Introduction

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The UDP-glucuronosyltransferases (UGTs¹) catalyze the transfer of glycosyl group from a nucleotide sugar to an aglycone. The transfer of glucuronic acid from UDP-glucuronic acid (UDPGA) leads to glucuronidation, a major conjugation reaction, which, in general, results in inactivation and excretion of endogenous compounds such as bilirubin and steroids as well as a wide variety of xenobiotics including drugs, carcinogens, and other environmental pollutants (Dutton, 1980). The past decades have witnessed significant progress in substrate specificity identification in several species, especially in rats and humans, with the availability of purified and/or cloned UGTs, and sequence analysis (Burchell, 1999). For instance, morphine glucuronidation was found to be catalyzed by human UGT2B7 (King et al., 1996; Coffman et al., 1997), and diclofenac glucuronidation is believed to be primarily catalyzed by rat UGT2B1 and human UGT2B7 (King et al., 2001).

Interestingly, in addition to substrate specificity, there exists the specificity of UGTs toward UDP-sugars (cosubstrates). As early as in 1977, Fevery et al. demonstrated that although UDPGA is the primary UDP-sugar cosubstrate used for the bilirubin esterification in mammals, two other UDP-sugars, UDP-glucose (UDPG), and UDP-xylose, also are potential cosubstrates (Fevery et al., 1977). Studies by Motoyama (1979) with 50 human liver samples showed a significant correlation (r² = 0.974) between bilirubin UGT and bilirubin xylosyltransferase activity, suggesting that a single enzyme was responsible for these activities. Later on, UDP-sugar specificity of bilirubin UGT has been studied in purified rat liver preparations, and it was proposed that a single bilirubin UGT could accept different sugar nucleotides (Burchell and Blanckaert, 1984). Similarly, Senafi et al. (1994) demonstrated that a cloned-expressed human bilirubin UGT (UGT1A1) could use UDPGA, UDPG, and UDP-xylose as a substrate with the catalytic potential (V_max/K_{m (bilirubin)}) for UDPGA 2- and 10-fold greater than that for UDP-xylose and UDPG, respectively. Apparently, it was believed that a single enzyme, at least a common subunit, was responsible for the transfer of sugars from respective sugar nucleotides to bilirubin. However, formation of hyodeoxycholic acid (a 6-hydroxylated bile acid) glucoside was thought to be catalyzed by a novel UDPG-specific glucosyltransferase because the patterns of photo-affinity labeling by 5-[-³²P]azido-UDPGA and 5-[-³²P]azido-UDPG in the 50- to 56-kDa range were significantly different and cloned hyodeoxycholic acid-specific UGT (UGT2B4) failed to generate the glucoside conjugate (Radominska et al., 1993). Therefore, the authors suggested the presence of a novel UDPG-specific glucosyltransferase catalyzing the biosynthesis of 6-O-glucoside of bile acid in human liver microsomes. The different scenarios may stem from the combination of substrate dependence and the involvement of different UGT isoforms. For instance, an aglycone may undergo conjugation with more than one sugar nucleotide but catalyzed by different UGT isoforms, in which no correlation should be observed among different conjugations. A correlation could be observed in which a UGT isoform is capable of transferring different sugars from different sugar nucleotides to the same aglycone. Although a considerable number of drugs and other xenobiotics, as well as some endogenous compounds, have been reported to be conjugated with UDPGA, UDPG, and/or UDP-xylose in different tissue microsomes from different species (Senafi et at, 1994; Chmela et al., 2001; Shipkova et al., 2001), the specificity of UGTs toward sugar nucleotides has received scant attention.

In the present study, we found that compound A [(+)-(5S,6R,7R)-2-isopropylamino-7-[4-methoxy-2-((2R)-3-methoxy-2-methylpropyl)-5-(3,4-methylenedioxyphenyl) cyclopenteno [1,2-b] pyridine 6-carboxylic acid, Fig. 1], a new selective and potent endothelin ET_A receptor antagonist (Okada et al., 2000), underwent significant acyl glucuronidation and acyl glucosidation in human liver microsomes. To determine the mechanism of both conjugations, we evaluated the kinetics of the glucuronide and glucoside formation and mutual inhibition of UDPGA and UDPG on conjugation reactions. Additionally, by using recombinant UGTs, we also evaluated UGT isoforms responsible for both conjugations and demonstrate that multiple UGTs are involved in the glucuronidation but essentially only UGT2B7 is responsible for the glucosidation.

View larger version (20K): [in this window] [in a new window]   Fig. 1.   Structures of compound A, its glucoside and glucuronide, and compound B (internal standard).

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

Chemicals and Reagents. Compound A and its analog compound B [(5S,6R,7R)-5-(1,3-benzodioxol-5-yl)-7-[2-(3-hydroxy-2-methylpropyl)-4-(methyloxy)phenyl]-2-[(1-methylethyl)amino]-6,7-dihydro-5H-cyclopenta[b]pyridine-6-carboxylic acid] used as an internal standard (Fig. 1) were synthesized by Banyu Pharmaceutical Co. (Ibaraki, Japan). UDPGA, UDPG, alamethicin, and Brij 58 were obtained from Sigma-Aldrich (St. Louis, MO). Solvents used for liquid chromatography were of analytical or HPLC grade. A pool of human liver microsomes from 10 individuals (HHM-0259) were obtained from IIAM (Scranton, PA). A bank of human liver microsomes (n = 16 different organ donors) was purchased from Xenotech LLC (Kansas City, KS). Recombinant human UGT1A1, 1A3, 1A4, 1A6, 1A9, 2B7, and 2B15 (Supersomes) were obtained from the BD Gentest Corporation (Woburn, MA) and baculosomes of UGT1A7 and 1A10 from PanVera (Madison, WI).

Incubation Conditions. For the quantitation of conjugate formation, incubations were carried out at 37°C in a Fisher shaking water bath, employing 1.1 ml of polypropylene disposable deep well tubes purchased from Matrix Technologies Corp. (Hudson, NH). The incubation mixture (final volume of 250 µl) consisted of the following: 0.1 M potassium phosphate buffer (pH 7.4), MgCl₂ (10 mM), EDTA (1 mM), UDPGA or UDPG (fixed concentration of 5 mM or varied concentrations), human liver microsomes or expressed UGTs (0.1 mg of protein/ml), and compound A with desired concentrations (100-fold concentrated stock solution in 50% acetonitrile aqueous solution). The concentration of alamethicin and Brij 58, when applied, was 50 and 100 µg/mg of protein, respectively. The reaction was started by the addition of the UDPGA or UDPG and terminated with acetonitrile (200 µl) following a 15-min incubation. The internal standard (compound B) solution (water/acetonitrile, 50:50) was added to the samples (50 µl, 2.5 µM). Following brief vortexing and centrifugation (10 min at 2000 rpm), the supernatant was transferred to 96-well microtiter plates for LC-MS assay.

LC-MS Analysis. For structure identification, the chromatographic separation of compound A and its metabolites was performed on a reverse phase C₁₈ column (DBS Hypersil, 2.0 × 150 mm, 5 µm; Thermo Hypersil, Keystone Scientific Operations, Bellefonte, PA) with a HP 1050 LC system (Hewlett-Packard, Palo Alto, CA). Solvent A consisted of 0.02% aqueous acetic acid with pH adjusted to 4.5 with NH₄OH and acetonitrile (90:10) and solvent B of acetonitrile and water (90:10) and were delivered at a constant total flow rate of 0.2 ml/min. The initial mobile phase consisted of 10% of solvent B, which was linearly increased to 55% over 20 min, then to 80% in another 2 min and held for additional 3 min. The column was then equilibrated under initial conditions for 5 min.

HPLC-UV Conditions. The chromatographic separation of compound A and the two conjugates generated in the large scale incubation was performed on a reverse phase C₁₈ column (DBS Hypersil, 4.6 × 150 mm, 5 µm; Thermo Hypersil, Keystone Scientific Operations) with a Shimadzu LC-10AD HPLC system (Shimadzu Scientific Instruments, Inc., Columbia, MD). The same mobile phase for LC-MS analysis was used and delivered at a constant flow rate of 1.0 ml/min. The initial mobile phase consisted of 50% of solvent B, which increased linearly to 75% over 5 min and then to 85% in 0.1 min. The value was then held for an additional 2 min, and the column was equilibrated for 5 min. The elution of the analytes was monitored by UV detection (286 nm). Under these conditions, the glucuronide, glucoside, and compound A eluted at 4.3, 7.8 ,and 9.6 min, respectively. The fractions of the glucuronide and glucoside were collected, dried in a lyophilizer, and stored at 20°C until further use. The purity of each conjugate isolated was >95% confirmed by HPLC-UV analysis.

NMR Analysis. The ¹H ¹D and total correlation spectroscopy NMR spectra for compound A and the isolated conjugates were obtained in CD₃OD (99.96% deuterium content; Isotec Inc., Miamisburg, OH) at 25°C on a Varian Inova 500 MHz spectrometer (Varian Medical Systems, Palo Alto, CA) equipped with a MIDG-3 probe (Nalorac Corp., Martinez, CA). The ¹H chemical shifts (in parts per million) are relative to the solvent CD₂HOD signal, which is set at 3.33 ppm.

Quantitation of Glucuronide and Glucoside of Compound A. The purified conjugates were dissolved in a given volume of buffered 50% acetonitrile aqueous solution (pH 6.0). A small quantity of the solution was added to phosphate buffer at pH 10 and incubated at 37°C for 2 h. Analysis by LC-MS confirmed the complete hydrolysis of the conjugates to compound A, which was then calibrated with its standard curve. The quantified stock solution of each conjugate was kept at 20°C for the generation of standard curves.

Data Analysis. The apparent enzyme kinetic parameters were determined by fitting the reaction velocities versus substrate concentrations to eq. 1 or eq. 2 (GraFit; Erithacus Software Ltd., Staines, UK), which describe Michaelis-Menten kinetics alone or Michaelis-Menten kinetics coupled with uncompetitive substrate inhibition, respectively.

Where v, S, V_max, K_m and K_s are the velocity of conjugations, compound A concentration, the maximal conjugation velocity, apparent Michaelis constant, and substrate inhibition constant, respectively.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

Structure Characterization of Compound A Conjugates. Compound A appeared as its protonated molecule (MH⁺) at m/z 533 under positive ion electrospray ionization. In a pilot study where compound A was incubated in fresh human hepatocytes, two major metabolites were detected with MH⁺ at m/z 709 and 695 (data not shown). They were also generated in human liver microsomes supplemented with either UDPGA (MH⁺ at m/z 709) or UDPG (MH⁺ at m/z 695). Upon tandem mas spectrometry fragmentation, the metabolite with MH⁺ at m/z 709 gave rise to a prominent fragment at m/z 533 via loss of 176 (Fig. 2A), whereas that with MH⁺ at m/z 695 generated the same fragment (m/z 533) via loss of 162 (Fig. 2B). The involvement of UDP-sugars and fragmentation pattern suggest the metabolites as a glucuronide and glucoside of compound A, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 2.   Tandem mass spectrometry spectra of compound A glucuronide (A) and glucoside (B) generated in a pool of human liver microsomes (HHM-0259) in the presence of UDPGA or UDPG.

Kinetic Properties of Compound A Acyl Glucuronidation and Glucosidation in Human Liver Microsomes. Optimal assay conditions were established using pooled human liver microsomes with varying concentrations of compound A, UDPGA, UDPG, microsomal protein, and time of incubation. Both reactions were shown to be linear up to 30 min, and all subsequent incubations were carried out for 15 min and contained 0.1 mg/ml of microsomal protein and 5 mM of either UDPGA or UDPG.

View larger version (19K): [in this window] [in a new window]   Fig. 3.   Concentration-dependent rate of formation of compound A glucuronide (A) and glucoside (B) in a pool of human live microsomes (HHM-0259) in the presence and absence of alamethicin or Brij 58.  Data are expressed as mean ± S.E. of triplicates.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Inhibition of glucuronidation by UDPG (A) and glucosidation by UDPGA (B). A, double reciprocal plot for the inhibition of glucuronidation by UDPG; B, double reciprocal plot for the inhibition of glucosidation by UDPGA. Data are expressed as mean ± S.E. of triplicates.

Acyl Glucuronidation and Glucosidation in Different Human Liver Microsomes. The rates of compound A glucuronidation and glucosidation varied in human liver microsomes of 16 donors with a ratio of glucuronide to glucoside of 1.2 to 3.5, 2.9 to 7.2, and 1.6 to 4.9 in native, almethicin-treated and Brij 58-treated human liver microsomes, respectively. There was no correlation between these two conjugations (Fig. 5). The correlation coefficients and the reaction rates of glucuronidation and glucosidation are summarized in Table 4. It appeared that glucosidation was more resistant than glucuronidation to the effect of alamethicin and Brij 58 in all microsomal preparations.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Comparison of the rates of glucuronidation and glucosidation of compound A determined in 16 human liver microsomal preparations. Concentration of compound A was 50 µM. The correlation coefficient was determined by linear least square method.

Acyl Glucuronidation and Glucosidation by Stably Expressed UGTs. The screening for compound A glucuronidation and glucosidation activity in all commercially available microsomes expressing specific UGT isoforms revealed that compound A was glucuronidated by multiple UGTs (1A1, 1A3, 1A9, 2B7 and 2B15), whereas it was glucosidated essentially only by UGT2B7, which gave rise to a comparable activity of both glucuronidation and glucosidation (Fig. 6). It was also found that the activity of UGTs toward both conjugations was decreased in the presence of alamethicin and Brij 58. As a result, no latency-disrupting agents were used in studies using expressed UGTs. There was no activity of glucuronidation and very low level of glucosidation [< 2 pmol/(min · mg of protein)] in control microsomes from cells infected with wild-type vector.

View larger version (15K): [in this window] [in a new window]   Fig. 6.   Rate of compound A glucuronidation and glucosidation catalyzed by stably expressed UGT isoforms. Data are expressed as mean ± S.E. of triplicates.

Kinetic Properties of Compound A Acyl Glucuronidation and Glucosidation by Expressed UGTs. Glucuronidation by four UGT isoforms (UGT1A1, 1A3, 2B7 and 2B15) and glucosidation by UGT2B7 displayed typical Michaelis-Menten kinetics. However, glucuronidaiton by UGT1A9 showed a more complex kinetics with decreasing velocity at higher substrate concentration (Fig. 7), suggesting the effect of substrate inhibition. Fitting the data points to the eq. 1 or 2 yielded the kinetic parameters listed in Table 5. As for glucuronidation, relatively low apparent K_m values ( 3 µM) for compound A were observed with all three isoforms from the 1A family, especially UGT1A9 (0.24 µM), whereas a greater value (>2.5-fold) was obtained with UGT2B7 and 2B15. The kinetic parameters of glucosidation by UGT2B7 were close to those of glucuronidation by the same isoform (Table 5).

View larger version (19K): [in this window] [in a new window]   Fig. 7.   Concentration-dependent rate of glucuronide formation of compound A by UGT1A9. Data are expressed as mean ± S.E. of triplicates.

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

Glucuronidation reactions are catalyzed by the microsomal UGT. Consistent with its broad substrate profile, UGT is known to exist as a superfamily of enzymes (Mackenzie et al., 1997). Parallel to the progress in expression and characterization of UGT isoforms in human and laboratory animals (Tukey and Strassburg, 2000), recognition of UGT isoforms responsible for glucuronidation of given compounds has received increasing attention recently (Coffman et al., 1997; King et al., 2001; Vashishtha et al., 2001). However, after Senafi et al. (1994) used a cloned human bilirubin UGT (UGT1A1) to investigate UDP-sugar specificity, little effort has been reported on the UDP-sugar specificity of UGT isoforms in phase II metabolism of drugs. Using stably expressed UGTs in the present study, we demonstrate that a single UGT isoform can catalyze both glucuronidation and glucosidation of an endothelin ET_A receptor antagonist in human liver microsomes. Although multiple UGTs (1A1, 1A3, 1A9, 2B7, and 2B15) were involved in its glucuronidation, only UGT2B7 was responsible for its glucosidation as well as glucuronidation. Clearly, UGT2B7 can use both UDPGA and UDPG as cosubstrates for metabolism of this compound to transfer the respective sugar moiety to this compound with a comparable catalytic capacity (V_max) for both UGPGA and UDPG (Table 5), but other isoforms can only have UGPGA as a cosubstrate. This finding may explain the lack of correlation between these two conjugations among 16 subjects (Fig. 5). Possible involvement in the glucosidation of other isoforms that are not commercially available cannot be ruled out at the present time.

Glucuronidation has been considered as the most common phase II reaction for a wide variety of endogenous substrates and xenobiotics in mammals. Usually, if glucuronidation is possible, the remaining types of glycosidations would be of minor importance in animals, although they are common in plants and invertebrates (Tang, 1990). Interestingly, we found that compound A significantly underwent both acyl glucuronidation and acyl glucosidation in human liver microsomes with the glucoside formation accounting for ~30 to 80% of glucuronide conjugation in 16 native human liver microsomal preparations. In contrast to the cases of bilirubin (Senafi et al., 1994) and benzo(a)pyrene-3,6-quinol (Gschaidmeier et al., 1995), where the formation of glucoside conjugate was much lower than their respective glucuronide conjugate (UGT1A1 for bilirubin and UGT1A6 and 1A7 for benzo(a)pyrene-3,6-quinol), the present study revealed a comparable catalytic capacity in the formation of both glucuronide and glucoside conjugates by UGT2B7. In addition, compound A glucoside was one of the major metabolites generated in a fresh preparation of human hepatocytes (data not shown). Collectively, these findings suggest that acyl glucosidation of compound A may not be a minor pathway in vivo. Increasing evidence has shown that glucosidation could be predominant in some species. As early as in 1970s, N-glucosides of a triazole drug and phenobarbital were identified as major metabolites in dogs (Duggan et al., 1974) and in man (Tang et al., 1979). Recently, a number of endogenous compounds and drugs have been found to be preferentially or exclusively converted to O--D-glucosides in animals. Boberg et al. (1998) found that cerivastatin was preferentially metabolized to O--D-glucoside conjugate in dogs, as compared with production of glucuronide. Bohemine and pantothenic acid were exclusively transformed to O--D-glucosides in mice (Chmela et al., 2001) and dogs (Nakano et al., 1986a), respectively. Glucosidation may serve as an alternative detoxification pathway when levels of UDPGA are depleted due to toxicological or pathological consequences or in cases where aglycones preferentially undergo glucosidation.

The relative significance of glucuronidation and glucosidation of compound A may depend on a couple of factors. The concentration of the nucleotide sugars is an important determinant. These two conjugations may compete for the UDP-sugars based on the fact that UDPGA and UDPG competitively inhibited their counterpart conjugations with comparable K_i values (Fig. 4), and similar K_m values were observed for both UDPGA and UDPG (Table 3). Therefore, the actual concentrations of individual UDP-sugars are critical to the ratio of these two conjugations. It has been found that in a number of vertebrates, hepatic UDPGA concentration is not significantly different from UDPG (except for guinea pig liver where UDPGA level exceeds about two times that of UDPG), but UDPG is synthesized at a higher rate than UDPGA in rat, guinea pig, and chicken livers (Zhivkov and Tosheva, 1986). It is worth noting that considerable fluctuations in UDP-sugar concentration have been observed after drug and hormone treatment (Tosheva and Zhivkov, 1976, 1977). Therefore, the change in relative significance of compound A glucuronidation and glucosidation is likely where UDPGA and UDPG are altered due to toxicological and pathological impacts.

The relative activity of UGT2B7 in different subjects may also be a source of variability. The variation of the rate of compound A glucoside formation in liver microsomal preparations from different donors may be derived mainly from the different activity of UGT2B7, which was characterized in the present study as the only UGT isoform catalyzing acyl glucosidation of compound A in human liver microsomes. Of human isoforms comprising the UGT2B subfamily, UGT2B7 is of particular significance in drug metabolism. Notably, it glucuronidates numerous carboxylic acid-containing drugs (nonsteroidal anti-inflammatory drugs, clofibric acid and valproic acid) and opioids (Jin et al., 1993; Coffman et al., 1998). Compound A, also a carboxylic acid, was found to be comparably conjugated with UDPG and UDPGA by UGT2B7. Since its glucuronidation was mediated by multiple UGT isoforms (Fig. 6), any change in UGT2B7 activity would exert more significant impact on its glucosidation than its glucuronidation. Beside liver, UGT2B7 also has been found to a varying degree in extrahepatic tissues (gastrointestinal tract, brain, and kidney) in human (King et al., 2000). As a result, it is reasonable to expect different glucosidation activity for compound A in different tissues. Interestingly, several studies have shown higher glucosidation activity in kidney microsomes than in liver microsomes (Chmela et al., 2001; Shipkova et al., 2001). Whether it could be the same case with compound A needs to be explored in future studies.

While for P450 catalyzed reactions, where more than one isoforms with different Michaelis-Menten kinetics participate, the primary determinant is concentration of a substrate at the active site of the isoform (Chesne et al., 1998; Ko et al., 1998). Such is not a case with compound A conjugations by either sugar due to the comparable apparent K_m values determined in human liver microsomes (Table 2). The relatively low K_m values (~10 µM) for compound A in both conjugations suggest that compound A is a good substrate for UGTs, whereas most compounds reported for glucuronidation and glucosidation tend to show much higher K_m values (Senafi et al., 1994; Innocenti et al., 2001; Shipkova et al., 2001). Interestingly, much lower K_m values for compound A were obtained in the expressed UGT1A isoforms, especially in UGT1A9 (only 0.23 µM), but the values obtained in the expressed UGT2B isoforms remained comparable to those measured in human liver microsomes.

There have been several reports of the formation of ether glucosides in the literature. To the best of our knowledge, this is the first description of an acyl glucoside conjugate of a drug generated by human liver microsomes. Another drug acyl glucoside formed in human previously reported is mycophenolic acid glucoside, but it was only generated in human kidney microsomes at a low level (Shipkova et al., 2001). Chemically, acyl glucuronides and acyl glucosides belong to the same class of electrophilic metabolites. Acyl glucuronides are known for their nonenzymatic reactions including acylation and/or glycation of endogenous macromolecules, hydrolysis to reform the parent aglycone, and intramolecular rearrangement (Sallustio et al., 2000). However, the reactivity of acyl glucosides has attracted little attention probably due to their rare occurrence, especially in man. At present, our laboratory is active in evaluating the relative reactivity of acyl glucuronide and acyl glucoside conjugates of compound A, and this will be the subject of a separate manuscript.

In summary, the present study demonstrates that UGTs in human liver microsomes catalyze the transfer of glucuronic acid and glucose from UDPGA and UDPG, respectively, to compound A, a new, selective and potent endothelin ET_A antagonist, resulting in extensive acyl glucuronidation and acyl glucosidation. Using commercially available microsomes expressing UGT isoforms, it is found that the glucosidation is mediated by a single isoform (UGT2B7), whereas the glucuronidation is catalyzed by multiple isoforms (UG1A1, 1A3, 1A9, 2B7 and 2B15). UGT2B7 possesses a comparable catalytic capacity for both conjugations.