title: Comparative evaluation of two dye probes in the rat everted gut sac model for unambiguous classification of P-gp substrate and inhibitor.
abstract: BACKGROUND
  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 10μM Ab-HiLyte with or without 50μM of MC18 or MC266. Ab-HiLyte concentrations in mucosal fluid were measured spectrophotometrically at 594nm at each appropriate time.
  
  
  RESULTS
  The Ab-HiLyte P-gp mediated efflux had a K=1.00×10(-2)min(-1) and t(1/2)=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(-3)min(-1)) and the half life is extremely increased (t(1/2)=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(-2)min(-1) and t(1/2)=38.28min).
  
  
  CONCLUSIONS
  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.
authors.affiliation: Dipartimento di Farmacia- Scienze del Farmaco, Università degli Studi di Bari Aldo Moro, via Orabona 4, 70125, Bari, Italy. mariagrazia.perrone@uniba.it
authors.forename: Maria Grazia
authors.initials: MG
authors.lastname: Perrone
authors.forename: Carmela
authors.initials: C
authors.lastname: Inglese
authors.forename: Francesco
authors.initials: F
authors.lastname: Berardi
authors.forename: Marcello
authors.initials: M
authors.lastname: Leopoldo
authors.forename: Roberto
authors.initials: R
authors.lastname: Perrone
authors.forename: Nicola Antonio
authors.initials: NA
authors.lastname: Colabufo
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000037:antagonists & inhibitors* / Q000378:metabolism*; D016229:Amyloid beta-Peptides / Q000378:metabolism* / Q000494:pharmacology; D000818:Animals; D004353:Drug Evaluation, Preclinical / Q000379:methods; D005456:Fluorescent Dyes / Q000378:metabolism* / Q000494:pharmacology; D007413:Intestinal Mucosa / Q000187:drug effects / Q000378:metabolism*; D007583:Jejunum / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D010446:Peptide Fragments / Q000378:metabolism* / Q000494:pharmacology; D051381:Rats; D017208:Rats, Wistar; D012235:Rhodamines / Q000378:metabolism* / Q000494:pharmacology; D013379:Substrate Specificity / Q000502:physiology
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D016229:Amyloid beta-Peptides; D005456:Fluorescent Dyes; D010446:Peptide Fragments; D012235:Rhodamines; C078592:amyloid beta-protein (1-40)