Pharmacokinetics of two monounsaturated metabolites of valproic acid in the rat
Creators: name: Singh, Kuldeep
Publisher: name: University of British Columbia
Year: 2010

description: 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 (&gt;200 µg/mL) in the rat are two orders of magnitude higher than 4-ene VPA levels (&lt;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.