Ribociclib (RBC, Kisqali®) 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™ module and the Xenosite web predictor tool, respectively. Later, in vitro metabolites and adducts of RBC were characterized from rat liver microsomes using LC-MS/MS. Subsequently, in silico data was used as a guide for the in vitro work. Finally, in silico toxicity assessment of RBC metabolites 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 vitro phase I metabolic profiling of RBC was 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 of RBC were characterized. The proposed metabolic pathways involved in generation of these metabolites are hydroxylation, oxidation and reduction. The reactive intermediate generation mechanism of RBC may 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 of RBC in 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 for RBC and its proposed derivatives at the active site of the human CDK6 enzyme. Methyl-RBC exhibited the best ADMET and docking analysis and fewer side effects compared to RBC and fluoro-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 of RBC in vitro metabolic profiling and structural characterization and toxicological properties of the generated metabolites.

Vandetanib (Caprelsa tablets, VNT) is an orally inhibitor of vascular endothelial growth factor receptor 2. The current research reports the characterization and identification of in vitro, in vivo and reactive intermediates of VNT. In vitro metabolites of VNT were performed by incubation with rat liver microsomes (RLMs). Extraction of vandetanib and its in vitro metabolites from the incubation mixtures were done by protein precipitation. In vivo metabolism was done by giving one oral dose of vandetanib (30.8 mg/kg) to Sprague Dawley rats in metabolic cages by using oral gavage. Urine was gathered then filtered at certain time intervals (0, 6, 12, 18, 24, 48, 72, 96 and 120 h) from vandetanib dosing. A similar volume of ACN was added to each collected urine sample. Both layers (organic and aqueous) were injected into liquid chromatography electro spray ionization tandem mass spectrometry (LC–ESI–MS/MS) to detect in vivo vandetanib metabolites. N-methyl piperidine ring of vandetanib is considered a cyclic tertiary amine that undergoes metabolism forming iminium intermediates that are very reactive toward nucleophilic macromolecules. Incubation of vandetanib with RLMs in the presence of 1.0 mM KCN was made to check reactive metabolites as it is usually responsible for noticeable idiosyncratic toxicities including phototoxicity and QT interval prolongation. Four in vivo phase I, one in vivo phase II metabolites, six in vitro phase I metabolites and four cyano conjugates of vandetanib were detected by LC–MS/MS. In vitro and in vivo phase I metabolic reactions were N-oxide formation, N-demethylation, α-carbonyl formation and α-hydroxylation. In vivo phase II metabolic reaction was direct conjugation of vandetanib with glucuronic acid. All metabolic reactions occurred in N-methyl piperidine of vandetanib which causes toxicity and instability of vandetanib.

Olmutinib (OTB, Olita™) is an orally available third-generation epidermal growth factor receptor tyrosine kinase inhibitor (EGFR TKI). It was developed by Boehringer Ingelheim and Hanmi Pharmaceutical Co. Ltd for the cure of non-small cell lung cancer (NSCLC). In May 2016, OTB was approved in South Korea for the treatment of patients suffering from metastatic or locally advanced EGFR T790M mutation-positive NSCLC. A LC-MS/MS methodology was validated for OTB quantification in human plasma. An extended application for this validated LC-MS/MS is OTB metabolic stability evaluation. Chromatographic separation of OTB and ponatinib (PNT, IS) was attained using a reversed phase with isocratic elution. The linearity of the developed LC-MS/MS method ranged from 5.00 to 500.00 ng mL⁻¹ with r² ≥ 0.9999 in human plasma. LOD and LOQ were 1.12 and 3.39 ng mL⁻¹, respectively. The intra-day and inter-day precision and accuracy were 1.17 to 2.75% and 97.86 to 101.48%, respectively. The intrinsic clearance (CL_int) was 2.71 mL min⁻¹ kg⁻¹ and the in vitro half-life (t_1/2) was 48.80 min. A review of the literature revealed that there are no previous articles about the quantification of OTB in human plasma using LC-MS/MS or its metabolic stability assessment.