New Human Organotypic Corneal Tissue Model for Ophthalmic Drug Delivery Studies
PURPOSE. The purpose of the current work was to develop a physiologically relevant, in vitro human three-dimensional (3D) corneal epithelial tissue model for use in ophthalmic drug development. METHODS. Normal human corneal epithelial cells were cultured at the air–liquid interface to produce the 3D corneal tissue model. Corneal barrier was determined by measuring transepithelial electrical resistance (TEER). Quantitative PCR arrays were utilized to investigate expression of 84 phase I/II metabolizing enzymes and 84 drug transporter genes. Permeability was evaluated using model compounds with a wide range of hydrophobicity, molecular weight, and excipients. Finally, different formulations of latanoprost and bimatoprost were administered and drug absorption and tissue viability and integrity were investigated. RESULTS. Histologic assessment and TEER of the corneal tissue model revealed tissue structure, thickness, and barrier formation (1000 ± 146 X Ωcm2) comparable to native human corneal epithelium. The 3D corneal tissue expressed tight junctions, mucins, and key corneal epithelial detoxification enzymes. Drug-metabolizing enzyme and transporter gene expression in 3D corneal tissue and excised human corneal epithelium were highly correlated (r2 = 0.87). Coefficients of permeation for model drugs in the tissue model and excised rabbit corneas also showed a high correlation (r2 = 0.94). As expected, latanoprost and bimatoprost free acids had much lower permeability (Papp = 1.2 X 10-6 and 1.9 X 10-6) than the corresponding prodrugs (Papp = 2.5 X 10-5 and 5.6 X 10-5), respectively. The presence of 0.02% benzalkonium chloride in ophthalmic formulations significantly affected tissue barrier and viability.
CONCLUSIONS. The newly developed 3D corneal tissue model appears to be very useful for evaluation of corneal drug permeability and safety during ophthalmic drug development.
corneal epithelium, in vitro reconstructed tissue model, organotypic tissue model, biocompatibility, ocular drug delivery, ocular permeation, ocular drug permeability, alternative to animal testing; ophthalmic drug development, transepithelial electrical resistance (TEER), gene expression, drug metabolizing enzymes, drug transporters, immunofluorescent staining: antibodies to ZO-1, occludin, claudin-1, MUC-1, cytokerains CK3/12, and DAPI nuclear stain
immunofluorescent staining: antibodies to ZO-1, occludin, claudin-1, MUC-1, cytokerains CK3/12, and DAPI nuclear stain, Latanoprost, latanoprost free acid, bimatoprost, bimatoprost free acid, Lumigan, Benzalkonium chloride, BAC, Sodium Fluorescein, Fluorescein isothiocyanate-dextran (FD4), Lycifer Yellow, Rhodamine B, Ofloxacin
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