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Abstract:

To characterize the in vitro metabolism of TMC125 in hepatocytes and liver subcellular fractions of male and female mice (CD-1 and rasH2) and rats, female rabbit, male dog and man.14C-TMC125 (5 mM) was incubated in hepatocytes (suspension and primary cell cultures) and liver sub-cellular fractions (microsomes and 12,000 g supernatants). Samples were taken at 60 and 120 min after the start of the incubation for all preparations except for the primary hepatocyte culture where they were taken at 12 and 24h. The samples were analyzed by radio-HPLC and the metabolites were identified using LC-MS/MS techniques, co-chromatography with authentic substances and enzyme hydrolysis.Extensive metabolism of TMC125 was observed in hepatocyte cultures of all animal species, while metabolism occurred to a significantly lower extent in human hepatocytes. In all species, metabolism was less extensive in the liver subcellular fractions compared to hepatocytes. Methylhydroxylation, at one of the methyl groups of the di-methylbenzonitrile moiety, in combination with glucuronide conjugation was the major metabolic pathway in all species. Aliphatic hydroxylation at both methyl groups of the same moiety, resulting in di-hydroxymethyl metabolite, also occurred in all species but the glucuronide conjugate of this metabolite was more important in man than in other species. Aromatic hydroxylation, at the benzonitrile moiety, was a minor pathway and metabolites resulting from this pathway alone or in combination with glucuronide conjugate occurred in some species but not in man.TMC125 metabolism was extensive in all species. All metabolites identified in human matrices were also found in incubates of at least one preclinical species.