title: Does P-glycoprotein contribute to amphotericin B epithelial transport in Caco-2 cells?
abstract: BACKGROUND
  Amphotericin B (AmB) is a highly efficacious therapeutic for invasive fungal infections and protozoal diseases. Increasing prevalence of these conditions warrants the development of an oral AmB formulation. Efflux transporters, such as the ABCB1 gene product P-glycoprotein, affect the oral bioavailability and disposition of a range of clinically relevant compounds. At present, it remains to be determined whether AmB is a substrate of P-glycoprotein mediated efflux. The objective of this study was to determine whether P-glycoprotein contributes to the epithelial transport of AmB in a Caco-2 cell model.
  
  
  METHODS
  Stimulation of P-glycoprotein ATPase activity was assessed using membranes containing human recombinant P-glycoprotein. An ABCB1 knockdown Caco-2 cell model was employed to determine non-toxic concentrations of AmB. AmB cellular association, following a 180 min incubation, was determined using an high performance liquid chromatography-ultraviolet (HPLC-UV) assay.
  
  
  RESULTS
  At the concentrations investigated, AmB did not stimulate P-glycoprotein ATPase activity. Non-toxic concentrations of AmB were 1 μg/mL-5 μg/mL; these were used in subsequent experiments. No significant difference in AmB cellular association was observed for ABCB1 small interfering ribonucleic acid transfected and non-transfected Caco-2 cells, following a 180 min incubation with 1 μg/mL and 2.5 μg/mL AmB. However, significantly greater AmB was associated with transfected cells as compared to non-transfected cells, when cells were incubated with 5 μg/mL AmB.
  
  
  CONCLUSIONS
  These results suggest that AmB is not a substrate of P-glycoprotein mediated efflux in this Caco-2 cell model. P-glycoprotein is not expected to be a major barrier to the oral absorption and disposition of AmB.
authors: Faculty of Pharmaceutical Sciences, University of British Columbia , Vancouver, BC , Canada.
authors: Jo-Ann
authors: JA
authors: Osei-Twum
authors: Kishor M
authors: KM
authors: Wasan
mesh_terms: D018435:ATP Binding Cassette Transporter, Subfamily B / Q000235:genetics; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism; D000666:Amphotericin B / Q000493:pharmacokinetics*; D000935:Antifungal Agents / Q000493:pharmacokinetics*; D001692:Biological Transport; D018938:Caco-2 Cells; D002851:Chromatography, High Pressure Liquid; D055785:Gene Knockdown Techniques; D006801:Humans; D007413:Intestinal Mucosa / Q000378:metabolism*; D013056:Spectrophotometry, Ultraviolet; D014162:Transfection
chemical_list: C513055:ABCB1 protein, human; D018435:ATP Binding Cassette Transporter, Subfamily B; D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000935:Antifungal Agents; D000666:Amphotericin B; D000251:Adenosine Triphosphatases
keywords: Amphotericin B; Caco-2 cells; P-glycoprotein; epithelial transport