In Vitro Kinetic Characterization of Axitinib Metabolism

N-Methyl-2-[3-(( E )-2-pyridin-2-yl-vinyl)-1 H -indazol-6-ylsulfanyl]-benzamide (axitinib) is an oral inhibitor of vascular endothelial growth factor receptors 1–3, which is approved for the treatment of advanced renal cell cancer. Human [14C]-labeled clinical studies indicate axitinib’s primary route of clearance is metabolism. The aims of the in vitro experiments presented herein were to identify and characterize the enzymes involved in axitinib metabolic clearance. In vitro biotransformation studies of axitinib identified a number of metabolites including an axitinib sulfoxide, several less abundant oxidative metabolites, and glucuronide conjugates. The most abundant NADPH- and UDPGA-dependent metabolites, axitinib sulfoxide (M12) and axitinib N -glucuronide (M7) were selected for phenotyping and kinetic study. Phenotyping experiments with human liver microsomes (HLMs) using chemical inhibitors and recombinant human cytochrome P450s demonstrated axitinib was predominately metabolized by CYP3A4/5, with minor contributions from CYP2C19 and CYP1A2. The apparent substrate concentration at half-maximal velocity ( K m) and V max values for the formation of axitinib sulfoxide by CYP3A4 or CYP3A5 were 4.0 or 1.9 µ M and 9.6 or 1.4 pmol·min−1·pmol−1, respectively. Using a CYP3A4-specific inhibitor (Cyp3cide) in liver microsomes expressing CYP3A5, 66% of the axitinib intrinsic clearance was attributable to CYP3A4 and 15% to CYP3A5. Axitinib N -glucuronidation was primarily catalyzed by UDP-glucuronosyltransferase (UGT) UGT1A1, which was verified by chemical inhibitors and UGT1A1 null expressers, with lesser contributions from UGTs 1A3, 1A9, and 1A4. The K m and V max values describing the formation of the N -glucuronide in HLM or rUGT1A1 were 2.7 µ M or 0.75 µ M and 8.9 or 8.3 pmol·min−1·mg−1, respectively. In summary, CYP3A4 is the major enzyme involved in axitinib clearance with lesser contributions from CYP3A5, CYP2C19, CYP1A2, and UGT1A1.