Elucidating CYP2D6-driven metabolism and hepatotoxic bioactivation of metoprolol in plateable human and animal hepatocytes: Original scientific paper

Jiang Pu; Mei Yang; Min Zhang; Ruiqi Gao; Yue Xiao; Lingyu Liu; Chuanjing Zhang; Wennuo Xu; Kaifang Li; Wanyong Feng

doi:10.5599/admet.2961

Authors

Jiang Pu Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0003-7324-118X
Mei Yang Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0008-7637-9332
Min Zhang Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0000-0002-1741-5785
Ruiqi Gao Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0006-2962-3959
Yue Xiao Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0001-5088-2254
Lingyu Liu Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0008-3360-4069
Chuanjing Zhang Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0006-8817-5076
Wennuo Xu Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0003-9491-4099
Kaifang Li Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0007-1176-3563
Wanyong Feng Bioduro Biologics Co., Ltd., Shanghai, China https://orcid.org/0009-0002-7254-2389

DOI:

https://doi.org/10.5599/admet.2961

Keywords:

Metabolite, cytotoxicity, recombinant, low-clearance, glutathione, CYP2D6

Abstract

Background and purpose: As a classic β-blocker with low systemic clearance, metoprolol has been linked to rare but clinically significant hepatotoxicity, yet its hepatic metabolic fate remains poorly characterized. Experimental approach: Metoprolol was incubated individually in plateable human and animal hepatocytes, and recombinant cytochrome (CYP) P450 enzymes, followed by sample processing for cytotoxicity assessment, stability analysis, phenotyping and metabolite identification studies. Key results: In vitro cytotoxicity assessment revealed distinct species-specific responses to metoprolol exposure. Metoprolol showed no observable cytotoxicity across the tested concentration range (0 to 500 µM) in human hepatocytes, whereas it was cytotoxic only at a concentration of 500 µM in rat hepatocytes. Metabolic characterization showed low intrinsic clearance in human hepatocytes (0.56±0.12 µL min^-1 per million cells) over a 72-hour incubation. Comprehensive mass spectrometer analysis identified 22 metabolites across four species (rat, dog, monkey, and human) and fifteen metabolites were identified as the new ones, with CYP2D6-mediated biotransformation pathways (including mono-oxygenation, O-demethylation, and oxidation) accounting for the generation of four major metabolites (M1, M10, M13, M17). Notably, species-specific metabolism was observed for a-hydroxy-metoprolol (M10). It served as the predominant metabolite in rat hepatocytes and underwent subsequent bioactivation to a reactive glutathione (GSH) conjugate. Inhibition studies with 1-aminobenzotriazole (a non-specific CYP inhibitor) confirmed the CYP-dependent nature of this hepatotoxic metabolic pathway. Conclusion: The sustained metabolic activity of plateable hepatocytes facilitated a comprehensive metabolic profiling of metoprolol, including direct observation of GSH-mediated bioactivation. Integrating with cytotoxicity data, these findings offered crucial insights into its hepatic adverse effects.

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