Evaluating barriers to bioavailability in vivo: validation of a technique for separately assessing gastrointestinal...

The purpose of this study was to develop and validate a method for separately evaluating the roles of gastrointestinal absorption and hepatic extraction as barriers to oral bioavailability (BA). The method was validated using five reference compounds known to have different absorption and hepatic extraction properties. Dose-dependence was also investigated for one reference compound.

Five reference compounds, amoxicillin, antipyrine, atenolol, propranolol, and testosterone, were administered as a cassette intravenouly (IV), via the hepatoportal vein (IPV), intraduodenally (ID), and intracolonically (IC) to male Sprague-Dawley rats. Blood samples were taken at nine time points, and the compounds were extracted from plasma using solid phase extraction. Plasma concentrations of each compound were determined using Liquid Chromatography Tandem Mass Spectrometry (LC/MS/MS). Pharmacokinetic parameters including bioavailability were calculated for each compound for each route of administration.

Testosterone BA was less than 10% by ID, IC, and IPV routes, due to high hepatic extraction, consistent with its high systemic clearance (63 ml x min(-1) x kg(-1)) and short terminal plasma half-life (23 min). The IPV BA of amoxicillin was 95%+/-6% indicating the absence of hepatic extraction in the rat, but with an ID BA of approximately 39% suggesting incomplete GI absorption to be the main barrier to bioavailability. Absorption was poor from the colon, demonstrating site-dependence consistent with literature reports of site-dependent absorption. Low oral BA of propranolol was due in part to first-pass hepatic extraction (IPV BA of 36%). The IPV BA of propranolol was dose-dependent, most likely due to saturation of the P450 enzymes. Atenolol was incompletely bioavailable due to incomplete intestinal absorption, with no contribution of hepatic first-pass metabolism. Antipyrine was highly bioavailable by all routes.

This in vivo rat model is demonstrated to be useful for identifying and quantifying the causes of incomplete bioavailabilty. It separately evaluates intestinal absorption, hepatic extraction, and site-dependent absorption. Concentration-dependence of saturable processes can also be examined.