Frank S. Abbott

Oxidative stress as a mechanism of valproic acid-associated hepatotoxicity.

Valproic acid (2-n-propylpentanoic acid; VPA) has several therapeutic indications, but it is used primarily as an anticonvulsant. VPA is a relatively safe drug, but its use is associated with idiosyncratic hepatotoxicity, which in some cases may lead to fatality. The underlying mechanism responsible for the hepatotoxicity is still not well understood, but various hypotheses have been proposed, including oxidative stress. This article discusses the experimental evidence on the effect of VPA on the various indices of oxidative stress and on the potential role of oxidative stress in VPA-associated hepatotoxicity.

Influence of CYP2C9 genotypes on the formation of a hepatotoxic metabolite of valproic acid in human liver microsomes

ABSTRACTThe present study investigated the effect of cytochrome P450 2C9 (CYP2C9) genetic polymorphism on the biotransformation of valproic acid (VPA) to its hepatotoxic metabolite, 4-ene-VPA, and compared that to the formation of the inactive 4-OH-VPA and 5-OH-VPA. cDNA-expressed CYP2C9*2 and CYP2C9*3 variants were less efficient than the CYP2C9*1 wild type in catalyzing the formation of these metabolites, as assessed by the ratio of Vmax and apparent Km (in vitro intrinsic clearance). The reduced efficiency by CYP2C9*2 was due to a reduced Vmax, whereas, in the case of CYP2C9*3, it was the result of increased apparent Km. The formation rates of 4-ene-VPA, 4-OH-VPA, and 5-OH-VPA in human liver microsomes were reduced by 29, 28, and 31%, respectively, in samples with one mutated CYP2C9 allele, and by 61, 73, and 58%, respectively, in samples with two mutated CYP2C9 alleles. Overall, the homozygote and heterozygote CYP2C9*2 and CYP2C9*3 genotypes may compromise hepatic VPA biotransformation.

The effect of aspirin on valproic acid metabolism

Urinary valproic acid (VPA) and VPA metabolite profiles were determined before (day 1) and after (day 2) the administration of antipyretic doses of acetylsalicylic acid (ASA) to seven subjects with steady‐state levels of VPA. Of the 13 metabolites assayed by GC/MS, levels of (E)‐2‐ene VPA and 3‐keto VPA were significantly decreased on day 2, whereas those of the VPA conjugates (glucuronide) and 4‐ene VPA were significantly increased. The β‐oxidation pathway consisting of (E)‐2‐ene VPA, 3‐OH VPA, and 3‐keto VPA was decreased from 24.5% ± 10.3% of total metabolites excreted on day 1 to 8.3% ± 4.2% on day 2, a decrease of 66% (P < 0.05). VPA glucuronide content increased from 50.5% ± 12.6% on day 1 to 65.5% ± 14% of total excreted on day 2, an increase of 30% (P < 0.05). The day 2/day 1 ratios of VPA glucuronide correlated significantly with the day 2/day 1 ratios of VPA mean free fraction (r = 0.9424; P = 0.005) in six of the seven subjects. Inhibition of VPA β‐oxidation by salicylate was sufficient to counterbalance the increased elimination of VPA as its conjugates and explains why total clearance of VPA after salicylate remains unchanged even though the free fraction of VPA is increased. Metabolic profiles indicate that salicylate likely inhibits VPA β‐oxidation by reducing valproyl‐coenzyme A formation.

Methadone maintenance: Effect of urinary pH on renal clearance in chronic high and low doses

The subjects were 12 male patients stabilized on methadone for many months or years. A comparison was made of the plasma levels and renal clearance of methadone between patients on “high” doses (80 to 110 mg/day) and those on “low” doses (15 to 40 mg/day). A general trend to higher renal clearance was seen in the “high”‐dose group, but on more detailed examination there was a direct correlation only when the patients were categorized by urinary pH. At low pHs, there was nearly a 3‐fold increase in renal clearance which was associated with a decreased major metabolite to methadone ratio. No evidence for a difference in rate of metabolism between the two groups was found nor were there differences in hepatic function. It was concluded that urinary pH was a major factor in renal clearance of methadone.

Effects of Age and Polytherapy, Risk Factors of Valproic Acid (VPA) Hepatotoxicity, on the Excretion of Thiol Conjugates of (E)‐2,4‐diene VPA in People with Epilepsy Taking VPA

Summary:  Purpose: Valproic acid (VPA) is an antiepileptic drug (AED) used for generalized and absence seizures. It has a rare but potentially fatal hepatotoxicity side effect, and many researchers believe that reactive metabolites of VPA could be involved. We demonstrated here that the thiol conjugates of (E)‐2,4‐diene VPA were significantly elevated in a high‐risk group of patients.

Interaction between valproic acid and aspirin in epileptic children: Serum protein binding and metabolic effects

In five of six epileptic children who were taking 18 to 49 mg/kg/day valproic acid (VPA), the steady‐state serum free fractions of VPA rose from 12% to 43% when antipyretic doses of aspirin were also taken. Mean total VPA half‐life (t½) rose from 10.4 ± 2.7 to 12.9 ± 1.8 hr and mean free VPA t½ rose from 6.7 ± to 2.1 to 8.9 ± 3.0 hr when salicylate was present in the serum. The in vitro albumin binding association constant (ka) for VPA was decreased by salicylate, but the in vivo ka value was not affected. The 12‐hr (trough) concentrations of both free and total VPA were higher in the presence of serum salicylate in five of six patients. Renal excretion of unchanged VPA decreased in five of six patients, but the VPA carboxyl conjugate metabolite–excretion patterns were not consistently affected. Salicylate appeared to displace VPA from serum albumin in vivo, but the increased VPA t½ and changes in VPA elimination patterns suggest that serum salicylate also altered VPA metabolism.

The effect of valproic acid on hepatic and plasma levels of 15-F2t-isoprostane in rats

The mechanism by which valproic acid (VPA) induces liver injury remains unknown, but it is hypothesized to involve the generation of toxic metabolites and/or reactive oxygen species. This studys objectives were to determine the effect of VPA on plasma and hepatic levels of the F(2)-isoprostane, 15-F(2t)-IsoP, a marker for oxidative stress, and to investigate the influence of cytochrome P450- (P450-) mediated VPA biotransformation on 15-F(2t)-IsoP levels in rats. In rats treated with VPA (500 mg/kg), plasma 15-F(2t)-IsoP was increased 2.5-fold at t(max) = 0.5 h. Phenobarbital pretreatment (80 mg/kg/d for 4 d) in VPA-treated rats increased plasma and liver levels of free 15-F(2t)-IsoP by 5-fold and 3-fold, respectively, when compared to control groups. This was accompanied by an elevation in plasma and liver levels of P450-mediated VPA metabolites. Pretreatment with SKF-525A (80 mg/kg) or 1-aminobenzotriazole (100 mg/kg), which inhibited P450-mediated VPA metabolism, did not attenuate the increased levels of plasma 15-F(2t)-IsoP in VPA-treated groups. Plasma and hepatic levels of 15-F(2t)-IsoP were further elevated after 14 d of VPA treatment compared to single-dose treatment. Our data indicate that VPA increases plasma and hepatic levels of 15-F(2t)-IsoP and this effect can be enhanced by phenobarbital by a mechanism not involving P450-catalyzed VPA biotransformation.

CYP2E1-mediated modulation of valproic acid-induced hepatocytotoxicity

OBJECTIVES To determine the cytotoxicity of valproic acid (VPA) and its metabolite, 4-ene-valproic acid (4-ene-VPA) in human hepatoblastoma cells (Hep G2), and to study the modulatory effect of cytochrome P450 2E1 induction in this model. METHODS Cells were exposed to VPA or 4-ene-VPA in the presence of either ethanol (EtOH), or EtOH combined with disulphiram (DS). Some cells were exposed to alpha-fluoro-VPA or to alpha-fluoro-4-ene-VPA in the absence of CYP2E1 inducers. Apoptosis and necrosis were measured by analyzing 6000 cells per sample using transmission electron microscopy, while cytokine release and apoptosis were quantitated by ELISA. RESULTS VPA + EtOH increased VPA cytotoxicity. 4-ene-VPA + EtOH significantly increased toxicity, while DS + EtOH significantly reduced this toxicity. Alpha-fluorinated analogues reduced cytotoxicity compared to the corresponding VPA compounds. Neither VPA nor alpha-fluorinated VPA increased the release of IL-6 or TNF-alpha in media. A significant increase in the release of TNF-alpha was observed in cells exposed to 4-ene-VPA that further increased with EtOH exposure. CONCLUSIONS Cells exposed to 4-ene-VPA experience greater cytotoxicity than those treated with VPA. Cytochrome P450 2E1 inducers enhance toxicity in VPA-exposed cells, while alpha-fluorination of VPA diminishes cytotoxicity by directly interfering with the beta-oxidation of the 4-ene-VPA metabolite.

Analysis of methadone and metabolites in biological fluids with gas chromatography—mass spectrometry

The analysis of methadone and its metabolites in biological fluids by gas chromatography--mass spectrometry is described with deuterated methadone and metabolites as internal standards. The method allowed the determination of 20 ng methadone in 0.5 ml of plasma or saliva. Mean saliva to plasma ratio of methadone for two patients was determined to be 0.51 +/- 0.13. Methadone and 2-ethylidene-1,5-dimethyl-3,3-diphenylpyrrolidine (EDDP) in urine were measured by selected ion monitoring. Gas chromatography--mass spectrometry was found to have advantages over conventional gas chromatographic methods in terms of ratio analysis. 1,5-Dimethyl-3,3-diphenyl-2-pyrrolidone previously reported as a metabolite was shown to result primarily from the decomposition of EDDP free base.

Chemistry and biotransformation

Since the serendipitous discovery of the anticonvulsant activity of valproic acid (VPA) by Munier in 1963, and its introduction to the clinic in 1967, the study of the metabolic fate and diverse pharmacological activity of VPA metabolites have progressively continued for three decades. Up to fifty VPA metabolites were identified by 1987 [1], with a few of these demonstrating significant biological activity. As a result, VPA metabolites have been studied in detail not only with respect to their antiepileptic potential ([2] and see below), but also as model compounds to characterize the structural requirements for induction of teratogenic or hepatotoxic effects (see Radatz and Nau, this volume) and also to elucidate the biochemical nature of novel pathways of biotransformation, e.g. cytochrome P450-mediated desaturation and (ω-2)-hydroxylation [3]. In light of the need for rational drug-design of new efficacious antiepileptic drugs having minimal sideeffects and free of the undesired interactions with other antiepileptic agents, structure-activity relationship studies of VPA analogues and metabolites have played a pivotal role towards the development of new antiepileptic agents [4–7]. The current chapter describes the chemistry and structure-activity relationships (SAR) of VPA analogues and related metabolites, followed by an overview on the metabolic pathways involved in VPA biotransformation.