Molecular identification and functional characterization of P-glycoprotein in cornea and ocular pharmacokinetics of erythromycin in rabbits

Multidrug resistance cell lines express high amounts of an energy dependent efflux pump responsible for reduced accumulation of drugs into the cells. Such efflux pumps like P-glycoprotein are believed to be a major barrier to drug delivery. The substrates of P-glycoprotein include anticancer drugs, HIV protease inhibitors, cardiac drugs, and antibiotics. A majority of topically applied drugs including steroids, beta-blockers, antibiotics, and non-steroidal anti-inflammatory agents are substrates of P-glycoprotein. The process of corneal drug absorption has proven to be extremely inefficient. Only about 1--10% of the topically applied dose is absorbed intraocularly. Lower ocular bioavailability so far was attributed primarily to the inability of drug molecules to cross the lipoidal membrane and not attributed to any efflux proteins that might efflux molecules out of corneal epithelium. Such efflux pumps have been identified in the eye. In the eye, P-glycoprotein is expressed in the retina, conjunctiva, lens and iris and ciliary muscle cells. The broad overall objective of this present study was to determine if human and rabbit cornea expresses the functionally active P-glycoprotein efflux pump. Primary cultured rabbit corneal epithelial cells and a corneal cell line were selected as the model. Rhodamine-123 and erythromycin (known P-glycoprotein substrates), was used as a P-glycoprotein probe. RT-PCR was done using conserved primers for rabbit and human P-glycoprotein. Subcloning, sequencing and protein sequence determination was performed to confirm P-glycoprotein. A topical well infusion model was used to determine the role of P-glycoprotein in restricting ocular drug absorption. Permeability of cyclosporine A in apical-to-basolateral direction was 3 times larger as compared to basolateral-to-apical direction. Uptake of rhodamine-123 across both cell types was inhibited significantly in presence of a series of inhibitors. Western blot and RT-PCR studies confirmed the presence of P-glycoprotein. Protein translation of the fragment indicated 89% homology with human P-glycoprotein. Ocular pharmacokinetics of erythromycin was determined in the presence and absence of inhibitors. Testosterone increased the aqueous erythromycin concentration significantly. Thus P-glycoprotein was found to be functionally expressed in human and rabbit cornea which restricts in vivo ocular drug absorption.