Does P-glycoprotein contribute to amphotericin B epithelial transport in Caco-2 cells?

Abstract

Introduction: 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.

• Amphotericin B
• Caco-2 cells
• epithelial transport
• P-glycoprotein

Declaration of interest

This study was supported with an Operating Grant from the Canadian Institutes of Health Research. The authors report no declarations of interest. J. O. carried out the experiments presented in this article, performed the statistical analysis, and drafted the article. All authors have read and given approval to the final version of the article.