title: Lipid excipients Peceol and Gelucire 44/14 decrease P-glycoprotein mediated efflux of rhodamine 123 partially due to modifying P-glycoprotein protein expression within Caco-2 cells.
abstract: OBJECTIVE
  The objective of this study was to determine the influence of two lipid excipients, Peceol(c) and Gelucire(c) 44/14 on P-glycoprotein (Pgp) activity and protein expression in human colon adenocarcinoma cells (Caco-2). Lipid excipients are increasingly used as drug delivery systems for hydrophobic drugs to increase their bioavailability by overcoming the barrier of low absorption. This study will probe a novel mechanism by which lipid excipients reduce Pgp-mediated efflux and thereby increase bioavailability of orally administered therapeutics.
  
  
  METHODS
  Non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 were determined for 24-hour treatments of Caco-2 cells using integrity of the cell membranes and mitochondrial respiration as markers. Pgp activity after treatment with non-cytotoxic concentrations of Peceol(c) and Gelucire(c) 44/14 was measured with a fluorescent Pgp substrate, rhodamine 123 (Rh123). The activity of Pgp was ascertained by measuring accumulation and the directional flux of Rh123 using the Transwell(c) semi-permeable cell culture support system. To assess the effect of Peceol(c) and Gelucire(c) 44/14 on Pgp protein expression, Western blotting with a specific Pgp antibody was performed. RESULTS. The two assays for cytotoxicity were in agreement and showed that concentrations of less than 0.5% (v/v) Peceol(c) and less than 0.02% (w/v) Gelucire(c) 44/14 were not toxic to Caco-2 cells. Rh123 accumulation was increased up to 3-fold in cells treated with sub-toxic concentrations of the excipients. The flux of Rh123 across the cell monolayer was unaffected by treatment in the absorptive (apical to basolateral) direction but the efflux transport was reduced after treatment with Peceol(c), Gelucire(c) 44/14 or the positive control , 100microM verapamil. Some of the reduction in Pgp efflux activity can be explained by the reduction in protein expression after treatment with the lipid excipients; treatment with 0.25% (v/v) and 0.5% (v/v) Peceol(c) reduced Pgp protein levels to 62.4% and 68.4% of the control respectively while Gelucire(c) 44/14 treatments of 0.01% (w/v) and 0.02% (w/v) reduced Pgp to 64.5% and 51.8% respectively.
  
  
  CONCLUSIONS
  In this study we utilized established methodologies to assess the inhibitory effect of the excipients on the Pgp-mediated efflux of the probe, Rh123 and tested the hypothesis that long-term treatment of Caco-2 cells with the lipid excipients, Peceol(c) and Gelucire(c) 44/14, decreased Pgp protein expression. The results suggest a new mechanism which may contribute to the improved bioavailability seen for drugs formulated with lipid-based excipients.
authors.affiliation: Division of Pharmaceutics and Biopharmaceutics, Faculty of Pharmaceutical Sciences, The University of British Columbia, Vancouver, British Columbia, Canada.
authors.forename: Kristina
authors.initials: K
authors.lastname: Sachs-Barrable
authors.forename: Andrea
authors.initials: A
authors.lastname: Thamboo
authors.forename: Stephen D
authors.initials: SD
authors.lastname: Lee
authors.forename: Kishor M
authors.initials: KM
authors.lastname: Wasan
mesh_terms: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000187:drug effects* / Q000378:metabolism; D000251:Adenosine Triphosphatases / Q000378:metabolism; D001682:Biological Availability; D001692:Biological Transport / Q000187:drug effects; D018938:Caco-2 Cells; D002462:Cell Membrane / Q000187:drug effects / Q000378:metabolism; D002470:Cell Survival / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D005079:Excipients / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D005786:Gene Expression Regulation / Q000187:drug effects; D006801:Humans; D007408:Intestinal Absorption / Q000187:drug effects; D008055:Lipids / Q000008:administration & dosage / Q000494:pharmacology / Q000633:toxicity; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism; D009829:Oleic Acids / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D011092:Polyethylene Glycols / Q000008:administration & dosage / Q000494:pharmacology* / Q000633:toxicity; D020112:Rhodamine 123 / Q000008:administration & dosage / Q000493:pharmacokinetics* / Q000633:toxicity
chemical_list: D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D005079:Excipients; D008055:Lipids; D009829:Oleic Acids; C482692:Peceol; C104509:gelucire 44-14; D020112:Rhodamine 123; D011092:Polyethylene Glycols; D000251:Adenosine Triphosphatases