Abstract
5-(4′-Hydroxyphenyl)-5-phenylhydantoin (4′-HPPH), a major metabolite of phenytoin in human, is exclusively metabolized to a glucuronide. 4′-HPPH has a chiral center. (S)-4′-HPPH is a predominant form produced from phenytoin in humans, and (R)-4′-HPPH is an extremely toxic form with respect to gingival hyperplasia. In the present study, we investigated stereoselective 4′-HPPH O-glucuronide formation in human liver microsomes. Human liver microsomes predominantly formed (S)-4′-HPPH O-glucuronide rather than (R)-4′-HPPH O-glucuronide from racemic 4′-HPPH. Among human UDP-glucuronosyltransferase (UGT) enzymes, UGT1A1, UGT1A9, and UGT2B15 showed 4′-HPPH O-glucuronide formation. Interestingly, UGT1A1 stereoselectively formed (R)-4′-HPPH O-glucuronide, whereas UGT1A9 and UGT2B15 stereoselectively formed (S)-4′-HPPH O-glucuronide from racemic 4′-HPPH. By using UGT1A double-expression systems in HEK293 cells that we previously established, the effects of UGT-UGT interactions on 4′-HPPH O-glucuronide formation were investigated. It was demonstrated that coexpression of UGT1A4 increased the Vmax values of (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A1 but decreased the Vmax values of (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A9. Coexpression of UGT1A6 increased the S50 values and decreased the Vmax values of (S)- and (R)-4′-HPPH glucuronide formation catalyzed by UGT1A1 and UGT1A9. However, the interaction did not alter the stereoselectivity. In conclusion, we found that 4′-HPPH O-glucuronide formation in human liver microsomes is catalyzed by UGT1A1, UGT1A9, and UGT2B15 in a stereoselective manner, being modulated by interaction with other UGT1A isoforms.
Phenytoin, 5,5-diphenylhydantoin, is a widely used anticonvulsant drug. It is metabolized to 5-(4′-hydroxyphenyl)-5-phenylhydantoin (4′-HPPH) mainly by cytochrome P450 CYP2C9 and, to a small extent, by CYP2C19 in humans (Giancarlo et al., 2001). 4′-HPPH has an asymmetric carbon atom. CYP2C9 preferentially catalyzes the formation of the (S)-enantiomer of 4′-HPPH, whereas CYP2C19 is not stereoselective (Bajpai et al., 1996; Yasumori et al., 1999). It has been reported that 98% of circulating 4′-HPPH after phenytoin administration is the (S)-enantiomer in humans (Ieiri et al., 1995; Yasumori et al., 1999). 4′-HPPH has no anticonvulsant properties, but it is associated with side effects such as gingival hyperplasia, somnolence, dry month, and general fatigue (Ieiri et al., 1992). The (R)-enantiomer, although it is a minor metabolite, has been reported to be extremely toxic with respect to gingival hyperplasia (Ieiri et al., 1995). A number of studies suggested that 4′-HPPH is bioactivated by peroxidase to a free radical intermediate, which can oxidize lipids, proteins, and DNA (Kim and Wells, 1996; Parman et al., 1998). However, 4′-HPPH is exclusively metabolized to glucuronide, which is a major metabolite of phenytoin in human (Yamanaka et al., 2005). Thus, glucuronidation is an important detoxification pathway of 4′-HPPH.
Glucuronidation of a variety of xenobiotics and endogenous compounds is catalyzed by UDP-glucuronosyltransferases (UGTs). In humans, the UGT superfamily of genes is divided into two families, UGT1 and UGT2, based on sequence similarity at the amino acid level (Mackenzie et al., 2005). In our previous study, we found that 4′-HPPH O-glucuronide formation in human liver microsomes is catalyzed by UGT1A enzyme(s), although we could not determine which UGT1A enzymes make the major contribution (Nakajima et al., 2002). The limitation of the previous study was that the sensitivity was too low to detect the activity by recombinant UGTs. In our recent study, we improved the assay procedure and the HPLC condition to increase the sensitivity for the detection of 4′-HPPH O-glucuronide. Moreover, we could successfully separate the (S)- and (R)-4′-HPPH glucuronide in the improved HPLC condition. In the present study, we sought to determine the catalytic activities for the (S)- and (R)-4′-HPPH glucuronide formation by each human UGT enzyme. Recently, we reported that UGT1A enzymes interact with each other, possibly by heterodimerization, changing the kinetics of the enzymatic activity (Fujiwara et al., 2007a,b). Extending these studies, we investigated the effects of UGT-UGT interactions on the stereoselective formation of 4′-HPPH O-glucuronides.
Materials and Methods
Materials. 4′-HPPH, UDP-glucuronic acid (UDPGA), and alamethicin were purchased from Sigma-Aldrich (St. Louis, MO). Phenytoin was purchased from Wako Pure Chemicals (Osaka, Japan). Pooled human liver microsomes were from BD Gentest (Woburn, MA). Racemic 4′-HPPH was separated to (S)- and (R)-enantiomers according to a method of Yasumori et al. (1999) using a Chiralcel OJ column (4.6 × 250 mm, 10 μm; Daicel Chemical, Osaka, Japan). All other chemicals and solvents were of the highest grade commercially available.
Recombinant Human UGT and Immunoblot Analysis. Microsomes from baculovirus-infected insect cells expressing human UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, UGT2B17, and CYP2C9 (Supersomes) were from BD Gentest. Single- and double-expression systems of UGT1A1, UGT1A4, UGT1A6, and UGT1A9 stably expressed in HEK293 cells were previously established in our laboratory (Fujiwara et al., 2007a, b). For double-expression systems, the clones expressing two isoforms almost equally were selected in this study. Total cell homogenates were prepared as described previously (Fujiwara et al., 2007b). The expression levels of UGT1A proteins in the homogenates were determined by immunoblot analysis using rabbit anti-human UGT1A polyclonal antibody (BD Gentest) and were defined on the basis of a standard curve using the UGT1A1 single-expression system (1 unit/1 mg of cell homogenates), as described previously (Fujiwara et al., 2007b).
(S)- and (R)-4′-HPPH O-Glucuronide Formation. A typical incubation mixture (100 μl total volume) contained 50 mM Tris-HCl buffer (pH 7.4), 5 mM MgCl2, 2.5 mM UDPGA, 25 μg/ml alamethicin, 0.5 mg/ml human liver microsomes or 1 mg/ml recombinant UGTs (single- or double-expression systems in HEK293 cells and Supersomes), and racemic 4′-HPPH or each enantiomer. Because it was confirmed that alamethicin increased 4′-HPPH O-glucuronide formation both in human liver microsomes (2.2- to 2.5-fold) and by recombinant UGTs (1.0- to 1.8-fold), alamethicin was included in all incubation mixtures. 4′-HPPH was dissolved in methanol, and the final concentration of the organic solvent in the incubation mixture was set at 1%. It was confirmed that the inhibitory effects of 1% methanol on 4′-HPPH O-glucuronidation were negligible. The reactions were initiated by the addition of UDPGA and were then incubated at 37°C for 60 min. The reactions were terminated by 100 μl of 10% perchloric acid. After the centrifugation at 10,000g for 5 min, the supernatant (50 μl) was injected into the HPLC system. HPLC analyses were performed using a PC-980 pump (Jasco, Tokyo, Japan), a UV-970 intelligent UV/visible detector (Jasco), an AS-950-10 autosampler (Jasco), a D-2500 integrator (Hitachi, Tokyo, Japan), and a CTO-6A column oven (Shimadzu, Kyoto, Japan) equipped with an YMC-Pack ODS-AM column (4.6 × 150 mm; 5 μm; YMC, Kyoto, Japan). The mobile phase was 10% acetonitrile-50 mM potassium dihydrogenphosphate. The flow rate was 0.7 ml/min, and the column temperature was 35°C. The eluent was monitored at 214 nm with a noise-base clean Uni-3 (Union, Gunma, Japan). The Uni-3 can reduce the noise by integrating the output and increase the signal 3-fold by differentiating the output and 5-fold by further amplification with an internal amplifier, resulting in a maximum 15-fold amplification of the signal. The retention times of (R)- and (S)-4′-HPPH O-glucuronides and racemic 4′-HPPH were 16.8, 17.6, and 109 min (Fig. 1A). The detection limit of (S)- and (R)-4′-HPPH O-glucuronides was 0.1 pmol.
Quantification of 4′-HPPH O-Glucuronides. The incubation mixtures with human liver microsomes including 4′-HPPH O-glucuronides were extracted with diethyl ether to exclude the large amount of unconjugated 4′-HPPH. Peak heights of the 4′-HPPH O-glucuronides in the residual water phase were determined with the HPLC condition described above (Fig. 1B). 4′-HPPH in the residual water phase was also quantified (Fig. 1D) by comparing it with the peak height of the authentic standard (Fig. 1F), using the modified HPLC condition (the mobile phase was 20% acetonitrile-50 mM potassium dihydrogenphosphate, and the flow rate was 1 ml/min). The retention time of 4′-HPPH was 13.0 min. A part of the water phase was incubated with 800 U/ml β-glucuronidase at 37°C for 24 h. 4′-HPPH formed by the hydration of glucuronides was quantified (Fig. 1E). Once we determined the peak height per known content of 4′-HPPH O-glucuronide, it was applied to the calculation of the 4′-HPPH O-glucuronide formed in the incubation mixtures.
Kinetic Analyses of (S)- and (R)-4′-HPPH Glucuronide Formation. 4′-HPPH glucuronide formation in human liver microsomes and recombinant UGTs was determined with 5 to 500 μM racemic 4′-HPPH. To measure the activity in human liver microsomes at 1 mM racemic 4′-HPPH, 2 μl of 50 mM substrate in methanol was added to 100 μl of the incubation mixture, resulting in a 2% methanol concentration. Therefore, the observed activity was corrected with the inhibition percentage [5% for (S)-4′-HPPH O-glucuronide formation and 12% for (R)-4′-HPPH O-glucuronide formation]. Kinetic parameters were estimated by fitting untransformed experimental data to the Hill equation using a KaleidaGraph computer program (Synergy Software, Reading, PA) designed for nonlinear regression analysis.
The Hill equation, which describes sigmoidal kinetics, is
where V is the velocity of the reaction, S is the substrate concentration, Vmax is the maximum velocity, S50 is the substrate concentration showing the half Vmax, and n is the Hill coefficient. Maximum clearance (CLmax), which has been proposed as an appropriate parameter for sigmoid kinetic data instead of intrinsic clearance (Houston and Kenworthy, 2000; Uchaipichat et al., 2004), was calculated by the following equation:
Data are expressed as means ± S.D. of three independent determinations.
Statistical Analyses. Statistical significances of the kinetic parameters were determined by analysis of variance followed by Dunnett's test. A value of P < 0.05 was considered statistically significant.
Results
(S)- and (R)-4′-HPPH O-Glucuronide Formation in Human Liver Microsomes. When the pooled human liver microsomes were incubated with racemic 4′-HPPH and UDPGA, two peaks with retention times of 16.8 and 17.6 min were observed (Fig. 1A). Both peaks appeared to correspond to 4′-HPPH O-glucuronide, because these peaks disappeared by treatment with β-glucuronidase. The formation of these glucuronides increased linearly with an incubation time up to 90 min and with a protein concentration up to 2 mg/ml. To identify which of the two peaks corresponds to (S)-4′-HPPH O-glucuronide, (S)-4′-HPPH prepared from the incubation of phenytoin with recombinant CYP2C9 and an NADPH-generating system was used as the substrate. Incubation of the prepared (S)-4′-HPPH with the human liver microsomes and UDPGA showed only a peak with the retention time of 17.6 min. Accordingly, two peaks at 16.8 and 17.6 min were determined to correspond to (R)- and (S)-4′-HPPH O-glucuronides, respectively. For the quantification of the 4′-HPPH O-glucuronides, the decrease of 4′-HPPH O-glucuronides and the increase of 4′-HPPH were compared by the treatment of β-glucuronidase (Table 1). Repetitive estimations with an incubation mixture using racemic 4′-HPPH or each enantiomer as the substrate demonstrated that the peak height of 4′-HPPH O-glucuronide per 1 pmol was 881 ± 42 (coefficient of variation was 4.8%). These results suggest that the approach to quantify the 4′-HPPH O-glucuronides was sufficiently reliable.
Kinetic analyses of the (S)- and (R)-4′-HPPH O-glucuronide formation in the pooled human liver microsomes were performed with racemic 4′-HPPH. The kinetics was fitted to the Hill equations (Fig. 2). The abscissa denotes the concentrations of (S)- or (R)-4′-HPPH enantiomer, namely half concentrations of racemic 4′-HPPH. For (S)-4′-HPPH O-glucuronide formation, S50 was 256 ± 30 μM, Vmax was 3.9 ± 0.2 nmol/min/mg, and the Hill coefficient was n = 1.3 ± 0.0. For the (R)-4′-HPPH O-glucuronide formation, S50 was 236 ± 17 μM, Vmax was 1.4 ± 0.0 nmol/min/mg, and Hill coefficient was n = 1.5 ± 0.1. Thus, the CLmax of (S)-4′-HPPH O-glucuronide formation (2.3 ± 0.2 μl/min/mg) was 2.9-fold higher than that of (R)-4′-HPPH O-glucuronide formation (0.8 ± 0.1 μl/min/mg) in pooled human liver microsomes. To investigate whether one enantiomer affects the activity or the sigmoidal kinetics of the other, (S)- and (R)-4′-HPPH enantiomers were isolated from racemic 4′-HPPH. Although the activities could be measured only up to 50 μM concentrations of the (S)- and (R)-4′-HPPH enantiomers because of their limited quantity, they were almost the same as the activities with racemic 4′-HPPH at twice the concentration. Therefore, racemic 4′-HPPH was used as the substrate in the subsequent studies.
(S)- and (R)-4′-HPPH O-Glucuronide Formation by Recombinant UGT Enzymes. Recombinant human UGT enzymes expressed in baculovirus-infected insect cells were screened for 4′-HPPH O-glucuronide formation. As shown in Fig. 3A, UGT1A1, UGT1A9, and UGT2B15 showed 4′-HPPH O-glucuronide formation. UGT1A1 dominantly formed (R)-4′-HPPH O-glucuronide (43 pmol/min/mg) rather than (S)-4′-HPPH O-glucuronide (5 pmol/min/mg). In contrast, UGT1A9 and UGT2B15 dominantly formed (S)-4′-HPPH O-glucuronide (26 and 91 pmol/min/mg, respectively) rather than (R)-4′-HPPH O-glucuronide (9 and 15 pmol/min/mg, respectively). The HEK293 cell homogenates expressing human UGT1A1, UGT1A4, UGT1A6, and UGT1A9 were also used to determine the 4′-HPPH glucuronidation. UGT1A1 and UGT1A9 selectively formed (R)- and (S)-4′-HPPH O-glucuronides, respectively (Fig. 3B). To normalize the UGT1A expression levels, immunoblot analysis using anti-human UGT1A antibodies was performed. The expression levels of UGT1A1 and UGT1A9 in Supersomes were previously determined to be 15.9 and 10.4 units/mg, respectively (Fujiwara et al., 2007b). The expression levels of UGT1A1 and UGT1A9 in HEK293 expression systems were determined to be 1.00 ± 0.03 and 0.14 ± 0.02 unit/mg, respectively (Table 2). By the correction of the activity with the UGT1A expression level, the HEK293 expression systems showed higher activity than Supersomes (Fig. 3, C and D). In the HEK293 expression systems, (S)- and (R)-4′-HPPH O-glucuronide formation by UGT1A1 was 4 and 36 pmol/min/unit, respectively; the (S)- and (R)-4′-HPPH O-glucuronide formation by UGT1A9 was 51 and 10 pmol/min/unit, respectively.
Kinetics of (S)- and (R)-4′-HPPH O-Glucuronide Formation by Recombinant Human UGT Enzymes. Kinetic analyses of 4′-HPPH O-glucuronide formation by the UGT1A1, UGT1A9, and UGT2B15 Supersomes were performed with racemic 4′-HPPH. All of the kinetics was fitted to the Hill equation without (R)-4′-HPPH O-glucuronide formation by UGT1A9 (Table 3). UGT1A1 showed an S50 of 81 μM and Vmax of 14 pmol/min/mg for (S)-4′-HPPH O-glucuronide formation and showed an S50 of 110 μM and Vmax of 179 pmol/min/mg for (R)-4′-HPPH O-glucuronide formation. UGT1A9 showed an S50 of 25 μM and Vmax of 38 pmol/min/mg for (S)-4′-HPPH O-glucuronide formation and showed an S50 of 33 μM and Vmax of 16 pmol/min/mg for (R)-4′-HPPH O-glucuronide formation. UGT2B15 showed an S50 of 91 μM and Vmax of 504 pmol/min/mg for (S)-4′-HPPH O-glucuronide formation and showed an S50 of 96 μM and Vmax of 121 pmol/min/mg for (R)-4′-HPPH O-glucuronide formation.
Kinetics analyses of 4′-HPPH O-glucuronide formation by the recombinant UGT1A1 and UGT1A9 in HEK293 cells were also performed with racemic 4′-HPPH. The kinetics were fitted to the Hill equation (Fig. 4; Table 3). UGT1A1 showed an S50 of 48 μM and normalized Vmax of 9 pmol/min/unit for (S)-4′-HPPH O-glucuronide formation and showed an S50 of 74 μM and normalized Vmax of 110 pmol/min/unit for (R)-4′-HPPH O-glucuronide formation. UGT1A9 showed an S50 of 23 μM and normalized Vmax of 67 pmol/min/unit for (S)-4′-HPPH O-glucuronide formation and showed an S50 of 56 μM and normalized Vmax of 22 pmol/min/unit for (R)-4′-HPPH O-glucuronide formation.
Effects of Coexpression of Other UGT1A Isoforms on (S)- and (R)-4′-HPPH O-Glucuronide Formation Catalyzed by UGT1A1 or UGT1A9. Double-expression systems of human UGT1A in HEK293 cells were used to determine the effects of other isoforms on 4′-HPPH O-glucuronide formation catalyzed by UGT1A1 or UGT1A9. The activities of the double-expression systems were normalized with the expression levels of UGT1A1 and UGT1A9 proteins summarized in Table 1. Coexpression of UGT1A4 significantly increased the normalized Vmax values of both (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A1 (Table 3). Coexpression of UGT1A6 increased the S50 values and significantly decreased the normalized Vmax values of both (S)- and (R)-4′-HPPH O-glucuronide formation-catalyzed UGT1A1. In contrast, coexpression of UGT1A4 decreased the normalized Vmax values or both (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A9. Coexpression of UGT1A6 significantly increased the S50 value and decreased the normalized Vmax value of (S)-4′-HPPH O-glucuronide formation and decreased the normalized Vmax value of (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A9. Thus, coexpression of UGT1A4 showed opposite effects toward UGT1A1- and UGT1A9-catalyzed 4′-HPPH O-glucuronide formation, but coexpression of UGT1A6 showed similar effects on UGT1A1- and UGT1A9-catalyzed 4′-HPPH O-glucuronide formation. In any case, the UGT-UGT interaction did not alter the stereoselectivity of the 4′-HPPH O-glucuronide formation.
The double-expression system UGT1A1/UGT1A9 was also used to investigate the interaction of UGT1A1 and UGT1A9 (Fig. 5). Because both isoforms have catalytic activity toward 4′-HPPH O-glucuronide formation, the activity by the double-expression system UGT1A1/UGT1A9 was predicted to be the sum of activities by UGT1A1 and UGT1A9. For (S)-4′-HPPH O-glucuronide formation, the observed activities were lower than the predicted activities based on the expression levels of UGT1A1 and UGT1A9 proteins (Fig. 5). In contrast, for (R)-4′-HPPH O-glucuronide formation, the observed activities were higher than the predicted activities at enantiomer concentrations >25 μM. These results suggested that (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A1 may be increased by the coexpression of UGT1A9, and (S)-4′-HPPH O-glucuronide formation catalyzed by UGT1A9 may conversely be decreased by the coexpression of UGT1A1.
Discussion
It has been demonstrated that some UGT enzymes stereoselectively catalyze glucuronidation (Court et al., 2002; Tougou et al., 2004; Bichlmaier et al., 2006; Sten et al., 2006). In the present study, we characterized different human UGT enzymes for glucuronide formation using racemic 4′-HPPH. We found that UGT1A1 dominantly formed (R)-4′-HPPH O-glucuronide, but UGT1A9 and UGT2B15 dominantly formed (S)-4′-HPPH O-glucuronide. In human liver microsomes, (S)-4′-HPPH O-glucuronide formation was predominant compared with (R)-4′-HPPH O-glucuronide formation. Which UGT isoform has a major contribution to the (S)- and (R)-4′-HPPH O-glucuronide formation in human liver microsomes? To address the issue, the absolute protein levels of each UGT isoform in human liver microsomes should be determined. Alternatively, specific substrates or inhibitors for each UGT isoform can be used to estimate the contribution quantitatively. Unfortunately, the lack of a methodology for the quantification of the absolute UGT protein levels beyond UGT1A and UGT2B subfamilies as well as a lack of specific substrates or inhibitors prevents us from accomplishing a quantitative estimation.
Concerning stereoselectivity, the question is raised whether the preference for the (S)- and (R)-enantiomers is determined by the binding affinity of substrates or by the rate of transfer of glucuronic acid to the already-bound substrate. Kinetic analyses for UGT1A1 and UGT2B15 activities revealed that the S50 values for the two enantiomers differed much less than the corresponding Vmax values (Table 3). On this basis, it appears that the differences in the rate of glucuronic acid transfer to the aglycone are the major determinant of the stereoselectivity. In the case of UGT1A9, both differences in the rate of glucuronic acid transfer to the aglycone and in the affinity of the enzyme for 4′-HPPH enantiomers may be involved.
When the activities normalized with the UGT expression level were compared between two recombinant systems (Fig. 3), the activities by the recombinant UGT1A expressed in the baculovirus-infected insect cells were much lower (1/10-1/20) than those by recombinant UGT1A expressed in HEK293 cells. The phenomenon was not specific for 4′-HPPH O-glucuronide formation, because similar results were obtained with other substrates such as estradiol, serotonin, and propofol (data not shown). The differences between the recombinant systems might be due to differences in the membrane environment or lipid components in the host cells. Furthermore, the differences in the post-translational modulation of UGT such as glycosylation or phosphorylation between the insect cells and HEK293 cells might be responsible, as such modulations have been reported to affect the UGT activity (Barbier et al., 2000; Basu et al., 2005).
It has been reported that UGTs form homo- and heterodimers or oligomers (Matern et al., 1982; Meech and Mackenzie, 1997; Ghosh et al., 2001; Kurkela et al., 2003). In our recent studies (Fujiwara et al., 2007a, b), the glucuronidations of a variety of typical substrates were determined using the double-expression systems of human UGT1A enzymes to investigate the effects of the heterodimerization on the enzymatic activity. These previous studies demonstrated that coexpression of other UGT1A isoforms differently changed the kinetics of specific activities depending on the substrates as well as the UGT isoforms. Extending our studies, we found that the coexpression of UGT1A4 and UGT1A6 differently affected the kinetics of 4′-HPPH O-glucuronide formation by UGT1A1 or UGT1A9. Thus, the UGT-UGT interactions, which may also occur in human liver microsomes, affected the kinetics of 4′-HPPH O-glucuronide formation. The kinetic analyses revealed sigmoid curves in both (S)- and (R)-4′-HPPH O-glucuronide formation by recombinant UGT1A1 and UGT1A9. The sigmoid curve may indicate positive cooperativity owing to the binding of multiple substrate molecules to a single enzyme active site or to the existence of multiple conformations of the enzyme (Cornish-Bowden, 1995). In particular, allosteric effects would be feasible, because UGT forms a dimer or oligomer. It is interesting that the cooperativity was not affected by the coexpression of other isoforms, possibly heterodimerization. The S50 values of (S)- and (R)-4′-HPPH O-glucuronide formation in human liver microsomes were unexpectedly higher than those by recombinant UGT enzymes. Although the coexpression of other isoforms substantially increased the Km values for certain substrates (Fujiwara et al., 2007a, b), prominent effects on the S50 values were not observed in the case of 4′-HPPH. We are also interested in interactions between UGT1A and UGT2B. Double-expression systems of UGT1A and UGT2B isoforms are now being constructed in our laboratory to investigate the effects of the interaction on the enzymatic activity. In addition, the possibility that other enzymes such as cytochrome P450 might also interact with UGT1A to affect the kinetics cannot be excluded. Further studies will be worth pursuing.
In the present study, we characterized the stereoselective 4′-HPPH O-glucuronide formation by human UGT1A1, UGT1A9, and UGT2B15. Furthermore, it was demonstrated that interaction between UGT1A enzymes differently affected the kinetics of (S)- and (R)-4′-HPPH O-glucuronide formation catalyzed by UGT1A1 and UGT1A9 but did not affect the stereoselectivity.
Acknowledgments
We acknowledge Brent Bell for reviewing the manuscript.
Footnotes
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H.Y. is supported as a Research Fellow of the Japan Society for the Promotion of Science.
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doi:10.1124/dmd.107.015909.
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ABBREVIATIONS: 4′-HPPH, 5-(4′-hydroxyphenyl)-5-phenylhydantoin; UGT, UDP-glucuronosyltransferase; HPLC, high-performance liquid chromatography; UDPGA, UDP-glucuronic acid.
- Received March 26, 2007.
- Accepted June 13, 2007.
- The American Society for Pharmacology and Experimental Therapeutics