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Purpose: To investigate the effect of the peptide NC-1059 on riboflavin (RF) diffusion across an intact corneal epithelium into the stroma., Methods: NC-1059 peptide was synthesized by solid-phase synthesis with 9-fluorenylmethoxycarbonyl chemistry, characterized by reversed-phase HPLC, and matrix-assisted laser desorption ionization time-of-flight mass spectroscopy. The diffusion of RF across embryonic day 18 chick corneal epithelium ex vivo was monitored using confocal microscopy. The depth distributions of RF in the corneal stroma were calculated using a group of linear equations based on the relationship between RF fluorescence intensity and concentration., Results: Data presented in this study demonstrate that the NC-1059 peptide can transiently open the intact epithelial barrier to allow the permeation of RF into the stroma. The effect of NC-1059 peptide on RF diffusion across the corneal epithelium was concentration and time dependent. The amount of RF reaching a 50-μm depth of chick corneal stoma increased dramatically after exposure to NC-1059 for 10 minutes, reaching a plateau by 30 minutes. The concentrations of RF in the presence of NC-1059 at corneal stromal depths of 50, 100, and 150 μm were significantly higher than in the absence of the peptide, and almost as high as in corneas in which the epithelium first had been physically removed. In addition, a cell viability assay indicated that the NC-1059 peptide did not kill corneal epithelial cells., Conclusions: NC-1059 peptide significantly enhances the diffusion of RF across intact corneal epithelium into the stroma.

Purpose: The goal of this study was to determine whether a synthetic peptide, NC-1059, can modulate the corneal epithelium to increase the permeation of therapeutic agents across this barrier., Methods: An in vitro system employing transformed human corneal epithelial (THCE) cells was optimized for this study. Culture conditions were identified to promote formation of a confluent monolayer that rapidly develops a substantial transepithelial electrical resistance. Electrical parameters were measured with a modified Ussing flux chamber, and solute flux was quantified with fluorescently labeled compounds., Results: NC-1059 causes a concentration-dependent increase in short-circuit current and an increase in transepithelial electrical conductance when assessed in modified Ussing chambers. The effect of NC-1059 on transepithelial electrical resistance was reversible. To test for paracellular permeability and size exclusion, FITC-labeled dextran ranging in size from 10 to 70 kDa was used. Dextran permeated the corneal cell monolayer in the presence, but not the absence, of NC-1059. Fluorescein sodium and carboxyfluorescein were then used as low molecular weight markers with similar NC-1059-modulated kinetics being observed. Maximum permeation for the fluorescein derivatives occurred 30 to 90 minutes after a 5-minute NC-1059 exposure. A prototypical drug, methotrexate, also exhibited increased permeation in the presence of NC-1059., Conclusions: NC-1059 enhances drug permeation across cultured corneal epithelial cell monolayers by transiently affecting the paracellular pathway. Thus, NC-1059 is a lead compound for development of cotherapeutic agents to enhance access and effectiveness of ophthalmic compounds.