The metabolism of midazolam and comparison with other CYP enzyme substrates during intestinal absorption: in vitro studies with rat everted gut sacs.

PURPOSE The purpose of this study was to quantify the intestinal metabolism of midazolam, a CYP P450 substrate, usually used as a probe for the activity of the isoform CYP3A4/1 and to compare it with previous results obtained for other P450 substrates such as testosterone, dextromethorphan and bupropion, which show some specificities for different CYP isoforms. The aim was to shed light on the role of metabolism in the intestinal tissues and the relationship with efflux mechanisms, such as by P-glycoprotein (P-gp) and the influence of metabolism on bioavailability. METHODS We used the improved everted rat gut sac model to study in vitro the absorption and metabolism of the different CYP isoenzyme probes: midazolam, testosterone, bupropion and dextromethorphan. This method enables drug metabolism to be studied during absorption, conditions which mimic the in vivo situation. The drugs and their metabolites were measured by LC-MS in the mucosal and serosal media and in the mucosal tissue, to give a complete picture of the transport and metabolism. RESULTS Midazolam, as with the other CYP probes, was metabolized in everted gut sacs. The metabolites were detected in the same proportions in both the serosal and mucosal compartments for midazolam, testosterone and bupropion. In the case of dextromethorphan, the metabolite methoxymorphinan was found at a higher concentration in the mucosal compartment, indicating efflux from the cells. The transport of dextromethorphan and its metabolite was not modified in the presence of verapamil, a P-gp inhibitor, thus demonstrating that dextromethorphan and methoxymorphinan were not P-gp substrates. CONCLUSION Given that the rat is a widely used species for pre-clinical studies, the everted gut sac model provides a useful tool to assess the role of metabolism during drug absorption by the intestine and is also capable of demonstrating P-glycoprotein mediated transport.