Received for publication October 20, 2004.
Revised December 6, 2004.
Accepted for publication December 16, 2004.

Regioselective glucuronidation of denopamine: Marked species differences and identification of human UDP-glucuronosyltransferase isoform

Abstract

Denopamine is one of the oral 1-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 were found. In contrast, in human the alcoholic glucuronide was exclusively detected. 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 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 formations of denopamine glucuronidation by human liver, jejunum and recombinant UGT2B7 microsomes were 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 phenolic hydroxyl group, occurs regioselectively in human.

Key words: enzyme kinetics, extrahepatic drug metabolism, glucuronidation, HPLC, liver microsomes, microsomes, phase II drug metabolism, UDP glucuronyltransferases

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