In this study, we determined that a synthetic peptide, NC-1059, modifies the barrier function of THCE cells grown as a monolayer culture. Initially, experiments were conducted to verify that THCE cells cultured for fewer than 10 days form an epithelial barrier in culture that is capable of separating fluid compartments with distinctly different compositions. After the utility of this in vitro system was demonstrated, experiments were conducted to show that NC-1059 caused a concentration-dependent enhancement of gte that is paralleled by an increase in passive, gradient-driven permeation rates for either large uncharged solutes (dextran), low-molecular-weight markers (fluorescein), or a prototypical low-molecular-weight drug (methotrexate). Of importance, the results showed that short-term (5 minutes) exposure to NC-1059 enhanced permeation of fluorescein for 60 to 90 minutes, after which no effect on permeation rates was observed. The rate of methotrexate permeation was likewise enhanced over the first 2 hours after NC-1059 exposure. Equally important is the observation that gte, the most sensitive indicator of epithelial barrier integrity, returned to pretreatment values and became responsive to repeated exposure. Taken together, these results indicate that NC-1059 enhanced drug permeation across a model corneal epithelium by transiently affecting the paracellular pathway.
This study identified THCE cells cultured on permeable supports for ~1 week as a robust in vitro system to evaluate corneal drug permeation. Many human corneal cell culture models exist today including primary cell cultures, immortalized cell lines,12,13,24–26
and whole corneal models.27–29
At this time, whole corneal models are being developed and show great promise as tools for drug-permeation studies, but currently data on the barrier function of these models are sparse. Immortalized cell lines offer many advantages over primary corneal cells including minimizing the expense in culturing and maintaining the cells, absence of individual-to-individual variation, control of experimental design, and the opportunity to use tissue initially derived from humans. Immortalized cell lines also offer the opportunity for rapid screening before ex vivo experiments. Many of the corneal epithelial models that exist24–26
were developed for use with in vitro toxicity testing and cell physiology, therefore the barrier function of these cell lines has not been examined, and it is not known whether these cell models could play a role in drug-permeation studies. Transepithelial electrical resistance has long been used as a gold standard to quantify barrier integrity. An early report indicated that freshly excised rabbit cornea exhibited resistances of 5,000 to 15,000 Ω cm2
More recent reports, however, indicate values in the range of 800 to 1000 Ω cm2
for rabbit cornea and approximately 500 Ω cm2
for human cornea. 12,31
In an elegant and systematic study, Becker et al.12
compared both drug permeation and Rte
across numerous corneal models and reported that THCE cells exhibited barrier parameters that were similar to freshly excised human corneas and to cultured human corneal epithelial cells obtained from a commercial source.12
Various culture conditions that promote barrier function for this cell line have been identified and the system has been used by others to examine drug permeation. 32–34
Becker et al. reported that THCE cells could be cultured in standard culture medium and that the apical fluid could be removed allowing for an apical air interface with a resulting multilayer architecture.12
Experiments conducted in the present study identified culture conditions that decreased further the time needed to reach the maximum Rte
, which was similar to the value reported by Becker et al.12
Thus, THCE cells are an ideal system for permeation studies, and although they differ in this case, from the intact cornea by being only a single cell layer thick, the superficial surface of the corneal epithelium contributes over half of the total electrical resistance of the entire cornea,35
making this system the best experimental model for this study. Experiments conducted in this study further validate this assumption, in that THCE monolayers are able to form a resistive monolayer and function as an epithelial barrier separating distinct fluid compartments.
For adequate vision, the eye must uphold specialized barriers that regulate the uptake of materials into the eye. These barriers also inhibit the access to deeper eye tissues of therapeutic compounds resulting in poor drug permeation and potential treatment failure and hinder the development of new therapeutic agents for ocular tissues. Bioavailability is limited by the volume that can be held by the human eye (~30 µL) and the rapid rate of tear turnover (~1 µL/min).36
Thus, only 1% to 5% of an eye drop–instilled dose is delivered to anterior sections of the eye.2,36
To overcome the problem of insufficient drug delivery through the cornea, new advances in ocular drug delivery are needed.
Numerous attempts at improving ocular bioavailability of topically applied drugs have been reported. Penetration enhancers such as surfactants,37,38
have been used to promote corneal permeation of ophthalmic agents. However, these enhancers generally exhibit their effects by inducing morphologic changes in the corneal structure38,43,44
and occasionally lead to adverse effects such as irritation, hypersensitivity, cellular damage,45,46
and reduction in corneal wound healing.47
Clearly, new technologies are needed to optimize ocular drug delivery.
Reversibly modulating the tight junctions of epithelial barriers for therapeutic considerations provides many benefits. One of the techniques currently undergoing investigation is the zonula occludens toxin (ZOT) elaborated from Vibrio cholerae48
and the biologically active component of ZOT, ΔG.49
ZOT has been shown to reversibly open the tight junctions between intestinal epithelial cells and bovine brain microvessel endothelial cells resulting in enhanced permeability of compounds 50,51
and early studies suggest that ΔG has similar effects although it must be protected from enzymatic degradation. 52
ZOT and ΔG provide a lead structure to act as an absorption enhancer for therapeutic agents although a major limitation is that the ZOT “receptor” has only been located in the intestine, nasal epithelium, heart, and the blood–brain barrier endothelium. This differs from NC-1059 in which the effects have been observed with epithelia from a variety of tissues8
and which is now shown to be effective with corneal epithelial cells.
In this study, NC-1059 induced a concentration-dependent increase in Isc
across THCE monolayers with a concurrent and delayed transient increase in gte
. Peak gte
was observed ~15 minutes after NC-1059 exposure. Previous studies suggested that the magnitude of increase in gte
after the addition of NC-1059 is due to an opening of the paracellular pathway, as well as the introduction of an ion channel into the apical membrane.7
This was confirmed in the present study by the use of FITC-dextran. After the addition of NC-1059, there was an increase in the amount of permeation seen for all sizes of FITC-dextran used compared with the control, although the increment for 40- and 70-kDa dextran was modest. Nonetheless, THCE cells do not show the same degree of size exclusion for FITC-dextran as reported previously with MDCK cells.7
EDTA enhanced the corneal permeation for all sizes of FITC-dextran to a much greater extent and is believed to cause structural damage to the epithelial cell layer, which accounts for the greater effect that was observed with this chelating agent.
Sodium fluorescein and carboxyfluorescein were used to test the effects of NC-1059 on permeation of low-molecular-weight compounds as labeled surrogates for drugs. Data suggest that the permeation of both sodium fluorescein and carboxyfluorescein increases over a 30-minute period whether in the presence of NC-1059 or after a 5-minute exposure. Ninety minutes after NC-1059 washout, the barrier function of THCE monolayers, as assessed by fluorescein permeation, is indistinguishable from untreated controls. These results indicate that NC-1059 does not function by exerting a toxic effect on THCE monolayers since the cells are able to regain the functional barrier in a short time frame. The practical inference from these observations is that a single dose of NC-1059 might be used to “prime” an eye for the uptake of a therapeutic agent that is administered 15 to 30 minutes later. NC-1059 would need to stay in contact with the eye for 5 minutes or less, which is consistent with tear turnover rate. A second drug applied 15 to 30 minutes later would be expected to achieve two- to threefold greater uptake into the eye, which ultimately might reduce the number of times that the therapeutic drug must be applied.
Both FITC-dextran and fluorescein derivatives were used as surrogate drugs to look at the affect NC-1059 had on the permeation across THCE monolayers. A prototypical drug was then selected to look at the effect of NC-1059 on its permeation. In recent years methotrexate has been used experimentally as a treatment for systemic lymphoma with ocular involvement, 53–55
and corneal angiogenesis.57
The outcomes suggest that topical application of methotrexate would be beneficial for the treatment of many ocular diseases. The permeation of methotrexate in the presence of NC-1059, EDTA, and BAC was determined. NC-1059 enhanced the permeation of methotrexate with the most robust difference relative to untreated controls observed in the first 2 hours. The permeation rate in control conditions began to approach the permeation rate of NC-1059 treated monolayers during the second half of the 4-hour monitoring period. Both EDTA and BAC also increased the permeation rate of methotrexate, 20 and 6 times that of the control, respectively, but both of these compounds have deleterious effects on the corneal epithelium. The quaternary ammonium compound BAC is one of the most frequently used preservatives in ophthalmic formulations. It can alter corneal permeability; however, the BAC damages the corneal epithelium, resulting in the loss of the protective barrier. 22,23,58
Damage to the cornea has been shown to increase with increased concentrations and exposure time of BAC.22
A perceived benefit with NC-1059 is that the epithelial barrier is fully recovered within 90 minutes.
Although first synthesized to form anion-selective channels in epithelial monolayers as a potential therapeutic for cystic fibrosis, NC-1059 has also been shown to modulate epithelial tight junctions.7,8
This characteristic of NC-1059 provides the potential to increase the permeation of therapeutic agents across epithelial monolayers via modulation of the tight junctions. In this study, NC-1059 had a significant effect on the corneal permeability to surrogate compounds such as FITC-dextran, sodium fluorescein, and carboxyfluorescein. Permeation of methotrexate, a prototypical drug, was also greater for monolayers that were concurrently or previously exposed to NC-1059. Thus, NC-1059 should be considered a leading compound for development of co-therapeutic agents to enhance access and effectiveness of ophthalmic compounds.