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

摘要

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.