Download trimethoprim and sulfamethoxazole are selective inhibitors of cyp2c8

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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
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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
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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
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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
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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.
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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
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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.
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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.
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