Abstract

Gemfibrozil more potently inhibits CYP2C9 than CYP2C8 in vitro, and yet the opposite inhibitory potency is observed in the clinic. To investigate this apparent paradox, we evaluated both gemfibrozil and its major metabolite, an acyl-glucuronide (gemfibrozil 1-O-β-glucuronide) as direct-acting and metabolism-dependent inhibitors of the major drug-metabolizing cytochrome P450 enzymes (CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4) in human liver microsomes. Gemfibrozil most potently inhibited CYP2C9 (IC₅₀ of 30 μM), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC₅₀ of 24 μM). Unexpectedly, gemfibrozil glucuronide, but not gemfibrozil, was found to be a metabolism-dependent inhibitor of CYP2C8 only. The IC₅₀ for inhibition of CYP2C8 by gemfibrozil glucuronide decreased from 24 μM to 1.8 μM after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a K_I (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 μM and a k_inact (maximal rate of inactivation) of 0.21 min^–1. Potent inhibition of CYP2C8 was also achieved by first incubating gemfibrozil with alamethicin-activated human liver microsomes and UDP-glucuronic acid (to form gemfibrozil glucuronide), followed by a second incubation with NADPH. Liquid chromatography-tandem mass spectrometry analysis established that human liver microsomes and recombinant CYP2C8 both convert gemfibrozil glucuronide to a hydroxylated metabolite, with oxidative metabolism occurring on the dimethylphenoxy moiety (the group furthest from the glucuronide moiety). The results described have important implications for the mechanism of the clinical interaction reported between gemfibrozil and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone.