In vitro and in vivo efficacy, toxicity, bio-distribution and resistance selection of a novel antibacterial drug candidate

Abstract

A synthetic antimicrobial peptide was identified as a possible candidate for the development of a new antibacterial drug. The peptide, SET-M33L, showed a MIC90 below 1.5 μM and 3 μM for Pseudomonas aeruginosa and Klebsiella pneumoniae, respectively. In in vivo models of P. aeruginosa infections, the peptide and its pegylated form (SET-M33L-PEG) enabled a survival percentage of 60-80% in sepsis and lung infections when injected twice i.v. at 5 mg/Kg, and completely healed skin infections when administered topically. Plasma clearance showed different kinetics for SET-M33L and SET-M33L-PEG, the latter having greater persistence two hours after injection. Bio-distribution in organs did not show significant differences in uptake of the two peptides. Unlike colistin, SET-M33L did not select resistant mutants in bacterial cultures and also proved non genotoxic and to have much lower in vivo toxicity than antimicrobial peptides already used in clinical practice. The characterizations reported here are part of a preclinical development plan that should bring the molecule to clinical trial in the next few years.

Figure 1. In vivo antibacterial activity of SET-M33L and SET-M33L-PEG peptides in sepsis animal model.

10 BALB/c neutropenic mice/group were injected i.p. with a lethal amount of P. aeruginosa PAO1 and then treated twice i.v. with SET-M33L or SET-M33-PEG (5 mg/Kg), 24 and 72 hours post-infection. Percentage survival (y-axis) is plotted as a function of time (x-axis); p < 0.02.

Figure 2. In vivo antibacterial activity of SET-M33L and SET-M33L-PEG peptides in lung infection.

(A) Survival of neutropenic BALB/c mice after peptide treatment. All animals were injected i.t. with a lethal amount of P. aeruginosa PAO1. One group of animals was treated i.v. with 5 mg/Kg SET-M33L and one group with 5 mg/Kg SET-M33L-PEG, 1 and 16 hours post-infection. The control group (CTR) only received vehicle. The groups are indicated as described in the internal legend. Percentage survival (y-axis) is plotted as a function of time (x-axis); p < 0.05. (B) Scatter plots representing the CFUs/lung (y-axis) in treated and untreated non-neutropenic mice (each circle corresponds to one mice). All animals were injected i.t. with P. aeruginosa PAO1. One group was treated i.t. with a single 5 mg/Kg dose of SET-M33L (white circles). The control group only received vehicle (black circles). The horizontal lines represent the median and the difference between medians is indicated as a percentage. p = 0.05. There were 10 mice/group.

Figure 3. In vivo antibacterial activity of SET-M33L peptide in skin infection.

15 neutropenic mice per group (BALB/c) were infected on abraded skin with P. aeruginosa P1242 and then treated every day with 10 mg/ml SET-M33L-lotion (SET-M33L) or with SET-M33L free-lotion (CTR). (A) Example of images of five animals at day 2. (B) Scatter plots representing the photon flux per second (p/s) emitted by bacteria at day 2 from all mice (each circle corresponds to one animal). The horizontal lines indicate the median value. p < 0.03.

Figure 4. Acute in vivo toxicity of SET-M33L, SET-M33L-PEG and colistin at 40 mg/Kg, 20 mg/Kg and 10 mg/Kg, given in a single dose.

Ten mice/group (each circle represents one mouse) were inoculated i.v. with SET-M33L, SET-M33L-PEG or colistin and were monitored for 96 hours. Different scales of grey indicate severity of signs as described in the legend. Toxicity scores were rated as described in Material and Methods.

Figure 5. Accumulation of [¹²⁵I]SET-M33L and [¹²⁵I]SET-M33L-PEG, expressed as % of injected dose per gram of tissue (%ID/g) into the different organs and plasma after intravenous administration and organ dissection or plasma collection.

(A–G) Peptide bio-distribution in organs indicated above each panel. The Y axis scale is different in every graph. (H) Plasma clearance. Fitted curves are exponential for SET-M33L and bi-exponential for SET-M33L-PEG. The graph was obtained using GraphPad Prism software.

Figure 6. In vivo dose-response activity of SET-M33L.

5 BALB/c mice per group were injected i.p. with 1 × 10⁷ CFU/mouse of P. aeruginosa PAO1 and after 15 minutes the antimicrobial peptide SET-M33L was inoculated i.p. at different doses (0.03–1.2 mg/Kg). Five hours after bacterial challenge, peritoneal fluid was analyzed for bacteria and CFU count (y-axis). The scales of the axes are logarithmic.