<b>Gut bacterial</b> <i> <b>O</b> </i> <b>-demethylation modulates systemic exposure to oral etoposide</b>

Gut microbial <i>O</i>-demethylation has been reported for plant-derived dietary compounds containing <i>O</i>-methylated aromatic(s). However, the significance of gut microbial <i>O</i>-demethylation in drug metabolism and disposition remains unexplored. This study examined 64 clinically used oral drugs containing one or more methoxylated aromatics for gut microbial <i>O</i>-demethylation using high-resolution mass spectrometry (HRMS). For 35 of the tested drugs, including the anticancer agent etoposide, we detected metabolites corresponding to <i>O</i>-demethylation (i.e., a mass difference of -14 and its multiples) when individual drugs were incubated with mouse cecal contents. We confirmed that the <i>O</i>-demethylated metabolite (M1) of the model drug etoposide is etoposide catechol using HRMS and proton nuclear magnetic resonance spectroscopy. By testing an in-house collection of 56 gut bacteria individually, we identified seven previously unknown gut bacterial species that exhibit etoposide <i>O</i>-demethylating activity. Etoposide anticancer therapy has been associated with an increased risk of acute myeloid leukemia. We demonstrated that M1 is more genotoxic to myeloid cells when it is orally administered to mice, whereas M1 is less cytotoxic against MCF-7 and HeLa cancer cells than the parent etoposide, suggesting that the gut microbiota may contribute to the secondary genotoxicity of etoposide via <i>O</i>-demethylation. Comparative pharmacokinetic analysis of orally administered etoposide in control and antibiotic-treated mice showed that systemic exposure to etoposide increased 1.9-fold, while M1 exposure decreased 3.7-fold in antibiotic-treated mice, suggesting that gut microbial <i>O</i>-demethylation is a significant determinant of etoposide metabolism and disposition. Collectively, our study reveals the prevalence of gut bacteria with <i>O</i>-demethylation activity, illustrates the contribution of gut microbial <i>O</i>-demethylation to altering drug efficacy and toxicity with the model drug etoposide, and provides a knowledge basis for in-depth characterization of other drugs identified as being susceptible to gut microbial <i>O</i>-demethylation.