Metabolic Profiling of Canertinib: A Comprehensive Cross-Species Investigation Using Advanced UPLC-MS/MS and...

Canertinib is an EGFR tyrosine kinase inhibitor intended for the treatment of leukemia and non-small cell lung cancer. This study described a UPLC-MS/MS method for quantitatively assessing the metabolic stability of canertinib in liver microsomes. The developed method showed excellent linearity over the concentration range of 10-1000 nM, which is suitable for in vitro high-throughput screening. Canertinib showed marked species-dependent metabolism, with CL_{in vitro, mic} following the order: human (28.3 μL/min/mg protein) < rat (48.2 μL/min/mg protein) < monkey (77.8 μL/min/mg protein). An LC-Orbitrap-HRMS facilitated structural characterization of the metabolites via accurate mass measurements and MS/MS fragmentation interpretation. Post-acquisition data-mining strategies, specifically high-resolution extracted ion chromatograms and multimass defect filtering, were employed to screen the putative metabolite candidates. Sixteen NADPH-dependent metabolites and one GSH conjugate were structurally characterized. Cross-species comparative analysis revealed notable interspecies variations: metabolites M9 and M17 were identified as human-specific, while M15 and M16 demonstrated monkey-specificity. The metabolic pathways of canertinib included oxidative defluorination, O-dealkylation, oxidative deamination, piperidine ring opening, lactam formation, and GSH conjugation. This work represents the first cross-species metabolic investigation of canertinib, providing critical insights into interspecies metabolic disparities. The elucidated metabolic framework advances mechanistic understanding of the compound's pharmacological activity and toxicity profiles.