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

J Pharmacol Toxicol Methods. 2012 Dec 9;

Authors: Perrone MG, Inglese C, Berardi F, Leopoldo M, Perrone R, Colabufo NA


INTRODUCTION: P-glycoprotein (P-gp) plays a crucial role in beta-amyloid efflux from 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 is not always confirmed by in 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 intestine was 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: Ab-HiLyte P-gp mediated efflux had a K = 1.00 x 10(-2) min(-1) and t(1/2) = 68.74 min, while in the presence of MC18, Ab-HiLyte efflux resulted to be reduced of an order of magnitude (K = 1.65 x 10(-3) min(-1)) and the half life is extremely increased (t(1/2) = 419 min). A P-gp substrate, like MC266, determines no change in the efflux of Ab: kinetic constant and the half life result to be unmodified (K = 1.81 x 10(-2) min(-1) and t(1/2) = 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 inhibitor. This is particularly important as different groups obtain a controversial classification of the same compound.

PMID: 23234641 [PubMed - as supplied by publisher]