Role of Gut Microbiota in Etoposide Systemic Exposure in a Mouse Model

Etoposide is the first line of defense against many cancers. Etoposide exhibits a narrow therapeutic index and high variability in inter and intrapatient bioavailability. Sub- and supra- therapeutic dosing leads to low therapeutic effect and drug toxicity, respectively. However, major contributing (genetic and non-genetic) factors to the variability remain unclear. The human gut harbors trillions of bacteria that can mediate diverse biochemical reactions. The objective of this study was to examine the role of gut microbiota in etoposide disposition. Upon incubation of etoposide with C57BL6/J mouse cecal contents, small intestinal content, and human stool samples, a significant loss of etoposide was observed, accompanied by the appearance of a new metabolite peak (M1) in HPLC-UV chromatogram. Subsequent isolation, purification, and structure elucidation of M1 revealed that M1 is an O-demethylated product of etoposide (i.e., etoposide catechol). As compared to etoposide, M1 exhibited lower cytotoxicity in cancer cells (in-vitro) but higher mutagenicity in mice when treated in-vivo. One-day antibiotic treatment in drinking water significantly increased etoposide systemic exposure and depleted M1 exposure in mice when administered etoposide orally while showing no difference when etoposide was administered via i.v. route. In our study design, antibiotic treatment significantly depleted mouse gut bacteria while having no effects on etoposide absorption or elimination, suggesting changes in etoposide PK were the results of gut bacterial depletion. Upon screening with 48 abundant human gut bacterial species, most bacteria belonging to the Eubacterium genus were identified as etoposide metabolizers. We also identified Enterococcus faecalis, an abundant bacterium human gut bacterium, as an etoposide metabolizer, a novel finding since the O-demethylase activity of E. faecalis is not known to date. In conclusion, our study's findings suggest gut microbiota-mediated metabolism as a previously unrecognized elimination route for etoposide, which may affect etoposide exposure after oral dosing.