Abstract
Purpose :
Available treatments for bacterial keratitis which include antibiotic eye drops and topical corticosteroids have many limitations due to the lack of efficacy of these treatments and the risk for serious complications. The most serious threat to a corneal abrasion or injury is an infection caused by multi-drug resistant (MDR) bacteria that do not respond to many of the currently available antibiotics. Our overall goal is to develop ocular peptide therapeutics that will be effective against antibiotic resistant pathogens that are the causative agents of keratitis. Here, we characterize a peptide derived from the defense protein CAP37 for antimicrobial, anti-inflammatory, and corneal wound-healing activities.
Methods :
Antibacterial activity against MDR bacteria was determined for peptide alone, and peptide combined with antibiotic partners, in vitro using a checkerboard assay and in a mouse model of bacterial keratitis. Anti-inflammatory activity of the peptide was determined in vitro using the HEK-Blue hTLR4 reporter cell line, and in a mouse model of corneal abrasion. Wound healing activity was also determined in the corneal abrasion model. Cytotoxicity was determined by measuring red blood cell hemolysis.
Results :
Minimum bactericidal concentrations (MBCs) of peptide determined in vitro, were between 12.5 and >50 μg/ml, depending on the pathogen. The panel included both Gram positive and Gram negative organisms. The combination studies of peptide with standard of care of antibiotics demonstrated that significant potentiation occurred when the peptide was combined with a fluoroquinolone and a cephalosporin. Further corroboration of these findings was observed in our mouse studies, where this peptide potentiated the killing of MDR Pseudomonas aeruginosa by ofloxacin. In cells, the peptide inhibited activation of TLR4 by S100A9, a pro-inflammatory mediator strongly up regulated in the cornea by injury and infection. In a mouse model of corneal abrasion, the peptide inhibited induction of the neutrophil chemokine C-X-C motif ligand 1 (CXCL1), and accelerated wound healing. Finally, this peptide had low cytotoxicity (<2% red blood cell hemolysis) at doses up to 1 mg/ml.
Conclusions :
This study uncovers an innovative research path to combat multi-drug resistant infections. We are developing this peptide as a new product with a unique set of effects for the rapid resolution of vision-threatening bacterial keratitis.
This is a 2020 ARVO Annual Meeting abstract.