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

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 μM Ab-HiLyte with or without 50 μM 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=1.00×10−2 min−1 and t1/2=68.74 min, while in the presence of MC18, the Ab-HiLyte efflux turned out to be reduced by an order of magnitude (K=1.65×10−3 min−1) and the half life is extremely increased (t1/2=419 min). A P-gp substrate, likeMC266, determines no change in the efflux of Ab: the kinetic constant and the half life turned out to be unmodified (K=1.81×10−2 min−1 and t1/2=38.28 min). 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.