Abstract

In vitro studies were conducted to identify the major metabolites of eplerenone (EP) and the cytochrome P450 (P450) isozymes involved in its primary oxidative metabolism in humans and dogs. The major in vitro metabolites were identified as 6β-hydroxy EP and 21-hydroxy EP in both humans and dogs. EP was metabolized by cDNA-expressed human CYP3A4 and dog CYP3A12 but only minimally by human CYP3A5. In human microsomes, inhibition of total metabolism by the CYP3A-selective inhibitors ketoconazole, troleandomycin, and 6′,7′-dihydroxybergamottin, each at 10 μM concentration, was 83 to 95%, whereas inhibition with inhibitors selective for other P450 isozymes was minimal. In dog liver microsomes, the percentages of inhibition were 53 to 76% with the CYP3A-selective inhibitors. A monoclonal anti-CYP3A4 antibody inhibited EP metabolism by 84%, whereas other monoclonal antibodies had minimal effects. The formation of 6β-hydroxy and 21-hydroxy metabolites in human liver microsomes was best correlated with CYP3A-selective dextromethorphanN-demethylation and testosterone 6β-hydroxylation activities. EP moderately inhibited only CYP3A (testosterone 6β-hydroxylase) activity in human liver microsomes by 23, 34 and 45% at concentrations of 30, 100, and 300 μM, respectively. With human microsomes, the V_max andK_m for 6β-hydroxylation and 21-hydroxylation were 0.973 nmol/min/mg and 217 μM, and 0.143 nmol/min/mg and 211 μM, respectively. The human hepatic clearance calculated from total in vitro EP metabolism was 2.30 ml/min/kg, which agrees with in vivo data. In conclusion, 6β- and 21-hydroxylation of EP is primarily catalyzed by CYP3A4 in humans and CYP3A12 in dogs. Also, it is unlikely that EP would substantially inhibit the metabolism of other drugs that are metabolized by CYP3A4 or other P450 isoforms.