Peptide-Enhanced Corneal Drug Delivery

Reference Number: 05-07

Inventor: John Tomich, Takeo Iwamoto, James Broughman, and Bruce Schultz


Researchers with Na+celle Therapeutics (startup company at K-State) and Kansas State University have developed peptides that have demonstrated (in vitro, ex vivo and in vivo) an ability to reversibly increase the paracellular permeability of ocular tissues for enhanced delivery of drugs to the eye. Ocular conditions such as glaucoma, ocular inflammation and infection often require ocular administration of drugs for most efficient treatment. Unfortunately, the current state of the art for drug transport across ocular barrier membranes is inefficient, owing to the fact that the intrinsic conjunctional epithelia forms tight junctions with high resistance to ocular delivery. Moreover tear washout and the blinking reflex dilutes and washes out the administered drugs.

The broad spectrum of immunological and inflammatory responses of the eye and ocular epithelium pose a significant problem in the effective treatment of ocular conditions that remain largely unresolved. What is needed is a delivery system for reversibly modifying epithelial tight junctions safely to permit absorption or transport of drugs or other desirable compounds.

This group of Na+celle’s peptides potentially overcome the problems outlined above and provides improved methods and preparation for administration to ocular tissues. The technology serves to open paracellular pathways and reversibly modify tight junctions in the epithelial tissue, thereby facilitating drug transfer.


Delivery of drugs or other desirable compounds needed to treat eye conditions and/or infections.


Potential advantages of this novel technology over current methods:

  • Increased efficiency of drug delivery to the eye
  • Decrease the amount of drug needed to treat conditions or infections in the eye
  • Reversibly modify epithelial tight junctions without damaging or injuring the epithelial cells forming the tight junctions
  • Decreases transepithelial resistance in increasing ion transport across epithelial cells regulated by tight junctions
Technology Readiness

In vitro, ex vivo and in vivo studies have been completed and information provided below.

In vitro
style="text-decoration: underline;">Paracellular Permeation Studies
The palindromic sequence, p1059- KKKKAARVGLGITTVLVTTIGLGVRAA, forms non-selective ion channels that reversibly alter epithelial barrier function. This modulation occurs through the reorganization of epithelial tight junctions in a reversible and repeatable manner. This peptides has therapeutic potential: facilitating the delivery of hydrophilic or larger drugs past barrier membranes. The sequence induced a transient reduction in barrier integrity of, cultured epithelial monolayers derived from canine kidney cells (MDCK) and multi-layered SV40-immortalized human corneal epithelial cell line (THCE) provided by Araki-Sasak. The effect of the peptide on conductance and paracellular permeation was both transient and repeatable. Thirty minutes after exposing corneal monolayers to the peptide, the conductance and paracellular permeation start to decrease.

By 90 min the paracellular pathway is no longer accessible to carboxyfluorescein (CF) even though gte remained high (Fig.1A, THCE cells). Addition of more peptide at this time does not re establish paracellular permeation.

This treatment resulted in a two to four fold increase in dextran permeation compared to vehicle-treated monolayers (Fig. 1B). However, between 24 and 48 hr after returning the cells to fresh peptide-free media, the conductance returned to pretreatment values indicating a time dependent process (Fig.2, MDCK cells) and the cells again become sensitive to addition of the peptide. MDCK monolayers, when subsequently retreated with p1059 showed increases in gte that were statistically identical to the initial treatment.These results suggest the peptide is effective in opening the paracellular pathway for a period that is sufficient for enhancing drug delivery.

The peptide-triggered appearance of paracellular permeability suggested a mechanism that includes changes in the organization of molecules that compose or regulate tight junctions. Studies were initiated to elucidate the mechanism by which p1059, increased epithelial paracellular permeability. The increases in gte and paracellular permeability were accompanied by a reorganization/depolymerization of F-actin, which began within the first 15 minutes of exposure and pronounced effects were observed in less than 60 minutes. A change was also observed in the distribution and abundance of the tight junction proteins-- occludin and ZO-1. The ZO-1 re localization followed a similar time course as F-actin disruption. Other cellular proteins that showed peptide induced changes in distribution and cellular abundance were the adheren junction proteins, E-cadherin and ß-catenin (Fig. 4). The peptide caused a time and concentration dependent decrease in the abundance of E-cadherin, due to degradation (based on Western analyses).

We hypothesize that breakdown of E-cadherin, redistribution of ß-catenin, F-actin rearrangement and disruption of tight junction proteins ZO-1 and occludin are inter-related events that are regulated either by identical mechanisms or through pathways with significant cross talk. These results suggest that the p1059 mediated increase in paracellular permeability might activate biochemical pathways common to several molecules that modulate barrier function. Small G proteins have a significant role in regulating F-actin organization and junctional proteins. The reorganization of actin accompanied by disruption of tight junction proteins caused by activation or inactivation of Rho and Rac also affects localization of E-cadherin in MDCK monolayers. It is possible that the reorganization of actin and possibly subsequent loss in junctional proteins induced by p1059, could involve Rho/Rac activity.

As previously described, gte recovers to pretreatment values within 48 hours in MDCK monolayers exposed to p1059. The effects of the peptides were reversible with a progressive recovery of the apical cell junction components occludin and ZO-1 at 24 and 48 hr post exposure. The most significant observation was that the effects of p1059 on epithelial permeability were only temporary with the majority of the gte recovering within 24 hours along with the localization of junctional proteins particularly ZO-1. In studies where E-cadherin was degraded as seen with ATP depletion or Bacteroides fragilis toxin (BFT), the effects were reversible and resynthesis of E-cadherin were involved in recovery. The reversible nature of the peptide’s effect on junctional proteins is an encouraging and important aspect in the development of compound that can facilitate the uptake of therapeutic agents across barrier membranes.

In vivo and Ex vivo
Animal Model: RabbitNumber: 3
Translation work with ocular epithelium Ex vivo and in vivo rabbit corneal tissues were tested with p1059. These studies were performed in collaboration with Dr. Henry Edelhauser at Emory University Medical School. The cornea and conjunctiva are high resistance barrier membranes that could be exploited for drug delivery to the eye for treatment of both infection and inflammatory diseases. In these unpublished studies, we systematically studied p1059’s dose-dependent modulation of corneal transepithelial model drug uptake to determine the minimal effective dose, time course of p1059 effects and optimal exposure time to test compounds and generated clear and unambiguous data that support the hypothesis that p1059 can increase the paracellular permeation of normally impermeable hydrophilic molecules. We observed using both freshly excised and in vivo corneas of rabbits that p1059 transiently opened the tight junctions thereby increasing the delivery of small molecules into and across the cornea. Most of the initial studies were performed substituting two less expensive fluorescent dyes, sodium fluorescein (NaF) and carboxyfluorescein (CF) whose abilities to cross the cornea have been well documented by Dr. Edelhauser and others . In Figure 5 the peptide (200 µM) induced fold increase for paracellular CF permeation shows a biphasic profile: up to 90’ there is a rapid increase in CF transport that then levels off to a steady state rate that is equal to that observed in the absence of peptide (data not shown). This result is consistent with our earlier studies that showed the transient nature of the peptide’s ability to open the paracellular pathway.

Three other derivative peptides were produced- a biotinylated form of p1059 with the biotin added at the N-terminus, the second variant was the all D-isomer of the peptide and lastly, a fluorescently labeled version, with 6-carboxytetra-methylrhodamine (6-TAMRA) added to the N-terminus. All three have passed in our epithelial monolayer assay system.

The biotinylated and TAMRA peptides are available for use in ultrastructure studies to determine cellular uptake and the depth of penetration of the peptide into corneal tissues.

To document the in situ opening of the tight junctions, peptide treated corneas were chemically fixed at the end of a dye transport assay for morphology studies. The control and test samples were treated with the dye ruthenium red, which binds to the plasma membrane of epithelium and cannot pass the tight junctions. The transmission electron micrographs prepared are shown in Figure 6. In the peptide treated tissue (Fig 6B), the multilayered epithelium is apparent due to the binding of the ruthenium red which now has access to the paracellular pathway. The morphology of the cells is unaltered suggesting that the opening of the junctions is biochemical rather than mechanical as would be expected with agents that cause the cells to swell or shrink. As a positive control, the ophthalmic preservative benzalkonium chloride (BAC) was used in place of the peptide. BAC is known to disrupt the corneal epithelium barrier function and cause cellular damage at concentrations greater than 0.005%. Severe disruption of surface cell layers occurred simultaneously with decreased resistance (Fig. 6C).

The cells appear swollen and the villi have disappeared. In a separate study 15 min of preincubation with either p1059 or BAC the induced transport rates for CF in the paired corneas appear identical for BAC and the peptide. With a longer preincubation time, 30 minutes (the BAC begins to promote increased CF translocation over peptide after the three-hour test point (data not shown).

A series of in vivo corneal epithelial permeability experiments in unanaesthetized rabbits was also conducted at Emory. P1059 is prepared in BSS (200 µM) and placed inside the lower eyelid of one eye with the other eye receiving an equal amount of BSS alone. The solutions are held in place for 3 minutes and then washed with BSS. After the indicated incubation periods both eyes receive 300 µM NaF or CF and the dye is kept in place for 3 or 5 minutes.

After the contact period the eyes are washed with 40 mL of BSS solution. Total uptake was measured for both the control and treated eyes with an OcuMetrics, Ocular Fluorophotometer. The fold changes between the peptide treated and untreated eyes were calculated (Fig. 7). The peptide treatment followed by a 3 min exposure to the dye resulted in a 2.2 ? 0.46 (n = 3) fold increase. With the 5 minute treatment of CF added at 0 and 30 minutes after the rinse resulted in 24.7 ? 8.6 (n=3) and 14.4 ? 5.6 (n=2) fold increases, respectively. These results show that the peptide was able to open tight junctions in vivo in hand held animals. Also these results showed that increasing the exposure time of the dye, in peptide treated eyes, significantly increases corneal uptake. It would also appear that adding the dye immediately after peptide exposure results in the highest permeation.

In the fourth column, the eyes that were previously monitored at the 30-minute time point (showing a 2.7-fold increase) were challenged a second time with CF at 6 hours, and showed only a 1.1-fold increase. The reduced uptake at the 6-hour time point futher documents the transient effects of the peptide in vivo. This transient effect has been observed under all assay conditions: in vitro, in situ and in vivo.
Patent Status
  • U.S. patent #7,592,341 issued on September 22, 2009.

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