Characterization of the in vitro, ex vivo, and in vivo Efficacy of the Antimicrobial Peptide DPK-060 Used for Topical Treatment

Abstract

Antimicrobial peptides, also known as host defense peptides, have recently emerged as a promising new category of therapeutic agents for the treatment of infectious diseases. This study evaluated the preclinical in vitro, ex vivo, and in vivo antimicrobial activity, as well as the potential to cause skin irritation, of human kininogen-derived antimicrobial peptide DPK-060 in different formulations designed for topical delivery. We found that DPK-060 formulated in acetate buffer or poloxamer gel caused a marked reduction of bacterial counts of Staphylococcus aureus in vitro (minimum microbicidal concentration <5 μg/ml). We also found that DPK-060 in poloxamer gel significantly suppressed microbial survival in an ex vivo wound infection model using pig skin and in an in vivo mouse model of surgical site infection (≥99 or ≥94% reduction in bacterial counts was achieved with 1% DPK-060 at 4 h post-treatment, respectively). Encapsulation of DPK-060 in different types of lipid nanocapsules or cubosomes did not improve the bactericidal potential of the peptide under the applied test conditions. No reduction in cell viability was observed in response to administration of DPK-060 in any of the formulations tested. In conclusion, the present study confirms that DPK-060 has the potential to be an effective and safe drug candidate for the topical treatment of microbial infections; however, adsorption of the peptide to nanocarriers failed to show any additional benefits.

Keywords: DPK-060; antimicrobial peptides; cubosomes; lipid nanocapsules; skin infections.

Figure 1

Schematic presentation of the experimental design of the study. LNC/ML-LNC particles were constructed and DPK-060 was adsorbed to the particle surface; DPK060-loaded LNCs/ML-LNCs were then added to the poloxamer gel. A cubic liquid crystalline gel was formed by mixing glycerol monooleate with DPK-060; cubosomes were then prepared by dispersing the cubic gel in poloxamer solution. In most experiments, DPK-060 dissolved in poloxamer gel was included as a reference group. The in vitro/ex vivo/in vivo antimicrobial efficacy of the formulations was characterized, and the release profile and skin irritancy were assessed. GMO, glycerol monooleate.

Figure 2

Time-kill assay for DPK-060 formulated in acetate buffer (A), poloxamer gel (B), or in different nanocarriers in poloxamer gel (C–E) against S. aureus. Data are mean ± SD (n = 3). Samples were considered as bactericidal if there was at least 3 log reduction in the CFU number compared with the starting inoculum.

Figure 3

The release of DPK-060 from the formulations after different storage times (0, 7, or 14 days) investigated by dialysis method. Data are mean ± SD (n = 2).

Figure 4

The release of DPK-060 investigated by Franz diffusion cell studies. Data are mean ± SD (n = 2).

Figure 5

Antibacterial effect of DPK-060 formulated in poloxamer gel, or in different nanocarriers in poloxamer gel, in an ex vivo wound infection model using pig skin. The bacterial survival was assessed 4 h (A) or 24 h (B) after the treatment. Schematic presentation of the experimental setup is shown. Data on bacterial survival of S. aureus are mean ± SEM (n = 4–5 wounds per treatment group). ^** P < 0.01 (only the comparisons with the corresponding vehicle are shown). Vh, vehicle.

Figure 6

Antibacterial effect of DPK-060 formulated in poloxamer gel, or in different nanocarriers in poloxamer gel, in an in vivo mouse model of surgical site infection. Schematic presentation of the experimental setup is shown. Results are presented as average relative bacterial survival (%) of S. aureus compared to sham ± SEM (n = 5 mice per each treatment group). ^*P < 0.05 (only the comparisons with the corresponding vehicle are shown). Vh, vehicle.