Vol. 29, Issue 10, 1290-1295, October 2001

Quaternary Ammonium-Linked Glucuronidation of 1-Substituted Imidazoles: Studies of Human UDP-Glucuronosyltransferases Involved and Substrate Specificities

Sarvesh C. Vashishtha, Edward M. Hawes, Gordon McKay, and Denis J. McCann

Drug Metabolism and Drug Disposition Group, College of Pharmacy and Nutrition, University of Saskatchewan, Saskatoon, Saskatchewan, Canada (S.C.V., E.M.H., G.M.); and Drug Disposition and Metabolism Department, AstraZeneca, Wilmington, Delaware (D.J.M.).

A series of eight 1-substituted imidazoles was investigated as model substrates for glucuronidation at an aromatic tertiary amine of polyaza heterocyclic ring systems. The human UDP-glucuronosyltransferases (UGTs) involved and substrate specificities were investigated. Nine expressed enzymes (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A7, UGT1A9, UGT1A10, UGT2B7, and UGT2B15) were examined, but only UGT1A4 catalyzed the formation of a quaternary ammonium-linked glucuronide metabolite for six of the substrates. UGT1A3 also catalyzed the glucuronidation of the previously investigated 1-phenylimidazole but none of the newly investigated compounds. No glucuronidation was observed with 1-(4-nitrophenyl)imidazole, the compound with the 4-phenyl substituent with the largest electron withdrawing effect. The incubation conditions for the determination of the kinetic constants for UGT1A4 catalysis of six substrates were optimized and included incubation at pH 7.4 with alamethicin at 10 µg/mg of protein. Latency disrupting agents, including alamethicin and sonication, enhanced glucuronidation 1.25-fold at most. There were 17.5- and 2.2-fold variations in the apparent K_m (range, 0.18-3.15 mM) and V_max values (range, 0.16-0.35 nmol/min/mg of protein). Linear correlation analyses between UGT1A4 kinetics and substrate physicochemical parameters showed significant correlation between V_max and both the partition coefficient (log P, n-octanol/water) and pK_a and between K_m and pK_a, thereby indicating that the lipophilicity and the ease of availability of the tertiary amine lone pair of electrons of the substrate are important with respect to enzyme catalysis.