The antimicrobial peptide SET-M33: in vivo efficacy and toxicity in different animal species and encapsulation in inhalable polymeric nanoparticles for pulmonary delivery

SET-M33 is a synthetic peptide that is being developed as a new antibiotic against major Gram-negative bacteria. In this Ph.D. thesis the results about in vivo efficacy and toxicity in different animal species are reported, concluding the preclinical development of the peptide. Furthermore, the construction of a biocompatible nanosystem where the antimicrobial peptide SET-M33 is encapsulated into polymeric nanoparticles (NPs) of poly(lactide-co-glycolide) (PLGA) conjugated with polyethylene glycol (PEG) is also described. In the first study SET-M33 was administered to CD-1 mice by snout inhalation exposure for 1 h/day for 7 days at doses of 5 and 20 mg/kg/day. The results showed adverse clinical signs and effects on body weight at the higher dose, as well as some treatment-related histopathology findings (lungs and bronchi, nose/turbinates, larynx and tracheal bifurcation). On this basis, the no observable adverse effect level (NOAEL) was considered to be 5 mg/kg/day. In the same study, an efficacy evaluation of the peptide in an endotoxin (LPS)-induced pulmonary inflammation model. Intratracheal administration of SET-M33 at 0.5, 2 and 5 mg/kg significantly inhibited BAL neutrophil cell counts after an LPS challenge was presented. A significant reduction in pro-inflammatory cytokines, KC, MIP-1α, IP-10, MCP-1 and TNF-α was also recorded after SET-M33 administration. In the second study, dose range finding experiments determined the doses to use in toxicokinetic evaluation, clinical biochemistry analysis, necroscopy and in neurological and respiratory measurements. Clinical laboratory investigations in dogs and rats showed a dose-related increase in creatinine and urea levels, indicating that the kidneys are the target organ. This was also confirmed by necroscopy studies of animal tissues, where signs of degeneration and regeneration were found in the kidney when SET-M33 was administered at the highest doses in the two animal species. Neurological toxicity measurements by the Irwin method and respiratory function evaluation in rats did not reveal any toxic effect even at the highest dose. Finally, repeated administration of SET-M33 by short infusion in dogs revealed a NOAEL of 0.5 mg/kg/day. In the third study, the system peptide-NPs was conceived for the better delivery of the peptide to the lungs by aerosol. The encapsulated peptide showed prolonged antibacterial activity, due to its controlled release, and much lower toxicity than the free molecule. The peptide-based nanosystem killed Pseudomonas aeruginosa in planktonic and sessile forms in a dose-dependent manner, remaining active up to 72 hours after application. The encapsulated peptide showed no detectable cytotoxicity when incubated with human bronchial epithelial cells from healthy individuals and from cystic fibrosis patients, unlike the free peptide which showed an EC50 of about 22 µM. In vivo acute toxicity studies in experimental animals showed that the peptide nanosystem did not cause any appreciable side effects, and confirmed its ability to mitigate the toxic and lethal effects of free SET-M33.