NC-1059: a channel-forming peptide that modulates drug delivery across in vitro corneal epithelium

Drug delivery of ocular therapeutics presents several challenges. There are many difficulties in attaining and sustaining adequate therapeutic levels in the eye while avoiding systemic toxicity. Although there are several routes of administration available for the eye (e.g., topical, subconjunctival, retrobulbar, intracameral, and systemic), topical application of therapeutic agents to the eye offers several advantages including localized drug effects with limited entry into systemic circulation, better accessibility than can be achieved by systemic delivery, avoidance of first-pass hepatic metabolism, convenience, and simplicity. Despite the benefits of topical application, many factors limit the bioavailability of therapeutic agents. For instance, the maximum volume that can be contained within the conjunctival cul-de-sac and precorneal tear film is approximately 30 µL, and the solutes are eliminated rapidly from the precorneal area by lacrimal secretions. The cornea is the outermost transparent portion of the eye and provides the primary barrier through which ocular absorption must occur.1 The anatomic structure of the cornea results in inefficient delivery of drugs to the deeper structures of the eye due to the relatively small surface area and its intrinsic nature as a barrier. Three main strategies are used to increase the bioavailability of ophthalmic drugs including increasing the contact time of the drug with the cornea, improving drug penetration characteristics, and enhancement of the corneal permeability (reviewed in Ref. 2). Corneal epithelial cells constitute the major rate-limiting barrier to drug absorption after topical application. There are two major routes for the movement of compounds through the corneal epithelium: transcellular and paracellular. Since epithelial cell membranes are barriers rich in lipid, lipophilic agents show the best intrinsic penetration through the transcellular pathway. In the absence of an active transport process, however, hydrophilic compounds are limited substantially to permeation through the paracellular pathway where movement is blocked by tight junctions that join adjacent cells near their mucosal surface. The tight junctions define the apical and basolateral membrane components and they serve as a semi-permeable barrier to the flow of solutes through the paracellular pathway. In some tissues, the permeation rates across the tight junctions change in conjunction with various events such as leukocyte transmigration and nutrient uptake in the intestine. 3,4 Tight junctions are also regulated by hormones in some tissues, such as transforming growth factor, glucocorticoids, and progesterone in mammary epithelium.5,6 Thus, therapeutic modulation of corneal tight junctions may allow alteration of the paracellular pathway that would enhance ophthalmic drug concentrations. A synthetic channel-forming peptide, designated NC-1059, has been shown to transiently modulate the barrier function of tight junctions in Madin-Darby canine kidney (MDCK) epithelial cell monolayers.7 This functionality was also observed with epithelial cells derived from the urogenital duct, gut, and air-way of other species.8 In initial studies that targeted the development of a synthetic ion channel, the peptide used was based on the second transmembrane segment of the glycine receptor α-subunit (M2GlyR). This peptide forms a selective pathway in epithelial cells that allows for anion secretion,9 but exhibits relatively low bioavailability due to solution aggregation. Modification of the M2GlyR peptide increased aqueous solubility while maintaining the channel-forming properties as well as anion selectivity.10,11 A peptide was then designed in which the transmembrane segment had an N-terminal tetra-lysine segment and a central leucine that was flanked by a palindrome that is based on the N-terminal portion and was designated NC-1059 (KKKK-AARVGLGITTVLVTTIGLGVRAA). NC-1059 elicited an increase in short circuit current (Isc, a sensitive indicator of net ion transport), remains monomeric in aqueous solution,10 and exhibits no obvious indication of cytotoxicity.7 Thus, NC-1059 represents a possible means of manipulating the epithelial barrier of the cornea at the tight junctions. Studies that assess ophthalmic bioavailability can be conducted in a variety of assay systems. Historically, drug permeation studies have been conducted in vivo with the use of rabbits. However, an in vitro system has many advantages over conventional techniques including the opportunity to use tissues derived from humans and the opportunity to minimize time-consuming, expensive, and controversial animal studies. 12 THCE cells13 provide a model system to asses drug permeation, as has been reported recently.12 These cells have been used to assess tight junctions,14,15 to characterize corneal wound healing,16–18 and to test for cytotoxicity.19 Additional electrophysiological studies verified that THCE cells provided a viable option for the assessment of drug permeation across a confluent epithelial layer. The goal of this study was to determine whether a synthetic channel-forming peptide, NC-1059, can modulate the barrier function of corneal epithelium in vitro using THCE cells. After establishing critical basal parameters for the cultured corneal epithelial cells, experiments were conducted to test for an NC-1059-associated enhancement in gradient driven drug permeation across confluent monolayers. The outcomes show that methotrexate and surrogate drugs of various sizes and chemistries more readily gain access across the corneal epithelial barrier in the presence of or after brief exposure to NC-1059.