Novel Therapy for Chronic Non-healing Wounds

描述(由申请人提供)：摘要：由于糖尿病和代谢综合征日益流行，目前估计有2000万美国人(约占总人口的7%！)受到影响，因此迫切需要解决与其相关的并发症。伤口修复受损是血糖控制不佳的主要并发症，每年要花费100亿美元。本实验室首先鉴定并克隆了兔(Larrick et al.，1991)阳离子抗微生物蛋白(HCAP18)，然后根据其与内毒素的结合和抗微生物活性鉴定并克隆了人阳离子抗微生物蛋白(HCAP18)。在过去的10年里，hCAP18已被证明是天然免疫系统的重要组成部分，其C末端片段LL-37具有广泛的抗微生物活性。HCAP18在白细胞中有成分地产生，在炎症和感染时在屏障器官(如肺、胃肠道、皮肤)中被诱导。最近的研究表明，hCAP18在皮肤创伤的血管形成和再上皮化中起着关键作用。伤后皮肤中hCAP18水平较高，伤后48h达高峰，闭合后降至伤前水平。HCAP18在炎性浸润液和移行于伤口床上的上皮细胞中均可检测到。例如，使用由器官培养的人皮肤组成的非炎性体外伤口愈合模型，海尔布伦等人。(2003)显示，用抗LL-37抗体治疗抑制了再上皮化。在慢性不可愈合创面中，hCAP18水平较低，溃疡边缘上皮无hCAP18/LL-37免疫反应。我们推测，IL-37在伤口闭合中起着关键作用，在慢性伤口中它的缺失会损害再上皮化，促进细菌定植，并导致伤口无法愈合。因此，这项提案的总体目标是开发一种具有蛋白酶抗性的LL-37形式，作为一种治疗慢性、不可愈合伤口的新方法。为此，在第一阶段，我们将制备PrLL-37，一种由D-氨基酸组成的抗蛋白酶形式，展示对蛋白酶降解的抗性，并在使用糖尿病小鼠的伤口愈合模型中评估PrLL-37的活性。在第二阶段，我们将开展临床前研究，以支持IND的提交。这种疗法将解决烧伤和慢性无法愈合、糖尿病足、卧位症和静脉淤积性溃疡患者的主要未得到满足的需求。摘要：随着糖尿病和代谢综合征日益流行，目前估计有2000万美国人(约占总人口的7%！)受到影响，因此迫切需要解决与其相关的并发症。伤口修复受损是血糖控制不佳的主要并发症，每年要花费100亿美元。本实验室首先鉴定并克隆了兔(Larrick et al.，1991)阳离子抗微生物蛋白(HCAP18)，然后根据其与内毒素的结合和抗微生物活性鉴定并克隆了人阳离子抗微生物蛋白(HCAP18)。在过去的10年里，hCAP18已被证明是天然免疫系统的重要组成部分，其C末端片段LL-37具有广泛的抗微生物活性。HCAP18在白细胞中有成分地产生，在炎症和感染时在屏障器官(如肺、胃肠道、皮肤)中被诱导。最近的研究表明，hCAP18在皮肤创伤的血管形成和再上皮化中起着关键作用。伤后皮肤中hCAP18水平较高，伤后48h达高峰，闭合后降至伤前水平。HCAP18在炎性浸润液和移行于伤口床上的上皮细胞中均可检测到。例如，使用由器官培养的人皮肤组成的非炎性体外伤口愈合模型，海尔布伦等人。(2003)显示，用抗LL-37抗体治疗抑制了再上皮化。在慢性不可愈合创面中，hCAP18水平较低，溃疡边缘上皮无hCAP18/LL-37免疫反应。我们推测，IL-37在伤口闭合中起着关键作用，在慢性伤口中它的缺失会损害再上皮化，促进细菌定植，并导致伤口无法愈合。因此，这项提案的总体目标是开发一种具有蛋白酶抗性的LL-37形式，作为一种治疗慢性、不可愈合伤口的新方法。为此，在第一阶段，我们将制备PrLL-37，一种由D-氨基酸组成的抗蛋白酶形式，展示对蛋白酶降解的抗性，并在使用糖尿病小鼠的伤口愈合模型中评估PrLL-37的活性。在第二阶段，我们将开展临床前研究，以支持IND的提交。这种疗法将解决烧伤和慢性无法愈合、糖尿病足、卧位症和静脉淤积性溃疡患者的主要未得到满足的需求。

DESCRIPTION (provided by applicant): 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. 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.