Orbitrap HRMS-Based Comprehensive Metabolite Characterization of Duvelisib in Cross-Species Hepatocytes.
RATIONALE Duvelisib (DVB), a selective PI3K-δ/γ inhibitor approved for chronic lymphocytic leukemia, requires detailed metabolic characterization to support early drug development and selection of appropriate toxicology species. Because drug metabolism varies across species, an integrated system such as primary hepatocytes containing both Phase I and Phase II enzymes is critical for accurately defining metabolic pathways and identifying potential reactive metabolites. METHODS DVB metabolism was investigated using primary hepatocytes from human, monkey, dog, rat, and mouse. Incubated samples were analyzed using high-resolution mass spectrometry (HRMS) to detect and characterize metabolites. Structural elucidation was performed based on MS/MS fragmentation patterns. Mechanistic studies were conducted using a selective aldehyde oxidase (AO) inhibitor to assess the role of AO in DVB metabolism. RESULTS DVB underwent diverse metabolic transformations, including oxidation, N-dealkylation, N-glucuronidation and glutathione (GSH) conjugation, leading to the identification of 18 putative metabolites. Thirteen metabolites, including several glucuronide and GSH conjugates were newly identified. AO-mediated oxidation on the purine ring emerged as a major pathway and was significantly reduced in the presence of an AO inhibitor, confirming its involvement. Two previously unreported GSH conjugates were also characterized, suggesting potential sites of reactive metabolite formation and associated toxicity risk. Comparative analysis revealed distinct species-dependent metabolic profiles, with both shared and species-specific metabolites identified among human, mouse, rat, dog, and monkey hepatocytes. CONCLUSIONS The present study provides a comprehensive cross-species metabolic profile of DVB, highlighting key pathways and novel metabolites, including those linked to potential toxicity. Mouse hepatocytes are recommended for future toxicological studies.