Received for publication April 1, 2005.
Revised July 22, 2005.
Accepted for publication July 22, 2005.

IN VITRO METABOLISM OF THE CALMODULIN ANTAGONIST DY-9760e BY HUMAN LIVER MICROSOMES: INVOLVEMENT OF CYTOCHROME P450s IN ATYPICAL KINETICS AND POTENTIAL DRUG INTERACTIONS

Abstract

Human cytochrome P450 (CYP) isozyme(s) responsible for metabolism of the calmodulin antagonist DY-9760e and kinetic profiles for formation of its 6 primary metabolites (M3, M5, M6, M7, M8, and DY-9836) were identified using human liver microsomes and recombinant CYP enzymes. In vitro experiments, including an immunoinhibition study, correlation analysis, and reactions with recombinant CYP enzymes, revealed that CYP3A4 is the primary CYP isozyme responsible for the formation of the DY-9760e metabolites, except for M5, which is metabolized by CYP2C9. Additionally, at clinically relevant concentrations, CYP2C8 and 2C19 make some contribution to the formation of M3 and M5, respectively. The formation rates of DY-9760e metabolites except for M8 by human liver microsomes are not consistent with a Michaelis-Menten kinetics model, but are better described by a substrate inhibition model. In contrast, the enzyme kinetics for all metabolites formed by recombinant CYP3A4 can be described by an autoactivation model or a mixed model of autoactivation and biphasic kinetics. Inhibition of human CYP enzymes by DY-9760e in human liver microsomes was also investigated. DY-9760e is a very potent competitive inhibitor of CYP2C8/9 and 2D6 (K_i 0.25-1.7 µM); a mixed competitive and non-competitive inhibitor of CYP2C19 (K_i 2.4 µM); and a moderate inhibitor of CYP1A2 and 3A4 (K_i 11.4-20.1 µM), suggesting a high possibility for human drug-drug interaction.

Key words: CYP inhibition, cytochrome P450, enzyme kinetics, human CYP enzymes, microsomes