Mitigation of nitrogen mustard mediated skin injury by a novel indomethacin bifunctional prodrug.

Nitrogen mustard (NM) is a bifunctional alkylating agent that is highly reactive in the skin causing extensive tissue damage and blistering. In the present studies, a modified cutaneous murine patch model was developed to characterize NM-induced injury and to evaluate the efficacy of an indomethacin pro-drug in mitigating toxicity. NM (20µmol) or vehicle control was applied onto 6mm glass microfiber filters affixed to the shaved dorsal skin of CD-1 mice for 6min. This resulted in absorption of approximately 4µmol of NM. NM caused localized skin damage within 1 d, progressing to an eschar within 2-3 d, followed by wound healing after 4-5 d. NM-induced injury was associated with increases in skin thickness, inflammatory cell infiltration, reduced numbers of sebocytes, basal keratinocyte double stranded DNA breaks, as measured by phospho-histone 2A.X expression, mast cell degranulation and increases in inducible nitric oxide synthase (iNOS) and cyclooxygenase-2 (COX-2). Wound healing was characterized by epidermal hyperplasia and marked increases in basal cells expressing proliferating cell nuclear antigen. A novel indomethacin-anticholinergic prodrug (4338) designed to target cyclooxygenases and acetylcholinesterase (AChE), was found to markedly suppress NM toxicity, decreasing wound thickness and eschar formation. The prodrug also inhibited mast cell degranulation, suppressed keratinocyte expression of iNOS and COX-2, as well as markers of epidermal proliferation. These findings indicate that a novel bifunctional pro-drug is effective in limiting NM mediated dermal injury. Moreover, our newly developed cutaneous patch model is a sensitive and reproducible method to assess the mechanism of action of countermeasures.