The long-term goal of the research described in this grant proposal is to understand the mechanistic basis for inhibitory drug interactions involving human CYP3A4. This is important for the avoidance of adverse events with the numerous drugs in clinical use today that are either a substrate or inhibitor of the enzyme. It will also aid greatly in predicting the in vivo inhibitory potential for new molecular entities under develop. We hypothesize that effects of several clinically important inhibitory drugs on the first-pass clearance of CYP3A substrate occurs predominantly with the intestinal mucosa, and they can last well beyond the period of inhibitor absorption. This will be investigated with the following Specific Aims: I. To determine whether the inhibitory effect of azole anti-fungals on the first-pass metabolism of the CYP3A marker midazolam occurs predominantly within the intestinal mucosa rather than liver, and whether this preferential inhibition persists will beyond the period of inhibitor absorption due to sequestration of inhibitor in the mucosa. II. To determine whether inhibition of intestinal rather than hepatic first-pass is the predominant mechanism by which dialkylamine inhibitors elevate the systemic availability of orally administered midazolam, and to determine whether the time-course of inhibition parallels the formation of a slowly reversible MI-CYP3A complex. III. To determine if the in vivo effect during multiple dosing of a prototype macrolide inhibitor, erythromycin, an oral midazolam bioavailability, depends on the amount of CYP3A4 expressed in the intestinal mucosa and the accumulation over time of the di-desmethyl erythromycin metabolite in that tissue. We will employ three experimental paradigms; pharmacokinetic studies in healthy human volunteers; in vitro metabolic studies in human-derived Caco-2 cell culture monolayers; and in vivo intestinal extraction studies in a domestic pig model. This three-tiered approach should allow us to identify the contribution of readily predictable, reversible interactions between inhibitor and substrate, and current unpredictable, slowly reversible phenomena such as intracellular inhibitor sequestration and MI complex formation.
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