Multidrug resistance-associated protein (MRP1, 2, 4 and 5) expression in human corneal cell culture models and animal corneal tissue.
Preclinical studies addressing the transcorneal absorption of ophthalmic drugs are mainly performed using ex vivo animal corneas and in vitro corneal cell culture models, leaving open the question of transferability to humans in an in vivo situation. While passive drug absorption through corneal tissue is well understood, little is known about the expression of transporter proteins and active drug transport in human and animal corneas as well as corneal cell culture models. Therefore, the aim of this study was to conduct an expression analysis of four multidrug resistance-associated proteins (MRP1, 2, 4 and 5) in various in vitro and ex vivo corneal models, leading to a better understanding of the comparability of different corneal models regarding drug absorption and transferability to humans. Two well-established in vitro human corneal models, the HCE-T epithelial model and the more organotypic Hemicornea construct, both of which are based on the SV40 immortalized human corneal epithelial cell line HCE-T, were analyzed, as were excised rabbit and porcine cornea. Specimens of abraded epithelia from human donor corneas were also tested. MRP mRNA expression was determined via reverse transcriptase polymerase chain reaction. Protein expression was examined using Western blot experiments and immunohistochemistry. The functional activity of the MRP efflux transporter was detected in transport assays using specific marker and inhibitor substances. The functional expression of all of the tested MRP transporters was detected in the HCE-T epithelial model. Hemicornea constructs displayed a similar expression pattern for MRP1, 4 and 5, whereas no MRP2 protein expression or activity was detected. However, excised animal corneas exhibited different expression profiles. In porcine cornea, no functional expression of MRP1, 2, or 5 was observed, and we failed to detect MRP4 expression in rabbit cornea. The results suggest that MRP1, 2, 4, and 5 are expressed in the human corneal epithelium and confirm that the transfer of data obtained from animal experiments to an in vivo situation in humans should be performed with caution.
Institut für Pharmazeutische Technologie, Technische Universität Braunschweig , Braunschweig, Germany.
Jessica
J
Verstraelen
Stephan
S
Reichl
D000818:Animals; D018938:Caco-2 Cells; D045744:Cell Line, Tumor; D002478:Cells, Cultured; D003315:Cornea / Q000378:metabolism*; D004847:Epithelial Cells / Q000378:metabolism; D006801:Humans; D026901:Membrane Transport Proteins / Q000235:genetics / Q000378:metabolism; D027425:ATP-Binding Cassette, Sub-Family C Proteins / Q000235:genetics* / Q000378:metabolism*; D012333:RNA, Messenger / Q000235:genetics; D011817:Rabbits; D013552:Swine / Q000235:genetics / Q000378:metabolism; D059467:Transcriptome / Q000235:genetics
D026901:Membrane Transport Proteins; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D012333:RNA, Messenger