Metabolic profiling of TRPV1 antagonists of the benzothiazole amide series: implications for in vitro genotoxicity assessment.

In vitro metabolic profiling and in vitro genotoxicity assessment are important aspects of the drug discovery program as they eliminate harmful compounds from further development. In standard in vitro genotoxicity testing, induced rat liver S9 is used as an exogenous bio-activation system for detecting promutagens. In this study we show that rat liver S9 is an insufficient system regarding the conversion of TRPV1 antagonists of the benzothiazole amide series into relevant in vivo metabolites. Human and rat hepatocyte experiments demonstrated generation of an aryl amine metabolite that was subsequently N-acetylated. The hydrolyzed metabolites as well as the parent compound were also metabolized into glutathione (GSH) conjugates. Rat liver S9 exhibited a very low amide hydrolysis capacity and no formation of GSH conjugates when supplemented with NADPH and GSH. The discrepancy in metabolic capability between hepatocytes and rat liver S9 led to confounding results in in vitro genotoxicity assessment for this chemical class as judged by the results of Ames test, mouse lymphoma assay, SOS/umu test and Comet assay in rat hepatocytes. This study highlights the pivotal role that understanding the mechanism of metabolite formation has in interpreting as well as designing reliable and relevant in vitro genotoxicity experiments.

Novel bioactivation mechanism of reactive metabolite formation from phenyl methyl-isoxazoles.

Recently, we described a series of phenyl methyl-isoxazole derivatives as novel, potent, and selective inhibitors of the voltage-gated sodium channel type 1.7 (Bioorg Med Chem Lett 21:3871-3876, 2011). The lead compound, 2-chloro-6-fluorobenzyl [3-(2,6-dichlorophenyl)-5-methylisoxazol-4-yl]carbamate, showed unprecedented GSH and cysteine reactivity associated with NADPH-dependent metabolism in trapping studies using human liver microsomes. Additional trapping experiments with close analogs and mass spectra and NMR analyses suggested that the conjugates were attached directly to the 5'-methyl on the isoxazole moiety. We propose a mechanism of bioactivation via an initial oxidation of the 5'-methyl generating a stabilized enimine intermediate and a subsequent GSH attack on the 5'-methylene. Efforts to ameliorate reactive metabolite generation were undertaken to minimize the potential risk of toxicity. Formation of reactive metabolites could be significantly reduced or prevented by removing the 5'-methyl, by N-methylation of the carbamate; by replacing the nitrogen with a carbon or removing the nitrogen to obtain a carboxylate; or by inserting an isomeric 5'-methyl isoxazole. The effectiveness of these various chemical modifications in reducing GSH adduct formation is in line with the proposed mechanism. In conclusion, we have identified a novel mechanism of bioactivation of phenyl 5-methyl-isoxazol-4-yl-amines. The reactivity was attenuated by several modifications aimed to prevent the emergence of an enimine intermediate. Whether 5'-methyl isoxazoles should be considered a structural alert for potential formation of reactive metabolites is dependent on their context, i.e., 4'-nitrogen.

Tissue distribution, pharmacokinetics and identification of roscovitine metabolites in rat.

The pharmacokinetics, biodistribution and the metabolic pathway of roscovitine were investigated in Sprague-Dawley rats after a single intravenous dose of 25 mg/kg. Blood, lungs, kidney, liver, testis, adipose tissue, spleen and brain were removed at different time-points. Plasma and tissue samples were analyzed using high performance liquid chromatography. The metabolites were identified using liquid chromatography/tandem mass spectrometry and nuclear magnetic resonance spectroscopy. Roscovitine (MW=354) was cleared rapidly from circulation and highly distributed to the tissues. The elimination half-life of roscovitine in plasma and tissues was short (<30 min). A major metabolite (M1) was observed mainly in plasma and in lower levels in all other tissues. M1 was identified as conversion of the hydroxyl-group at C2 to carboxylic acid (MW=368). A second metabolite (M2) was observed mainly in liver and kidney and identified as a hydroxylation product of the C8 of the purine-ring (MW=370). A third metabolite (M3) was found in several organs and corresponded to N-dealkylation of the N9-isopropyl side-chain (MW=312). Roscovitine concentrations in the brain were 30% of that observed in plasma, however no metabolites were detected in brain. In this investigation, three major metabolites of roscovitine were isolated and identified. Also, it was shown that roscovitine eliminates rapidly from both blood and tissues.