Differential In Vitro and In Vivo Toxicities of Antimicrobial Peptide Prodrugs for Potential Use in Cystic Fibrosis

Abstract

There has been considerable interest in the use of antimicrobial peptides (AMPs) as antimicrobial agents for the treatment of many conditions, including cystic fibrosis (CF). The challenging conditions of the CF patient lung require robust AMPs that are active in an environment of high proteolytic activity but that also have low cytotoxicity and immunogenicity. Previously, we developed prodrugs of AMPs that limited the cytotoxic effects of AMP treatment by rendering the antimicrobial activity dependent on the host enzyme neutrophil elastase (NE). However, cytotoxicity remained an issue. Here, we describe the further optimization of the AMP prodrug (pro-AMP) model for CF to produce pro-WMR, a peptide with greatly reduced cytotoxicity (50% inhibitory concentration against CFBE41o-cells, >300 M) compared to that of the previous group of pro-AMPs. The bactericidal activity of pro-WMR was increased in NE-rich bronchoalveolar lavage (BAL) fluid from CF patients (range, 8.4% ؎ 6.9% alone to 91.5% ؎ 5.8% with BAL fluid; P ‫؍‬ 0.0004), an activity differential greater than that of previous pro-AMPs. In a murine model of lung delivery, the pro-AMP modification reduced host toxicity, with pro-WMR being less toxic than the active peptide. Previously, host toxicity issues have hampered the clinical application of AMPs. However, the development of application-specific AMPs with modifications that minimize toxicity similar to those described here can significantly advance their potential use in patients. The combination of this prodrug strategy with a highly active AMP has the potential to produce new therapeutics for the challenging conditions of the CF patient lung.

The Antimicrobial Peptide lin-SB056-1 and Its Dendrimeric Derivative Prevent Pseudomonas aeruginosa Biofilm Formation in Physiologically Relevant Models of Chronic Infections

“…Although (lin-SB056-1) 2 -K at the active concentrations exhibited a certain degree of cytotoxicity against both erythrocytes and lung epithelial cells, different strategies could be exploited in the attempt to reduce peptide adverse effects against mammalian cells, while maintaining its antimicrobial properties. A number of different approaches have been described to control cell selectivity of AMPs, including modulation of a range of physicochemical parameters (e.g., hydrophobicity, helicity, and amphipathicity), design of pro-peptides, and insertion of D -amino acids and/or non-natural residues in the peptide primary sequence (Matsuzaki, 2009; Takahashi et al, 2010; Forde et al, 2016). Among these strategies, peptide entrapment in suitable nanocarriers able to deliver the active molecules to the infection site, while increasing their stability and minimizing side effects, appears one of the most promising approaches, and is currently under evaluation in our laboratory (Piras et al, 2015; Sandreschi et al, 2016).…”

Host defense antimicrobial peptides as antibiotics: design and application strategies

“…Forde et al described the optimization of the AMP prodrug model for cystic fibrosis (CF) to produce pro-WMR, a peptide with greatly reduced cytotoxicity. The bactericidal activity of pro-WMR is restored in neutrophil elastase-rich bronchoalveolar lavage fluid from the CF patients [44]. …”

Synergies with and Resistance to Membrane-Active Peptides

“…Activation of MAP prodrugs by naturally expressed enzymes is crucial for their activity. The creation or utility of peptide prodrugs is an elegant strategy to evade the off-target MAP associated cytotoxic effects [ 125 , 126 ]. This is a form of naturally occurring synergy; peptide prodrugs and microbial enzymes work synergistically to activate the peptides.…”