Identification of Reactive Metabolites of Acetaminophen and Saxagliptin in Human Hepatocytes and Hepatic Organoids

Objectives: This study aims to identify the reactive metabolite of acetaminophen (AAP) and the cyanopyrrolidine metabolite of saxagliptin in human induced pluripotent stem cell-derived hepatic organoids (HHOs) and to compare them with human liver microsomes (HLMs) and plateable cryopreserved human hepatocytes (CHHs) to evaluate the feasibility of HHOs for reactive metabolite screening and metabolite profiling. Methods: AAP (50 μM) or sax-agliptin (50 μM) was incubated for 1 h at 37 °C in HLMs with or without NADPH-generating solution and 0.5 mM reduced glutathione (GSH). AAP (50 μM) was incubated for 24 h in HHOs and CHHs at 37 °C in a CO₂ incubator. AAP and saxagliptin metabolites in the reaction mixtures were analyzed using ultra-performance liquid chromatography coupled with tandem mass spectrometry. Results: N-acetyl-p-benzoquinone imine (NAPQI) was identified in the incubation mixture of HLMs with AAP, and its levels were reduced in the presence of GSH, accompanied by increased formation of AAP–GSH adduct. Incubation of AAP with HHOs for 24 h resulted in the formation of NAPQI, AAP–GSH, AAP–glucuronide, and AAP–sulfate. Moreover, CYP1A2 induction using omeprazole treatment increased the formation of AAP and AAP–GSH conjugate from phenacetin, reflecting enhanced CYP1A2 activity in both CHHs and HHOs. The findings indicate that HHOs are a suitable platform for reactive metabolites, such as NAPQI and AAP–GSH adducts, under chronic exposure and metabolic modulator intervention. Additionally, CHHs and HHOs exhibited similar saxagliptin metabolite profiles after incubation with saxagliptin and generated cysteine conjugates of saxagliptin and its hydroxylated metabolite. Conclusions: HHOs system can be used as an in vitro model for screening reactive metabolites, comparable to those obtained with CHHs.