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

磷脂抗代谢物脂醚胺，用于局部治疗慢性伤口和相关生物膜。

• 批准号: 10384660
• 负责人: DANIEL J GIBSON
• 金额: $ 29.97万
• 依托单位: INTEGUMED, LLC
• 依托单位国家: 美国
• 财政年份: 2021
• 资助国家: 美国
• 起止时间: 2021-09-10 至 2023-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10384660
• 关键词: Acinetobacter baumannii; Acute; Affect; Affinity; Alabama; Amines; Amputation; Antibiotic Resistance; Antibiotic Therapy; Antimetabolites; Antimicrobial Effect; Area; Bacteria; Bacterial Adhesins; Bacterial Antibiotic Resistance; Bacterial Infections; Bilateral; Binding; Bioinformatics; Biological Assay; Blood Glucose; Cell membrane; Cells; Clinical; Cluster Analysis; Colony-forming units; Confocal Microscopy; Cutaneous; Data; Dermis; Development; Diabetes Mellitus; Diabetic Foot Ulcer; Diabetic mouse; Diffuse; Docking; Dose; Drug resistance; ESKAPE pathogens; Endotoxins; Enterobacter; Enterococcus faecium; Enzyme-Linked Immunosorbent Assay; Ethers; Exotoxins; Extracellular Matrix; Family suidae; Fluorescence; Formulation; Functional disorder; Goals; Growth; Health; Healthcare; Healthcare Systems; High Pressure Liquid Chromatography; Histology; Human; Image; Impaired wound healing; In Situ; In Vitro; Inflammation; Inflammatory; Insulin; Keratin; Kidney Diseases; Klebsiella pneumoniae; Lead; Ligands; Lipase; Lipids; Lower Extremity; Machine Learning; Mammals; Mass Spectrum Analysis; Membrane; Metabolic dysfunction; Microbial Biofilms; Modeling; Modification; Mus; Mutation; Obesity; Outcome Measure; Pathogenicity; Pathway interactions; Patients; Phase; Phospholipase; Phospholipids; Plasma; Plasmids; Polysaccharides; Property; Proteins; Pseudomonas aeruginosa; Pseudomonas aeruginosa infection; Punch Biopsy; Resistance; Resolution; Safety; Sampling; Signal Transduction; Skin; Skin wound healing; Stains; Staphylococcus aureus; Sterile coverings; Supervision; Systems Biology; Technology; Testing; Therapeutic; Therapeutic Effect; Thick; Topical application; Toxic effect; Universities; Vascular Diseases; Virulence; Virulence Factors; Wound models; amphiphilicity; analog; antimicrobial; bacterial metabolism; bactericide; candidate selection; chronic infection; chronic wound; clinically relevant; comorbidity; conditioning; culture plates; cytokine; cytotoxic; density; diabetic ulcer; high risk; in vivo; in vivo Model; in vivo imaging; involucrin; knock-down; lead candidate; lipid metabolism; lipid nanoparticle; lipidome; lipidomics; metabolomics; microbial; microbicide; migration; mouse model; novel; pathogen; primary outcome; receptor; resistance mechanism; secondary outcome; skin regeneration; standard of care; treatment effect; wound; wound biofilm; wound closure; wound dressing; wound healing; wound treatment

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.

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，并且经常复发 60-70%的患者。这些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合并症。这些伤口经常携带细菌感染， 混合的革兰氏阴性菌（例如铜绿假单胞菌，PA）和革兰氏阳性菌生物膜（例如S.金黄色葡萄球菌，SA），赋予 对抗生素治疗的抗性和抑制消退。由脂质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生理化学屏障和基质。 嗜酸性细菌向不同生物膜状态的转变需要一种合适的底物用于特异性粘附素 受体，如在宿主膜和细胞外基质（ECM）中发现的那些，或非特异性受体， 物理化学结合，使聚集和生物膜ECM组织。这包括以下方面的变化： 微生物的脂质组成，分泌脂质群体信号和脂肪酶毒力因子，并转向氧化 脂质代谢，使持久的殖民和高密度增殖。超高分子量聚乙烯的研究进展 高效液相色谱法、高分辨率质谱法和生物信息学技术 能够详细和准确地定义细菌脂质组，有助于识别脂质途径， 目标的抗微生物活性使用系统生物学方法。 促炎性内源性磷脂酶活性使伤口愈合进一步复杂化。我们提出 脂质醚胺（莱亚）代表了一种用磷脂抗- 代谢物活性，抵消宿主和病原体致病机制。作为初步数据，我们 在多个体外培养的抗生素耐药细胞中显示>3-log（> 1000 x）菌落形成单位减少 生物膜和抗代谢活性的优化。第一阶段将综合其他候选人，筛选 生物膜减少和宿主细胞中的活性，选择导致确定体内伤口愈合效果的平台 使用生物膜感染的db/db糖尿病小鼠模型。LC-MS/MS分析将用于表征细菌或 宿主脂质体和治疗效果。在目标I中，将筛选莱亚候选化合物， 使用临床相关的耐药ESKAPE（E.屎斑S.金黄色葡萄球菌K. pneumoniae、A. 鲍曼不动杆菌、铜绿假单胞菌、肠杆菌属）病原体在培养的生物膜，包括一个可翻译的受伤 离体猪皮外植体模型。在Aim II中，将使用以下方法评估莱亚对迁移和增殖的影响： 体外全层皮肤构造。在目标III中，先导化合物将应用于糖尿病小鼠伤口 感染PA或SA生物膜的愈合模型，以评估体内杀微生物和伤口闭合效果。 在第二阶段，我们将通过混合细菌生物膜的筛选， 使用体外结合和敲低/敲除机制，评估潜在的适应性抗性机制， 开展全面的ADME-T研究，并开始大型哺乳动物体内概念验证，用于IND申报。