Vol. 30, Issue 11, 1250-1256, November 2002

Involvement of Multiple UDP-glucuronosyltransferase 1A Isoforms in Glucuronidation of 5-(4'-hydroxyphenyl)-5-phenylhydantoin in Human Liver Microsomes

Miki Nakajima, Nozomi Sakata, Noriko Ohashi, Toshiyuki Kume, and Tsuyoshi Yokoi

Division of Drug Metabolism, Faculty of Pharmaceutical Sciences, Kanazawa University, Kanazawa, Japan (M.N., N.S., T.Y.); and Discovery Research Laboratory, Tanabe Seiyaku Co., Ltd., Saitama, Japan (N.O., T.K.)

In humans, orally administered phenytoin, 5,5-diphenylhydantoin, is mainly excreted as 5-(4'-hydroxyphenyl)-5-phenylhydantoin (4'-HPPH) O-glucuronide. Phenytoin is oxidized to 4'-HPPH by CYP2C9 and to a minor extent by CYP2C19, and then 4'-HPPH is metabolized to 4'-HPPH O-glucuronide by UDP-glucuronosyltransferase (UGT). In the present study, 4'-HPPH O-glucuronidation in human liver microsomes was investigated. The metabolite formed by incubation with human liver microsomes, 4'-HPPH, and UDP-glucuronic acid was identified as 4'-HPPH O-glucuronide by liquid chromatography-tandem mass spectrometry analysis. The 4'-HPPH O-glucuronosyltransferase activity in human liver microsomes was not saturated at concentrations up to 500 µM of 4'-HPPH. Any commercially available recombinant human UGTs (UGT1A1, UGT1A3, UGT1A4, UGT1A6, UGT1A9, UGT2B7, and UGT2B15) expressed in baculovirus-infected insect cells did not show detectable 4'-HPPH O-glucuronide. The 4'-HPPH O-glucuronidation in pooled human liver microsomes was inhibited by -estradiol as a typical substrate for UGT1A1 (IC₅₀ = 21.1 µM) and imipramine as a typical substrate for UGT1A4 (IC₅₀ = 57.7 µM). The inhibitory effects of propofol as a specific substrate for UGT1A9 (IC₅₀ = 167.1 µM) and emodin as a substrate for UGT1A8 and UGT1A10 (IC₅₀ = 287.6 µM) were not prominent. The interindividual difference in the 4'-HPPH O-glucuronidation in 14 human liver microsomes was 28.5-fold (0.023-0.656 nmol/min/mg of protein). The 4'-HPPH O-glucuronosyltransferase activity in 11 human liver microsomes was significantly (r = 0.609, P < 0.05) correlated with the 4-nitrophenol glucuronosyltransferase activity, which is catalyzed by UGT1A6 and UGT1A9. These results suggest that multiple UGT1As such as UGT1A1, UGT1A4, UGT1A6, and UGT1A9 are involved in 4'-HPPH O-glucuronidation in human liver microsomes, although the percentage contribution of each UGT1A could not be estimated. Large interindividual differences in the glucuronidation of 4'-HPPH might be responsible for the nonlinearity of the phenytoin plasma concentration or adverse reactions in humans.