The eye is the most sensitive organ to SM,24,25
being irritated by an exposure 10 times less than that which would irritate the respiratory tract (for review, see Smith and Dunn1
). The vesicant causes blistering of skin, whereas microseparations at the BMZ between the epithelium and stroma are observed in the cornea. SM exposure causes a range of injuries to the eye, from mild conjunctivitis to advanced corneal disease. The SM injuries from the Iran-Iraq war of the 1980s recorded severe ocular damage in about 10% of those exposed,6,23,26
and this has emphasized the need for potential therapies. To date, there is no FDA-approved therapy. Further, the ease with which SM can be synthesized suggests that it should be taken very seriously as a potential terrorist agent.
To identify therapies against SM is no minor task. This vesicant cannot be used except in designated restricted facilities in the United States. Using these facilities makes it difficult to do extensive drug screening because animal studies using the vesicant are costly. Because air–liquid interface corneal organ cultures have been used for a variety of experimental purposes,13,16,17,27–31
we adapted an organ culture model system and tested whether it could serve as a preliminary screening method for potential SM therapies. SM exposures cause variable injury according to the exposure time, concentration of the vesicant, and the exposed individual's susceptibility.32–34
By exposing the rabbit corneal organ cultures to CEES and NM, we were able to induce a range of 24-h postexposure injuries at the BMZ that resembled those occurring 24
h after in vivo
exposure of rabbit eyes to SM. Both light and electron microscopic analyses highlighted the similarity of the injury at the BMZ with the 3 vesicants and suggested the organ cultures would serve our purpose. The rabbit corneal organ culture system allowed us to inexpensively test 4 tetracycline derivatives on several corneas for their ability to preserve the BMZ at 24
h after exposure. The most promising candidate was then transitioned to the New Zealand White rabbit eye model.
The decision of what drugs to test were based on data showing MMP activity at the BMZ after injury. Recurrent corneal erosions, where the corneal epithelium is loosely adherent and is able to separate from the stroma, express enhanced levels of MMP-2 and MMP-9.35
Tetracyclines have anti-MMP activity,36–38
and the antibiotic tetracycline derivative, doxycycline, was found to improve the outcome of recurrent corneal erosions.39
Doxycycline also improved the outcome of SM-exposed eyes.15
Therefore, we tested 4 tetracyclines as therapies against vesicants. Three tetracycline derivatives with antibiotic activity and one deaminated tetracycline, DDMT, which does not possess antibiotic activity, but does retain antimetalloproteinase activity,36,40
were employed. The same number of nanomoles of each drug was used in experiments because the goal of this pilot project was to identify the best drug in the group quickly and inexpensively. Our results showed that all of the tetracycline derivatives inhibited the NM-induced expression of corneal epithelial MMP-9. The surprise, however, was that only doxycycline was truly effective at the selected concentration for preserving the BMZ at 24
h after NM exposure. Therefore, doxycycline may not be effective against SM exposure because of its anti-MMP activity, but because of some other, as yet unidentified, activity.
Ocular exposure to SM is very painful and can persist from days to months. We reasoned that patients might not be compliant with delivering a drug dropwise to injured eyes 3–4 times per day. Because of this, we used vesicant-exposed organ cultures to test whether a hydrogel releasing doxycycline over a 24-h period worked as well at preserving the BMZ as dropwise application of the drug and we found that this was the case. Proceeding on to animal testing, hydrogel delivery of doxycycline was compared with dropwise delivery of the drug on rabbit eyes exposed to SM in vivo. Because of the severity of SM compared with CEES and NM, treatment was extended beyond 1 day. Based on the fact that a month is not an unreasonable healing time for a human ocular SM exposure, our experiment ran for 28 days. Our data demonstrated that the hydrogel was effective. Corneal thickness did not decrease as much with the hydrogel as it did with the drops, but by 7 days, animals in both treatment groups had transparent corneas, whereas the untreated SM-exposed corneas were still cloudy.
In SM-exposed humans, the delayed effects (1–2 weeks after exposure) often include neovascularization.41
Our experiments showed this also, detecting neovascularization at 7 and 28 days time points. Interestingly, the hydrogel formulation appeared to reduce the amount of neovascularization, although this must be studied further considering the small number of animals tested. However, the trend is apparent. Also, with our small sample size, it cannot be verified conclusively which delivery method for doxycycline permits a better recovery. Although our results suggest that corneas treated with doxycycline in hydrogels may correct edema more slowly than those treated with doxycycline drops, the reduction of neovascularization may make the hydrogel delivery more desirable. The corneal epithelium has a higher nerve density than skin and lung. A definite advantage of the hydrogel is that the patient needs to apply the treatment to a painful injury only once per day and to the eyelid pocket. Avoiding multiple daily applications should help with patient compliance. These issues need to be addressed in a large-scale study.
Hydrogels have been used in various biomaterial and biotechnology applications such as drug delivery.17,42–44
Our work employing doxycycline in a hydrogel is an attempt to identify a drug and a delivery method that might have a beneficial impact on the chronic effect of conjunctivalization of the cornea after ocular SM exposure. Blindness from conjuctivalization is attributed to limbal stem cell deficiency. When a large region of the corneal epithelium is lost, as in SM exposure, limbal epithelial stem cells must be used to replace it. We hypothesize that by preventing the loss of the entire corneal epithelium in the first 24
h after SM exposure with a drug such as doxycycline, the corneal epithelium has an opportunity to recover in situ
and allows any remaining healthy basal epithelial cells to proliferate and repair the injury. If this is true, doxycycline treatment could prevent the depletion of the limbal stem cells. Ophthalmologists routinely prescribe doxycycline for ocular injuries, although it is not FDA approved for such use. We conclude that doxycycline as drops and hydrogels should be pursued as potential countermeasures against SM exposure. However, hydrogels may prove to be a better therapy because of the lower level of neovascularization. This may be due to the more continuous release of doxycycline over each 24-h period. A combination therapy with doxycycline and an antiangiogenic in a hydrogel also warrants investigation as a potential therapy.