Gut bacterial O-demethylation modulates systemic exposure to oral etoposide.

Gut microbial O-demethylation was detected for 35 of 64 clinically used oral drugs containing methoxylated aromatics.

• 64 clinically used oral drugs containing one or more methoxylated aromatics were screened for gut microbial O-demethylation using high-resolution mass spectrometry (HRMS).
• Individual drugs were incubated with mouse cecal contents.
• O-demethylation was identified by detection of metabolites with a mass difference of -14 and its multiples.
• 35 of the tested drugs, including the anticancer agent etoposide, showed O-demethylated metabolites.

The O-demethylated metabolite (M1) of etoposide was confirmed to be etoposide catechol.

• Confirmation was achieved using both HRMS and proton nuclear magnetic resonance (NMR) spectroscopy.
• Etoposide was used as the model drug for in-depth characterization of gut microbial O-demethylation.

Seven previously unknown gut bacterial species with etoposide O-demethylating activity were identified.

• An in-house collection of 56 gut bacteria was tested individually for etoposide O-demethylating activity.
• Seven species were identified as exhibiting etoposide O-demethylating activity, all previously unreported for this activity.

Etoposide catechol (M1) is more genotoxic to myeloid cells than the parent etoposide when orally administered to mice.

• M1 was demonstrated to be more genotoxic to myeloid cells upon oral administration in mice.
• This finding suggests the gut microbiota may contribute to the secondary genotoxicity (increased risk of acute myeloid leukemia) associated with etoposide anticancer therapy via O-demethylation.
• Etoposide anticancer therapy has been previously associated with an increased risk of acute myeloid leukemia.

Etoposide catechol (M1) is less cytotoxic against MCF-7 and HeLa cancer cells than the parent etoposide.

• Cytotoxicity comparisons were made between M1 and parent etoposide in MCF-7 (breast cancer) and HeLa (cervical cancer) cell lines.
• M1 showed lower cytotoxicity than etoposide in both cancer cell lines tested, suggesting O-demethylation may reduce anticancer efficacy.

Antibiotic treatment of mice increased systemic exposure to etoposide 1.9-fold and decreased exposure to M1 by 3.7-fold.

• Comparative pharmacokinetic analysis was performed in control versus antibiotic-treated mice following oral etoposide administration.
• Systemic exposure to etoposide increased 1.9-fold in antibiotic-treated mice compared to control mice.
• M1 exposure decreased 3.7-fold in antibiotic-treated mice compared to control mice.
• These results indicate that gut microbial O-demethylation is a significant determinant of etoposide metabolism and disposition.