Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor

摘要

Introduction: P-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from the blood-brain barrier thus becoming a promising pharmacological target in the treatment of Alzheimer's disease (AD). The increase of P-glycoprotein expression and activity by a P-gp inducer could be an effective pharmacological strategy in slowing or halting the progression of AD. Commonly used. in vitro methods to classify a P-gp interacting molecule as substrate, inhibitor, modulator or inducer are not always confirmed by. in vivo experiments. Here we validate the new dye-probe beta-amyloid (1-40) HiLyte Fluor? TR-labeled (Ab-HiLyte) (Anaspec) P-gp mediated transport in the ex vivo rat everted gut sac assay by using. MC18 or. MC266, a fully characterized P-gp inhibitor and substrate, respectively, and compare it with the commonly used dye rhodamine. Methods: Male Wistar rats' everted intestines were divided into sacs, each sac was filled with 10. ??M Ab-HiLyte with or without 50 ??M of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594. nm at each appropriate time. Results: The Ab-HiLyte P-gp mediated efflux had a K=1.00??10-2min-1 and t1/2=68.74min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K=1.65??10-3min-1) and the half life is extremely increased (t1/2=419min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K=1.81??10-2min-1 and t1/2=38.28min). Discussion: The results demonstrate that the new dye probe, Ab-HiLyte, could be a probe of choice to unequivocally distinguish between a P-gp substrate and an inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound. ? 2013 Elsevier Inc.