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REGIOSELECTIVE GLUCURONIDATION OF DENOPAMINE: MARKED SPECIES DIFFERENCES AND IDENTIFICATION OF HUMAN UDP-GLUCURONOSYLTRANSFERASE ISOFORM

Exploratory Toxicology & DMPK Research Laboratories, Tanabe Seiyaku Co., LTD., Saitama, Japan

(Received October 19, 2004; accepted December 16, 2004)

	Abstract

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Denopamine is one of the oral ß₁-adrenoceptor-selective partial agonists. Denopamine glucuronide is the most abundant metabolite in human, rat, and dog urine when administered orally. Species differences in denopamine glucuronidation were investigated with liver microsomes obtained from humans and experimental animals. In rat and rabbit, only the phenolic glucuronide was detected, whereas in dog and monkey, not only the phenolic glucuronide but also the alcoholic glucuronide was found. In contrast, in humans, the alcoholic glucuronide was detected exclusively. The kinetics of denopamine glucuronidation in human liver microsomes showed a typical Michaelis-Menten plot. The K_m and V_max values accounted for 2.87 ± 0.17 mM and 7.29 ± 0.23 nmol/min/mg protein, respectively. With the assessment of denopamine glucuronide formation across a panel of recombinant UDP-glucuronosyltransferase (UGT) isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17), only UGT2B7 exhibited high denopamine glucuronosyltransferase activity. The K_m value of denopamine glucuronidation in recombinant UGT2B7 microsomes was close to those in human liver and jejunum microsomes. The formation of denopamine glucuronidation by human liver, jejunum, and recombinant UGT2B7 microsomes was effectively inhibited by diclofenac, a known substrate for UGT2B7. The denopamine glucuronidation activities in seven human liver microsomes were significantly correlated with diclofenac glucuronidation activities (r² = 0.685, p < 0.05). These results demonstrate that the denopamine glucuronidation in human liver and intestine is mainly catalyzed by UGT2B7 and that glucuronidation of the alcoholic hydroxyl group, but not the phenolic hydroxyl group, occurs regioselectively in humans.

Denopamine, (–)-(R)-(p-hydroxyphenyl)-2-[(3,4-dimethoxyphenethyl)amino]ethanol (Fig. 1), is one of the oral ß₁-adrenoceptor-selective partial agonists (Nagao et al., 1984; Thormann et al., 1985). It has been reported that the majority of the parent and its metabolites is excreted into urine (human, 30% of dose; rat and dog, 60–70% of dose) when denopamine is administered orally (Suzuki et al., 1983; Furuuchi et al., 1985). Rats excreted the drug almost entirely as the phenolic glucuronide, whereas in the dog, the major metabolites were the phenolic and alcoholic glucuronides (Fig. 1), at a ratio of approximately 3:1. Although denopamine glucuronide is the most abundant metabolite in human urine, the position of its conjugation has not been elucidated. Until now, denopamine glucuronide formed by in vivo studies had been determined by thin-layer chromatography using ¹⁴C-labeled denopamine (Furuuchi et al., 1985) or gas chromatography-MS analyses after hydrolysis with ß-glucuronidase (Suzuki et al., 1983). Therefore, the purpose of the present study was, first, to develop a simple HPLC-UV method for determining denopamine glucuronides directly and to elucidate the position of its conjugation using human liver as well as jejunum microsomes. Second, to identify the human UGT isoform(s) responsible for the denopamine glucuronidation, the activities and kinetics in recombinant UGT microsomes from baculovirus-insect cells were determined. Furthermore, we performed the inhibition analyses and correlation analyses with typical substrates for UGT isoforms to confirm the contribution of the enzyme on denopamine glucuronidation.

View larger version (13K): [in this window] [in a new window]   FIG. 1. Structures of denopamine and its glucuronides.

	Materials and Methods

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Materials. Denopamine was synthesized at Tanabe Seiyaku Co., LTD. (Saitama, Japan). UDP-glucuronic acid, alamethicin, trifluoperazine, and ß-glucuronidases (type H-5 from Helix pomatia, type B-3 from bovine liver, and type VII-A from Escherichia coli) and 4-trifluoromethyl-7-hydroxycoumarin glucuronide were purchased from Sigma-Aldrich (St. Louis, MO). 7-Hydroxy 4-trifluoromethylcoumarin (7-HFC) was obtained from BD Gentest (Woburn, MA). Diclofenac was obtained from SAFC (Manchester, UK). Pooled human liver microsomes from donors (13 males, 4–62 years old; 9 females, 40–74 years old) and microsomes from seven individual human livers (HG32, HG74, HH18, HH47, HH91, HH74, and HK25) were purchased from BD Gentest. The glucuronosyltransferase activities of ß-estradiol (for the 3-hydroxy position), trifluoperazine, and propofol in these human liver microsomes were provided as typical activities for UGT1A1, UGT1A4, and UGT1A9, respectively, by the manufacturer. Recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) expressed in baculovirus-infected insect cells (Supersomes) were also obtained from BD Gentest. Pooled human jejunum microsomes from donors (five males, 19–55 years old; five females, 16–51 years old) were obtained from Tissue Transformation Technologies (Edison, NJ). For animal species comparison, pooled male rat liver microsomes (pool of 82 Sprague-Dawley rats), pooled male dog liver microsomes (pool of five beagle dogs), pooled male monkey liver microsomes (pool of six cynomolgus monkeys), and pooled male rabbit liver microsomes (pool of six New Zealand White rabbits) were purchased from BD Gentest. The protein contents were used as described in the data sheets provided by the manufacturer. All other chemicals and solvents were of the highest grade commercially available.

Denopamine Glucuronidation Assay. A typical incubation mixture (100-µl total volume) contained 50 mM Tris-HCl buffer, pH 7.5, 8 mM MgCl₂, 5 mM UDP-glucuronic acid, 25 µg/ml alamethicin, 0.5 mg/ml human liver microsomes (human jejunum microsomes, recombinant UGTs, or experimental animal liver microsomes), and 100 µM denopamine. Denopamine was dissolved in dimethyl sulfoxide. The final concentration of dimethyl sulfoxide in the reaction mixture was 1% (v/v). After preincubation at 37°C for 5 min, the reaction was initiated by the addition of denopamine. The reaction mixture was incubated at 37°C for 30 min (10 min in the case of rat and rabbit liver microsomes) and the reaction was stopped with 50 µl of ice-cold acetonitrile. After removal of the protein by centrifugation at 10,000 rpm for 3 min, a 100-µl portion of the supernatant was added to 100 µl of water, and then a portion of the sample was subjected to HPLC. Chromatography was performed using a Waters Alliance HPLC system (model 2695; Waters, Milford, MA), equipped with a model 2996 photodiode array detector (Waters), and an Atlantis dC₁₈ column (4.6 x 150 mm, 3 µm; Waters). The column temperature was 40°C and the eluate was monitored at 275 nm. Mobile phase A was 0.1% formic acid in water (v/v) and mobile phase B was acetonitrile. The gradient, expressed as changes in mobile phase B, was as follows: 0 to 4 min, hold at 5% B; 4 to 10 min, a linear increase from 5% to 40% B; 10 to 12 min, hold at 90% B. The mobile phase flow rate for the analysis of denopamine and its glucuronides was 1.0 ml/min. The retention times of the phenolic glucuronide (G1), the alcoholic glucuronide (G2), and denopamine were 9.1, 9.8, and 10.0 min, respectively.

Due to the absence of authentic standards for denopamine glucuronides, quantification of the glucuronides in the incubation mixtures was accomplished using standard curves for denopamine. In addition, for the quantification of denopamine glucuronides, the eluate of the HPLC from the incubation mixture with dog liver microsomes including both G1 and G2 was collected separately. A part of each eluate was hydrolyzed with ß-glucuronidase, as described below. The completely hydrolyzed G1 and G2 were quantified as denopamine by HPLC. We determined the peak areas at 275 nm and calculated their apparent factors according to the known contents of G1 and G2. These factors were applied to the calculation of G1 and G2 formed in the incubation mixture.

Hydrolysis with ß-Glucuronidase. The eluates corresponding to G1 and G2 were evaporated in vacuo and reconstituted in 1.0 ml of 0.5 M sodium acetate buffer (pH 5.0) or 0.1 M sodium phosphate buffer (pH 6.8). Each sample was incubated in the absence (control) and presence of 1000 units of ß-glucuronidase (at pH 5.0 for type H-5 from H. pomatia and type B-3 from bovine liver; at pH 6.8 for type VII-A from E. coli) at 37°C for 20 h. Samples were withdrawn for analysis at 0, 1, 4, and 20 h after the addition of the enzyme preparation as follows. Aliquots (100 µl) of each incubation were stopped with 50 µl of ice-cold acetonitrile. After removal of the protein by centrifugation, the supernatant was subjected to HPLC as described above.

Identification of Denopamine Glucuronide by LC-MS/MS Analysis. The incubation of denopamine with human or dog liver microsomes for structure identification was carried out as described above. Detection of G1 and G2 was achieved with injection of 5 µl of the centrifugal supernatant onto the LC-MS/MS system. LC-MS/MS analysis was performed using a Q-TOF Ultima API (Waters) mass spectrometer coupled to an Agilent model 1100 series HPLC system (Agilent Technologies, Palo Alto, CA). Ionization of the analytes was achieved by electrospray in the positive ion mode. An Atlantis dC₁₈ column (2.1 x 150 mm, 3 µm; Waters) was used for the LC separation. The HPLC separation was carried out at 45°C using a gradient composed of mobile phase A [0.1% formic acid in water (v/v)] and mobile phase B (acetonitrile). The gradient, expressed as changes in mobile phase B, was as follows: 0 to 10 min, hold at 2% B; 10 to 25 min, a linear increase from 2% to 30% B; 25 to 27 min, hold at 90% B. The mobile phase flow rate was 0.3 ml/min. MS conditions used were as follows: capillary voltage, 3.5 kV; cone voltage, 35 V; cone gas, 50 l/h; desolvation gas, 500 l/h; desolvation temperature, 350°C; source temperature, 100°C. MS and MS/MS spectra were obtained in the range of m/z 100 to 700. Collision-induced dissociation was performed using argon as collision gas, and collision energy was 25 eV. The retention times of G1, G2, and denopamine were 19.3, 21.5, and 21.7 min, respectively.

Effects of Organic Solvents on Denopamine Glucuronidation. The effects of methanol and dimethyl sulfoxide on the denopamine glucuronidation in human and dog liver microsomes were determined. Incubations were performed as described above. Each organic solvent was added separately to incubations to give final concentrations of 0.5, 1, and 2%, v/v. Data were compared with activities determined in the presence of 0.5% (v/v) methanol, and the inhibitory effects of organic solvents were indicated as percentage of control activity for duplicate determinations.

Kinetic Analyses. The kinetics studies were performed using the liver microsomes obtained from human, rat, dog, monkey, and rabbit, and human jejunum microsomes, and recombinant UGT2B7 expressed in microsomes. In determining the kinetic parameters, the denopamine concentration ranged from 15.6 µM to 4000 µM except for the case of rat and rabbit liver microsomes, which ranged from 0.78 µM to 200 µM. The concentrations of microsomal protein and incubation time were 0.5 mg/ml and 30 min, respectively, except for the case of rat and rabbit liver microsomes, which were 0.1 mg/ml and 10 min, respectively. Kinetic parameters were estimated from the fitted curves using the Prism computer program (GraphPad Software Inc., San Diego, CA), designed for nonlinear regression analysis. The following equation was applied, assuming a Michaelis-Menten equation: V = V_max x [S]/(K_m + [S]), where V is the rate of reaction, V_max is the maximum velocity, K_m is Michaelis constant (substrate concentration at 0.5 V_max), and [S] is the substrate concentration.

Determination of Enzyme Activity of Recombinant UGTs and Microsomes. The glucuronosyltransferase activities of recombinant UGTs (UGT1A1, UGT1A3, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B7, UGT2B15, and UGT2B17) and human liver and jejunum microsomes were determined in assays using HPLC and 7-HFC as substrate. An incubation mixture (100-µl total volume) contained 50 mM Tris-HCl buffer, pH 7.5, 8 mM MgCl₂, 2 mM UDP-glucuronic acid, 25 µg/ml alamethicin, 0.01 mg/ml microsomes, and 100 µM 7-HFC. 7-HFC was dissolved in methanol. The final concentration of methanol in the reaction mixture was 2% (v/v). After preincubation at 37°C for 5 min, the reaction was initiated by the addition of 7-HFC. The reaction mixture was incubated at 37°C for 10 min and the reaction was stopped with 50 µl of acetonitrile. After removal of the protein by centrifugation at 10,000 rpm for 3 min, a 50-µl portion of the sample was subjected to HPLC. Chromatography was performed using a Waters Alliance HPLC system and a Symmetry C₁₈ column (4.6 x 150 mm, 3.5 µm; Waters). The column temperature was 40°C and the eluate was monitored at 325 nm. Mobile phase A was 0.1% formic acid in water (v/v), and mobile phase B was acetonitrile. A linear gradient of 5 to 65% mobile phase B over 10 min was used for the analysis of 7-HFC and its glucuronide, at a flow rate of 1.0 ml/min. The retention times of 7-HFC and its glucuronide were 10.2 and 7.9 min, respectively. The formation of glucuronide was quantified by comparing to the peak areas in the incubations to a standard curve for 4-trifluoromethyl-7-hydroxycoumarin glucuronide. Recombinant UGT1A4 microsomes do not form glucuronides with 7-HFC as a substrate. Therefore, the activity of UGT1A4 was determined in assays using HPLC and trifluoperazine as a substrate according to the UGT Batch Data Sheet provided by BD Gentest.

Inhibition Analysis of Denopamine Glucuronosyltransferase Activity in Human Liver and Jejunum Microsomes, and Recombinant UGT2B7. Diclofenac, a known substrate for UGT2B7 (Miners et al., 1997; King et al., 2001), was tested for inhibitory effects on denopamine glucuronosyltransferase activity. Diclofenac was dissolved in dimethyl sulfoxide, with the solvent being used as the control. The final concentration of organic solvents in the reaction mixture was 1% (v/v). The diclofenac concentrations for IC₅₀ determinations in human liver, jejunum microsomes, or recombinant UGT2B7 were 1 to 300 µM. Each incubation contained 0.5 mg/ml microsomal protein. The glucuronosyltransferase activities in all microsomes at 100 µM denopamine were determined as described above. The IC₅₀ values were estimated graphically.

Diclofenac Glucuronidation Assay. Diclofenac glucuronosyltransferase activities in microsomes from seven individual human livers were determined in the assay using HPLC. An incubation mixture (100-µl total volume) contained 50 mM Tris-HCl buffer, pH 7.5, 8 mM MgCl₂, 2 mM UDP-glucuronic acid, 25 µg/ml alamethicin, 0.1 mg/ml microsomes, and 100 µM diclofenac. After preincubation at 37°C for 5 min, the reaction was initiated by the addition of diclofenac. The reaction mixture was incubated at 37°C for 10 min and the reaction stopped with 80 µl of ice-cold acetonitrile containing 6% (v/v) acetic acid. After removal of the protein by centrifugation at 10,000 rpm for 3 min, an 80-µl portion of the sample was subjected to HPLC. Chromatography was performed using a Waters Alliance HPLC system and a Symmetry C₁₈ column. The column temperature was 40°C and the eluate was monitored at 275 nm. Mobile phase A was 0.1% formic acid in water (v/v), and mobile phase B was acetonitrile. A linear gradient of 20 to 80% mobile phase B over 10 min was used for the analysis of diclofenac and its glucuronide, at a flow rate of 1.0 ml/min. The retention times of diclofenac and its glucuronide were 10.1 and 7.9 min, respectively. Due to the absence of authentic standard for diclofenac glucuronide, quantification of the glucuronide in the incubation mixtures was accomplished using standard curves for diclofenac. The absorbance spectrum of diclofenac glucuronide was consistent with that of diclofenac, for which the wavelength of maximum absorbance of diclofenac glucuronide and diclofenac was 275 nm. The peak area ratio of diclofenac to the converted diclofenac glucuronide in the HPLC conditions was 1.0, after which an apparent correction factor was applied to the calculation of diclofenac glucuronide formed in the microsomal incubation mixture.

Correlation Analysis. Correlation analysis between denopamine glucuronidation and the other glucuronidation activities was determined by Pearson's moment method. A p value less than 0.05 was considered statistically significant.

	Results

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Denopamine Glucuronide Formation in Liver Microsomes. Figure 2 shows representative HPLC chromatograms of the denopamine glucuronide formation in liver microsomes obtained from rat, dog, and human. After incubation of denopamine with rat and rabbit liver microsomes in the presence of UDP-glucuronic acid, only one metabolite (G1) was observed (rat data shown in Fig. 2A). Incubation of denopamine with dog and monkey liver microsomes demonstrated the formation of two different glucuronides (G1 and G2) (dog data shown in Fig. 2B). In contrast, with respect to human liver microsomes, only one metabolite (G2) was observed (Fig. 2C).

View larger version (24K): [in this window] [in a new window]   FIG. 2. Representative HPLC chromatograms of the formation of denopamine glucuronides in rat (A), dog (B), and human liver (C) microsomes. All the liver microsomes (0.5 mg/ml protein) were incubated with 100 µM denopamine and 5 mM UDP-glucuronic acid at 37°C for 10 min.

In all species, the glucuronide formation (G1 and G2) was linear up to 1.0 mg/ml microsomal protein and 45 min of incubation time (data not shown). Methanol tested up to 2% (v/v) had no effect on the formation of denopamine glucuronides. Dimethyl sulfoxide tested up to 1% (v/v) had no effect on the formation of denopamine glucuronides, but 2% (v/v) of dimethyl sulfoxide reduced the activities of the denopamine glucuronidation in both human and dog liver microsomes by 6 to 8% (data not shown). Due to the limited water solubility of denopamine, dimethyl sulfoxide was used as an organic solvent in kinetics and inhibition studies, in which the final concentration was 1% (v/v) in the reaction mixture.

LC-MS/MS Analyses of Denopamine Glucuronide. Figure 3A shows the representative product ion spectrum of denopamine. The electrospray ionization mass spectra of two peaks (G1 and G2) typically formed by incubation of denopamine with dog liver microsomes in the presence of UDP-glucuronic acid had [M + H]⁺ ion at m/z 494 corresponding to denopamine glucuronide. G1 initially would fragment from the parent ion to lose the hydroxyl group at the phenethyl moiety (m/z 476) or to lose the glucuronic acid element (176 Da) to leave the aglycon (m/z 300) (Fig. 3B). The product ion spectrum of G2 showed that a loss of the glucuronic acid element yields the dehydrated aglycon ion at m/z 300 (Fig. 3C). From these observations, it was concluded that G1 and G2 formed by the incubation of denopamine in dog liver microsomes supplemented with UDP-glucuronic acid were, respectively, the phenolic glucuronide and the alcoholic glucuronide of denopamine. The fragment pattern of the metabolite corresponding to G2 in human liver microsomes was consistent with that of G2 in dogs, suggesting that the glucuronide conjugation occurred at the alcoholic hydroxyl group of denopamine in humans.

View larger version (20K): [in this window] [in a new window]   FIG. 3. Electrospray ionization-MS/MS spectrum of denopamine (A) and the two peaks (G1 and G2) (B and C) formed by the incubation of denopamine with dog liver microsomes in the presence of UDP-glucuronic acid.

Hydrolysis with ß-Glucuronidase. The eluates corresponding to G1 and G2 were incubated with various ß-glucuronidases (Fig. 4). G1 was relatively easily hydrolyzed with all of these ß-glucuronidases and converted to parent denopamine. G2 was hydrolyzed with ß-glucuronidase from E. coli and completely converted to denopamine, whereas it was considerably resistant to hydrolysis by two ß-glucuronidases from H. pomatia and bovine liver. No change was observed in the glucuronides over the 20 h of incubation without ß-glucuronidase. Therefore, all detected denopamine in the presence of the ß-glucuronidase can be attributed to enzymatic hydrolysis. The apparent factors calculated from the peak areas over the known contents of G1 and G2 were 1.3 and 1.0, respectively. Thus, each factor was applied to the quantification of G1 and G2 formed in the microsomal incubation mixture.

View larger version (11K): [in this window] [in a new window]   FIG. 4. Hydrolysis of denopamine glucuronides (G1 and G2) with ß-glucuronidase from three different sources. Denopamine glucuronides and converted denopamine were analyzed by HPLC as described under Materials and Methods.

Kinetics of Denopamine Glucuronidation in Liver Microsomes. Kinetic analysis of denopamine glucuronidation was performed in liver microsomes obtained from humans and experimental animals. As shown in Fig. 5A, the denopamine glucuronidation (G2) by human liver microsomes displayed typical Michaelis-Menten kinetics. Additionally, an Eadie-Hofstee plot was monophasic. With respect to rat, rabbit, dog, and monkey liver microsomes, these kinetic patterns of G1 and G2 were similar to those of G2 in humans. Fitting the data points to the Michaelis-Menten equation yielded the kinetic parameters listed in Table 1. Incubation of various concentrations of denopamine with human liver microsomes demonstrated that the K_m, V_max, and CL_int (V_max/K_m) values for G2 were 2.87 ± 0.17 mM, 7.29 ± 0.23 nmol/min/mg protein, and 2.54 µl/min/mg protein, respectively. In contrast, very little G1 formation was detected in all concentrations of denopamine determined (<5.2 pmol/min/mg protein). On the other hand, incubation of various concentrations of denopamine with rat and rabbit liver microsomes demonstrated that the CL_int values for G1 were 1220 and 152 µl/min/mg protein, respectively. In contrast, very little G2 formation was detected in all concentrations of denopamine determined in both microsomes (<4.0 pmol/min/mg protein). In dog liver microsomes the CL_int values for G1 and G2 were estimated to be 2.14 and 1.62 µl/min/mg protein, respectively. In monkey liver microsomes the CL_int values for G1 and G2 were estimated to be 1.96 and 3.40 µl/min/mg protein, respectively.

View larger version (15K): [in this window] [in a new window]   FIG. 5. Kinetics of denopamine glucuronidation in human liver (A), jejunum (B), and recombinant UGT2B7 (C) microsomes. The concentration of denopamine ranged from 15.6 to 4000 µM. The formation of denopamine glucuronide (G2) was determined as described under Materials and Methods. Each inset shows the Eadie-Hofstee plot of the experimental data. Each incubation was performed by triplicate determinations.

Activities of Recombinant UGTs, Human Liver, and Jejunum Microsomes (Positive Control). The results of activity determination of 12 recombinant UGT isoforms expressed in baculovirus-infected insect cells, human liver, and jejunum microsomes are presented in Table 2. These results served as positive control for the glucuronosyltransferase activities and demonstrated that the recombinant UGTs and microsomes were active.

Denopamine Glucuronidation in Recombinant UGT Isoforms. All recombinant UGTs were used to determine their denopamine glucuronosyltransferase activities (Fig. 6). Only UGT2B7 exhibited remarkably high denopamine glucuronidation (G2) activities (0.25 ± 0.01 nmol/min/mg protein). All other isoforms (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A8, UGT1A9, UGT1A10, UGT2B4, UGT2B15, and UGT2B17) had very low denopamine glucuronidation (G1 and G2) activities (<4.0 pmol/min/mg protein) or no activities.

View larger version (9K): [in this window] [in a new window]   FIG. 6. Denopamine glucuronosyltransferase activity in recombinant human UGTs. The formation of denopamine glucuronide (G2) in recombinant UGTs was determined at 100 µM denopamine. Each column represents the mean ± S.D. of triplicate determinations. The lower limit of quantitation of the assay under these conditions was 4.0 pmol/min/mg protein.

Kinetics of Denopamine Glucuronidation in Human Jejunum Microsomes and Recombinant UGT2B7. Kinetics analyses of the denopamine glucuronidation in human jejunum microsomes and recombinant UGT2B7 were performed (Fig. 5, B and C). Denopamine glucuronidation (G2 formation) by human jejunum microsomes and recombinant UGT2B7 also displayed typical Michaelis-Menten kinetics, similar to that by human liver microsomes. Additionally, each Eadie-Hofstee plot was monophasic. Fitting the data points to the Michaelis-Menten equation yielded the kinetic parameters listed in Table 1. Incubation of various concentrations of denopamine with human jejunum microsomes and recombinant UGT2B7 demonstrated that the K_m, V_max, and CL_int values for G2 were 2.99 ± 0.30 and 1.48 ± 0.05 mM, 1.17 ± 0.06 and 3.58 ± 0.06 nmol/min/mg protein, and 0.39 and 2.43 µl/min/mg protein, respectively. In contrast, very little G1 formation was detected in all concentrations of denopamine determined in both microsomes (<5.2 pmol/min/mg protein).

Inhibition Analyses of Denopamine Glucuronidation in Human Liver and Jejunum Microsomes, and Recombinant UGT2B7. The inhibitory effects of diclofenac on the denopamine glucuronosyltransferase activities in human liver and jejunum microsomes, and recombinant UGT2B7 were investigated. As shown in Fig. 7, A and B, the denopamine glucuronosyltransferase activities in human liver and jejunum microsomes were prominently inhibited by diclofenac (IC₅₀ = 41.5 and 29.7 µM, respectively). In addition, the denopamine glucuronosyltransferase activities in UGT2B7 were also inhibited by diclofenac, with an IC₅₀ value (24.0 µM) equivalent to that for human liver and jejunum microsomes (Fig. 7C).

View larger version (16K): [in this window] [in a new window]   FIG. 7. Inhibitory effects of diclofenac on denopamine glucuronosyltransferase activity in human liver (A), jejunum (B), or recombinant UGT2B7 (C) microsomes. Denopamine glucuronosyltransferase activities at 100 µM denopamine in human liver, jejunum, and UGT2B7 microsomes were determined in the presence or absence of diclofenac as described under Materials and Methods. Corresponding control activities were 197.9 ± 1.43, 27.7 ± 0.39, and 203.8 ± 1.74 pmol/min/mg protein, respectively. Each value represents the mean ± S.D. of triplicate determinations.

Interindividual Variability of Denopamine Glucuronosyltransferase Activity in Seven Human Livers and Correlation Analyses. Denopamine glucuronosyltransferase activities in microsomes from seven human livers were determined at 100 µM denopamine. The interindividual difference in denopamine glucuronosyltransferase activity was at most 2.7-fold (0.12 ± 0.03 to 0.33 ± 0.05 nmol/min/mg protein). Correlation analyses were performed between the denopamine glucuronosyltransferase activity and ß-estradiol (UGT1A1), trifluoperazine (UGT1A4), propofol (UGT1A9), or diclofenac (UGT2B7) glucuronosyltransferase activities. As shown Fig. 8, A and D, the denopamine glucuronosyltransferase activities in seven human liver microsomes were significantly correlated with the ß-estradiol 3-glucuronosyltransferase activities (r² = 0.796, p < 0.01) and diclofenac glucuronosyltransferase activities (r² = 0.685, p < 0.05), although the denopamine glucuronosyltransferase activities did not correlate with the trifluoperazine (r² = 0.175, p = 0.350) and propofol (r² = 0.501, p = 0.076) glucuronosyltransferase activities (Fig. 8, B and C).

View larger version (31K): [in this window] [in a new window]   FIG. 8. Correlation between denopamine glucuronosyltransferase activity and other glucuronosyltransferase activities in microsomes from seven human livers. Denopamine glucuronosyltransferase activities in microsomes from seven human livers were determined at 100 µM denopamine. ß-Estradiol 3-glucuronosyltransferase activity (A), trifluoperazine N-glucuronosyltransferase activity (B), and propofol glucuronosyltransferase activity (C) in human liver microsomes were provided by the manufacturer. Diclofenac glucuronosyltransferase activity (D) was determined in the present studies as described under Materials and Methods.

	Discussion

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It has been reported that denopamine is metabolized to its glucuronide in humans, rats, and dogs (Suzuki et al., 1983; Furuuchi et al., 1985). In the structure of denopamine, there are phenolic and alcoholic hydroxyl groups, which can possibly be linked to glucuronic acid. Although denopamine glucuronide is the most abundant metabolite in human urine, the position of its conjugation has not been elucidated. Since denopamine glucuronide detected on in vivo studies had been determined by the thin-layer chromatography or gas chromatography-MS analyses, we developed a simple HPLC method for the determination of denopamine glucuronides, which was able to separate the two regioisomers directly. First, two kinds of metabolites (G1 and G2) formed by the incubation of denopamine with dog liver microsomes in the presence of UDP-glucuronic acid (Fig. 2B) were identified as the phenolic glucuronide and the alcoholic glucuronide, respectively, by the LC-MS/MS analyses. We also confirmed that the metabolite formed in rat liver microsomes was only the phenolic glucuronide. The regioselectivity of glucuronidation in vitro was consistent with the previous report of in vivo experiments using rats and dogs (Furuuchi et al., 1985). To elucidate the regioselectivity of denopamine glucuronidation in humans, we conducted in vitro studies using human liver and jejunum microsomes. The incubation of denopamine with both microsomes resulted in the formation of only one metabolite (Fig. 2C), which was identified as the alcoholic glucuronide by the HPLC and LC-MS/MS analyses. In addition, to characterize species differences of regioselectivity on the glucuronidation of denopamine, we determined the glucuronosyltransferase activities using liver microsomes obtained from several experimental animals. After incubation of denopamine with rabbit liver microsomes, only the phenolic glucuronide was observed, as was the case with rats, whereas incubation of denopamine with monkey liver microsomes as well as dog liver microsomes demonstrated the formation of both phenolic and alcoholic glucuronides. The metabolic activity (V_max/K_m) of denopamine to the phenolic glucuronide was in the following order: rat >> rabbit >> dog = monkey. In contrast, the formation of the alcoholic glucuronide was detected in human, monkey, and dog, never in rat and rabbit. Thus, there are drastic species differences in the overall glucuronosyltransferase activities along with the regioselectivity. Although glucuronidation on a phenolic hydroxyl group seems to be common across species, the phenolic glucuronide of denopamine was not detected in human liver and jejunum microsomes. To our knowledge, the free phenolic hydroxyl group is preferred to the alcoholic hydroxyl group on the glucuronidation of xenobiotics and endogenous substrates (Hoffmann et al., 1982; Coffman et al., 1997; Patrick et al., 2002; Ghosal et al., 2004). For example, it has been reported that ezetimibe, a cholesterol absorption inhibitor, underwent phase II metabolism predominantly to the phenolic glucuronide, but not to the alcoholic glucuronide, after oral administration to humans (Patrick et al., 2002). Ghosal et al. (2004) have recently reported that human jejunum microsomes supplemented with UDP-glucuronic acid converted ezetimibe to the phenolic and alcoholic glucuronides; however, the phenolic glucuronide was the major metabolite in human liver microsomes. It is interesting that the present results regarding denopamine are obviously different from these previous reports on the regioselectivity of glucuronidation. However, it has been reported that UGT enzymes do not possess regioselectivity based on functional group (Radominska-Pandya et al., 1999). Therefore, it would more likely be based on molecular shape of denopamine, as is the case with the regioselectivity in the present study.

The alcoholic glucuronide of denopamine was resistant to hydrolysis by two kinds of ß-glucuronidases from H. pomatia and bovine liver. In contrast, it was completely hydrolyzed with ß-glucuronidase from E. coli, although it was considerably resistant to hydrolysis in comparison with the phenolic glucuronide (Fig. 4). A high degree of resistance to the hydrolysis of denopamine glucuronide by ß-glucuronidase has been reported in a previous paper (Furuuchi et al., 1985). Although the glucuronide has not been hydrolyzed completely, under their conditions, we found the complete hydrolysis conditions using another type of ß-glucuronidase. Our results were similar to those of the previous report (Ghosal et al., 2004) in which the alcoholic glucuronide was resistant to hydrolysis by ß-glucuronidase compared with the phenolic glucuronide.

To identify the human UGT isoform(s) responsible for the formation of the alcoholic glucuronide of denopamine, we first examined the activities and kinetics in recombinant UGT microsomes from baculovirus-insect cells. In consequence, only UGT2B7 exhibited high denopamine glucuronosyltransferase activity among the UGT isoforms examined. The kinetics of denopamine glucuronidation in human liver and jejunum microsomes was monophasic, suggesting that a single UGT isoform is responsible for the glucuronidation. Furthermore, it was confirmed that the K_m value of denopamine glucuronidation in recombinant UGT2B7 was similar to those in human liver and jejunum microsomes (Table 1). These results suggest that UGT2B7 would mainly catalyze the denopamine glucuronidation both in human liver and intestine. To clarify the contribution of UGT2B7 for denopamine glucuronidation, inhibition studies and correlation analyses with typical substrates for UGT isoforms were performed using human liver and jejunum microsomes. Diclofenac, a substrate for UGT2B7 (Miners et al., 1997; King et al., 2001), inhibited the denopamine glucuronosyltransferase activities equivalently in human liver, jejunum, and recombinant UGT2B7 microsomes, with similar IC₅₀ values (41.5, 29.7, and 24.0 µM, respectively) (Fig. 7). The denopamine glucuronosyltransferase activities in seven human liver microsomes were significantly correlated with the diclofenac glucuronosyltransferase activities (Fig. 8D). These results indicate that UGT2B7 is the major UGT isoform catalyzing denopamine glucuronidation in humans. Incidentally, the denopamine glucuronosyltransferase activities were also significantly correlated with the ß-estradiol 3-glucuronosyltransferase activities, which were mainly catalyzed by UGT1A1 (Senafi et al., 1994; King et al., 1996). It has been reported that glucuronidation of phenols may be catalyzed by a dimeric form of UGT (Gschaidmeier and Bock, 1994). Homooligomer formation of rat UGT2B1 (Meech and Mackenzie, 1997), and hetero-oligomer formation of rat UGT2B1 and UGT1A families (Ikushiro et al., 1997) have been reported. Additionally, Ishii et al. (2001) reported that hetero-oligomer formation of guinea pig UGT2B21 and UGT2B22 leads to altered substrate specificity. In recombinant UGTs used in most studies, including the present studies, only homo-oligomers would be formed. Although denopamine might also be catalyzed by hetero-oligomers, which had activities like UGT1A1, it is considered that the contribution of UGT1A1 on the denopamine glucuronidation would be minor, based on our finding that recombinant UGT1A1 had no activity on the glucuronidation of denopamine (Fig. 6). There might be one way to explain this phenomenon.

UGT2B7 has the capacity to conjugate a wide range of xenobiotics and hydroxy-steroids (Jin et al., 1993), and its transcripts are found in human liver, intestine, esophagus, brain, kidney, and pancreas (Tukey and Strassburg, 2000). Coffman et al. (1997, 1998) indicate that UGT2B7 is the major isoform responsible for the glucuronidation of opioids of the morphinan and oripavine class. Among them, morphine is glucuronidated at the phenolic 3-hydroxyl group as well as the alcoholic 6-hydroxyl group, at a ratio of approximately 7:1, by recombinant UGT2B7 (Coffman et al., 1997). It has been reported that morphine is not converted to the more potent morphine metabolite, morphine 6-O-glucuronide, thus forming only the 3-O-glucuronide, when administered to rats (Oguri et al., 1990). It was found that rat UGT2B1 is qualitatively similar to the human UGT2B7 in overall substrate specificity, with one important exception: UGT2B1 catalyzes only the formation of morphine 3-O-glucuronide (King et al., 1997). In contrast, monkey UGT2B9 is 89% similar to human UGT2B7 in primary amino acid sequence analysis, and it catalyzes the glucuronidation of morphine at both the 3-hydroxyl and 6-hydroxyl positions (Green et al., 1997). From these reports, regioselective species differences in the alcoholic glucuronidation of denopamine between humans and experimental animals would also be attributed to the conformational difference of the substrate-enzyme complex due to the displacement by amino acid at the catalytic site of the UGT2B family.

Concerning UGT2B7 polymorphism, a C to T transversion at nucleotide 802 of the UGT2B7 coding region gives rise to enzymes with either His (UGT2B7*1) or Tyr (UGT2B7*2) at residue 268 (Bhasker et al., 2000). Although the frequencies of the two alleles are almost equal in white people, the UGT2B7*1 allele is three times more prevalent in Japanese. It has been reported that the glucuronosyltransferase activities of UGT2B7*1 were slightly higher than those of UGT2B7*2 for some substrates such as zidovudine; however, differences were not apparent in a large number of UGT2B7 substrates (Coffman et al., 1998; Barbier et al., 2000). In addition, statistically significant intergenotypic differences in microsomal glucuronosyltransferase activities were not apparent for androsterone, menthol, morphine, and even zidovudine (Bhasker et al., 2000; Court et al., 2003). The interindividual difference in the denopamine glucuronosyltransferase activities in seven human liver microsomes was at most 2.7-fold (Fig. 8). These in vitro data are compatible with the previous in vivo data (n = 5), which reported a 1.9-fold variation on the excretion rate of denopamine glucuronide in human urine (Suzuki et al., 1983). These reports and the present results suggest that there would be little interindividual variation in glucuronidation of denopamine. Additionally, there are other metabolic pathways consisting of 4'-demethylation and 3-hydroxylation, followed by glucuronidation at each hydroxyl group, in which 4'-demethyl-denopamine has been found as the distinct metabolite in human plasma (Suzuki et al., 1983). Even if there were any polymorphisms in glucuronidation of denopamine, the drug would be metabolized effectively in other metabolic pathways described above. To further address this issue, the correlation between the interindividual differences in denopamine pharmacokinetic profiles and genotypes of UGT2B7 in these human subjects needs to be investigated.

In conclusion, there are remarkable species differences along with regioselectivity in denopamine glucuronidation in liver microsomes obtained from humans and experimental animals. In particular, denopamine is glucuronidated exclusively at the alcoholic hydroxyl group, but not the phenolic hydroxyl group, in humans. In addition, we found that this unique glucuronidation was mainly catalyzed by UGT2B7 in both human liver and intestine.

	Acknowledgments

 
We express deep gratitude to Dr. Toru Ishizuka, Director of Exploratory Toxicology & DMPK Research Laboratories, for interest and encouragement. The technical assistance of Hiroyuki Hashimoto is gratefully acknowledged.

	Footnotes

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

doi:10.1124/dmd.104.002667.

ABBREVIATIONS: UGT, UDP-glucuronosyltransferase; 7-HFC, 7-hydroxy-4-trifluoromethylcoumarin; MS, mass spectrometry; HPLC, highperformance liquid chromatography; LC-MS/MS, liquid chromatography-tandem mass spectrometry.

Address correspondence to: Hidefumi Kaji, Exploratory Toxicology & DMPK Research Laboratories, Tanabe Seiyaku Co., LTD., 2-2-50 Kawagishi, Toda-Saitama 335-8505, Japan. E-mail: h-kaji{at}tanabe.co.jp

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