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Molecular Evidence and Functional Expression of P-Glycoprotein (MDR1) in Human and Rabbit Cornea and Corneal Epithelial Cell Lines

¹From the Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri-Kansas City, Kansas City, Missouri; and the ²Medical College of Georgia, Department of Biochemistry and Molecular Biology, Augusta, Georgia.

PURPOSE. Efflux pumps such as P-glycoprotein (P-gp; MDR1) are believed to be a major barrier to drug delivery. The purpose of this work was to determine whether cornea and corneal epithelial cells expresses the functionally active P-gp efflux pump.

METHOD. Cultured rabbit primary corneal epithelial cells (rPCECs) and a corneal cell line (Statens Seruminstitut rabbit cornea [SIRC] cells) were selected as the model. Rhodamine-123 (Rho-123), a P-gp substrate, was used as a P-gp probe. To confirm gene expression, RT-PCR was performed with appropriate pairs of primers for rabbit and human MDR1. Subcloning, sequencing, and protein sequence determination were performed to confirm P-gp.

RESULTS. Permeability of [³H] cyclosporin A (CsA) across SIRC cells was found at 1.74 x 10^-6 cm/s in the apical-to-basolateral and 5.1 x 10^-6 cm/s in the basolateral-to-apical directions. Uptake of Rho-123 across both SIRC cells and rPCECs was time and temperature dependent. Rho-123 uptake in SIRC cells was 14.4 picomoles/mg protein and in the presence of CsA (10 µM) was 70.8 picomoles/mg protein at 3 hours. Uptake in rPCECs was the highest at 3 hours. Western blot analysis indicated a 170-kDa band confirming the presence of P-gp. Human cornea was also checked for the presence of P-gp. RT-PCR data indicated one single band, which was subcloned and sequenced to confirm the presence of P-gp. The protein sequence deduced from the fragment product indicated more than 89% homology with human MDR1.

CONCLUSIONS. Functional and molecular characterization showed the existence of P-gp in human cornea, rabbit cornea, and a rabbit corneal cell line. This knowledge of the existence of P-gp will help in development of better ocular drug delivery strategies.

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