Testosterone metabolism of equine single CYPs of the 3A subfamily compared to the human CYP3A4

Cytochrome P450 enzymes (CYPs) are responsible for the phase I metabolism of drugs, xenobiotics and endogenous substances. Knowledge of single CYPs and their substrates is important for drug metabolism, helps to predict adverse effects and may prevent reduced drug efficacy in polypharmacy. In this study, three equine isoenzymes of the 3A subfamily, the equine flavoprotein NADPH-P450 oxidoreductase (POR), and the cytochrome b5 (CYB5) were cloned, sequenced and heterologously expressed in a baculovirus expression system. Testosterone, the standard compound for characterization of the human CYP3A4, was used to characterize the newly expressed equine CYPs. The metabolite pattern was similar in equine and the human CYPs, but the amounts of metabolites were isoform-dependent. All equine CYPs produced 2-hydroxytestosterone (2-OH-TES), a metabolite never described in equines. The main metabolite of CYP3A4 6β-hydroxytestosterone (6β-OH-TES) was measured in CYPs 3A95 and 3A97 with levels close to the detection limit. Ketoconazole inhibited 2-OH-TES in the human CYP3A4 and the equine CYP3A94 and CYP3A97 completely, whereas a 70% inhibition was found in CYP3A95. Testosterone 6β- and 2-hydroxylation was significantly different in the equine CYPs compared to CYP3A4. The expression of single equine CYPs allows characterizing drug metabolism and may allow prevention of drug-drug interactions.