Mechanistic studies of valproic acid hepatotoxicity : identification and characterization of thiol conjugates

The severe hepatotoxicity of the antiepileptic drug valproic acid (VPA) is believed to be mediated through chemically reactive metabolites. The monounsaturated metabolite 4-ene VPA is steatogenic in the rat, and in a similar fashion to the hepatotoxin 4-pentenoic acid, is thought to be oxidized by mitochondria to a highly reactive α,β-unsaturated ketone, 3-keto-4-ene VPA. The tripeptide thiol, glutathione (GSH), is known to react with a variety of electrophilic compounds that have the potential to interact with cellular macromolecules. The identification and structural characterization of GSH conjugates provides a means of identifying short-lived unstable electrophiles and thus an insight into the mechanisms of toxicity. This thesis describes the synthesis and characterization of thiol conjugates of reactive metabolites derived from the in vivo metabolism of VPA, 4-ene VPA, (E)-2,4-diene VPA, and 4-pentenoic acid. A negative ion chemical ionization gas chromatographic/mass spectrometric (NICI/GC/MS) method for the determination of VPA and 14 of its metabolites in a single chromatographic run was developed. A combination of pentafluorobenzyl and trimethylsilyl derivatization resulted in the [M-181]̄̄̄ ̄anion as the base peak for all the metabolites measured. When these ions were monitored sensitivities in the low picogram range were achieved. The VPA metabolite profile was determined in pediatric patients on VPA monotherapy and on combined therapy with either carbamazepine or clobazam. 4-Ene- and (E)-2,4-diene-VPA were found to be minor metabolites with serum levels below 1% that of VPA. In patients on combined therapy with carbamazepine, the ω and ω-l pathways of VPA metabolism were induced, while products of β-oxidation were significantly decreased. Polytherapy had no significant effect on the serum levels of 4-ene- or (E)-2,4-diene-VPA. Rats were dosed intraperitoneally with 100 mg/kg of the sodium salts of VPA, 4-ene-, (E)-2,4-diene-VPA, 4-pentenoic or (E)-2,4-pentadienoic acids. Methylated bile extracts were analyzed by high pressure liquid chromatography and liquid chromatography/tandem mass spectrometry (LC/MS/MS) for GSH conjugates while urine samples were analyzed by GC/MS and LC/MS for N-acetylcysteine (NAC) conjugates and other metabolites. The GSH conjugate of (E)-2,4-diene VPA was detected in the bile of rats treated with 4-ene- and (E)-2,4-diene-VPA. The NAC conjugate was a major urinary metabolite of rats given (E)-2,4-diene VPA and was a prominent urinary metabolite of those animals given 4-ene VPA. The structures of these metabolites were confirmed by comparing GC/MS or LC/MS properties of the isolated metabolites to those of synthetic standards. The GSH and NAC conjugates of (E)-2,4-diene VPA were chemically synthesized and their structures established to be (E)-5-(glutathion-S-yl)-3-ene VPA and (E)-5-(N-acetylcystein-S-yl)-3-ene VPA by nuclear magnetic resonance spectroscopy and mass spectrometry. In contrast to the very slow reaction of the free acid of (E)-2,4-diene VPA with GSH, the methyl ester reacted rapidly with GSH to yield the adduct. In vivo it appears the diene forms an intermediate with enhanced electrophilic reactivity to GSH as indicated by the facile reaction of the diene with GSH in vivo (about 40% of the (E)-2,4-diene VPA administered to rats was excreted as the NAC conjugate in 24 hr). In rats treated with 4-pentenoic and/or (E)-2,4-pentadienoic acids the following conjugates were identified and characterized by synthesis: GSH and cysteine conjugates of 3-oxo-4-pentenoic acid, GSH and NAC conjugates of (E)-2,4-pentadienoic acid, and the NAC conjugate of acrylic acid. The results thus provided the first direct biochemical evidence for the in vivo formation of the metabolite of 4-pentenoic acid considered responsible for the irreversible inhibition of fatty acid metabolism. The results also revealed basic differences between the mitochondrial metabolism of 4-ene VPA and 4-pentenoic acid. The 3-keto-4-ene VPA and its GSH and NAC conjugates were synthesized in order to facilitate the in vivo identification of these compounds following the administration of VPA, 4-ene-, or (E)-2,4-diene-VPA to rats. However, neither the 3-keto-4-ene VPA nor its thiol derivatives were evident in any of the treatments. The NAC conjugate of (E)-2,4-diene VPA was also found to be a metabolite of VPA in patients. The level of the conjugate appeared to be higher in two patients who recovered from VPA-induced liver toxicity. The characterization of GSH and NAC (in humans and rats) conjugates of (E)-2,4-diene VPA suggests that VPA is metabolized to a chemically reactive intermediate that may contribute to the hepatotoxicity of the drug.Pharmaceutical Sciences, Faculty ofGraduat

Negative ion chemical ionization GCMS analysis of valproic acid and its metabolites

Valproic acid (VPA) is a major anticonvulsant drug widely used in the treatment of absence seizures. VPA is extensively metabolized in humans. Several VPA metabolites possess anticonvulsant activity and other metabolites are implicated in rare but fatal cases of hepatotoxicity. A highly sensitive and more specific analytical method was required to analyze the large number of VPA metabolites, some of which are present at trace levels. The objective of this study was to develop such a method and to make a preliminary application of the method to the determination of trace VPA levels and to search for new VPA metabolites. The suitability of analyzing halogenated derivatives of VPA and its metabolites by negative ion chemical ionization (NICI) GCMS was evaluated for the desired sensitivity and specificity. An assay was thus developed for VPA in serum and saliva based on NICI-GCMS of the pentafluorobenzyl (PFB) derivative. The NICI spectrum of the PFB ester of VPA was dominated by a single fragment ion, the m/z 143 ([M-181]⁻) ion. When the m/z 143 ion was monitored the lower limit of detection was 2 ng/mL of VPA in serum or saliva. Using [²H₆]-VPA as the internal standard, the intra- and inter-assay variations were less than 10 % at serum VPA concentrations of 10 to 800 ng/mL. Linearity was observed over the concentration range of 10 ng/mL to 25 µg/mL. The NICI assay was employed to quantitate VPA in serum (total and free) and saliva in five healthy volunteers who took part in a drug interaction study between VPA and carbamazepine (CBZ). A total of 63 paired saliva and serum samples were analyzed by NICI-GCMS; 33 before the administration of CBZ and 30 after CBZ. The % decrease in the average VPA concentration after CBZ was 27.91 ± 3.48, 36.85 ± 13.64, and 48.13 ± 7.70, for serum total, serum free and saliva VPA, respectively. There was a significant reduction (p<0.025) in the average VPA concentration in all three biological fluids. The average saliva to serum free VPA ratio was 18.92% ± 6.25 before CBZ and 16.37% ± 2.82 following CBZ. The average saliva to serum total VPA ratio was 2.43% ± 0.86 before CBZ and 1.67% ± 0.50 following CBZ, indicating that the saliva to serum total VPA ratio was concentration dependent. A strong correlation was found between saliva and both serum free (r = 0.9035 ± 0.0784) and serum total VPA (0.9058 ± 0.0450) (after CBZ). The free fraction of VPA did not increase after CBZ administration suggesting that the decrease in VPA concentration after CBZ was not related to changes in the free fraction of VPA. PFB derivative formation of VPA metabolites was facile and resulted in uniform derivatization of all metabolites studied. In the NICI mass spectra most of the ion current was carried by the [M-181]⁻ fragment ion, the only exception being that of 3-keto VPA. The base peak in the NICI spectrum of PFB derivatized 3-keto VPA was [M-181-C0₂]⁻. Isolated metabolites were identified with the help of twin ions (deuterated and undeuterated) in the mass spectra and by comparison of mass spectra and retention times with synthetic reference compounds. Urine or serum metabolites were analyzed in one chromatographic run and SIM chromatograms obtained. Serum and urine controls showed no interfering peaks and the analytical method appears suitable for a sensitive assay of VPA metabolites. The NICI method employing PFB derivatives was sensitive enough to detect VPA metabolites in saliva. Seven metabolites were detected. The ratio of Z to E isomers of 2-ene VPA was much greater in saliva than in serum (3.82 vs. 0.458), suggesting differences in the transport or plasma protein binding properties of these two isomers. A new VPA metabolite, assigned the structure 4¹-keto-2-ene VPA was detected in urine. The mass spectrum and retention time of this new metabolite matched that of one compound which was present in a synthetic mixture containing 4¹-keto-2-ene VPA. Another new metabolite which appears to be 2-(2¹-propenyl)-glutaric acid was also detected in urine. The synthesis of 4¹-keto-2-ene VPA was attempted using two different synthetic methods. The first method which involved the dehydrogenation of the 0-TMS dialkyl ketene acetal of ethyl 2-propyl-4-oxopentanoate apparently resulted in the formation of the positional isomer, 4-keto-2-ene VPA. The second synthetic route was based on the dehydration of 4-carboethoxy-2-ethylenethioketal-5-hydroxyheptane and produced 4¹-keto-2-ene VPA. However, it was not possible to isolate sufficient product for NMR characterization.Pharmaceutical Sciences, Faculty ofGraduat

DMD #37184 1 Pharmacokinetics and Metabolism in Rats, Dogs and Monkeys of the Cathepsin K Inhibitor Odanacatib: Demethylation of a Methylsulfonyl Moiety as a Major Metabolic Pathway DMD #37184 2 Running Title: Pharmacokinetics and Metabolism of Odanacatib

ABSTRACT Odanacatib is a potent cathespin K inhibitor that is being developed as a novel therapy for osteoporosis. The disposition and metabolism of odanacatib were evaluated in rats, dogs and rhesus monkeys after IV and oral administration of [ 14 C]odanacatib. Odanacatib was characterized by low systemic clearance in all species and by long plasma half-life in monkeys (18 hr) and dogs (64 hr). The oral bioavailability was dependent on the vehicle used and ranged from 18% (monkey) to ~100% (dog) at doses of 1-5 mg/kg, using non-aqueous vehicles. After IV and oral administration to intact rats and monkeys &gt;90% of the dose was recovered mainly in the feces. Studies in bile-duct cannulated animals indicated biliary secretion was the major mode of elimination of radioactivity; odanacatib also underwent some intestinal secretion. In monkeys, odanacatib was almost completely eliminated by metabolism; metabolism also played a major role in the clearance of odanacatib in rats and dogs. The major metabolic pathways were: methyl hydroxylation (formation of M8 and its derivatives), methyl sulfone demethylation (formation of M4 and its derivative M5) and glutathione conjugation (formation of the cyclized cysteinylglycine adduct M6 following addition of glutathione to the nitrile group of odanacatib). The major metabolites in rats (M4, parent-14 Da and M5, oxygenated derivative of M4) were determined to arise from a novel pathway that involved oxidative demethylation of the methylsulfonyl moiety of odanacatib. Overall, odanacatib displayed species-dependent metabolism which at least in part explains the divergent plasma half-life observed

Pharmacokinetics and Metabolism in Rats, Dogs, and Monkeys of the Cathepsin K Inhibitor Odanacatib: Demethylation of a Methylsulfonyl Moiety as a Major Metabolic Pathway □ S

ABSTRACT: Odanacatib is a potent cathespin K inhibitor that is being developed as a novel therapy for osteoporosis. The disposition and metabolism of odanacatib were evaluated in rats, dogs, and rhesus monkeys after intravenous and oral administration of [ 14 C]odanacatib. Odanacatib was characterized by low systemic clearance in all species and by a long plasma half-life in monkeys (18 h) and dogs (64 h). The oral bioavailability was dependent on the vehicle used and ranged from 18% (monkey) to ϳ100% (dog) at doses of 1 to 5 mg/kg, using nonaqueous vehicles. After intravenous and oral administration to intact rats and monkeys &gt;90% of the dose was recovered, mainly in the feces. Studies in bile duct-cannulated animals indicated that biliary secretion was the major mode of elimination of radioactivity; odanacatib also underwent some intestinal secretion. In monkeys, odanacatib was almost completely eliminated by metabolism; metabolism also played a major role in the clearance of odanacatib in rats and dogs. The major metabolic pathways were methyl hydroxylation (formation of M8 and its derivatives), methyl sulfone demethylation (formation of M4 and its derivative M5), and glutathione conjugation (formation of the cyclized cysteinylglycine adduct M6 after addition of glutathione to the nitrile group of odanacatib). The major metabolites in rats [M4 (parent-14 Da) and M5 (oxygenated derivative of M4)] were determined to arise from a novel pathway that involved oxidative demethylation of the methylsulfonyl moiety of odanacatib. Overall, odanacatib displayed species-dependent metabolism, which explains, at least in part, the divergent plasma half-life observed