An Ultra-Fast Validated Green UPLC-MS/MS Approach for Assessing Revumenib in Human Liver Microsomes: In Vitro Absorption, Distribution, Metabolism, and Excretion and Metabolic Stability Evaluation

Abstract

Background and Objectives: Revumenib (SNDX-5613) is a powerful and specific inhibitor of the menin–KMT2A binding interaction. It is a small molecule that is currently being researched to treat KMT2A-rearranged (KMT2Ar) acute leukemias. Revumenib (RVB) has received Orphan Drug Designation from the US FDA for treating patients with AML. It has also been granted Fast Track designation by the FDA for treating pediatric and adult patients with R/R acute leukemias that have a KMT2Ar or NPM1 mutation. Materials and Methods: The target of this research was to create a fast, precise, green, and extremely sensitive UPLC-MS/MS technique for the estimation of the RVB level in human liver microsomes (HLMs), employing an ESI source. The validation procedures were carried out in accordance with the bioanalytical technique validation requirements established by the US Food and Drug Administration that involve linearity, selectivity, precision, accuracy, stability, matrix effect, and extraction recovery. The outcome data of the validation features of the UPLC-MS/MS approach were acceptable according to FDA guidelines. RVB parent ions were formed in the positive ESI source and its two fragment ions were estimated employing multiple reaction monitoring (MRM) mode. The separation of RVB and encorafenib was achieved using a C8 column (2.1 mm, 50 mm, and 3.5 µm) and isocratic mobile phase. Results: The RVB calibration curve linearity ranged from 1 to 3000 ng/mL (y = 0.6515x − 0.5459 and R2 = 0.9945). The inter-day precision and accuracy spanned from −0.23% to 11.33%, while the intra-day precision and accuracy spanned from −0.88% to 11.67%, verifying the reproducibility of the UPLC-MS/MS analytical technique. The sensitivity of the developed methodology demonstrated its capability to quantify RVB levels at an LOQ of 0.96 ng/mL. The AGREE score was 0.77, confirming the greenness of the current method. The low in vitro t1/2 (14.93 min) and high intrinsic clearance (54.31 mL/min/kg) of RVB revealed that RVB shares similarities with medications that have a high extraction ratio. Conclusions: The present LC-MS/MS approach is considered the first analytical approach with the application of metabolic stability assessment for RVB estimation in HLMs. These methods are essential for advancing the development of new pharmaceuticals, particularly in enhancing metabolic stability.