Benzylic Oxidation of Gemfibrozil-1-O-β-Glucuronide by P450 2C8 Leads to Heme Alkylation and Irreversible Inhibition

Gemfibrozil-1-O-β-glucuronide (GEM-1-O-gluc), a major metabolite of the antihyperlipidemic drug gemfibrozil, is a mechanism-based inhibitor of P450 2C8 in vitro, and this irreversible inactivation may lead to clinical drug−drug interactions between gemfibrozil and other P450 2C8 substrates. In light of this in vitro finding and the observation that the glucuronide conjugate does not contain any obvious structural alerts, the current study was conducted to determine the potential site of GEM-1-O-gluc bioactivation and the subsequent mechanism of P450 2C8 inhibition (i.e., modification of apoprotein or heme). LC/MS analysis of a reaction mixture containing recombinant P450 2C8 and GEM-1-O-gluc revealed that the substrate was covalently linked to the heme prosthetic heme group during catalysis. A combination of mass spectrometry and deuterium isotope effects revealed that a benzylic carbon on the 2′,5′-dimethylphenoxy group of GEM-1-O-gluc was covalently bound to the heme of P450 2C8. The regiospecificity of...