In silicoandin vitrometabolism of ribociclib: a mass spectrometric approach to bioactivation pathway elucidation and metabolite profiling

Ribociclib (RBC, Kisqali (R)) is a highly selective CDK4/6 inhibitor that has been approved for breast cancer therapy. Initially, prediction of susceptible sites of metabolism and reactivity pathways were performed by the StarDrop WhichP450 (TM) module and the Xenosite web predictor tool, respectively. Later,in vitrometabolites and adducts ofRBCwere characterized from rat liver microsomes using LC-MS/MS. Subsequently,in silicodata was used as a guide for thein vitrowork. Finally,in silicotoxicity assessment ofRBCmetabolites was carried out using DEREK software and structural modification was proposed to reduce their side effects and to validate the bioactivation pathway theory using the StarDrop DEREK module.In vitrophase I metabolic profiling ofRBCwas performed utilizing rat liver microsomes (RLMs). Generation of reactive metabolites was investigated using potassium cyanide (KCN) as a trapping nucleophile for the transient and reactive iminium intermediates to form a stable cyano adduct that can be identified and characterized using mass spectrometry. Nine phase I metabolites and one cyano adduct ofRBCwere characterized. The proposed metabolic pathways involved in generation of these metabolites are hydroxylation, oxidation and reduction. The reactive intermediate generation mechanism ofRBCmay provide an explanation of its adverse reactions. Aryl piperazine is considered a structural alert for toxicity as proposed by the DEREK report. We propose that the generation of only one reactive metabolite ofRBCin a very small concentration is due to the decreased reactivity of the piperazine ring compared to previous reports of similar drugs. Docking analysis was performed forRBCand its proposed derivatives at the active site of the human CDK6 enzyme.Methyl-RBCexhibited the best ADMET and docking analysis and fewer side effects compared toRBCandfluoro-RBC. Further drug discovery studies can be conducted taking into account this concept allowing the development of new drugs with enhanced safety profiles that were confirmed by using StarDrop software. To the best of our knowledge, this is the first literature report ofRBCin vitrometabolic profiling and structural characterization and toxicological properties of the generated metabolites.