Hybrid Derivative of Cathelicidin (LL-37) and Human Beta-Defensin Against Gram-Positive Bacteria: A Novel Approach for the Treatment of Bacterial Keratitis

Abstract

Purpose : Bacterial keratitis (BK) represents a leading cause of corneal blindness worldwide. This study aimed to generate potent hybridized human-derived host defense peptides (HDPs) as novel topical antimicrobial therapy for BK.

Methods : Hybrid peptides were rationally designed through combination of functional amino acids in parent HDPs, including human cathelicidin (LL-37) and human beta-defensin (HBD)-1 to -3. Minimal inhibitory concentrations (MICs) were determined using broth microdilution method, and cytotoxicity was evaluated against human corneal epithelial cells (HCE-2) and human erythrocytes. Time- and concentration-dependent antimicrobial activity was examined using time-kill kinetics assay. In vivo safety and efficacy of the most promising peptide was examined using corneal wound healing and Staphylococcus aureus (ATCC SA29213) keratitis murine models, respectively.

Results : A second-generation hybrid peptide (HDP23) demonstrated good efficacy against methicillin-sensitive and methicillin-resistant S. aureus (MIC=12.5-25.0μg/ml or 5.2-10.4μM) and S. epidermidis (MIC=3.1-12.5μg/ml or 1.3-5.2μM), and moderate efficacy against P. aeruginosa (MIC=50μg/ml or 20.9μM). HDP23 (2x MIC) killed all the bacteria within 30 mins, which was 8 times faster than amikacin (20x MIC). At 200μg/ml (16x MIC), HDP23 was shown to be relatively safe against HCE-2 (<30% toxicity) and erythrocytes (<10% toxicity). Pre-clinical murine studies showed that HDP23 0.05% (500μg/ml) achieved a median reduction of S. aureus bacterial viability by 94% (or 1.2 log₁₀ CFU/ml) while not impeding corneal healing.

Conclusions : Rational modification of human-derived HDPs, via hybridization of LL-37 and HBD, has led to the generation of an efficacious and safe topical antimicrobial agent that has potential for treatment of Gram-positive BK in humans.

This is a 2021 ARVO Annual Meeting abstract.