Comprehensive In Vitro Metabolic Characterization of Eudesmin in Human and Mouse Hepatocytes

Background/Objectives: Eudesmin is a tetrahydrofurofuranoid lignan known for its diverse pharmacological activities, including anti-tumor, anti-inflammatory, and neuroprotective effects. However, its metabolism has not been well characterized. Methods: This study examined the in vitro metabolism of eudesmin using human and mouse hepatocytes, human liver microsomes, and recombinant drug-metabolizing enzymes. Liquid chromatography–high-resolution mass spectrometry combined with ion identity molecular networking enabled the comprehensive visualization and annotation of eudesmin metabolites. Results: Eudesmin exhibited moderate metabolic stability in human and mouse hepatocytes, with half-lives of 181.0 min and 132.9 min, and intrinsic clearance values of 27.7 mL/min/kg and 154.0 mL/min/kg, respectively. Incubation of eudesmin with human hepatocytes resulted in the formation of 13 metabolites, including five phase I metabolites (M1–M5) and eight phase II conjugates. Phase I metabolism was dominated by O-demethylation of the 3,4-dimethoxyphenyl moieties, yielding mono-O-demethylated (M1 and M2) and di-O-demethylated metabolites (M3 and M4), as well as a hydroxylated metabolite (M5). Enzyme phenotyping, kinetic analyses, and chemical inhibition experiments identified cytochrome P450 2C9 (CYP2C9) as the major contributor to O-demethylation, with additional contributions from CYP2C19, CYP2C8, CYP3A4, and CYP3A5, whereas hydroxylation was mediated primarily by CYP3A4 and CYP3A5. The O-demethylated metabolites subsequently underwent phase II metabolism, forming glucuronide conjugates of M1–M4 and sulfate conjugates of M1–M3, including a disulfate of M3. Uridine 5′-diphospho-glucuronosyltransferase and sulfotransferase screening revealed the involvement of multiple conjugative enzymes, indicating extensive and distributed phase II metabolism. Specifically, di-O-demethylated metabolites and their conjugates were detected in human hepatocytes but not in mouse hepatocytes, suggesting that the sequential O-demethylation pathway is limited in mice. Conclusions: This study characterizes eudesmin metabolism, with CYP2C9-mediated O-demethylation and significant species differences between humans and mice, and provides a basis for its further pharmaceutical development.