The long-term objective of the proposed research is to determine the impact of intestinal clearance on drug plasma level variability. In preliminary studies, substantial appearance of the sulfoxide metabolite of a thioether imidazole drug, cimetidine, was observed in the intestinal lumen from jejunal but not ileal perfusion in rats. The thioether amino acid, methionine, and the anion-exchange inhibitor, DIDS, were shown to inhibit metabolite appearance in rats. Appearance of cimetidine sulfoxide was also confirmed in jejunal perfusions in human subjects. Based on these preliminary findings, cimetidine will be used as a probe for 1) identifying intestinal flavin monooxygenase (FMO) isozymes as a function of intestinal region in rat and human; 2) examining mechanisms of intestinal secretion of drug and metabolite; 3) delineating the role of intestinal drug and metabolite clearance in contributing to variability in drug plasma level profiles. Cimetidine's special value as a probe for these oral drug delivery factors stems from 1) its capacity to inhibit cytochrome P450 providing for oxidation dominated by FMO; 2) its interactions with intestinal membrane transporters; 3) its pharmacokinetic properties which dictate a double peak in drug plasma level versus time plots, respectively. Species and regional differences in intestinal metabolism will be investigated in rat and human tissue homogenates as well as in rat in situ perfusion studies. Drug-drug and drug-nutrient interactions utilizing intestinal cimetidine sulfoxidation will be explored mechanistically in rat intestinal perfusions and investigated for pharmacokinetic impact in human perfusion studies. Drug oxidation-reduction kinetics will be detailed in intestinal subcellular fractions and directionality of drug and metabolite transport will be studied in intestinal tissue mounted in Ussing chambers. The potential for paracellular bypass to reduce the extent of intestinal clearance will be examined both in vitro and in situ. Contributions to variable clearance as a function of drug concentration and intestinal residence time will be studied in situ with the controlled input afforded in intestinal perfusion experiments. Studies to uncouple cellular metabolism from export kinetics will be performed with drug and metabolite-loaded membrane vesicles. The intestinal transport and metabolite data obtained in these studies will be coupled with other pharmacokinetic parameters in a physiologic model to project the impact of intestinal clearance on drug plasma levels. The model will be used to predict the influence of intestinal clearance on pharmacokinetics of new and existing drugs which show inhibition of cimetidine clearance in these in situ and in vitro systems of study. Intestinal metabolism/export of xenobiotics is an area of investigation that has received little attention. It is the goal of this research proposal to expand this knowledge base in animal and human studies. The tools developed in this study will be used to explore the intestinal metabolism of other drug classes. The biochemical mechanisms underlying these preliminary in situ observations may have more general implications for the GI tract as an organ of drug elimination.
