Metabolism of the Tyrosine Kinase Inhibitor Masitinib In Vitro

Masitinib is an orally bioavailable inhibitor of protein kinases such as c-kit, Lyn, Fyn and MCSFR-1. These pathways play a key role in cell survival and proliferation of mast cells and microglia. Due to the selectivity of masitinib for these pathways, this drug is under investigation for treating neurodegenerative disorders, inflammatory disorders, as well as pancreatic and prostate cancer. Hepatotoxicity has been observed in some patients taking masitinib during clinical trials and is thought to arise from reactive metabolites formed by cytochrome P450 (CYP) enzymes in the liver. The purpose of this study was to elucidate the metabolism and bioactivation pathways of masitinib using in vitro phenotyping methods to identify the enzymes involved in masitinib metabolic activation. Masitinib (2 μM) was incubated with pooled human liver microsomes in the presence and absence of NADPH. Potassium cyanide was added to trap reactive metabolites as cyano adducts. Metabolites were analyzed using reversed-phase liquid chromatography - tandem mass spectrometry (LC-MS/MS). To determine which enzymes are involved in forming masitinib metabolites and reactive metabolites, masitinib was incubated with human liver microsomes in the presence of P450-selective reversible inhibitors as well as time-dependent inhibitors. To validate the inhibitor panel findings, masitinib was also incubated with recombinant P450 enzymes. From these assays we found that the primary metabolite, N-desmethyl masitinib, was primarily formed by CYP2C8 and CYP3A4, with contributions from CYP2D6 and CYP3A5. Other primary metabolites produced by monooxygenation of masitinib, M515a, M515b, and M515d, were formed by CYP3A4 and CYP3A5, while M515c was formed by CYP3A4 with minor contribution from CYP2D6. We also found that reactive metabolite cyano adducts MCN510 and MCN538 were formed by CYP3A4 and CYP3A5. MCN524 and MCN526 were generated by CYP3A4 with contributions from CYP2D6, CYP2C8, and CYP3A5. By elucidating these pathways, we gain further insight into the routes that generate reactive and potentially toxic metabolites of masitinib. Given the polymorphic nature of the enzymes involved, future studies are needed to determine the impact of CYP genetic polymorphisms on masitinib metabolism. This information could ultimately provide evidence to inform precision prescribing for masitinib to ensure safety and efficacy for future patients.