A One-Min, FDA-Validated UPLC-MS/MS Method for the AML Smoothened Inhibitor Glasdegib Quantification and Metabolic Stability Assessment in Human Liver Microsomes: In Silico Metabolic Lability and Toxicity Screening.

Glasdegib (GSB) is an orally bioavailable Smoothened (Hedgehog pathway) inhibitor approved, in combination with low-dose cytarabine, for the treatment of newly diagnosed acute myeloid leukemia (AML) in patients who are unfit for intensive induction chemotherapy. Although GSB is predominantly cleared by hepatic CYP3A4-mediated oxidation, no human liver microsomes (HLM) metabolic stability method has been reported; existing assays are limited to plasma matrices. Here, we describe an ultra-fast validated UPLC-MS/MS method for GSB and apply it to the first assessment of metabolic stability in HLM. StarDrop WhichP450 (CSL = 0.8928) and DEREK alerts were performed to localize metabolic soft spots and safety liabilities. Chromatography was performed on an SB-C18 column in 1 min. The method was linear over 1-4000 ng/mL (y = 1.2753x + 0.4567; r2 = 0.9992), with accuracy (-3.10 to +10.25 %E) and precision (≤ 3.56 %RSD). GSB exhibited an in vitro t1/2 of 44.15 min and an intrinsic clearance of 18.37 mL/min/kg (moderate-clearance compound). The predicted soft spots (the N-methylpiperidine ring [65%] and the benzimidazole moiety [29%]) are consistent with the known human N-desmethyl metabolite and CYP3A4 dominance. The combined dataset provides a soft-spot-guided rationale for the metabolic optimization of future Hedgehog-pathway inhibitors.