Developing and validating a sensitive and fast UPLC-MS/MS method for estimating the in vitro metabolic stability of crenolanib in HLMs: identification of structural alarms related to the in silico toxicity and metabolic lability

Abstract

Crenolanib (CP-868,596) is an orally accessible tyrosine kinase inhibitor that selectively inhibits PDGFR relative to other receptor tyrosine kinases. Crenolanib (CLB) was developed by AROG Pharmaceuticals, Dallas, TX. This study developed a swift, dependable, and eco-friendly UPLC-MS/MS method for the estimation of CLB in human liver microsomes (HLMs), and the in vitro metabolic stability of CLB in HLMs was evaluated. The validation guidelines for the UPLC-MS/MS methodology followed the US-FDA guidelines for bioanalytical approaches. CLB and encorafenib (EFB as the internal standard, IS) were separated utilizing an isocratic mobile phase method on a reversed-phase (SB–C18) column. The CLB calibration graph demonstrated linearity at concentrations ranging from 1 to 3000 ng mL⁻¹. This investigation determined the accuracy and precision of the UPLC-MS/MS approach for intra- and inter-day evaluations, yielding ranges of −1.74% to 9.00% and −3.96% to 9.67%, respectively. The StarDrop program package involved WhichP450 and Deductive Estimation of Risk from Existing Knowledge (DEREK) modules that were applied for assessing metabolic lability and characterizing CLB structural alarms, respectively. The intrinsic clearance of CLB was computed to be 26.79 mL min⁻¹ kg⁻¹, while the in vitro half-life was estimated to be 30.27 min. In silico analysis indicated that slight structural changes to the piperidin-4-amine moiety (99%) during drug design may increase the metabolic stability and improve the safety compared to the case with CLB.