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

Background and Objectives: Revumenib (SNDX-5613) is a potent and selective inhibitor of the menin-KMT2A interaction, currently under investigation for the treatment of KMT2A-rearranged (KMT2Ar) acute leukemias. As a small molecule, Revumenib has received Orphan Drug Designation from the US FDA for the treatment of acute myeloid leukemia (AML) and has also been granted Fast Track designation for use in pediatric and adult patients with relapsed or refractory (R/R) acute leukemias harboring a KMT2Ar or NPM1 mutation. This study aimed to develop a fast, accurate, environmentally friendly, and highly sensitive UPLC-MS/MS method for quantifying RVB levels in human liver microsomes (HLMs) using an ESI source.

Materials and Methods: The research focused on creating a UPLC-MS/MS technique to measure RVB levels in HLMs. The validation followed the bioanalytical method validation criteria set by the FDA, including linearity, selectivity, precision, accuracy, stability, matrix effects, and extraction recovery. The validation results met FDA guidelines, with RVB parent ions formed in the positive ESI source and quantified through multiple reaction monitoring (MRM). Separation of RVB from encorafenib was achieved using a C8 column (2.1 mm, 50 mm, 3.5 µm) and an isocratic mobile phase.

Results: The calibration curve for RVB was linear within the range of 1 to 3000 ng/mL (y = 0.6515x – 0.5459, R² = 0.9945). Inter-day precision and accuracy ranged from -0.23% to 11.33%, while intra-day precision and accuracy ranged from -0.88% to 11.67%, confirming the reproducibility of the UPLC-MS/MS method. The developed method demonstrated a low limit of quantification (LOQ) of 0.96 ng/mL, showing excellent sensitivity. The AGREE score of 0.77 confirmed the method’s environmentally friendly profile. Additionally, RVB exhibited a low in vitro half-life (14.93 minutes) and high intrinsic clearance (54.31 mL/min/kg), suggesting similarities to drugs with a high extraction ratio.

Conclusions: This LC-MS/MS method represents the first analytical approach for assessing the metabolic stability of RVB in HLMs. The technique is essential for advancing the development of new pharmaceuticals, particularly in improving metabolic stability during drug development.