Received for publication January 5, 2006.
Revised January 26, 2006.
Accepted for publication January 27, 2006.

N-Glucuronidation of the PDGF Receptor Tyrosine Kinase Inhibitor 6,7-(Dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)-(3-fluoro-phenyl)-amine by Human UDP-Glucuronosyltransferases

Abstract

Abstract The potential cancer therapeutic agent, 6,7-(dimethoxy-2,4-dihydroindeno[1,2-c]pyrazol-3-yl)-(3-fluoro-phenyl)-amine (JNJ-10198409), formed three N-glucuronides that were positively identified by LC-MS/MS and NMR as N-amine-glucuronide (Glu-A), 1-N-pyrazole-glucuronide (Glu-B), and 2-N-pyrazole-glucuronide (Glu-C). All three N-glucuronides were detected in rat liver microsomes, whereas only Glu-A and B were found in monkey and human liver microsomes. In contrast to common glucuronides, Glu-B was completely resistant to -glucuronidase. Kinetic analyses revealed that glucuronidation of JNJ-10198409 in human liver microsomes exhibited atypical kinetics that may be described by a two-site binding model. For the high affinity binding, K_m values were 1.2 and 5.0 µM, and V_max values were 2002 and 2403 nmole min^-1 mg^-1 for Glu-A and Glu-B, respectively. Kinetic constants of low affinity binding were not determined due to low solubility of the drug. Among the human UDP-glucuronosyltransferases (UGTs) tested, UGT1A9, 1A8, 1A7 and 1A4 were the most active isozymes to produce Glu-A; for the formation of Glu-B, UGT1A9 was the most active enzyme followed by UGT1A3, 1A7 and 1A4. Glucuronidation of JNJ-10198409 by those UGT1A enzymes followed classic Michaelis-Menten kinetics. In contrast, no glucuronides were formed by all UGT2B isozymes tested, including UGT2B4, 2B7, 2B15 and 2B17. Collectively, these results suggested that glucuronidation of JNJ-10198409 in human liver microsomes is catalyzed by multiple UGT1A enzymes. Since UGT1A enzymes are widely expressed in various tissues, it is anticipated that both hepatic and extra-hepatic glucuronidation will likely contribute to the elimination of the drug in humans. Additionally, conjugation at the nitrogens of the pyrazole ring represents a new structural moiety for UGT1A-mediated reactions.

Key words: metabolite identification, phase II drug metabolism, structure elucidation, UDP glucuronyltransferases

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