An ultra-fast validated green UPLC-MS/MS approach for the evaluation of zanubrutinib in vitro metabolic stability in human liver microsomes: Screening for in silico metabolic lability, ADME parameters, and DEREK toxic alerts

Zanubrutinib (Brukinsa, BeiGene) is a FAD approved second-generation Bruton's tyrosine kinase inhibitor that has more selectivity and reduced off-target action, resulting in mitigated cardiotoxicity compared to ibrutinib. The target of this investigation was to establish a fast, precise, green, and extremely sensitive UPLC-MS/MS approach for quantifying the level of zanubrutinib (ZNB) in human liver microsomes (HLMs). The separation of ZMB and revumenib was achieved using C8 column and isocratic gradient of the mobile phase. The linearity of the constructed ZNB calibration curve was ranged from 1 to 3,000 ng mL-1. The StarDrop software package including DEREK and WhichP450 modules was used in screening for the toxic alerts the in silico metabolic lability of ZNB. The AGREE score was 0.76 that approves the greenness of the established method. The low in vitro t1/2 (17.61 min) and high intrinsic clearance (46.03 mL min-1 kg-1) of ZNB revealed that ZNB shares similarities with medications that have a high extraction ratio. The present LC-MS/MS approach is considered the first analytical methodology for assessment of ZNB metabolic stability in HLMs matrix. In silico data from WhichP450 and DEREK modules suggest that making small structural changes (bioisosteric replacement) to the carboxamide moiety during drug design can potentially improve the metabolic safety and stability of novel compounds relative to ZNB. These methods are essential for advancing the development of new pharmaceuticals, particularly in enhancing metabolic stability.