Abstract

Phase I biotransformation of Trichostatin A (TSA), a histone deacetylase inhibitor with promising antifibrotic and antitumoral properties, was investigated in rat and human liver microsomes and in suspensions of rat hepatocytes. TSA (50 μM) was readily and completely metabolized by rat hepatocytes in suspension (2 × 10⁶ cells/ml), whereafter its phase I metabolites were separated by high-performance liquid chromatography and detected with simultaneous UV and electrospray ionization mass spectrometry (ESI-MS). ESI tandem mass spectrometry (ESI-MS/MS) was used to identify the metabolites. Two major phase I biotransformation pathways in rat hepatocytes were shown to be N-demethylation and reduction of the hydroxamic acid function to its corresponding amide.N-monodemethylated TSA and TSA amide were preferentially formed during the first 20 min of exposure, andN-monodemethylated TSA amide appeared as the main metabolite after a 30 min incubation period. At this time, virtually all TSA had been metabolized. Trichostatic acid,N-monodemethylated Trichostatic acid, andN-didemethylated TSA were identified as minor metabolites. Longer incubation led to the formation ofN-didemethylated TSA amide as the main metabolite. Lower concentrations of TSA (5 and 25 μM) formed relatively higher amounts of N-demethylated, nonreduced metabolites. Incubations of TSA with rat and human microsomal suspensions, however, led to an incomplete biotransformation with the formation of two major metabolites, N-mono- and N-didemethylated TSA. Traces of Trichostatic acid, TSA amide, N-mono- andN-didemethylated TSA amide were also detected. This study is the first to show that TSA undergoes intensive phase I biotransformation in rat hepatocytes. This has important consequences for its potential development as a drug, since rapid biotransformation resulting in a short exposure to the pharmacologically active parent compound, and a complex mixture of metabolites is usually not desired. Further biotransformation studies of TSA and structural analogs with antitumoral and antifibrotic properties need to be performed in cultured intact hepatocytes, in particular since one of the major phase I biotransformation pathways is catalyzed by nonmicrosomal enzymes.