. administration of 75, 150 or 300 mg/kg of (E,Z)-2,3'-diene V P A to rats. Each value represents the mean concentration obtained from 5 animals. 52 Table 6. Apparent pharmacokinetic parameters in serum for three dose levels of (E,Z)-2,3'-diene V P A in rats. Values are expressed as mean ± SD for 5 animals at each dose. Parameter 75 mg/kg 150 mg/kg 300 mg/kg C m a x (pg/mL) tmax (min) AUC 0-i2h Oig-h/mL) p1 (min"1) ti/21 (min) 232.4 + 33.6 15.0 286.2 + 48.2 0.0275 ± 0.0076 27.1 ±7.1 387.1 ±50.5 22.5 605.0 ± 125.4 0.0248 ±0.0031 28.5 ±3 .9 730.8** ± 176.1 22.5 1852.5** ±319.2 0.0121** ±0.0027 60.0** ± 12.9 P was estimated from the slopes of the linear portion of the serum drug concentration-time profile ** Significantly different from the next lower dose at/? < 0.05 using one-way A N O V A Table 7. Calculated1 pharmacokinetic parameters in serum for three dose levels of (E,Z)-2,3'-diene V P A in rats. Values are expressed as mean ± SD for 5 animals at each dose. Parameter 75 mg/kg 150 mg/kg 300 mg/kg ka (min1) 0.1077 ±0.0261 0.1147 ±0.0308 0.1289 ±0.0324 km (min"1) 0.0251* ±0.0047 0.0179* ±0.0024 0.0106* ±0.0024 kn (min"') 0.0065 ±0.0018 0.0049 ±0.0018 0.0017** ±0.0001 k 2i (min1) 0.0268 ±0.0137 0.0406 ±0.0188 0.0147 ±0.0076 V c (mL/kg) 191.91* ± 14.36 250.87* ± 22.59 289.80* ±13.47 Cleff2 (mL/min-kg) 6.02 ± 1.04 5.72 + 0.53 3.56** ±0.66 Clnet3 (mL/min-kg) 4.78 ±0.75 4.47 ±0.50 3.07** ±0.64 1 Using a non-linear least squares regression program MULTI (RUNGE) 2 Cl e ff = V c (k,0 + k,2) Clnet = V c * kio * Significantly different from other doses, p < 0.05 ** Significantly different from next lowest dose, p < 0.05 53 3.3.2 Excretion of (E,Z)-2,3'-diene VPA and Conjugated (E,Z)-2,3'-diene VPA in Urine The urinary recoveries of (E,Z)-2,3'-diene VPA and its conjugates were measured in rats after administration of the three different doses of the drug in order to determine if the dose modified the excretion of the drug or its conjugates. Figure 10 displays a graphical representation of the excretion of free and conjugated (E,Z)-2,3'-diene VPA in rat urine following i.p. administration of 75, 150, or 300 mg/kg of the drug. The fraction of parent and conjugated (E,Z)-2,3'-diene VPA excreted in the urine were 5.5, 6.8 and 5.9 % and 11.4, 25.6 and 26.3 %, at doses of 75, 150 and 300 mg/kg, respectively. For the parent drug, urinary recovery remained relatively unchanged as dose increased. In contrast, the fraction of the dose excreted in the urine as conjugates increased as dose increased, although the extent of the excretion of conjugated (E,Z)-2,3'-diene VPA at doses of 150 and 300 mg/kg remained relatively unchanged. 3.3.3 Excretion of (E,Z)-2,3'-diene VPA and Conjugated (E,Z)-2,3'-diene VPA in Bile The biliary excretion of (E,Z)-2,3'-diene VPA in bile-exteriorized rats was investigated to characterize drug excretion by this excretory route and establish whether drug excretion could be related to choleresis. Biliary excretion of (E,Z)-2,3'-diene VPA was rapid following a 150 mg/kg i.p. injection of the drug. Approximately 10.5% of the dose given was excreted as parent (E,Z)-2,3'-diene VPA with most of this occurring within the first 2 h. Figure 11 illustrates a comparison of a choleretic effect after i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene VPA in the rat and elimination of (E,Z)-2,3'-diene VPA as conjugates. About 23.2% of the dose given was secreted as (E,Z)-2,3'-diene VPA conjugates, with 54 approximately 78.0% of this excreted within the first 2 h. The extensive conjugation of the diene appeared to parallel bile flow production, which also increased markedly within the first two hours. Total biliary excretion of (E,Z)-2,3'-diene VPA (free and conjugated) in 6 h was approximately 33.7% of the dose. 55 Legend 75 mg/kg 150 mg/kg 300 mg/kg Dose Administered Figure 10. Amount of (E,Z)-2,3'-diene V P A excreted in urine as unchanged and conjugated drug following i.p. injection of 75, 150 or 300 mg/kg of (E,Z)-2,3'-diene VPA. Each value represents the mean concentration obtained from 3 animals. 56 = 1 H I LEGEND Bile flow rate (E,Z)-2,3'-diene conjugates I -30 - 0 0 - 1 1 - 2 2 - 3 3 - 4 4 - 5 5 - 6 Collection T i m e Interval (h) r- 5 4 & h 2 Figure 11. (E,Z)-2,3'-diene V P A conjugate excretion and bile flow rate following the i.p. administration of 150 mg/kg (E,Z)-2,3'-diene VPA. Each value represents the average concentration obtained from 3 animals. 57 3.4 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF (E,Z)-2,3'-DIENE VPA IN BRAIN, SERUM AND LIVER The purpose of the pharmacokinetic and tissue distribution study was to establish whether (E,Z)-2,3'-diene VPA reflects desirable attributes of an anticonvulsant medication, namely rapid absorption and distribution to brain followed by sustained brain levels over an extended period of time, accumulation of the drug in selected brain regions, and low liver distribution. It should be noted that several attempts were made to fit the data obtained from this investigation into a time-lag four compartment model, as described in section 2.5.2. Unfortunately, all attempts were unsuccessful. The reasons for the inability to model our data are discussed in section 4.4. 3.4.1 Pharmacokinetics of (E,Z)-2,3 '-diene VPA in Serum The concentration-time plot of (E,Z)-2,3'-diene VPA in serum over a 12 h period after i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene VPA in the rat is illustrated in figure 12. Apparent pharmacokinetic parameters in serum are listed in table 8. The drug was rapidly absorbed and attained peak serum concentrations of 336.9 Ug/mL in 15 min. The serum concentration elimination profile declined rapidly with a tm of 35.5 min. A plateau in the serum concentration was observed at approximately 120-240 min followed by a slower elimination phase. The AUC0-i2h value for (E,Z)-2,3'-diene VPA in serum determined from 0 to 12 h was calculated to be 518.9 p:g*h/mL. 58 When the serum data from the current study was compared with the dose-dependent study at the 150 mg/kg dose, similar features were observed. Both the dose-dependent and the current studies exhibited similar serum C m a x (387.1 |lg/mL and 336.9 u,g/mL, respectively), AUC0-i2h (604.9 and 518.9 |0.g-h/mL, respectively) and tl/2 values (28.5 and 35.5 min, respectively). One difference observed when comparing the two studies was the apparent increase in serum concentration at 720 min for the dose-dependent study which was not seen with the latter study. The mean serum concentration values for the dose-dependent and the current study at 720 min were 3.4 ± 6.2 and 0.29 ± 0.05 (ig/mL, respectively. Upon investigating this observation, our results indicated one serum sample from the dose-dependent study that was significantly higher in concentration than the others, resulting in the higher average serum concentration when compared to the present study. When calculating the mean serum concentration from 4 samples without the "outlier", the resulting concentration is 0.34 ± 0.42 which compares well with the 720 min serum concentration for the present pharmacokinetic study. 59 1000.00 LEGEND serum • liver A whole brain 0 i 1 1 1 1 i i i i r 60 120 180 240 300 360 420 480 540 600 660 720 Time (min) Figure 12. Concentration-time curve of (E,Z)-2,3'-diene V P A in rat serum, liver and brain following the i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene V P A . Each value represents an average of 8 animals (with the exception of the 720 min timepoint for liver as discussed in the text). 60 Table 8. Apparent pharmacokinetic parameters in serum, whole remaining brain and liver following i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene V P A in rats. C m a x values are expressed as mean ± SD. Parameter Serum Whole Remaining Brain Liver C m a x (pg/mL or /g) 336.9 + 39.9 47.1 ±8 .6 247.3 ±57.2 tmax (min) 15.0 22.5 7.5 AUC 0-i2h (pg-h/mL or /g) 518.9 43.7 241.5 p1 (min1) .0195 .0327 .0196 ti/2' (min) 35.5 21.2 35.4 Cl 2 (mL/min-kg) 4.82 1 P was estimated from the slope of the linear portion of the concentration-time profiles 2 Cl= Dose/ AUC 0-i2h 61 3.4.2 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Whole Remaining Brain and Selected Brain Regions The brain concentration-time plots of (E,Z)-2,3'-diene VPA in whole remaining brain and the 11 selected brain regions (cerebellum, medulla, pons, olfactory bulbs, inferior colliculus, superior colliculus, hippocampus, corpus callosum, corpus striatum, frontal cortex, and substantia nigra) after i.p. administration of 150 mg/kg of the drug is illustrated in figure 13. The elimination profiles in all brain regions including whole remaining brain displayed similar characteristics, exhibiting a rapid initial elimination phase followed by a decrease in the rate of decline (or a plateau) in brain concentration. The plateau in the whole remaining brain profile appeared to parallel that observed for serum, with the plateau occurring at approximately 120-240 min. In contrast, the elimination profiles for the 11 brain regions displayed the transient increase at approximately 240-480 min. Tables 8 and 9 display the apparent pharmacokinetic parameters for whole remaining brain. Table 9 displays the apparent pharmacokinetic parameters for the 11 brain regions. Maximum whole remaining brain and regional brain concentrations ranging from 42.5-55.4 pg/g were attained within 15-22.5 min. The half-life values for whole remaining brain and the brain regions ranged from 17.6 to 27.9 min. The regional distribution of (E,Z)-2,3'-diene VPA in the brain was found to be relatively uniform as the AUC 0-i2h values for each brain region (ranging from 38.7 to 51.0 pg-h/g) were similar to that obtained for whole remaining brain (43.7 pg-h/g). Based on the AUCo-i2h values for whole remaining brain and the 11 brain regions, distribution of the drug to brain was approximately 11.5-fold less than serum. 62 3.4.3 Pharmacokinetics of (E,Z)-2,3 '-diene VPA in Liver The distribution of (E,Z)-2,3'-diene VPA into the liver after i.p. administration of the drug is displayed in figure 12. The apparent pharmacokinetic parameters calculated for liver are listed in table 8. Peak liver concentrations of 247.3 (ig/mL were attained within 7.5 min. The elimination of (E,Z)-2,3'-diene VPA from the liver was rapid with a tm of 35.4 min and appeared to parallel the decline of the drug from serum. In addition, a slight plateau at about 240 min was observed in the liver which was similar to that observed in the serum and brain elimination profiles. At 12 hours following the dose, (E,Z)-2,3'-diene VPA levels fell below the LOQ for 1 of the 8 rat livers sampled and 3 samples were undetected; therefore, the reported mean concentration at tj2h is based on the mean from the 4 detectable liver samples. The mean liver concentration of (E,Z)-2,3'-diene VPA was 8.5 times lower at tm, than found in serum. Distribution of (E,Z)-2,3'-diene VPA to liver was less than 1/2 that found in serum based on the AUC 0-i2h values (241.5 Ug-h/g and 518.9 |lg-h/mL, respectively). 63 LEGEND 1000.000 100.000 -i #o 2 10.000 = a = 0 U «U > c s 1 • N 1.000 d 0.100 0.010 • C E R 0 C C • M E D • F C A PONS A IC • CS V S C • HIP 0 W R B O B 0 SN 0—0 SER 1 1 1 1 1 1 I I I 0 6 0 120 180 2 4 0 3 0 0 3 6 0 4 2 0 4 8 0 5 4 0 6 0 0 6 6 0 7 2 0 Time (min) Figure 13. Concentration-time curves of (E,Z)-2,3'-diene V P A in rat serum (SER), cerebellum (CER), medulla (MED), pons (PONS), corpus striatum (CS), hippocampus (HIP), olfactory bulb (OB), corpus callosum (CC), frontal cortex (FC), inferior colliculus (IC), superior colliculus (SC), whole remaining brain (WRB) and substantia nigra (SN) following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene V P A . Error bars for SER and W R B have been omitted for clarity. (WRB and SER: n=8/time point; brain regions: pooled from 8 rats). 64 c #o et s-VI '8 1 a OU a % o a '5c u et u 13 a et et u Xi DX) C '3 "8 £ — u <u V E rt et a 93 < OH C CU 0> c o u OJ E s* et JA a s cu u et a a < » CI_I H © r o • WD : 5 -$* •to? :::.a:;::; 00 co T-H oo oo (N cN 1 ON rt r~ oo VO cn od CN oo oo oo O CN r t ON o r -r t oo cn CN r t r -r t in r t in r t rt" d r r r t r t in IT) ON r t CN r t cn vD r t oo • ON r--oo r t O r t VO r t vo r t rt 00 r i ON m r t i n i n i n i o CN CN CN CN CN CN CN CN CN m CN CN in CN rt CN CN CN CN CN CN CN ON 00 t - oo 00 oo r t CN r t oo ON oo o VO CN vo r - r t oo ON ON r - 00 VO r t VO cn cn cn <N CO CO CO co co CO co CO o O O O o •O o o o o O O oo CN r t r t vq r t in CN CN S-l ba a 'a 0 O e 1 o rt 1 3 o a 3 O o 3 & O o X o o 3 e o O o S-l o o o op ' r*\ O ™ o> a 3 3 65 3.4.4 Brain:Serum Concentration Ratios and the Relationship Between Brain, Free Serum and Total Serum Concentrations Figure 14 displays the brain: total serum concentration ratios at tmax and tJ2h for whole remaining brain and the 11 brain regions. Braimtotal serum ratios remained below unity at both tmax and ti2h- The concentration ratios for the diene ranged from 0.13-0.17 at tmax and 0.12-0.99 at ti2h- The concentration ratios increased by up to 6.2x for 10 of 11 brain regions at tnh when compared to their corresponding concentration ratios at tmax. However, the ratio for whole remaining brain:total serum, which was used as a reference, also increased in magnitude, and therefore the most noteworthy increase was observed in the inferior colliculus (0.99) when compared to whole remaining brain (0.35). Similar to the whole remaining brain:total serum concentration ratio, the whole remaining brain:free serum ratio was also found to increase over time (figure 15). The braimfree serum ratio of (E,Z)-2,3'-diene VPA increased from 0.33 at tmax to 3.93 at ti2h-Figure 16 displays the correlation between whole remaining brain concentration and free serum concentration of (E,Z)-2,3'-diene VPA. The free serum levels of the drug increased linearly with whole remaining brain concentration levels as indicated by the coefficient of determination of 0.900. In contrast, the relationship between whole remaining brain concentration and total serum concentration was not linear but instead was best described as a non-linear or polynomial fit, exhibiting a slight curvature at the lower brain and serum concentrations (figure 17). Whole remaining brain:total serum concentration ratios remained well below unity at each timepoint, consistent with the results obtained from the regional brain:total serum concentration ratios. 66 0.20 0.18 0.16 0.14 -0.12 -0.10 -0.08 0.06 -\ 0.04 0.02 0.00 cer med pon cs hip ob cc fc ic sc sn wrb 1.0 - , 0.9 .2 0.8 « § 0 - 7 I 0.6 1 0.5 1 °-4 u 0.3 0.2 0.1 0.0 l12h cer med pon cs hip ob cc fc ic sc sn wrb Figure 14. Brain:total serum ratios of (E,Z)-2,3'-diene V P A in cerebellum (CER), medulla (MED), pons (PONS), corpus striatum (CS), hippocampus (HIP), olfactory bulb (OB), corpus callosum (CC), frontal cortex (FC), inferior colliculus (IC), superior colliculus (SC), substantia nigra (SN) and whole remaining brain (WRB) of rats at tm a x and tnh following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene V P A (WRB: mean value from n=8; brain regions: pooled from 8 rats). * tm a x ranged from 15 to 22.5 min. 67 S E R W R B L I V Figure 15. Tissue concentrations relative to total and free serum concentrations of (E,Z)-2,3'-diene V P A in whole remaining brain (WRB) and liver (LIV) at t m a x (ranging from 7.5 to 22.5 min) and t ] 2 h following 150 mg/kg i.p. administration of the compound (n=8 for total serum and n=5 for free serum, with the exception of liver at tub as discussed in the text; error bars = SD). 68 0 5 0 1 0 0 1 5 0 2 0 0 Free Serum Concentration (|Lig/mL) Figure 16. Rat whole remaining brain concentrations versus free serum concentrations of (E,Z)-2,3'-diene V P A following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene V P A to rats. Each value represents an average of 5 animals. Free serum values were corrected for drug binding to the Amicon® ultrafiltration apparatus with Y M T membrane. 69 Figure 17. Rat whole remaining brain concentration versus total serum concentration of (E,Z)-2,3*-diene V P A following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene V P A to rats. Each value represents an average of 8 animals. 70 3.4.5 Relationship Between Liver, Free Serum and Total Serum Concentrations Figure 18 displays the relationship between liver concentration and free serum concentration of (E,Z)-2,3'-diene VPA. The free serum levels of the drug increased linearly with corresponding liver concentrations as indicated by the coefficient of determination of 0.901. In contrast, the liver concentration vs. total serum concentration plot was a polynomial fit, exhibiting a slight curvature at the lower liver and serum concentrations (figure 19), similar to that reported with whole remaining brain vs. total serum. The livenserum concentration ratios, as illustrated in figure 15, decreased in magnitude over time. The liventotal and livenfree serum concentrations of (E,Z)-2,3'-diene VPA decreased from 1.09 and 3.77 at tmax to 0.08 and 0.68 at ti2h, respectively. It should be noted that the liver:serum ratios at ti2h were based on the mean derived from 4 liver and total or free serum samples, as 1 of 8 liver samples sampled fell below the LOQ and 3 samples were undetected. 71 Free Serum Concentration (|i.g/mL) Figure 18. Rat liver concentrations versus free serum concentrations of (E,Z)-2,3'-diene V P A following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene V P A to rats. Each value represents the average concentration from 5 animals. Free serum values were corrected for drug binding to the Amicon® ultrafiltration apparatus with Y M T membrane. 7 2 250 -] 0 50 100 150 200 250 300 350 Total Serum Concentration (jug/mL) Figure 19. Rat liver concentrations versus total serum concentrations of (E,Z)-2,3'-diene V P A following 150 mg/kg i.p. administration of (E,Z)-2,3'-diene V P A to rats. Each value represents an average of 8 animals. 73 4. D I S C U S S I O N Much concern over the risk of VPA-induced hepatotoxicity and teratogenicity have stimulated efforts to investigate other candidates as potential alternatives to VPA. Since the properties of VPA are believed to be related to its branched-chain fatty acid-like structure, various analogues of VPA have been developed and investigated for their potential as alternative anticonvulsants without the toxic effects. Preliminary work in our laboratory with (E,Z)-2,3'-diene VPA, a serum metabolite of VPA, demonstrated equivalent anticonvulsant activity to VPA in rats (Lee, 1991) and mice (Palaty and Abbott, 1995) using the pentylenetetrazole-induced convulsion test. In addition, (E,Z)-2,3'-diene VPA was reported to exhibit niinimal muscle spasticity and sedative effects that were similar to that reported for VPA (Lee, 1991). The favorable anticonvulsant potency and low neurotoxicity results obtained for (E,Z)-2,3'-diene VPA from the preliminary study suggest that (E,Z)-2,3'-diene VPA could potentially be used as an alternative to VPA. Several investigations were therefore conducted to further assess the diene's potential as a viable alternative to VPA. 4.1 ANALYSIS OF (E,Z)-2,3-DIENE VPA BY EI-GCMS 74 A reliable GCMS assay of ?-BDMS derivatives of VPA analogues has been developed and reported previously (Acheampong et al, 1984; Abbott et al, 1986; Yu et al, 1995). We report here that the GCMS assay used provided the conditions necessary for the resolution of the ?-BDMS derivatives of (E,Z)-2,3'-diene VPA and the internal standard, [2H7]-2-ene VPA. A mass selective detector, operated under EI mode, provided adequate sensitivity for the detection of the compounds of interest. Selected ion monitoring was used to monitor the two ions, m/z 197 and 206, corresponding to (E,Z)-2,3'-diene VPA and [2H7]-2-ene VPA, respectively. The assay demonstrated good sensitivity which could be partly attributed to the highly intense m/z [M-57]+ fragment ion, corresponding to the loss of the tert-butyl fragment of the t-BDMS derivatives. The f-BDMS derivatives of (E,Z)-2,3'-diene VPA and [2H7]-2-ene VPA demonstrated sharp and symmetrical peaks on all GCMS chromatograms. Our results demonstrated that the assay is reliable. All calibration curves gave satisfactory linearity over the concentration ranges used for all biological tissues, with coefficients of determination ranging from 0.994 to 0.999. In addition, the reliability of the assay was demonstrated from the slopes of calibration curves measured over a 12 month period. The inter-assay variation remained below 15% for all biological tissues analyzed. In addition, the accuracy and precision of the assay was determined to be less than 17% CV for anLOQ of 0.01 pg/mL. 75 Extraction efficiencies for both brain and liver samples ranged from 56.0 to 73.5% which are considered to be low. Reasons for such low extraction efficiencies are not clear but could be due to the fact that samples were extracted with ethyl acetate only once, a step designed to shorten the duration of handling a large number of samples. However, the entire assay has been demonstrated to have satisfactory accuracy and precision, suggesting that the low extraction efficiency did not interfere with the reliability of the assay and therefore was acceptable. 76 4.2 SERUM PROTEIN BINDING OF (E,Z)-2,3-DIENE VPA The binding of a drug to serum proteins is important therapeutically as it can affect tissue distribution, volume of distribution and clearance (Tozer, 1981). Because the pharmacological effect of a drug correlates with free serum concentration in the body, it is important therapeutically to determine the binding characteristics of the drug and corresponding free drug concentrations. Drugs that are highly bound to serum or plasma proteins are subject to unpredictable variations in free drug levels. Because VPA has been reported to be highly serum bound (discussed below), and tissue distribution of VPA is dependent on free levels, the serum protein binding of (E,Z)-2,3'-diene VPA was investigated to assess whether the diene reflects similar binding characteristics as VPA. In addition, the data from the current study was compared to studies performed with other VPA analogues. The results from the ex vivo serum protein binding suggest that the protein binding of (E,Z)-2,3'-diene VPA is concentration dependent. The protein binding data presented here indicate that as total diene serum concentration increases from 14.3 to 53.7 |0,g/mL to over 167 |J<g/rnL, free fraction increases from less than 17% to over 30%. It would appear that drug binding becomes saturated at approximately 53.7 )ig/mL, which may be attributed to the saturation of protein binding sites for (E,Z)-2,3'-diene VPA, thereby leaving a greater percentage of drug unbound as the serum concentration of (E,Z)-2,3'-diene VPA increased. Ex vivo protein binding of VPA has also been reported to be concentration-dependent. After the administration of 1000, 2000 or 3000 mg of VPA to healthy volunteers, the mean free fraction of VPA was found to increase with dose (Gomez Bellver et al, 1993). In 77 addition, Cramer et al. (1986) reported that the average unbound fraction of VPA in humans ranged between 7 and 9% at concentrations below 75 p,g/mL and increased to 15 and 30% at concentrations of 100 and 150 pg/mL, respectively. Non-linear protein binding for VPA has also been reported in other species such as guinea pigs (Yu and Shen, 1992) and dogs (Loscher, 1978). Rats have also been reported to exhibit saturable serum protein binding over the range of 10 to 400 pg/mL of VPA (Brouwer et al, 1993) although other investigators did not report non-linear binding of VPA in rats (Loscher, 1978). This discrepancy for the latter study, in part, may be due to the fact that Loscher (1978) reported protein binding at only two serum concentrations of 105 and 190 pg/mL, respectively, thus only measuring above saturable protein concentration of 70 to 80 pg/mL. It would appear that the ex vivo serum protein binding characteristics of the diene are similar to that of VPA. In accordance with the ex vivo studies, the in vitro protein binding experiments exhibited a similar trend in that the degree of binding decreased with increasing diene concentration. (E,Z)-2,3'-Diene VPA appeared to be highly bound (91.3 to 96.2%) at concentrations between 15.5 and 64.9 pg/mL and decreased to 75% bound at 123.5 p.g/mL. In comparison to in vitro data obtained for VPA, Semmes and Shen (1990) reported similar in vitro results where VPA exhibited concentration-dependent binding to rat plasma proteins that was most apparent between VPA plasma concentrations of 100 and 400 p,g/mL. Similar results were obtained over the total concentration range of 8 to 2400 pg/mL where saturation of serum protein binding by VPA occurred at approximately 100 p.g/mL (Haberer and Pollack, 1994). In vitro plasma protein binding studies with guinea-pigs over a wide range of steady-state plasma concentration also demonstrated similar concentration-dependent binding 78 characteristics, with the unbound fraction increasing from 14 to 79% with an increase in VPA concentration of 10 to 1000 |_Lg/mL (Yu and Shen, 1992). In addition, studies performed in humans reported similar binding behavior with VPA reaching binding saturation at approximately 80 |ig/mL (Cramer and Mattson, 1979). Based on the results from the current ex vivo and in vitro serum protein binding studies for the diene and from the results obtained for VPA from the literature, it would appear that both (E,Z)-2,3'-diene VPA and VPA display similar saturable protein binding characteristics. The serum protein binding of (E,Z)-2,3'-diene VPA appears to be greater than VPA at concentrations below 65 (ig/mL, and this observation has also been made for two other unsaturated VPA metabolites, (E)-2-ene VPA (Semmes and Shen, 1990) and (E,E)-2,3'-diene VPA (Lee, 1991). It is interesting to note that enhanced serum protein binding is observed with those unsaturated metabolites with a double bond in the C-2 position. This observation suggests that the degree of serum protein binding may be structurally dependent, although additional studies would be required to confirm this hypothesis for (E,Z)-2,3'-diene VPA. 79 4.3 DOSE-DEPENDENT PHARMACOKINETICS AND METABOLIC STUDY OF (E,Z)-2,3' -DIENE VPA 4.3.1 Pharmacokinetics of (E,Z)-2,3'-diene VPA in Serum Dose-related pharmacokinetics of VPA in various species have been reported by several investigators (Dickinson et al., 1979; Yu et al., 1987) whereby changes in one or more of the pharmacokinetic parameters during the absorption, distribution, metabolism and excretion of the drug occurred due to increasing dose levels. Deviations from linearity may be due to several factors, such as protein binding to serum or tissues, saturable metabolism, and capacity-limited elimination of a drug. This investigation was designed to examine the serum profiles of (E,Z)-2,3'-diene VPA after i.p. administration of three different doses of the drug, and to compare any dose-related changes in kinetic parameters among the doses with data from similar dose-related investigations of VPA and its analogues. The present data suggest that the pharmacokinetics of (E,Z)-2,3'-diene VPA are complex and do not follow a first-order process. Following a single dose of either 75, 150 or 300 mg/kg i.p. of (E,Z)-2,3'-diene VPA, peak serum concentrations were attained within 15 to 22.5 min, indicating rapid absorption of the drug into the general circulation. Each elimination profile exhibited a secondary increase or a plateau in serum concentration which occurred at approximately the same time for the two lower doses (120 min) and at a later time (300 min) for the highest dose. The secondary increases in concentration are most likely due to enterohepatic circulation (EHC), a phenomenon which is a result of reabsorption of free drug from the intestine into the systemic circulation. Several studies examining the 80 pharmacokinetics of VPA have reported that VPA undergoes enterohepatic recycling in rats (Dickinson et al, 1979; Ogiso et al, 1986; Liu et al, 1990). Additional studies indicated that the observed secondary increase in VPA serum concentration was eliminated in bile-exteriorized rats which diverted a large amount of the drug out of the rats (Dickinson et al, 1979; Ogiso et al, 1986). Taken together, the observed secondary increase in serum concentration for (E,Z)-2,3'-diene VPA was most likely due to enterohepatic circulation. The observed differences in the pharmacokinetic parameters suggest that the pharmacokinetics of (E,Z)-2,3'-diene VPA are dose-dependent. The elimination ti/2 of (E,Z)-2,3'-diene VPA remained relatively unchanged at the lower doses but doubled in magnitude when the dose was increased from 150 to 300 mg/kg. It should be noted that the Un was measured from the linear portion of the serum concentration-time profile (from 45 to 120 min for the low and intermediate doses, and 60 to 240 min for the 300 mg/kg dose), due to the observed secondary serum concentration increases. Accordingly, the elimination rate constant, (3, exhibited a dramatic decrease in magnitude at the 300 mg/kg dose level when compared to the values obtained at the lower dose levels. The AUC increased 2-fold when the dose level doubled from 75 to 150 mg/kg, yet increased more than 3-fold as the dose doubled to 300 mg/kg. Similar dose-related trends for ti /2, p\ and AUC have been reported for VPA for rats and guinea-pigs. Dickinson et al. (1979) investigated dose-related changes after VPA administration in rats and reported half-lives of 11.7, 41 and 125 min after administration of 15, 150 and 600 mg/kg, respectively. Guinea pigs of different ages that were administered doses of 20, 200, and 600 mg/kg exhibited similar increases in ti / 2 with increasing dose (Yu et al, 1987). In addition, AUC increased almost proportionately with 81 increasing doses from 20 to 200 mg/kg, but increased out of proportion (5 to 6 x greater in magnitude) as the dose increased to 600 mg/kg (Yu et al, 1987). The dose-related parameter changes have been interpreted to result from concentration-dependent protein binding and/or metabolic saturation of elimination pathways. Our results from the serum protein binding experiment (section 4.2) indicated that the serum protein binding of the diene is concentration dependent, and would therefore be the most likely explanation for this occurrence. However, saturable metabolism may also be a contributing factor to the dose-related changes. The results obtained from the study of the excretion of conjugated (E,Z)-2,3'-diene VPA in urine (section 4.3.2) provided some evidence of saturable metabolism, namely the glucuronide pathway. The excretion of the diene as glucuronides for the intermediate and high dose levels remained relatively unchanged, suggesting a capacity-limited or saturable glucuronidation. The serum data were fitted into a two compartment time-lag pharmacokinetic model, a model originally proposed by Steimer et al. (1982) for drugs susceptible to hepatobiliary recycling. The microconstants generated from the MULTI(RUNGE) program suggest that (E,Z)-2,3'-diene VPA is moderately excreted from bile and highly reabsorbed into the general circulation because the reabsorption rate (k 2i) is greater than the biliary excretion rate (k)2) for all three dose levels. Elimination from the central compartment (depicted by the microconstant, ki0) decreased with increasing dose, which is consistent with our contention of saturable metabolism with increasing dose. The values for the volume of the central compartment (Vc) were also generated from the computer model. The V c increased as the dose level increased, and this trend has also been reported in rats for the VPA analogue 1-methyl-1-cyclohexanecarboxylic acid (MCCA) 82 (Liu and Pollack, 1993) and in guinea pigs for VPA (Yu and Shen, 1996). It has been suggested that a dose-dependent increase in V c may be due in part to dose-dependent saturation of serum protein binding, resulting in redistribution of free drug from serum to tissues (Liu and Pollack, 1993; Yu and Shen, 1996). In section 4.2, we reported that the diene exhibited concentration-dependent serum protein binding that attained an apparent ex vivo saturation of binding at approximately 53.7 pg/mL. Because serum concentrations remained well above this concentration after administration of 300 mg/kg of the compound, it is possible that unbound drug may have been redistributed to tissues, resulting in an increased V c at this dose level when compared to the V c obtained with lower doses. Another possible explanation for the dose-related changes in V c may be related to the degree of recirculation produced with increasing dose. Studies performed by Pollack and Brouwer (1991) using rats dosed with VPA indicated that an increase in V c with increasing dose may be due to an apparent dose-dependent increase in enterohepatic recirculation. Because the recirculatory peaks observed in our serum (E,Z)-2,3'-diene VPA studies followed a similar pattern as reported for VPA and appeared to increase with increasing dose (which was most apparent at the 300 mg/kg dose level), it is possible that hepatobiliary recycling may be contributing to the dose-dependent changes in V c. Nevertheless, it is difficult to assess the exact mechanisms involved in the dose-related changes in V c for (E,Z)-2,3'-diene VPA. The contention of saturable serum protein binding may be only partially responsible for the changes in V c with dose. More likely, the increase in V c is a combination of saturable serum protein binding, increased volume of distribution with an apparent increase in the recirculatory peak and perhaps other unknown mechanisms. 83 Clearance, namely effective clearance (Cleff) and net clearance (Clnet), were used to determine the clearance characteristics of (E,Z)-2,3'-diene VPA in rats. It was shown by using the microconstants generated from computer modelling, namely kio and ki 2, that the calculated clearance values would best reflect that of compounds recycled in the bile. Therefore, Cleff, which was used to describe the intrinsic ability of the liver to remove drug from the blood, could be determined using the equation Cleff = V c (k i0 + k,2). Furthermore, Cl n e t , described as the estimate of the permanent elimination of drug from the body, could be determined from Cl n et - V c • kio (Colburn, 1984). The Cleff and Cl n e t after (E,Z)-2,3'-diene VPA administration were found to be dose-dependent. Cleff and Clnet were determined to decrease with increasing dose, which was most significant for the transition from 150 to 300 mg/kg. Similar dose-related changes in clearance for VPA in rats (Liu and Pollack, 1993) and guinea-pigs (Yu et al, 1987) have been reported previously. Yu et al (1987) reported an apparent decrease of total clearance following increasing doses of 20, 200 and 600 mg/kg of VPA, and suggested that the dose-dependent changes may be due in part to concentration-dependent protein binding or saturable hepatic elimination. Saturable elimination of VPA has been reported previously. Vree et al. (1977) reported dose-dependency of VPA at doses higher than 5 mg/kg in monkeys and discovered that conjugation of VPA was a Umiting factor for the elimination of VPA. In addition, investigations performed by Liu and Pollack (1993) using rats that were administered 75, 150 and 350 mg/kg demonstrated the same trend in clearance that was also attributed to capacity-limited elimination processes, although other contributing factors such as end-product inhibition and concentration-dependent enterohepatic recirculation were not 84 ruled out. It is possible that one or more of these factors may indeed.be responsible for the dose-related changes in clearance for (E,Z)-2,3'-diene VPA. 85 4.3.2 Excretion of (E,Z)-2,3'-diene VPA and Conjugated (E,Z)-2,3 '-diene VPA in Urine The urinary recoveries of (E,Z)-2,3'-diene VPA and its conjugate (presumably glucuronide) were measured in rats after administration of the three different doses of the drug. Total urine was collected for 24 h. The amount of conjugated (E,Z)-2,3'-diene VPA in urine was determined by base hydrolysis, a method previously used for determination of VPA conjugates and has been shown to produce similar results as incubation with (3-glucuronidase (Dickinson et al., 1985; Liu et al, 1992). The formation of conjugate, presumably the glucuronide, were determined by the degree of increase of the chromatographic peak corresponding to the parent drug, following incubation with NaOH. Urinary recovery of parent (E,Z)-2,3'-diene VPA remained relatively unchanged as dose increased, which is consistent with results reported for VPA (Dickinson et al, 1979; Liu et al, 1992). Based upon the recovery of base-labile conjugate in the urine, the fraction of the dose excreted in the urine as glucuronide increased as dose increased, and a similar trend has been reported by other investigators for VPA-glucuronide in urine (Dickinson et al, 1979). However, the extent of glucuronide secretion for VPA following administration of a low (15 mg/kg) and high (150 mg/kg) dose was larger than observed for (E,Z)-2,3'-diene VPA (23 and 51% vs. 11.4 and 25.6%, respectively), suggesting other metabolic pathways may be responsible for the metabolism of (E,Z)-2,3'-diene VPA. The excretion of (E,Z)-2,3'-diene VPA glucuronide for the 150 and 300 mg/kg dose levels remained relatively unchanged, which may suggest that elimination of the diene via glucuronide conjugation may be capacity-limited or saturable. 86 4.3.3 Excretion of (E,Z)-2,3'-diene VPA and Conjugated (E,Z)-2,3 '-diene VPA in Bile It has been well documented that VPA undergoes extensive conjugation with endogenous D-glucuronic acid, yielding VPA-glucuronide which can subsequently be excreted into the bile. The conjugate that is excreted into the bile would then be susceptible to hydrolysis by [^ -glucuronidase enzymes in the gastrointestinal tract, and the liberated drug could be reabsorbed into the systemic circulation. Dickinson et al. (1979) reported findings in rats whereby 54% of the 150 mg/kg administered dose of VPA was excreted in the bile as VPA-glucuronide, and secondary serum concentration peaks that were observed in normal rats were eliminated upon exteriorization of the bile. Our data indirectly supports the contention that the apparent secondary rise in (E,Z)-2,3'-diene VPA serum concentration is most likely due to formation and cleavage of (E,Z)-2,3'-diene VPA glucuronides and subsequent reabsorption of parent drug. Like VPA, (E,Z)-2,3'-diene VPA was also excreted as glucuronide into bile (23% of the administered dose) and secondary serum concentration peaks were observed. Although bile-exteriorization with concurrent serum sampling studies were not performed with (E,Z)-2,3'-diene VPA, the observed secondary increase in serum concentration of (E,Z)-2,3'-diene VPA appears to be a result of the formation and recycling process of (E,Z)-2,3'-diene VPA glucuronide. The extensive conjugation of (E,Z)-2,3'-diene VPA appeared to parallel bile flow production, suggesting a correlation between choleretic effect and glucuronide excretion of (E,Z)-2,3'-diene VPA in bile. This phenomenon is consistent with observations made for VPA (Dickinson et al, 1979), although the extent and magnitude of the choleretic effect for VPA was significantly greater than that observed for (E,Z)-2,3'-diene VPA. A similar choleretic 87 effect was observed after administration of both VPA and MCCA, and it was shown that a strong correlation existed between choleresis and the biliary excretion rate of conjugate (Liu et al., 1992). Together, our data supports the notion of a relationship between choleresis and biliary excretion of glucuronide conjugate. Total biliary excretion of (E,Z)-2,3'-diene VPA was substantially smaller than that reported for VPA (33.7 vs. 58.0% of dose administered, respectively) (Dickinson et al., 1979), suggesting that other metabolic routes play a significant role in the metabolism of (E,Z)-2,3'-diene VPA. Extensive metabolic profiling of this diene will be necessary to confirm this hypothesis. 88 4.4 PHARMACOKINETICS AND TISSUE DISTRIBUTION OF (E,Z)-2,3'-DIENE VPA IN SERUM, BRAIN, AND LIVER Because (E,Z)-2,3'-diene VPA has demonstrated its potential in preliminary studies to be an alternate anticonvulsant medication, we investigated the pharmacokinetics and tissue distribution following i.p. administration of 150 mg/kg of the diene to rats for comparative purposes with VPA. The pharmacokinetics and tissue distribution of VPA has been well documented in many species including rats (Dickinson et al, 1979; Lee, 1991; Aly and Abdel-Latif, 1980), but to our knowledge, pharmacokinetics and tissue distribution of (E,Z)-2,3-diene VPA have not been previously determined in any species. The distribution of the diene in several brain areas was performed to assess if any significant variations exist between selected brain regions. Because VPA has been shown to be potentially hepatotoxic, the elimination kinetics of diene from the liver were investigated and potential affinity of drug for liver tissue was evaluated. The results from this study will be used to further assess the potential of the diene as a future anticonvulsant medication. Due to the nature of (E,Z)-2,3'-diene VPA's elimination profile, a time-lag four compartment pharmacokinetic model was chosen to fit the serum, brain and liver data obtained from this investigation. The four-compartment time-lag model was chosen as it best described the data, with serum, brain, liver and gut lumen (indicative of EHC) as individual compartments. Unfortunately, all attempts to fit the data were unsuccessful. One reason for the inability to fit the data may be that the data was averaged from eight animals as opposed to obtaining serial samples over a period of 12 h (as performed in the dose-dependent study) and 89 the resultant data from each rat modelled as a separate profile. The timepoint of onset of enterohepatic cycling as well as the magnitude of each rat's hepatobiliary cycling peak may be different for each animal. Therefore, when the data was averaged from eight animals, the magnitude and onset of the recycling peak may appear less than that from animals that were sampled separately, resulting in a plateau in the concentration-time profiles and an inadequate fitting of the data by the modelling program. The computer generated fit (appendix 2) modelled an enterohepatic peak which adequately fit the data from the previous dose-dependent study, because the magnitude of the enterohepatic peaks (from the individual rat serum profiles) were either apparent or the initial decline in serum drug concentration was not as steep when compared to that observed for the current study. Perhaps if additional serum and tissue concentrations were determined in the current study over the course of "apparent" enterohepatic cycling, hepatobiliary cycling would become apparent and the data could then be modelled. Furthermore, additional studies with bile-exteriorized rats would provide more information regarding the rates of transfer between central and gut lumen compartments, depicted as the microconstants k ] 2 and k 2i (figure 4), and the amount of drug excreted into the gut lumen (Pollack and Brouwer, 1991). If this data was known, perhaps a new EHC model could be used, that would overcome the current problems associated with apparent secondary increases in concentration. 90 4.4.1 Profile of (E,Z)-2,3 '-diene VPA in Serum Both the serum profile from the current study and the dose-dependent study exhibited almost identical serum profiles, with the exception of the 720 min timepoint for the dose-dependent study which was markedly higher in concentration than the current study. However, upon closer examination of the data, an "outlier" was discovered (described in section 3.3.1). Upon removal of the outlier, the two serum profiles were virtually identical, although the serum profile in the current study exhibited a slightly steeper decrease in the rate of decline when compared to the individual serum profiles from the dose-dependent study, which subsequently resulted in an inability to model the data using the ADAPTII program (discussed in section 4.4). In addition, both the serum profile from the current study and the dose-dependent study exhibited similar C m a x , AUC 0-i2h, CI, and Un values. This suggests that the serum data from both studies following i.p. administration of an equivalent dose are consistent despite the differences in methodology. The details pertaining to the apparent decrease in the rate of decline (or a plateau) and dose-related trends have been described in detail in section 4.2; therefore, the following discussion will focus on comparing the pharmacokinetics of the diene with known literature values for VPA and its analogues. The serum elimination profile of (E,Z)-2,3'-diene VPA exhibited a decrease in the rate of decline (or a plateau) in serum concentration which occurred at approximately 120 to 240 min. This observation has also been reported for VPA, although the elimination profiles of VPA exhibited an apparent secondary increase in serum concentration (Dickinson et al, 1979; Ogiso et al, 1986), attributed to enterohepatic cycling. (E,Z)-2,3'-Diene VPA attained peak serum levels of 336.9 |lg/mL in 15 min, indicating relatively rapid absorption into the systemic 91 circulation. Our findings indicate that (E,Z)-2,3'-diene VPA is more rapidly absorbed and attained higher concentration levels than VPA when compared to a reported C m a x of 142.7 pg/mL achieved in 30 min after single dose administration with VPA (Lee, 1991). It should be noted that these studies were conducted from 0 to 12h. Due to the presence of enterohepatic circulation, the apparent elimination constant, (3, could not be used to calculate the area under the curve after the last sample measured as this (3 was determined only from the initial linear portion of the serum elimination profile. Therefore, the apparent and calculated pharmacokinetic parameters AUC and Cl are calculated from time 0 to 12h. (E,Z)-2,3'-Diene VPA was eliminated with a ti / 2 of 35.5 min. It should be noted that ti/2 was determined from the linear portion of the elimination profiles, due to the secondary increases in serum concentration. This value compares well with a literature value of 41 min for VPA following i.v. administration of 150 mg/kg of VPA to rats (Dickinson et al, 1979). In contrast, the AUC0-i2h determined for (E,Z)-2,3'-diene VPA in this study of 518.9 p.g-h/mL was slightly higher than literature values of approximately 383.1 pg^h/mL following i.v. administration of 150 mg/kg of VPA to rats (Dickinson et al, 1979). Our AUC value compares well with another study that obtained an AUC0-ioh of 455 pg-h/mL after i.p. administration of 150 mg/kg VPA in rats (Lee, 1991). Clearance of (E,Z)-2,3'-diene VPA from serum was calculated from the dose administered and the apparent AUCo-i2h value. The calculated Cl0-i2h after administration of the diene was 4.82 mL/min-kg, which is similar to the computer generated clearance values obtained from the dose-dependent study following the administration of an equivalent dose. 92 This value was comparable to a literature value reported for VPA of 3.39 ± 0.70 mL/min-kg after i.v. administration of 150 mg/kg of VPA (Liu and Pollack, 1993). 93 4.4.2 Profile of (E,Z)-2,3 '-diene VPA in Whole Remaining Brain and Selected Brain Regions A study of the pharmacokinetics and tissue distribution of (E,Z)-2,3'-diene VPA in the brain and eleven brain regions (namely the cerebellum, medulla, pons, corpus striatum, hippocampus, olfactory bulbs, corpus callosum, frontal cortex, inferior and superior colliculi, and substantia nigra) was conducted to determine the pharmacokinetic parameters in brain tissue as well as to assess whether the diene persists or accumulates in specific brain regions. The eleven brain regions were selected based on previous investigations where discrete brain areas were investigated for their differences in GABA content (Hariton et al, 1984; Loscher et al., 1988; Loscher, 1993). Of particular interest were the substantia nigra and superior colliculus, which in previous experiments were shown to demonstrate varying effects on GABA and GABAergic neurons in these areas (Iadorola and Gale, 1982; Gale, 1992). As illustrated in figure 13, the distribution of (E,Z)-2,3'-diene VPA into the 11 brain regions was found to be relatively uniform when compared to whole remaining brain. Peak regional brain concentration, tmax, AUC 0-i2h and ti / 2 values were similar to that obtained for whole remaining brain (table 9). The brain C m a x concentrations and tmax values are comparable to previous results obtained from a similar study performed with VPA and two of its unsaturated metabolites, (E)-2-ene VPA and (E,E)-2,3'-diene VPA, in rats (Lee, 1991). In addition, AUC values were reported in the range of 37-56 pg-h/g and 43-54 pg-h/g for (E,E)-2,3'-diene VPA and (E)-2-ene VPA, respectively, as illustrated in table 10 (Lee, 1991) which agrees well with the AUC values obtained in the current study for (E,Z)-2,3'-diene VPA. However, the reported AUC values for VPA are 2-fold greater (ranging from 105-134 94 |Xg-h/g) than that found in the current study for (E,Z)-2,3'-diene VPA and for the other two metabolites from the previous investigation (Lee, 1991). Lower distribution of (E,Z)-2,3'-diene VPA and the other two metabolites compared to that of VPA may be due in part to their concentration-dependent serum protein binding characteristics, transport mechanisms other than passive diffusion into the brain, or perhaps some unknown mechanism which is structurally dependent and selective for the unsaturated compounds. It has been suggested that the accumulation of metabolites of VPA may contribute to the anticonvulsant properties of the parent drug (Loscher and Nau, 1984; Pollack et al, 1986) which may explain the delayed anticonvulsant effect following discontinuation of VPA administration (Lockard and Levy, 1976; Harding et al, 1978). In one study from our laboratory in which levels of VPA and its metabolites were determined in selected brain regions following administration of 150 mg/kg of VPA, (E,Z)-2,3'-diene VPA was the predominant diene detected in rat brain, suggesting that the diene may persist in brain tissue (Lee, 1991). The results from the current investigation support this contention. Braimtotal serum concentration ratios determined at tmax and ti2h suggest that (E,Z)-2,3'-diene VPA may accumulate in specific brain regions over time. All regions except the frontal cortex exhibited an increase in the braimtotal serum ratio over time. Taking into account that whole remaining brain is used as a reference for the brain regions, the most notable increase was observed for the inferior colliculi, exhibiting a 2.9-fold increase in tissue:serum ratio when compared to whole remaining brain. A 7-fold increase in the ratio for the inferior colliculi at ti 0 n was observed in rats dosed with the diene's isomer, (E,E)-2,3'-diene VPA, and selectivity for other brain regions was also reported in the substantia nigra and superior colliculi (Lee, 1991). 95 Interestingly, selectivity in brain tissue over time in rats dosed with VPA was not observed (Lee, 1991). In addition, other studies with rats (Nau and Loscher, 1982) and humans (Shen et al, 1992) did not find evidence that VPA accumulated in brain tissue following VPA administration. However, evidence exists that two of VPA's unsaturated metabolites, namely 2,3'-diene VPA and (E)-2-ene VPA, may persist in brain tissue after administration of VPA to rats. Firstly, a slow accumulation of (E)-2-ene VPA has been reported in the substantia nigra, superior and inferior colliculus, hippocampus and medulla of the rat brain after administration with VPA (Loscher and Nau, 1983). Secondly, Loscher et al. (1988) reported increased levels of a 2,3'-diene VPA in the substantia nigra after VPA administration to rats for three consecutive days. The apparent selectivity of (E,Z)-2,3'-diene VPA for discrete brain regions is noteworthy and further studies may reveal the reason for the apparent affinity for brain tissue. However, it should be noted that this phenomenon occurred at concentrations well below effective anticonvulsant concentrations and is therefore unlikely to contribute to prolonged anticonvulsant action. It is noteworthy that at high free serum concentrations and at all observed serum concentrations of (E,Z)-2,3'-diene VPA, braimtotal serum and free serum concentration ratios remained below unity. In particular, braimtotal serum concentration ratios, determined at tmax and tnh, ranged from 0.13 to 0.17 and 0.12 to 0.99, respectively. The low brain concentrations of (E,Z)-2,3'-diene VPA relative to total serum concentration have also been reported for VPA in rats (Cornford et al, 1985; Loscher et al, 1989; Semmes and Shen, 1991), mice (Nau and Loscher, 1982) and humans (Vajda et al, 1981; Shen et al, 1992; Adkison et al, 1995). The low brain levels of (E,Z)-2,3'-diene VPA relative to the 96 corresponding total serum drug concentration may indicate asymmetric transport of diene between blood and brain. This is consistent with the contention of an efficient fatty-acid transport mechanism by which the efflux of the drug from the brain exceeds the transport of drug from blood into the brain as described by other investigators (Cornford et al, 1985; Adkison et al, 1994; Naora and Shen, 1995; Adkison and Shen, 1996). Several investigations provided evidence to suggest the uptake of VPA is mediated via the monocarboxylic acid (MCA) carrier system (Terasaki et al, 1991; Adkison et al, 1994). Adkison et al. (1994) provided evidence that VPA efflux from the CNS occurred almost completely from a probenecid-sensitive transporter situated at the blood-brain barrier. Cornford et al. (1985) reported that the kinetic rate constant for the efflux of VPA from rat brain was greater than that for the influx into the brain, supporting the contention of an asymmetric transport mechanism between blood and brain. Our total drug distribution data suggest that the diene may indeed share the same transport mechanisms as reported for VPA, although further investigation is required to substantiate this hypothesis. It is interesting to note that the brain:free serum levels increased dramatically to 3.93 at t,2h when compared to 0.33 at tmax. In contrast, VPA braimfree serum concentrations have not been reported to increase over time or at low free serum concentrations (Semmes and Shen, 1991; Adkison et al, 1995). However, braimfree serum concentration ratios greater than unity have been reported previously for various unsaturated analogues of VPA. Semmes and Shen (1991) reported that brain:free plasma concentrations were highest at low free plasma concentrations for rats dosed with (E)-2-ene VPA. The braimfree plasma concentrations decreased markedly from 1.8 to below 1 as free plasma concentrations 97 increased from 7 to 100 (ig/mL (Semmes and Shen, 1991). It was suggested that the high degree of protein binding of (E)-2-ene VPA may play a role in this observation, in that as free levels increase with increasing serum levels, a saturation in the uptake of free drug into brain may result. In addition, studies performed by Adkison et al. (1995) revealed that at free serum levels less than 0.1 |lg/mL, steady-state braimfree serum ratios were greater than unity for 5 of 6 unsaturated metabolites detected in cortical brain samples that were obtained from patients undergoing neurosurgery. The unsaturated metabolites with a double-bond in the C-2 position, namely (E)-2-ene VPA, (E,E)-2,3'-diene VPA, and (E)-2,4-diene VPA, gave the highest braimfree serum ratio, with (E)-2-ene VPA exhibiting the highest ratio of 9.73 ± 1.90, It was theorized that structurally-dependent differences between the unsaturated metabolites and VPA may be responsible for these observations, and that either a higher degree of serum protein binding, a higher affinity for the MCA carrier into the brain, or a low affinity for the transporter out of the brain may play a role in these observations (Adkison et al., 1995). It is possible that (E,Z)-2,3'-diene VPA may share similar structurally-related serum protein binding or brain transport mechanisms as the aforementioned unsaturated metabolites. A plateau in the brain concentration-time profile or a secondary concentration peak was observed in all brain regions and for whole remaining brain. One interesting observation was that the decline of drug concentration in brain did not appear to parallel that in serum at approximately 240 to 480 min. Following a slight plateau in the serum elimination profile at 240-360 min, serum concentrations continued to decline. In contrast, almost all regional brain profiles exhibited an increase in concentration or a plateau from 240-540 min before brain concentrations decreased. This suggests that an equilibration may not exist between brain and 98 capillary blood at these timepoints, which corresponds to low serum and brain concentrations. Interestingly, this observation has not been reported for VPA. In fact, studies have reported that the subsequent decline of drug VPA concentration from brain parallels that in plasma (Hariton et al, 1984; Lee, 1991). This observation is highly unusual and the mechanisms involved are uncertain. The proposed carrier-mediated transport of the diene may play a role in the circumstances behind this observation, and additional studies would be required to confirm this hypothesis. It is also possible that the diene may exhibit a high affinity for brain tissue at low serum concentrations, perhaps as a consequence of concentration-dependent protein binding of the diene. Nevertheless, it is difficult to interpret this phenomenon, and therefore the aforementioned possibilities remain purely speculative. A nonlinear distribution pattern of drug was observed for whole remaining brain when compared to total serum concentrations of (E,Z)-2,3'-diene VPA. In contrast, (E,Z)-2,3'-diene VPA concentrations in brain increased linearly with corresponding free serum levels. Similar trends for brain, free plasma and total plasma concentrations for VPA were obtained in rats and were attributed to saturable plasma protein binding (Semmes and Shen, 1991). Our data indicates that the serum protein binding of (E,Z)-2,3'-diene VPA was concentration-dependent; therefore, the protein binding of (E,Z)-2,3'-diene VPA may be a contributing factor in the brain distribution of the diene in rats and may be responsible for the nonlinear distribution patterns in rat brain. 99 3 1/3 C s o U o 4* > U 3 o CD 0) S 3 93 0) S> 93 c 93 E U C #o 93 U 5 <u u e o u 93 tU 6 £ 3 S 93 s s xi H 93 H C 0> ro of I o 5 £ CD c CD • < OH CD J 3 "3 a 5P o o ce CD o -a c '3 C O c o -00 3 « 3 'S >» 93 CD d * • 93 ' Q • s D C 93 U a t/2 OX) c o u 3 o O o 4-1 e U on ^ "3 c O (D S 6 O ca ° 3 • c a a -O 3 C3 o 0 3 3 I o O &< a. <u c o CQ T 3 <u "o o :S a _o u X> JO "3 <u u X W T3  Q W T3 s 0 0 cm "S C3 a -.3 c 3 U u o 3 & O o o J3 w u 1) <u IU o in 0 0 rf n- T t t ^ r t - v o i n c o T t v o c o m r f < n r f n - r | - - * > o O N OO rj- . cn rt- rl- u-> rt O in m p CN » n v O r t < / - ) O i / " > o o r l - c o r - -O O C - l r t C N O r t C N m O vo 0 0 O N r-m rj- <N cs m o T t cs 0 0 in co cs cn co ' o o r- o\ cs *—1 in co m co -<t co in • " H C S c s i n O N O N v o i / - ) m m i n i n v o i n n - r t -in o O O O in in in T t m O *0 * H H H O lO cn rt o o o o o o i n o m o cn c n c n c n c n c n r - H c n r t c n O o in o m o cn cn co r-n cn m in in o r-i ^+ en in o 0 V & < > a co cn o f W W 0 < PL, > a .1 OJ 1 © rt &H ON > ^ a OJ (D "S D O c O a o <U •fei rt a - p i cd o < CO 100 4.4.4 Profile of (E,Z)-2,3'-diene VPA in Liver Since VPA has been demonstrated to be potentially hepatotoxic (Zimmerman and Ishak, 1982) development of VPA analogues without the hepatotoxic effects would be therapeutically beneficial. The distribution of (E,Z)-2,3'-diene VPA in the liver was therefore investigated in this respect in an effort to characterize the transfer of drug to the liver. Since (E,Z)-2,3'-diene VPA is highly serum protein bound, transfer of the drug to the liver may be significantly compromised, and may reflect a potentially lower liver toxicity. Elimination of (E,Z)-2,3'-diene VPA from liver was rapid and appeared to parallel the decline of drug from serum. The liver profile exhibited a slight plateau at approximately 240 min, similar to that observed in serum, but to a lesser extent. The elimination constant and half-life values were almost identical for liver and serum, indicating similar elimination characteristics for both tissues. A few interesting differences were noted between the serum and liver profiles. Firstly, the absorption of (E,Z)-2,3'-diene VPA into the liver was more rapid than serum, attaining peak liver concentrations in 7.5 min. This time to peak concentration is much shorter than that reported for VPA in liver, with times ranging from 30 to 45 min (Aly and Abdel-Latif, 1980; Lee, 1991). However, one study reported rapid absorption within 2-5 min of VPA after oral administration of 50 mg/kg of VPA to mice (Nau and Loscher, 1985). Secondly, the liver concentrations were consistently lower than corresponding serum concentrations. Subsequently, distribution of (E,Z)-2,3'-diene VPA to liver was less than 1/2 of that found in serum, based on the AUCo-i2h values of 241.5 ug'h/g and 518.9 |ig-h/mL for liver and serum, respectively. In contrast, liver distribution after VPA administration in rats has been reported 101 to be twice that obtained for plasma, based on the AUC0-ioh values of 455 (ig-h/mL and 854 u. g-h/g, respectively (Lee, 1991). The low liver concentrations and distribution of (E,Z)-2,3-diene VPA, when compared to that of VPA, may be attributed to the more extensive serum protein binding of (E,Z)-2,3'-diene VPA. High serum protein binding, in theory, would effectively limit the availability of free drug, thus restricting transfer of drug into liver. The low liver distribution that was observed for (E,Z)-2,3'-diene VPA has also been reported for other unsaturated metabolites. Liver distribution of (E)-2-ene VPA has been reported in the mouse (Nau and Loscher, 1985) and the rat (Lee, 1991) and both investigations revealed that liver concentrations were consistently less than corresponding plasma concentrations. Similar results with rats dosed with (E,E)-2,3'-diene VPA have been reported, with a liver distribution of 3-fold less than that found in plasma, based on the AUC 0 . ioh values of 133 ilg'h/g and 401 (ig-h/mL for liver and serum, respectively (Lee, 1991). It was suggested that higher plasma protein binding of the two unsaturated metabolites may be a contributing factor to the low liver concentrations observed for both compounds. (E,Z)-2,3'-Diene VPA concentrations in liver were found to increase proportionately with corresponding free serum levels, whereas the relationship between liver and total serum concentrations of the diene was nonlinear. This trend is similar to the results obtained from the brain studies (section 3.4.4). This observation suggests that the distribution of the drug into the liver is dependent on the concentration of (E,Z)-2,3'-diene VPA in serum and that distribution may be restricted to the unbound form of the drug. The livenserum concentration ratios of (E,Z)-2,3'-diene VPA decreased with decreasing drug concentration in serum. When compared to values obtained at tmax, the 102 liventotal and livenfree serum ratios decreased by more than 5 fold at t12n. In contrast to our findings, the livenplasma VPA concentration ratio determined after an i.p. dose of VPA in mice increased as VPA plasma concentrations decreased (Loscher and Nau, 1984). Similar findings were reported in studies performed with rats following i.p. injection of VPA (Lee, 1991). Livenplasma ratios of VPA increased from 1.8 at tmax to 4.6 at ti0h. In the current study, the low levels of diene in liver relative to serum levels may suggest a lower affinity of the diene for liver tissues over time and/or a strong influence of the concentration dependent serum protein binding of the diene. It is noteworthy that the liver: serum ratio of the diene was significantly less than that reported for VPA at t^h- From a toxicological point of view, high levels of VPA in liver have been associated with drug-induced hepatotoxicity (Nau and Loscher, 1985). In light of the current investigation, the low liver: serum ratio at t ] 2 h as well as the low liver distribution determined from the AUC 0 - i 2 h values suggest that (E,Z)-2,3'-diene VPA may be less hepatotoxic than VPA. Extensive metabolic profiling as well as toxicological studies are necessary to establish whether the low liver concentrations of the diene represent a lower liver toxicity. 103 5. CONCLUSIONS The results from these studies have provided a greater understanding into the pharmacokinetics and disposition of (E,Z)-2,3'-diene VPA in rats. The following conclusions were made based on these results. The serum protein binding characteristics of (E,Z)-2,3'-diene VPA were investigated in order to assess whether the diene reflects similar binding attributes as VPA and its analogues. The data indicated that for both ex vivo and in vitro studies, the binding behaviour of (E,Z)-2,3'-diene VPA was similar to that reported for VPA and is dependent on concentration. The serum.protein binding of the diene appeared to be higher than that reported for VPA at concentrations below 65 |ig/rnL. After three different i.p. doses of (E,Z)-2,3'-diene VPA, the pharmacokinetic profiles in serum were characterized. Enterohepatic cycling was observed following administration of all three doses, with a delay in the appearance of the secondary peak at the highest dose level. Serum data were fit into a time-lag two compartment model and pharmacokinetic parameters were calculated. The observed differences in the pharmacokinetic parameters of (E,Z)-2,3'-diene VPA suggest that the kinetics of (E,Z)-2,3'-diene VPA were dose-dependent. Several factors may be attributable to the dose-related parameter changes, including concentration-dependent serum protein binding, saturable metabolism, and hepatobiliary recycling. Urinary recoveries of parent and conjugated (E,Z)-2,3'-diene VPA were determined after administration of three different doses of the compound. The fraction of the dose of (E,Z)-2,3'-diene VPA excreted as parent drug remained relatively unchanged as dose levels 104 increased, but the fraction of the dose excreted as glucuronides increased with increasing dose. The excretion of glucuronide conjugates for the intermediate and high dose levels remained relatively unchanged, suggesting saturable elimination of the diene via the glucuronide pathway. The excretion of (E,Z)-2,3'-diene VPA and conjugated (E,Z)-2,3'-diene VPA was also determined in the bile. The diene was found to be excreted as glucuronide conjugates and induced a choleretic effect which was related to the amount of glucuronide excreted in the bile. Totally biliary excretion of the diene was markedly less than that reported for VPA, suggesting that other metabolic routes may play a significant role in the metabolism of (E,Z)-2,3'-diene VPA. The pharmacokinetics and tissue distribution profiles were determined in serum, liver and various brain regions of rats after i.p. administration of 150 mg/kg (E,Z)-2,3'-diene VPA to rats. Serum concentrations were consistently higher than corresponding brain and liver concentrations. Evidence of enterohepatic cycling was observed in the serum and brain elimination profiles but to a lesser degree in the liver profile. The brain distribution studies indicated that the diene is rapidly absorbed and distributed to brain, which are desired attributes for an anticonvulsant medication. At very low serum concentrations, the diene was seen to accumulate in 10 of 11 brain regions, but brain levels were significantly less than effective anticonvulsant concentrations. Braimserum concentrations ratios remained well below unity, suggesting the involvement of an efficient fatty-acid transport mechanism by which the efflux of the diene from the brain exceeds the transport into the brain. 105 The diene was rapidly distributed into liver tissue and liver distribution was less than 1/2 of that found in serum based on the apparent AUC 0-i2h values. The livenserum concentration ratios were found to decrease over time. The liventotal serum ratio at t\2h and liver distribution were significantly lower than that reported for VPA, suggesting that (E,Z)-2,3'-diene VPA may be potentially less hepatotoxic than VPA. Nonlinear distribution patterns were observed for whole remaining brain and liver tissues when compared to total serum concentration, indicating that the distribution of (E,Z)-2,3'-diene VPA into central and peripheral tissues is dependent on concentration. (E,Z)-2,3'-Diene VPA concentrations in brain and liver increased linearly with the corresponding free serum levels, suggesting that serum protein binding plays a significant role in the tissue distribution of (E,Z)-2,3'-diene VPA in rats. The favorable anticonvulsant potency and low neurotoxicity results obtained for (E,Z)-2,3'-diene VPA in previous investigations from our laboratory suggest that the diene could potentially be used as a VPA alternative (Lee, 1991). The results obtained from these investigations indicate that (E,Z)-2,3'-diene VPA reflects certain desired attributes for anticonvulsant therapy, namely rapid absorption and distribution to brain, accumulation in selected brain sections as well as low liver distribution. These observations further strengthen the contention that the diene may be a suitable compound for development as an anticonvulsant. Future investigations may include extensive metabolic studies to determine whether potential hepatotoxic metabolites are formed, additional neurotoxic studies using the rotorod test in rats, as well as multiple dose studies to investigate whether the diene accumulates in brain tissue over an extended period. 106 6. REFERENCES Abbott, F.S. and Acheampong, A. A., Quantitative structure-anticonvulsant activity relationships of valproic acid, related carboxylic acids and tetrazoles, Neuropharmacology, 27 (1988)287-294. Abbott, F., Kassam, J., Acheampong, A., Ferguson, S., Panesar, S., Burton, R., Farrell, K., Orr, J., Capillary gas chromatography-mass spectrometry of valproic acid metabolites in serum and urine using tert-butyldimethylsilyl derivatives, J. Chromatogr., 375 (1986) 285-298. Acheampong, A., Abbott, F., Burton, R., Identification of valproic acid metabolites in human serum and urine using hexadeuterated valproic acid and gas chromatographic mass spectrometric analysis, Biomed. Mass Spectr., 10 (1983) 586-595. Acheampong, A.A., Abbott, F.S., Orr, J.M., Ferguson, S.M., Burton, R.W., Use of hexadeuterated valproic acid and gas chromatography-mass spectrometry to determine the pharmacokinetics of valproic acid, J. Pharm. Sci., 73 (1984) 489-494. Adkison, K.D.K., Artru, A.A., Powers, K.M., and Shen, D.D. Contribution of probenecid-sensitive anion transport processes at the brain capillary endothelium and choroid plexus to the efficient efflux of valproic acid from the central nervous system, J. Pharmacol. Exp. Ther., 268 (1994) 797-805. Adkison, K.D.K., Ohemann, G.A., Rapport, R.L., Dills, R;L., and Shen, D.D., Distribution of unsaturated metabolites of valproate in human and rat brain—pharmacological relevance? Epilepsia, 36 (1995) 772-782. Adkison, K.D.K., and Shen, D.D., Uptake of valproic acid into rat brain is mediated by a medium-chain fatty acid transporter, J. Pharmacol. Exp. Ther., 276 (1996) 1189-1200. Aly, M.I. and Abdel-Latif, A.A., Studies on distribution and metabolism of valproate in rat brain, liver, and kidney, Neurochem. Res., 5 (1980) 1231-1242. Baillie, T.A., Metabolic activation of valproic acid and drug-mediated hepatotoxicity. Role of the terminal olefin, 2-n-propyl-4-pentenoic acid, Chem. Res. Toxicol., 1 (1988) 195-199. 107 Baillie, T.A., and Rettenmeier, A.W.: Valproate: Biotransformation. In Antiepileptic Drugs, R.H. Levy, R.H. Mattson, B.S. Meldrum and J.K. Penry, (Eds.) 601-619, Raven Press, New York, 1989. Baillie, T.A., Metabolism of valproate to hepatotoxic intermediates, Pharm. Weekbl. Sci. Ed., 14 (3A) (1992) 122-125. Bialer, M., Hussein, Z., Raz, I., Abramsky, O, Herishanu, Y., and Pachys, F., Pharmacokinetics of valproic acid in volunteers after a single dose study, Biopharm. Drug Dispos., 6 (1985) 33-42. Bjorge, S.M., and Baillie, T.A., Inhibition of medium-chain fatty acid (3-oxidation in vitro by valproic acid and its unsaturated metabolite, 2-n-propyl-4-pentenoic acid, Biochem. Biophys. Res. Commun., 132 (1985) 245-252. Bjorge, S.M. and Baillie, T.A., Studies on the (3-oxidation of valproic acid in rat liver mitochondrial preparations, Drug Metab. Dispos., 19 (1991) 823-829. Bowdle, T.A., Patel, I.H., Levy, R.H., Wilensky, A.J., Valproic acid dosage and plasma protein binding and clearance, Clin. Pharmacol. Ther., 28 (1980) 486-492. Brouwer, K.L.M., Hall, E.S., and Pollack, G.M., Protein binding and hepatobiliary distribution of valproic acid and valproate glucuronide in rats, Biochem. Pharmacol., 45 (1993) 735-742. Bruni, J., Wilder, J., Valproic Acid: Review of a new antiepileptic drug, Arch. Neurol, 36 (1979) 393-398. Bryant, A.E., and Dreifuss, F.E., Valproic acid hepatic fatalities. III. U.S. experience since 1986, Neurology, 46 (1996) 465-469. Chapman, A., Keane, P.E., Meldrum, B.S., Simiand, J., Vernieres, J.C., Mechanism of anticonvulsant action of valproate, Prog. Neurobiol, 19 (1982) 315-359. 108 Cloyd, J.C., Kriel, R.L., Fischer, J.H., Sawchuk, R.J., Eggerth, R.M., Pharmacokinetics of valproic acid in children: 1. Multiple antiepileptic drug therapy, Neurology, 33 (1983) 185-191. Colburn, W.A., Pharamcokinetic analysis of concentration-time data obtained following administration of drugs that are recycled in the bile, 7. Pharm. Sci., 73 (1984) 313-317, Cornford, E.M., Diep, CP., and Pardridge, W.M., Blood-brain barrier transport of valproic acid, J. Neurochem., 44 (1985) 1541-1550. Cramer, J.A., and Mattson, R.H., Valproic Acid: In vitro plasma protein binding and interaction with pheytoin, Ther. Drug Monitor., 1 (1979) 105-116. Cramer, J.A., Mattson, R.H., Bennett, D.M., Swick, C.T., Variable free and total valproic acid concentrations in sole- and multi-drug therapy, Ther. Drug. Monitor., 8 (1986) 411-415. Darius, J., Meyer, F.P., Sensitive capillary gas chromatographic-mass spectrometric method for the therapeutic drug monitoring of valproic acid and seven of its metabolites in human serum. Application of the assay for a group of pediatric epileptics, J. Chromatogr., 656 (1994) 342-351. Davis, R., Peters, D.H., McTavish, D., Valproic acid: A reappraisal of its pharmacological properties and clinical efficacy in epilepsy, Drugs, 47 (1994) 332-372. Dickinson, R.G., Harland, R.C., Ilias, A.M., Rodgers, R.M., Kaufman, S.N., Lynn, R.K., Gerber, N., Disposition of valproic acid in the rat: Dose-dependent metabolism distribution, enterohepatic recirculation and choleretic effect, J. Pharmacol. Exp. Ther., 211 (1979) 583-595. Dickinson, R.G., Taylor, S.M., Kaufman, S.N., Rodgers, R.M., Lynn, R.K., Gerber, N., and Baughman, W.L., Nonlinear elimination and choleretic effect of valproic acid in the monkey, J. Pharmacol. Exp. Ther., 213 (1980) 38-48. Dickinson, R.G., Hooper, W.D., and Eadie, M.J., pH-dependent rearrangement of the biosynthetic ester glucuronide of valproic acid to P-glucuronidase-resistant forms, Drug Metab. Dispos., 12 (1983) 247-252. 109 Dickinson, R.G., Eadie, M.J., and Hooper, W.D., Glucuronidase-resistant glucuronides of valproic acid: consequences to enterohepatic recirculation of valproate in the rat, Biochem. Pharmacol, 34 (1985) 407-408. Dickinson, R.G., Kluck, R.M., Eadie, M., and Hooper, W.D., Disposition of (3-glucuronidase-resistant "glucuronides" of valproic acid after intrabiliary administration in the rat: intact absorption, fecal excretion and intestinal hydrolysis, J. Pharmacol. Exp. Ther., 233 (1985) 214-221. ! Dickinson, R.G., Hooper, W.D., Dunstan, P.R., and Eadie, M.J., Urinary excretion of valproate and some metabolites in chronically treated patients, Then Drug Monitor., 11 (1989) 127-133. Dreifuss, F.E., Santilli, N., Langer, D.H., Sweeney, K.P., Moline, K.A., Valproic acid hepatic fatalities: a retrospective review, Neurology, 37 (1987) 379-385. and Menander, K.B., Dreifuss, F.E., Langer, D.H., Moline, K.A., and Maxwell, J.E., Valproic acid hepatic fatalities. II. US experience since 1984, Neurology, 39 (1989) 201-207. Elmazar, M.M.A., Hauck, R.S., and Nau, H., Anticonvulsant and Neurotoxic activities of Twelve analogues of Valproic acid, J. Pharm. Sci, 82 (1993), 1255-1258. Farrell, K., Abbott, F.S., Orr, J.M., Applegarth, D.A., Jan, J.E., and yong, P.K., Free and total serum valproate concentration: their relationship to seizure control, liver enzymes and plasma ammonia in children, Can. J. Neurol. Sci., 13 (1986) 252-255. I Fisher, E., Wittfoht, W., and Nau, H., Quantitative determination of valproic acid and 14 metabolites in serum and urine by gas chromatography/mass spectrometry, Biomed. Chromatogr., 6 (1992), 24-29. Fisher, E., Siemes, H., Pund, R., Wittfoht, W. and Nau, H., Valproate and urine during antiepileptic therapy in children with infantile spasms: metabolites in serum Abnormal metabolite pattern associated with reversible hepatotoxicity, Epilepsia, 33 (1992) 165-171. Frey, H.H., and Loscher, W., Distribution of valproate across the interface between blood and cerebrospinal fluid, Neuropharmacology, 17 (1978) 637-642. | i 110 Friel, P.N., Ojemann, G.A., Rapport, R.L., Levy, R.H., Van Belle, G., Human brain phenytoin: correlation with unbound and total serum concentrations, Epilepsy Res., 3 (1989) 82-85. Gale, K., GABA and epilepsy: Basic concepts from preclinical research, Epilepsia, 33 (Suppl. 5) (1992) S3-S12. Gibaldi, M., and Perrier, D., Pharmacokinetics. 2nd ed., Marcel Dekker, New York, 1982. Godin, Y., Heiner, L., Mark, J., Mandel, P., Effects of di-n-propylacetate, an anticonvulsive compound, on GABA metabolism, J. Neurochem., 16 (1969) 869-873. Gofflot, F., van Maele-Fabry, G., Picard, J.J., Cranial nerves and ganglia are altered after in vitro treatment of mouse embryos with valproic acid (VPA) and 4-en-VPA, Develop. Brain Res., 93 (1996) 62-69. Gomez Bellver, M.J., Sanchez, M.J.G., Gonzalez, C.A., Buelga, S., and Dominguez-Gil, A., Plasma protein binding kinetics of valproic acid over a broad dosage range: therapeutic implications, J. Clin. Phar. Ther., 18 (1993) 191-197. Granneman, G.R., Wang, S.I., Machinist, J.M., Kesterson, J.W., Aspects of the metabolism of valproic acid, Xenobiotica, 14 (1984) 375-387. Gugler, R., Schell, A., Eichelbaum, M., Froscher, W., and Schulz, H.U., Disposition of valproic acid in man, Eur. J. Clin. Pharmacol., 12 (1977) 125-132. Gugler, R., and von Unruh, G.E., Clinical pharmacokinetics of valproic acid, Clin. Pharmacokin., 5 (1980) 67-83. Hall, K., Otten, N., Johnston, B., Irvine-Meek, J., Leroux, M., A multivariable analysis of factors governing steady-state pharmacokinetics of valproic acid in 52 young epileptics, J. Clin. Pharmacol., 25 (1985) 261-268. Harberer, L.J., and Pollack, G.M., Disposition and protein binding of valproic acid in the developing rat, Drug Metab. Dispos., 22 (1994) 113-119. Ill Harding, G.F.A., Herrick, C.E., Jeavons, P.M., A controlled study of the effect of sodium valproate on photosensitive epilepsy and its prognosis, Epilepsia, 19 (1978) 555-565. Hariton, C , Ciesielski, L., Simler, S., Valli, M., Jadot, G., Gobaille, S., Mesdjian, E., and Mandel, P., Distribution of sodium valproate and gaba metabolism in CNS of the rat, Biopharm. Drug Dispos., 5 (1984) 409-414. Hauck, R., and Nau, H., Asymmetric synthesis and enantioselective teratogenicity of 2-n-propyl-4-pentenoic acid (4-en-VPA), an active metabolite of the anticonvulsant drug, valproic acid, Toxicology Letters, 49 (1989) 41-48. Hoffmann, F., von Unruh, G.E., and Jancik, B.C., Valproic acid disposition in epileptic patients during combined antiepileptic maintenance therapy, Eur. J. Clin. Pharmacol, 19 (1981) 383-385. Horton, R.W., Anlezark, G.M., Sawaya, M.C.B., and Meldrum, B.S., Monoamine and GABA metabolism and the anticonvulsant action of di-n-propylacetate and ethanolamine-o-sulphate, Eur. J. Pharmacol, 41 (1977) 387-397. Iadarola, M.J., and Gale, K., Substantia nigra: Site of anticonvulsant activity mediated by y-aminobutyric acid, Science, 218 (1982) 1237-1240. Jeavons, P.M., and Clark, J.E., Sodium valproate in the treatment of epilepsy, Brit. Med. Journal, 2 (1974) 584-586. Kaneko, S., Otani, K., Fukushima, Y., Ogawa, Y., Nomura, Y., Ono, T., Nakane, Y., Teranishi, T., Goto, M., Teratogenticity of antiepileptic drugs: Analysis of possible risk factors, Epilepsia, 29 (1988) 459-467. Kassahun, K., Burton, R., and Abbott, F.S., Negative ion chemical ionization gas chromatography/mass spectrometry of valproic acid metabolites, Biomed. Environ. Mass Spec, 18 (1989) 918-926. Kassahun, K., Farrell, K., Zheng, J., Abbott, F., Metabolic profiling of valproic acid in patients using negative-ion chemical ionization gas chromatograpy-mass spectrometry, J. Chromatogr., 527 (1990) 327-341. 112 Kassahun, K . , Farrell, K . and Abbott, F. , Identification and characterization of the glutathione and N-acetylcysteine conjugates of (E)-2-propyl-2,4-pentadienoic acid, a toxic metabolite of valproic acid, in rats and humans, Drug Metab. Dispos., 19 (1991) 525-535. Keane, P .E . , Simiand, J., Morre, M . , Comparison of the pharmacological and biochemical profiles of valproic aicd ( V P A ) and its cerebral metabolite (2-en-VPA) after oral administration in mice, Meth. Find. Exp. Clin. Pharmacol., 7 (1985) 83-86. Kesterson, J.W., Granneman, G.R. , Machinist, J . M . , The hepatotoxicity of valproic acid and its metabolites in rats. I. Toxicologic, biochemical, and histopathologic studies, Hepatology, 4(1984) 1143-1152. Klotz , U . , and Antonin, K . H . , Pharmacokinetics and bioavailability of sodium valproate, Clin. Pharmacol. Ther., 21 (1977)736-743. Klug , S., Lewandowski, C. , Zappel, F. , Merker, H . , and Neubert, D . , Effects of valproic acid, some of its metabolites and analogues on prenatal development of rats in vitro and comparison with effects in vivo, Arch. Toxicol, 64 (1990) 545-553. Kobayashi, S., Takai, K . , Iga, T. and Hanano, M . , Effect of pregnancy on the disposition of valproate in rats, J. Pharmacobio-Dyn., 13 (1990) 533-542. Kondo, T., Otani, K . , Hirano, T., Kaneko, S., and Fukushima, Y . , The effects of phenytoin and carbamazepine on serum concentrations of mono-unsaturated metabolites of valproic acic, J. Clin. Pharmac, 29 (1990) 116-119. Kondo, T., Kaneko, S., Otani, K . , Ishida, M . , Hirano, T., Fukushima, Y . , Muranaka, H . , Koide, N . , and Yokoyama, M . , Associations between risk factors for valproate hepatotoxicity and altered valproate metabolism, Epilepsia, 33 (1992) 172-177. Lawyer, C . H . , Gerber, N . , Lynn, R . K . , and Dickinson, R . G . , Application of the two-compartment open model with Michaelis-Menten elimination kinetics to valproic acid in the bile-exteriorized rat, Res. Comm. Chem. Path. Pharmacol, 27 (1980) 469-484. Lee, R . D . , Kassahun, K . , Abbott, F.S. , Stereoselective synthesis of the diunsaturated metabolites of valproic acid, / . Pharm. Sci., 78 (1989) 667-671. 113 Lee, R.D., (1991): Pharmacokinetics, tissue distribution, and pharmacodynamics of valproic acid and its unsaturated metabolites in rats, Ph.D. Dissertation, The University of British Columbia, 306 p. Levy, R.H., and Koch, K.M., Drug interactions with valproic acid, Drugs, 24 (1982) 543-556. Levy, R.H., CSF and plasma pharmacokinetics: relationship to mechanisms of action as exemplified by valproic acid in monkey. In Epilepsy: A Window to Brain Mechanisms, J.S. Lockard and A.A. Ward (Eds.), Raven Press, New York, 1980. Lin, J.M.H., O'Connor-Semmes, L., Shen, D.D., Levy, R.H., Formation of hepatotoxic metabolites from 2(E)-ene-VPA in rats. Epilepsia, 32 (Suppl. 3) (1991) 3. Liu, M., Scott, K.R., and Pollack, G.M., Pharmacokinetics and pharmacodynamics of valproate analogues in rats. I. Spiro[4.6]Undecane-2-carboxylic acid, Epilepsia, 31 (1990) 465-473. Liu, M., Brouwer, K., and Pollack, G.M., Pharmacokinetics and pharmacodynamics of valproate analogs in rats. III. Pharmacokinetics of valproic acid, cyclohexanecarboxylic acid, and 1-methyl-1-cyclohexanecarboxylic acid in the bile-exteriorized rat, Drug Metab. Dispos., 20(1992) 810-815. Liu, M.J., Pollack, G.M., Pharmacokinetics and pharmacodynamics of valproate analogues in rats. II. Pharmacokinetics of octanoic acid, cyclohexanecarboxylic acid, and 1-metyl-l-cyclohexanecarboxylic acid, Biopharm. Drug Dispos., 14 (1993) 325-339. Liu, M. and Pollock, G.M., Pharmacokinetics and pharmacodynamics of valproate analogues in rats. IV. Anticonvulsant action and neurotoxicity of octanoic acid, cyclohexanecarboxylic acid, and 1-methyl-1-cyclohexanecarboxylic acid, Epilepsia, 35 (1994) 234-243. Lockard, J.S. and Levy, R.H., Valproic acid: reversibly acting drug?, Epilepsia, 17 (1976) 477-479. Loscher, W., Serum protein binding and pharmacokinetics of valproate in man, dog, rat and mouse, J. Pharmacol. Exp. Ther., 204 (1978) 255-261. 114 Loscher, W., Effect of inhibitors of GABA transaminase on the synthesis, binding, uptake and metabolism of GABA, J. Neurochem., 34 (1980) 1603-1608. Loscher, W., Anticonvulsant activity of metabolites of valproic acid, Arch. Int. Pharmacodyn., 249 (1981) 158-163. Loscher, W. and Nau, H., Valproic acid: Metabolite concentrations in plasma and brain, anticonvulsant activity, and effects on GABA metabolism during subacute treatment in mice, Arch. Int. Pharamcodyn., 257 (1982) 20-31. Loscher, W. and Nau, H., Distribution of valproic acid and its metabolites in various brain areas of dogs and rats after acute and prolonged treatment, 7. Pharmacol. Exp. Ther., 226 (1983) 845-854. Loscher, W., and Frey, H., Kinetics of penetration of common antiepileptic drugs into cerebrospinal fluid, Epilepsia, 25 (1984) 346-352. Loscher, W., Nau, H., Marescaux, C , Vergnes, M., Comparative evaluation of anticonvulsant and toxic potencies of valproic acid and 2-en-valproic acid in different animal models of epilepsy, Eur. J. Pharmacol., 99 (1984) 211-218. Loscher, W. And Vetter, M., In vivo effects of aminooxyacetic acid and valproic acid on nerve terminal (synaptosomal) GABA levels in discrete brain areas of the rat, Biochem. Pharmacol, 34 (1985) 1747-1756. Loscher, W. and Nau, H., Pharmacological evaluation of various metabolites and analogues of valproic acid. Anticonvulsant and toxic potencies in mice, Neuropharmacology, 24 (1985) 427-435. Loscher, W., Fisher, J.E., Nau, H., Honack, D., Marked increase in anticonvulsant activity but decrease in wet-dog shake behaviour during short-term treatment of amygdala-kindled rats with valproic acid, Eur. J. Pharmacol, 150 (1988) 221-232. Loscher, W., Nau, H. and Siemes, H., Penetration of valproate and its active metabolites into cerebrospinal fluid of children with epilepsy, Epilepsia, 29 (1988) 311-316. 115 Loscher, W., Fisher, J.E., Nau, H. and Honack, D., Valproic acid in amygdala-kindled rats: Alterations in anticonvulsant efficacy, adverse effects and drug and metabolite levels in various brain regions during chronic treatment, J. Pharmacol. Exp. Ther., 250 (1989) 1067-1078. Loscher, W., Honack, D., Nolting, B. and Fassbender, C , Tran.y-2-en-valproate: reevaluation of its anticonvulsant efficacy in standardized seizure models in mice, rats and dogs, Epilepsy Res., 9(1991) 195-210. Loscher, W., Wahnschaffe, U . , Honack, D., Wittfoht, W., and Nau, H., Effects of valproate and E-2-en-valproate on functional and morphological parameters of rat liver. I. Biochemical, histopathological and pharmacokinetic studies, Epilepsy Res., 13 (1992) 187-198. Loscher, W., Effects of the antiepileptic drug valproate on metabolism and function of inhibitory and excitatory amino acids in the brain, Neurochem. Res., 18 (1993) 485-502. Loscher, W., Nau, H., Wahnschaffe, U . , Honack, D., Rundfeldt, C , Wittfoht, W., and Bojic, U . , Effects of valproate and E-2-en-valproate on functional and morphological parameters of rat liver, n. Influence of phenobarbital comedication, Epilepsy Res., 15 (1993) 113-131. Loscher, W., Wahnschaffe, U . , Honack, D., Drews, E., and Nau, H., Effects of valproate and E-2-en-valproate on functional and morphological prarmeters of rat liver. III. Influence of fasting, Epilepsy Res., 16 (1993) 183-194. Loscher, W., In vivo administration of valproate reduces the nerve terminal (synaptosomal) activity of GABA aminotransferase in discrete brain areas of rats, Neuroscience Letters, 160 (1993) 177-180. McLean, M.J., and MacDonald, R.L., Sodium valproate, but not ethosuximide, produces use-and voltage-dependent limitation of high frequency repetitive firing of action potentials of mouse central neurons in cell culture, J. Pharmacol. Exp. Ther., 237 (1986) 1001-1011. McNamara, J.O., Drugs effective in the treatment of epilepsies. In The Pharmacological Basis of Therapeutics, Goodman Gilman, A., et al., (Eds.), 461-486, McGraw-Hill, New York, 1996. May, T., and Rambeck, B., Serum concentrations of valproic acid: influence of dose and comedication, Ther. Drug Monitor., 7 (1985) 387-390. 116 Meshki Baf, M.H., Subhash, M.N., Madepalli Lakshmana, K., and Sridhara Rama Rao, B.S., Sodium valproate induced alterations in monoamine levels in different regions of the rat brain, Neuwchem. Int., 24 (1994) 67-72. Meunier, H., Carraz, G., Meunier, Y., and Eymard, P., Proprietes pharmacodynamiques de l'acide n-dipropylacetique, Therapie, 18 (1963) 435-438. Naora, K., and Shen, D.D., Mechanism of valproic aid uptake by isolated rat brain microvessels, Epilepsy Res., 22 (1995) 97-106. Nau, H. and Loscher, W., Valproic acid: Brain and plasma levels of the drug and its metabolites, anticonvulsant effects and 7-aminobutyric acid (GABA) metabolism in the mouse, J. Pharmacol. Exp. Ther., 220 (1982) 654-659. Nau, H., and Loscher, W., Valproic acid and metabolites: Pharmacological and toxicological studies, Epilepsia, 25 (Suppl. 1) (1984) S14-S22. Nau, H. and Loscher, W., Valproic acid and active unsaturated metabolite (2-EN): Transfer to mouse liver following human therapeutic doses, Biopharm. Drug Disp., 6 (1985) 1-8. Nau, H., Transfer of valproic acid and its main active unsaturated metabolite to the gestational tissue: Correlation with neural tube defect formation in the mouse, Teratology, 33 (1986) 21-27. Nau, H., and Loscher, W., Pharmacologic evaluation of various metabolites and analogs of valproic acid: teratogenic potencies in mice, Fund. Appl. Toxicol., 6 (1986) 669-676. Nau, H., Hauck, R., and Ehlers, K., Valproic acid-induced neural tube defects in mouse and human: aspects of chirality, alternative drug development, pharmacokinetics and possible mechanisms, Pharm. Toxicol., 69 (1991) 310-321. Ogiso, T., Ito, Y., Iwaki, M., Yamahata, T., Disposition and pharmacokinetics of valproic acid in rats, Chem. Pharm. Bull., 34 (1986) 2950-2956. 117 Omtzigt, J.G.C., Los, R.J., Grobbee, D.E., Pijpers, L., Jahoda, M.G.J., Brandenburg, H., Stewart, P.A., Gaillard, H.L.J., Sachs, E.S., Wladimiroff, J.W., and Lindhout, D., The risk of spina bifida aperta after first-trimester exposure to valproate in a prenatal cohort, Neurology, 42(1992) 119-125. Palaty, J., and Abbott, F.S., Structure-activity relationships of unsaturated analogues of valproic acid, J. Med. Chem., 38 (1995) 3398-3406. Paxinos, G., Watson, C., The rat brain in stereotaxic coordinates, North Ryde, N.S.W.: Academic Press Australia, 1982. Perucca, E., Gatti, G., Frigo, G.M., and Crema, A., Pharmacokinetics of valproic acid after oral and intravenous administration, Br. J. Clin. Pharamacol., 5 (1978) 313-318. Perucca, E., Gatti, G., Frigo, G.M., and Crema, A., Disposition of sodium valprotate in epileptic patients, Br. J. Clin. Pharmacol., 5 (1978) 495-499. Perucca, E., Hedges, A., Makki, A., Ruprah, M., Wilson, J.F., Richens, A., A comparative study of the relative enzyme inducing properties of anticonvulsant drugs in epileptic patients, Br. J. Clin. Pharmac, 18 (1984) 401-410. Phillips, N.L, and Fowler, L.J., The effects of sodium valproate on y-aminobutyrate metabolism and behaviour in naive and ethanolmaine-O-sulphate pretreated rats and mice, Biochem. Pharmacol, 31 (1982) 2257-2261. Pollack, G.M., McHugh, W.B., Gengo, F.M., Ermer, J.C., Shen, D.D., Accumulation and washout kinetics of valproic acid and its active metabolites, J. Clin. Pharmacol, 26 (1986) 668-676. Pollack, G.M., and Brouwer, K.L.R., Dose-dependent metabolism and biliary excretion of valproic acid (VPA) and valproate glucuronide (VPA-G) in the rat, Pharm. Res., 5 (1988) S-163. Pollack, G.M., and Brouwer, K.L.R., Physiologic and metabolic influences on enterohepatic recirculation: simulations based upon the disposition of valproic acid in the rat, J. Pharmacokin. Biopharm., 19 (1991) 189-225. 118 Rettenmeier, A.W., Prickett, K.S., Gordon, P., Bjorge, S.M., Chang, S., Levy, R.H., and Baillie, T.A., Studies on the biotransformation in the perfused rat liver of 2-n-propyl-4-pentenoic acid, a metabolite of the antiepileptic drug valproic acid, Drug Metab. Dispos., 13 (1985) 81-96. Rettenmeier, A.W., Gordon, W.P., Barnes, FL, Baillie, T.A., Studies on the metabolic fate of valproic acid in the rat using stable isotope techniques, Xenobiotica, 17 (1987) 1147-1157. Rettenmeier, A.W., Howald, W.N., Levy, R.H., Witek, D.J., Gordon, W.P., Porubek, D.J., Baillie, T.A., Quantitative metabolic profiling of valproic acid in humans using automated gas chromatography/mass spectrometric techniques, Biomed. Environ. Mass Spec, 18 (1989) 192-199. Rettie, A.E., Rettenmeier, A.W., Howald, W.N., and Baillie, T.A., Cytochrome P-450-Catalyzed formation of A^-VPA, a toxic metabolite of valproic acid, Science, 235 (1987) 890-893. Rowan, A.J., Binnie, CD. , Warfield, C.A., Meinardi, H., Meijer, J.W.A., The delayed effect of sodium valproate on the photoconvulsive response in man, Epilepsia, 20 (1979) 61-68. Royer-Morrot, M., Zhiri, A., Jacob, F., Necciari, J., Lascombes, F., and Royer, R., Influence of food intake on the pharmacokinetics of a sustained release formulation of sodium valproate, Biopharm. Drug Dispos., 14 (1993) 511-518. Schafer, H., and Liirhrs, R., Responsibility of the metabolite pattern for potential side-effect in the rat being treated with valproic acid, 2-propylpenten-2-oic acid, and 2-propylpenten-4-oic acid, in Levy, R.H., Pitlick, W.H., Eichelbaum, M., Meijer, J., (eds), Metabolism of Antiepileptic Drugs, New York: Raven Press (1984) 73-81. Scheffner, D., Konig, S., Rautergerg-Ruland, I., Kochen, W., Hofmann,.W.J., Unkelbach, S., Fatal liver failure in 16 children with valproate therapy, Epilepsia, 29 (1988) 530-542. Schobben, F., van der Kleihn, E., Gabreels, F.J.M., Pharmacokinetics of di-n-propylacetate in epileptic patients, Eur. J. Clin. Pharmacol, 8 (1975) 97-105. Schulz, H., Metabolism of 4-pentenoic acid and inhibition of thiolase by metabolites of 4-pentenoic acid, Biochemistry, 22 (1983) 1827-1832. 119 Semmes, R. and Shen D., Nonlinear binding of valproic acid (VPA) and E-A2-valproic acid to rat plasma proteins, Pharm. Res., 1 (1990) 461-467. Semmes, R. and Shen, D., Comparative pharmacodynamics and brain distribution of E-A 2-valproate and valproate in rats, Epilepsia, 32 (1991) 232-241. Shen, D.D., Ojemann, G.A., Rapport, R.L., Dills, R.L., Friel, P.N. and Levy, R.H., Low and variable presence of valproic acid in human brain, Neurology, 42 (1992) 582-585. Singh, K., Orr, J. and Abbott, F., Pharmacokinetics and enterohepatic circulation of (E)-2-ene valproic acid in the rat, J. Pharmacobio-Dyn., 13 (1990) 622-627. Singh, K., Orr, J.M., and Abbott, F.S., Pharmacokinetics and enterohepatic circulation of 2-n-propyl-4-pentenoic acid in the rat, Drug Metab. Dispos., 16 (1988), 848-852. Steimer, J., Plusquellec, Y., Guillaume, A., and Boisvieux, J., A time-lag model for pharmacokinetics of drugs subject to enterohepatic circulation, J. Pharm. Sci., 71 (1982) 297-302. Tang, W., Borel, A.G., Fujimiya, T., and Abbott, F.S., Fluorinated analogues as mechanistic probes in valproic acid hepatotoxicity: Hepatic microvesicular steatosis and glutathione status, Chem. Res. Toxicol, 8 (1995) 671-682. Terasaki, T., Takakuma, S., Moritani, S., and Tsuji, A., Transport of monocarboxylic acids at the blood-brain barrier: studies with monolayers of primary cultured bovine brain capillary endothelial cells, /. Pharmacol. Exp. Ther., 258 (1991) 932-937. Thompson, R., A behavioural atlas of the rat brain, New York, Oxford University Press, 1978. Tozer, T.N., Concepts basic to pharmacokinetics, Pharmac. Ther., 12 (1981) 109-131. Vajda, F.J.E., Donnan, G.A., Phillips, J., and Bladin, P.F., Human brain, plasma, and cerebrospinal fluid concentration of sodium valproate after 72 hours of therapy, Neurology, 31 (1981)486-587. 120 van der Laan, J.W., De Boer, T., Bruinvels, J., Di-n-propylacetate and GABA degradation. Preferential inhibition of succinic semialdehyde dehydrogenase and indirect inhibition of GABA-transaminase, J. Neurochem., 32 (1979) 1769-1780. Vorhees, C.V., Acuff-Smith, K.D., Weisenburger, W.P., Minck, D.R., Berry, J.S., Setchell, K.D.R. and Nau, H., Lack of teratogenicity of fr-an.s-2-ene-valproic acid compared to valproic acid in rats, Teratology, 43 (1991) 583-590. Vree, T.B., and Van der Kleihn, E., Pharmacokinetics and renal excretion of 2-n-propyl pentanoate (Depakine®) in man, dog and Rhesus monkey, Pharmaceut. Week., 112 (1977) 290-292. Wieser, H., Comparison of valproate concentrations in human plasma, CSF and brain tissue after administration of different formulations of valproate or valpromide, Epilepsy Res., 9 (1991) 154-159. Yamaoka, K., and Nakagawa, T., A nonlinear test squares program based on differential equations, MULTI(RUNGE), for microcomputers, /. Pharmacobio-Dyn, 6 (1983) 595-606. Yu, H., Shen Y., Sugiyama, Y., and Hanano, M., Dose-dependent pharmacokinetics of valproate in guinea pigs of different ages, Epilepsia, 28 (1987) 680-687. Yu, H., and Shen, Y., Dose-dependent inhibition in plasma protein binding of valproic acid during contined treatment in guinea-pigs, J. Pharm. Pharmacol., 44 (1992) 408-412. Yu, D., Gordon, J.D., Zheng, J.J., Panesar, S.K., Riggs, K.W., Rurak, D.W., and Abbott, F.S., Determination of valproic acid and its metabolites using gas chromatography with mass-selective detection: application to serum and urine samples from sheep, 7. Chromatogr., 666 (1995) 269-281. Yu, H., and Shen, Y., Dose-dependent distribution volumes of total and unbound valproate in guinea-pigs: consequence of non-linear plasma protein binding, Biopharm. Drug Dispos., 17 (1996) 237-247. Zaccara, G., Messori, A., and Moroni, F., Clinical pharmacokinetics of valproic acid, 1988. Clin. Pharmacokin., 15 (1988) 367-389. 121 Zeman, W., and Innes, J.R.M., Craigie's neuroanatomy of the rat, New York, Academic Press, 1963. Zheng, J.J., (1993): Metabolism and pharmacokinetic studies of valproic acid using stable isotope techniques, M.Sc. Thesis, The University of British Columbia, 147 p. Zimmerman, H.J., and Ishak, K.G., Valproate-induced hepatic injury: analysis of 23 fatal cases, Hepatology, 2 (1982) 591-597. 122 7. APPENDICES Appendix 1. •i The mass balance differential equations for the four-compartment time-lag model were: *dCi / dt = -(kio + k n + ki2 + k u) x Ci + (k3, x C3) + (L,, x C4) + (k2ixC2) + (kaxC in j) *dC i n j/dt = -(k axC i n j) dCi / dt = -(kio + k I 3 + k,2 + k,4) xC, + (k3i x A 3 / V c ) + (Lu x A , / V c ) + (k2, x A 2 / V c) + (ka x DOSE x e"kat / V c ) *dC2/dt = -(k21 xC 2 )+ (k, 2xC,) dA2 / dt = -(k2i x A2) +( k 1 2 x Ci x V c ) *dC 3/dt = (k 1 3 xC,) - (k 3 1 xC 3 ) dA3 / dt = ( k i 3 x C, x V c ) - (k31 x A3) *dC4 / dt = (kn x CO - (L,i x C4) dAj/dt = (kn x Ci x V c ) - (Lu x A4) where Q , C 2 , C 3 and C 4 are the drug concentrations in compartment 1, 2, 3, and 4, respectively, at time t; Cinj is the concentration of the injection "compartment"; A 2 is the amount of drug in compartment 2 after time T (where T is the time-lag); A 3 and A4 are the amounts of drug in compartment 3, and 4, respectively; ki 2, k 2i, ki3, k 3i, ki4, and Lu are first-order rate constants for the transfer of drug between the various compartments; kio is the first-order rate constant for the elimination of drug from compartment 1; ka is the first-order absorption rate constant for compartment 1; and Vi is the volume of distribution for compartment 1. The time-lag (T) was chosen at the apparent onset of enterohepatic cycling which corresponded to approximately 150 min. * differential equations used in the ADAPTII program 123 1 0 0 0 . 0 0 -g 0 6 0 1 2 0 180 2 4 0 3 0 0 3 6 0 4 2 0 4 8 0 5 4 0 6 0 0 6 6 0 7 2 0 Time (min) Appendix 2. A computer simulation (ADAPT2) of a serum concentration-time profile with a secondary increase in serum concentration (indicative of enterohepatic cycling) based on data from the dose-dependent study (section 3.3.1). All data points (closed circles and line of best fit) were generated based on the microconstants determined after i.p. administration of 150 mg/kg of (E,Z)-2,3'-diene VPA to rats (table 7). 

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