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.

抽象的 仅在美国就有超过 250 万名慢性伤口影响患者，平均持续 12-13 个月并反复发作 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。这些伤口经常存在细菌感染，例如 混合革兰氏阴性（例如铜绿假单胞菌，PA）和革兰氏阳性生物膜（例如金黄色葡萄球菌，SA），赋予 对抗生素治疗产生耐药性并抑制消退。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理化屏障和基质。 浮游细菌向独特生物膜状态的转变需要特定粘附素的合适底物 受体，例如在宿主膜和细胞外基质 (ECM) 中发现的受体，或非特异性受体 理化结合，实现聚集和生物膜 ECM 组织。这包括以下方面的变化 微生物脂质组成、脂质群体信号和脂肪酶毒力因子的分泌以及向氧化的转变 脂质代谢以实现持续定植和高密度增殖。超高能领域的最新进展 高效液相色谱、高分辨率质谱和生物信息学技术 能够详细而准确地定义细菌脂质组，有助于识别脂质途径和 使用系统生物学方法确定抗菌活性目标。 促炎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）代表了一种新型局部伤口治疗平台，具有磷脂抗 代谢活性，抵消宿主和病原体的致病机制。作为初步数据，我们 证明多个体外培养的抗生素抗性菌落形成单位减少 >3-log (>1000x) 生物膜和抗代谢活性的优化。第一阶段将综合更多候选者，筛选 宿主细胞中生物膜的减少和活性，选择平台来确定体内伤口愈合效果 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 每个目标中的宿主脂质组和治疗效果。在目标 I 中，将筛选 LEA 候选化合物 使用临床相关的耐药 ESKAPE（屎肠球菌、金黄色葡萄球菌、肺炎克雷伯菌、金黄色葡萄球菌、肺炎克雷伯菌、 鲍曼不动杆菌、铜绿假单胞菌、肠杆菌属）培养生物膜中的病原体，包括可翻译的伤口 离体猪皮肤外植体模型。在目标 II 中，将使用以下方法评估 LEA 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，以评估体内杀菌和伤口闭合效果。 在第二阶段，我们将通过接种混合细菌生物膜来提高体内模型的复杂性，完善 使用体外结合和击倒/淘汰机制，评估潜在的适应性耐药机制， 进行全面的 ADME-T 研究，并开始大型哺乳动物体内概念验证以提交 IND。