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Characterization of Cyclosporin A Transport in Cultured Rabbit Corneal Epithelial Cells: P-Glycoprotein Transport Activity and Binding to Cyclophilin

From the Santen Pharmaceutical Co., Ltd., Nara Research and Development Center, Ophthalmic Research Division, Ikoma-chi, Japan.

PURPOSE. The purpose of this study was to characterize cyclosporin A (CsA) uptake and transport in cultured rabbit corneal epithelial cells (RCECs) .

METHODS. CsA uptake was evaluated by measuring time-dependent ³H-CsA accumulation in confluent RCECs. Bidirectional ³H-CsA fluxes were measured across the RCEC layers grown on Transwell-COL culture plate inserts. The anti-P-gp monoclonal antibody C219 was used in western blot analysis to probe for the presence of P-gp in these cells.

RESULTS. The accumulation of ³H-CsA was time and temperature dependent. Steady state was reached by 60 minutes. The initial uptake was saturable and was suppressed as a function of increases in preloading with unlabeled CsA. This uptake process was enhanced by metabolic inhibition with either 3-O-methylglucose, MG, or 10 mM NaN₃ and 3-O-MG. The largest increase was obtained with 10 mM NaN₃ in combination with 3-O-MG. In their presence, uptake increased by 40%. A multidrug-resistance (MDR)–reversing agent (i.e., 500 µM verapamil, 100 µM vincristine, 100 µM progesterone, 100 µM testosterone, 500 µM quinidine, or 100 µM chlorpromazine) significantly increased ³H-CsA accumulation. The largest increase was obtained with 500 µM quinidine (i.e., 36%). Conversely, verapamil and vincristine produced the largest inhibition of ³H-CsA efflux (i.e., 19% and 28%, respectively). However, in the presence of 10 µM unlabeled CsA, ³H-CsA efflux increased. ³H-CsA flux across RCEC layers showed marked directional asymmetry. The stromal (S) to tear (T) side transcellular ³H-CsA permeability coefficient (P_trans) was approximately seven times higher than that in the T-to-S direction. The S-to-T P_trans was reduced by an MDR-reversing agent by up to 40%. Western blot analysis of lysates revealed a 170-kDa membrane protein band.

CONCLUSIONS. These results suggest that in RCEC the tear-side–facing membrane has a P-gp–mediated drug efflux pump. In addition, there is suggestive evidence for the presence of the cytosolic protein, cyclophilin. The presence of P-gp in these cells could help protect them from being damaged by the uptake of toxic substances.

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