| Summary: | Background:
Amphotericin B (AmB) is a highly efficacious therapeutic for invasive fungal infections (IFIs) and protozoal diseases. Increasing prevalence of these conditions necessitates the development of an oral AmB formulation, with reduced toxicity, improved tissue distribution, and improved affordability. Efflux transporters, such as the ABCB1 gene product P-glycoprotein (P-gp), affect the oral bioavailability and disposition of a diverse range of compounds. It remains to be ascertained whether AmB is a substrate of P-gp mediated efflux. Therefore, the objective of this study was to determine whether P-gp contributes to the epithelial transport of AmB in the enterocyte-like Caco-2 cell model.
Methods:
Transient knockdown of ABCB1 was achieved in Caco-2 cells using small interfering RNA. The effect of this knockdown on Caco-2 cell differentiation, transporter expression, and P-gp function were investigated using the measurement of trans-epithelial electrical resistance, Western blot analysis, bi-directional transport and accumulation of Rhodamine 123, a fluorescent P-gp substrate. The interaction between AmB and P-gp was examined using a P-gp ATPase activity and a LDH assay. The ABCB1 transient knockdown Caco-2 cell model and a cell-based AmB HPLC-UV assay were employed to quantify AmB cellular association.
Results:
Transfected and non-transfected Caco-2 cells formed confluent and differentiated monolayers. Transient knockdown of ABCB1 reduced P-gp expression and efflux efficiency by approximately 63% compared to non-transfected cells. AmB did not stimulate P-gp ATPase activity and AmB cytotoxicity did not differ between transfected and non-transfected cells. Increased association of AmB was demonstrated for ABCB1 siRNA transfected cells compared to non-transfected cells, at the highest tested AmB concentration (5 μg/mL).
Conclusions:
Small interfering RNA is a possible alternative to the physical inhibition of P-gp, as the transient knockdown of ABCB1 in Caco-2 cells decreased P-gp expression and function. Although there was an increased cellular association of AmB with ABCB1 siRNA transfected cells, differences were not observed for P-gp ATPase activity or between AmB cytotoxicity profiles. The data suggested that P-gp has a minimal contribution to the epithelial transport of AmB in Caco-2 cells.
Significance:
Clinically, it is unlikely that the oral bioavailability and drug disposition of AmB will be affected by P-gp mediated efflux.
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