The peptide sequence KKIRVRLSA was synthesized in a dimeric structure (SET-M33DIM) and evaluated as a candidate drug for infections due to multidrug-resistant (MDR) Gram-negative pathogens.SET-M33DIM showed significant antibacterial activity against MDR strains of Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli (Minimal Inhibitory Concentration [MICs], 1.5-11 µM), and less activity against Pseudomonas aeruginosa (MICs, 11-22 µM).It showed very low toxicity in vitro, ex vivo, and in vivo; in cytotoxicity tests, its EC 50 was as much as 22 times better than that of SET-M33, a peptide with the same amino-acid sequence, but synthesized in tetra-branched form (638 vs 28 µM).In in vivo and ex vivo experiments, SET-M33DIM cleared P. aeruginosa infection, significantly reducing signs of sepsis in animals, and restoring cell viability in lung tissue after bacterial challenge.It also quelled inflammation triggered by LPS and live bacterial cells, inhibiting expression of inflammatory mediators in lung tissue, cultured macrophages, and bronchial cells from a cystic fibrosis patient.

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.

The tetra-branched peptide M33 (Pini et al. in FASEB J 24:1015–1022, 2010) is under evaluation in animal models for its activity as antimicrobial agent in lung infections and sepsis. The preclinical development of a new drug requires medium-scale manufacture for tests of efficacy, biodistribution, pharmacokinetics and toxicity. In order to produce the most suitable peptide form for these purposes, we evaluated the behaviour of the peptide M33 obtained with different counter-ions. We compared activity and toxicity in vitro and in vivo of the peptide M33 produced as trifluoroacetate salt (TFacetate) and as acetate salt. The two forms did not differ substantially in terms of efficacy in vitro or in vivo but showed different toxicities for human cells and in animals. M33-TFacetate proved to be 5–30% more toxic than M33-acetate for cells derived from normal bronchi and cells carrying ΔF508 mutation in the CFTR gene, the most frequent variant in cystic fibrosis. M33-TFacetate produced manifest signs of in vivo toxicity immediately after administration, whereas M33-acetate only generated mild signs, which disappeared within a few hours. The peptide M33-acetate proved more suitable for the development of a new drug, and was therefore chosen for further characterization.

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.

We describe the nonnatural antimicrobial peptide KKIRVRLSA (M33) and its capacity to neutralize LPS-induced cytokine release, preventing septic shock in animals infected with bacterial species of clinical interest. M33 showed strong resistance to proteolytic degradation when synthesized in tetra-branched form with 4 peptides linked by a lysine core, making it suitable for use in vivo. HPLC and mass spectrometry demonstrated its stability in serum beyond 24 h. M33 was found to be very selective for gram-negative bacteria. Minimal inhibitory concentration (MIC) ranged from 0.3 to 3 mu M for multidrug resistant clinical isolates of several pathogenic species, including Pseudomonas aeruginosa, Klebsiella pneumoniae, and Acinetobacter baumannii. M33 neutralized LPS derived from P. aeruginosa and K. pneumoniae, and prevented TNF-alpha release from LPS-activated macrophages, with an EC50 of 3.8e-8 M and 2.8e-7 M, respectively, as detected by sandwich ELISA. M33 activity was also tested in sepsis animal models. It averted septic shock symptoms due to Escherichia coli and P. aeruginosa in doses compatible with clinical use (5-25 mg/kg). These properties make tetrabranched M33 peptide a good candidate for the development of a new antibacterial drug.-Pini, A., Falciani, C., Mantengoli, E., Bindi, S., Brunetti, J., Iozzi, S., Rossolini, G. M., Bracci, L. A novel tetrabranched antimicrobial peptide that neutralizes bacterial lipopolysaccharide and prevents septic shock in vivo. FASEB J. 24, 1015-1022 (2010). www.fasebj.org

3982 The finding that receptors for different endogenous regulatory peptides, like the neuropeptide neurotensin (NT), are over-expressed in different human cancers, has opened new perspectives on the use of synthetic peptides for tumor-selective targeting (1). Peptide ligands carrying cytotoxic moieties or radiotracers might act as drugs or diagnostics. Nonetheless, the use of peptides as drugs has largely been limited by their short half-life. Peptides are physiologically hydrolysed by proteases and peptidases and the chemical modifications necessary to stabilize endogenous sequence can deeply modify peptide affinity and specificity. We demonstrated that NT, like several other endogenous peptides, retains its biological activity and becomes resistant to proteolysis when synthesized in a branched form (2). NT receptors are over-expressed in different human tumors with high incidence and mortality rate, like small cell lung cancer, colon, prostate and pancreas carcinomas. For this reason, high affinity and stable NT peptides can have important applications as specific tumor-targeting agents. We synthesized the short functional fragment NT(8-13) in a tetra-branched form (NT4), which was coupled during the synthesis to different units designed for tumor tracing or therapy. Cell internalization of branched NT4 molecules together with NT receptor regulation and trafficking, were followed by confocal microscopy. NT4 conjugated to fluorescent probes was also used for NT receptor tracing in human specimens from colon cancer surgical resections. Comparison with normal tissues from the same patients showed a clear over-expression of NT receptor in a high percentage of tumors even at early differentiation stages. Cytotoxicity of NT4 conjugated to methotrexate or to the photosensitizer chlorine e6, was tested on human colon adenocarcinoma cell lines. In both cases, conjugation to branched NT dramatically decreased non-specific toxicity of the drugs by inducing receptor-specific uptake of the toxic moiety by target cells. NT4 conjugated to methotrexate was also tested in in vivo experiments using HT29 human colon adenocarcinoma xenografts in nude mice. Animals treated with NT4 conjugated to methotrexate showed a clear decrease in tumor growth with respect to untreated mice or mice treated with equimolar amount of the unconjugated drug. Branched NT molecules are good candidates for the development of peptide tumor-targeting agents that might have the dual use of specific NT receptor tracing followed by targeted therapy of NT receptor-expressing tumors. Reubi JC. Peptide receptors as molecular targets for cancer diagnosis and therapy. Endocr Rev. 2003; 24(4): 389-427. Bracci L. et al. Synthetic peptides in the form of dendrimers become resistant to protease activity. J Biol Chem. 2003; 278(47): 46590-5.