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Antimicrobial Agents and Chemotherapy, June 2006, p. 1967-1972, Vol. 50, No. 6
0066-4804/06/$08.00+0 doi:10.1128/AAC.01483-05
Copyright © 2006, American Society for Microbiology. All Rights Reserved.
Department of Anesthesiology and Intensive Care, Turku University Hospital, Turku, Finland,1 Department of Pharmacology and Clinical Pharmacology, University of Turku, Turku University Hospital, Turku, Finland,2 Department of Medical Laboratory Sciences and Technology, Division of Clinical Pharmacology at Karolinska Institute, Huddinge University Hospital, Stockholm, Sweden,3 Department of Clinical Pharmacology, University of Helsinki, and Helsinki University Central Hospital, Helsinki, Finland4
Received 17 November 2005/ Returned for modification 29 January 2006/ Accepted 17 March 2006
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ABSTRACT |
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INTRODUCTION |
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Fluconazole is another azole antifungal agent and a well-documented potent inhibitor of CYP2C9-catalyzed reactions both in vitro (3, 20) and in vivo (4, 18) and a weaker inhibitor of CYP3A4-catalyzed reactions (23). It has also been found to inhibit CYP2C19-catalyzed reactions both in vitro (34) and in vivo (17). However, there are few data on the possible interactions between fluconazole and nonsteroidal anti-inflammatory drugs (NSAIDs). Treatment with fluconazole significantly increased the AUC of the CYP2C9 substrate celecoxib (7), and recently, it has been shown that coadministration of fluconazole and a novel cyclooxygenase 2 (COX-2)-selective inhibitor, lumiracoxib, metabolized by CYP2C9, caused a small (18%) increase in the mean AUC of lumiracoxib (32).
Ibuprofen is a chiral NSAID widely used worldwide, and it is available as an over-the-counter remedy in many countries. Ibuprofen is a nonselective inhibitor of COX-1 and COX-2. Most preparations contain the racemic mixture of R-(–)- and S-(+)-ibuprofen, with the S-(+) enantiomer of ibuprofen possessing the majority of pharmacological activity (1, 9). After administration of racemic ibuprofen, about 60% of R-(–)-ibuprofen is inverted to the S-(+)-enantiomer in the human body, but there is no measurable inversion in the other direction (2, 16). The major pathway for ibuprofen elimination is oxidative metabolism to hydroxylated metabolites. In vitro studies have indicated the stereoselectivity of ibuprofen hydroxylation, with CYP2C9 being the main enzyme involved in the hydroxylation of S-(+)-ibuprofen and CYP2C8 being the main enzyme involved in the hydroxylation of R-(–)-ibuprofen (12).
Both CYP2C9 and CYP2C8 exhibit genetic polymorphism, with interethnic differences in the frequency of mutant alleles. The frequencies of mutant alleles CYP2C9*2 and CYP2C9*3 have been reported to range from 8 to 19% and from 3.3 to 16.2% in Caucasian populations, respectively (35), and the mutant allele CYP2C8*3 has an 8.9 to 13% allelic frequency in Caucasians (6, 24). It has been found that the mean S-(+)-ibuprofen clearance was significantly decreased in individuals who are hetero- or homozygous for CYP2C9*3 compared with that in individuals with the wild-type genotype, whereas the CYP2C9*2 variant had no significant effect on S-(+)-ibuprofen clearance (19). In addition, CYP2C9 polymorphism had no significant influence on R-(–)-ibuprofen metabolism.
Although ibuprofen is considered a well-tolerated drug, it is not free of adverse events. Gastritis, peptic ulcer, minor or major upper intestinal bleeding, analgesic nephropathy, and fluid retention are typical concentration-dependent adverse effects of ibuprofen. The coadministration of voriconazole or fluconazole and ibuprofen may lead to increased levels of ibuprofen and increase the risk for ibuprofen-related adverse events. However, no clinical studies have addressed the effect of either voriconazole or fluconazole on the pharmacokinetics of ibuprofen. Therefore, the aim of this study was to clarify the possible interaction between these azole antifungals and ibuprofen.
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MATERIALS AND METHODS |
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Protocol. This study had an open, randomized, crossover design with three phases, with a washout period of 2 weeks between the phases. In the control phase, the subjects received a single 400-mg oral dose of racemic ibuprofen (Burana; Orion, Helsinki, Finland) with no pretreatment. In the second and third phases, the subjects ingested as pretreatment either fluconazole (Fluconazol ratiopharm; Merkle GmbH, Blauburen, Germany) at 400 mg as a single daily dose on the first day and 200 mg on the second day or voriconazole (Vfend; Pfizer, Sandwich, United Kingdom) at 400 mg twice daily on the first day and 200 mg twice daily on the second day. The oral dose of 400 mg racemic ibuprofen was given 1 h after the last dose of voriconazole or fluconazole at 9 a.m. with 150 ml of water.
To determine the plasma concentrations of S-(+)- and R-(–)-ibuprofen, a 10-ml blood sample was drawn via a venous forearm cannula before and at 0.5, 1, 1.5, 2, 2.5, 3, 4, 6, 8, 12, and 24 h after the ibuprofen dose. The plasma was separated and stored at –70°C until the S-(+)- and R-(–)-ibuprofen concentrations were analyzed by high-performance liquid chromatography (HPLC).
During all phases, the subjects fasted overnight before administration of ibuprofen and continued fasting until a standardized lunch was served 4 h after ibuprofen ingestion. The subjects did not use any other medication or natural products for 14 days before and during the study and no drug known to cause enzyme induction or inhibition for a period of 30 days prior the study. Caffeine, grapefruit juice, and alcohol-containing beverages were not allowed during the study. All fluconazole and voriconazole doses except for the last doses were self-administered by the subjects; the last doses were administered by the study personnel. The intake of the premedication by the subjects was verified by having the subjects send a short message to the investigators by mobile phone right after they took each dose of voriconazole or fluconazole. Compliance was also ascertained by quantifying plasma voriconazole and fluconazole levels on the study days before the last administration of these drugs.
Genotyping. Genotyping for CYP2C9*2, CYP2C9*3, and CYP2C8*3 was performed by a 5'-nuclease assay. For CYP2C9*2, primers CAA TGG AAA GAA ATG GAA GGA GAT and AAG ATA GTA GTC CAG TAA GGT CAG TGA TAT G (Cybergene AB, Novum Research Park, Sweden) were used with minor grove binding dark quencher MGB probes VIC-TTG AAC ACA GTC CTC A-MGB and FAM-TTG AAC ACG GTC CTC-MGB (Applied Biosystems, Cheshire, United Kingdom), where boldface indicates the specific places of the probes and VIC and FAM are the fluorescent dyes used to differentiate each probe. For CYP2C9*3, primers CAG GAA GAG ATT GAA CGT GTG ATT and CTA TGA ATT TGG GGA CTT CGA AA and probes FAM-AGA TAC CTT GAC CTT CT-MGB and VIC-AGA GAT ACA TTG ACC TTC-MGB were used. For CYP2C8*3, the primers were ATG TCC ACT ACT TCT CCT CAC TTC TG and AAA GTG GCC AGG GTC AAA GA, and the probes were FAM-TG ATG ACA GAG AAT TT and VIC-ATG ATG ACA AAG AAT TT. Each reaction used the primers at a final concentration of 450 nM and the probes at a final concentration of 75 nM. The reaction volume used was 25 µl, and the PCR setting was 50°C for 10 min and 95°C for 10 min, followed by 35 times cycling of 95°C for 15 s, after which the PCR setting was 60°C for 1 min. Each sample was run in duplicate.
Bioanalysis of ibuprofen. The plasma concentrations of R-(–)- and S-(+)-ibuprofen were determined by HPLC with UV detection at 220 nm (22, 26). Briefly, R-(–)- and S-(+)-ibuprofen and the internal standard, fenoprofen, were extracted from plasma with ethyl acetate. After evaporation, the residue was reconstituted with the mobile phase and the supernatant was used for injection. The mobile phase was a mixture of 2-propanol (0.25%) and 50 mM sodium hydrogen phosphate buffer (pH 7.0; 99.75%). Chromatographic analysis was performed on a Chiral AGP analytical column (150 by 4 mm [inner diameter]; Chrom Tech Ltd., Congleton, United Kingdom) protected with a Chiral AGP Guard column (10 by 4 mm [inner diameter]; Chrom Tech Ltd.). Because a voriconazole metabolite interfered with the HPLC analysis of R-(–)-ibuprofen, the concentrations of R-(–)-ibuprofen during the voriconazole phase were quantified with a liquid chromatography-tandem mass spectrometry (LC/MS/MS) system (MDS SCIEX; Q Trap LC/MS/MS system; Applied BioSystems, Foster City, CA) by using a mixture of 2-propanol (0.25%) and 10 mM ammonium acetate (pH 6.5; 99.75%) as the mobile phase. The mass spectrometer was operated in the atmospheric pressure chemical ionization mode with negative ion detection. The ion transitions monitored were a mass-to-charge ratio (m/z) 204.9 to m/z 161.2 for R-(–)- and S-(+)-ibuprofen and m/z 241.0 to m/z 93.1 for the internal standard, fenoprofen. These transitions represent the product ion of the [M-H]– ion. The day-to-day coefficients of variation (CVs) were less than 12% for both enantiomers at concentrations of 500 ng/ml, 5,000 ng/ml, and 25,000 ng/ml (n = 11 for the HPLC method; n = 5 for the LC/MS/MS method). The limit of quantification for R-(–)- and S-(+)-ibuprofen was 250 ng/ml. The HPLC and LC/MS/MS methods used gave identical results at the relevant concentrations for both enantiomers during all other study phases except during the voriconazole phase for R-(–)-ibuprofen.
Bioanalysis of voriconazole and fluconazole. After a solid-phase extraction of voriconazole from plasma, its concentration was determined by HPLC with UV detection at 255 nm by using a fluconazole analog (UK 54373) as the internal standard (10, 25). The limit of voriconazole quantification was 0.05 mg/liter, and the CV was less than 4% at the relevant concentrations (0.05 mg/liter, 1.0 mg/liter, and 10.0 mg/liter; n = 7). The plasma fluconazole concentrations were determined after a solid-phase extraction by HPLC with UV detection at 210 nm by using UK 54373 as the internal standard (15). The limit of fluconazole quantification was 0.2 mg/liter. The CV was less than 2% at the relevant concentrations (3 mg/liter and18 mg/liter; n = 6 to 7).
Data analysis. The pharmacokinetic parameters for S-(+)- and R-(–)-ibuprofen were calculated by standard noncompartmental methods (WinNonlin; Pharsight Co., Mountain View, CA). The maximum plasma concentration (Cmax) and the time to Cmax (Tmax) for each subject were derived directly from the plasma concentration data. The AUC was calculated from time zero to infinity by use of the linear trapezoidal rule. The elimination half-life (t1/2) was calculated by least-squares regression analysis of the terminal linear part of the log concentration-time curve. The absolute changes in the pharmacokinetic parameters were tested by use of the analysis of variance model for repeated measurements (SPSS 11.0 for Windows 2001; SPSS Inc., Chicago, IL), and Tukey's test was used for post-hoc testing. Tmax was analyzed by Friedman's test, and the Wilcoxon signed rank test was used for pairwise comparisons. The correlation between the voriconazole and the fluconazole trough concentrations and the change in the S-(+)-ibuprofen AUC were tested by the Pearson correlation test when the data were normally distributed, and the Spearman rank test was used for nonnormally distributed data. The chosen statistical significance level was a P value of <0.05. The results are presented as means ± standard deviations in the text and Table 1 and for clarity are presented as the standard error of the mean in Fig. 1. For Tmax, the median with the range is shown. The percent differences between treatments were calculated within subjects. Since pharmacokinetic drug interactions can also be assessed statistically by the same methods that are standard for investigation of bioequivalence, we calculated the 90% confidence intervals (CIs) for treatment ratios (coadministration/ibuprofen alone) using log-transformed values of AUC and Cmax. Bioequivalence (i.e., the lack of an interaction) was concluded if the 90% CI of the geometric mean ratios for both Cmax and AUC were within the acceptance limit of 0.8 to 1.25.
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RESULTS |
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S-(+)-Ibuprofen pharmacokinetics. Voriconazole significantly increased the plasma S-(+)-ibuprofen concentrations (Fig. 1a). The AUC of S-(+)-ibuprofen was increased by 105% (P < 0.001) compared with that in the control phase (Table 1). Importantly, the increase was evident in all subjects (Fig. 2a). Similarly, the mean Cmax of S-(+)-ibuprofen was 22% higher (P < 0.01) and there was a prolongation of the S-(+)-ibuprofen elimination half-life by 43% (P < 0.01) compared with the values in the control phase (Table 1; Fig. 2b and c). In addition, a significant correlation was seen between the voriconazole trough plasma concentration and the increase in the S-(+)-ibuprofen AUC (Spearman r = 0.82; P < 0.01). No significant difference in Tmax between the voriconazole phase and the control phase was noticed.
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R-(–)-Ibuprofen pharmacokinetics. The AUC of R-(–)-ibuprofen was increased by 20% (P < 0.05) in the voriconazole phase compared with that in the control phase (Table 1; Fig. 1b and 3a), whereas the elimination t1/2 was slightly shortened by 7% (P < 0.01) (Table 1). The Cmax and Tmax of R-(–)-ibuprofen were unaffected by voriconazole pretreatment (Table 1; Fig. 3b). Fluconazole had no significant effects on the AUC, Cmax, elimination t1/2, or Tmax of R-(–)-ibuprofen (Table 1; Fig. 3).
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In the control phase, the mean AUCs of S-(+)- and R-(–)-ibuprofen in the two subjects with the CYP2C9*1/*3 and CYP2C8*1/*1 haplotype were 75.8 and 41.1 µg · h ml–1, respectively, which were similar to the mean AUCs of S-(+)- and R-(–)-ibuprofen in that phase (Table 1). However, these two subjects had the longest elimination half-lives of S-(+)-ibuprofen in the control phase, and the prolongation of the elimination t1/2 of S-(+)-ibuprofen in the voriconazole and the fluconazole phases was the shortest with these subjects (Fig. 2c). The one subject with the CYP2C9*1/*1 and CYP2C8*1/*3 haplotype had the highest Cmax and AUC of S-(+)-ibuprofen in the control phase. In addition, he seemed to have the strongest inhibitory effect by both voriconazole and fluconazole. The pharmacokinetic parameters of S-(+)- and R-(–)-ibuprofen in individuals with different CYP2C9 and CYP2C8 haplotypes are illustrated in Fig. 2 and 3.
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DISCUSSION |
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The most likely mechanism for the interaction between voriconazole or fluconazole and S-(+)-ibuprofen is the inhibition of CYP2C9, as this isoenzyme is mainly responsible for the metabolism of S-(+)-ibuprofen (12). Voriconazole has been shown to be an inhibitor of the CYP3A4 (31), CYP2C9 (28), and CYP2C19 (http://www.emea.eu.int./humandocs/Humans/EPAR/vfend/vfend.htm) isoenzymes. For example, pretreatment with voriconazole at 400 mg twice daily increased by 80% the mean steady-state AUC of phenytoin, a substrate for CYP2C9 and CYP2C19 (30). Fluconazole is another well-known potent inhibitor of CYP2C9 and CYP2C19 and a moderate inhibitor of CYP3A4 (4, 17, 23). Treatment with fluconazole (400 mg daily) for 6 days inhibited the CYP2C9-dependent hydroxylation of S-warfarin by 70% (4). Neither voriconazole nor fluconazole is known to inhibit CYP2C8, the principal enzyme responsible for R-(–)-ibuprofen hydroxylation (13, 33). This is supported by the present results, which indicate that these azole antifungals have very little effect on the oral pharmacokinetics of R-(–)-ibuprofen. Furthermore, our results suggest that CYP2C9, CYP2C19, and CYP3A4 have little role in the metabolism of R-(–)-ibuprofen.
In our study we used the clinically recommended dosages of azole antifungals, which are 200 mg to 400 mg every 12 h and 50 mg to 400 mg every 24 h for voriconazole and fluconazole, respectively. In both phases, a significant correlation between the azole antifungal trough concentrations and the increase in the level of exposure to S-(+)-ibuprofen was seen. When it is taken into account that the voriconazole pretreatment consisted of only four doses and that the fluconazole pretreatment consisted of only two doses, it is possible that this kind of short pretreatment did not produce maximal hepatic enzyme inhibition and a longer pretreatment period would have led to an even greater interaction with S-(+)-ibuprofen.
Kirchheiner et al. have identified an association between the CYP2C9 polymorphism and decreased S-(+)-ibuprofen clearance but not R-(–)-ibuprofen clearance (19). Nevertheless, it should be kept in mind that the unidirectional conversion of R-(–)-ibuprofen to S-(+)-ibuprofen occurs in vivo (2, 16). The apparent oral clearance of S-(+)-ibuprofen was approximately halved in carriers of the CYP2C9*3/*3 genotype, an effect that is similar in magnitude to those found with voriconazole or fluconazole in the present study. Interestingly, it was recently found that the clearance for S-(+)-ibuprofen is influenced by CYP2C8*3 and CYP2C9*3 alleles to similar extents (11), which suggests the involvement of CYP2C8 on the hydroxylation of S-(+)-ibuprofen as well. In our study, 2 of the 12 subjects were carriers of the CYP2C9*1/*3 and CYP2C8*1/*1 haplotype. Their elimination t1/2 of S-(+)-ibuprofen was the longest among the 12 subjects in the control phase. In one of these two subjects, pretreatment with azole antifungals did not produce any prolongation of his S-(+)-ibuprofen elimination t1/2, and in the other subject, pretreatment with azole antifungals produced only a slight prolongation of his elimination t1/2 of S-(+)-ibuprofen, which suggests that these two individuals were less prone to the inhibitory effect of CYP2C9 by voriconazole or fluconazole, probably due to lower baseline CYP2C9 activity. The haplotype CYP2C8*1/*3 and CYP2C9*1/*1 was found in one of the subjects. He had the highest Cmax and AUC of S-(+)-ibuprofen in the control phase, and he had also the greatest AUC values for both S-(+)- and R-(–)-ibuprofen in the voriconazole phase and in the fluconazole phase. Accordingly, it seems that the CYP2C8*1/*3 genotype affects the metabolism of S-(+)-ibuprofen, too, as assumed by Garcia-Martin et al. (11).
Coadministration of voriconazole or fluconazole and ibuprofen is likely in a clinical setting. It has been shown that increased S-(+)-ibuprofen concentrations due to a lack of functional CYP2C9 enzyme leads to increased inhibition of both cyclooxygenase type 1 and cyclooxygenase type 2, as evidenced by reduced thromboxane B2 and prostaglandin E2 formation (19). Accordingly, increased exposure to ibuprofen due to inhibition of CYP2C9 by voriconazole or fluconazole might increase the risk of the concentration-dependent renal, cardiovascular, or gastrointestinal adverse drug reactions caused by ibuprofen. This assumption is supported by the findings of a study reporting that the impaired metabolism of ibuprofen due to mutations in the CYP2C9 gene increases the risk of acute gastrointestinal bleeding (21). On the basis of the results of earlier studies and those of the present study, the use of a reduced dose of ibuprofen should be considered when it is coadministered with voriconazole or fluconazole.
Visual disturbances have been associated with voriconazole treatment, and the appearance of this adverse effect is dependent on the CYP2C19 genotype, the principal enzyme responsible for the metabolic clearance of voriconazole (14; Mikus et al., Abstr. 7th Congr. Eur. Assoc. Clin. Pharmacol. Ther., 2005). In this open study, one-third of the subjects complained of visual disturbances during voriconazole pretreatment. That proportion is similar to that found in previous studies (29). All the events were mild and resolved within 1 h without any medical intervention. No other drug-related adverse events were recognized during the study.
In conclusion, voriconazole and fluconazole treatments increased the level of exposure to S-(+)-ibuprofen 2- and 1.8-fold, respectively. This was likely caused by the inhibition of CYP2C9-mediated biotransformation of S-(+)-ibuprofen by voriconazole and fluconazole. The clinical significance of the present fluconazole-ibuprofen and voriconazole-ibuprofen interactions remains unclear. However, patients using high doses of ibuprofen daily are exposed to considerable ibuprofen concentrations if they are treated with voriconazole or fluconazole. Thus, caution should be exercised when treatment with voriconazole, fluconazole, or some other potent CYP2C9 inhibitor is initiated in patients who receive high repeated doses of ibuprofen.
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ACKNOWLEDGMENTS |
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This work was supported by Turku University Hospital research fund EVO 13693 and EVO 13390 and Swedish Science Council (Medicine 04496).
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FOOTNOTES |
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REFERENCES |
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