Valproic acid (VPA), an antiepileptic drug, possesses a delay in maximum pharmacological response upon initial drug administration, and a prolonged duration of activity following discontinuation of the drug. Metabolites of VPA are thought to be involved as evidence from previous studies in mice demonstrated that (E)-2-ene VPA and (E,E)-2,3'-diene VPA, major products of VPA metabolism in serum, exerted some degree of anticonvulsant activity against pentylenetetrazole (PTZ)-induced seizures. Also associated with VPA therapy is a fatal idiosyncratic hepatotoxicity possibly involving two metabolites, 4-ene VPA and (E)-2,4-diene VPA. Preliminary tissue distribution studies had suggested that (E)-2-ene VPA may not be as hepatotoxic as VPA based solely on (E)-2-ene VPA concentrations in liver. The main objectives of this study were to investigate the kinetic and metabolic profiles, disposition, and anticonvulsant activity of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA in rats. Results of these experiments were intended to provide insight into the possible contributions of these metabolites towards VPA activity or toxicity. Synthesis of (E)-2-ene VPA and (E,E)-2,3'-diene VPA was accomplished by the regiospecific addition of propionaldehyde to an ester enolate, followed by nucleophilic elimination of the mesylate ester with l,8-diazabicyclo[5.4.0]undec-7-ene or potassium hydride. The synthesis provided good yields and was stereoselective. The isomeric purity of the synthesized compounds was found to be 95 - 97% based on nuclear magnetic resonance and gas chromatographyc-mass spectrometric data. The assay of VPA and its metabolites in rat plasma and tissue homogenate extracts was achieved by negative ion chemical ionization gas chromatography-mass spectrometry. This method proved to be selective, sensitive, reproducible, and amenable to automation. In order to compare the disposition and pharmacokinetics of VPA and its analogues, VPA was administered intraperitoneally to rats and the kinetic profiles in plasma, liver, heart, lungs, and nine brain regions were determined. Selective binding of VPA to liver was observed with the liver/plasma ratio at 10 hours after dosing being 4.6. VPA did not persist in brain and the distribution in brain tissue appeared uniform. Metabolites of VPA also were not retained in brain. A most interesting observation was the absence of (E,E)-2,3'-diene VPA in brain while a minor plasma metabolite, (E,Z)-2,3'-diene VPA, was the only detectable diene. A stereoselective active transport mechanism could account for this unusual result. Present in plasma but not detected in liver was (E)-2,4-diene VPA, the hepatotoxic metabolite of VPA. It was proposed that the diene may be covalently bound to liver tissue. Following single dose administration to rats, (E)-2-ene VPA appeared to persist in all tissues assayed following an initial decline phase. The prolonged terminal elimination phase may be attributed to the extensive plasma protein binding of (E)-2-ene VPA (>99%). No selective binding of (E)-2-ene VPA in brain was observed. Brain/plasma ratios at 10 hours after dosing did not exceed 0.03. Metabolites of (E)-2-ene VPA were mainly products of β-oxidation and reduction. Both hepatotoxic metabolites were observed in plasma with concentrations of 4-ene VPA in liver higher than normally seen following VPA administration. Questions arise regarding the potential hepatotoxicity of (E)-2-ene VPA. After single dose administration of (E,E)-2,3'-diene VPA to rats, clearance of the diene was rapid compared to that of VPA or (E)-2-ene VPA. Selective binding of the diene was observed in the superior and inferior colliculus and substantia nigra but the concentrations were too low to be considered clinically significant. Reduction of (E,E)-2,3'-diene VPA appeared to be the main route of metabolism. 4-Ene VPA and (E)-2,4-diene VPA were not detected in plasma or tissues suggesting (E,E)-2,3'-diene VPA may have a lower potential for liver toxicity. The anticonvulsant activities of VPA, (E)-2-ene VPA, and (E,E)-2,3'-diene VPA were compared in rats by the PTZ-induced seizure test. Based on ED50 values, the anticonvulsant potencies of VPA and (E)-2-ene VPA were comparable and significantly greater than (E,E)-2,3'-diene VPA. The detection of (E,Z)-2,3'-diene VPA in brain following VPA administration led to the testing of this diene isomer. The potency of the (E,Z)-isomer was found to be equivalent to VPA and (E)-2-ene VPA. Sedation was a severe side effect of (E)-2-ene VPA and the (E,E)-2,3'-diene VPA was stereoselectively unique in causing skeletal muscle rigidity. Sedation was minimal and muscle rigidity was not a property of the (E,Z)-isomer over the dose range studied. Based on the results of these studies, it can be concluded that neither (E)-2-ene VPA nor (E,E)-2,3'-diene VPA is responsible for the pharmacodynamic effects of VPA. From the metabolism of (E,E)-2,3'-diene VPA and the results of anticonvulsant testing, it was proposed that (E,Z)-2,3'-diene VPA may have potential as a relatively safe and useful anticonvulsant drug. / Pharmaceutical Sciences, Faculty of / Graduate

Valproic acid (VPA) is a broad spectrum antiepileptic agent used widely in the treatment of absence and tonic-clonic seizures. VPA is extensively metabolized and forms 17 metabolites in man. A monounsaturated metabolite, (E)-2-ene VPA, is at least as potent as the parent drug VPA in several animal models of epilepsy. Moreover, (E)-2-ene VPA appears to be free of two serious side effects of VPA, namely hepatotoxicity and teratogenicity. Another monounsaturated metabolite of VPA, 4-ene VPA, has been incriminated in the pathogenesis of fatal hepatic failure in children on VPA therapy. This thesis describes the synthesis of (E)-2-ene VPA and 4-ene VPA and the development of a simple and sensitive capillary gas chromatographic-mass spectrometric (GCMS) assay method for the estimation of (E)-2-ene VPA and 4-ene VPA in the biological fluids of the rat. This thesis also describes the pharmacokinetics of (E)-2-ene VPA and 4-ene VPA at two dose levels of 20 and 100 mg/kg in normal and bile exteriorized rats. A simple capillary GCMS assay method was developed that involves a single extraction of 80 µL of plasma, urine or bile with ethyl acetate followed by derivatization with MTBSTFA (N-tertiarybutyldimethylsilyl-N-methyl-trifluoroacetamide). For an 80 µL biological sample employed for extraction, the lowest detection limit for (E)-2-ene VPA was 60 ng/mL and for 4-ene VPA, 100 ng/mL. The calibration curves for (E)-2-ene VPA were linear over a fairly wide concentration range of 0.4-35 /µg/mL in plasma and 2-200 µg/ml in urine of the rat. Standard curves for 4-ene VPA were prepared in concentration ranges of 0.5-45 µg/mL in plasma and 2-80 µg/ml in urine. The assay method is reliable, reproducible, and is able to separate the diene metabolites of (E)-2-ene VPA. For pharmacokinetic studies, a single intravenous (IV) bolus dose of either (E)-2-ene VPA or 4-ene VPA was administered to normal or bile-exteriorized rats. On increasing the dose from 20 to 100 mg/kg in normal rats, the apparent plasma clearance of (E)-2-ene VPA changed from 4.9 ± 1.7 (SD) to 3.0 ± 0.3 mL/min.kg, and of 4-ene VPA decreased from 8.7 ± 0.6 to 5.9 + 0.5 mL/min.kg. A total (conjugates and unconjugates) of 32 + 6% of the low dose and 50 ± 11% of the high dose of (E)-2-ene VPA was recovered in the urine of the rat. The second metabolite, 4-ene VPA, was eliminated in the urine to a relatively smaller extent (22 ± 3% of the low dose and 28 ± 6% of the high dose). In bile-duct cannulated rats, the apparent plasma clearance of (E)-2-ene VPA was 7.7 ± 1.8 mL/min.kg at the low dose and 6.0 ± 1.1 mL/min.kg at the high dose. The corresponding values for 4-ene VPA were 11 ± 1.8 mL/min.kg and 7.4 ± 1.1 mL/min.kg, respectively. The apparent elimination half-life of (E)-2-ene VPA remained unchanged at 20-21 min at the two dose levels, compared to a 1.5 fold increase in the t½ °f 4-ene VPA from 13 ± 2 to 19 ± 3 min. The fraction of the low dose (29 ± 5%) eliminated in bile was significantly larger than at the high dose (21 ± 4%), when calculated as the sum of conjugated and unconjugated 4-ene VPA. The biliary elimination of (E)-2-ene VPA showed a non-significant change from 38 ± 10 to 31 ± 9% on increasing the dose. Like the parent drug VPA, (E)-2-ene VPA and 4-ene VPA showed enterohepatic recirculation in the rat which produced secondary plasma peaks in normal animals. Moreover, both (E)-2-ene VPA and 4-ene VPA showed a rapid but transient choleretic effect in the rat. The plasma protein binding of 4-ene VPA was apparently low (14-25%), in the concentration range of 20-350 µg/mL. The results indicate that 4-ene VPA is cleared much faster from the plasma than (E)-2-ene VPA in the rat. The plasma levels of 4-ene VPA required to show a non-linear decline (>200 µg/mL) in the rat are two orders of magnitude higher than 4-ene VPA levels (<1 µg/ml) seen in patients on VPA therapy. It is, therefore, unlikely that 4-ene VPA is eliminated more slowly than VPA in man. On the other hand, the plasma elimination t½ of (E)-2-ene VPA in bile-exteriorized rats is longer than that reported for VPA, indicating that (E)-2-ene VPA may have a longer lasting pharmacologic effect than VPA. / Pharmaceutical Sciences, Faculty of / Graduate