Designed Host Defense Peptides for the Treatment of Bacterial Keratitis
L. Edward Clemens, Jesse Jaynes, Alison McDermott, Kathryn Woodburn
Bacterial keratitis must be treated aggressively so as to limit corneal damage and potential loss of vision. Bacterial keratitis is presently treated with antibiotics; however, innovation in antimicrobial agents is needed due to the rapid emergence of bacterial resistance. Designed host defense peptides (dHDPs) are synthetic analogues of naturally occurring HDPs that provide the first line of defense against invading pathogens. These studies identify novel dHDPs for the topical treatment of bacterial keratitis.
Because bacterial eradication is the primary goal of antibiotic treatment with bacterial load the main determinant of therapeutic outcome, the criteria for selecting potential therapeutic agents were rapid bactericidal activity and reduced inflammatory cell recruitment. Four novel dHDPs were identified from in vitro minimum inhibitory concentration (MIC) and time-kill assays that have bactericidal activity against both G+ and G- bacterial strains. The most active dHDP was selected for in vivo evaluation in a murine model of P. aeruginosa induced bacterial keratitis.
In the in vitro time-kill assay, dHDPs were able to reduce bacterial counts by 100% within 30 minutes at 2 µg/ml. Additionally the dHDPs demonstrated preferential destruction of bacterial membranes over eukaryotic membranes in time-kill assays with L929 fibroblasts. The lead dHDP, RP444, demonstrated significant in vivo efficacy in reducing clinical scores in the murine model of bacterial keratitis. A significant dose-dependent decrease in ocular clinical scores was observed on Day 5 post injection with scores of 2.56±0.176, 1.80±0.374, 1.56±0.176, and 1.20±0.200 obtained for the PBS-control, 0.0002%, 0.0064% and 0.064% groups, respectively. In addition to improving the clinical scores RP444 significantly reduced the bacterial load per eye (CFU) and the inflammatory response as measured by corneal MPO (myeloperoxidase) levels.
The topical application of RP444 reduced the severity of P. aeruginosa keratitis in mice as reflected in an improved clinical score, reduced CFU and reduced inflammatory cell infiltration. The proposed membrane disruption reduces the opportunity to induce antibiotic resistance. These studies demonstrate that a novel dHDP may have the potential to treat patients with bacterial keratitis.