TY - JOUR T1 - UDP-Glucuronosyltransferase 1A1 Is the Principal Enzyme Responsible for Etoposide Glucuronidation in Human Liver and Intestinal Microsomes: Structural Characterization of Phenolic and Alcoholic Glucuronides of Etoposide and Estimation of Enzyme Kinetics JF - Drug Metabolism and Disposition JO - Drug Metab Dispos SP - 371 LP - 380 DO - 10.1124/dmd.106.012732 VL - 35 IS - 3 AU - Zhiming Wen AU - Melanie N. Tallman AU - Shazia Y. Ali AU - Philip C. Smith Y1 - 2007/03/01 UR - http://dmd.aspetjournals.org/content/35/3/371.abstract N2 - Etoposide, an important anticancer agent, undergoes glucuronidation both in vitro and in vivo. In this study, three isomeric glucuronides of etoposide, including one phenolic (EPG) and two alcoholic glucuronides (EAG1 and EAG2), were biosynthesized in vitro with human liver microsomes (HLMs), and identified by liquid chromatography-electrospray ionization-mass spectrometry and confirmed by β-glucuronidase cleavage. In vitro UDP-glucuronosyltransferase (UGT) reaction screening with 12 recombinant human UGTs demonstrated that etoposide glucuronidation is mainly catalyzed by UGT1A1. Although UGT1A8 and 1A3 also catalyzed the glucuronidation of etoposide, their activities were approximately 10 and 1% of UGT1A1. Enzyme kinetic study indicated that the predominant form of etoposide glucuronide in HLMs and human intestinal microsomes (HIMs) was EPG, whereas EAG1 and EAG2 were the minor metabolites, with approximately an 8 to 10% glucuronidation rate of EPG. For the formation of EPG, the Vmax of HLMs (110 pmol/min/mg protein) was very similar to that of recombinant UGT1A1 (124 pmol/min/mg protein), whereas the Vmax of HIMs (54.4 pmol/min/mg protein) was 2-fold lower than those of HIMs and UGT1A1. The Km values of HLMs (530 μM) and HIMs (608 μM) were 2-fold higher than that of UGT1A1 (285 μM). The Vmax/Km values for the formation of EPG were 0.21 and 0.09 μl/min/mg protein for HLMs and HIMs, respectively. The data indicated that UGT1A1 is principally responsible for the formation of etoposide glucuronides, mainly in the form of phenolic glucuronide, suggesting that etoposide can be used as a highly selective probe substrate for human UGT1A1 in vitro. The American Society for Pharmacology and Experimental Therapeutics ER -