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P. K. Karla, A. K. Mitra; Identification of Drug Efflux Pumps in Human Corneal Epithelial Cells - Potential Role in Drug Efflux. Invest. Ophthalmol. Vis. Sci. 2007;48(13):2730.
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Cornea forms the major barrier for ocular drug delivery. Poor permeation is responsible for low ocular bioavailability of various topically applied drugs resulting in treatment failure. The main objective of this study was to explore barriers for corneal drug delivery at molecular level and to evaluate their functional significance.
Transfected Human Corneal Epithelial Cells (HCEC), rabbit corneal epithelial cells (rPCEC) were employed as an in vitro model. Excised rabbit cornea was used for ex vivo evaluation. RT-PCR, Western blot were used for molecular expression studies. ACV a drug to treat (HSV) keratitis, CsA, an immunosuppressant used to prevent corneal graft rejection and E17ß-g, an organic anion a proven substrate for efflux pumps like MRP, BCRP and LRP were employed.
Westernblot analysis indicated unique bands, MRP4 (~150 kDa), MRP5 (~160 kDa), and BCRP (~ 70 kDa) in HCEC. RT-PCR indicated distinct bands MRP4 (~277 bp), MRP5 (~228 bp), BCRP (~429 bp) and LRP (~500 bp). Uptake studies of ACV in the presence of MK571 revealed a significant increase in uptake in both HCEC (~173.11)% and rPCEC (~220)% as compared to control (~100)%. Significant increase in uptake of CsA was also noticed in HCEC (~164)% and rPCEC (~173.26)% as compared to control (~100)%. Transport of ACV and CsA across excised rabbit cornea resulted in a significant increase in transport from epithelial to endothelial side. Decrease in efflux of E17ß-g was noticed in the presence of specific inhibitors like MK571 and FTC.
For the first time we have demonstrated the expression of novel nucleoside/nucleotide efflux transporters MRP4 and MRP5 at protein level in human corneal epithelial cells. Significant increase in uptake and transport results in the presence of MK571, a specific MRP inhibitor indicated that MRP4, MRP5 might be responsible for treatment failure of topical ACV in HSV keratitis patients. Increase in uptake of CsA indicated the possible role MRP in cyclosporine efflux. Also, significant decrease in efflux of E17ß-g in the presence of FTC a specific BCRP inhibitor indicated the over expression of BCRP in transfected human corneal epithelial cells. Preliminary results in our laboratory indicate that various antiglaucoma drugs currently in the market are good substrates for MRP (unpublished data) indicating potential clinical significance of this discovery. Employing specific or a combination of inhibitors might result in a significant increase in ocular bioavailability of anterior chamber of eye.
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