Short Communication GEMFIBROZIL IS A POTENT INHIBITOR OF HUMAN CYTOCHROME P450 2C9

The in vitro inhibitory effects of gemfibrozil on cytochrome P450 (CYP) 1A2 (phenacetin O-deethylation), CYP2A6 (coumarin 7-hydroxylation), CYP2C9 (tolbutamide hydroxylation), CYP2C19 (Smephenytoin 4 -hydroxylation), CYP2D6 (dextromethorphan O-deethylation), CYP2E1 (chlorzoxazone 6-hydroxylation), and CYP3A4 (midazolam 1 -hydroxylation) activities were examined using pooled human liver microsomes. The in vivo drug interactions of gemfibrozil were predicted in vitro using the [I]/([I] Ki) values. Gemfibrozil strongly and competitively inhibited CYP2C9 activity, with a Ki (IC50) value of 5.8 (9.6) M. In addition, gemfibrozil exhibited somewhat smaller inhibitory effects on CYP2C19 and CYP1A2 activities, with Ki (IC50) values of 24 (47) M and 82 (136) M, respectively. With concentrations up to 250 M, gemfibrozil showed no appreciable effect on CYP2A6, CYP2D6, CYP2E1, and CYP3A4 activities. Based on [I]/([I] Ki) values calculated using peak total (or unbound) plasma concentration of gemfibrozil, 96% (56%), 86% (24%), and 64% (8%) inhibition of the clearance of CYP2C9, CYP2C19, and CYP1A2 substrates could be expected, respectively. In conclusion, gemfibrozil inhibits the activity of CYP2C9 at clinically relevant concentrations, and this is the likely mechanism by which gemfibrozil interacts with CYP2C9 substrate drugs, such as warfarin and glyburide. Gemfibrozil may also impair clearance of CYP2C19 and CYP1A2 substrates, but inhibition of other CYP isoforms is unlikely. Gemfibrozil is a fibric acid derivative, which is used in the treatment of patients with lipid disorders (Dollery, 1999). A combined use of gemfibrozil and a statin can result in severe myopathy and rhabdomyolysis (Pierce et al., 1990; Tal et al., 1997). Recently, gemfibrozil has been found to elevate markedly plasma simvastatin acid and lovastatin acid levels, indicating a pharmacokinetic mechanism in the gemfibrozil-statin interactions (Backman et al., 2000; Kyrklund et al., 2001). In addition, gemfibrozil has been reported to enhance the effects of some other drugs, such as warfarin and glyburide (Ahmad, 1990; Ahmad, 1991; Rindone and Keng, 1998). However, the mechanisms of these gemfibrozil-related drug interactions are unclear. We have investigated the effects of gemfibrozil on the major cytochrome P450 (CYP) isoform activities in human liver microsomes in vitro, using selective marker reactions for CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 to clarify the mechanisms of the drug-drug interactions of gemfibrozil. Experimental Procedures Materials. Gemfibrozil, dextromethorphan, and dextrorphan were obtained from Orion Pharma (Espoo, Finland). Phenacetin, paracetamol, coumarin, 7-hydroxycoumarin, tolbutamide, chlorzoxazone, and NADPH were purchased from Sigma (St. Louis, MO). Hydroxytolbutamide, 6-hydroxychlorzoxazone, S-mephenytoin, and 4 -hydroxymephenytoin were purchased from Ultrafine Chemicals (Manchester, UK). Midazolam and 1 -hydroxymidazolam were kindly provided by Hoffmann-La Roche (Basel, Switzerland). Pooled human liver microsomes (prepared from five male and five female human liver microsomal samples) containing representative activities of CYP1A2, CYP2A6, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were obtained from GENTEST (Woburn, MA). Other chemicals and reagents were obtained from Merck (Darmstadt, Germany). Inhibition Studies. The effects of gemfibrozil on seven different CYP isoform-specific marker reactions were studied: phenacetin O-deethylation for CYP1A2, coumarin 7-hydroxylation for CYP2A6, tolbutamide hydroxylation for CYP2C9, S-mephenytoin 4 -hydroxylation for CYP2C19, dextromethorphan O-deethylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and midazolam 1 -hydroxylation for CYP3A4. The incubation conditions used to study the metabolism of the various substrates and the effects of specific inhibitors have been reported elsewhere (Wen et al., 2001). The time of incubation and concentration of microsomal protein (100 g/ml) used in each assay were determined to be in the linear range for the rate of metabolite formation. All incubations were performed in duplicate, and the mean values were used. Briefly, gemfibrozil was dissolved in acetonitrile. After evaporation of acetonitrile to dryness, gemfibrozil was reconstituted in an incubation medium (final concentrations, 0–250 M) containing 0.1 M sodium phosphate buffer, pH 7.4, 5 mM MgCl2, and 1.0 mM NADPH. The reaction was started by the addition of the marker substrate either without or after preincubation of the sample at 37°C for 15 min. After a specific period of time, the reactions were terminated by adding the appropriate chemicals to precipitate the proteins (Wen et al., 2001). After centrifugation at 10,000g for 5 min, an aliquot of the supernatant was subjected to analysis of the metabolites by high-performance liquid chromatography, as described previously (Wen et al., 2001). The intraand interday coefficients of variation were 7% at relevant concentrations (n 6). Data Analysis. The IC50 values were determined graphically. The apparent inhibitory constant (Ki) values were calculated by nonlinear regression analysis by fitting different models of enzyme inhibition to the kinetic data using SYSTAT for Windows 6.0.1 (SPSS, Inc., Chicago, IL). Results and Discussion Gemfibrozil strongly inhibited CYP2C9-catalyzed tolbutamide hydroxylation (Fig. 1A). The double reciprocal plots, Dixon plots, and This study was supported by grants from the Helsinki University Central Hospital Research Fund and the National Technology Agency of Finland (Tekes). 1 Abbreviation used is: CYP, cytochrome P450. Address correspondence to: Dr. Janne T. Backman, Department of Clinical Pharmacology, University of Helsinki, Haartmaninkatu 4, FIN-00290 Helsinki, Finland. E-mail: [email protected] 0090-9556/01/2911-1359–1361$3.00 DRUG METABOLISM AND DISPOSITION Vol. 29, No. 11 Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 442/936256 DMD 29:1359–1361, 2001 Printed in U.S.A. 1359 at A PE T Jornals on Jne 1, 2017 dm d.aspurnals.org D ow nladed from