Phospholipid antimetabolite lipid ether amines for topical treatment of chronic wounds and associated biofilms.

摘要 仅在美国，慢性伤口就影响了超过250万名患者，平均持续12-13个月，并反复出现 在60%-70%的患者中。这些疾病主要发生在下肢和血管疾病、糖尿病、肾病、 和代谢功能障碍是常见的并存疾病。这些伤口经常有细菌感染，因为 混合革兰氏阴性菌(如铜绿假单胞菌，PA)和革兰氏阳性生物膜(如金黄色葡萄球菌，SA)， 对抗生素治疗的耐药性和抑制消退。由脂类组成的异质生物膜屏障， 蛋白质和多糖为持续感染提供了强大的物理化学屏障和底物。 浮游细菌向不同生物膜状态的转变需要特定粘附素的合适底物 受体，如在宿主膜和细胞外基质(ECM)中发现的那些，或非特异性的 物理化学结合，使聚集和生物膜ECM组织。这包括在 微生物的脂质组成、分泌的脂质定额信号和脂肪酶毒力因子，以及向氧化转变 脂代谢，以实现持久的定植和高密度增殖。超高功率激光技术的最新进展 高效液相色谱、高分辨率质谱学和生物信息学技术 能够对细菌脂体进行详细和准确的定义，帮助识别脂质途径和 使用系统生物学方法的抗微生物活性目标。 促炎症的内源性磷脂酶活性使伤口愈合进一步复杂化。我们建议 脂醚胺(LEA)代表了一种新型磷脂抗创伤局部治疗的平台 代谢产物活性，对抗寄主和病原体的致病机制。作为初步数据，我们 证明&gt；3-log(&gt；1000x)集落形成单位在多个体外培养的抗生素耐药的情况下减少 生物被膜与抗代谢活性的优化。第一阶段将综合其他候选人，筛选 生物膜在宿主细胞中的减少和活性，选择一个平台来确定体内伤口愈合的效果 采用生物被膜感染db/db糖尿病小鼠模型。将使用LC-MS/MS分析来表征细菌或 各靶点的宿主脂体及治疗效果。在AIM I中，将筛选LEA候选化合物 使用临床相关的、耐药的ESKAPE(粪肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、A. 鲍曼氏杆菌、铜绿假单胞菌、肠杆菌属培养生物膜中的病原体，包括可翻译的创伤 体外猪皮肤移植模型。在AIM II中，LEA对移徙和扩散的影响将使用 体外全厚皮肤构造。在AIM III中，先导化合物将应用于糖尿病小鼠的伤口 建立感染PA或SA生物膜的愈合模型，评价体内杀菌及创面闭合效果。 在第二阶段，我们将通过接种混合细菌生物膜来升级体内模型的复杂性，提炼 利用体外结合和敲除/敲除机制，评估潜在的适应性抗性机制， 进行全面的ADME-T研究，并开始在体内验证大型哺乳动物提交IND的概念。

ABSTRACT Chronic wounds affect over 2.5 million patients in the US alone, lasting on average 12-13 months and recurring in 60-70% of patients. These occur primarily in lower extremities and vascular disease, diabetes, nephropathy, and metabolic dysfunction are prevalent co-morbidities. These wounds frequently harbor bacterial infections as mixed Gram-negative (e.g. P. aeruginosa, PA) and Gram-positive biofilms (e.g. S. aureus, SA), conferring resistance to antibiotic therapy and inhibiting resolution. The heterogenous biofilm barrier, composed of lipids, proteins, and polysaccharides, presents a robust physiochemical barrier and substrate for persistent infection. Transition of planktonic bacteria to the distinct biofilm state requires a suitable substrate for specific adhesin receptors, such as those found in host membranes and extracellular matrix (ECM), or nonspecific physiochemical binding, enabling aggregation and biofilm ECM organization. This includes changes in microbial lipid composition, secretion of lipid quorum signals and lipase virulence factors, and shift to oxidative lipid metabolism to enable persistent colonization and high-density proliferation. Recent advances in ultra-high- performance liquid chromatography, high resolution mass spectrometry, and bioinformatics technologies have enabled detailed and accurate definition of the bacterial lipidome, aiding in identification of lipid pathways and targets for antimicrobial activity using a systems biology approach. Wound healing is further complicated by pro-inflammatory endogenous phospholipase activities. We propose that lipid ether amines (LEA) represent a platform for novel topical wound treatments with phospholipid anti- metabolite activity, counteracting both host and pathogen pathogenic mechanisms. As preliminary data, we demonstrate >3-log (>1000x) colony forming unit reductions in multiple in vitro cultured, antibiotic resistant biofilms and optimization of anti-metabolite activity. Phase I will synthesize additional candidates, screen for biofilm reduction and activity in host cells, selecting a platform lead to determine in vivo wound healing effects using biofilm infected db/db diabetic mouse model. LC-MS/MS analysis will be used to characterize bacterial or host lipidomes and treatment effect in each Aim. In Aim I, LEA candidate compounds will be screened for antimicrobial effect using clinically relevant, drug resistant ESKAPE (E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa, Enterobacter spp.) pathogens in cultured biofilms, including a translatable wounded ex vivo pig skin explant model. In Aim II, LEA effects on migration and proliferation will be assessed using in vitro full thickness skin construct. In Aim III, the lead compound will be applied in a diabetic mouse wound healing model infected with PA or SA biofilms, to assess in vivo microbicidal and wound closure effects. In Phase II, we will escalate in vivo model complexity by inoculating with mixed bacterial biofilms, refine mechanism using in vitro binding and knock-down/out, evaluate potential adaptive resistance mechanisms, conduct comprehensive ADME-T study, and begin in vivo large mammal Proof of Concept for IND submission.