title: Molecular evidence and functional expression of a novel drug efflux pump (ABCC2) in human corneal epithelium and rabbit cornea and its role in ocular drug efflux.
abstract: Cornea is considered as a major barrier for ocular drug delivery. Low ocular bioavailability of drugs has been attributed primarily to low permeability across corneal epithelium, thus leading to sub-therapeutic concentrations of drug in the eye and treatment failure. The role of drug efflux proteins, particularly the P-glycoprotein (P-gp) in ocular drug bioavailability has been reported. The objective of this research was to determine whether human corneal epithelium expresses multidrug resistance associated proteins (MRPs) contributing to drug efflux by employing both cultured corneal cells and freshly excised rabbit cornea. SV40-HCEC and rPCEC were selected for in vitro testing. SV40-HCEC and freshly excised rabbit corneas were utilized for transport studies. [(3)H]-cyclosporine-A and [(14)C]-erythromycin, which are known substrates for ABCC2 and MK-571, a specific inhibitor for MRP were applied in this study. RT-PCR indicated a unique and distinct band at approximately 272 bp corresponding to ABCC2 in HCEC, SV40-HCEC, rabbit cornea, rPCEC, and MDCKII-MRP2 cells. Also RT-PCR indicated a unique band approximately 181 bp for HCEC and SV40-HCEC. Immunoprecipitation followed by Western Blot analysis revealed a specific band at approximately 190 kDa in membrane fraction of SV40-HCEC, MDCKII-MRP2 and no band with isotype control. Uptake of [(3)H]-cyclosporine-A and [(14)C]-erythromycin in the presence of MK-571 was significantly enhanced than control in both SV40-HCEC and rPCEC. Similarly a significant elevation in (A-->B) permeability of [(3)H]-cyclosporine-A and [(14)C]-erythromycin was observed in the presence of MK-571 in SV40-HCEC. A-->B transport of [(3)H]-cyclosporine-A was elevated in the presence of MK-571 in freshly excised rabbit cornea indicating potential role of this efflux transporter and high clinical significance of this finding.
authors.affiliation: Division of Pharmaceutical Sciences, School of Pharmacy, University of Missouri, Kansas City, MO 64110, United States.
authors.forename: Pradeep K
authors.initials: PK
authors.lastname: Karla
authors.forename: Dhananjay
authors.initials: D
authors.lastname: Pal
authors.forename: Tim
authors.initials: T
authors.lastname: Quinn
authors.forename: Ashim K
authors.initials: AK
authors.lastname: Mitra
mesh_terms: D000818:Animals; D001692:Biological Transport / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002478:Cells, Cultured; D003315:Cornea / Q000187:drug effects / Q000378:metabolism*; D016572:Cyclosporine / Q000378:metabolism; D004285:Dogs; D004847:Epithelial Cells / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D019573:Epithelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D004917:Erythromycin / Q000378:metabolism; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008297:Male; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism*; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D004364:Pharmaceutical Preparations / Q000378:metabolism*; D011422:Propionates / Q000494:pharmacology; D011804:Quinolines / Q000494:pharmacology; D011817:Rabbits
chemical_list: D016572:Cyclosporine; D004917:Erythromycin; D026901:Membrane Transport Proteins; D000089762:Multidrug Resistance-Associated Protein 2; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D004364:Pharmaceutical Preparations; D011422:Propionates; D011804:Quinolines; C000715087:ABCC2 protein, human; C059141:verlukast; C421109:multidrug resistance-associated protein 1