Nonadditivity in human microsomal drug metabolism revealed in a study with coumarin 152, a polyspecific cytochrome P450 substrate

Abstract

We closely characterized 7-Dimethylamino-4-trifluromethylcoumarin (Coumarin 152, C152), a substrate metabolized by multiple P450 species, to establish a new fluorogenic probe for the studies of functional integration in the cytochrome P450 ensemble. Scanning fluorescence spectroscopy and LC/MS-MS were used to characterize the products of N-demethylation of C152 and optimize their fluorometric detection. The metabolism of C152 by the individual P450 species was characterized using the microsomes containing cDNA-expressed enzymes. C152 metabolism in human liver microsomes (HLM) was studied in a preparation with quantified content of eleven P450 species. C152 is metabolized by CYP2B6, CYP3A4, CYP3A5, CYP2C19, CYP1A2, CYP2C9, and CYP2C8 listed in the order of decreasing turnover. The affinities exhibited by CYP3A5, CYP2C9, and CYP2C8 were lower than those characteristic to the other enzymes. The presumption of additivity suggests the participation of CYP3A4, CYP2B6, and CYP2C19 to be 84, 8, and 0.2%, respectively. Contrary to this prediction, inhibitory analysis identified CYP2C19 as the principal C152-metabolizing enzyme. We thoroughly characterize C152 for the studies of drug metabolism in HLM and demonstrate the limitations of the proportional projection approach by providing an example, where the involvement of individual P450 species cannot be predicted from their content.