Elsevier

Biochemical Pharmacology

Volume 67, Issue 7, 1 April 2004, Pages 1269-1278
Biochemical Pharmacology

Uridine diphosphate sugar-selective conjugation of an aldose reductase inhibitor (AS-3201) by UDP-glucuronosyltransferase 2B subfamily in human liver microsomes

https://doi.org/10.1016/j.bcp.2003.11.010Get rights and content

Abstract

N-Glucosidation is known as a major metabolic reaction for barbiturates in humans. However, the enzyme(s) involved in this N-glucosidation has not been clarified yet. Thus, to clarify the enzyme(s) involved in the N-glucosidation in human liver microsomes, we investigated the N-glucosyltransferase activity in recombinant UDP-glucuronosyltransferases (UGTs) using AS-3201, an aldose reductase inhibitor, as a substrate. AS-3201 was found to be biotransformed to both N-glucoside and N-glucuronide in human liver microsomes. The N-glucosyltransferase activities were detectable with multiple UGT isoforms (UGT1A1, UGT1A3, UGT1A4, UGT2B4, UGT2B7, and UGT2B15). In contrast, the N-glucuronyltransferase activities for the same substrate were seen with UGT1A (UGT1A1, UGT1A3, UGT1A4, and UGT1A9) but not UGT2B isoforms. We then determined the relative activity factor of each recombinant UGT and estimated the contribution of each UGT isoform to the N-glucosidation in human liver microsomes. The results showed that UGT2B isoforms mainly contribute to AS-3201 N-glucosidation in human liver microsomes. In addition, the activity of AS-3201 N-glucosyltransferase significantly correlated with that of amobarbital N-glucosyltransferase in microsomes from sixteen human livers (r=0.964, P<0.01), indicating that UGT2B isoforms were also involved in the barbiturate N-glucosidation in humans. The findings of this study clearly show that UGT2B specifically utilizes UDP-glucose but not UDP-glucuronic acid as a sugar donor for the conjugation of AS-3201 in human liver microsomes.

Introduction

UGTs play an important role in the metabolism of xenobiotics and endogenous compounds. UGTs have been classified into two families, UGT1 and UGT2; the latter being subdivided into the UGT2A and UGT2B subfamilies [1]. In humans, the UGT1 genes are located on chromosome 2q37, and are encoded by at least 12 unique first exons, which are spliced to common exons from 2 to 5 [2]. UGT2Bs are encoded by separate genes, and are clustered on chromosome 4q13 [3], [4].

The UGTs catalyze the conjugation of a variety of substrates with a sugar using UDP-sugar as a sugar donor. The UDP-sugar consists of UDP-GA, UDP-galactose, UDP-glucose, or UDP-xylose. Glucuronidation is one of the most important phase II drug-metabolizing reactions. A number of exogenous as well as endogenous compounds have been shown to undergo glucuronidation in humans. The UGT1 has been reported to catalyze the glucuronidation of bilirubin and various phenols and amines [5], [6], whereas the UGT2B has been reported to catalyze the glucuronidation of opioids, bile acids, and steroids [6].

In contrast, glucosidation is known as a metabolic reaction for a relatively limited number of compounds, such as phenobarbital [7], amobarbital [8], sulphadimidine, sulphamerazine and sulphamethoxazole [9]. Accordingly, the N-glucosides of barbiturates have been reported as the major urinary metabolites in humans (Fig. 1A), whereas the N-glucuronides have not been detected in human urine [7], [8], [10], [11]. Thus, it was theorized that the enzyme(s) involved in barbiturate N-glucosidation might specifically utilize UDP-glucose as a sugar donor. However, to date, this enzyme(s) has not yet been clarified.

(R)-(−)-2-(4-bromo-2-fluorobenzyl)-1,2,3,4-tetrahydropyrrolo[1,2-a]pyrazine-4-spiro-3′-pyrrolidine-1,2′,3,5′-tetrone (AS-3201, Fig. 1B) is a structurally novel and potent aldose reductase inhibitor containing a succinimide ring [12], [13]. The nitrogen atom in the succinimide ring is located between carbonyl groups, which is particularly similar to the target nitrogen atom for the glucosidation of barbiturates. N-Glucuronide and N-glucoside have been isolated and identified as the major metabolites of AS-3201 in humans, and it has been clarified that these conjugates formed through unstable intermediates [14].

In this paper, the enzyme(s) involved in the AS-3201 N-glucosidation was investigated, and compared with the enzyme(s) involved in the N-glucuronidation. We found that UGT2B enzymes catalyzed the N-glucosidation but not the N-glucuronidation of this chemical. Also, an application of RAF for recombinant human UGTs is reported for the first time.

Section snippets

Materials

AS-3201 and [14C]AS-3201 (1.41 MBq/mg) were synthesized at Dainippon Pharmaceutical. The radiochemical purity of [14C]AS-3201 was more than 98%. Amobarbital, bilirubin, estradiol, estradiol 3-glucuronide, trifluoperazine, 4-methylumbelliferone, 4-nitrophenol, hyodeoxycholic acid, and azidothymidine were obtained from Sigma. UDP-GA and UDP-glucose were purchased from Wako Pure Chemical, recombinant UGTs (human UGT supersomes) and control microsomes (insect cell control supersomes) from Gentest,

AS-3201 N-glucosidation in human liver microsomes

The formation of AS-3201 N-glucuronide and N-glucoside was detectable after incubation of AS-3201 with human liver microsomes in the presence of UDP-GA or UDP-glucose (Fig. 2A and B). The retention times of AS-3201 N-glucuronide and N-glucoside were 13.0 and 15.8 min, respectively. Peaks of m/z 568, 570, 604, and 606 were detected in the LC/MS spectrum of the N-glucuronide (Fig. 2C). The peaks of m/z 568 and 570, or 604 and 606 are isotopic clusters. The peaks of m/z 568 and 570 were considered

Discussion

In the present study, we described several lines of evidence that the UGT2B isoforms are responsible for AS-3201 N-glucosidation but not N-glucuronidation in human liver microsomes. Our results indicated that UGT2B15 mostly contributes to AS-3201 N-glucosidation in human liver microsomes. UGT2B15 is known to catalyze the glucuronidation of a number of steroids, including 5α-dihydrotestosterone and androstane-3α,17β-diol [26], [27]. A polymorphism of the UGT2B15 gene in the coding region that

Acknowledgements

We thank Mr. Glen Argyle (Dainippon Pharmaceutical) for critical reading of the manuscript. This study was supported in part by a Grant-in-Aid (No. 99-2) from The Organization for Pharmaceutical Safety and Research (OPSR), Ministry of Education, Science, Sports and Culture of Japan, and Ministry of Health, Labour and Welfare of Japan.

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