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P.K. Karla, D. Pal, A.K. Mitra; Molecular Evidence and Functional Expression of Multi Drug Resistance Associated Protein (MRP) in Human Corneal Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):65.
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Drug resistance proteins as, multidrug resistance associated protein (MRP) and P–glycoprotein (P–gp; MDR1) are found to be a major barrier in drug delivery. Decrease in therapeutic concentration is found to correlate with the expression of a glycoprotein encoded by MDR1 gene (P–gp) and a transmembrane phosphoglycoprotein (MRP). The major objective of the work was to determine whether human corneal epithelial cells (HCEC) express multidrug resistance associated protein (MRP) and study the role of MRP in corneal drug efflux. Also, the role of MRP in efflux of guanosine analogues as Acyclovir was studied.
Human primary corneal epithelial cells and SV40 transfected human corneal epithelial cells were used. MK–571, a potent and specific inhibitor for MRP, Ketoconazole, a specific P–gp inhibitor, C–14 Erythromycin–a proven substrate for both P–gp and MRP and H3–Acyclovir – a guanosine analogue were used. Cells were grown in twelve well cell culture plates as per the protocol established in our lab. The uptake experiments of C–14 Erythromycin and H3–Acyclovir in the presence of MK–571 and Ketoconazole were performed after 10–12 days of growth. Cytotoxicity tests were performed to check of cell membrane integrity. RT–PCR and western blot were performed to confirm the expression of MRP.
Significant uptake of C–14 Erythromycin and H3–Acyclovir was observed in the presence of both MK–571. Uptake of erythromycin increased by >40% with the use of ketoconazole and >100% with the use of MK–571. Combined use of Ketoconazole and MK–571 resulted in a cumulative increase in the uptake of erythromycin 150% (app.) indicating collective role of MRP and P–gp in corneal drug efflux. Uptake of H3–Acyclovir increased by >100% as compared to control. Studies are in progress on MRP mediated efflux of Acyclovir and the results can give us a new insight into understanding of corneal epithelium as a barrier for hydrophilic drug molecules. RT–PCR indicated the presence of MRP–1, MRP–2, MRP–3, MRP–4, MRP–5, MRP–6 and MRP–7 in human corneal epithelium. The results of RT–PCR are confirmed by sequencing analysis. Western blot analysis using monoclonal MRP–2 antibody confirmed the presence of MRP–2 in membrane fraction of human corneal epithelium. For the first time we have demonstrated the presence of MRP in human corneal epithelium and significant role it can play in drug efflux.
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