Antimicrobial peptides

Abstract

The invention relates to an antimicrobial peptide consisting of the amino acid sequence: RX1WILX2WLRTWX3X4 wherein X1, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or solvate thereof. The use of said antimicrobial peptide in the treatment of an infection caused by Gram negative bacteria, Gram positive bacteria, fungi, and / or yeasts, as well as the use of said peptide as an antimicrobial agent for the prevention of contamination of an article or a living tissue by and / or for the decontamination of an article or a living tissue from Gram negative bacteria, Gram positive bacteria, fungi, and / or yeasts are further aspects of the invention.

Description

[0001] Antimicrobial peptides

[0002] Field of the invention

[0003] The present invention relates to antimicrobial peptides.

[0004] State of the art

[0005] The treatment of bacterial infections with antibiotic therapies has been one of the foundations of medicine since the discovery of penicillin to the present day. Unfortunately, the effectiveness of many antibiotics is declining due to increased resistance of some bacterial strains, linked to an improper and excessive use of these drugs, and decreased success in the search for new antibiotics. Today, infectious diseases are the second cause of death worldwide and the largest cause of premature deaths and loss of work productivity in industrialized countries. Some studies show that by 2050, deaths caused by bacterial infections, particularly by antibiotic-resistant strains, could exceed 10 million per year, exceeding the number of deaths from cancer (O’Neill, J. (2016) Tackling Drug-Resistant Infections Globally: Final Report and Recommendations. Review on Antimicrobial Resistance. Wellcome Trust and HM Government).

[0006] A major limitation to the development of antibiotics is the difficulty of finding new molecules having properties equivalent to conventional antibiotics, namely low host toxicity and broad spectrum of action against pathogens.

[0007] A promising type of compounds consists of antimicrobial peptides, i.e., a group of molecules active in the innate immune response that constitutes the first line of defence against pathogens (Alberts B, Johnson A, Lewis J, et al. Innate immunity, in Molecular Biology of the Cell. 4thedition. New York: Garland Science; 2002). These molecules, also known by the acronym AMPs (“AntiMicrobial Peptides”), are produced in many tissues and cell types of organisms such as bacteria, plants, insects, amphibians, and in general, all higher organisms. Their amino acid composition and structure-related chemical-physical characteristics allow them to selectively interact with the lipid bilayer of the bacterial membrane, thereby causing the death of microorganisms.

[0008] Antimicrobial peptides appear to have high potential for activity on many bacterial strains pathogenic for humans, both Gram negative and Gram positive; furthermore, these peptides, unlike the drugs currently in use, do not easily select resistant mutants and therefore do not induce antibiotic-resistance phenomena; finally, they exhibit a synergistic effect with conventional antibiotics and can often activate the host's innate immune response without showing immunogenicity (Browne, K.; Chakraborty, S.; Chen, R.; Willcox, M.D.; Black, D.S.; Walsh, W.R.; Kumar, N. A New Era of Antibiotics: The Clinical Potential of Antimicrobial Peptides. Int. J. Mol. Sci. 2020, 21, 7047. https: / / doi.org / 10.3390 / ijms21197047).

[0009] In recent years, numerous antimicrobial peptides have been identified through various techniques ranging from in silico analysis to peptide library screening.

[0010] In particular, patent application WO2015 / 038339 describes the structure of 753 peptides of 7 to 12 amino acids in length, which are indicated as having an anti-biofilm and / or immunomodulatory activity. Some of the peptides described therein were tested for their ability to inhibit biofilms formed by Klebsiella pneumoniae, Staphylococcus aureus, E. coli, Acinetobacter baumannii, Salmonella enterica ssp. Typhimurium, Burkholderia cenocepacia, respectively. The decapeptide named HE10 is described in this context (SEQ ID NO: 33 in WO2015 / 038339).

[0011] Patent application W02019 / 012158 discloses the use of some antimicrobial peptides, including IDR-1018-K6, as antibacterial agents for the prevention and / or treatment of contamination of a product or surface by a specific bacterium, Listeria monocytogenes,' this document further discloses the use of the peptides in the treatment of infections caused in a subject by Listeria monocytogenes .

[0012] International patent application WO2021 / 191851 describes antimicrobial decapeptides characterized by the structural motif W-X-X-X-W which gives them the ability to act against certain Gram-positive and Gram-negative pathogenic bacteria, yeasts, and fungi. An example of these antimicrobial decapeptides is RiLKl (SEQ ID NO: 1 in WO2021 / 191851).

[0013] In light of what is reported in the state of the art, there is still a need to provide additional antimicrobial peptides having a broader spectrum of antimicrobial activity, including effective activity against the so-called ‘eskape’ pathogens, i.e., the pathogens accountable for the majority of nosocomial, highly virulent and antibiotic-resistant infections (multi-drug resistance).

[0014] Identifying additional AMP amino acid sequences is not an easy task. In fact, although over the years numerous researches have pointed out a relationship between the antimicrobial activity of AMP peptides and the chemical-physical properties thereof, such as in particular the amino acid composition, net charge, spatial arrangement of the charge, hydrophobicity, hydrop athi city, and hydrophilicity (Shuqin Li, Yajie Wang, Zihan Xue, Yanan Jia, Ruilin Li, Chengwei He, Haixia Chen, The structure-mechanism relationship and mode of actions of antimicrobial peptides: A review, Trends in Food Science & Technology, Volume 109, 2021, Pages 103-115, ISSN 0924-2244, https: / / doi.Org / 10.1016 / j.tifs.2021.01.005), at the same time studies carried out to date have shown that, in many cases, the peptides tend to assume the structure (predominantly a-helical domains) associated with the antimicrobial activity only after contact with cell membranes. Therefore, a clear structure / function relationship cannot be established for this category of antimicrobial molecules. Consequently, it is extremely difficult to predict their antimicrobial activity.

[0015] A very important additional need, intrinsically linked to the possible applications of AMPs, is that related to structural stability, namely the ability to preserve the antimicrobial activity over time and under different environmental conditions in terms of pH, temperature, and ionic strength.

[0016] Yet another need concerns the absence of toxicity to eukaryotic cells resulting from the use of AMPs in all those applications involving contact with the human or animal organism, such as for example the coating on surfaces of materials, including work surfaces, straws, cannulas or catheters, in order to prevent microbial contamination, or even the use of these compounds as disinfectants. Lastly, a further need concerns the cost of producing these molecules, which should be kept as low as possible.

[0017] Summary of the invention

[0018] These and other needs are now met by the present invention, which provides a new set of antimicrobial peptides, which combine strong activity against various Gram-positive and Gram -negative pathogenic bacteria species, yeasts and fungi, including ‘eskape’ pathogens; significant structural stability over time under different pH, temperature and ionic strength conditions; the absence of toxicity to eukaryotic cells; and an amino acid sequence of only 13 amino acids, which helps to keep their production cost low.

[0019] The amino acid sequence of the antimicrobial peptides (AMPs) of the present invention is selected from the amino acid sequences represented by the following general formula:

[0020] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration.

[0021] Salts and solvates of the aforementioned peptides also fall within the scope of the invention.

[0022] In the general formula above and throughout the description below, the amino acid sequence of the antimicrobial peptides of the invention is represented using the one-letter code.

[0023] The AMP peptides of the invention have in common the fact that they include the motif “WXXXW” in tandem (WXXXWXXXW). The presence of tryptophan residues and their arrangement allow this structural element to promote the formation of a stable alpha-helix structure capable of interacting effectively and quickly with cell membranes, thus providing the whole new set of AMPs of the invention with advantageous and unpredictable chemicalphysical properties in terms of stability and antimicrobial potency. The structural motif “WXXXWXXXW” is characterized by the presence of three tryptophan (W) residues separated by three amino acids of various kinds indicated by the symbol X. This specific structural motif makes all the AMP peptides of the invention rich in tryptophan and identifiable as Trp-rich peptides. Tryptophan, in general, is an amino acid sometimes found in antimicrobial peptides, which promotes interaction with cell membranes. In fact, although tryptophan is a non-polar amino acid, the indole ring in its side chain contains both polar and non-polar components, making it an amphipathic molecule. This specific nature allows tryptophan to interact with both the hydrophobic lipid tails and the polar heads of the phospholipids that make up cell membranes, facilitating their adhesion and interaction. Tryptophan can therefore penetrate membranes by positioning itself at the interface of the lipid bilayer, thus promoting interactions of the peptides of which it is part with the cell membranes of the target microorganisms. This interaction leads to disruption of the membrane structure and, accordingly, of cellular functions, causing damage that can lead to cell lysis.

[0024] However, it is generally agreed that the presence of tryptophan residues in the sequence of an antimicrobial peptide causes an increase in hydrophobicity of the molecule, which, especially when occurring on the non-polar face of amphipathic alpha-helical peptides, appears to be related to increased toxicity to human cells, compromising its possible applications. Nevertheless, the inventors have surprisingly found that the AMPs of the invention, although amphipathic and rich in tryptophan, do not follow this behaviour.

[0025] An antimicrobial peptide as defined above, for use as a medicament, also falls within the scope of the invention.

[0026] A specific therapeutic application of the antimicrobial peptides of the invention relates to the therapeutic treatment of an infection caused by Gram negative bacteria, Gram positive bacteria, fungi, and / or yeasts.

[0027] The scope of the invention also includes a pharmaceutical formulation comprising an antimicrobial peptide as defined above and at least one pharmaceutically acceptable excipient and / or carrier.

[0028] For the reasons explained below, and as shown by the in vitro toxicity tests carried out by the inventors (see the experimental section of the present description), the antimicrobial peptides of the invention do not pose risks to human health.

[0029] Bacterial and eukaryotic membranes exhibit fundamental differences in their structure and molecular composition, which underlie the selectivity of the peptide molecules of the present invention and hence their antibacterial action. In detail, Gram-positive bacteria are composed of a cytoplasmic membrane and lipoteichoic acids bound to a thick peptidoglycan layer, while Gram-negative bacteria are characterized by the presence of a cell wall that includes a thin peptidoglycan layer located between the inner cytoplasmic membrane and the outer membrane. The outer membrane is particularly distinctive, being composed of a lipid bilayer containing lipopolysaccharides. Although structural differences exist, both peptidoglycan and lipopolysaccharides are characterized by their amphiphilic and anionic nature. In addition, the contribution of lipids such as phosphatidyl glycerol and cardiolipin is crucial in giving the bacterial membrane its negative charge. The membranes of eukaryotic cells, on the other hand, are mainly composed of sphingomyelin and phosphatidylcholine, which are zwitterionic molecules (i.e., electrically neutral, with an equal number of positive and negative formal charges). Anionic lipids are sequestered into the monolayer facing the inside of the cell, making the surface of the eukaryotic membrane not as negatively charged as the bacterial membrane.

[0030] These differences affect the selectivity of the antibacterial agents, which tend to bind to the negatively charged surface of bacterial membranes, while they do not interact with the zwitterionic surface of eukaryotic membranes.

[0031] Consumers’ attention to the possible effects on their health related to food quality is having a great impact on food processing and preservation industries. Compared to the past, today the consumer is increasingly aware and attentive to the quantity and quality of chemical preservatives in food and to the effectiveness of both conventional preservation methods and innovative preservation technologies in extending the shelf life and improving the safety of a wide range of food products, while preserving the taste / flavour thereof. Several microorganisms are recognized today as responsible for food infections related to food consumption; Listeria monocytogenes, a Gram-positive bacterium causing listeriosis, and Salmonella typhimurium, a Gram-negative bacterium causing salmonellosis, are certainly among these. These microorganisms can grow in a wide variety of foods, usually raw foods, such as undercooked meat, raw vegetables, fish products, cheese prepared with unpasteurized milk, or ready-to-use food products that are industrially processed and require low temperature storage. Foodborne listeriosis is a rather rare but serious disease, with a mortality rate (20-30%) comparable or higher to that of other foodborne diseases, such as, for example, salmonellosis.

[0032] Therefore, the present invention also includes the use of an antimicrobial peptide as defined above as an antimicrobial agent for the prevention of contamination of an article (e.g., a food, a material, a container or a tool) by and / or for the decontamination of an article (e.g., a food, a material, a container or a tool) from Gram negative bacteria, Gram positive bacteria, fungi, and / or yeasts.

[0033] The scope of the invention also includes an antimicrobial composition comprising at least one antimicrobial peptide as defined above, a carrier and optionally an additional antimicrobial agent.

[0034] The invention further relates to an article, such as, for example, a food, a material, a container or a tool, having an antimicrobial peptide covalently bound to a surface thereof, or having an antimicrobial peptide contained in a coating adhered to said surface, wherein said antimicrobial peptide is as defined above.

[0035] Finally, the invention relates to the use of an antimicrobial peptide of the invention as a disinfectant or antiseptic, excluding methods for surgical or therapeutic treatment of the human or animal body and diagnostic methods applied to the human or animal body. In this regard, it is specified that an antiseptic is a product intended to prevent or stop the growth or action of microorganisms on living tissues, while a disinfectant is a product intended to eliminate microorganisms found on inert material.

[0036] Antiseptic or disinfectant formulations for personal and environmental hygiene are known per se and are, for example, solution, spray or gel formulations. According to the invention, such antiseptic or disinfectant formulations comprise an antimicrobial peptide of the invention alone or in combination with one or more additional antimicrobial agents, together with a carrier, and optionally one or more complementary ingredients having the purpose of enhancing the efficacy and tolerability of the formulation. In addition, it is possible to supplement the antiseptic or disinfectant formulations of the invention with additives having a specific action, such as perfumes and essential oils.

[0037] A formulation according to the invention for gynecological applications aimed at fighting fungal infections, such as Candida, may comprise an antimicrobial peptide of the invention alone or in combination with an antifungal agent, such as clotrimazole, miconazole or fluconazole, suitably combined with a water-based moisturizing solution, with or without perfume. This formulation can be administered in a variety of ways, such as for example by spray, cream, ointment or soap, in order to ensure a full range of therapeutic options.

[0038] Brief description of the figures

[0039] Figure 1A shows the CD spectra of 50 pM RKW without SDS and in the presence of different concentrations of SDS (3-150 mM).

[0040] Figure IB shows the CD spectra of 50 pM RKW without SDS and incubated for 24 h at 25 °C with 50 mM SDS.

[0041] Figure 2A shows the CD spectra of 50 pM RKW without SDS and incubated for 48 h at 25 °C in pH 2.0 buffer solution with 50 mM SDS.

[0042] Figure 2B shows the CD spectra of 50 pM RKW without SDS and incubated for 48 h at 25 °C in pH 11.0 buffer solution with 50 mM SDS.

[0043] Figure 3 A shows the CD spectra of 50 pM RKW without SDS and incubated for 48 h at 4 °C with 50 mM SDS.

[0044] Figure 3B shows the CD spectra of 50 pM RKW without SDS and incubated for 48 h at 90 C with 50 mM SDS.

[0045] Figure 4 shows the CD spectra of 50 pM RKW without SDS and incubated for 48 h at 25 °C in a saline solution (150 mM NaCl) with 50 mM SDS.

[0046] Detailed description of the invention

[0047] A first object of the present invention is an antimicrobial peptide consisting of an amino acid sequence represented by the following general formula:

[0048] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or solvate thereof.

[0049] In the general formula above and throughout the description below, the amino acid sequence of the antimicrobial peptides of the invention is represented using the one-letter code.

[0050] It should also be noted that all amino acid sequences are represented in the present description by formulas whose orientation from left to right is in the conventional direction, i.e., from the amino terminus to the carboxyl terminus.

[0051] The antimicrobial peptide of the invention can be synthesized by any one of the solid phase or solution peptide synthesis procedures known in art.

[0052] According to a preferred embodiment, the amino acids in the above general formula are all in the D configuration or all in the L configuration.

[0053] Within the scope of the invention, the following specific amino acid sequences are preferred:

[0054] RI<WILI<WLRTWI<I< (SEQ ID NO: 1) RRWILRWLRTWRR (SEQ ID NO:2) RRWILRWLRTWKK (SEQ ID NO:3) RRWILRWLRTWRK (SEQ ID NO:4) RRWILRWLRTWKR (SEQ ID NO: 5) RKWILKWLRTWRR (SEQ ID NO: 6) RKWILKWLRTWRR (SEQ ID NO: 7) RKWILKWLRTWRR (SEQ ID NO: 8) RKWILRWLRTWRR (SEQ ID NO: 9) RKWILRWLRTWRR (SEQ ID NO: 10) RKWILRWLRTWRR (SEQ ID NO: 11) RRWILRWLRTWKR (SEQ ID NO: 12) RRWILKWLRTWRR (SEQ ID NO: 13) RRWILKWLRTWRR (SEQ ID NO: 14) RRWILKWLRTWRR (SEQ ID NO: 15) RRWILKWLRTWRR (SEQ ID NO: 16) wherein each amino acid is independently in the D or L configuration.

[0055] Among them, the most preferred sequence is RKWILKWLRTWKK (SEQ ID NO: 1), which is representative of all the above sequences.

[0056] Within the scope of the present invention, the term "solvate" or "solvates" refers to complexes of the peptides of the invention with the solvents in which the synthesis reaction takes place or in which they are precipitated or crystallized. For example, a complex with water is known as a "hydrate". Solvates suitable for the purposes of the invention are those that do not lead to a change in the conformation or stability of the peptides according to the invention, and therefore, do not interfere with their biological activity.

[0057] Preferred salts according to the invention are pharmaceutically acceptable salts that do not lead to a change in the conformation or stability of the peptides according to the invention. By way of illustrative and non-limiting examples, pharmaceutically acceptable acid addition salts include those formed with the hydrochloric, hydrobromic, acetic, phosphoric, lactic, pyruvic, acetic, trifluoroacetic, succinic, perchloric, fumaric, maleic, glycolic, lactic, salicylic, oxaloacetic, methanesulfonic, ethanesulfonic, p-toluenesulfonic, formic, benzoic, malonic, naphthalene-2-sulfonic, benzenesulfonic and isethionic acids. Other acids, such as oxalic acid, although not pharmaceutically acceptable per se, can be useful as intermediates for obtaining the peptides of the invention and pharmaceutically acceptable salts thereof. Acceptable base salts include ammonium salts, alkali metal salts, for example potassium and sodium salts, alkaline earth metal salts, for example calcium and magnesium salts, and salts with organic bases, for example di cyclohexyl amine and N-methyl-D-glucosamine.

[0058] A second object of the present invention is an antimicrobial peptide consisting of the amino acid sequence represented by the following general formula:

[0059] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or a solvate thereof, for use as a medicament.

[0060] A third object of the present invention is an antimicrobial peptide consisting of the amino acid sequence represented by the following general formula:

[0061] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or a solvate thereof, for use in the therapeutic treatment of an infection caused by Gram negative bacteria, Gram positive bacteria, yeasts, and / or fungi.

[0062] Within the scope of the invention, the Gram negative bacterium is preferably selected from Campylobacter such as, for example, Campylobacter coli, Campylobacter concisus, Campylobacter jejuni, Campylobacter C. rectus; Arcobacter such as, for example, Arcobacter butzleri, Arcobacter cryaer ophilus; Citrobacter such as, for example, Citrobacter amalonaticus, Citrobacter braakii, Citrobacter farmeri, Citrobacter freundii, Citrobacter gillenii, Citrobacter koseri; Enterobacter such as, for example, Enterobacter aerogenes, Enterobacter agglomerans, Enterobacter cloacae, Enterobacter cowanii, Enterobacter gergoviae; Escherichia such as, for example, Escherichia coli; Klebsiella; Morganella such as, for example, Morganella Morganii; Proteus such as, for example, Proteus vulgaris, Proteus mirabilis; Shigella such as, for example, Shigella dysenteriae; Salmonella such as, for example, Salmonella typhi, Salmonella typhimurium; Yersinia such as, for example, Yersinia pestis, Yersinia pseudotuberculosis, Yersinia enterocolitica; Serratia marcescens; Aerobacter aerogenes; Enterobacter sakazakii; Acinetobacter, such as, for example, Acinetobacter baumannii, Acinetobacter beijerinckii, Acinetobacter bereziniae, Acinetobacter boissieri; Moraxella such as, for example, Moraxella catarrhalis (synonym Branhamella catarrhalis) Neisseria such as, for example, Neisseria meningitidis; Haemophilus such as, for example, Haemophilus influenzae; Pasteurella such as, for example, Pasteurella multocida; Pseudomonas such as, for example, Pseudomonas aeruginosa; Vibrio such as, for example, Vibrio cholerae, Vibrio fischeri, Stenotrophomonas maltophilia.

[0063] More preferably, the Gram negative bacterium is selected from Salmonella typhimurium and Escherichia coli.

[0064] Within the scope of the invention, the Gram positive bacterium is preferably selected from: Actinobacteria such as, for example, Tropheryma whipplei; Bacillus; Car nobacterium; Clostridium; Corynebacterium diphtheria; Enterococcus such as, for example, Enterococcus faecalis; Gardnerella vaginalis; Lactobacillus; Lactococcus; Listeria such as, for example, Listeria monocytogenes; Micrococcus; Staphylococcus such as, for example, Staphylococcus aureus; Streptococcus such as, for example, Streptococcus agalactiae, Streptococcus pneumoniae, Streptococcus pyogenes, Streptococcus viridans. More preferably, the Gram positive bacterium is selected from Listeria monocytogenes and Staphylococcus aureus.

[0065] Within the scope of the invention, the fungus preferably is Aspergillus brasiliensis and the yeast preferably is Candida albicans.

[0066] A fourth object of the present invention is a pharmaceutical formulation comprising an antimicrobial peptide consisting of the amino acid sequence represented by the following general formula:

[0067] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or a solvate thereof, and at least one pharmaceutically acceptable excipient and / or carrier.

[0068] The pharmaceutical formulations according to the invention include, by way of example, formulations suitable for oral, parenteral (including subcutaneous, intradermal, intramuscular, intravenous and intra-articular) and topical use, or for inhalation (including particularly small particle powders or mists that may be generated by means of various types of pressurized aerosol meters, nebulizers or insufflators). However, the most suitable route of administration may depend, for example, on the condition of the subject and the disease to be treated. The person skilled in the art is capable of identifying the most suitable route of administration and pharmaceutical formulation, based on the specific circumstances of the case.

[0069] The pharmaceutical formulations of the invention can be conveniently presented in unit dosage form and can be prepared by means of any one of the protocols well known in the pharmaceutical art. All the methods involve the step of combining the active ingredient (i.e., the peptide) with a carrier, including one or more pharmaceutically acceptable excipients. Generally, pharmaceutical formulations are prepared in a uniform way by combining the active ingredient with liquid carriers or finely divided solid carriers, or both, and then, if necessary, shaping the product into the desired formulation.

[0070] The pharmaceutical formulations of the present invention suitable for oral administration can be presented in the form of discrete units such as, for example, capsules, cachets or tablets, each containing a predetermined amount of the active ingredient; powder or granules; a solution or suspension in an aqueous liquid or in a non-aqueous liquid; or an oil- in-water liquid emulsion or a water-in-oil liquid emulsion. The active ingredient can also be presented as a bolus, electuary or paste. Several pharmaceutically acceptable excipients and carriers are described in standard formulation texts, for example Remington's

[0071] Pharmaceutical Sciences by E. W. Martin. See also Wang, Y.J. and Hanson, M. A., Journal of Parenteral Science and Technology, Technical Report No. 10, Suppl.

[0072] Formulations for oral administration include suspensions which may contain, for example, microcrystalline cellulose for imparting bulkiness, alginic acid or sodium alginate as a suspending agent, methylcellulose as a viscosifier, and sweeteners or flavouring agents such as those already known in the pharmaceutical sector; immediate release tablets, which may contain, for example, microcrystalline cellulose, calcium phosphate, starch, magnesium stearate and / or lactose and / or other excipients, binders, extenders, disintegrants, diluents and lubricants such as those known in the pharmaceutical art.

[0073] Formulations for parenteral administration include, for example, sterile aqueous and nonaqueous solutions for injection which may contain antioxidants, buffers, bacteriostats and solutes which make the formulation isotonic; sterile aqueous and non-aqueous suspensions which may include surfactants and thickeners. The formulations can be presented in singledose containers or in multi-dose containers, such as vials and sealed vials, and can be stored in lyophilized form, only requiring the addition of the sterile liquid carrier immediately prior to use.

[0074] Formulations for topical administration may include creams, ointments, gels, lotions, emulsions, transdermal patches and other forms that can be applied directly to the skin or mucous membranes. These formulations may contain, for example, emulsifiers, stabilizers, penetrating agents, preservatives and other substances suitable for facilitating local absorption of the active ingredient and enhancing the stability of the formulation. Commonly used excipients may include cetyl alcohol, glycerin, stearic acid, liquid paraffin and other substances known in the field of topical formulation.

[0075] Formulations for nasal aerosol or inhalation administration include, for example, saline solutions that may contain benzyl alcohol or other appropriate preservatives, which promote absorption to enhance bioavailability, and / or other solubilizing agents such as those known in the pharmaceutical art. In formulations for nasal aerosol or inhalation administration, the peptide of the invention is administered in the form of an aerosol from a pressurized container or a nebulizer, using a suitable propellant, for example, dichlorodifluoromethane, trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide, or other suitable gas. In the case of a pressurized aerosol, the dosage unit can be determined by providing a valve to deliver a metered quantity. Preferred unit dosage formulations are those containing an effective dose, as previously described, or an appropriate ratio, of the active ingredient.

[0076] In addition to the ingredients specifically mentioned above, the formulations of the present invention can also include other conventional agents used in the pharmaceutical art, taking into account the type of formulation in question; for example, those suitable for oral administration may include flavouring agents.

[0077] The antimicrobial peptides of the invention are also appropriately administered as sustained and controlled release systems. Suitable examples of sustained release systems usable with the peptides of the invention include suitable polymeric materials, such as semi-permeable polymer matrices, for example, films or microcapsules; specific hydrophobic materials, such as emulsions in suitable oils or ion exchange resins; and derivatives of the peptides of the invention, such as, for example, a salt or solvate.

[0078] A fifth object of the present invention is an antimicrobial composition comprising a carrier and an antimicrobial peptide consisting of the amino acid sequence represented by the following general formula:

[0079] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or solvate thereof.

[0080] According to a preferred embodiment, the antimicrobial composition comprises at least one further antimicrobial peptide according to the invention and / or at least one further antimicrobial agent.

[0081] A sixth object of the present invention is the use of an antimicrobial peptide consisting of the amino acid sequence represented by the following general formula: RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or a solvate thereof, as a disinfectant or an antiseptic, i.e., respectively, for the prevention of contamination of an article (e.g., a food, a material, a container or a tool) or of a living tissue (preferably the epidermis) by and / or for the decontamination of an article (e.g., a food, a material, a container or a tool) or of a living tissue (preferably the epidermis) from Gram negative bacteria, Gram positive bacteria, fungi, and / or yeasts.

[0082] A seventh object of the invention is an article having an antimicrobial peptide covalently bound to a surface thereof, or having an antimicrobial peptide contained in a coating adhered to said surface, wherein said antimicrobial peptide consists of the amino acid sequence represented by the following general formula:

[0083] RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or solvate thereof.

[0084] In the event that the peptide is covalently bound to the surface of the article, the binding occurs with reactive groups on the surface itself.

[0085] Preferably, the article is a container, a tool or a material for the storage or processing of food products, or a food.

[0086] According to a preferred embodiment, the antimicrobial peptide is contained in a coating applied to the surface of the article. In this case, the application provides that a liquid antimicrobial composition containing the peptide according to the invention is applied to the surface of said article and then left to dry. This composition can optionally comprise filmforming agents which form a film on the surface of the article and favour the permanence of the peptide thereon. Therefore, the aforementioned coating preferably comprises a filmforming polymer. According to another preferred embodiment, the peptide is bound with a covalent chemical bond to the surface of the article. In this case, the peptide is bound by the formation of a covalent bond with reactive groups on the surface of the article. This makes it possible to obtain a surface characterized by a stable bactericidal activity for long periods of time.

[0087] Techniques for binding peptides or proteins to solid supports are known to those skilled in the art and vary depending on the material used.

[0088] For example, in the case of materials having metal (gold, silver, platinum) and semiconductor (titanium, zinc, tin, zirconium, germanium) surfaces, a silanization process can be used. This involves, for example, reacting the material to be treated with a mixture of sulfuric acid (H2SO4) and oxygen peroxide (H2O2), which are able to activate the aforementioned surfaces by creating bonds of surface atoms and hydroxyl groups (-OH) easily replaceable by more stable bonds such as Si-C or Au-S. The activated surfaces can covalently bind the peptides of the invention following treatment with a silanizing agent, such as aminopropyldimethylethoxysilane or aminopropyltriethoxysilane and with a compound having two functional groups capable of forming the peptide covalent bond with the amino groups of the peptide, such as glutaraldehyde or bis-succinimide. These treatments are typical of the chemistry of aqueous solutions and for this reason they are referred to as wet processes, which are advantageous because they do not require particular technological equipment but only, preferably rigid, materials which can be wetted and dried without difficulty.

[0089] In the case of plastic or polymeric surfaces, these can instead be functionalized to link the peptides of the invention either by applying the wet processes described above, or by applying high energy electromagnetic radiation (for example, laser, ultraviolet radiation, gamma rays). For the so-called soft materials such as non-rigid plastics, wet processes may not be completely effective; therefore, the so-called dry functionalization processes based on the interaction of the surface with a gas plasma or electromagnetic radiation are preferred. The interaction of the surface of a polymer with electromagnetic radiation causes surface activation through the breaking of the accessible polymer bonds; therefore, the C=C bonds become -C-C-, thereby allowing subsequent chemical modification of the surface itself. A similar operating principle applies to the other polymer surface activation method, which consists in the treatment thereof with ionized gas (gas plasma). This method is particularly advantageous because, with the cold plasma technique, the temperature of the treated material does not reach high values with respect to room temperature. This method requires low pressure (0.1-100 Pa) and the presence of a working gas (usually N2, O2 or Ar, CF4). [Hegemann, Dirk, Herwig Brunner, and Christian Oehr. "Plasma treatment of polymers for surface and adhesion improvement." Nuclear instruments and methods in physics research section B: Beam interactions with materials and atoms 208 (2003): 281-286],

[0090] Further antimicrobial applications of the antimicrobial peptides of the invention include, for example, the sanitization of plants or machinery intended for the processing of food products, or the use as a preservative in food products, for example for the prevention of contamination thereof by and / or the elimination of contamination thereof from Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Staphilococcus aureus, Aspergillus brasiliensis and Candida albicans. This use involves the use of the peptide on the surface and / or inside the food or on the surface of its packaging materials.

[0091] Preferably, the antimicrobial peptide is used as a preservative agent in food products. Alternatively, the peptide is used for application to the surface of food packaging materials, e.g., for the prevention of contamination by and / or the elimination of contamination from Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Staphilococcus aureus, Aspergillus brasiliensis and Candida albicans of said materials and of the foods contained therein.

[0092] In all embodiments described above, peptides consisting of the amino acid sequences SEQ ID NO: 1 through SEQ ID NO: 16 described above, and salts and solvates thereof are preferred.

[0093] Experimental Section

[0094] The experimental section below reports the results of the studies on conformation, structural stability, toxicity and antimicrobial activity carried out by the present inventors with the peptide consisting of the amino acid sequence RKWILKWLRTWKK (referred to by brevity as “RKW”, SEQ ID NO: 1), which is representative of the new set of AMP peptides forming the object of the invention, as it includes the motif “WXXXW” in tandem (WXXXWXXXW) common to all the AMP peptides of the invention.

[0095] The synthesis of the above-mentioned representative RKW peptide is also illustrated.

[0096] 1. Conformation and Structural Stability

[0097] In particular, the results obtained by the inventors in studies concerning conformation and structural stability are reported in the accompanying figures. Figures 1-4 refer to RKW peptide analysis to define its conformation (Figure 1) and structural stability over time and under different environmental conditions in terms of pH (Figure 2), temperature (Figure 3), and ionic strength (Figure 4). These analyses were carried out using two different spectroscopic techniques, namely Circular Dichroism (CD) and fluorescence spectroscopy which, by exploiting different approaches, can provide information on the structural rearrangements that the molecule assumes in space and time under the different assay conditions. CD spectroscopy is generally used to determine the secondary and tertiary structure of proteins. Its principle is based on the differential absorption of laevorotatory and dextrorotatory polarized light. Since optically active molecules absorb laevorotatory and dextrorotatory circular polarized light differently, this becomes the basis for determining the structural elements of the biomolecule. The examples show, for simplicity and relevance, only the dichroic spectra of RKW under the different experimental conditions. The analyses were performed on a JASCO J-810 spectropolarimeter (JASCO, Tokyo, Japan) using a quartz cuvette with an optical path of 0.1 cm (Hellma Analytics, Milan, Italy) recording spectra in the far-UV region (195 and 250 nm) setting a scanning speed of 20 nm / min and 5 accumulations.

[0098] Figure 1A shows that, in the absence of Sodium Dodecyl Sulfate (SDS), i.e., a detergent capable of forming micelles that mimic the anion component of bacterial membranes, RKW has the typical spectra (CD) of an unfolded peptide molecule. However, following the addition of the detergent, a folding process is observed through which the peptide assumes a typical a-helix structure. In the presence of all tested concentrations of SDS - that is, a 3 mM concentration which is lower than the Critical Micelle Concentration (CMC) and two concentrations of 50 mM and 150 mM, respectively, which are both higher than the CMC - the process of folding of the RKW molecule, which assumes an a-helix structure, is observed. Micelles are aggregates that form in aqueous solution by spontaneous association of amphiphilic molecules such as SDS, which are formed by the joining of a hydrophobic group with a hydrophilic group; in reality, the formation of micelles (structures having the hydrophobic regions inside and the hydrophilic regions on the surface exposed to water) occurs only if the concentration of surfactant is above a critical value referred to as "CMC".

[0099] Figure IB also shows that the structure of the peptide remains virtually unchanged over the course of 24 hours.

[0100] Figure 2 shows that in all pH conditions investigated (pH 2.0; pH 7.0; pH 11.0), following the addition of the SDS detergent, a structuring of the RKW peptide occurs, which always assumes an a-helix conformation. What is observed under these experimental conditions is that, while the conformation is maintained almost unchanged (for 48 hours) at pH 7.0 (not shown) and pH 11.0 (Figure 2B), under acidic conditions (pH 2.0) the conformation is partially maintained at 24 hours, but the molecule is destructured at 48 hours (Figure 2A). This demonstrates an excellent conformational stability of the RKW peptide at neutral and basic pH values but a vulnerability in the long term (48 hours) at acidic pH.

[0101] Figure 3 shows that when RKW is incubated at temperatures of 4 and 90 °C for different times (0 - 24 - 48 h) in the absence and presence of SDS, no changes in peptide conformation are observed, showing a stable a-helix structure throughout the incubation time (Figures 3 A and 3B). This shows that temperature does not affect the conformational stability of the molecule.

[0102] Figure 4 shows that in a saline solution (150 mM NaCl) RKW remains stably structured throughout the time interval (48 h) investigated (Figure 4).

[0103] 2. Antimicrobial activity Experiments were also carried out to assess the antimicrobial activity of the RKW peptide.

[0104] 2.1 Minimum Bactericidal Concentration (MBC) and Minimum Fungicidal Concentration (MFC)

[0105] The Minimum Bactericidal Concentration (MBC) of RKW (i.e., the lowest concentration of antimicrobial substance required to cause the death of more than 99.9 % of a given microbial population) was calculated for some of the main food-contaminating pathogenic bacterial strains (Salmonella typhimurium isolated from chicken, Listeria monocytogenes isolated from fish, Escherichia coli isolated from mussels, and Staphylococcus aureus isolated from pastry products). RKW was also tested against two Salmonella strains isolated from food (Salmonella monofasica and Salmonella Napoli) and against Campylobacter jejuni and Pseudomonas aeruginosa. The results shown in Table 1 demonstrate that RKW is active against all strains tested, in some cases at very low concentrations in both samples with starting bacterial concentrations of 3 log and 5 log. Table 1 also shows the activity data of RKW against an important fish pathogen, Vibrio harvey. The results obtained show extreme efficiency towards Vibrio harvey. In addition, the Minimum Fungicidal Concentration (MFC) of RKW was assessed for two fungal strains pathogenic to humans, namely Aspergillus brasiliensis ATCC 9341 and Candida albicans ATCC 14053, and the peptide was shown to be active against both strains (Table 1).

[0106] Table 1. Calculated MBC and MFC values for some of the main food-borne pathogens - contact time: 6 h

[0107] The efficacy of the peptide was assessed in accordance with the International Standard UNI CEI EN ISO / IEC 17025:2005 registered under No 1043. The analyses were carried out by applying the UNI EN ISO 11290-2:2017 plate enumeration method, concerning the “Microbiologia della catena alimentare” (Microbiology of the food chain). Specifically, concentrations of 103cfu / ml of different food pathogenic bacterial strains [Salmonella typhimurium isolated from chicken according to UNI EN ISO 6579-1 and serotyped with the ISO / TR 6579-3:2014 method, L. monocytogenes isolated from fish according to UNI EN ISO 11290-1, E. coll from mussels according to UNI EN ISO 16649-3, and S. aureus from pastry products according to UNI EN ISO 6888-2) were prepared in peptone water (BPW) and placed in contact with the peptide at a concentration of 15 pM. The different bacterial cultures in the absence of the antimicrobial molecule, which represent the control cultures (CTRL), and in the presence of the RKW peptide were incubated for 6 h at 37 °C. After the established time, 50 pl were collected from each sample and spread on selective chromogenic plates (Agar Listeria acc. to Ottaviani & Agosti (ALOA) - Biolife Italiana for

[0108] L. monocytogenes,' Salmonella Chromogenic agar - Oxoid UK for S. typhimurium,' Baird Parker agar base - Biolife Italiana for S. aureus,' pseudomonas agar base with CFC supplement-Oxoid for P. aeruginosa, TBX agar — Biolife Italiana for E. coli). The plates thus prepared were incubated at 37 °C for 24 ± 2 h for L. monocytogenes, S. typhimurium and S. aureus, and at 44 °C overnight for E. coli. Subsequently, the counting was carried out as required by the current standard. To test the ability of the peptide to counteract a higher bacterial load, the same experiment was repeated with an inoculum of 105cfu / ml of the different bacterial strains listed above by incubating them for 6 h at two different concentrations of the molecule (10 and 20 pM).

[0109] In addition, the antimicrobial peptide object of the patent was also tested against two food- isolated Salmonella strains (Salmonella monofasica and Salmonella Napoli) and Campylobacter jejuni at two different concentrations, i.e., 40 and 80 pM. The different bacterial strains in BPW at a concentration of 103cfu / ml were incubated at a temperature of 37 °C for 6 h, in the absence (CTRL) and in the presence of different peptide concentrations. After the established time, 50 pl were collected and spread on selective chromogenic plates (Salmonella Chromogenic agar - Oxoid UK for S. typhimurium and Campylobacter Karmali agar CF / 20 - Liofilchem for C. jejuni).

[0110] In order to define the molecule’s performance against other microbial species, its antifungal activity was investigated using two strains from the American Type Culture Collection (ATCC, Manassas, VA, USA): Aspergillus brasiliensis ATCC 9341 and Candida albicans ATCC 14053. For both fungal species, an inoculum of 105cfu / ml in BPW was prepared to which the peptide was added or not (CTRL). The fungal suspension as such kept as a control and the one added with the different concentrations of peptide molecule (4 - 8 - 15 pM) were incubated for 6 h at 37 °C. Subsequently, 100 pl of the culture was plated on DG18 plates (Dichloran 18% Glycerol Agar — ISO 21527-2) which were incubated at 25 °C for 7 days.

[0111] 2.2 Activity against ESKAPE pathogens

[0112] The term ESKAPE is an acronym resulting from the initials of the six nosocomial pathogens that exhibit multidrug resistance and virulence, namely E. faecium, S. aureus, K. pneumoniae, A. baumannii, P. aeruginosa and Enterobacter spp. The ESKAPE pathogens tested were S. aureus MRSA (Methicillin resistant), E. faecium (resistant to high-dose aminoglycosides, gentamicin, streptomycin), A. baumannii (resistant to fluoroquinolones, carbapenems, aminoglycosides), Pseudomonas spp. (a group of bacteria comprising over 100 species, including P. fluorescens and P. putida), K. pneumoniae (resistant to carbapenems, cephalosporins, aminoglycosides, fluoroquinolones) - the pathogenicity of these strains is obviously aggravated by their drug resistance and relative ease with which they acquire new resistance to antibiotics.

[0113] Table 2 provides data on the activity of the RKW peptide against some “ESKAPE” pathogens isolated from human patients, as provided by the Istituto Zooprofilattico Sperimentale del Mezzogiorno (IZSM). As reported in Table 2, the RKW peptide showed antimicrobial activity that could be defined as “species-specific” because substantial differences in sensitivity towards the different strains used were perceived. In detail, all Staphylococcus aureus strains were found to be sensitive to the activity of the peptide, with MIC values coinciding with the MBC values and close to the concentration of 50 pM, for both inocula (3 Log and 5 Log). The Enterococcus faecium strains showed particular sensitivity to the peptide even at very low concentrations, recording a MIC of 20 pM at both microbial load values. As to the MBC values, on the other hand, differences were found depending on the concentration of the inoculum; in fact, the MBC value was 20 pM towards an inoculum of 3 Log (cfu / mL) and 50 pM when the peptide was tested with an inoculum of 5 Log (cfu / mL). The molecule exhibited a different sensitivity towards the Acinetobacter baumannii strains, with MIC values of 50 pM for both the 3 Log (cfu / mL) and 5 Log (cfu / mL) inoculum tests, while for the MBC values, the active concentration was found to be higher than that verified for the above microbial species, being 75 pM towards the 3 Log (cfu / mL) inoculum and higher than 100 pM towards the 5 Log (cfu / mL) inoculum. The Pseudomonas aeruginosa strains were shown to be sensitive to the action of the peptide even at very low concentrations (MIC=20 pM). However, higher concentrations are required to cause bacteriolysis (MBC < 100 pM). Lastly, the Klebsiella pneumoniae strains were found to be resistant to the highest peptide concentration tested. For these reasons, the MIC and MBC values could not be determined.

[0114] Table 2. MIC and MBC values calculated for some pathogenic bacterial strains characterized by multi -re si stance to different classes of antibiotics.

[0115] The antimicrobial molecule was tested at different concentrations against a total of 28 strains, of which 11 belonged to the species Staphylococcus aureus (SA), 6 to Klebsiella pneumoniae (E-KP), 5 to Pseudomonas aeruginosa (PA), 3 to Acinetobacter baumannii (AC) and 3 to

[0116] Enterococcus faecium (EF). The strains, stored in Tryptone Soya Broth (TSB) and glycerol at -20 °C, were plated in Tryptone Soya agar (TSA) and incubated for 24 h at 37 °C with the exception of Pseudomonas aeruginosa, for which a temperature of 25 °C was set. The assay was performed according to the guidelines of the European Committee on Antimicrobial Susceptibility Testing (EUCAST, 2023) and ISO 20776-1, using the broth microdilution method to determine the Minimum Inhibitory Concentrations (MIC), i.e., the lowest concentration of an antimicrobial agent capable of preventing the appearance of visible growth of a microorganism within a defined period of time. For each ESKAPE strain included in the test, bacterial inocula were prepared in 10 mL of TSB medium and incubated for 18+2 h at their respective growth temperatures until they reached a turbidity value of 0.5

[0117] McFarland, i.e., 1-2x10 cfu / mL, adjusting it with the addition of physiological saline or broth if the recorded value was higher. As per the reference methodology, the inoculum was further diluted to a value of 5 * 105cfu / mL, but as additional data, an inoculum was also prepared with a lower microbial load (5 x 103cfu / ml). For each strain, decreasing concentrations of peptide from 100 to 6.25 pM were added to both inocula, keeping a condition in the absence of antimicrobial molecule as a control. The samples thus prepared were incubated at 37 °C for 18+2 h. After determining the MIC, defined as the first inoculum appearing clear to the naked eye, in order to assess the MBC values, 100 pL of bacterial suspension, in which no visible bacterial growth was observed, were plated on Nutrient Agar plates and incubated for 24 h at 37 °C. The antimicrobial test was replicated at least three times for all strains.

[0118] 2.3 Evaluation of use as a disinfectant or antiseptic

[0119] The experimental data collected on the antimicrobial action of RKW were validated by a certified analytical laboratory commissioned to carry out activity tests against some bacterial and fungal strains, reported in EFSA case studies among the main contaminants in food and food processing / production environments. The suitability of the RKW peptide for use as a disinfectant or antiseptic has been evaluated. The results obtained are shown in Tables 3 and 4.

[0120] Tables 3 and 4 indicate that RKW is suitable for use as a disinfectant and an antiseptic, as it is active at all concentrations tested against the bacterial strains Pseudomonas aeruginosa ATCC 15442 and Staphylococcus epidermidis ATCC 12228 (Table 3) and against Candida albicans ATCC 10231 at a concentration of at least 50 pM (Table 4).

[0121] Table 3. BS EN 1276:2019 test procedure for the evaluation of chemicals classifiable as chemical disinfectants and antiseptics. Quantitative test for the evaluation of the bactericidal activity of chemical disinfectants and antiseptics used in the food, industrial, domestic and institutional sectors. Incubation time 60 min; test temperature 37°C. No= number of cfu / ml in the test mixture at the start (time zero) of incubation. R=decrease in viability.

[0122] The BS EN 1276:2019 protocol is a quantitative suspension test to evaluate the efficacy of formulations of chemical disinfectants and antiseptics against a wide range of bacteria. In this case it was used to test the bactericidal activity of the antimicrobial peptide at different concentrations (100 pM, 50 pM, 20 pM, 10 pM and 5 pM) against standard strains of Staphylococcus aureus (ATCC 6538), Pseudomonas aeruginosa (ATCC 15442) and Staphylococcus epidermidis (ATCC 12228). During the preparation of the test, the peptide, stored in the lyophilized form at room temperature and shielded from light, was diluted in distilled water and assumed a clear and colourless appearance. According to the BS EN 1276:2019 protocol, the peptide was added to a bacterial test suspension with the addition of an interfering substance (0.3 g / L bovine albumin). This suspension was kept in incubation at a temperature of 37 °C for a contact time of 60 minutes. The activity assessment was conducted following the dilution and neutralization method, using peptone saline solution as the diluent, Tryptone soy agar as the culture medium, and a neutralizer consisting of polysorbate 80, sodium thiosulfate, lecithin, tryptone, NaCl, phosphate buffer, and water. The stability and appearance of the mixture during the procedure were evaluated as homogeneous and without precipitates. At the end of the contact time, an aliquot was taken and immediately added to the neutralizing solution to stop the bactericidal activity. After a 5-minute neutralization period, 1 mL of the mixture was plated to check for any bacterial growth. The plates were incubated for 20-24 hours and the point-wise logarithmic reduction in CFUs (Colony Forming Units) was calculated relative to control plates.

[0123] Table 4. BS EN 1650:2019 test procedure for the evaluation of chemicals classifiable as chemical disinfectants and antiseptics. Quantitative test for the evaluation of the anti-yeast and mould activity of chemical disinfectants and antiseptics used in the food, industrial, domestic and institutional sectors. Incubation time 60 min; test temperature 37°C. N0= number of cfu / ml in the test mixture at the start (time zero) of incubation. R=decrease in viability.

[0124] The BS EN 1650:2019 protocol is a quantitative suspension test that evaluates the fungicidal or yeasticidal activity of chemical disinfectants and antiseptics. In this case it was used to test the fungicidal and yeasticidal activity of the antimicrobial peptide at different concentrations (100 pM, 50 pM, 20 pM, 10 pM and 5 pM) against standard strains of Candida albicans (ATCC 10231) and Aspergillus brasiliensis (ATCC 16404). During the preparation of the test, the peptide, stored in the lyophilized form at room temperature and shielded from light, was diluted in distilled water and assumed a clear and colourless appearance. According to the BS EN 1650:2019 protocol, the peptide was added to a test suspension of fungi or yeasts with the addition of an interfering substance (0.3 g / L bovine albumin). This suspension was kept in incubation at a temperature of 37 °C for a contact time of 60 minutes. The activity assessment was conducted following the dilution and neutralization method, using peptone saline solution as the diluent, malt extract agar as the culture medium, and a neutralizer consisting of polysorbate 80, sodium thiosulfate, lecithin, tryptone, NaCl, phosphate buffer, and water. The stability and appearance of the mixture during the procedure were evaluated as homogeneous and without precipitates. At the end of the contact time, an aliquot was taken and immediately added to the neutralizing solution to stop the bactericidal activity. After the neutralization period, 1 mL of the mixture was plated to check for any bacterial growth. The plates were incubated for 48-72 hours and the point-wise logarithmic reduction in CFUs (Colony Forming Units) was calculated relative to control plates.

[0125] 4. Toxicity

[0126] Cell viability tests were performed by incubating mouse embryonic fibroblasts in the presence of 25 pM, 10 pM or 5 pM RKW. RKW was found to be non-cytotoxic at all concentrations tested, as a cell viability of 93.4%, 97.7% and 99.9%, respectively, was observed. The results are given in Table 5 and demonstrate the full biocompatibility of the peptide.

[0127] Table 5. ISO 10993-5:2009 certified protocol; in vitro assessment of cytotoxicity on Mus musculus cell lines (embryonic fibroblasts). The RKW test result is considered negative (cytotoxic) if the viability is less than 70%.

[0128] The protocol according to ISO 10993-5:2009 (https: / / nhiso.com / wp- provided for the use of B ALB 3T3 mouse embryonic fibroblast cell lines, clone A31 (Kiyoshi Sasaki et al., Recommended protocol for the BALB / c 3T3 cell transformation assay, Mutation Research / Genetic Toxicology and Environmental Mutagenesis, Volume 744, Issue 1, 2012, Pages 30-35, ISSN 1383-5718, https: / / doi.Org / 10.1016 / j.mrgentox.2011.12.014) from ATCC CCL-163. The BALB / c 3T3 cell line is widely used in the Cell Transformation Assay (CTA) to assess how exposure to test substances induces morphological transformations in cultures of these mouse fibroblasts. The BALB / c 3T3 CTA test is particularly useful for screening the cytotoxicity of individual chemicals or complex mixtures, including food contaminants and additives. It is considered an alternative method to animal testing, as it allows for the identification of potential toxic and / or carcinogenic agents in a more ethical and cost-effective way.

[0129] The day before the test, the cells were plated and incubated to allow the formation of subconfluent monolayers. These cells were grown under controlled conditions at a temperature of 37°C ± 1°C, with a concentration of 5% CO2 and a relative humidity of 90% ± 10%. Growth occurred in a specific culture medium, Dulbecco's modified Eagle's medium (DMEM), containing 10% fetal bovine serum and 4 mM glutamine. Several materials and reagents were used for the test, including sodium dodecyl sulfate (SDS) solution as a positive control and Dulbecco's phosphate-buffered saline (D-PBS) solution as a washing solution. After 24 hours, the growth medium was removed and replaced with the peptide concentrations to be tested, the positive control, the negative control, (6 replicates). The peptide was prepared at different concentrations (5 pM, 10 pM, 25 pM) and added to the cells in 96-well plates. The plates were then incubated for an additional 24 hours at a temperature of 37°C ± 1°C, with a concentration of 5% CO2. The Neutral Red Uptake (NRU) test was subsequently performed to assess cell viability and any morphological abnormalities caused by cytotoxicity. Each well was washed with 150 pl of D-PBS and treated with 100 pl of Neutral Red (NR) solution for 3 hours at a temperature of 37°C ± 1°C, with a concentration of 5% CO2. The plates were then washed with 150 pl of D-PBS and stirred in the dark for 10 minutes. Finally, the optical density of the extracted NR solution was measured at 540 nm using a spectrophotometer. This measurement provides a quantitative assessment of the cell viability and cytotoxicity of the tested materials, as the amount of Neutral Red taken up by the viable cells is proportional to their viability. The data obtained were processed to calculate the relative cell viability compared to the control.

[0130] 5. Comparison of the antibacterial activity of RKW peptide (invention) and RiLKl peptide (prior art)

[0131] Table 6 below shows a comparison between the RKW peptide of the invention and the known RiLKl peptide, limited to the bactericidal activity on two commercially important bacterial strains as these organisms may cause food spoilage and food poisoning. Table 6 shows that RKW has an overall better performance against the bacterial strains used, although the RiLKl peptide, in other studies aimed at validating its activity, has shown remarkable specificity and efficiency, for example, against L. monocytogenes . For these and other reasons, the RKW peptide represents a new, extremely promising tool that can be advantageously used alone or in combination with other antimicrobial peptides, such as RiLKl, depending on specific needs.

[0132] Table 6. Comparison of the antibacterial activity of RKW peptide (invention) and RiLKl peptide (prior art). Contact time: 60 min; temperature: 37°C; No= number of cfu / ml in the test mixture at the start of the contact time; R= decrease in viability.

[0133] 6. Synthesis of the RKW peptide

[0134] The peptide representative of the invention RKW was synthesized by solid phase peptide synthesis using the Fluoromethoxycarbonyl (Fmoc) protective group.

[0135] Rink- Amide MBHA resin with a degree of substitution of 0.5 mmol / g was used as the solid support. The resin has a linker that provides an amide bond and releases the peptide amidated at the C-terminus.

[0136] At the end of the synthesis, the protective group was removed by treatment with a 40% (v / v) solution of piperidine in DMF, while the detachment from the resin and the removal of the protective groups from the amino acid side chains were obtained by treatment with an acidic solution consisting of 95% trifluoroacetic acid, 2.5% triisopropylsilane, and 2.5% H2O (v / v / v).

[0137] After the detachment from the solid support, the peptide was precipitated in cold ethyl ether, at -20°C. The sample was centrifuged at 3500 rpm for 5 minutes in order to collect the precipitate. The precipitate was dissolved in a mixture of CH3CN / H2O (95:5), frozen and lyophilized.

Claims

CLAIMS1. An antimicrobial peptide consisting of an amino acid sequence of formula:RX1WILX2WLRTWX3X4 wherein Xi, X2, X3 and X4 are independently selected from K and R, and wherein each amino acid is independently in the L or D configuration, or a salt or solvate thereof.

2. The antimicrobial peptide according to claim 1, wherein the amino acid sequence is selected from the group consisting of:RRWILRWLRTWRR (SEQ ID NO: 1)RRWILRWLRTWRR (SEQ ID NO:2)RRWILRWLRTWRR (SEQ ID NO:3)RRWILRWLRTWRR (SEQ ID NO:4)RRWILRWLRTWRR (SEQ ID NO: 5)RRWILRWLRTWRR (SEQ ID NO: 6)RRWILRWLRTWRR (SEQ ID NO: 7)RRWILRWLRTWRR (SEQ ID NO: 8)RRWILRWLRTWRR (SEQ ID NO: 9)RRWILRWLRTWRR (SEQ ID NO: 10)RRWILRWLRTWRR (SEQ ID NO: 11)RRWILRWLRTWRR (SEQ ID NO: 12)RRWILRWLRTWRR (SEQ ID NO: 13)RRWILRWLRTWRR (SEQ ID NO: 14)RRWILRWLRTWRR (SEQ ID NO: 15)RRWILRWLRTWRR (SEQ ID NO: 16) wherein each amino acid is independently in the L or D configuration, or a salt or solvate thereof.

3. The antimicrobial peptide according to claim 1 or 2, for use as a medicament.

4. The antimicrobial peptide for use according to claim 3, wherein the medicament isan antiseptic.

5. The antimicrobial peptide according to claim 1 or 2, for use in the therapeutic treatment of an infection caused by Gram negative bacteria, Gram positive bacteria, fungi, and / or yeasts.

6. A pharmaceutical formulation comprising at least one antimicrobial peptide according to claim 1 or 2 and at least one pharmaceutically acceptable carrier and / or excipient.

7. An antimicrobial composition comprising at least one antimicrobial peptide according to claim 1 or 2, a carrier, and optionally at least one further antimicrobial agent.

8. The use of an antimicrobial peptide according to claim 1 or 2 as a disinfectant for the prevention of contamination of an article by and / or for the decontamination of an article from Gram-negative bacteria, Gram-positive bacteria, fungi, and / or yeasts, wherein said article is preferably selected from a food, material, container, and tool.

9. The use of an antimicrobial peptide according to claim 1 or 2 as an antiseptic, for the prevention of contamination of a living tissue by and / or for the decontamination of a living tissue from Gram-negative bacteria, Gram-positive bacteria, fungi, and / or yeasts, wherein the living tissue is preferably the epidermis, excluding use in any method for the surgical or therapeutic treatment of the human or animal body and in any diagnostic method applied to the human or animal body.

10. An article having an antimicrobial peptide covalently bound to one surface thereof, or having an antimicrobial peptide contained in a coating adhered to said one surface, wherein the antimicrobial peptide is an antimicrobial peptide according to claim 1 or 2, and wherein the article is preferably selected from a food, material, container, and tool.

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