PROJECT SUMMARY Mustard gas causes severe epithelial and deep tissue injury resulting in blisters, pain, delayed wound healing, and scarring. The complex mechanism of tissue damage following nitrogen mustard (NM) and sulfur mustard (SM) exposure involves an initial phase of rapid damage. This phase is characterized by acute damage to epidermal keratinocytes and to the basement membrane attaching the top layers of the skin with the underlying dermis. This initiates a cascade of reactions including the release of inflammatory cytokines that signal for the migration of immune cells into the skin from the circulation. The activity of tissue-infiltrating immune cells constitutes a secondary ‘hit’ to the initial, chemical-induced burn. This culminates in prolonged skin inflammation leading to protracted healing and scarring. In a clinical trial, we acquired skin tissue from human subjects experimentally exposed to NM. From those studies we collected ‘omics’ data, which we have utilized to guide experimental models and translationally relevant milestones in the development of therapeutic strategies. In preliminary studies, we hypothesized that the skin’s response to NM would be blunted via the topical application of a persistent, stable radical scavenger in the form of a synthetic mimetic of human eumelanin. That is, by mimicking the natural function of melanin as a skin protective antioxidant, we would set the wound site on a path towards reparative healing. Indeed, in studies in mice and in human skin explants, we observed that topically applied synthetic melanin particles (SMPs) significantly increase the rate of repair. Therefore, in the UG3 phase, we will leverage our experience with mustard and our abundant access to fresh human skin tissue, to evaluate a select group of SMPs for their ability to mitigate the initial injury and to accelerate wound healing. The first selection criterion will be the ability of a given SMP to prevent sub-epidermal separation (blistering) in human skin explants. The second criterion is mitigation of keratinocyte necrosis. The third is a demonstration of inhibition of key biomarkers of epidermal damage. These criteria, tested in human skin explants, will lead to the advancement of six candidates for in vivo evaluation in our established NM mouse model. Here, the first criterion is a demonstration of reduced wound area size (early-stage) and rapid re-epithelialization (late-stage). Next, a successful SMP will lead to reduced scar formation and inhibition of hallmark skin damaging factors. This algorithm will lead to endpoint-driven prioritization and selection of two SMPs; a lead and back-up candidate to bring forward to the next phase. In the UH3 phase, we will evaluate the two SMPs in a NM porcine model. This model is adapted from FDA-enabling studies that includes a demonstration of in vivo 21-day wound reduction coupled with assessment using a 15-point histology grading instrument. Studies in this phase will involve...