We previously showed that a modified CPP, TAT-Cd0
, had antiviral activity in cell culture and was nontoxic to cells at concentrations several times the in vitro EC50
is hydrophilic and contains nine positive charges that are important for the antiviral activity.14
Here we have shown that TAT-Cd0
significantly reduced disease in a mouse ocular model of herpes keratitis, when treatment was initiated within 4 hours of infection. We tested TAT-Cd0
in four different delivery vehicles, to determine if the delivery vehicles affected the antiviral activity of TAT-Cd0
. The results showed that TAT-Cd0
reduced ocular disease in all of the vehicles tested. However, the data suggest that PBS or an artificial tear solution is a more effective delivery vehicle for TAT-Cd0
than gel or cream formulations.
Of the four vehicles used, aquaphor cream had the poorest performance against blepharitis, with higher disease scores and a higher percentage of mice presenting with blepharitis. This is not surprising given the highly cationic nature of TAT-Cd0
. The highly viscous nature of the cream may prevent TAT-Cd0
from reaching the eyelids in effective concentrations where swelling is most prominent and we have seen this effect before when testing other antivirals in a cream vehicle.20
Although the cream vehicle affected blepharitis scores, it did not appear to impair the effect of TAT-Cd0
against neovascularization and stromal keratitis.
We found that after the treatment regimen ended on day 7, neovascularization and stromal keratitis began to increase beginning on day 11, indicating that TAT-Cd0 delayed the onset of stromal keratitis and neovascularization regardless of the vehicle used. Based on these findings, treating with TAT-Cd0 for more than 7 days may provide greater therapeutic effect.
There was a statistically significant 1-log drop in tear film titers at 24 hours postinfection for TAT-Cd0
–treated groups regardless of vehicle and virus cleared by day 9 in all of the TAT-treatment groups, except the cream vehicle. Previous work with other antivirals has shown that as little as a 1-log reduction in viral titer could reduce the severity of ocular disease17,19–21
and this study suggests that even reducing the viral titers in the first 24 hours can have a significant effect on the disease. The data from this study reinforce the conclusion that achieving a significant therapeutic effect does not necessarily require complete inhibition of viral replication. In addition, the titer data indicate that treatment with less viscous vehicles resulted in a more rapid clearance of virus compared with the untreated infected controls.
Treatment with the tears and PBS vehicles reduced the severity of vascularization and corneal clouding, although the differences were not significant compared with the untreated control. Shortly after infection with HSV-1, IL-1α and IL-6 are quickly produced and both cytokines have been shown to induce vascular endothelial growth factor A (VEGF-A) production in nearby uninfected cells. The rise in VEGF-A leads to blood vessel formation in the cornea.6,22
IL-6 has also been shown to stimulate MIP-2 and MIP-1α production in cells, which aids in the recruitment of neutrophils to the site of infection.23
Along with neutrophils, CD4+
T cells are also recruited to the site of infection due to the upregulation of IL-6 and IL-12 early in infection. The lesion formation and clouding of the eye indicative of stromal keratitis are thought to be primarily due to the actions of CD4+
T cells, particularly the Th1 cell subset; however, recent evidence has shown that the Th17 cell subset plays a role in both the beginning and later phases of immunopathology and that IL-6 and TGF-β are responsible for Th17 cell differentiation through the production of IL-17.7,22–24
The invasion of neutrophils and T cells to the infected area is made easier through VEGF-A–induced neovascularization. In turn, the invading immune cells stimulate further vascularization through VEGF-A production and stimulate continued immune cell recruitment through the release of proinflammatory cytokines, resulting in tissue damage and lesion formation. Treatment with vehicle would flush cytokines from the eye and this might account for the slight effect on disease severity seen in the PBS and tears groups.
Treatment with 10- and 100-fold lower concentrations of TAT-Cd0 did not reduce the severity of ocular disease. The 10-fold lower concentration (0.1 mg/mL) appeared to reduce disease severity somewhat, suggesting a dose–response, but the differences were not significant. Pharmacokinetic studies to determine the amount of TAT-Cd0 in corneal tissue are needed to establish if, at lower doses, the concentrations are sufficient to achieve an antiviral effect.
When we delayed the initiation of TAT-Cd0 treatment by 24 hours, we found that there was no therapeutic effect. This is consistent with the titer data showing that significant reductions in viral titers were achieved only on day 1 postinfection. This suggests that inhibition of viral replication very early in infection interrupts critical events in the pathology. Whether this is due to reductions in the synthesis and release of critical proinflammatory mediators will require further testing.
The blepharitis scores were more severe on days 13 and 15 (P < 0.05) in the mice where treatment was delayed 24 hours. Disease scores were also slightly higher for stromal keratitis (but not significantly different). It is not clear how TAT-Cd0 treatment could lead to enhancement of disease, but this observation emphasizes that treatment should be initiated as soon as possible. At the present time, the possibility that delayed onset of treatment would exacerbate disease, reduces the potential clinical utility of TAT-Cd0.
The eye poses a unique challenge to effective drug delivery. Topical delivery of drugs or other compounds are preferred due to the ease of administration and patient compliance; however, the corneal and conjunctival epithelia serve as a static barrier to topical instillation of compounds, and have lower rates of diffusion compared with other tissues. Dynamic mechanisms in the precorneal tear film can clear the eye of compounds at a rate of 0.5 to 2.2 μL/min, with many topical drop solutions persisting on the cornea for only 5 minutes before being flushed away into the nasolacrimal duct.25–27
Many groups have found that therapeutic concentrations of drugs in the anterior segment of the eye (cornea, anterior chamber, iris, crystalline lens, and ciliary body) are still achievable and that higher concentrations of topically instilled drugs can help to increase the bioavailability of drugs to the anterior segments of the eye.9,25–28
In addition, the viscosity of the drug vehicle has been shown to play a role in increasing residence time on the cornea.29,30
Small proteins and organic solutes can passively diffuse into the cornea and conjunctiva, whereas active transport mechanisms for amino acids, peptides, larger proteins, and other solutes exist in the cornea for a number of different organisms. A number of groups have identified peptides and other small molecules with therapeutic activity when topically applied to the cornea, showing that with some modification to delivery vehicle or formulation, many types of compounds can be effective in treating corneal disease.17,27,28,31–35
A synthetic retrocyclin, RC-2, is a cationic, synthetic θ-defensin (molecular weight: 2041 daltons [Da]) that functions as an inhibitor of HSV infection.36
θ-Defensins are circular octadecapeptides consisting of two antiparallel β-sheets with six cysteines connecting the β-sheets to each other, forming a ladder-like array, and certain θ-defensins have shown activity against multiple viruses in cell culture.36–40
In our murine model, we previously reported that RC-2 had only a modest effect on keratitis when administered postinfection. RC-2 protected mice from infection when incubated with the virus prior to infection. Previous work has shown that θ-defensins act primarily as both attachment and entry inhibitors, with little effect on the virus after entry.36,38,40,41
differs from RC-2 in that TAT-Cd0
predominantly exists as a dimer in solution and through this study has been shown to reduce disease when applied postinfection. In the dimer form, TAT-Cd0
has a molecular weight that is 956 Da greater than that of RC-2 and has 18 positive charges, whereas RC-2 has 5 positive charges and 4 hydrophobic isoleucine residues. Consequently, TAT-Cd0
has a higher charge-to-mass ratio than that of RC-2. Previous work with other derivatives of the TAT peptide showed that antiviral activity decreased if positively charged amino acids were substituted with negatively charged residues14,17
and it is possible that the higher charge-to-mass ratio of TAT-Cd0
contributes to increased efficacy when compared with RC-2. In addition, there may be sequence-specific attributes that distinguish TAT-Cd0
as a more effective antiviral than RC-2 when applied postinfection, and warrants further investigation.
In summary, we have shown that TAT-Cd0 is an effective antiviral when treatment is initiated shortly after infection and identified potential delivery vehicles for TAT-Cd0. Additional studies are needed to define the mechanisms by which TAT-Cd0 inhibits infection and pharmacokinetic studies are needed to determine tissue concentrations and half-life of TAT-Cd0 in the eye. Additional studies are also needed to determine why treating with TAT-Cd0 at 24 hours postinfection was not effective and may, in fact, exacerbate the disease.