A significant need exists to address complications associated with the growing pandemic of diabetes mellitus and metabolic syndrome now estimated to affect >20 million people in the United States (~7% of the population!). Impaired wound repair, a major complication of poor glycemic control, costs >$10 billion annually. Our laboratory first identified and cloned rabbit (Larrick et al., 1991) and then human Cationic Anti- microbial Protein (hCAP18) based on its LPS binding and anti-microbial activities. Over the past 10 years hCAP18 has been shown to be an important component of the innate immune system with broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs (e.g. lung, GI, skin) upon inflammation and infection. Recent work demonstrated a key role of hCAP18 in vascularization and re-epithelialization of skin wounds. High levels of hCAP18 are produced in skin in vivo upon wounding with a peak at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. For example, using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, Heilbron et al. (2003) showed that treatment with anti-LL-37 antibodies inhibits re-epithelialization. In chronic non-healing wounds, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. We hypothesize that LL-37 plays a critical role in wound closure and that its absence in chronic wounds impairs re-epithelialization, promotes bacterial colonization and contributes to failure of the wounds to heal. Hence, the overall goal of this proposal is to develop a protease resistance form of LL-37 as a novel therapy for chronic, non-healing wounds. To this end in phase I we will prepare prLL-37, a protease resistant form comprised of D-amino acids, demonstrate resistance to degradation by proteases, and evaluate activity of prLL-37 in a wound healing model using diabetic mice. In phase II we will carry out preclinical studies to support submission of an IND. This therapy will address a major unmet need among patients suffering from burn injuries and chronic non-healing, diabetic foot, decubitus and venous stasis ulcers. ABSTRACT: A significant need exists to address complications associated with the growing pandemic of diabetes mellitus and metabolic syndrome now estimated to affect >20 million people in the United States (~7% of the population!). Impaired wound repair, a major complication of poor glycemic control, costs >$10 billion annually. Our laboratory first identified and cloned rabbit (Larrick et al., 1991) and then human Cationic Anti- microbial Protein (hCAP18) based on its LPS binding and anti-microbial activities. Over the past 10 years hCAP18 has been shown to be an important component of the innate immune system with broad anti-microbial activity conferred by its C-terminal fragment LL-37. hCAP18 is constitutively produced in leukocytes and is induced in barrier organs (e.g. lung, GI, skin) upon inflammation and infection. Recent work demonstrated a key role of hCAP18 in vascularization and re-epithelialization of skin wounds. High levels of hCAP18 are produced in skin in vivo upon wounding with a peak at 48 h post-injury, declining to pre-injury levels upon wound closure. hCAP18 is detected in the inflammatory infiltrate and in the epithelium migrating over the wound bed. For example, using a noninflammatory ex vivo wound healing model, composed of organ-cultured human skin, Heilbron et al. (2003) showed that treatment with anti-LL-37 antibodies inhibits re-epithelialization. In chronic non-healing wounds, hCAP18 levels are low and immunoreactivity for hCAP18/LL-37 is absent in ulcer edge epithelium. We hypothesize that LL-37 plays a critical role in wound closure and that its absence in chronic wounds impairs re-epithelialization, promotes bacterial colonization and contributes to failure of the wounds to heal. Hence, the overall goal of this proposal is to develop a protease resistance form of LL-37 as a novel therapy for chronic, non-healing wounds. To this end in phase I we will prepare prLL-37, a protease resistant form comprised of D-amino acids, demonstrate resistance to degradation by proteases, and evaluate activity of prLL-37 in a wound healing model using diabetic mice. In phase II we will carry out preclinical studies to support submission of an IND. This therapy will address a major unmet need among patients suffering from burn injuries and chronic non-healing, diabetic foot, decubitus and venous stasis ulcers. ? ? ?
