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Characterization of N-Glucuronidation of 4-(5-Pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile (FYX-051): A New Xanthine Oxidoreductase Inhibitor

Research Laboratories 2 (K.O., T.N., T.I., O.N.) and Research Laboratories 1 (T.S.), Fuji Yakuhin Co., Ltd., Nishi-ku, Saitama, Japan

(Received June 14, 2007; accepted August 27, 2007)

	Abstract

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In humans, orally administered 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile (FYX-051) is excreted mainly as triazole N₁- and N₂-glucuronides in urine. It is important to determine the enzyme(s) that catalyze the metabolism of a new drug to estimate individual differences and/or drug-drug interactions. Therefore, the characterization and mechanism of these glucuronidations were investigated using human liver microsomes (HLMs), human intestinal microsomes (HIMs), and recombinant human UDP-glucuronosyltransferase (UGT) isoforms to determine the UGT isoform(s) responsible for FYX-051 N₁- and N₂-glucuronidation. FYX-051 was metabolized to its N₁- and N₂-glucuronide forms by HLMs, and their K_m values were 64.1 and 72.7 µM, respectively; however, FYX-051 was scarcely metabolized to its glucuronides by HIMs. Furthermore, among the recombinant human UGT isoforms, UGT1A1, UGT1A7, and UGT1A9 catalyzed the N₁- and N₂-glucuronidation of FYX-051. To estimate their contribution to FYX-051 glucuronidation, inhibition analysis with pooled HLMs was performed. Mefenamic acid, a UGT1A9 inhibitor, decreased FYX-051 N₁- and N₂-glucuronosyltransferase activities, whereas bilirubin, a UGT1A1 inhibitor, did not affect these activities. Furthermore, in the experiment using microsomes from eight human livers, the N₁- and N₂-glucuronidation activity of FYX-051 was found to significantly correlate with the glucuronidation activity of propofol, a specific substrate of UGT1A9 (N₁: r² = 0.868, p < 0.01; N₂: r² = 0.775, p < 0.01). These results strongly suggested that the N₁- and N₂-glucuronidation of FYX-051 is catalyzed mainly by UGT1A9 in human livers.

Glucuronidation is one of the most important phase II metabolic reactions and is catalyzed by UDP-glucuronosyltransferase (UGT). UGTs are classified into two families (UGT1 and UGT2) on the basis of their primary amino acid sequences. To date, 19 human UGT isoforms have been identified (Mackenzie et al., 2005). Identification of enzymes involved in drug metabolism is important to predict drug-drug interactions. Therefore, it is preferable to identify the UGT isoforms that catalyze glucuronidation during the early stage of development of a new drug.

Several examples of xenobiotic N-glucuronidations have been reported thus far (Smith and Williams, 1949; Green and Tephly, 1998). Xenobiotic N-glucuronidation is classified into that involving aliphatic and aromatic conjugations; the latter includes N-glucuronidation of conjugates such as pyridine, pyridazine, pyrimidine, imidazole, triazole, and tetrazole. Some cases of triazole N-glucuronidation such as that of methylbiphenyl triazole and posaconazole have been documented (Huskey et al., 1994a; Krieter et al., 2004). However, N-glucuronidation occurring at two positions in one aromatic ring is unique and extremely rare (Huskey et al., 1994a; Yan et al., 2006).

The present study was performed to identify the human UGT isoforms responsible for the N₁- and N₂-glucuronidation of FYX-051. Furthermore, kinetics and inhibition analysis using human liver microsomes (HLMs) and recombinant human UGT-expressing in baculovirus-infected insect cells were also investigated.

	Materials and Methods

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FYX-051 (Fig. 1) was synthesized by Fuji Yakuhin Co., Ltd. (Saitama, Japan). Two FYX-051 N-glucuronides (triazole N₁- and N₂-glucuronide) were purified from human urine by Fuji Yakuhin Co., Ltd., as described previously (Nakazawa et al., 2006) (Fig. 1). Bilirubin, eugenol, 7-hydroxy-4-trifluoromethylcoumarin (7-HFC), and mefenamic acid were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Trifluoperazine and estradiol were purchased from Sigma-Aldrich (St. Louis, MO). Propofol was purchased from Tokyo Kasei Kogyo Co., Ltd (Tokyo, Japan). Pooled and eight individual HLMs were purchased from Tissue Transformation Technologies (Edison, NJ). Pooled human intestinal microsomes (HIMs) were purchased from XenoTech, LLC (Lenexa, KS). Recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cells (Supersomes), UGT cofactor mixture A (containing 25 mM UDP-glucuronic acid in water), and UGT cofactor mixture B (containing 250 mM Tris-HCl, 40 mM MgCl₂, and 0.125 mg/ml alamethicin in water) were purchased from BD Gentest (Woburn, MA). The activity of each UGT isoform was determined using the typical corresponding substrate: estradiol for UGT1A1 (376 pmol/min/mg protein) and UGT1A3 (220 pmol/min/mg protein); trifluoperazine for UGT1A4 (527 pmol/min/mg protein); 7-HFC for UGT1A6 (5370 pmol/min/mg protein), UGT1A7 (4892 pmol/min/mg protein), UGT1A8 (698 pmol/min/mg protein), UGT1A9 (2883 pmol/min/mg protein), UGT1A10 (249 pmol/min/mg protein), UGT2B4 (289 pmol/min/mg protein), UGT2B7 (1160 pmol/min/mg protein), and UGT2B15 (1443 pmol/min/mg protein); and eugenol for UGT2B17 (838 pmol/min/mg protein) according to the instructions provided by BD Gentest. All other reagents were of the highest grade commercially available and were purchased from Kishida Chemical Co. (Osaka, Japan).

View larger version (8K): [in this window] [in a new window]   FIG. 1. Chemical structure of FYX-051 (A) and its N1-glucuronide (B) and N2-glucuronide (C) metabolites

Glucuronidation of FYX-051. A typical incubation mixture (300-µl total volume) contained 50 mM Tris-HCl buffer (pH 7.5), 8 mM MgCl₂, 2 mM UDP-glucuronic acid, 25 µg/ml alamethicin, and FYX-051 with 1 mg/ml HLMs, 1 mg/ml HIMs, or 0.5 mg/ml recombinant human UGTs. FYX-051 was dissolved in dimethyl sulfoxide (DMSO), and the final concentration of DMSO in the reaction mixture was 1% (v/v). After preincubation at 37°C for 5 min, the reactions were initiated by the addition of UDP-glucuronic acid. The reaction mixtures were incubated at 37°C for 60 min, and the reaction was terminated by adding 3 times the volume of ice-cold methanol containing the FYX-051 analog as an internal standard. Standard curves were prepared as described above, except that incubation was not included and N₁- and N₂-glucuronide standards were used instead of FYX-051. Both glucuronides were quantified from these standard curves ranging from 0.012 to 12 µM (limit of quantitation, 0.2 pmol/min/mg protein). After centrifugation at 19,000g for 10 min, the supernatants obtained were assayed by liquid chromatography tandem mass spectrometry (LC-MS/MS).

LC-MS/MS Analysis of N₁- and N₂-Glucuronides of FYX-051. The FYX-051 glucuronides were quantified by LC-MS/MS, according to a previously described method (Nakazawa et al., 2006) with a slight modification. A 5-µl aliquot of the supernatant was injected into a LC-MS/MS unit consisting of a 1100 series high-performance liquid chromatography system (Agilent Technologies, Palo Alto, CA) and API 3000 (Applied Biosystems/MDS Sciex, Foster City, CA). High-performance liquid chromatography separations were carried out using a Mightysil RP-18 GP column (i.d., 2.0 mm x150 mm; particle size, 5 µm; Kanto Chemical Co. Inc., Tokyo, Japan) at a flow rate of 0.2 ml/min with 0.5% acetic acid-acetonitrile (83:17, v/v). The column temperature was maintained at 35°C. Ionization was conducted in the turbo ion spray and negative ion modes at 450°C. N₁- and N₂-glucuronides were analyzed as [M–H]^– ions in the selected reaction monitoring mode (N₁- and N₂-glucuronide: m/z 423 247) and detected at 2.5 min for N₂-glucuronide and 5.0 min for N₁-glucuronide. All LC-MS/MS data were integrated using Analyst (version 1.4; Applied Biosystems/MDS Sciex).

Kinetic Analysis of N₁- and N₂-Glucuronidation in HLMs and Recombinant Human UGT1A9. Kinetic studies were performed using pooled HLMs and recombinant human UGT1A9. N₁- and N₂-glucuronosyltransferase activities with FYX-051 concentrations ranging from 1 to 200 µM for pooled HLMs and from 0.5 to 100 µM for recombinant human UGT1A9 were determined. The kinetic parameters were estimated as follows from the fitted curves by the Michaelis-Menten equation by using WinNolin (version 2.1; Pharsight, Mountain View, CA): v = V_max x [S]/(K_m + [S]), where v, V_max, [S], and K_m are the rate of reaction, maximum velocity, substrate concentration, and Michaelis-Menten constant, respectively.

Inhibition Analysis of N₁- and N₂-Glucuronidation in HLMs. Bilirubin and mefenamic acid were tested for their inhibitory effects on the FYX-051 N₁- and N₂-glucuronosyltransferase activities in pooled HLMs. Bilirubin is a well known typical substrate, and it was used for the inhibition analysis of UGT1A1 (Bosma et al., 1994; King et al., 1996; Williams et al., 2002). Mefenamic acid was used for inhibition analysis of UGT1A9 (McGurk et al., 1996; Wynalda et al., 2003; Tachibana et al., 2005). Bilirubin and mefenamic acid were dissolved in DMSO, and their concentrations in the reaction mixture were adjusted to 20 and 100 µM, respectively. Glucuronosyltransferase activities at 50 µM FYX-051 were determined in a manner similar to that described above.

Interindividual Variability and Correlation Analysis of N₁- and N₂-Glucuronidation in HLMs. In the correlation analysis, propofol was tested as a substrate for UGT1A9 (Burchell et al., 1995; McGurk et al., 1998). FYX-051 N₁- and N₂-glucuronosyltransferase activities with 10 µM FYX-051 and propofol glucuronosyltransferase activity with 100 µM propofol were measured in the microsomes obtained from eight human livers. Correlation analysis between the FYX-051 N₁- or N₂- and propofol glucuronidation activities was carried out using Pearson's moment method. p < 0.05 was considered statistically significant.

	Results

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N₁- and N₂-Glucuronidation of FYX-051 by HLMs and HIMs. Kinetic analysis of the N₁- and N₂-glucuronidation of FYX-051 in pooled HLMs was performed. N₁- and N₂-glucuronidation was fitted to the Michaelis-Menten kinetics that manifests as a linear Eadie-Hofstee plot, as shown in Fig. 2 and Table 1. The apparent K_m and V_max values were 64.1 µM and 87.5 pmol/min/mg protein, respectively, for N₁-glucuronidation and 72.7 µM and 46.5 pmol/min/mg protein, respectively, for N₂-glucuronidation. The K_m/V_max value of N₁-glucuronidation was approximately 2.1-fold higher than that of N₂-glucuronidation. On the other hand, glucuronidation of FYX-051 was scarcely observed in HIMs (<0.2 pmol/min/mg protein), whereas that of 7-HFC, a substrate of intestinal UGTs, was observed (863.7 pmol/min/mg protein).

View larger version (16K): [in this window] [in a new window]   FIG. 2. Kinetics of FYX-051 N1-glucuronidation (A) and N2-glucuronidation (B) in pooled human liver microsomes. FYX-051 N-glucuronosyltransferase activities were determined as described under Materials and Methods. Each point represents the mean of duplicate determinations. Each inset shows the Eadie-Hofstee plot of the experimental data.

N₁- and N₂-Glucuronidation of FYX-051 by Recombinant Human UGT Isoforms. The FYX-051 N₁- and N₂-glucuronosyltransferase activities of 12 recombinant UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cells were determined. UGT1A9 exhibited an extremely high N₁-glucuronosyltransferase activity of 85.6 pmol/min/mg protein and N₂-glucuronosyltransferase activity of 29.5 pmol/min/mg protein (Fig. 3). UGT1A1 and UGT1A7 exhibited low N₁- and N₂-glucuronosyltransferase activities (UGT1A1: N₁ and N₂, 0.4 and 0.3 pmol/min/mg protein, respectively; UGT1A7: N₁ and N₂, 0.9 and 0.3 pmol/min/mg protein, respectively). No other UGT isoforms showed N₁- or N₂-glucuronosyltransferase activity (<0.2 pmol/min/mg protein).

View larger version (8K): [in this window] [in a new window]   FIG. 3. Screening of UGT isoforms for the formation of an N-glucuronide conjugate from FYX-051 at a concentration of 10 µM. Each column represents the mean of duplicate determinations. The lower limit of quantitation of the assay under these conditions was 0.2 pmol/min/mg protein.

Inhibition Analysis of N₁- and N₂-Glucuronidation in HLMs. The effects of bilirubin and mefenamic acid on the catalysis of FYX-051 N₁- and N₂-glucuronidation in pooled HLMs were tested. Mefenamic acid inhibited both N₁- and N₂-glucuronidation of FYX-051, and the inhibition ratios were 60.6 and 63.7% at 20 µM and 93.9 and 93.4% at 100 µM, respectively (Fig. 4). However, the inhibition ratios of bilirubin for N₁- and N₂-glucuronidation were 9.1 and 13.3%, respectively, at a concentration of 100 µM (Fig. 4).

View larger version (15K): [in this window] [in a new window]   FIG. 4. Effects of bilirubin and mefenamic acid on the N1- and N2-glucuronidation of FYX-051 in pooled human liver microsomes. FYX-051 N-glucuronosyltransferase activities were measured at an FYX-051 concentration of 50 µM coincubated with either 20 µM bilirubin or 100 µM mefenamic acid. The control activities for FYX-051 N1- and N2-glucuronidation in the pooled human liver microsomes in the absence of inhibitors were 33.0 ± 3.8 and 16.6 ± 1.6 pmol/min/mg protein, respectively. Each column represents the mean + S.D. of triplicate determinations.

Interindividual Variability and Correlation Analysis of N₁- and N₂-Glucuronidation in HLMs. The N₁- and N₂-glucuronosyltransferase activities in microsomes from eight human livers were determined at an FYX-051 concentration of 10 µM. The interindividual variabilities were 5.5 to 16.0 (11.1 ± 3.9) and 2.6 to 8.1 (5.6 ± 2.0) pmol/min/mg protein for N₁- and N₂-glucuronidation, respectively (Fig. 5). The mean N₁-glucuronidation activity was 2.0-fold higher than the N₂-glucuronidation activity. The activities of N₁- and N₂-glucuronosyltransferase toward FYX-051 in the HLMs correlated well with the propofol glucuronosyltransferase activities (Fig. 6) (N₁-glucuronidation: r² = 0.868, p < 0.01; N₂-glucuronidation: r² = 0.775, p < 0.01).

View larger version (20K): [in this window] [in a new window]   FIG. 5. Interindividual variability in FYX-051 N1- and N2-glucuronosyltransferase activities in microsomes from eight human livers. FYX-051 N1- and N2-glucuronosyltransferase activities were determined at 10 µM FYX-051. Each column represents the mean + S.D. of triplicate determinations.

View larger version (18K): [in this window] [in a new window]   FIG. 6. Correlation between FYX-051 N1-glucuronosyltransferase (A) or N2-glucuronosyltransferase (B) activities and propofol glucuronosyltransferase activities in microsomes from eight human livers. FYX-051 N1- and N2-glucuronosyltransferase activities and propofol glucuronosyltransferase activities were determined at 10 µM FYX-051 and 100 µM propofol, respectively.

View larger version (19K): [in this window] [in a new window]   FIG. 7. Kinetics of FYX-051 N1-glucuronidation (A) and N2-glucuronidation (B) in recombinant human UGT1A9. FYX-051 N-glucuronosyltransferase activities were determined as described under Materials and Methods. Each point represents the mean of duplicate determinations. Each inset shows the Eadie-Hofstee plot of the experimental data.

	Discussion

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The present study was performed to identify the human UGT isoforms that are responsible for the N₁- and N₂-glucuronidation of FYX-051. First, the glucuronidation of FYX-051 by HLMs and HIMs was examined. FYX-051 was metabolized to its N₁- and N₂-glucuronides by HLMs; their respective K_m values were 64.1 and 72.7 µM, and their respective V_max values were 87.5 and 46.5 pmol/min/mg protein (Fig. 2). On the other hand, the glucuronidation of FYX-051 by HIMs was scarcely observed. These results suggested that both the N₁- and N₂-glucuronides of FYX-051 were generated mainly in the liver. Therefore, we focused on the hepatic UGT isoforms that are involved in the glucuronidation of FYX-051.

Next, the glucuronidation of FYX-051 by 12 commercially available recombinant human UGTs was examined. Among them, the FYX-051 N₁- and N₂-glucuronidation activities of UGT1A1 (0.4 and 0.3 pmol/min/mg protein), UGT1A7 (0.9 and 0.3 pmol/min/mg protein), and UGT1A9 (85.6 and 29.5 pmol/min/mg protein) were observed, but the FYX-051 N₁- and N₂-glucuronidation activities of other UGTs were less than 0.2 pmol/min/mg protein at 10 µM FYX-051 (Fig. 3). It is well known that UGT1A1 is predominantly expressed in the liver and intestine and that it mainly catalyzes the O-glucuronidation of substances such as bilirubin and catechol estrogens (Radominska-Pandya et al., 1999). In addition, UGT1A9 is reported to have a broad spectrum of action and is widely expressed in the kidney, colon, and reproductive organs as well as in the liver (Albert et al., 1999; Radominska-Pandya et al., 1999; Tukey and Strassburg, 2000). On the other hand, UGT1A7 is expressed mainly in the gastric epithelium and not in the liver (Strassburg et al., 1998). Therefore, because the UGT isoforms involved in the glucuronidation of FYX-051 were thought to be UGT1A1 and/or UGT1A9, the contribution of each UGT isoform in the glucuronidation of FYX-051 was investigated using HLMs.

At 100 µM, bilirubin, an inhibitor of UGT1A1, did not affect the HLM activities of FYX-051 N₁- and N₂-glucuronidation; however, at 100 µM mefenamic acid, an inhibitor of UGT1A9, decreased these activities to less than 10% of the control (Fig. 4). Furthermore, the experiment with HLMs demonstrated a significant correlation between the UGT1A9 glucuronidation activities for FYX-051 and propofol as specific substrates (Burchell et al., 1995; McGurk et al., 1998) (Fig. 6). These results strongly suggested that both the N₁- and N₂-glucuronidation of FYX-051 are catalyzed mainly by UGT1A9.

The present study demonstrated that the K_m values of N₁- and N₂-glucuronidation by recombinant UGT1A9 were 13.2 and 12.9 µM, respectively, and these values are lower than those of N₁- and N₂-glucuronidation by HLMs (Table 1). These results appeared to be in conflict with the conclusion that both N₁- and N₂-glucuronidation of FYX-051 is catalyzed mainly by UGT1A9. It was thought that the difference between the protein binding of FYX-051 between recombinant UGT1A9 and HLMs might account for this discrepancy because the apparent K_m value is increased by high protein binding. However, protein binding of FYX-051 at 1 to 100 µM was scarcely observed for both UGT1A9 and HLMs (<5%). On the other hand, Soars et al. (2003) reported that the K_m values obtained for propofol using expressed UGT1A9 were almost 10 times lower than those observed using HLMs. Fujiwara et al. (2007) indicated that this difference might result from protein-protein interaction as the K_m value of propofol O-glucuronide formation by UGT1A9 was significantly increased from 59.8 to 173.1 µM by its coexpression with UGT1A1 or UGT1A6. Thus, our result might be explained by the phenomenon of protein-protein interaction.

For the safe usage of a drug, it is helpful to determine the interindividual variability in its metabolic activity. In this study, the FYX-051 N₁- and N₂-glucuronosyltransferase activities in microsomes from eight human livers ranged from 5.5 to 16.0 and from 2.6 to 8.1 pmol/min/mg protein, respectively; this range represented 3-fold variability (Fig. 5). Therefore, the interindividual variability in FYX-051 N₁- and N₂-glucuronidation was thought to be relatively small. However, several single nucleotide polymorphisms, one of the causes of interindividual differences, have been identified in the UGT1A9 gene. For example, it has been reported that the SN-38 glucuronidation activity of UGT1A9*3 (M³³T) was 3.8% that of UGT1A9*1 allele (Villeneuve et al., 2003) and that the SN-38 glucuronidation efficiency of UGT1A9*5 (D256N) was less than 5% that of wild-type UGT1A9 (Jinno et al., 2003). Furthermore, Yamanaka et al. (2004) reported that the transcriptional activity of UGT1A9 promoter constructs containing A(T)₁₀AT (UGT1A9*22) was increased by 2.6-fold compared with that of constructs containing A(T)₉AT. Because the N₁- and N₂-glucuronidation of FYX-051 is catalyzed mainly by UGT1A9, we should exercise caution while administering FYX-051 to patients with these single nucleotide polymorphisms.

In conclusion, we characterized the triazole N₁- and N₂-glucuronidations of FYX-051 in HLMs and found that both are catalyzed mainly by UGT1A9.

	Footnotes

 
Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.

doi:10.1124/dmd.107.017251.

ABBREVIATIONS: FYX-051, 4-(5-pyridin-4-yl-1H-[1,2,4]triazol-3-yl) pyridine-2-carbonitrile; UGT, UDP-glucuronosyltransferase; HLM, human liver microsome; 7-HFC, 7-hydroxy-4-trifluoromethylcoumarin; HIM, human intestinal microsome; DMSO, dimethyl sulfoxide; LC-MS/MS, liquid chromatography-tandem mass spectrometry; SN-38, 7-ethyl-10-hydroxycamptothecin.

Address correspondence to: Dr. Koichi Omura, Research Laboratories 2, Fuji Yakuhin Co., Ltd., 636-1 Iidashinden, Nishi-ku, Saitama 331-0068, Japan. E-mail: k-omura{at}fujiyakuhin.co.jp

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