Accelerated Communication GLUCURONIDATION CONVERTS GEMFIBROZIL TO A POTENT , METABOLISM-DEPENDENT INHIBITOR OF CYP 2 C 8 : IMPLICATIONS FOR DRUG-DRUG INTERACTIONS

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-Oglucuronide) 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 M), whereas gemfibrozil glucuronide most potently inhibited CYP2C8 (IC50 of 24 M). 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 M to 1.8 M after a 30-min incubation with human liver microsomes and NADPH. Inactivation of CYP2C8 by gemfibrozil glucuronide required NADPH, and proceeded with a KI (inhibitor concentration that supports half the maximal rate of enzyme inactivation) of 20 to 52 M and a kinact (maximal rate of inactivation) of 0.21 min . 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. There have been several reports of clinical interactions between gemfibrozil (e.g., Lopid, Parke-Davis) and CYP2C8 substrates such as cerivastatin, repaglinide, rosiglitazone, and pioglitazone (Backman et al., 2002; Niemi et al., 2003a,b; Jaakkola et al., 2005). Reports on the in vitro inhibitory potential of gemfibrozil demonstrated that this lipid-lowering drug is a more potent inhibitor of CYP2C9 than of CYP2C8 (Wen et al., 2001; Wang et al., 2002; Fujino et al., 2003). However, in the clinic, gemfibrozil is a more potent inhibitor of CYP2C8 than of CYP2C9. Coadministration of gemfibrozil with the CYP2C9 substrate warfarin does not increase the plasma concentrations of either Ror S-warfarin (in fact, it actually decreases them) (Lilja et al., 2005). An important step in providing a potential explanation for why gemfibrozil is a more potent inhibitor of CYP2C9 than CYP2C8 in vitro but is a more potent inhibitor of CYP2C8 than CYP2C9 in vivo was provided by Shitara et al. (2004), who demonstrated that gemfibrozil 1-O-glucuronide is a more potent inhibitor than gemfibrozil of CYP2C8. These same authors demonstrated that gemfibrozil 1-O-glucuronide inhibits in vitro the CYP2C8-mediated metabolism of cerivastatin, as well as the OATP2-mediated uptake of cerivastatin (Shitara et al., 2004). Other recent reports of glucuronide metabolites interacting directly with CYP2C8 have been published, including the CYP2C8-mediated hydroxylation of the acyl glucuronide of diclofenac, and the 17 -O-glucuronide of estradiol (Kumar et al., 2002; Delaforge et al., 2005). This study was designed to compare the in vitro inhibitory potential of gemfibrozil and its 1-O-glucuronide toward the major human drug-metabolizing P450 enzymes, namely CYP1A2, 2B6, 2C8, 2C9, 2C19, 2D6, and 3A4. In addition to confirming that gemfibrozil glucuronide is a more potent inhibitor of CYP2C8 than is gemfibrozil, we discovered that gemfibrozil glucuronide is a metabolism-dependent inhibitor of CYP2C8, suggesting that the clinical interactions between gemfibrozil and CYP2C8 substrates involve, at least in part, the conversion of gemfibrozil to an acyl glucuronide followed by the NADPH-dependent inactivation of CYP2C8 (Fig. 1). The conversion of gemfibrozil to its acyl glucuronide, and the subsequent metabolism-dependent inhibition of CYP2C8 by gemfibrozil glucuronide, could also be demonstrated in vitro by incubating Article, publication date, and citation information can be found at http://dmd.aspetjournals. org. doi:10.1124/dmd.105. 007633. ABBREVIATIONS: P450, cytochrome P450; IC50, inhibitor concentration that causes 50% inhibition; HPLC, high-performance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry; KI, inhibitor concentration that supports half the maximal rate of inactivation; kinact, maximal rate of enzyme inactivation. 0090-9556/06/3401-191–197$20.00 DRUG METABOLISM AND DISPOSITION Vol. 34, No. 1 Copyright © 2006 by The American Society for Pharmacology and Experimental Therapeutics 7633/3082235 DMD 34:191–197, 2006 Printed in U.S.A. 191 at A PE T Jornals on D ecem er 4, 2017 dm d.aspurnals. org D ow nladed from