Voriconazole increases while itraconazole decreases plasma meloxicam ACCEPTED

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1 AAC Accepts, published online ahead of print on 17 November 08 Antimicrob. Agents Chemother. doi:.1128/aac Copyright 08, American Society for Microbiology and/or the Listed Authors/Institutions. All Rights Reserved. Voriconazole increases while itraconazole decreases plasma meloxicam concentrations 5 V.V. Hynninen 1,2*, K.T. Olkkola 2, L. Bertilsson 3, K.J. Kurkinen 4, T. Korhonen 1, P.J. Neuvonen 4, and K. Laine 1,5 Department of Pharmacology, Drug Development and Therapeutics, University of Turku, Turku, Finland 1 ; Department of Anesthesiology, Intensive Care, Emergency Care and Pain Medicine, Turku University Hospital, Turku, Finland 2 ; Department of Laboratory Medicine, Division of Clinical Pharmacology at Karolinska Institute, Karolinska University Hospital, Huddinge, Stockholm, Sweden 3 ; Department of Clinical Pharmacology, University of Helsinki and Helsinki University Hospital, Helsinki, Finland 4 ; TYKSLAB, Unit of Clinical Pharmacology, Turku, Finland 5 Running title: Interactions of meloxicam with two azole antimycotics Corresponding author: Mailing address: Department of Pharmacology, Drug Development and Therapeutics, University of Turku, Itäinen Pitkäkatu 4B, FIN-5, Finland. Phone: Fax: vilhyn@utu.fi 1

2 ABSTRACT This study investigated the effect of voriconazole, an inhibitor of cytochrome P450 2C9 (CYP2C9) and CYP3A4, and itraconazole, an inhibitor of CYP3A4, on the pharmacokinetics and pharmacodynamics of meloxicam. Twelve healthy volunteers ingested in a cross-over study mg meloxicam, without pretreatment (control), after voriconazole pretreatment, and after itraconazole pretreatment. Plasma concentrations of meloxicam, voriconazole, itraconazole and thromboxane B 2 (TxB 2 ) generation were followed. Compared to the control phase, voriconazole increased the mean area under the plasma concentration-time curve (AUC 0-72 ) of meloxicam by 47% (P < 0.001) and prolonged its mean half-life (t ½ ); by 51% (P < 0.01), without affecting its mean peak concentration (C max ). In contrast, itraconazole decreased the mean AUC 0-72 and C max of meloxicam by 37% (P < 0.001) and by 64% (P < 0.001), respectively and prolonged its t ½ and time to C max. Plasma protein unbound fraction of meloxicam was unchanged by voriconazole and itraconazole. Lowered plasma meloxicam concentrations during the itraconazole phase were associated with decreased pharmacodymic effects of meloxicam, as observed by weaker inhibition of TxB 2 synthesis compared to the control and voriconazole phases. Voriconazole increases plasma concentrations of meloxicam, whereas itraconazole, unexpectedly, decreases plasma meloxicam concentrations, possibly by impairing its absorption. 2

3 Meloxicam is a nonsteroidal anti-inflammatory drug (NSAID) of the oxicam class with selectivity towards cyclo-oxygenase (COX)-2 compared to COX-1 (8, 19). It is widely used in the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis and other rheumatological conditions. Meloxicam has an oral bioavailability of 89% and its maximum plasma concentrations (C max ) are achieved within 4-11 h (, 26). It is extensively bound to plasma proteins (> 99%), mainly to albumin (24). The elimination half-life (t ½ ) of meloxicam ranges from 13 to h and it is suitable for once daily dosing (24, 26). Meloxicam is extensively metabolized in the liver, primarily by polymorphic cytochrome P450 2C9 (CYP2C9) enzyme and to a minor extent by CYP3A4 enzyme, to four pharmacologically inactive metabolites (6, 24). Only negligible amounts of the parent drug are found in urine and in faeces (24). The effect of different genotypes on the pharmacokinetics of meloxicam is not known. Voriconazole is a triazole antifungal agent used both intravenously and orally to treat invasive fungal infections. Voriconazole undergoes extensive oxidative metabolism involving CYP enzymes CYP2C19, CYP2C9, and CYP3A4 (13). Voriconazole is also an inhibitor of CYP2C9, CYP3A4, and CYP2C19 catalyzed reactions both in vitro and in vivo (17, 21, 23, 25). Another triazole antifungal, itraconazole, is a potent inhibitor of CYP3A4 (18, 28), but it is without effect on CYP2C9 in humans (, 27). Because both voriconazole and itraconazole inhibit CYP enzymes involved in the metabolism of meloxicam, we hypothesized that they might interact with meloxicam leading to its increased concentrations in plasma. Therefore, we found it important to investigate the effects of voriconazole and itraconazole on the pharmacokinetics of meloxicam. In addition, we wanted to study whether possibly changed plasma meloxicam concentrations are reflected in its effects on 3

4 the generation of thromboxane B 2 (TxB 2 ) during whole blood clotting, which is a validated index of COX-1 activity ex vivo (3). 4

5 MATERIALS AND METHODS 5 Subjects. Twelve healthy male volunteers (age range, years; weight range, kg) participated in the study. Each subject was ascertained to be in healthy as assessed by medical history, clinical examination, and routine laboratory tests. All subjects were nonsmokers and used no continuous medication. The subjects received both verbal and written information on the study and written informed consent was obtained. The study protocol was approved by the Ethics Committee of the Hospital District of Southwest Finland as well as by National Agency for Medicines, Finland. Study design. The study was carried out in a Latin-square, open-label, randomized, 3-phase crossover design, with a washout period of 4 weeks between the phases. The volunteers were given either no pretreatment (control phase) or oral voriconazole (voriconazole phase) for 2 days or oral itraconazole (itraconazole phase) for 4 days in a randomized order. The dose of voriconazole (Vfend 0 mg tablet, Pfizer, Heinrich Mack Nachf. GmbH & Co, Illertissen, Germany) was 400 mg every 12 hours for one day and then 0 mg every 12 hours for one additional day. The dose of itraconazole (Sporanox 0 mg capsule, Janssen-Cilag, Latina, Italy) was 0 mg once a day at 8 AM for four days. Voriconazole and itraconazole were selfadministered by subjects except for the last doses, which were administered by the study personnel. The intake of the premedication by the subjects was verified by use of mobile phone short message service. One hour after the last dose of voriconazole or itraconazole was ingested, all volunteers received a mg oral dose of meloxicam (Mobic mg tablet, Boehringer Ingelheim, Ingelheim, Germany) at 9 AM with 0 ml of water. During all phases, the subjects fasted overnight before administration of meloxicam and continued fasting until a standardized 5

6 lunch was served 4 hours after meloxicam ingestion. The subjects were forbidden to use any other medication for 14 days before and during the study and any drugs 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. Blood sampling and drug analysis. Venous blood samples were drawn from the cannulated forearm vein immediately before and at 1, 2, 3, 4, 5, 6, 7, 8,, 12, 24, 48, and 72 h after meloxicam administration. Plasma was separated within 30 minutes and stored at -70 o C until analysis. To study the protein binding of meloxicam, protein-free ultrafiltrates were prepared with the Amicon Centrifree filter YM30 (Amicon, Beverly, MA, USA) at the room temperature from the plasma samples of each subject taken 5 h after meloxicam ingestion, in each three phases. The free fraction of plasma meloxicam was prepared from 1 ml plasma (taken 5 h after meloxicam ingestion) by centrifugation using a fixed-angle rotor at 10 g and 25ºC for 30 min. There was no detectable binding of meloxicam to filter even when a low concentration (2.5 ng/ml) of meloxicam and a protein-free solution were used in the ultrafiltration. Determination of drug concentrations. Plasma concentrations of meloxicam were measured, as described earlier (14), using piroxicam as an internal standard and a Q Trap liquid chromatography-tandem mass spectrometry system (Sciex Division of MDS, Toronto, Ontario, Canada). The mass spectrometer was operated in positive TurboIonSpray (Sciex Division of MDS) mode, and the transition of the [M+H] + precursor ion to a product ion was monitored. The ion transitions monitored were: mass-to-charge ratio (m/z) to m/z 1.1 for meloxicam, and m/z to m/z 95.1 for the internal standard piroxicam. The interday coefficient of variation (CV) for total meloxicam was less than % at relevant concentrations (n=14). All 6

7 plasma samples taken after meloxicam ingestion were quantifiable for their total meloxicam, and all samples taken at 5 h for their free (protein unbound) meloxicam concentration. All plasma ultrafiltrate samples (altogether 36 samples from 5 h plasma) were analyzed in the same assay for their free meloxicam concentration. The meloxicam standard curve was linear over the concentration range used (standards 0.5, 1.0, 2.5, 5.0,, 25 and 50 ng/ml), and the intra-assay coefficient of variation was 9.7% at ng/ml (n=7). Voriconazole and itraconazole did not interfere with the determination of meloxicam. In the voriconazole assay, the internal standard (UK 54373) and 0.1 ml of perchloric acid were added to plasma (0.5 ml) samples. Then the samples were vortexed, centrifuged, and the supernatants were applied to Oasis MCX (30 mg, 1 ml) solid-phase extraction cartridges (Waters, Milford, MA, USA), which had been conditioned with 1 ml of methanol followed by 1 ml of water. The cartridges were then washed with 1 ml of 0.1 M hydrochloric acid and 1.0 ml of methanol-water (40:60 v/v). The cartridges were dried by a vacuum prior to the elution step with 1 ml 5% ammonium hydroxide in methanol. The samples were evaporated to dryness under nitrogen and reconstituted in 0.1 ml of mobile phase (72.5% ammonium phosphate 40 mm, ph 9.0 and 27.5% acetonitrile). The chromatographic conditions were otherwise as described previously (9), but we used a higher ph (ph 9.0) of the mobile phase instead of ph 6.0. Plasma concentrations of itraconazole were quantified by high-performance liquid chromatography as described earlier (11). The interday CV for voriconazole was 2.8% or less at relevant concentrations (n=3). The interaday CV for itraconazole was 6.1% or less at relevant concentrations (n=4). Genotyping. Blood samples were drawn and stored at - C. Genomic DNA was extracted from the leukocytes by using QiAmp DNA blood kit (Qiagen, Hilden,Germany). A TaqMan assay was used to determine the CYP2C9*2 and CYP2C9*3 alleles, as previously described (29). Alleles 7

8 containing no *2 or *3 are named CYP2C9*1. After an internal DNA denaturation at 95 C for minutes, the samples were run for 40 cycles at 95 C for seconds and at 60 C for 1 minute. 5 Pharmacokinetics. The C max and T max for each subject were obtained directly from the plasma concentration data. The elimination rate constant (k el ) was determined by a linear regression analysis of the log-linear part of the plasma concentration-time curve. The t ½ was calculated by equation: t ½ = ln 2/k el. The area under the plasma concentration-time curve was calculated from zero to 72 h postdose (AUC 0-72 ) using linear trapezoidal rule for the rising phase of the plasma concentration-time curves and the logarithmic trapezoidal rule for the descending phase. All pharmacokinetic calculations were performed with WinNonlin pharmacokinetic program (version 4.1; Pharsight, Mountain View, California). Pharmacodynamics. Pharmacodynamics of meloxicam were assessed by measuring the TxB 2, a stable metabolite of TxA 2, generation by platelets in spontaneously clotting whole blood (3). The decrease in TxB 2 generation is shown to reflect the degree of COX-1 inhibition. The decrease in TxB 2 generation was calculated by comparing TxB 2 concentration at different time points to the individual baseline value. On study days, blood samples for TxB 2 assay were drawn before, and at 5, 8, 12, 24, and 48 hours after meloxicam administration. Blood samples were collected into glass tubes containing no anticoagulant and were immediately incubated for 1 hour at 37 C to stimulate the TxB 2 production in platelets during coagulation. Next, serum was collected and centrifuged and stored at -70 C until assayed for TxB 2. Serum TxB 2 concentrations were determined by enzyme immunoassay kit (Amersham Thromboxane B 2 Enzymeimmunoassay Biotrak TM System, GE Healthcare, UK). The limit of detection was ng/ml, and the interassay CV was %. 8

9 Statistical analysis. The pharmacokinetic and pharmacodynamic variables were compared by use of analysis of variance (ANOVA) for repeated measures, and a posteriori testing was performed by use of the Tukey test. T max was analyzed with Friedman`s test and Wilcoxon signed rank test was used for pairwise comparisons. The correlation between voriconazole or itraconazole AUC and the ratio of meloxicam AUC in the voriconazole and in the itraconazole phase to the meloxicam AUC in the control phase as well as the correlation between meloxicam C max or AUC values and decrease in TxB 2 formation (AUC) was tested using Pearson correlation test. Statistical analyses were carried out using statistical program SYSTAT for Windows (version.2; Systat Software, Richmond, California). The chosen statistical significance level was P < Results are presented as mean ± standard deviation (SD) in the table, and for clarity, as mean ± standard error of the mean (SEM) on the figures. For T max median with range is shown in the table. The percentage differences between treatments were calculated within subjects and 95% confidence intervals (CIs) are given. 9

10 RESULTS 5 All twelve subjects completed the study according to the protocol. Mean plasma concentrations of meloxicam as a function of time in twelve volunteers during different phases of the study are illustrated in Figure 1. Effect of voriconazole. Voriconazole increased the mean AUC 0-72 of meloxicam by 47% (range, 11-1%; P < 0.001) compared to the control value and prolonged the mean t ½ of meloxicam by 51%, from 17.4 to 26.7 h (P < 0.01). The mean C max and median T max of meloxicam were unaffected by voriconazole (Table 1). The increase of the AUC 0-72 as well as the prolongation of t ½ of meloxicam was evident in all twelve subjects (Figure 2). Effect of itraconazole. During the itraconazole phase, the mean AUC 0-72 of meloxicam was 63% (range, 31-90%; P < 0.001) and its mean C max was 36% (range, 23-76%; P < 0.001) of the respective control values (Table 1). The decrease of AUC 0-72 and C max was seen in all subjects (Figure 2). The median T max of meloxicam was also reached significantly later (24 h in the itraconazole phase vs. 4 h in the control phase; P < 0.01) and t ½ was prolonged from 17.4 to 27 h (P < 0.01) (Table 1). Four subjects had T max of meloxicam as late as 48 hours after its ingestion, which made it impossible to determine meloxicam t ½ in these subjects, because the concentrations were measured for up to 72 hours and there were no measurements between 48 h and 72 h time points.

11 Protein binding. The plasma protein binding of meloxicam was 99.83% ± 0.05% (mean ± SD), 99.83% ± 0.04%, and 99.82% ± 0.07% during the control, voriconazole, and itraconazole phase, respectively, measured from the plasma samples taken 5 h after meloxicam ingestion. Effects of genotype. Two subjects had the *1/*2, one had *2/*2, and one had *1/*3 genotype for CYP2C9, the others were homozygous wild-type *1/*1 for CYP2C9. The meloxicam AUC 0-72 of subjects with *1/*2, *2/*2, and *1/*3 genotype in the control phase were 33.1 mg h/l (mean of two subjects), 26.5 mg h/l, and 40.1 mg h/l, respectively, and were comparable with the mean AUC 0-72 of meloxicam of wild type *1/*1 subjects (32.0 mg h/l) (Figure 2). Voriconazole and itraconazole seemed to have similar effect on the meloxicam AUC 0-72, C max, and t ½ in the subjects with *1/*2, and *2/*2 genotype compared to wild-type subjects. The subject with *1/*3 genotype had the longest t ½ in the control phase. In addition, the strongest inhibitory effect on meloxicam t ½ by voriconazole and itraconazole was observed with this subject (Figure 2). Pharmacodynamics. The absolute TxB 2 concentrations and percentage reduction from baseline TxB 2 concentrations at different time points are illustrated in Figure 3. The baseline serum TxB 2 levels were similar before each treatment; 145 ± 61 ng/ml, 144 ± 47 ng/ml, and 124 ± 48 ng/ml (mean ± SD) prior to ingestion of meloxicam, during the control phase, voriconazole phase and itraconazole phase, respectively. Meloxicam alone (control phase) inhibited the synthesis of TxB 2 from baseline significantly, i.e. at 5 h after ingestion by 30% (P < 0.05), at 8 h 37% (P < 0.05), at 12 h 56% (P < 0.001), at 24 h 28% (P < 0.05), and at 48 h 25% (P < 0.05). In the voriconazole phase, the corresponding values at the same time points were 37% (P < 0.01), 46% (P < 0.001), 49% (P < 0.001), 31% (P < 0.05), and 14% (P > 0.05). In the itraconazole phase, meloxicam did not cause any statistically significant inhibition of TxB 2 generation before 48 h after meloxicam 11

12 ingestion; at 48 h, 29% (P < 0.05) decline from baseline was seen. There was a significant correlation between both meloxicam C max values (P < 0.01, Pearson r = -0.46) and meloxicam AUC 0-48 values (P < 0.01; Pearson r = -0.47) and the inhibition of TxB 2 synthesis. No difference was observed in the inhibition of TxB 2 synthesis between the control and the voriconazole phase at any time point, whereas inhibition of TxB 2 synthesis was significantly greater, from 5 to 12 h after meloxicam ingestion in the control and in the voriconazole phase compared to the itraconazole phase. Plasma voriconazole and itraconazole. The mean C max, AUC (0- ), and t ½ of voriconazole were 2390 ng/ml (range, ng/ml), ng h/ml (range, ng h/ml), and h (range, h), respectively. The corresponding values for itraconazole were 385 ng/ml (range, ng/ml), 110 ng h/ml (range, ng h/ml), and 31 h (range, -49 h), respectively. Voriconazole AUC 0- of subjects with CYP2C9*1/*2, *2/*2, and *1/*3 genotype was 400 ng h/ml, ng h/ml, and ng h/ml, respectively, and itraconazole AUC (0- ) of subjects with *1/*2, *2/*2, and *1/*3 genotype was 170 ng h/ml, 80 ng h/ml, and ng h/ml, respectively. There were no significant correlations between the pharmacokinetic variables of voriconazole and the extent of interaction between voriconazole and meloxicam or between the pharmacokinetic variables of itraconazole and the extent of interaction between itraconazole and meloxicam. Adverse effects. During the voriconazole pretreatment, 3 of the 12 subjects reported visual disturbances, including photophobia and altered colour vision changes. No other clinically relevant adverse effects were observed or reported during the study. 12

13 DISCUSSION Our results reveal that the interactions of meloxicam with two triazole antifungals, voriconazole and itraconazole are opposite. Voriconazole significantly increased plasma concentrations of meloxicam, whereas, itraconazole, unexpectedly caused a notable decrease in the plasma concentrations of meloxicam. This was also associated with decreased pharmacodymic effects of meloxicam, as observed by weaker inhibition of TxB 2 synthesis during whole blood clotting, in the itraconazole phase compared to the control and voriconazole phases. The major primary metabolite of meloxicam is 5`-hydroxymethyl meloxicam, which is further oxidized to 5`-carboxy meloxicam. The oxidative cleavage of the benzothiazine ring of meloxicam creates two additional metabolites (6, 24). Based on in vitro studies, formation of 5`hydoxymethyl metabolite is catalyzed mainly by CYP2C9 (80%) and CYP3A4 appears to play a minor role (%) (6). There are only a few studies addressing the influence of other drugs on the pharmacokinetics of meloxicam. Cholestyramine has increased the clearance of i.v. meloxicam by 50%, suggesting that meloxicam undergoes enterohepatic circulation (4), whereas antacids, aspirin, furosemide and cimetidine have not affected its pharmacokinetics (5, 16). In this study, voriconazole, an inhibitor of both CYP2C9 and CYP3A4 (17), increased the meloxicam AUC 0-72 to about 1.5-fold and prolonged meloxicam t ½ from 17 h to 27 h, but had no effect on the C max or T max of meloxicam. Together, these observations suggest that the voriconazole-meloxicam interaction occurs principally during the meloxicam elimination phase and is likely due to the inhibition of CYP2C9, and to a lesser extent of CYP3A4 -mediated metabolism of meloxicam by voriconazole. The individual with the CYP2C9*1/*3 genotype, thus carrying low basal CYP2C9 activity, had the strongest effect by both the CYP3A4-inhibitory azoles on the meloxicam t ½, 13

14 suggesting a bigger role of CYP3A4 in the metabolism of meloxicam in individuals possessing impaired CYP2C9 activity. 5 In clinical situations, the voriconazole-meloxicam interaction can be stronger than that seen in the present study, where, for ethical reasons (to minimize the common visual disturbances), administration of voriconazole to healthy volunteers was not continued after meloxicam ingestion. As the half-life of voriconazole was about -11 h, its plasma concentrations were from 12 hours onwards lower than during its normal twice daily clinical use. This may have somewhat underestimated the extent of the interaction during the elimination phase of meloxicam, as meloxicam is eliminated more slowly than voriconazole. The changes in the pharmacokinetics of meloxicam during the itraconazole phase were surprising. The plasma concentrations of meloxicam were clearly lower during the first 24 h following the ingestion of meloxicam in the itraconazole phase compared to the control phase (Figure 1). The AUC 0-72 and C max, were considerably decreased, and the T max was greatly prolonged by itraconazole. The plasma protein binding of meloxicam was very high (99.8%), but there was no difference in its percentage binding between different phases of the study. Thus a displacement of meloxicam from plasma protein by itraconazole does not explain the observed interaction. The present findings strongly suggest that itraconazole decreased the exposure to meloxicam by impairing its gastrointestinal absorption. However, the exact mechanism of impaired absorption remains to be studied. Itraconazole could have inhibited some transport system in the gut wall, which is needed for the absorption of meloxicam. However, this theory is highly speculative, because there is no evidence for the participation of drug transporters in the absorption of meloxicam. 14

15 Due to the delayed absorption of meloxicam, our blood sampling period appeared to be too short for the reliable determination of the elimination rate constant (k el ) in 4 subjects in the itraconazole phase. Accordingly, because AUC values could not be extrapolated reliably to infinity in all 12 subjects in the itraconazole phase, AUC 0-72 was used for comparison of AUC values between the phases. It is therefore possible that the actual effect of itraconazole on the total exposition to meloxicam is somewhat smaller than that calculated in the present study. In the voriconazole phase, the magnitude of interaction between meloxicam and voriconazole remained essentially the same when checked by using the AUC values extrapolated to infinity (data not shown). The t ½ of meloxicam, calculated from 8 subjects, was significantly prolonged by itraconazole. However, the decline of plasma meloxicam concentration in the itraconazole phase seems to be determined by its rate of absorption. Therefore, if the absorption of meloxicam is prolonged as in the present study, also its apparent t ½ is prolonged (flip-flop phenomenon) and this can occur without a change in the rate of elimination. However, the CYP3A4 inhibition by itraconazole as a partial cause for prolonged t ½ can not be ruled out. In our study, voriconazole dosing was based on a previous study indicating that the steady state concentration of voriconazole can be achieved in 2 days, using a loading dose of 400 mg twice daily on the first day followed by 0 mg twice daily on the second day (). In contrast, fourday pretreatment with itraconazole was too short to achieve steady state concentrations of itraconazole, which are reached only -14 days after the beginning of treatment (12). Four-day pretreatment was selected, because it is not desirable to expose healthy volunteers to a 2 week pretreatment of itraconazole and because also 4-day pretreatment with similar doses of itraconazole as used in our study has been shown to produce a strong inhibition of CYP3A4 (1).

16 Typical adverse effects of NSAIDs include gastro-intestinal damage, inhibition of platelet function, and renal impairment. The first two are attributed mainly to the inhibition of COX-1, whereas renal impairment is also a prevalent side effect of COX-2-selective NSAIDs. Meloxicam is a preferential COX-2 inhibitor, which also dose-dependently inhibits COX-1 (2, 19, 22). In the present study, a 47% increase in the exposure to meloxicam by voriconazole was not associated with increased COX-1 inhibition. The maximum decline of the synthesis of TxB 2 was 56% and 49% in the control and voriconazole phase, respectively, which is comparable with other studies, in which TxB 2 synthesis was inhibited 35% to 66% by mg meloxicam (7, 19, 22). Accordingly, it is unlikely that a short-term concomitant use of voriconazole or other CYP2C9 inhibitors with meloxicam would increase its risk for gastro-intestinal adverse effects or bleeding. However, increased clinical alertness is recommended with the long term coadministration of meloxicam with CYP2C9 inhibitors, especially in the elderly or in patients having an increased risk for NSAID-related adverse effects. In the itraconazole phase, the decreased meloxicam concentrations were associated with clearly reduced pharmacodynamic effect. No reduction on TxB 2 synthesis was observed by 24 h after ingestion of mg meloxicam. At 48 h, TxB 2 synthesis was inhibited by 29%, which corresponds to the degree of inhibition reported previously 5 h after ingestion of 7.5 mg of meloxicam, without itraconazole (2). These findings suggest that the interaction between itraconazole and meloxicam is clinically significant and probably will cause a reduced meloxicam effect, at least in the short term. Because, both the AUC 0-72 and the C max of meloxicam correlated similarly with the degree of inhibition of TxB 2 formation it is difficult to evaluate whether it is lowered exposure or lowered peak concentration that is relevant for the interaction between meloxicam and itraconazole. 16

17 In conclusion, voriconazole moderately increases plasma concentrations of meloxicam, probably by inhibiting its metabolism via CYP2C9. In contrast to voriconazole, itraconazole causes a substantial decrease in plasma concentrations, especially in the C max, of meloxicam, probably by impairing its gastrointestinal absorption. The clinical efficacy of meloxicam can be reduced, at least in short term, when given during itraconazole treatment, but unchanged or increased when given during voriconazole treatment. Further studies are needed on the effect of itraconazole on the pharmacokinetics and pharmacodynamics of meloxicam during their longer concomitant administration. 17

18 ACKNOWLEDGMENTS This work was supported by Turku University Hospital research fund EVO (Laine) and EVO (Olkkola) and by Swedish Research Council, 3902 (Bertilsson). Mrs Elina Kahra is thanked for her skilful assistance in organizing the study. 18

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23 Figure Legends 5 1. Plasma concentrations (mean ± standard error of the mean) of meloxicam after a single mg oral dose in 12 healthy male subjects in control phase (open circles ) or after pretreatment with voriconazole (filled square ) or after pretreatment with itraconazole (filled circles ). The insert depicts the same data on a semilogarithmic scale. 2. Individual values for area under plasma concentration-time curves (AUC), maximum plasma concentrations (C max ), elimination half-lives (t ½ ), and time to maximum plasma concentrations (t max ), after the administration of mg meloxicam either alone (Control), or after pretreatment with voriconazole (Voric), or after pretreatment with itraconazole (Itrac). Symbols for CYP2C9*1/*1 genotype ( ), for CYP2C91*/2* genotype ( ), for CYP2C9*2/*2 genotype ( ), and for CYP2C9*1/*3 genotype ( ) 3. Mean percent inhibition of TxB 2 generation from baseline by mg meloxicam either given alone (open circles ) or after pretreatment with voriconazole (filled square ) or after pretreatment with itraconazole (filled circles ). 23

24 Table 1. The pharmacokinetic variables of meloxicam in twelve healthy subjects after a single oral dose of mg meloxicam without pretreatment (control) or after pretreatment with voriconazole or after pretreatment with itraconazole. Parameter Control Voriconazole AUC (mg h/l) % of control C max (mg/l) % of control t ½ (h) % of control 32.4 ± ± ± ±.9*** ± ± 11.6** 1 95% CI of difference from control Itraconazole 9.0 to.7.5 ± 7.7*** to ± 0.*** to ± 8.2** T max (h) 4 (4-7) 6 (4-24) 24 (4-48)** Free fraction (%) 0.18 ± ± ± % CI of difference from control to to to 16.1 The results are mean ± standard deviation (median with range for T max ). AUC, area under plasma concentration-time curve (0-72 h); C max, maximum plasma concentration; t ½, elimination halflife; T max, time to maximum plasma concentration; CI, confidence interval. Percent of control was calculated individually for each subject. The free fraction of plasma meloxicam was determined from plasma samples taken 5 hours after meloxicam ingestion *Significantly (P<0.05) different from control **Significantly (P<0.01) different from control ***Significantly (P<0.001) different from control 24

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