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

Abstract

1. 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.

2. 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.

3. 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.

4. 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.

5. 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.

Keywords:

• Cytochrome P450
• Coumarin 152
• fluorogenic substrates
• human liver microsomes
• drug metabolism
• inhibition
• CYP2C19
• CYP3A4

Acknowledgments

The authors are grateful to Jeffrey P. Jones (WSU) for research support, assistance in obtaining and analyzing LC-MS/MS data, and continuous interest in this study. The authors also acknowledge the support from The State Academies of Sciences of the Russian Federation provided for the analysis of HLM samples with mass-spectroscopy within the framework of the Fundamental Scientific Research Program for 2013–2020.

Disclosure statement

No potential conflict of interest was reported by the author(s).

Additional information

Funding

This research was supported by the National Institute On Alcohol Abuse And Alcoholism of NlH under Award Number R21AA024548.