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Abstract

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Abstract: Our objective was to identify the cytochrome P450 (CYP) enzymes that metabolise pioglitazone and to examine the effects of the CYP2C8 inhibitors montelukast, zafirlukast, trimethoprim and gemfibrozil on pioglitazone metabolism in vitro. The effect of different CYP isoform inhibitors on the elimination of a clinically relevant concentration of pioglitazone (1 μM) and the formation of the main primary metabolite M-IV were studied using pooled human liver microsomes. The metabolism of pioglitazone by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5 was investigated using human recombinant CYP isoforms. In particular, the inhibitors of CYP2C8, but also those of CYP3A4, markedly inhibited the elimination of pioglitazone and the formation of M-IV by HLM. Inhibitors selective to other CYP isoforms had a minor effect only. Of the recombinant isoforms, CYP2C8 (20 pmol/ml) metabolised pioglitazone markedly (56% in 60 min.), and also CYP3A4 had a significant effect (37% in 60 min.). Montelukast, zafirlukast, trimethoprim and gemfibrozil inhibited pioglitazone elimination in HLM with IC50 values of 0.51 μM, 1.0 μM, 99 μM and 98 μM, respectively, and the formation of the metabolite M-IV with IC50 values of 0.18 μM, 0.78 μM, 71 μM and 59 μM, respectively. In conclusion, pioglitazone is metabolised mainly by CYP2C8 and to a lesser extent by CYP3A4 in vitro. CYP2C9 is not significantly involved in the elimination of pioglitazone. The effect of different CYP2C8 inhibitors on pioglitazone pharmacokinetics needs to be evaluated also in vivo because, irrespective of their in vitro CYP2C8 inhibitory potency, their pharmacokinetic properties may affect the extent of interaction.

Pioglitazone is a thiazolidinedione compound used in the treatment of type 2 diabetes. It is extensively metabolised by hydroxylation and oxidation in the liver to form at least four primary metabolites (designated M-I, M-II, M-IV and M-V) and two secondary metabolites (M-III and M-VI; fig. 1) (Eckland & Danhof 2000). In addition, for example a putative dihydroxy metabolite (M-XI) has been identified in human urine (Jaakkola et al. 2006). M-IV is further metabolised to form a secondary metabolite M-III. The pharmacologically active M-IV and M-III are the main metabolites found in human serum and their circulating concentrations are equal to or greater than those of the parent pioglitazone (Eckland & Danhof 2000).

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Figure 1. The metabolism of pioglitazone to six major metabolites (M-I to M-VI) in human beings (Eckland & Danhof 2000) and a putative metabolite M-XI (Jaakkola et al. 2006). The structure of the internal standard rosiglitazone is shown on the bottom.

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According to review articles and the manufacturer of the drug, pioglitazone is metabolised by multiple cytochrome P-450 (CYP) enzymes, mainly by CYP2C8, CYP3A4 and CYP2C9 (Eckland & Danhof 2000; Hanefeld 2001). However, the information on the contribution of different CYP isoforms to the metabolism of pioglitazone seems to be based on unpublished data, and the information from different sources is discrepant. Thus, the European product information of Actos (EMEA 2004) states that the metabolism of pioglitazone occurs predominantly via CYP3A4 and CYP2C9, whereas the U.S. label states that the major CYP isoforms involved are CYP2C8 and CYP3A4 (FDA 1999). The lipid-lowering fibrate gemfibrozil, an inhibitor of CYP2C8 (Backman et al. 2002; Wang et al. 2002; Shitara et al. 2004; Ogilvie et al. 2006), has increased in human volunteers the mean area under the plasma concentration-time curve (AUC) of pioglitazone 3.2 times (Jaakkola et al. 2005). In contrast, the antimycotic agent itraconazole, a potent inhibitor of CYP3A4, had no effect on the pharmacokinetics of pioglitazone (Jaakkola et al. 2005). These findings suggest that pioglitazone is metabolised mainly by CYP2C8 in vivo.

The discrepant information regarding the role of different CYP enzymes in pioglitazone elimination prompted us to study in vitro the contributions of different CYP enzymes to the metabolism of pioglitazone at a clinically relevant concentration. Furthermore, we wanted to compare the effects of four CYP2C8 inhibitors, montelukast, zafirlukast, trimethoprim and gemfibrozil, on the metabolism of pioglitazone in human liver microsomes.

Materials and Methods

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Microsomes and recombinant enzymes. Pooled human liver microsomes (HLM) (Gentest new catalogue number 452161, lot 26), representing a pool from 46 individuals, and human recombinant CYP1A2 (new catalogue number 456203, lot 28), CYP2B6 (new catalogue number 456255, lot 13), CYP2C8 (new catalogue number 456252, lot 17461), CYP2C9 (new catalogue number 456258, lot 17460), CYP2C19 (new catalogue number 456259, lot 15466), CYP2D6 (new catalogue number 456217, lot 19853), CYP2E1 (new catalogue number 456206, lot 16), CYP3A4 (new catalogue number 456202, lot 18673) and CYP3A5 (new catalogue number 456256, lot 1) isoforms (SupersomesTM) and insect cell control SupersomesTM (new catalogue number 456201, lot 16908) were purchased from Gentest Corp. (Woburn, MA, USA). Human liver tissue had been collected in accordance with all pertinent regulations, and permissions from the donors' families had been obtained prior to organ collection. The procedures of organ collection had been reviewed and accepted by the respective institutional Human Subjects Committee.

Chemicals. Pioglitazone hydrochloride, diethyldithiocarbamate (DDC), omeprazole, quinidine, sulfaphenazole, trimethoprim, troleandomycin and β-NADPH (Sigma-Aldrich, Steinheim, Germany), itraconazole and ketoconazole (Janssen Biotech N.V., Olen, Belgium), clopidogrel sulphate, montelukast sodium and zafirlukast (Sequoia Research Products Ltd, Oxford, UK), furafylline (Ultrafine Chemicals, Manchester, UK), gemfibrozil (Gödecke AG, Freiburg, Germany) and rifampicin (Orion pharma, Espoo, Finland) were used in this study. Other chemicals were obtained from Merck (Darmstadt, Germany).

Incubation conditions. The incubations were carried out in 0.1 M sodium phosphate buffer (pH 7.4), containing 5.0 mM MgCl2, 1 μM pioglitazone, 1.0 mM β-NADPH and 0.3 mg/ml microsomal protein or 20 pmol/ml recombinant CYP. Pioglitazone, solvent with or without inhibitor, buffer and HLM or recombinant enzymes were premixed, and incubations were commenced by the addition of β-NADPH. Zafirlukast stock solution was prepared in acetonitrile, the stock solutions of other drugs were prepared in methanol. Final solvent concentration did not exceed 1% and it was equal in all control incubations. All incubations were conducted in duplicate at 37 ° in a shaking water bath and terminated by removing an aliquot (0.5 ml), adding to 100 μl perchloric acid (70%) and cooling on ice. The mean values of the duplicates were used in the calculations.

Pioglitazone metabolism and inhibition experiments. When substrate depletion as a function of incubation time was measured, aliquots were removed at 0, 8, 16, 30, 45 and 60 min. Human liver microsome concentration was chosen on the basis of preliminary experiments. The depletion of unchanged pioglitazone at 60 min. and the formation of the metabolite M-IV at 8 min. after incubation with human liver microsomes or recombinant CYP isoforms were used as the measure of pioglitazone metabolism in further experiments. The formation rate of M-IV was linear with respect to the microsomal protein concentration and incubation time up to 8 min.

To examine the effects of different CYP isoform inhibitors on the metabolism of pioglitazone in human microsomes, montelukast (1 μM) and trimethoprim (100 μM) were used as inhibitors of CYP2C8, gemfibrozil (100 μM) as an inhibitor of CYP2C enzymes, ketoconazole (1 μM), itraconazole (3 μM) and troleandomycin (100 μM) as inhibitors of CYP3A4, sulfaphenazole (10 μM) as an inhibitor of CYP2C9, omeprazole (10 μM) as an inhibitor of CYP2C19, furafylline (20 μM) as an inhibitor of CYP1A2, quinidine (10 μM) as an inhibitor of CYP2D6, diethyldithiocarbamate (100 μM) as an inhibitor of CYP2E1, clopidogrel (1 μM) as an inihibitor of CYP2B6 and rifampicin (100 μM) as an inhibitor of CYP2C8 and CYP3A4 (Baldwin et al. 1995; Newton et al. 1995; Bourriéet al. 1996; Ko et al. 1997; Eagling et al. 1998; Wang et al. 2002; Wen et al. 2002; Bidstrup et al. 2004; Isoherranen et al. 2004; Richter et al. 2004; Kajosaari et al. 2005; Walsky et al. 2005b). Troleandomycin, furafylline, diethyldithiocarbamate and clopidogrel were preincubated with human liver microsomes for 15 minutes, after which pioglitazone was added.

The metabolism of pioglitazone by CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5 was screened by using 20 pmol/ml recombinant CYP enzymes. The metabolism of pioglitazone by insect cell control SupersomesTM was also studied.

Determination of IC50 values of CYP2C8 inhibitors. Pioglitazone was coincubated with montelukast (0–3 μM), zafirlukast (0–10 μM), trimethoprim (0–240 μM) and gemfibrozil (0–180 μM) using human liver microsomes and the previously described incubation conditions. The depletion rate of unchanged pioglitazone and the formation rate of the metabolite M-IV were calculated. The inhibitor concentrations producing a 50% decrease in the original enzyme activity (IC50) were determined using nonlinear regression analysis with the program FigP (version 6.0, Biosoft, Cambridge, UK).

Measurement of pioglitazone and metabolite concentrations. After addition of internal standard (rosiglitazone 3 μg ml−1 in methanol-water 20:80 v/v), the samples were applied to the MCX solid phase extraction cartridges (Waters, Milford, Mass, USA), which had been conditioned with 1 ml of methanol and 1 ml of water. The cartridges were connected to Vac-Elut vacuum manifold (Varian, Harbor city, CA, USA) and the samples were allowed to pass through cartridges. The cartridges were washed with 1 ml of 100 mM hydrochloric acid and 1 ml of 70% methanol and finally the analytes were eluted with 1 ml of 2% ammonium hydroxide in methanol. The eluent was evaporated (at 50 °) to dryness under nitrogen stream and the residues were dissolved in a volume of 100 μl of acetonitrile-water (45:55 v/v) and transferred into autosampler vials.

The concentrations of pioglitazone and its metabolites were measured by use of SCIEX API 2000 liquid chromatography-tandem mass spectrometry system (Sciex Division of MDS Inc, Toronto, Ontario, Canada) (Lin et al. 2003). Chromatography was performed on XTerra RP C18 column (3.9×100 mm; Waters Corp., Milford, Massachusetts, USA) using mobile phase gradient consisting of 10 mM ammonium acetate (pH 9.50). The mass spectrometer was operated in positive atmospheric pressure chemical ionization (APCI) mode with selected reaction monitoring (SRM). The ion transitions monitored were m/z 357 to m/z 134 for pioglitazone, m/z 371 to m/z 148 for M-III, m/z 373 to m/z 150 for M-IV, m/z 387 to m/z 164 for M-V, m/z 389 to 166 for M-XI and m/z 358 to m/z 135 for rosiglitazone. These transitions represent the product ions of the [M+H]+ ions. The limit of quantification for pioglitazone was 0.3 nM and the coefficient of variation (CV) was 9.1% at 0.045 μM, 4.9% at 0.6 μM and 4.4% at 2.25 μM (n=12). A signal to noise ratio of 10:1 was used as the limit of quantification for pioglitazone metabolites. Their quantities are given in arbitrary units (AU) relative to the ratio of the peak height of the metabolite to the peak height of the internal standard.

Results

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

Incubation of pioglitazone (1 μM) with human liver microsomes resulted in time- and NADPH-dependent substrate consumption (64% in 60 min.) and metabolite formation. The decline in pioglitazone concentration was log-linear for 60 min. (fig. 2). Two major metabolites, M-IV (primary metabolite) and M-III (secondary metabolite), were found after incubation of pioglitazone with HLM for up to 60 min.; the formation of M-IV was linear up to at least 8 min. The metabolite M-XI (secondary metabolite) was also detectable, but its formation was very low compared to that of M-IV. In addition, trace amounts of the metabolite M-V were found (data not shown).

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Figure 2. Incubation time dependent metabolism (depletion) of pioglitazone (1 μM; left panel) and formation of the metabolite M-IV (AU/ml; right panel) by pooled human liver microsomes. Data represent the mean of duplicate incubations.

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The effects of 13 CYP inhibitors on the metabolism (depletion) of pioglitazone and the formation of M-IV by HML are shown in fig. 3. Among the inhibitors used, montelukast and trimethoprim (selective CYP2C8 inhibitors), gemfibrozil (CYP2C inhibitor), ketoconazole, itraconazole and troleandomycin (CYP3A4 inhibitors) and rifampicin (inhibits both CYP2C8 and CYP3A4) markedly inhibited the metabolism of pioglitazone. Montelukast (1 μM) was the most potent inhibitor, with 63% inhibition of pioglitazone metabolism and 85% inhibition of M-IV formation. In addition, diethyldithiocarbamate had a substantial effect, and sulfaphenazole, omeprazole, quinidine and clopidogrel a minor inhibitory effect on the metabolism of pioglitazone. Inhibitors of CYP2C8 and CYP3A4 reduced the formation of M-III and M-XI in parallel with their effects on M-IV (data not shown).

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Figure 3. Percent of inhibition of pioglitazone (1 μM) metabolic depletion (upper panel) and the formation of the metabolite M-IV (lower panel) by inhibitors of a range of CYP isoforms in human liver microsomes. Data represent the mean of duplicate incubations. Key: MO, montelukast (1 μM); GEM, gemfibrozil (100 μM); TMP, trimethoprim (100 μM); KET, ketoconazole (1 μM); ITR, itraconazole (3 μM); TAO, troleandomycin (100 μM); RIF, rifampicin (100 μM); DDC, diethyldithiocarbamate (100 μM); SZ, sulfaphenazole (10 μM); FF, furafylline (20 μM); OP, omeprazole (10 μM); QD, quinidine (10 μM); CLO, clopidogrel (1 μM).

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A 60 min. incubation of pioglitazone with recombinant CYP2C8 and CYP3A4 decreased the concentrations of pioglitazone by 56% and by 37%, respectively (fig. 4). Ten to 15% of pioglitazone was metabolised when incubated with recombinant CYP3A5, CYP1A2 or CYP2C19. Recombinant CYP2C9, CYP2D6, CYP2E1 and CYP2B6 did not metabolise pioglitazone to an appreciable degree, and control SupersomesTM had no effect. The formation rate of metabolite M-IV was highest in incubations with CYP2C8, while CYP3A4, CYP3A5 and CYP2C19 catalyzed the formation of M-IV at a low rate (fig. 4). Other CYP isoforms did not produce M-IV. Also metabolite M-III was formed by incubation of pioglitazone with CYP2C8, CYP2C19 and CYP1A2 (data not shown).

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Figure 4. Depletion of pioglitazone (% of initial concentration, 1 μM; incubation time 60 min.; upper panel) and formation of the metabolite M-IV (AU/ml, incubation time 8 min.; lower panel) by different human recombinant CYP isoforms (20 pmol/ml). Data represent the mean of duplicate incubations.

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All the used CYP2C8 inhibitors (montelukast, zafirlukast, trimethoprim and gemfibrozil) concentration-dependently inhibited pioglitazone metabolism in human liver microsomes (fig. 5). Montelukast was the most potent inhibitor with an IC50 value (± regression 95% confidence interval) of 0.51±0.44 μM for the elimination of pioglitazone and 0.18±0.09 μM for the formation of M-IV. The corresponding values for zafirlukast, trimethoprim and gemfibrozil were 1.0±0.3 μM (0.78±0.15 μM, for M-IV), 99±22 μM (71±14 μM) and 98±51 μM (59±13 μM), respectively. The CYP2C8 inhibitors concentration dependently inhibited also the formation of the metabolites M-III and M-XI (by >70% at highest inhibitor concentrations used), but no IC50 values were calculated because two metabolic steps are involved in their formation.

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Figure 5. Effect of montelukast, zafirlukast, trimethoprim and gemfibrozil on the metabolism of pioglitazone (1 μM, left panel) and the formation of metabolite M-IV (right panel) in human liver microsomes. Data represent the mean of duplicate incubations.

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Discussion

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

The aim of this in vitro study was to evaluate the contributions of different CYP enzymes to the metabolism of pioglitazone at a clinically relevant pioglitazone concentration. Our results strongly suggest that pioglitazone is in vitro metabolised principally by CYP2C8, and to a lesser extent by CYP3A4. Also the formation of the major metabolite M-IV is predominantly catalyzed by CYP2C8. Other CYP isoforms seem to play a minor role in the biotransformation of pioglitazone.

The potent CYP2C8 inhibitors montelukast (highly selective for CYP2C8) and zafirlukast (Walsky et al. 2005a & 2005b) strongly inhibited the depletion of the parent pioglitazone and the formation of the main primary metabolite M-IV. Trimethoprim, a moderately potent, selective inhibitor of CYP2C8 (Wen et al. 2002), and gemfibrozil, a nonselective CYP2C8 inhibitor (Wen et al. 2001; Wang et al. 2002), also markedly inhibited pioglitazone metabolism. In addition, in incubations of pioglitazone with recombinant CYP2C8 the depletion of pioglitazone was rapid and the rate of formation of M-IV was very high, consistent with a major role for CYP2C8 in the metabolism of pioglitazone.

The potent CYP3A4 inhibitors ketoconazole (1 μM) (Baldwin et al. 1995; Bourriéet al. 1996; Eagling et al. 1998), itraconazole (3 μM) (Back & Tjia 1991; Olkkola et al. 1994; Wang et al. 1999; Isoherranen et al. 2004) and troleandomycin (100 μM) (Newton et al. 1995) inhibited pioglitazone metabolism by 17% to 37%, and the formation of M-IV by 35% to 55%. While itraconazole and troleandomycin are rather selective inhibitors of CYP3A4, ketoconazole inhibits also CYP2C8 activity (by about 30%) at the concentration used (Ong et al. 2000). Thus, inhibition of CYP2C8 may partially explain the inhibitory effect of ketoconazole on the metabolism of pioglitazone. With recombinant CYP3A4 and CYP3A5 enzymes, the rate of pioglitazone depletion was approximately 70% and 50% of that with CYP2C8, and both these CYP3A forms catalysed the formation of M-IV at a low rate compared to CYP2C8. This finding suggests that the CYP3A forms can be more important in the formation of other pioglitazone metabolites than in the formation of M-IV.

In human liver microsomes, inhibitors of CYP2C8 and CYP3A4 had a slightly greater inhibitory effect on the formation of M-IV than on the depletion of pioglitazone (fig. 3, fig. 5). This suggests that enzymes other than CYP2C8 and CYP3A4 can be involved in the formation of other pioglitazone metabolites to a greater extent than in the formation of M-IV. Recombinant CYP2C19 catalysed pioglitazone depletion (10% in 60 min.) and M-IV formation to some extent, and the non-selective CYP2C19 inhibitor omeprazole (inhibits also CYP2C9 and CYP3A4) had a minor effect on the metabolism of pioglitazone. Recombinant CYP1A2 metabolised pioglitazone by 15% in 60 min., and catalysed the formation of a low but detectable amount of M-III. However, the potent, selective CYP1A2 inhibitor fyrafylline (Eagling et al. 1998) had practically no effect on pioglitazone elimination or metabolite formation. These findings suggest that CYP2C19 and CYP1A2 may be involved in the metabolism of pioglitazone, but to a much lesser degree than CYP2C8 and CYP3A4.

Recent studies demonstrate that rifampicin, in addition to its well known inducing effects, may also inhibit CYP2C8 and CYP3A4 enzymes (Bidstrup et al. 2004; Kajosaari et al. 2005). In the present study, rifampicin (100 μM) inhibited slightly pioglitazone elimination (by 14%), and, particularly, the (CYP2C8-mediated) formation of M-IV (by 51%). The CYP2E1 inhibitor diethyldithiocarbamate inhibits also CYP2C8 (Ong et al. 2000) and CYP3A4 activities (Eagling et al. 1998), which explains our finding that diethyldithiocarbamate inhibited pioglitazone metabolism (by 25%) but recombinant CYP2E1 did not metabolise pioglitazone to any appreciable extent. Similarly, the CYP2D6 inhibitor quinidine and the CYP2B6 inhibitor clopidogrel (Newton et al. 1995; Richter et al. 2004; Turpeinen et al. 2005) slightly inhibited pioglitazone metabolism, but the corresponding recombinant CYP did not metabolise pioglitazone. Quinidine can slightly inhibit CYP3A4 (Newton et al. 1995) and clopidogrel inhibits also CYP2C19 (Richter et al. 2004), which could explain the findings. CYP2C9 did not metabolise pioglitazone to any appreciable extent and the CYP2C9 inhibitor sulfaphenazole had only a marginal inhibitory effect, suggesting that CYP2C9 is not significantly involved in pioglitazone elimination.

Our results clarify the discrepancies between previous reports, concerning the roles of CYP2C8, CYP2C9 and CYP3A4 enzymes in the metabolism of pioglitazone (Eckland & Danhof 2000; Hanefeld 2001; Jaakkola et al. 2005). Overall, our results are consistent with a major role of CYP2C8 in the metabolism of pioglitazone, and a less significant role of CYP3A4. On the contrary, we found no evidence of a significant involvement of CYP2C9 in the elimination of pioglitazone. The results concerning the contribution of CYP2C8 are in agreement with recent in vivo findings showing that gemfibrozil (600 mg twice daily) inhibits the metabolism of pioglitazone in humans (Jaakkola et al. 2005). However, although itraconazole inhibited the biotransformation of pioglitazone in vitro, itraconazole (100 mg twice daily) did not significantly increase the AUC of pioglitazone in humans (Jaakkola et al. 2005). This could be explained e.g. by a minor contribution of CYP3A4 (< 10%) to the total elimination of pioglitazone in vivo, being not sufficient to result in a clinically relevant CYP3A4 inhibitory interaction.

According to a previous in vitro study, CYP2C8 is primarily responsible for the metabolism of rosiglitazone, another thiazolidinedione antidiabetic agent, with a minor contribution from CYP2C9 (Baldwin et al. 1999). In human beings, gemfibrozil has increased the AUC of rosiglitazone 2.3 times and that of pioglitazone 3.2 times (Niemi et al. 2003a; Jaakkola et al. 2005). Thus, although rosiglitazone and pioglitazone differ with respect to the minor CYP enzymes involved in their metabolism in vitro, they seem to be similar with respect to the major role of CYP2C8 in their metabolism in vivo.

The peak plasma concentrations of gemfibrozil (30 mg l−1=120 μM) during its normal daily dosing (600 mg twice daily) are only moderately higher than the in vitro IC50 values of gemfibrozil for pioglitazone elimination (98 μM) and M-IV formation (59 μM) observed in the present study. Furthermore, gemfibrozil is highly protein bound (> 95%) in plasma. Thus, inhibition of CYP2C8 by the parent gemfibrozil alone does not explain the strong effect of gemfibrozil on the pharmacokinetics of pioglitazone in humans (AUC increase 3.2 times) (Jaakkola et al. 2005). Recent studies have demonstrated that the main metabolite of gemfibrozil, gemfibrozil glucuronide, is a more potent inhibitor of CYP2C8 than the parent gemfibrozil, suggesting that in vivo the interactions between gemfibrozil and CYP2C8 substrates are, at least in part, due to the metabolite (Shitara et al. 2004; Kajosaari et al. 2005; Ogilvie et al. 2006). Although the parent gemfibrozil is a more potent inhibitor of CYP2C9 than of CYP2C8 in vitro (Wen et al. 2001; Wang et al. 2002; Ogilvie et al. 2006), the converse is true of gemfibrozil glucuronide (Ogilvie et al. 2006). In agreement with this, gemfibrozil inhibits the metabolism of CYP2C8 substrates in humans (Backman et al. 2002; Niemi et al. 2003a & 2003b), but does not reduce the clearance of the CYP2C9 substrate warfarin (Lilja et al. 2005).

In previous reports, montelukast and zafirlukast have potently inhibited CYP2C8 (amodiaquine N-deethylase) activity (Walsky et al. 2005a & 2005b). The inhibitory potency of montelukast has been highly dependent on the microsomal protein concentration, and the IC50 value of montelukast for CYP2C8 has been 0.18 μM at a microsomal protein concentration of 0.3 mg ml−1 (equals the protein concentration used in the present study) (Walsky et al. 2005b). The IC50 values of montelukast for other major CYP isoforms, e.g. CYP2C9 and CYP3A4, have been at least 50 times higher than that for CYP2C8 (Walsky et al. 2005b). The IC50 of zafirlukast for CYP2C8 has been 0.388 μM (microsomal protein concentration 0.025 mg ml−1) (Walsky et al. 2005a). In the present study, montelukast and zafirlukast were potent inhibitors of pioglitazone metabolism with IC50 values of 0.18–0.51 μM and 0.78–1.0 μM, respectively, which are very close to their IC50 for CYP2C8. According to the estimations of Walsky et al. (2005b), montelukast has a potential to cause CYP2C8-mediated drug-drug interactions in humans, although this has not been substantiated in vivo.

Trimethoprim is a selective inhibitor of CYP2C8, with an IC50 value of 54 μM for paclitaxel 6-α-hydroxylation (Wen et al. 2002). In vitro, trimethoprim has inhibited the metabolism of rosiglitazone, another thiazolidinedione compound and CYP2C8 substrate, with an IC50 value of 55 μM, and in vivo, it has increased the AUC of rosiglitazone by 31–37% (Niemi et al. 2004; Hruska et al. 2005). In the present study, trimethoprim inhibited pioglitazone elimination with an IC50 value of 99 μM and M-IV formation with an IC50 value of 71 μM. It is thus possible that, also in vivo, trimethoprim could moderately increase the concentrations of pioglitazone. Further studies are warranted to investigate the effects of montelukast, zafirlukast and trimethoprim on the metabolism of pioglitazone in vivo in humans.

The concentration of pioglitazone used in this study (1 μM, i.e. 357 ng ml−1) was chosen to correspond roughly to the peak plasma pioglitazone concentration in human beings (at a 15 mg dose, about 300–500 ng ml−1) (Eckland & Danhof 2000; Jaakkola et al. 2005). The use of a single substrate concentration has some limitations. However, our aim was to determine the role of different CYP enzymes in pioglitazone metabolism at a clinically relevant concentration in vitro, in order to resolve the discrepancy in earlier information (FDA 1999; Eckland & Danhof 2000; Hanefeld 2001; EMEA 2004) and to predict the risk of drug interactions in humans on the basis of in vitro investigations (Pelkonen et al. 2005). The concentrations of the metabolites were quantified as arbitrary units only, because the reference compounds were not available. However, the linearity of the detector response of the LC-MS-MS assay could be confirmed for the metabolites within the relevant concentration range by means of sample dilution. Accordingly, we were able to determine reliably the contribution of different CYP enzymes on the formation of the metabolites as relative changes in the metabolite concentrations.

To conclude, these in vitro findings strongly suggest that CYP2C8 is primarily responsible for the elimination of pioglitazone with CYP3A4 contributing to a lesser degree. CYP2C9 appears to be insignificant in the total elimination of pioglitazone. Montelukast, zafirlukast, trimethoprim and gemfibrozil markedly inhibit pioglitazone metabolism in vitro. Pioglitazone may be susceptible to CYP2C8 mediated drug interactions also in vivo. However, the pharmacokinetic properties of the inhibitors can be important determinants for their interaction potency in vivo. Accordingly, interaction studies in humans are needed before final conclusions can be made.

Acknowledgements

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References

This study was supported by grants from the Helsinki University Central Hospital Research Fund, the National Technology Agency and the Sigrid Jusélius Foundation, Finland.

References

  1. Top of page
  2. Abstract
  3. Materials and Methods
  4. Results
  5. Discussion
  6. Acknowledgements
  7. References
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