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Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan
(Received November 5, 2006; Accepted January 10, 2007)
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Abstract |
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) and the accumulation of collagen in granulation tissue (Isaji et al., 1987). Recently, a clinical trial regarding the prevention of restenosis after percutaneous transluminal coronary revascularization was performed in Western populations (Holmes et al., 2000). During that trial, it was found that hyperbilirubinemia might be induced by tranilast and the risk of hyperbilirubinemia was increased in individuals with Gilbert's syndrome (Danoff et al., 2004).
Major metabolic pathways of tranilast have been shown to be glucuronidation, 4-demethylation (N-3), and sulfation of N-3 in the data sheet of tranilast provided by Kissei Pharmaceutical (Fig. 1). Tranilast, N-3, and N-3 sulfate were reported to be detected in human urine (Slobodzian et al., 1985). Since the urine sample was hydrolyzed by glucuronidase and/or base in that article, the formation of glucuronide could be speculated by a comparison of the chromatograms before and after hydrolysis. The major metabolites of tranilast in urine in human were tranilast glucuronide and N-3 sulfate and their recoveries were almost the same (unpublished report from Kissei Pharmaceutical). Although phase I metabolism of tranilast was shown to be mainly catalyzed by CYP2C9 in humans (unpublished data from Kissei Pharmaceutical), tranilast metabolism in glucuronidation still remains uncertain.
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Materials and Methods |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tranilast Glucuronidation Assay. A typical incubation mixture (total volume, 0.2 ml) contained 50 mM Tris-HCl buffer (pH7.4), 5 mM MgCl2, 50 µg of alamethicin/mg microsomal protein, 5 mM UDP-GA, 0.5 mg/ml human liver microsomes, and tranilast. The reaction was initiated by the addition of UDP-GA and the mixture was then incubated for 60 min at 37°C. The reaction was terminated by boiling for 5 min. After removal of the protein by centrifugation at 9000g for 5 min, an 80-µl portion of the sample was subjected to high-performance liquid chromatography (HPLC) with a NovaPack Phenyl 4-µm analytical column (3.9 x 150 mm; Waters, Milford, MA). The product formation was measured as described previously (Slobodzian et al., 1985) with slight modifications. The mobile phase was methanol/50 mM sodium dihydrogen phosphate (pH 5.3), 40:60 (v/v) and the flow rate was 1.0 ml/min. The eluent was monitored at 335 nm with a noise-base clean Uni-3 (Union, Gunma, Japan). The retention times of tranilast glucuronide, tranilast, and 7-hydroxycoumarin (internal standard, IS) were 4.4, 10.0, and 3.6 min, respectively (Fig. 2). None of these chromatograms showed any interfering peaks with tranilast glucuronide. For the quantification of tranilast glucuronide, the eluate of the HPLC from the incubation mixture with human liver microsomes, including tranilast glucuronide, was collected with reference to the retention time. A part of the eluate was incubated with 1000 U/ml ß-glucuronidase at 37°C for 24 h. The hydrolyzed tranilast glucuronide was quantified as tranilast by HPLC. Once we determined the peak area per known content of tranilast glucuronide, the ratio was applied to the calculation of the tranilast glucuronide formed in the incubation mixtures.
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Kinetic Analyses. The kinetic studies were performed using human liver microsomes, human jejunum microsomes, and recombinant human UGT1A1 expressed in baculovirus-infected insect cells. When determining the kinetic parameters, the tranilast concentration ranged from 5 µM to 2 mM. The kinetic parameters were estimated from the fitted curves using a computer program, KaleidaGraph (Synergy Software, Reading, PA), designed for nonlinear regression analysis.
Correlation Analyses. The correlations between the tranilast glucuronosyltransferase activity and the other glucuronosyltransferase activities were determined by Pearson's product moment method. A p value of less than 0.05 was considered statistically significant.
Inhibition Analysis of Tranilast Glucuronosyltransferase Activity in Human Liver and Jejunum Microsomes. As described by Watanabe et al. (2002), bilirubin (UGT1A1), ß-estradiol (UGT1A1 and UGT1A9), 4-nitrophenol (UGT1A6 and UGT1A9), imipramine (UGT1A3 and UGT1A4), emodin (UGT1A8 and UGT1A10), and propofol (UGT1A9) are typical substrates for each UGT isoform. These six substrates were investigated for their inhibitory effects on the tranilast glucuronosyltransferase activity. For the determination of the IC50 values, the concentration of tranilast was set at 100 µM. The final concentration of the organic solvents in the reaction mixture was <2% (v/v). The tranilast glucuronosyltransferase activities in pooled human liver microsomes and human jejunum microsomes (HJM0040) at 100 µM tranilast were determined as described above.
Inhibition of N-3 and N-4 on Tranilast Glucuronosyltransferase Activity in Human Liver and Jejunum Microsomes. The inhibition of N-3, a phase I metabolite of tranilast, and N-4, a structural isomer of N-3, on the tranilast glucuronosyltransferase activity was also investigated in pooled human liver microsomes and individual human jejunum microsomes. For the determination of the IC50 values, the concentration of tranilast was set at 100 µM. For the determination of the Ki values in pooled human liver microsomes, the concentrations of tranilast, N-3, and N-4 ranged from 10 to 160 µM, 0 to 150 µM, and 0 to 30 µM, respectively. The Ki values were estimated from the fitted curve using a computer program (K cat; BioMetallics, Princeton, NJ) designed for nonlinear regression analysis.
Tranilast 4-Demethylation Assay. A typical incubation mixture (total volume, 0.2 ml) contained 100 mM Tris-HCl buffer (pH7.4), 0.2 mg/ml human liver microsomes, an NADPH-generating system (0.5 mM NADP+, 5 mM glucose 6-phosphate, 5 mM MgCl2, and 1 U/ml glucose-6-phosphate dehydrogenase), and tranilast. The reaction was initiated by the addition of the NADPH-generating system and was then incubated for 30 min at 37°C. The reaction was terminated by adding 100 µl of ice-cold methanol. 7-Hydroxycoumarin was added as an IS. After removal of the protein by centrifugation at 9000g for 5 min, an 80-µl portion of the sample was subjected to HPLC. The product formation was measured using the same method for tranilast glucuronide except the mobile phase. The mobile phase was methanol/50 mM sodium dihydrogen phosphate (pH 5.3), 33:67 (v/v). The retention times of N-3, tranilast, and 7-hydroxycoumarin (IS) were 9.8, 21.4, and 4.7 min, respectively. None of these chromatograms showed any interfering peaks with N-4 (data not shown).
The kinetic studies were performed using human liver microsomes. In determining the kinetic parameters, the tranilast concentration ranged from 2 to 500 µM. Kinetic parameters were estimated from the fitted curves using the computer program KaleidaGraph (Synergy Software) designed for nonlinear regression analysis.
Inhibition of Tranilast, N-3, and N-4 on Bilirubin Glucuronosyltransferase Activity in Human Live Microsomes. A typical incubation mixture (total volume, 0.2 ml) contained 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 50 µg of alamethicin/mg microsomal protein, 2 mM UDP-GA, 0.5 mg/ml human liver microsomes, 10 µM bilirubin, and tranilast (N-3 or N-4). The reaction was initiated by the addition of UDP-GA and was then incubated for 30 min at 37°C. The reaction was terminated by adding 100 µl of ice-cold methanol. After removal of the protein by centrifugation at 9000g for 5 min, a 50-µl portion of the sample was subjected to HPLC with a Develosil C30 5-µm analytical column (4.6 x 150 mm; Nomura Chemical, Aichi, Japan). The product formation was measured as described previously (Luquita et al., 2001) with slight modifications. The mobile phase was 55% methanol/50 mM potassium dihydrogen phosphate and the flow rate was 1.0 ml/min. The eluent was monitored at 450 nm with a noise-base clean Uni-3 (Union). The final concentration of the organic solvents in the reaction mixture was <2% (v/v).
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Results |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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Tranilast Glucuronosyltransferase Activity in Recombinant UGT Isoforms. The recombinant UGT isoforms expressed in baculovirus-infected insect cells were used to determine their tranilast glucuronosyltransferase activities (Fig. 4A). UGT1A1 exhibited the highest tranilast glucuronosyltransferase activity (13.5 pmol/min/mg protein). UGT1A3, UGT1A8, UGT1A9, and UGT1A10 exhibited low tranilast glucuronosyltransferase activities.
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Interindividual Variability of Tranilast Glucuronosyltransferase Activity from 22 Human Livers and Correlation Analyses. The tranilast glucuronosyltransferase activities in microsomes from 22 human livers were determined at 40 µM tranilast (Fig. 5, top). The tranilast glucuronosyltransferase activity ranged from 1.9 pmol/min/mg protein in HG93 to 18.3 pmol/min/mg protein in HH31. The interindividual variability in the tranilast glucuronosyltransferase activity was 9.5-fold. Correlation analyses were performed between the tranilast glucuronosyltransferase activity and bilirubin (UGT1A1), ß-estradiol (UGT1A1), etoposide (UGT1A1), trifluoperazine (UGT1A4), propofol (UGT1A9), or morphine glucuronosyltransferase activities (UGT2B7) provided by the manufacturer (Table 1). The etoposide glucuronosyltransferase activities (UGT1A1) were measured in our laboratory according to the method of Watanabe et al. (2002). Since we could not obtain those activities in all individual liver microsomes, correlation analyses among bilirubin, etoposide, and morphine glucuronosyltransferase activities were performed using 11 of 22 liver microsomes. The tranilast glucuronosyltransferase activities in individual human liver microsomes were significantly correlated with the ß-estradiol (r = 0.956, p < 0.0001), bilirubin (r = 0.937, p < 0.0001), and propofol (r = 0.449, p < 0.05) glucuronosyltransferase activities. The tranilast glucuronosyltransferase activities did not correlate with the trifluoperazine (r = 0.179) and morphine (r = 0.257) glucuronosyltransferase activities.
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Inhibition Analyses of Glucuronosyltransferase Activity in Human Liver or Jejunum Microsomes. The inhibitory effects of bilirubin, ß-estradiol, 4-nitrophenol, imipramine, emodin, and propofol on the tranilast glucuronosyltransferase activity in human liver and jejunum microsomes were determined. As shown in Fig. 6A, the tranilast glucuronosyltransferase activity in pooled human liver microsomes was inhibited by bilirubin (IC50 = 123.9 µM). As shown in Fig. 6B, the activity in human jejunum microsomes was strongly inhibited by bilirubin (IC50 = 81.1 µM) and ß-estradiol (IC50 = 75.3 µM).
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Inhibition of N-3 and N-4 on Tranilast Glucuronosyltransferase Activity in Human Liver and Jejunum Microsomes. The inhibitory effects of N-3 and N-4 on the tranilast glucuronosyltransferase activity in human liver and jejunum microsomes were determined. The tranilast glucuronosyltransferase activities in human liver and jejunum microsomes were strongly inhibited by both N-3 and N-4 (Fig. 6). The IC50 values of N-3 and N-4 were 141.7 and 81.3 µM, respectively, in liver microsomes and 82.8 and 45.9 µM, respectively, in jejunum microsomes. The inhibition pattern of N-3 was competitive. The Kis value of N-3 for the tranilast glucuronosyltransferase activity in human liver microsomes was 52.8 µM (Fig. 7). On the other hand, the inhibition pattern of N-4 was mixed. The Kis and Kii values of N-4 for the tranilast glucuronosyltransferase activity in human liver microsomes were 42.6 and 181.1 µM, respectively (Fig. 7).
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Interindividual Variability of Tranilast 4-Demethylase Activity from 22 Human Livers and Correlation Analyses. The tranilast 4-demethylase activities in microsomes from 22 human livers were determined at 40 µM tranilast. The interindividual variability in the tranilast glucuronosyltransferase activity was at most 16.4-fold (Fig. 5, bottom). The tranilast 4-demethylase activity ranged from 4.5 pmol/min/mg protein in HG32 to 73.1 pmol/min/mg protein in HG30. Correlation analyses were performed between the tranilast 4-demethylase activity and phenacetin O-deethylase activity (CYP1A2), coumarin 7-hydroxylase activity (CYP2A6), S-mephenytoin N-demethylase activity (CYP2B6), paclitaxel 6
-hydroxylase activity (CYP2C8), diclofenac 4'-hydroxylase activity (CYP2C9), S-mephenytoin 4'-hydroxylase activity (CYP2C19), bufuralol 1'-hydroxylase activity (CYP2D6), chlorzoxazone 6-hydroxylase activity (CYP2E1), testosterone 6ß-hydroxylase activity (CYP3A4), or lauric acid 12-hydroxylase activity (CYP4A) provided by the manufacturer. The tranilast 4-demethylase activities in the 22 human liver microsomes were significantly correlated with the diclofenac 4'-hydroxylase activities (r = 0.825, p < 0.0001) and the paclitaxel 6-hydroxylase activities (r = 0.576, p < 0.01).
Inhibition of Tranilast, N-3, and N-4 on Bilirubin Glucuronosyltransferase Activity in Human Liver Microsomes. The inhibitory effects of tranilast, N-3, and N-4 on the bilirubin glucuronosyltransferase activity in human liver microsomes were determined. The bilirubin glucuronosyltransferase activity was strongly inhibited by tranilast, N-3, and N-4. The IC50 values of tranilast, N-3, and N-4 were 28.7, 76.9, and 62.0 µM, respectively.
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Discussion |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
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The tranilast glucuronosyltransferase activity was reduced at high substrate concentrations (>500 µM) in human liver and jejunum microsomes and recombinant UGT1A1. The reason for this phenomenon was unclear, but substrate and/or metabolite inhibition may be involved. Further study is needed to clarify the mechanism of the inhibition. The maximum serum concentration of tranilast in humans has been reported to be 37.0 µM after single oral administration at the therapeutic dose of 100 mg (Slobodzian et al., 1985). As reported by Kissei Pharmaceutical, the maximum serum concentration of tranilast was 67.8 µM after taking 2.5 mg/kg tranilast three times per day for 5 days. In clinical practice, the concentration of tranilast is unlikely to reach 500 µM. Therefore, the kinetic parameters fitted to the Michaelis-Menten equation with <500 µM tranilast seems to be reasonable.
The in vitro intrinsic clearance (CLint) is calculated using the following equation (Obach et al., 1997; Soars et al., 2002): CLint = [Vmax/Km] x [microsomal protein/tissue (mg/g)] x [tissue/body weight (g/kg)]. Soars et al. (2002) reported that there were 45 mg of microsomal protein/g of liver and 20 g of liver/kg of body weight. The CLint in liver of tranilast was calculated to be 181.7 µl/min/kg. They also reported that there are 3 mg of microsomal protein/g of intestine and 30 g of intestine/kg of body weight (Soars et al., 2002). The CLint in intestine was estimated to be 76.3 µl/min/kg. The glucuronosyltransferase activity has been reported to differ according to regions of the intestine in humans (Strassburg et al., 2000). The UGT1A1 activity in humans was higher in upper intestine than lower intestine (Basu et al., 2004). Although the UGT activity in the intestine may differ from that in the liver, the tranilast glucuronosyltransferase activity in the intestine might be approximately 40% of that in the liver. UGT1A1 has shown polymorphic metabolism. Particularly, the relationship between the toxicity of irinotecan hydrochloride and the UGT1A1 genotype has been extensively studied (Ando et al., 2005). As well as irinotecan hydrochloride, UGT1A1 genetic polymorphism would affect the tranilast pharmacokinetics.
Another metabolic pathway of tranilast is N-3 formation catalyzed by CYP2C9 (unpublished data, Kissei Pharmaceutical). We first analyzed the kinetics of tranilast 4-demethylase activity in human liver microsomes. The Km value of N-3 formation in human liver microsomes was 37.1 ± 3.1 µM, which was similar to that of the tranilast glucuronosyltransferase activity. There was large interindividual variability (>16-fold) in the N-3 formation. In CYP2C9, several polymorphic alleles have been reported to have decreased enzyme activity in vitro and in vivo (Takahashi et al., 1998; Blaisdell et al., 2004). The frequency of poor metabolizing alleles such as CYP2C9*2 and CYP2C9*3 was different between ethnic groups (Sullivan-Klose et al., 1996; Blaisdell et al., 2004). Therefore, the genetic polymorphisms of CYP2C9 and UGT1A1 would play important roles in the pharmacokinetics of tranilast.
In the present study, N-3 was demonstrated to inhibit tranilast glucuronosyltransferase activity. Although the concentration of N-3 in the liver is unknown, N-3 could affect tranilast glucuronosyltransferase activity, leading to altered pharmacokinetics of tranilast. N-3 is further metabolized to be N-3 glucuronide and could be slightly detected in human urine (unpublished data, Kissei Pharmaceutical). Since the inhibition pattern of N-3 on tranilast glucuronosyltransferase activity was competitive, the glucuronidation of N-3 might be catalyzed by UGT1A1. N-4, a structural isomer of N-3, also inhibited tranilast glucuronosyltransferase activity. However, there are no reports that N-4 could be detected in humans. Because glucuronidation of a drug may be inhibited by its cytochrome P450 metabolites, the inhibition by metabolites should be kept in mind when estimating the pharmacokinetics.
Recently, the PRESTO (prevention of restenosis with tranilast and its outcomes) study was performed in Western populations because tranilast may have a benefit in preventing restenosis after percutaneous transluminal coronary revascularization (Holmes et al., 2000). During the phase III clinical trial, an increase in serum unconjugated bilirubin was observed in 12% of white subjects (Danoff et al., 2004). It was suspected that tranilast-induced hyperbilirubinemia might be caused by the inhibition by tranilast and N-3 of bilirubin glucuronosyltransferase activity. Danoff et al. (2004) suggested that tranilast-induced hyperbilirubinemia might be related to genetic polymorphisms of UGT1A1. As reported by Kissei Pharmaceutical, the frequencies of liver dysfunction and jaundice caused by tranilast were 0.62 and 0.008%, respectively, in Japanese. The frequency of hyperbilirubinemia in Japanese is unknown but seems to be lower than that in white subjects. The reason for this phenomenon may be interethnic variability in the UGT1A1 allelic frequency. In the case of UGT1A1*28, which is mainly responsible for Gilbert's syndrome, its allelic frequency has been shown to be 35.7 to 41.3% in whites (Monaghan et al., 1996, 1997) but 13.8% in Asians (Ando et al., 1998). In patients with Gilbert's syndrome, the unconjugated bilirubin concentration may be significantly elevated after tranilast administration because of the inhibition of UGT1A1 activity by tranilast and N-3. In addition, as mentioned above, the allele frequency of the CYP2C9 poor metabolizer was higher in whites than in Japanese (Nasu et al., 1997; Scordo et al., 2001). Thus, the genetic risk for the elevation of tranilast concentration would be higher in whites than in Japanese. The occurrence of adverse reactions from tranilast might be explained by the enzyme inhibition and the genetic polymorphisms of both phase I and phase II enzymes.
In conclusion, it was clarified that the tranilast glucuronosyltransferase activity was mainly catalyzed by UGT1A1 in human liver and intestine. N-3, a phase I metabolite of tranilast, inhibited the tranilast glucuronosyltransferase activity, suggesting that the inhibition by a phase I metabolite may be noteworthy when estimating the glucuronidation of a drug. The inhibition by tranilast and N-3 of the bilirubin glucuronosyltransferase activity may be partly responsible for tranilast-induced hyperbilirubinemia. We should keep in mind that UGT1A1 substrates may inhibit the bilirubin glucuronosyltransferase activity leading to hyperbilirubinemia, and that phase I metabolites can affect the glucuronidation of its parent drug.
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Acknowledgments |
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Footnotes |
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ABBREVIATIONS: N-3, 4-demethyltranilast; UGT, UDP-glucuronosyltransferase; N-4, 3-demethytranilast; UDP-GA, UDP-glucuronic acid; HPLC, high-performance liquid chromatography; IS, internal standard; CLint, intrinsic clearance.
Address correspondence to: Dr. Tsuyoshi Yokoi, Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kakuma-machi, Kanazawa 920-1192, Japan. E-mail: tyokoi{at}kenroku.kanazawa-u.ac.jp
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