Effect of -fluorination of Valproic Acid on Valproyl-s-acyl-coa Formation in Vivo in Rats

Studies designed to compare valproic acid (VPA) with its -fluorinated derivative (F-VPA) for their abilities to form acyl-CoA thioester derivatives in vivo are described. Recent studies have shown that -fluorination of a hepatotoxic metabolite of VPA ( -VPA) resulted in a nonhepatotoxic derivative. We hypothesize that the decrease in hepatotoxicity may be related to a lack of formation of the intermediary acyl-CoA thioester. To determine the effect of -fluoro substitution on acyl-CoA formation, we synthesized FVPA and compared it with VPA for its ability to form the acyl-CoA thioester derivative in vivo in rat liver. Thus, after dosing rats with VPA or F-VPA, animals were sacrificed (0.05-, 0.5-, 1-, 2-, and 5-h postadministration) for the analysis of liver tissue. High-performance liquid chromatography (HPLC) and electrospray ionization/ tandem mass spectrometry analysis of liver extracts from VPAdosed rats showed the presence of VPA-CoA that was maximal after 0.5 h (185 nmol/g of liver) and was still measurable 5-h postadministration (90 nmol/g of liver). In agreement with our hypothesis, F-VPA did not form the corresponding acyl-CoA derivative as determined by the absence of F-VPA-CoA upon HPLC analysis of liver extracts from F-VPA-dosed rats. Further examination of liver tissue for the presence of free acids revealed that the differences in acyl-CoA formation cannot be explained by differences in VPA and F-VPA free acid concentrations. From these observations and related studies showing the lack of toxicity due to -fluoro substitution, we propose that metabolism of VPA by acyl-CoA formation may mediate the hepatotoxicity of the drug. The use of valproic acid (2-n-propylpentanoic acid, VPA; Fig. 1) for the treatment of seizures has been associated with an idiosyncratic hepatotoxicity that is usually characterized by hepatic microvesicular steatosis (Zimmerman and Ishak, 1982). It has been proposed that a metabolite (or metabolites) of VPA induce the associated hepatotoxicity (Lewis et al., 1982; Zimmerman and Ishak, 1982; Baillie, 1992). -VPA, an unsaturated metabolite of VPA, elicits hepatic steatosis in rats to a greater degree than VPA or other metabolites tested (Kesterson et al., 1984). The mechanism by which -VPA causes its hepatotoxic effects is proposed to involve metabolic activation of -VPA by the enzymes of fatty acid -oxidation, resulting in the formation of reactive species that bind covalently to important enzymes involved in fatty acid metabolism (Fig. 2). Evidence for the formation of chemically reactive metabolites of VPA and -VPA comes from covalent binding studies in isolated rat hepatocytes. Covalent binding was abolished in cells pretreated with 4-pentenoic acid (a potent inhibitor of -oxidation) and increased in incubations with hepatocytes from rats pretreated with clofibrate (an inducer of -oxidation) (Porubek et al., 1989). In addition, similar studies in isolated rat liver mitochondria showed that -VPA covalently binds to proteins by a process that is dependent on the presence of the cofactors of -oxidation (CoA, ATP, L-carnitine, and Mg ) (Kassahun et al., 1994). Other experiments revealed that treatment of rats with -VPA leads to a depletion of total hepatic and mitochondrial glutathione (GSH), which supports a proposal that the depletion of GSH by reactive metabolites of -VPA may lead to the associated hepatotoxicity (Kassahun et al., 1991). In agreement with this finding are observations identifying the GSH conjugate of -VPA in the bile of -VPA-dosed rats and the excretion of the corresponding N-acetylcysteine conjugate in the urine of patients treated with VPA (Kassahun et al., 1991). The authors proposed that a reactive diene metabolite coming from the -oxidation of -VPA-CoA, namely -VPA-CoA, represents the reactive intermediate undergoing conjugation with GSH (Fig. 2). Acyl-CoA dehydrogenase enzymes catalyze the first step in mitochondrial fatty acid -oxidation by converting fatty acyl-CoA thioesters to their corresponding trans-2,3This work was supported in part by National Institutes of Health Grant GM36633. Preliminary accounts of this work were presented at the Millennial World Congress of Pharmaceutical Sciences, San Francisco, CA, April 2000. The University of California at San Francisco Mass Spectometry Facility is supported by the Biomedical Research Technology Program of the National Center for Research Resources, National Institutes of Health Grant RR01614, and National Science Foundation Grant DIR 8700766. 1 Present address: Pharmacia, Global Metabolism and Investigative Sciences, 301 Henrietta St., Kalamazoo, MI 49007. 2 Abbreviations used are: VPA, valproic acid; VPA-CoA, valproyl-S-acyl-CoA; F-VPA, -fluorovalproic acid; F-VPA-CoA, -fluorovalproyl-S-acyl-CoA; -VPA, unsaturated metabolite of VPA; F-VPA, -fluorinated analog of -VPA; VPA, hepatotoxic metabolite of -VPA; GSH, glutathione; BSTFA, bis(trimethylsilyl)trifluoroacetamide; CID, collisionally induced dissociation; THF, tetrahydrofuran; ESI/MS/MS, electrospray ionization/tandem mass spectrometry; GC/MS, gas chromatography/mass spectrometry; HPLC, high-performance liquid chro-