TRIMETHOPRIM AND SULFAMETHOXAZOLE ARE SELECTIVE INHIBITORS OF CYP 2 C 8 AND CYP 2 C 9 , RESPECTIVELY

To evaluate the inhibitory effects of trimethoprim and sulfamethoxazole on cytochrome P450 (P450) isoforms, selective marker reactions for CYP1A2, CYP2A6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were examined in human liver microsomes and recombinant CYP2C8 and CYP2C9. The in vivo drug interactions of trimethoprim and sulfamethoxazole were predicted in vitro using [I]/([I] Ki) values. With concentrations ranging from 5 to 100 M, trimethoprim exhibited a selective inhibitory effect on CYP2C8-mediated paclitaxel 6 -hydroxylation in human liver microsomes and recombinant CYP2C8, with apparent IC50 (Ki) values of 54 M (32 M) and 75 M, respectively. With concentrations ranging from 50 to 500 M, sulfamethoxazole was a selective inhibitor of CYP2C9-mediated tolbutamide hydroxylation in human liver microsomes and recombinant CYP2C9, with apparent IC50 (Ki) values of 544 M (271 M) and 456 M, respectively. With concentrations higher than 100 M trimethoprim and 500 M sulfamethoxazole, both drugs lost their selectivity for the P450 isoforms. Based on estimated total hepatic concentrations (or free plasma concentrations) of the drugs and the scaling model, one would expect in vivo in humans 80% (26%) and 13% (24%) inhibition of the metabolic clearance of CYP2C8 and CYP2C9 substrates by trimethoprim and sulfamethoxazole, respectively. In conclusion, trimethoprim and sulfamethoxazole can be used as selective inhibitors of CYP2C8 and CYP2C9 in in vitro studies. In humans, trimethoprim and sulfamethoxazole may inhibit the activities of CYP2C8 and CYP2C9, respectively. Trimethoprim is frequently combined with sulfamethoxazole as cotrimoxazole, a broad-spectrum antibacterial agent, to treat a wide range of infections. Although trimethoprim is mainly excreted unchanged in urine, a significant amount (20%) of the dose is metabolized by the hepatic cytochrome P450 (P450) isoforms (Gleckman et al., 1981). In individuals with severe liver damage, the elimination half-life of trimethoprim can be lengthened up to 2-fold (Rieder and Schwartz, 1975). Sulfamethoxazole is eliminated mainly by metabolism, and CYP2C9 plays an important role in its N4-hydroxylation (Cribb et al., 1995). Trimethoprim and sulfamethoxazole have increased the plasma concentrations or effects of drugs such as tolbutamide, phenytoin, warfarin, and glipizide, resulting in clinically significant drug-drug interactions (Hansen et al., 1979; O’Reilly, 1980; Wing and Miners, 1985; Johnson and Dobmeier, 1990). It has been suggested that inhibition of oxidative drug metabolism by trimethoprim and sulfamethoxazole is the likely mechanism of these drug-drug interactions (Wing and Miners, 1985). In previous in vitro studies, sulfamethoxazole has been shown to inhibit tolbutamide hydroxylation (a CYP2C9 marker reaction) with an apparent Ki value of about 250 M (Back et al., 1988; Komatsu et al., 2000a). However, it seems that there are no published in vitro studies investigating the effects of trimethoprim and sulfamethoxazole on different P450 isoforms. We have studied the inhibitory effect of trimethoprim and sulfamethoxazole on major P450 isoform activities in human liver microsomes and recombinant P450s using selective marker reactions. Experimental Procedures Materials. Dextromethorphan and dextrorphan were obtained from Orion Pharma (Espoo, Finland). Sulfamethoxazole, trimethoprim, phenacetin, paracetamol, coumarin, 7-hydroxycoumarin, tolbutamide, chlorzoxazone, paclitaxel, testosterone, and NADPH were purchased from Sigma-Aldrich (St. Louis, MO). Hydroxytolbutamide, 6-hydroxychlorzoxazone, S-mephenytoin, 4 -hydroxymephenytoin, 6 -hydroxytestosterone, and 6 -hydroxypaclitaxel were purchased from Ultrafine Chemicals (Manchester, UK). Midazolam and 1 -hydroxymidazolam were kindly provided by F. 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, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, and CYP3A4 were obtained from Gentest Corp. (Woburn, MA). Microsomes from baculovirus-infected cells engineered to express the cDNA encoding human CYP2C8 and CYP2C9 were also purchased from Gentest Corp. Other chemicals and reagents were obtained from Merck (Darmstadt, Germany). Inhibition Studies. The effects of trimethoprim and sulfamethoxazole on eight different P450 isoform-specific marker reactions were studied. Phenacetin O-deethylation was used to probe for CYP1A2, coumarin 7-hydroxylation for CYP2A6, paclitaxel 6 -hydroxylation for CYP2C8, tolbutamide hydroxylation for CYP2C9, S-mephenytoin 4 -hydroxylation for CYP2C19, dextromethorphan O-demethylation for CYP2D6, chlorzoxazone 6-hydroxylation for CYP2E1, and midazolam 1 -hydroxylation and testosterone 6 -hydroxylation This study was supported by grants from the Helsinki University Central Hospital Research Fund and the National Technology Agency of Finland (Tekes), Finland. 1 Abbreviations used are: P450, cytochrome P-450; HPLC, high-performance liquid chromatography. Address correspondence to: Dr. Janne T. Backman, M.D., Department of Clinical Pharmacology, University of Helsinki, Haartmaninkatu 4, FIN-00290 Helsinki, Finland. E-mail: janne.backman@hus.fi 0090-9556/02/3006-631–635$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 6 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 646/982016 DMD 30:631–635, 2002 Printed in U.S.A. 631 at A PE T Jornals on M ay 0, 2017 dm d.aspurnals.org D ow nladed from for CYP3A4. All incubations were performed in duplicate, and the mean values were used. Briefly, each incubation was performed with 20 g human liver microsomes or recombinant P450 isoforms in a final incubation volume of 0.2 ml, after diluting from their original concentrations (20 mg/ml, 3 mg/ml, 2.1 mg/ml for human liver microsomes, recombinant CYP2C8, and CYP2C9, respectively). The incubation medium contained 0.1 M sodium phosphate buffer (pH 7.4) and 5 mM MgCl2. To determine whether the inhibition of P450 isoforms by trimethoprim and sulfamethoxazole could be mechanism-based, trimethoprim (dissolved in 2 l of methanol, final concentration 5–500 M) and sulfamethoxazole (dissolved in 2 l of methanol, final concentration 50–1000 M) were preincubated with the incubation medium at 37°C for 15 min, either in the presence or absence of 1.0 mM NADPH. An equal volume (2 l) of methanol was added to the noninhibitor controls. After the preincubation, probe substrates were added either with or without 1.0 mM NADPH for measurement of the corresponding marker activities. Testosterone (dissolved in 2 l of methanol, final concentration 25 M) and paclitaxel (dissolved in 2 l of methanol, final concentration 1–5 M) were incubated with the incubation medium for 6 and 20 min, respectively. Acetonitrile (100 l) was used to terminate the reactions. For the other reactions, the incubation conditions including solvents, incubation times, quenching methods, and the effects of specific inhibitors have been reported elsewhere (Wen et al., 2001). The time of incubation and concentration of microsomes (100 g/ml) used in each assay were determined to be in the linear range for the rate of metabolite formation. After incubation at 37°C for a specific period of time, the reaction was quenched by adding an appropriate chemical to precipitate the proteins. The incubation mixtures were then centrifuged for 5 min at 10,000g. An aliquot of the supernatant fraction was subjected to analysis using highperformance liquid chromatography (HPLC). Incubations with the recombinant CYP2C8 and CYP2C9 isoforms were performed using the same conditions as the incubations with human liver microsomes, except that the incubation mixture contained 100 g/ml of CYP2C8 and CYP2C9 supersomes and was incubated for 20 min (CYP2C8) and 30 min (CYP2C9), respectively. HPLC Analysis. Assays for the respective products of P450 marker reactions were carried out using HPLC (Stewart and Carter, 1986; Harris et al., 1994; Wang et al., 2000; Wen et al., 2001). The HPLC system consisted of a Pharmacia LKB 2150 pump (LKB, Uppsala, Sweden), a Hewlett Packard 1050 autosampler (Hewlett Packard, Mississauga, ON), a Hewlett-Packard 3396 integrator (Hewlett Packard), a SPD-10AV Shimadzu UV detector (Shimadzu, Kyoto, Japan; for analysis of CYP1A2, CYP2C8, CYP2C9, CYP2C19, CYP2E1, and CYP3A4 activities), a RF-551 Shimadzu fluorescence detector (Shimadzu; for analysis of CYP2A6 and CYP2D6 activities) or model 5100A Coulochem electrochemical detector (ESA Inc., Bedford, MA; for analysis of the inhibitory effect of sulfamethoxazole on CYP2E1 activity). The intraday and interday coefficients of variation for all assays were less than 7% at relevant concentrations (n 6). Data Analysis. The IC50 values (concentration of inhibitor to cause 50% inhibition of original enzyme activity) were determined graphically. The apparent inhibitory constant (Ki) values were calculated by nonlinear regression analysis using Systat for Windows 6.0.1 (SPSS Inc., Chicago, IL). Different models of enzyme inhibition (i.e., competitive, noncompetitive, uncompetitive, and mixed-type inhibition) were fitted to the kinetic data (Segel, 1975). An assessment of goodness of fit of the models was made using the size of the residual sum of squares and the random distribution of the residuals, the standard error, and the 95% confidence interval of the parameter estimates.