Reevaluate In Vitro CYP3A Index Reactions of Benzodiazepines and Steroids between Humans and Dogs.

ABSTRACT

Cytochrome P450 3A (CYP3A), the most important class of drug-metabolizing enzymes, participates in the metabolism of half of clinically used drugs. The CYP3A index reactions of dogs, one of the most widely used preclinical nonrodent species, are still poorly understood. This work evaluated the activity and selectivity of 10 CYP3A index reactions, including midazolam (MDZ) 1'- and 4-hydroxylation, alprazolam (APZ) and triazolam (TRZ) α- and 4-hydroxylation, testosterone (T) 6ß-hydroxylation, lithocholate (LCA) 6α-hydroxylation, deoxycholate (DCA) 1ß- and 5ß-hydroxylation, with quantitative reaction phenotyping and kinetic analysis in human and canine recombinant CYP enzymes (rCYPs). In human studies, all reactions are reconfirmed as mixed index reactions of CYP3A with minor contributions from non-CYP3A isoforms. In canine studies, all reactions are also primarily catalyzed by CYP3A12 with lower contributions from CYP3A26. However, the canine CYP2B11 appreciably contributes to the hydroxylation of benzodiazepines except for APZ 4-hydroxylation. The canine CYP3A isoforms have lower activity than human isoforms toward T 6ß-hydroxylation and LCA 6α-hydroxylation and both substrates undergo non-CYP3A catalyzed side reactions. DCA 1ß- and 5ß-hydroxylation are validated as the CYP3A index reactions in both humans and dogs with limited non-CYP3A contributions and side reactions. In conclusion, this work provides a comprehensive overview for the selectivity and activity of in vitro CYP3A index reactions in humans and dogs. The validated CYP3A index reactions between humans and dogs may benefit future practices in drug metabolism and drug interaction studies. SIGNIFICANCE STATEMENT Dogs are one of the most important nonrodent animals with limited studies of cytochrome P450 enzymes than humans. This work provides the most comprehensive quantitative data to date for the selectivity and activity of CYP3A index reactions in humans and dogs. The canine CYP2B11 was found to appreciably contribute to hydroxylation of midazolam, alprazolam and triazolam, the well-known probes for human CYP3A. Deoxycholate 1ß- and 5ß-hydroxylation are validated as the CYP3A index reactions in both humans and dogs.