Comprehensive preclinical pharmacokinetics and metabolite profiling of a novel caspase inhibitor F573

A robust and versatile bioanalytical method is essential for the preclinical development of the novel caspase inhibitor F573 for acute liver failure.This endeavor faces significant challenges, including the need for high sensitivity to monitor rapid systemic clearance, the ability to handle diverse and complex biological matrices (e.g., plasma, tissues, excreta), and overcoming potential interferences from high plasma protein binding (>89 %). To address these challenges, a sensitive, selective, and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method was developed and validated for the quantification of F573 across these matrices, with additional capability for qualitative metabolite profiling. The method used a simple protein precipitation protocol optimized for high-throughput processing. It was rigorously validated per FDA guidelines, demonstrating a low limit of quantification (LLOQ of 2 ng mL^-1 in 50 μL plasma), excellent precision (RSD <15 %), and accuracy. The method effectively addressed challenges from high plasma protein binding and matrix complexity. Its application enabled the first comprehensive characterization of F573's preclinical profile: it exhibited rapid absorption and elimination in rats and dogs, extensive tissue distribution, and limited blood-brain barrier penetration. Approximately 56 % of the administered dose was excreted unchanged, primarily via urinary and biliary routes. Furthermore, eight distinct metabolites were identified and profiled in rat samples. The developed LC-MS/MS method thus serves as a critical and reliable tool for the comprehensive preclinical pharmacokinetic evaluation of F573, underscoring its value in supporting the development of this promising therapeutic candidate.