In previous papers, we observed that dendrimers of peptide mimotopes of the nicotinic receptor ligand site are strong antidotes against the lethality of the nicotinic receptor ligand alpha-bungarotoxin. Although their in vitro activity is identical to that of dendrimers, the corresponding monomeric peptide mimotopes are not effective in vivo. Because the higher in vivo efficiency of dendrimers could not in this case be related to polyvalent interaction, the stability to blood protease activity of monomeric versus tetrabranched dendrimeric mimotope peptides was compared here by incubating three different mimotopes with human plasma and serum. Unmodified peptides and cleaved sequences were followed by high pressure liquid chromatography and mass spectrometry. Tetrabranched peptides were shown to be much more stable in plasma and also in serum. To assess the notable stability of multimeric peptides, different bioactive neuropeptides, including enkephalins, neurotensin and nociceptin, were synthesized in monomeric and tetrabranched forms and incubated with human plasma and serum and with rat brain membrane extracts. All the tetrabranched neuropeptides fully retained biological activity and generally showed much greater stability to blood and brain protease activity. Some tetrabranched peptides were also resistant to trypsin and chymotrypsin. Our findings provide new insights into the possible therapeutic use of bioactive peptides.

A large 10-mer phage peptide library was panned against whole Escherichia coli cells, and an antimicrobial peptide (QEKIRVRLSA) was selected. The peptide was synthesized in monomeric and dendrimeric tetrabranched form (multiple antigen peptide [MAP]), which generally allows a dramatic increase of peptide stability to peptidases and proteases. The antibacterial activity of the dendrimeric peptide against E. coli was much higher than that of the monomeric form. Modification of the original sequence, by residue substitution or sequence shortening, produced three different MAPs, M4 (QAKIRVRLSA), M5 (KIRVRLSA), and M6 (QKKIRVRLSA) with enhanced stability to natural degradation and antimicrobial activity against a large panel of gram-negative bacteria. The MICs of the most potent peptide, M6, were as low as 4 to 8 μg/ml against recent clinical isolates of multidrug-resistant Pseudomonas aeruginosa and members of the Enterobacteriaceae . The same dendrimeric peptides showed high stability to blood proteases, low hemolytic activity, and low cytotoxic effects on eukaryotic cells, making them promising candidates for the development of new antibacterial drugs.

Introduction: Antibiotic-resistant bacteria kill 25,000 people every year in the EU. Patients subject to recurrent lung infections are the most vulnerable to severe or even lethal infections. For these patients, pulmonary delivery of antibiotics would be advantageous, since inhalation can achieve higher concentration in the lungs than iv administration and can provide a faster onset of action. This would allow for the delivery of higher doses and hence reduce the number of treatments required. We report here about a new nanosystem (M33-NS) obtained by capturing SET-M33 peptide on single-chain dextran nanoparticles. SET-M33 is a non-natural antimicrobial peptide synthesized in branched form. This form gives the peptide resistance to degradation in biological fluids. SET-M33 has previously shown efficacy in vitro against about one hundred of Gram-negative multidrug and extensively drug-resistant clinical isolates and was also active in preclinical infection models of pneumonia, sepsis and skin infections. Methods: The new nanosystem was evaluated for its efficacy in bacteria cells and in a mouse model of pneumonia. Toxicity and genotoxicity were also tested in vitro. Biodistribution and pharmacokinetic studies in healthy rats were carried out using a radiolabeled derivative of the nanosystem. Results: The M33-nanosystem, studied here, showed to be effective against Pseudomonas aeruginosa in time-kill kinetic experiments. Cytotoxicity towards different animal cell lines was acceptable. Lung residence time of the antimicrobial peptide, administered via aerosol in healthy rats, was markedly improved by capturing SET-M33 on dextran nanoparticles. M33-NS was also efficient in eradicating pulmonary infection in a BALB/c mouse model of pneumonia caused by P. aeruginosa. Discussion: This study revealed that the encapsulation of the antimicrobial peptide in dextran nanoparticles markedly improved lung residence time of the peptide administered via aerosol. The result has to be considered among the aims of the development of a new therapeutic option for patients suffering recurrent infections, that will benefit from high local doses of persistent antimicrobials.

The branched M33 antimicrobial peptide was previously shown to be very active against Gram-negative bacterial pathogens, including multidrug-resistant strains. In an attempt to produce back-up molecules, we synthesized an M33 peptide isomer consisting of D-aminoacids (M33-D). This isomeric version showed 4 to 16-fold higher activity against Gram-positive pathogens, including Staphylococcus aureus and Staphylococcus epidermidis, than the original peptide, while retaining strong activity against Gram-negative bacteria. The antimicrobial activity of both peptides was influenced by their differential sensitivity to bacterial proteases. The better activity shown by M33-D against S. aureus compared to M33-L was confirmed in biofilm eradication experiments where M33-L showed 12% activity with respect to M33-D, and in vivo models where Balb-c mice infected with S. aureus showed 100% and 0% survival when treated with M33-D and M33-L, respectively. M33-D appears to be an interesting candidate for the development of novel broad-spectrum antimicrobials active against bacterial pathogens of clinical importance.

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.