Benzydamine N-Oxygenation as a Measure of Flavin-Containing Monooxygenase Activity

Benzydamine is a nonsteroidal anti-inflammatory drug that undergoes flavin-containing monooxygenase (FMO)-dependent metabolism to a stable N -oxide. This metabolite can be quantified with high specificity and sensitivity by using a simple reverse-phase high-performance liquid chromatography (HPLC) assay with fluorescence detection. Studies with recombinant FMO enzymes demonstrate that FMO1 and FMO3 are the primary catalysts of benzydamine N -oxygenation, with minimal contributions from cytochrome P450 enzymes. Investigations conducted with human liver microsomes confirm that FMO3, in large part, is responsible for benzydamine N -oxide formation in this tissue. These features render benzydamine a useful in vitro probe for FMO activity in a wide range of tissues and cell types. In addition, benzydamine appears to be a suitable in vivo probe for human liver FMO3. This chapter provides a detailed account of the experimental protocol for determining rates of formation of benzydamine N -oxide by FMO-containing enzyme fractions.