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Sulfur mustard(SM) is a potent vesicant employed as a chemical weapon in various conflicts during the 20th century (1,2). It functions as a powerful alkylator and highly cytotoxic blisterogen in both humans and animals (1-9). Skin exposed to SM develops erythema within 30 minutes to several hours after exposure followed by edema, vesicle and blister formation, ulceration, necrosis and desquamation (2,7,8,10). The severity of lesions observed after exposure to SM has emphasized the need for an efficient pharmacological antidote against its vesicating activity. The powerful alkylating activity of SM(11-13) results from its conversion, in aqueous solution, to the highly electrophilic ethylene episulfonium derviative (2) which can be neutralized by nucleophilic agents.

Previous studies demonstrated the protective effect of our novel iodine formulation against SM-induced skin lesions. In order to improve the preparation we investigated the inflammatory processes occurring upon SM exposure and particularly those involve cyclooxygenase activity. To evaluate the role of cyclooxygenas-l and -2 (COX-l, COX-2) in sulfur mustard-induced skin toxicity, we applied the agent to the ears of wildtype (WI) and COX-l- and COX-2-deficient mice. In the latter, ear swelling 24 and 48 h after exposure was significantly reduced by 55% and 30%, respectively, compared to WT These findings were confirmed by histopathological evaluation. The COX-2 inhibitor celecoxib resulted in significant reductions of 27% and 28% in ear swelling at intervals of 40 and 60 min between exposure and treatment, respectively. These findings encouraged us to introduce celecoxib, together with the steroidal anti-inflammatory agent clobetasol, into our iodine preparation. After optimization procedures, the ointment was efficacious 60 min (or less) following exposure. The ability of low iodine concentrations to scavenge reactive oxygen species and to inhibit oxidative burst of activated mouse neutropils might explain the protective mechanism of action of iodine. Previous results have shown that prophylactic injection of H2A histone fragment protected against SM-induced skin lesions. In the present report we found that the T1/2 of the peptide in mouse and humans serum was 7 and 14 min. We also identified the cleavage sites of the peptide by mouse and human serum proteinases; and accordingly synthesized N-methylated analogs for stabilizing the peptide against this proteolytic activity. Injection of the peptide 30 min after exposure significantly reduced ear swelling in the mouse edema model., The original document contains color images. All DTIC reproductions will be in black and white.

The present study is a final report of the project. During the project we developed iodine formulation proved to be efficacious against SM in the guinea pig skin model at intervals of 15 and 30 rain between exposure and treatment. Incorporation of the antiinflammatory drug piroxicam and the steroidal antiinflamamtory agent clobetasol, caused the formulation to protect at intervals of 45 and 60 rain in the mouse ear swelling model and at 45 rain in the guinea pig skin irritation test. Introduction of ethanol to the formulation caused the preparation to be efficacious in the pig model; 20 rain interval between exposure and treatment caused significant protection. Longer intervals will be tested in the near future. The topical preparation was also effective against heat- and hydrofluoric acid-induced skin burns. Toxicokinetic studies with male, fur-covered and hairless guinea pigs showed that SM disappeared from the skin 60 rain after exposure whereas in the female, fur-covered guinea pig SM disappeared after 3 hours. Sodium hypochiorite inactivated SM in vitro but not in situ, namely, no significant inactivation by decontaminant applied on SM-exposed skin was observed. Iodine caused reduction in skin collagenase induced by mechlorethamine. COX2-deficient mice were less affected by SM that COX1-deficient end wild type mice. Iodine was also effective against dermally exposed organophosphate (paraoxon). The present study demonstrated that iodine induced skin protective peptides against noxious stimuli. Four peptides were identified and synthesized, of which two had protective activity when injected together with proteinase inhibitors to prevent degradation. Synthesis of N-methylated analogs produced metabolically stable peptides with protective activity against SM-induced skin lesions. Newly synthesized analogs showed protection in the mouse model when injected iv together with proinflammatory chemokine antibodies., Original contains color plates: All DTIC reproductions will be in black and white.