* Your assessment is very important for improving the workof artificial intelligence, which forms the content of this project
Download trimethoprim and sulfamethoxazole are selective inhibitors of cyp2c8
Drug discovery wikipedia , lookup
Plateau principle wikipedia , lookup
Environmental impact of pharmaceuticals and personal care products wikipedia , lookup
Development of analogs of thalidomide wikipedia , lookup
Discovery and development of proton pump inhibitors wikipedia , lookup
Toxicodynamics wikipedia , lookup
Neuropsychopharmacology wikipedia , lookup
Environmental persistent pharmaceutical pollutant wikipedia , lookup
Metalloprotease inhibitor wikipedia , lookup
Wilson's disease wikipedia , lookup
Discovery and development of integrase inhibitors wikipedia , lookup
Pharmacognosy wikipedia , lookup
Theralizumab wikipedia , lookup
Discovery and development of neuraminidase inhibitors wikipedia , lookup
Discovery and development of cyclooxygenase 2 inhibitors wikipedia , lookup
Pharmacokinetics wikipedia , lookup
Drug interaction wikipedia , lookup
Discovery and development of ACE inhibitors wikipedia , lookup
0090-9556/02/3006-631–635$7.00 DRUG METABOLISM AND DISPOSITION Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics DMD 30:631–635, 2002 Vol. 30, No. 6 646/982016 Printed in U.S.A. TRIMETHOPRIM AND SULFAMETHOXAZOLE ARE SELECTIVE INHIBITORS OF CYP2C8 AND CYP2C9, RESPECTIVELY XIA WEN, JUN-SHENG WANG, JANNE T. BACKMAN, JOUKO LAITILA, AND PERTTI J. NEUVONEN Department of Clinical Pharmacology, University of Helsinki and Helsinki University Central Hospital, Helsinki, Finland (Received December 5, 2001; accepted February 19, 2002) This article is available online at http://dmd.aspetjournals.org ABSTRACT: 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 (P4501) 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 This study was supported by grants from the Helsinki University Central Corp. Other chemicals and reagents were obtained from Merck (Darmstadt, Hospital Research Fund and the National Technology Agency of Finland (Tekes), Germany). Finland. Inhibition Studies. The effects of trimethoprim and sulfamethoxazole on 1 Abbreviations used are: P450, cytochrome P-450; HPLC, high-performance eight different P450 isoform-specific marker reactions were studied. Phenacliquid chromatography. etin O-deethylation was used to probe for CYP1A2, coumarin 7-hydroxylation for CYP2A6, paclitaxel 6␣-hydroxylation for CYP2C8, tolbutamide hydroxyAddress correspondence to: Dr. Janne T. Backman, M.D., Department of lation for CYP2C9, S-mephenytoin 4⬘-hydroxylation for CYP2C19, dextroClinical Pharmacology, University of Helsinki, Haartmaninkatu 4, FIN-00290 Helmethorphan O-demethylation for CYP2D6, chlorzoxazone 6-hydroxylation for sinki, Finland. E-mail: [email protected] CYP2E1, and midazolam 1⬘-hydroxylation and testosterone 6-hydroxylation 631 Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017 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] ⴙ 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 632 WEN ET AL. Results With concentrations ranging from 5 to 100 M, trimethoprim exhibited a selective inhibitory effect on CYP2C8-mediated paclitaxel 6␣-hydroxylation with an apparent IC50 (Ki) value of 54 M (32 M) in human liver microsomes and an IC50 of 75 M in recombinant CYP2C8 (Fig. 1, Table 1). The pattern of inhibition was competitive (Table 1). However, trimethoprim lost its isoform selectivity at concentrations higher than 100 M (Fig. 2). As much as 20 to 50% of CYP1A2-, CYP2C9-, CYP2C19-, CYP2D6- and CYP3A4- mediated activities were inhibited at concentrations of 250 and 500 M (Fig. 2). Sulfamethoxazole selectively and competitively inhibited tolbutamide hydroxylase activity with concentrations ranging from 50 to 500 M in human liver microsomes and recombinant CYP2C9, with apparent IC50 (Ki) values of 544 M (271 M) and 456 M, respectively (Fig. 1; Table 1). Very little (⬍20%) or no inhibition of other P450 isoforms was found in this concentration range. However, at concentrations higher than 500 M, sulfamethoxazole showed a modest (30 – 40%) inhibitory effect on CYP2A6-mediated coumarin 7-hydroxylation and CYP3A4-mediated midazolam 1⬘-hydroxylation (Fig. 2). Preincubation of trimethoprim and sulfamethoxazole with NADPH for 15 min prior to the addition of the specific substrates did not increase the degree of inhibition (data not shown). Discussion The results of the present study indicate that trimethoprim and sulfamethoxazole are selective inhibitors of CYP2C8 (Ki ⫽ 32 M) and CYP2C9 (Ki ⫽ 271 M), respectively, in human liver microsomes at concentrations ranging from 5 to 100 M trimethoprim and 50 to 500 M sulfamethoxazole. With concentrations higher than 100 M trimethoprim and 500 M sulfamethoxazole, both drugs lost their selectivity toward the P450 isoforms and became inhibitors of several P450 isoforms. The results are in agreement with previous in vitro studies showing that sulfamethoxazole competitively inhibited tolbutamide hydroxylase activity, with an apparent Ki value of 246 M (Back et al., 1988) or 283 M (Komatsu et al., 2000a) in human liver microsomes. Theoretically, drug-drug interactions based on inhibition of hepatic drug metabolism (i) can be predicted by the Ki value and the concentration of the inhibitor [I] around the metabolic enzyme in the liver using the following scaling model: i ⫽ [I]/([I] ⫹ Ki), assuming that the substrate concentration is much lower than its Km value (von Moltke et al., 1998). After oral administration of 200 mg trimethoprim and 800 mg sulfamethoxazole twice daily, the mean peak plasma concentrations of trimethoprim and sulfamethoxazole were approximately 20 and 250 M, respectively (Moore et al., 1996; Dollery, 1999). Although the exact liver/plasma partition ratios of trimethoprim and sulfamethoxazole in humans are unknown, animal experiments in monkeys have shown that the liver/plasma partition ratios are about 6.5 for trimethoprim and 0.15 for sulfamethoxazole (Craig and Kunin, 1973), which agree well with the relatively small volume of distribution of sulfamethoxazole (about 0.2 l/kg) in humans (Dollery, 1999). Accordingly, it can be estimated that the total liver concentrations of trimethoprim and sulfamethoxazole are around 130 and 40 M, respectively. Consequently, based on these total hepatic concentrations, one would expect approximately 80 and 13% inhibition of the metabolic clearance of CYP2C8 and CYP2C9 substrates by trimethoprim and sulfamethoxazole, respectively (Table 1). Alternatively, assuming that 55% of trimethoprim and 34% of sulfamethoxazole are unbound in plasma (Dollery, 1999) and that equal unbound concentrations are found in plasma and at the enzyme site, approximately 26 and 24% inhibition of CYP2C8 and CYP2C9 by trimethoprim and sulfamethoxazole would be expected, respectively. CYP2C8 is primarily responsible for the metabolism of e.g., taxol, cerivastatin, rosiglitazone and troglitazone and also involved in the metabolism of zopiclone, carbamazepine, verapamil, and amiodarone (Ohyama et al., 2000; Ong et al., 2000). However, most previous clinical drug-drug interaction studies involving trimethoprim have focused on substrates of CYP2C9. For example, trimethoprim used alone inhibited the metabolic clearance of tolbutamide (14%) and phenytoin (30%) (Hansen et al., 1979; Wing and Miners, 1985). As can be seen from Figs. 1 and 2, CYP2C8 is much more susceptible to Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017 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. TRIMETHOPRIM AND SULFAMETHOXAZOLE INHIBIT CYP2C8 AND CYP2C9 633 Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017 FIG. 1. Inhibitory effect of trimethoprim and sulfamethoxazole on P450-catalyzed reactions in human liver microsomes (A, B) and in recombinant CYP2C8 and CYP2C9 (C, D). Trimethoprim and sulfamethoxazole were incubated using conditions described under Experimental Procedures. The enzyme reactions evaluated were CYP1A2catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2C8-catalyzed paclitaxel 6␣-hydroxylation, CYP2C9-catalyzed tolbutamide hydroxylation, CYP2C19-catalyzed S-mephenytoin 4⬘-hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, CYP2E1-catalyzed chlorzoxazone 6-hydroxylation, CYP3A4-catalyzed midazolam 1⬘-hydroxylation, and testosterone 6-hydroxylation. Phenacetin, coumarin, paclitaxel, tolbutamide, S-mephenytoin, dextromethorphan, chlorzoxazone, midazolam, and testosterone were used at concentrations around their corresponding Km values (i.e., 50, 1, 5, 50, 40, 1.5, 25, 2, and 25 M, respectively). Each data point represents an average of duplicates. 634 WEN ET AL. TABLE 1 Inhibition of CYP2C8 and CYP2C9 isoforms by trimethoprim and sulfamethoxazole in human liver microsomes and recombinant P450 isoforms and the predicted in vivo inhibition of the metabolism of coadministered CYP2C8 and CYP2C9 substrates by trimethoprim and sulfamethoxazole, respectively, from in vitro data P450 Isoform Human Liver Microsomes Ki (IC50)a, Type of Inhibition CYP2C8 CYP2C9 32 (54), competitive 271 (544), competitive Recombinant P450 Isoforms IC50a Predicted in vivo Inhibition I/(I ⫹ Ki)b 75 456 80 (26) 13 (24) M Trimethoprim Sulfamethoxazole % a Ki values are derived using nonlinear regression analysis; IC50 are calculated graphically (see Experimental Procedures for details). Inhibitor concentration (I) of trimethoprim (130 M) and sulfamethoxazole (40 M) around the metabolic enzymes were calculated using the liver/plasma partition ratios of 6.5 and 0.15 for trimethoprim and sulfamethoxazole (Craig and Kunin, 1973), respectively. The plasma concentrations of trimethoprim (20 M) and sulfamethoxazole (250 M) were adopted from Moore et al. (1996) and Dollery (1999). Data in the parentheses were calculated using unbound plasma concentrations of trimethoprim (11 M) and sulfamethoxazole (85 M), assuming that 55% of trimethoprim and 34% of sulfamethoxazole are unbound in plasma (Dollery, 1999) and that equal unbound concentrations are found in plasma and at the enzyme site. b Each data point represents an average of duplicates. the inhibitory effect of trimethoprim than CYP2C9. Although tolbutamide and phenytoin are metabolized primarily by CYP2C9, they are metabolized to a minor extent also by CYP2C8 (Komatsu et al., 2000a,b). Therefore, inhibition of CYP2C8 by trimethoprim may explain the reported modest reduction in the metabolic clearance of tolbutamide and phenytoin. Trimethoprim at very high concentrations (i.e., 250 M and 500 M) inhibited 30 and 40% of CYP2C9 activity, respectively. Thus, because trimethoprim is distributed extensively into tissues such as liver, there is a possibility that trimethoprim may also slightly reduce the activity of hepatic CYP2C9. Consistent with our prediction, sulfamethoxazole used alone has modestly inhibited the metabolic clearance of the CYP2C9 substrates tolbutamide (14%) and phenytoin (10%) (Hansen et al., 1979; Wing and Miners, 1985). When coadministered with trimethoprim, sulfamethoxazole increased the area under the plasma concentration-time curve of S-warfarin, another CYP2C9 sub- strate, by 20% (O’Reilly, 1980). When combined with trimethoprim, sulfamethoxazole inhibited the metabolic clearance of tolbutamide more than when trimethoprim was used alone (25 versus 14%) (Wing and Miners, 1985). With the negligible inhibitory effects on the other P450 isoforms tested, trimethoprim and sulfamethoxazole with normal therapeutic doses are unlikely to produce clinically relevant interactions by inhibiting these P450 isoforms. In line with these findings, trimethoprim/sulfamethoxazole had no significant effects on the pharmacokinetics of theophylline (a CYP1A2 substrate) and nifedipine (a CYP3A4 substrate) (Jonkman et al., 1985; Edwards et al., 1990). No chemical has been previously identified as a selective inhibitor of CYP2C8 (Ong et al., 2000). Quercetin has been used as an inhibitor of CYP2C8, but it is also a potent inhibitor of CYP1A2 (Dierks et al., 2001). Also sulfaphenazole, a commonly used potent inhibitor of CYP2C9 (Ki ⫽ 0.3 M), has some inhibitory effects toward the other Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017 FIG. 2. Inhibitory effects of high concentrations of trimethoprim (A) and sulfamethoxazole (B) on CYP1A2-catalyzed phenacetin O-deethylation, CYP2A6-catalyzed coumarin 7-hydroxylation, CYP2C8-catalyzed paclitaxel 6␣-hydroxylation, CYP2C9-catalyzed tolbutamide hydroxylation, CYP2C19-catalyzed S-mephenytoin 4⬘hydroxylation, CYP2D6-catalyzed dextromethorphan O-demethylation, CYP2E1-catalyzed chlorzoxazone 6-hydroxylation, CYP3A4-catalyzed midazolam 1⬘hydroxylation, and testosterone 6-hydroxylation in human liver microsomes. TRIMETHOPRIM AND SULFAMETHOXAZOLE INHIBIT CYP2C8 AND CYP2C9 Acknowledgments. We thank Lisbet Partanen for skillful technical assistance. References Back DJ, Tjia JF, Karbwang J, and Colbert J (1988) In vitro inhibition studies of tolbutamide hydroxylase activity of human liver microsomes by azoles, sulphonamides and quinolines. Br J Clin Pharmacol 26:23–29. Craig WA and Kunin CM (1973) Distribution of trimethoprim-sulfamethoxazole in tissues of rhesus monkeys. J Infect Dis 128 (Suppl):575–579. Cribb AE, Spielberg SP, and Griffin GP (1995) N4-hydroxylation of sulfamethoxazole by cytochrome P450 of the cytochrome P4502C subfamily and reduction of sulfamethoxazole hydroxylamine in human and rat hepatic microsomes. Drug Metab Dispos 23:406 – 414. Dierks EA, Stams KR, Lim HK, Cornelius G, Zhang H, and Ball SE (2001) A method for the simultaneous evaluation of the activities of seven major human drug-metabolizing cytochrome P450s using an in vitro cocktail of probe substrates and fast gradient liquid chromatography tandem mass spectrometry. Drug Metab Dispos 29:23–29. Dollery C (1999) Sulfamethoxazole and trimethoprim combination, in Therapeutic Drugs (Dollery C ed) pp S136 –S140, Churchill Livingstone, Edinburgh. Edwards C, Monkman S, Cholerton S, Rawlins MD, Idle JR, and Ferner RE (1990) Lack of effect of co-trimoxazole on the pharmacokinetics and pharmacodynamics of nifedipine. Br J Clin Pharmacol 30:889 – 891. Gleckman R, Blagg N, and Joubert DW (1981) Trimethoprim: mechanisms of action, antimicrobial activity, bacterial resistance, pharmacokinetics, adverse reactions, and therapeutic indications. Pharmacotherapy 1:14 –20. Hansen JM, Kampmann JP, Siersbaek-Nielsen K, Lumholtz IB, Arroe M, Abildgaard U, and Skovsted L (1979) The effect of different sulfonamides on phenytoin metabolism in man. Acta Med Scand Suppl 624:106 –110. Harris JW, Rahman A, Kim BR, Guengerich FP, and Collins JM (1994) Metabolism of taxol by human hepatic microsomes and liver slices: participation of cytochrome P450 3A4 and an unknown P450 enzyme. Cancer Res 54:4026 – 4035. Johnson JF and Dobmeier ME (1990) Symptomatic hypoglycemia secondary to a glipizidetrimethoprim/sulfamethoxazole drug interaction. DICP-Ann Pharmacother 24:250 –251. Jonkman JH, Van der Boon WJ, Schoenmaker R, Holtkamp AH, and Hempenius J (1985) Lack of influence of co-trimoxazole on theophylline pharmacokinetics. J Pharm Sci 74:1103–1104. Komatsu K, Ito K, Nakajima Y, Kanamitsu Si, Imaoka S, Funae Y, Green CE, Tyson CA, Shimada N, Sugiyama Y (2000a) Prediction of in vivo drug-drug interactions between tolbutamide and various sulfonamides in humans based on in vitro experiments. Drug Metab Dispos 28:475– 481. Komatsu T, Yamazaki H, Asahi S, Gillam EM, Guengerich FP, Nakajima M, and Yokoi T (2000b) Formation of a dihydroxy metabolite of phenytoin in human liver microsomes/ cytosol: roles of cytochromes P450 2C9, 2C19, and 3A4. Drug Metab Dispos 28:1361–1368. Mancy A, Dijols S, Poli S, Guengerich P, Mansuy D (1996) Interaction of sulfaphenazole derivatives with human liver cytochromes P450 2C: molecular origin of the specific inhibitory effects of sulfaphenazole on CYP 2C9 and consequences for the substrate binding site topology of CYP 2C9. Biochemistry 35:16205–16212. Moore KH, Yuen GJ, Raasch RH, Eron JJ, Martin D, Mydlow PK, and Hussey EK (1996) Pharmacokinetics of lamivudine administered alone and with trimethoprim-sulfamethoxazole. Clin Pharmacol Ther 59:550 –558. Ohyama K, Nakajima M, Nakamura S, Shimada N, Yamazaki H, and Yokoi T (2000) A significant role of human cytochrome P450 2C8 in amiodarone N-deethylation: an approach to predict the contribution with relative activity factor. Drug Metab Dispos 28:1303–1310. Ong CE, Coulter S, Birkett DJ, Bhasker CR, and Miners JO (2000) The xenobiotic inhibitor profile of cytochrome P4502C8. Br J Clin Pharmacol 50:573–580. O’Reilly RA (1980) Stereoselective interaction of trimethoprim-sulfamethoxazole with the separated enantiomorphs of racemic warfarin in man. N Engl J Med 302:33–35. Rieder VJ and Schwartz DE (1975) Comparison of pharmacokinetics of the combination trimethoprim and sulfamethoxazole in patients with liver diseases and healthy persons. Arzneim-Forsch 25:656 – 666. Segel RH (1975) Simple inhibition system, in Enzyme Kinetics (Segel IH ed) pp100 –160, John Wiley & Sons, New York. Stewart JT and Carter HK (1986) High-performance liquid chromatography with electrochemical detection of chlorzoxazone and its hydroxy metabolite in serum using solid-phase extraction. J Chromatogr 380:177–183. von Moltke LL, Greenblatt DJ, Schmider J, Wright CE, Harmatz JS, and Shader RI (1998) In vitro approaches to predicting drug interactions in vivo. Biochem Pharmacol 55:113–122. Wang RW, Newton DJ, Liu N, Atkins WM, and Lu AY (2000) Human cytochrome P-450 3A4 in vitro drug-drug interaction patterns are substrate-dependent. Drug Metab Dispos 28:360 – 366. Wen X, Wang JS, Kivistö KT, Neuvonen PJ, and Backman JT (2001) In vitro evaluation of valproic acid as an inhibitor of human cytochrome P450 isoforms: preferential inhibition of cytochrome P4502C9 (CYP2C9). Br J Clin Pharmacol 52:547–553. Wing LM and Miners JO (1985) Cotrimoxazole as an inhibitor of oxidative drug metabolism: effects of trimethoprim and sulphamethoxazole separately and combined on tolbutamide disposition. Br J Clin Pharmacol 20:482– 485. Downloaded from dmd.aspetjournals.org at ASPET Journals on May 11, 2017 CYP2C isoforms, [e.g., CYP2C8 (Ki ⫽ 63 M) and CYP2C18 (Ki ⫽ 29 M)] (Mancy et al., 1996). Our results indicate that when used at concentrations lower than 100 M (trimethoprim) and 500 M (sulfamethoxazole), trimethoprim and sulfamethoxazole can be used as selective inhibitors of CYP2C8 and CYP2C9, respectively, in in vitro studies. In addition, even with a concentration reaching 1000 M, sulfamethoxazole is a very selective inhibitor of CYP2C9 among the CYP2C isoforms. However, the inhibitor concentration must be selected carefully. As noted above, at the concentration of 500 M, trimethoprim inhibited CYP1A2, 2C9, 2C19, 2D6, and 3A4 activities by about 25, 40, 50, 50, and 50%, respectively. Similarly, 1000 M sulfamethoxazole reduced the activities of CYP2A6 and 3A4 by 45 and 40%, respectively. In conclusion, our study demonstrated that trimethoprim (5–100 M) and sulfamethoxazole (50 –500 M) are selective inhibitors of CYP2C8 and CYP2C9 activities, respectively. At clinically relevant concentrations, trimethoprim strongly inhibits CYP2C8 activity, and sulfamethoxazole moderately inhibits CYP2C9 activity. The inhibition of CYP2C8 activity by trimethoprim and CYP2C9 by sulfamethoxazole may be the mechanisms involved in the drug-drug interactions between trimethoprim/sulfamethoxazole, and tolbutamide, phenytoin, warfarin, and glipizide. 635