Mammal milk-derived peptides with antimicrobial activity

Abstract

The present invention provides milk-derived peptide compounds and compositions having antimicrobial activity. These compounds, compositions, and methods are all useful as alternatives to existing antibiotics and antimicrobial agents.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims and enjoys the benefit of U.S. Provisional Application No. 63 / 343,536, filed on May 18, 2022, the entirety of which is incorporated by reference into this application. STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

[0002] This invention was made with government support under Grant No. 1R43 AI165105-01A1 awarded by the National Institute of Allergy and Infectious Diseases (NIAID). The government has certain rights in this invention. Field of the Invention

[0003] The present invention provides milk-derived peptide compounds and compositions with antimicrobial activity. The compounds, compositions and methods can be used as substitutes for existing antibiotics and antimicrobial agents. Background of the Invention

[0004] It is well known that existing antibiotics need to be replaced, and the problem continues to worsen. Mammalian milk is a source of antimicrobial compounds. It is reported that the milk of mammalian mothers was selected during evolution because it can protect infants from bacterial pathogens. Petherick, 2010, Nature, 468, S5-S7; Smilowitz et al., 2014, Annual Review of Nutrition, 34, 143-169; and Walker, 2010, Journal of Pediatrics, 156, S3-S7. It is worth noting that milk protein fragments released during infant digestion have antimicrobial properties. These peptides are usually inactive in the parent protein sequence and become active when released by proteolysis. Dallas et al., 2013, Journal of Proteome Research, 12, 2295-2304.

[0005] As resistance to existing antibiotic compositions increases, milk-derived peptides are a valuable starting point for formulating antimicrobial peptide compositions to meet the growing need for effective antimicrobial compounds. Summary of the Invention

[0006] The present invention provides milk-derived peptides, including antimicrobial compositions, which contain milk-derived peptides with antimicrobial activity. The present invention provides peptides, peptide compositions, peptide variants, and preparations thereof, wherein the peptides are derived from or derived from milk proteins of various mammals. The peptides disclosed in the present invention have antimicrobial activity and can be used for therapeutic purposes, including the treatment and prevention of primary or secondary diseases caused by bacteria, fungi, viruses, and / or other microorganisms. The peptides, compositions, and methods can all be used as substitutes for existing antibiotics and antimicrobial agents. Advantageously, the peptides, compositions, and methods disclosed in the present invention provide effective antibiotic peptide treatments for antibiotic-resistant strains. In addition, the antimicrobial peptides disclosed in the present invention can be used to preserve and stabilize cosmetic formulations.

[0007] In one embodiment, the peptide has an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. Each peptide may have an amino acid sequence consisting of one of SEQ ID NO: 1 to SEQ ID NO: 295.

[0008] The present invention provides a composition comprising a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. In another embodiment, the present invention provides an (antimicrobial) composition comprising one or more peptides or polypeptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295.

[0009] In other embodiments, the composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, the peptide being formulated with other excipients. Typically, the composition has antimicrobial activity. In typical embodiments, the antimicrobial composition comprises a peptide having an amino acid sequence selected from SEQ ID NO: 1 to SEQ ID NO: 217, SEQ ID NO: 219 to SEQ ID NO: 254, and SEQ ID NO: 256 to SEQ ID NO: 295. In an exemplary embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291. In an exemplary embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 256, and SEQ ID NO: 291.In yet other embodiments, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291. In certain embodiments, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 26, SEQ ID NO: 177, SEQ ID NO: 213, and SEQ ID NO: 291, formulated with an excipient disclosed herein. Examples of excipients include solvents and viscosity enhancers.

[0010] In other embodiments, the present invention provides an antimicrobial composition comprising one or more peptides, wherein the one or more peptides have an amino acid sequence selected from any one of the following: SEQ ID NO: 1 to SEQ ID NO: 217, SEQ ID NO: 219 to SEQ ID NO: 254, and SEQ ID NO: 256 to SEQ ID NO: 295. In other embodiments, the present invention provides an antimicrobial composition comprising one or more peptides, wherein the one or more peptides have an amino acid sequence selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291. In other embodiments, the present invention provides an antimicrobial composition comprising one or more peptides, wherein the one or more peptides have an amino acid sequence selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 256, and SEQ ID NO: 291.In yet other embodiments, the present invention provides an antimicrobial composition comprising at least two peptides, each of which has an amino acid sequence selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291.

[0011] In additional embodiments, the antimicrobial composition made from a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 comprises an N-terminal or C-terminal modification, PEGylation, glycosylation, and / or other suitable synthetic modifications. In additional embodiments, the antimicrobial composition comprising one or more peptides comprises an N-terminal or C-terminal modification, PEGylation, glycosylation, and / or other suitable synthetic modifications, wherein the one or more peptides each have an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295.

[0012] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made of a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 comprises a viscosity enhancer. In typical embodiments, the viscosity enhancer is one or more selected from hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone, dextran, and hyaluronic acid. In an exemplary embodiment, the concentration of the viscosity enhancer ranges from 0.4% to 6% based on the total weight or total volume of the composition.

[0013] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made of one or more peptides includes a viscosity enhancer, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. In typical embodiments, the viscosity enhancer is one or more selected from hydroxypropyl cellulose, hydroxypropyl methylcellulose, polyvinyl pyrrolidone, dextran, and hyaluronic acid. In an exemplary embodiment, the concentration of the viscosity enhancer ranges from 0.4% to 6% based on the total weight or total volume of the composition.

[0014] In additional or alternative embodiments, the antimicrobial composition comprises a solvent. In an exemplary embodiment, the solvent is selected from one or more of glycerol, ethanol, methanol, propylene glycol, and / or isopropanol. In an exemplary embodiment, the antimicrobial composition comprises a solvent selected from one or more of glycerol, propylene glycol, DMSO, and isopropanol in a concentration range of 0.1% to 30%; or the antimicrobial composition comprises a solvent selected from methanol and ethanol in a concentration up to 99%.

[0015] In an exemplary embodiment, the composition having antimicrobial activity comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, the peptide being formulated with a solvent and a viscosity enhancer. For example, the composition having antimicrobial activity comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, the peptide being formulated with a solvent and a viscosity enhancer. For example, the solvent may be selected from glycerol, propylene glycol and isopropanol, with a concentration ranging from 0.1% to 30%; or from methanol and ethanol, with a concentration up to 99%; and the viscosity enhancer may be selected from hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone, dextran, or hyaluronic acid. For example, the concentration of the viscosity enhancer is about 0.4% to 6% based on the total weight or total volume of the composition. In a further embodiment, the composition having antimicrobial activity comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, and the peptide is formulated with a solvent and carboxymethyl cellulose. More specifically, the composition having antimicrobial activity comprises a peptide having an amino acid sequence selected from one of SEQ ID NO: 26, SEQ ID NO: 177, SEQ ID NO: 213 and SEQ ID NO: 291, corresponding to peptides M6, M14, M16 and M22 disclosed in the present invention, respectively.

[0016] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 comprises one or more organic acids selected from citric acid, malic acid, tartaric acid, acetic acid, butyric acid, phosphoric acid, lactic acid, indole lactic acid, and / or indoleethanol. In some additional embodiments, the antimicrobial composition comprises an addition salt in combination with the one or more organic acids for a specific pH formulation.

[0017] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from one or more peptides comprises one or more organic acids selected from citric acid, malic acid, tartaric acid, acetic acid, butyric acid, phosphoric acid, indole lactic acid, and / or indoleethanol, wherein each of the one or more peptides has an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. In some additional embodiments, the antimicrobial composition comprises an addition salt in combination with the one or more organic acids for a specific pH formulation.

[0018] In an additional or alternative embodiment of the above disclosure, the antimicrobial composition made of a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 comprises a humectant. In an additional or alternative embodiment of the above disclosure, the humectant is one or more selected from sorbitol, glycerol, propylene glycol, sorbitol, seaweed, urea, peptides, amino acids, and honey. In an additional or alternative embodiment of the above disclosure, the humectant is sorbitol or glycerol. In an additional or alternative embodiment of the above disclosure, the antimicrobial composition comprises a humectant and a preservative selected from benzoic acid, benzyl alcohol, propylparaben, methylparaben, and / or phenoxyethanol.

[0019] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 comprises a humectant. In an exemplary embodiment, the humectant is sorbitol or glycerol. In an exemplary embodiment, the antimicrobial composition comprises a humectant and a preservative selected from benzoic acid, benzyl alcohol, propylparaben, methylparaben, and / or phenoxyethanol.

[0020] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 is formulated as a spray, a gel, an ointment, a lotion, a cream, or an insert tablet.

[0021] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 further comprises an acid, wherein the acid may be in salt form, free acid form, or any ester form thereof.

[0022] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 further comprises an acid, wherein the acid may be in salt form, free acid form, or any ester form thereof.

[0023] In additional or alternative embodiments of the above disclosure, the antimicrobial composition made from one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 further comprises a buffer. In an exemplary embodiment, the buffer is added in a range of 1 mM to 300 mM. In an exemplary embodiment, the buffer is selected from one of the following: acetate, succinate, lactate, fumarate, and any other monocarboxylate and dicarboxylate.

[0024] In some embodiments, the method of treating a microbial infection comprises administering a composition comprising one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 to a subject having a microbial infection. In typical embodiments, the method of treating a microbial infection comprises administering a composition comprising a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 140, SEQ ID NO: 142 to SEQ ID NO: 217, SEQ ID NO: 220 to SEQ ID NO: 254, and / or SEQ ID NO: 256 to SEQ ID NO: 295. For example, the method of treating a microbial infection in a subject comprises administering a composition comprising a peptide having an amino acid sequence selected from any one of SEQ ID NO: 26, SEQ ID NO: 177, SEQ ID NO: 213, and SEQ ID NO: 291 to a subject in need thereof. Microbial infections include bacterial infections, fungal infections, and / or viral infections. In some embodiments, the microbial infection is a bacterial infection.

[0025] In certain embodiments, the polypeptide sequence is combined with additional polypeptide sequences and one or more solvents, one or more viscosity enhancing agents, and one or more organic acids or salts to form a desired formulation.

[0026] In certain embodiments, the administration is via cells containing the polynucleotide sequence encoding the polypeptide sequence for delivery in human or non-human applications.

[0027] In some embodiments, the administration may be topical, intravenous, intraperitoneal, subcutaneous, intramuscular, rectal, nasal, oral, in direct association with a human or non-human animal, or directly on a surface (eg, a medical device or catheter).

[0028] In some embodiments, the peptide is a peptidomimetic of a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO:295. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 The amino acid sequence of camel kappa-casein is shown.

[0030] Figure 2 The amino acid sequence of human milk transferrin is shown.

[0031] Figure 3 The amino acid sequence of goat αS2-casein is shown.

[0032] Figure 4 The amino acid sequence of the human lactase-phlorizin hydrolase protein is shown.

[0033] Figure 5 The amino acid sequence of goat β-casein is shown.

[0034] Figure 6 The amino acid sequence of the bovine lysozyme C isozyme protein is shown.

[0035] Figure 7Depicted are the origin, sequence, and predicted charge of the first polypeptide of each of the polypeptide variant groups M1-M22 (SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 291) disclosed in the present invention.

[0036] Figure 8 shows images of a time-kill assay, indicating that the polypeptides (M1 (SEQ ID NO: 1); M2 (SEQ ID NO: 6); M3 (SEQ ID NO: 11); M4 (SEQ ID NO: 16); M5 (SEQ ID NO: 21); M6 (SEQ ID NO: 26); M7 (SEQ ID NO: 31); and M8 (SEQ ID NO: 36)) target G. vaginalis and L. iners, but not L. crispatus, as shown.

[0037] Fig. 9Each polypeptide (SEQ ID NO: 1 (M1); SEQ ID NO: 6 (M2); SEQ ID NO: 11 (M3); SEQ ID NO: 16 (M4); SEQ ID NO: 21 (M5); SEQ ID NO: 26 (M6); SEQ ID NO: 31 (M7); SEQ ID NO: 36 (M8); SEQ ID NO: 41 (M9); SEQ ID SEQ ID NO: 106 (M11); SEQ ID NO: 141 (M12); SEQ ID NO: 142 (M13); SEQ ID NO: 177 (M14); SEQ ID NO: 178 (M15); SEQ ID NO: 213 (M16); SEQ ID NO: 219 (M18); NO:220(M19); SEQ ID NO:256(M21); and SEQ ID The antibiotic activity of NO:291 (M22)) against Pseudomonas aeruginosa (P. aeruginosa) and Staphylococcus aureus (S. aureus) MRSA (peptide concentrations were 0.375 mg / ml (****), 0.75 mg / ml (***), 1.5 mg / ml (**), or 3 mg / ml (*), as shown in the figure), as shown in the figure.

[0038] Fig. 10A , Fig. 10B and Fig. 10C To depict the presence of Pseudomonas aeruginosa (P. aeruginosa) ( Fig. 10A )、S. aureus MRSA( Fig. 10B ) and Staphylococcus epidermidis (S. epidermidis) ( Fig. 10C Graph of bacterial viability in terms of colony forming units (CFU) per milliliter (ml) over time (minutes) in the presence of selected polypeptides (SEQ ID NO:26 (M6); SEQ ID NO:31 (M7); SEQ ID NO:177 (M14); SEQ ID NO:213 (M16); and SEQ ID NO:291 (M22)) compared to indomethacin (IND).

[0039] Fig.11A , Fig. 11B and Fig. 11CDepicts the polypeptides (SEQ ID NO: 26 (M6); SEQ ID NO: 31 (M7); SEQ ID NO: 177 (M14); SEQ ID NO: 213 (M16); or SEQ ID NO: 291 (M22)) in the presence of Pseudomonas aeruginosa (P. aeruginosa) ( Fig.11A )、S. aureus MRSA( Fig. 11B ) or Staphylococcus epidermidis (S. epidermidis) ( Fig. 11C ) as shown in the figure, wherein each of the indicated polypeptides is formulated in a composition together with 20% propylene glycol and 2% hydroxypropylcellulose (F is a formulation without peptide).

[0040] Fig. 12A , Fig. 12B , Fig. 12C and Fig.12D Depicts the peptides M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) and M22 (SEQ ID NO: 291), respectively, in the presence of G. vaginalis ( Fig. 12A )、Lactobacillus iners (L.iners)( Fig. 12B )、Lactobacillus crispatus (L. crispatus) ( Fig. 12C ) or Acinetobacter baumannii (A.baumannii) ( Fig.12D ) from 0 minutes (min) to 60 minutes of antimicrobial activity retention, wherein each of the indicated peptides was formulated in a composition with 20% propylene glycol and 2% hydroxypropylcellulose (F is a formulation without peptide), as shown in the figure, wherein the amount of growth is indicated as follows: 4 for complete growth; 3 for too many colonies to count; 2 for countable colonies; and 1 for no growth.

[0041] Fig.13 Depicted is the retention of antimicrobial activity of the indicated peptides M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) and M22 (SEQ ID NO: 291) in the presence of Staphylococcus aureus (S. aureus) MRSA from 0 minutes (min) to 60 minutes, wherein each indicated peptide was formulated in a composition with 20% propylene glycol and 2% hydroxypropyl cellulose (F is a formulation without peptide), as shown, wherein the amount of growth is indicated as follows: 4 is complete growth; 3 is too many colonies to count; 2 is countable colonies; and 1 is no growth.

[0042] Fig.14AThe eight most peptide-resistant strains of the MRSN panel of 100 P. aeruginosa strains are listed, along with their sensitive (S) or resistant (R) growth phenotype to the indicated antibiotics.

[0043] Fig. 14B Depicted Fig. 14B The growth of 8 peptide-resistant Pseudomonas aeruginosa (P. aeruginosa) strains in the presence of M14 (SEQ ID NO: 17) and / or M16 (SEQ ID NO: 213) at 0.003% (final concentration of 30 μg / ml) after 24 hours, where the growth amount is indicated as: 4 for complete growth; 3 for too many colonies to count; 2 for countable colonies; and 1 for no growth.

[0044] Figures 15A-15B Shows Fig.14A Peptide M16 (SEQ ID NO: 213) of Pseudomonas aeruginosa strain NR-51553 in 0.01XMH broth ( Fig.15A ) and peptide M16 ( Fig. 15B ) in a modified CLSI-based broth dilution test, where the amount of growth is represented by: 4 for complete growth; 3 for too many colonies to count; 2 for countable colonies; and 1 for no growth.

[0045] Fig.16 Depicted is the growth of S. aureus MRSA in a modified CLSI-based broth dilution assay in the presence or absence of each of the indicated peptides (M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), and M16 (SEQ ID NO: 213), wherein 250 μg / ml of the M14 peptide in 0.01X MH broth was more effective at 4 hours than 250 μg / ml of M16 at 24 hours, wherein the amount of growth is represented by: 4 for complete growth; 3 for too many colonies to count; 2 for countable colonies; and 1 for no growth.

[0046] Fig.17Depicted is the growth of A. baumannii in a modified CLSI-based broth dilution assay in the presence or absence of each of the indicated peptides (M14 (SEQ ID NO: 177) and M16 (SEQ ID NO: 213)), wherein 250 μg / ml of peptide M16 in 0.01X MH broth was more effective at 4 hours than 250 μg / ml of M14 at 24 hours, wherein the amount of growth is represented by: 4 for complete growth; 3 for too many colonies to count; 2 for countable colonies; and 1 for no growth.

[0047] Fig.18A Shown is the in vitro growth of Enterococcus faecalis (E. faecalis) in HBT double layer agar obtained from vaginal swabs of patients with symptoms of bacterial vaginosis, in the absence or presence of peptides M6 (SEQ ID NO:26) and M14 (SEQ ID NO:177) and standard of care (SOC) antibiotics metronidazole (MET) and clindamycin (CLIN) as indicated.

[0048] Fig.18B Shown is the in vitro growth of Candida albicans (C. albicans) on MRS agar obtained from vaginal swabs of patients in the absence or presence of peptides M6 (SEQ ID NO:26) and M14 (SEQ ID NO:177) and standard of care (SOC) antibiotics metronidazole (MET) and clindamycin (CLIN) as indicated.

[0049] Fig.19 It is a graph of cytotoxicity assays performed in vaginal epithelial cells (VK2 / E6E7) and cervical cells (HeLa) in the absence (None) or presence of peptides M6 (SEQ ID NO:26) and M14 (SEQ ID NO:177) at concentrations of 1 mg / ml, 5 mg / ml and 10 mg / ml, using ethanol as a positive control for cytotoxicity, wherein the number of viable cells is proportional to the calcein fluorescence unit (FU), or cell death is expressed as a decrease in calcein FU.

[0050] Fig. 20is a graph of biofilm disruption assays performed on G. vaginalis biofilms in the presence of peptide M6 (SEQ ID NO: 26), wherein a decrease in the amount of crystal violet dye corresponds to disruption of the biofilm, and as shown, disruption of G. vaginalis was observed at concentrations of 0.02 mg / ml, 0.2 mg / ml, and 2.0 mg / ml of M6 (SEQ ID NO: 26).

[0051] Fig.21A , Fig. 21B and Fig. 21C is a graph showing the percentage (%) of HIV infection in TZM-bl cells after exposure to 100 μg / ml, 1000 μg / ml or 5000 μg / ml of peptide M6 (SEQ ID NO: 26) in the presence or absence of HIV-1 for 48 hours, wherein in addition to peptide M6, the cells were: i) co-exposed to HIV alone or medium ( Fig.21A ); ii) pre-exposure to peptide M6 or medium for 6 hours; or iii) exposure to HIV for 2 hours followed by exposure to peptide M6, and then exposure to HIV and peptide M6 for 36 hours.

[0052] Fig. 22 is a graph showing the reduction in the amount of HSV DNA (%) in HSV-2G-infected HEC1A cells, which were infected with HSV-2G virus at the indicated multiplicity of infection (MOI), followed by virus removal, and incubation with 5 mg / ml of peptide M6 (SEQ ID NO: 26) for 3 days, wherein the amount of HSV DNA was then quantified relative to HSV-2G-infected HEC1A cells not incubated with peptide M6 (SEQ ID NO: 26). DETAILED DESCRIPTION OF THE INVENTION

[0053] The peptide sequences derived from mammalian milk proteins disclosed in the present invention or their analogs have activity against antimicrobial pathogens. These peptides are all useful anti-infective agents for inhibiting or preventing the growth of pathogens. These peptides can be applied alone or in the form of formulated compositions (e.g., with excipients). The formulated compositions containing the envisioned peptides include pharmaceutical compositions and / or cosmetic preparations. The envisioned methods include treating subjects (humans or animals) or treating surfaces (e.g., medical devices) with compositions containing the peptides disclosed in the present invention to inhibit or treat / treat (through cell death) the growth of pathogenic microorganisms on (or inside) the subjects or surfaces. The envisioned methods also include preserving (e.g., increasing the shelf life of) the cosmetic composition by adding one or more of the envisioned peptides to the cosmetic composition, thereby adding antimicrobial (e.g., bactericidal and / or antifungal) ingredients to the cosmetic composition. definition

[0054] As used in the present invention, with respect to the bacteria disclosed in the present invention, Pseudomonas aeruginosa refers to Pseudomonas aeruginosa, S. aureus refers to Staphylococcus aureus, S. aureus MRSA refers to methicillin-resistant S. aureus, S. epidermidis refers to Staphylococcus epidermidis, G. vaginalis refers to Gardnerella vaginalis, L. iners refers to Lactobacillus iners, L. crispatus refers to Lactobacillus crispatus, and L. spp. refers to Lactobacillus spp. crispatus, A. baumannii refers to Acinetobacter baumannii, and E. faecalis refers to Enterococcus faecalis.

[0055] As used herein, the 100 MRSN Panel P. aeruginosa refers to a reference panel of 100 P. aeruginosa strains collected by the Multidrug Resistant Organisms Repository and Surveillance Network (MRSN).

[0056] As used herein, with respect to the yeast disclosed herein, Candida albicans may be referred to as C. albicans.

[0057] As used herein, with respect to the viruses disclosed herein, HIV refers to human immunodeficiency virus and HSV refers to herpes simplex virus.

[0058] The term "polynucleotide" used in the present invention refers to oligonucleotides or nucleotides, and fragments or parts thereof, as well as DNA or RNA of genomic or synthetic origin, which can be single-stranded or double-stranded, representing a sense strand or an antisense strand. A single polynucleotide is translated into a single polypeptide.

[0059] As used herein, the terms "peptide" and "polypeptide" are used interchangeably to describe a single polymer in which the monomers are amino acid residues linked together by amide bonds. Polypeptide is intended to encompass any amino acid sequence, whether naturally occurring, recombinant or synthetically produced.

[0060] The terms "peptide mimetic" and "mimetic" all refer to synthetic chemical compounds that have substantially the same functional characteristics as a naturally or non-naturally occurring polypeptide, but have different (but usually similar) structural characteristics. Peptide analogs are commonly used in the art as non-peptide active compounds (e.g., drugs) having properties similar to those of the template peptide. Such non-peptide compounds are referred to as "peptide mimetics" or "mimetic peptides" (Fauchere, J. Adv. Drug Res. 15: 29 (1986); Veber and Freidinger, TINSp. 392 (1985); and Evans et al. J. Med. Chem. 30: 1229 (1987)). Peptide mimetics that are structurally similar to therapeutically useful peptides can be used to produce equivalent or enhanced therapeutic or preventive effects. In general, a peptide mimetic is structurally similar to a paradigm polypeptide (i.e., SEQ ID NO: 1) found in a polypeptide of interest, but has one or more peptide bonds that may be optionally replaced by a bond selected from the group consisting of, for example, —CH2NH—, —CH2S—, —CH2—CH2—, —CH═CH— (cis and trans), —COCH2—, —CH(OH)CH2—, and —CH2SO—. A mimetic may be composed entirely of synthetic non-natural amino acid analogs, or may be a chimeric molecule of partly natural peptide amino acids and partly non-natural amino acid analogs. A mimetic may also contain any number of conservative substitutions of natural amino acids, as long as such substitutions do not significantly change the structure and / or (e.g., antibacterial) activity of the mimetic.

[0061] The term "amino acid" refers to naturally occurring amino acids and synthetic amino acids, as well as amino acid analogs that function similarly to naturally occurring amino acids. Naturally occurring amino acids are the amino acids encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, i.e., an alpha carbon attached to a hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, methionine methylsulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Naturally encoded amino acids are the 20 common amino acids (alanine, arginine, asparagine, aspartic acid, cysteine, glutamine, glutamic acid, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine, and valine) as well as pyrrolysine, pyrro-carboxy-lysine, and selenocysteine.

[0062] As used herein, the terms "substantially identical" or "substantially identical" when used in the context of nucleic acids or polypeptides refer to sequences that have at least 50% sequence identity to a reference sequence. Alternatively, the identity percentage is any integer from 50% to 100%. In some embodiments, a sequence is substantially identical to a reference sequence if it has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to the reference sequence, as described using the standard parameters of BLAST. Thus, embodiments of the invention provide antimicrobial (e.g., antibacterial) peptides that are substantially identical to any one of SEQ ID NO: 1 to SEQ ID NO: 295 if they have at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0063] As used herein, a polynucleotide or polypeptide sequence is "heterologous" or "heterologous" to a cell if the sequence is derived from a different cell, or from the same cell but modified from its native form. For example, when a first amino acid sequence in a protein is referred to as being heterologous to a second amino acid sequence in the same protein, this means that the first amino acid is derived from a first cell or is non-naturally occurring, while the second amino acid is derived from a second cell or is modified from its native form.

[0064] The "pharmaceutically acceptable salt" used in the present invention includes salts formed with inorganic acids or organic acids. In some embodiments, salts formed with inorganic acids include, but are not limited to, hydrochlorides, hydrobromides, nitrates, sulfates, phosphates, and the like. In some embodiments, salts formed with organic acids include, but are not limited to, citrates, maleates, tartrates, acetates, butyrates, phosphates, indole lactate, lactate, formate, trifluoroacetate, propionate, oxalate, ascorbate, fumarate, malonate, methanesulfonate, benzosulfonate, p-toluenesulfonate, and the like.

[0065] The "cosmetically acceptable salts" used in the present invention include salts formed with inorganic acids or organic acids. In some embodiments, salts formed with inorganic acids include, but are not limited to, hydrochlorides, hydrobromides, nitrates, sulfates, phosphates, and the like. In some embodiments, salts formed with organic acids include, but are not limited to, citrates, maleates, tartrates, acetates, butyrates, phosphates, indole lactate, lactate, formate, trifluoroacetate, propionate, oxalate, ascorbate, fumarate, malonate, methanesulfonate, benzosulfonate, p-toluenesulfonate, and the like.

[0066] As used in this specification and the appended claims, references to "about" values ​​or parameters herein include (and describe) variations in the values ​​or parameters themselves. For example, references to "about X" include descriptions of "X".

[0067] Unless otherwise indicated, the terms "about" and "approximately" used in this specification and the appended claims, when used in conjunction with various terms such as temperature, dose, amount or weight percentage of an ingredient of a composition or dosage form, refer to, for example, a temperature, dose, amount or weight percentage recognized by a person of ordinary skill in the art to provide an effect equivalent to the effect obtained from the specified temperature, dose, amount or weight percentage. Specifically, when used in the present invention, the terms "about" and "approximately" encompass temperatures, doses, amounts or weight percentages, etc. within 15%, within 10%, within 5%, within 4%, within 3%, within 2%, within 1%, or within 0.5% of the specified temperature, dose, amount or weight percentage, etc.

[0068] As used in this specification and the appended claims, it is understood that a 1.0% concentration refers to weight per volume, for example a 1.0% concentration is 10 mg / ml, a 0.5% concentration is 5 mg / ml, and so forth.

[0069] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, "peptide" may mean "one or more peptides", each of which is independently selected; "solvent" may mean "one or more solvents"; "humectant" may mean "one or more humectants", and so on. Detailed description

[0070] The envisioned peptides disclosed in the present invention correspond to one of 22 peptide groups (i.e., 22 peptide families), which are referred to herein as M1 to M22. For example, the peptides represented by SEQ ID NO: 1 to SEQ ID NO: 295 encompass each of the peptides in M1 to M22 listed in Tables 1A-1L.

[0071] In some embodiments, the peptides are derived from naturally occurring milk proteins. Thus, the "derived" peptides are mutants, variants or unique fragments of naturally occurring proteins and therefore, as presently disclosed, do not occur in nature. Figure 1-6 The amino acid sequences of milk proteins derived from each of the envisioned peptides are shown. The sequences of camel kappa-casein, human milk transferrin, goat αS2-casein, human lactase-phlorizin hydrolase, goat β-casein, and bovine lysozyme C isozyme protein are shown, respectively.

[0072] The contemplated composition may include a combination of one or more peptides disclosed herein. For example, the composition may include at least two peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. More specifically, the contemplated composition includes at least two peptides having an amino acid sequence selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291.

[0073] Reference Figure 7 , the native proteins ("PROTEIN") of the peptide groups M1 to M22 are provided, and the amino acid sequence ("SEQUENCE") of the first peptide of each group is provided. If the first peptide sequence is modified from the native protein ("M") shown, the modified amino acid is underlined. As shown in the figure, the M1 peptide group corresponds to the camel κ-casein amino acid sequence, UniProtKB-P79139 (CASK_CAMDR) ( Figure 1 ). The M2 and M9-M22 peptide groups correspond to the amino acid sequence of human milk transferrin, UniProtKB-P02788 (TRFL_HUMAN) ( Figure 2 ). The M3 and M4 peptide groups correspond to the goat αS2-casein amino acid sequence, UniProtKB-P33049 (CASA2_CAPHI) ( Figure 3 ). The M5 and M6 peptide groups correspond to the amino acid sequence of human lactase-phlorizin hydrolase, UniProtKB-P09848 (LPH_HUMAN) ( Figure 4 ). The M7 peptide group corresponds to the goat β-casein amino acid sequence, UniProtKB-P33048 (CASB_CAPHI) ( Figure 5 ). The M8 peptide group corresponds to the amino acid sequence of bovine lysozyme C isozyme, UniProtKB-Q6B411 (LYSM_BOVIN) ( Figure 6 ).

[0074] Reference Figure 7 As well as the peptide sequences listed in Tables 1A-1L, specific differences of the envisioned peptides represented by SEQ ID NO: 1 to SEQ ID NO: 295 are disclosed. For example, the envisioned peptides may comprise an amino acid sequence having 0, 1, 2, 3 or 4 amino acid changes compared to the natural peptide sequence.

[0075] Contemplated milk-derived peptides may be at least 8 amino acids in length and up to 200 amino acids in length. In exemplary embodiments, the peptides are 8 to 200, 8 to 100, 8 to 50, 8 to 40, 8 to 30, 8 to 20, 8 to 15, 8 to 14, 8 to 13, 8 to 12, 8 to 11, 8 to 10, 8 to 9, or 8 amino acids in length. In exemplary embodiments, the peptides are 8 to 15 amino acids in length.

[0076] In addition, the milk-derived protein can be fused with an independent (non-milk-derived) heterologous protein or heterologous peptide (e.g., a marker or purification tag). For example, with respect to SEQ ID NO: 1 to SEQ ID NO: 295, each of these peptides with a length of 8 to 15 amino acids can be fused with a heterologous peptide or heterologous protein.

[0077] In some embodiments, the envisioned peptides have antimicrobial activity. The antimicrobial activity includes any reduction in microbial growth. In an exemplary embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. In a more typical embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 217, SEQ ID NO: 220 to SEQ ID NO: 254, and / or SEQ ID NO: 256 to SEQ ID NO: 295. In an exemplary embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291. In an exemplary embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 220, SEQ ID NO: 256, and SEQ ID NO: 291. In a further example, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 26, SEQ ID NO: 177, SEQ ID NO: 213, and SEQ ID NO: 291.

[0078] It is noteworthy that the antimicrobial peptide composition used to inhibit the growth of microorganisms on the surface or skin (for example, in a cosmetic composition or a wound composition) can be up to 50 mg / ml in concentration. Therefore, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, wherein the amount of the peptide in the antimicrobial composition ranges from about 0.020 mg / ml to up to 50 mg / ml of the peptide. In an exemplary embodiment, an antimicrobial composition for inhibiting or treating the growth of microorganisms on a surface or skin comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, wherein the amount of the peptide in the antimicrobial composition ranges from about 0.1 mg / ml to up to 45 mg / ml; about 0.5 mg / ml to about 40 mg / ml; 1 mg / ml to about 50 mg / ml; 1 mg / ml to about 40 mg / ml; about 1 mg / ml to about 30 mg / ml; about 1 mg / ml to about 20 mg / ml; about 5 mg / ml to about 50 mg / ml; about 5 mg / ml to about 25 mg / ml; about 5 mg / ml to about 20 mg / ml; about 5 mg / ml to about 15 mg / ml; about 5 mg / ml to about 10 mg / ml; or about 10 mg / ml to about 40 mg / ml.

[0079] For administration of an antimicrobial peptide composition to a subject, the peptide concentration may be from about 0.02 mg / ml (0.002%) up to 20 mg / ml (2.0%). In an exemplary embodiment, an antimicrobial composition for inhibiting or treating the growth of microorganisms in a subject comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, wherein the amount of the peptide in the antimicrobial composition ranges from about 0.1 mg / ml to up to 20 mg / ml; about 0.1 mg / ml to about 10 mg / ml; 0.1 mg / ml to about 8 mg / ml; 0.1 mg / ml to about 7 mg / ml; about 0.1 mg / ml to about 6 mg / ml; about 0.1 mg / ml to about 5 mg / ml; about 0.1 mg / ml to about 4 mg / ml; about 0.1 mg / ml to about 3.5 mg / ml; about 0.1 mg / ml to about 3.0 mg / ml; about 0.1 mg / ml to about 2.5 mg / ml; about 0.1 mg / ml to about 2.0 mg / ml; or about 0.10 mg / ml to about 1.0 mg / ml. In other exemplary embodiments, an antimicrobial composition for inhibiting or treating the growth of microorganisms in a subject comprises a peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, wherein the amount of the peptide in the antimicrobial composition ranges from about 0.02 mg / ml to up to about 5 mg / ml; about 0.02 mg / ml to about 4 mg / ml; about 0.02 mg / ml to about 3 mg / ml; about 0.02 mg / ml to about 2 mg / ml; about 0.50 mg / ml to about 10 mg / ml; about 0.5 mg / ml to about 8 mg / ml; about 0.5 mg / ml to about 6 mg / ml; about 0.5 mg / ml to about 4 mg / ml; or about 0.5 mg / ml to about 2 mg / ml.

[0080] An antimicrobial composition comprising a peptide having an amino acid sequence selected from SEQ ID NO: 26, SEQ ID NO: 177, SEQ ID NO: 213 and / or SEQ ID NO: 291 can be formulated with the viscosity-increasing agent disclosed herein.

[0081] In some embodiments, the envisioned peptides have antimicrobial activity. The antimicrobial activity includes any reduction in microbial growth. In an exemplary embodiment, the antimicrobial composition comprises one or more peptides or polypeptides, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. In a more typical embodiment, the antimicrobial composition comprises one or more peptides, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 217, SEQ ID NO: 220 to SEQ ID NO: 254, and / or SEQ ID NO: 256 to SEQ ID NO: 295. In an exemplary embodiment, the antimicrobial composition comprises one or more peptides, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291. In an exemplary embodiment, the antimicrobial composition comprises one or more peptides, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 220, SEQ ID NO: 256, and SEQ ID NO: 291.

[0082] In some embodiments, the envisioned peptides have favorable selective antimicrobial activity. Notably, as shown in Figure 8, exemplary peptides (SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31 and SEQ ID NO: 36) are all able to kill major pathogens, including Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus) (including MRSA), Staphylococcus epidermidis (S. epidermidis), vaginal Gardnerella (G. vaginalis), and advantageously, cannot kill the common strain Lactobacillus crispatus (L. crispatus) found in a healthy vagina.

[0083] In some embodiments, the contemplated peptides have antimicrobial activity against one or more pathogens, and / or the contemplated peptides have enhanced efficacy against one or more pathogens. Fig. 9 As shown, exemplary peptides M6, M7, M8, M14 and M15 (SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:177 and SEQ ID NO:178, respectively) are all more effective in reducing the viability of Pseudomonas aeruginosa (P. aeruginosa) than peptides M1, M2, M3, M4 and M5 (SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16 and SEQ ID NO:21, respectively).

[0084] The contemplated composition comprises a combination of at least two peptides disclosed herein. For example, the antimicrobial composition comprises at least two peptides, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. In some embodiments, the antimicrobial composition comprises at least two peptides, each of which has an amino acid sequence selected from any one of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256 and SEQ ID NO: 291. In some embodiments, the antimicrobial composition of at least two peptides disclosed herein can provide prolonged antimicrobial activity.

[0085] For the envisioned compositions having antimicrobial activity, the compositions comprise a peptide selected from any one of SEQ ID NOs: 1-295, formulated with a solvent and a viscosity enhancer. Non-limiting examples of suitable solvents include glycerol, ethanol, methanol, propylene glycol, and / or isopropanol. In an exemplary embodiment, the antimicrobial composition comprises a solvent selected from glycerol, propylene glycol, or isopropanol in a concentration range of 0.1% to 30% by volume; or the antimicrobial composition comprises a solvent selected from methanol or ethanol in a concentration up to 99% by volume.

[0086] In an exemplary embodiment, the antimicrobial composition comprises a peptide having an amino acid sequence selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291.

[0087] A method for inhibiting microbial growth or treating microbial infection in cells or subjects, comprising administering to infected cells or subjects a composition comprising a peptide selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295. The antimicrobial peptide composition can be prepared with a solvent and a viscosity increasing agent. The microorganism can be a bacterium, a fungus (e.g., a yeast), or a viral pathogen.

[0088] Reference Figures 10A-10C and Figures 11A-11C , the peptide or peptide composition disclosed in the present invention is applied in a method for inhibiting microbial growth or treating microbial infection of a bacterial pathogen (e.g., Pseudomonas aeruginosa (P.aeruoginosa), Staphylococcus aureus (S.aureus) or Staphylococcus epidermidis (S.epidermis)). The method for inhibiting the growth of Pseudomonas aeruginosa (P.aeruoginosa) or Staphylococcus epidermidis (S.epidermis) or treating its infection comprises applying an antimicrobial peptide selected from M6 (SEQ ID NO:26), M7 (SEQ ID NO:31), M14 (SEQ ID NO:177) and M16 (SEQ ID NO:213) to infected cells or subjects. The method for inhibiting the growth of Staphylococcus aureus (S.aureus) or treating its infection comprises applying M14 peptide (SEQ ID NO:177) or a composition comprising the M14 peptide (SEQ ID NO:177).

[0089] Reference Figures 12A-12D and Fig.17, the peptides or peptide compositions disclosed in the present invention are used in a method for inhibiting microbial growth or treating microbial infection of a bacterial pathogen (e.g., G. vaginalis, L. iners, or A. baumannii). The method for inhibiting the growth of G. vaginalis, L. iners, or A. baumannii or treating its infection comprises administering an antimicrobial peptide selected from M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213), and M22 (SEQ ID NO: 291). Advantageously, Lactobacillus crispatus is not sensitive to M6 (SEQ ID NO: 26) ( Fig. 12C In addition, the growth of A. baumannii was more sensitive to M16 (SEQ ID NO: 213) than to M22 (SEQ ID NO: 291) ( Fig.12D ). Therefore, the method of inhibiting the growth of vaginal Gardnerella vaginalis (G. Vaginalis), Lactobacillus iners (L. iners) or Acinetobacter baumannii (A. baumannii) or treating its infection comprises administering a peptide or peptide composition of M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) or M22 (SEQ ID NO: 291), wherein the administration of the M6 ​​peptide or peptide composition does not inhibit Lactobacillus crispatus (L. crispatus), and the administration of the M16 peptide or peptide composition can more effectively treat Acinetobacter baumannii (A. baumannii).

[0090] Reference Fig.13 A peptide composition comprising a peptide of M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) or M22 (SEQ ID NO: 291) is formulated with a solvent and a viscosity enhancer to inhibit the growth of or treat infection with Staphylococcus aureus (S. aureus) MRSA. The peptide concentration in the formulation may range from 0.10 wt % to 1.0 wt %. The addition of the solvent and viscosity enhancer disclosed in the present invention may enhance the antimicrobial activity of the peptide composition. For example, the peptide composition may comprise a solvent of 20% propylene glycol and 2% hydroxypropyl cellulose.

[0091] Reference Fig.14A , 14B, 15A and 15B, the peptides or peptide compositions disclosed in the present invention are applied in a method for inhibiting microbial growth or treating microbial infection of a bacterial pathogen (e.g., a strain of Pseudomonas aeruginosa). The method for inhibiting the growth of Pseudomonas aeruginosa (P. aeruginosa) or treating its infection comprises applying an antimicrobial peptide selected from M14 (SEQ ID NO: 177) and M16 (SEQ ID NO: 213). The peptide composition of M14 (SEQ ID NO: 177) or M16 (SEQ ID NO: 213) for treating Pseudomonas aeruginosa (P. aeruginosa) is formulated with a viscosity enhancer disclosed in the present invention to increase the microbial activity of the composition. For example, referring to Fig. 14B and Fig. 15B , M14 (SEQ ID NO: 177) or M16 (SEQ ID NO: 213) peptide compositions were formulated together with carboxymethyl cellulose (CMC).

[0092] In some embodiments, the peptides and polypeptides are synthesized by chemical synthesis of amino acids using a synthesizer, or by using an expression system (including but not limited to bacteria, yeast, insect or mammalian systems). The peptides can also be produced by enzymatic cleavage or any suitable chromatographic separation method.

[0093] For purification and / or separation, the peptide can be fused to any suitable tag. Examples of suitable tags include, but are not limited to, MBP, HIS6, protein A, protein G, GST, HSA, or fluorescent tags. In addition or alternatively, the peptide can be fused to a cleavage site.

[0094] Can be by selecting the preparation of antimicrobial peptide composition, described antimicrobial peptide composition is applied to mankind or non-human animal or is applied to surface to suppress or prevent the microbial growth therein or thereon.Therefore, described peptide composition can comprise one or more peptides, and described peptide is in salt form or as the ester of peptide disclosed by the present invention.Peptide composition disclosed by the present invention can comprise the acid selected from salt, free acid or any ester.

[0095] Administration to humans and non-human animals can be achieved using formulations comprising one or more solvents, one or more acids, one or more peptides in salt form, one or more peptide esters, one or more buffers, one or more viscosity enhancers, stabilizers, and humectants.

[0096] The contemplated compositions disclosed herein may include a stabilizer. In an exemplary embodiment, the stabilizer is selected from one or more of carboxymethyl cellulose, pectin, guar gum, and gelatin.

[0097] The composition disclosed in the present invention may include a viscosity enhancer. For example, the viscosity enhancer is independently selected from one or more of hydroxypropyl cellulose, hydroxypropyl methylcellulose, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone, dextran and hyaluronic acid. In an exemplary embodiment, the concentration range of the viscosity enhancer is about 0.4 volume % to about 6 volume % of the composition.

[0098] The contemplated compositions disclosed herein may include one or more organic acids selected from citric acid, malic acid, tartaric acid, acetic acid, butyric acid, phosphoric acid, lactic acid, indole lactic acid, and / or indoleethanol. In some additional embodiments, the antimicrobial composition includes an addition salt in combination with the one or more organic acids for a specific pH formulation.

[0099] Contemplated compositions disclosed herein may include N-terminal or C-terminal modifications, PEGylation, ethylene glycolation, and / or other suitable synthetic modifications thereof.

[0100] The envisioned compositions disclosed herein may include a humectant. In some embodiments, the humectant is selected from one or more of sorbitol, glycerol, propylene glycol, sorbitol, seaweed, urea, peptides, amino acids, and honey. In some embodiments, the envisioned compositions disclosed herein may include a humectant. In some embodiments, the humectant is sorbitol or glycerol. In some embodiments, the antimicrobial composition includes a humectant and one or more preservatives independently selected from benzoic acid, benzyl alcohol, propylparaben, methylparaben, and phenoxyethanol.

[0101] The peptides and peptide compositions disclosed in the present invention can be formulated into sprays, gels, ointments, lotions, creams, or insert tablets (for vaginal delivery). In some embodiments, the antimicrobial composition includes a solvent. In an exemplary embodiment, each solvent is independently selected from glycerol, ethanol, methanol, propylene glycol, and isopropanol. In an exemplary embodiment, the antimicrobial composition includes one or more solvents, and the one or more solvents are independently selected from glycerol, propylene glycol and isopropanol, and the concentration range is about 0.1% to about 30% based on the gross weight or total volume of the composition; or the antimicrobial composition includes a solvent, and the solvent is selected from methanol and ethanol, and the concentration is up to about 99% based on the gross weight or total volume of the composition.

[0102] Contemplated compositions of the present disclosure may include an acid selected from a salt, a free acid, or any ester.

[0103] The contemplated compositions disclosed herein may include a buffer. In an exemplary embodiment, the buffer is added in an amount of about 1 mM to about 300 mM. In an exemplary embodiment, the buffer is one or more substances independently selected from acetate, succinate, lactate, fumarate, or any other monocarboxylate and dicarboxylate.

[0104] In some embodiments, the method of treating a microbial infection comprises administering a composition comprising one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295 to a subject suffering from a microbial infection. In an exemplary embodiment, the method of treating a microbial infection comprises administering a composition comprising one or more peptides having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 140, SEQ ID NO: 142 to SEQ ID NO: 217, SEQ ID NO: 220 to SEQ ID NO: 254, and / or SEQ ID NO: 256 to SEQ ID NO: 295. In an exemplary embodiment, the method of treating a microbial infection comprises administering a composition comprising one or more peptides having an amino acid sequence selected from any one of M6 (SEQ ID NO: 26), M7 (SEQ ID NO: 31), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) and M22 (SEQ ID NO: 291). Microbial infections include bacterial infections, fungal infections, and / or viral infections. In exemplary embodiments, the microbial infection comprises a bacterial infection or a viral infection.

[0105] In certain embodiments, the administration is via cells containing a polynucleotide sequence encoding a polypeptide sequence for delivery in human or non-human applications.

[0106] Antimicrobial compositions disclosed in the present invention also include cosmetic compositions, and the cosmetic compositions include one or more peptides disclosed in the present invention. For example, the antimicrobial compositions disclosed in the present invention can be combined with cosmetic compositions or formulated into cosmetic compositions. In some embodiments, compared with cosmetic compositions without antimicrobial compositions, the combination of the envisioned antimicrobial compositions (e.g., one or more peptides represented by SEQ ID NO:1 to SEQ ID NO:295) and any suitable cosmetic compositions prolongs the stability and shelf life of the cosmetic compositions. The types of cosmetic compositions are not limited and may include creams, lotions, cleansers, solutions (e.g., toners, water), essences, ointments, and makeup products (blush, pressed powder, eyeliners, and mascara).

[0107] In addition, the topical skin composition for improving skin health may include one or more peptides disclosed in the present invention. Adding antimicrobial peptides (e.g., having bactericidal and / or antifungal activity) to the skin can reduce inflammation, improve wound healing, and / or enhance the integrity of the skin barrier. Therefore, the topical skin composition for improving skin health includes one or more peptides independently selected from SEQ ID NO:1 to SEQ ID NO:295. The topical skin composition for enhancing skin health may include peptides disclosed in the present invention, and its concentration is about 0.1mg / ml to 50mg / ml of peptides. The topical skin composition may include a peptide concentration selected from the following ranges: about 0.1mg / ml to 50mg / ml; about 1mg / ml to about 50mg / ml; about 2mg / ml to about 45mg / ml; about 5mg / ml to about 40mg / ml; about 5mg / ml to about 30mg / ml; about 5mg / ml to about 25mg / ml; about 5mg / ml to about 20mg / ml; about 5mg / ml to about 15mg / ml; and about 10mg / ml to about 30mg / ml. The topical skin composition may comprise from about 0.25 mg / ml to about 10 mg / ml; from about 0.25 mg / ml to about 5 mg / ml; from about 0.50 mg / ml to about 10 mg / ml; from about 1 mg / ml to about 10 mg / ml; from about 2 mg / ml to about 10 mg / ml; from about 3 mg / ml to about 10 mg / ml; from about 4 mg / ml to about 10 mg / ml; from about 5 mg / ml to about 10 mg / ml; from about 6 mg / ml to about 10 mg / ml; from about 7 mg / ml to about 10 mg / ml; from about 8 mg / ml to about 10 mg / ml; from about 9 mg / ml to about 10 mg / ml; from about 0.50 mg / ml to about 5 mg / ml; from about 1 mg / ml to about 8 mg / ml; from about 2 mg / ml to about 8 mg / ml; and from about 2 mg / ml to about 6 mg / ml. Specifically, the topical skin composition comprises a peptide selected from M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) and M22 (SEQ ID NO: 291). The method of enhancing skin health comprises applying a topical composition to the skin of a subject (e.g., a human or an animal), the topical composition comprising a peptide selected from M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) and M22 (SEQ ID NO: 291), and the peptide concentration is within the range disclosed herein. M1-M22 and variant peptides (SEQ ID NO: 1 to SEQ ID NO: 295)

[0108] Table 1A. M1-M2 peptide variants <![CDATA[ M1 ]]> <![CDATA[ M2 ]]> SEQ ID NO:1 PKVERRPRPR SEQ ID NO:6 GKRKPVTRAR SEQ ID NO:2 PKVERRPRP SEQ ID NO:7 GKRKPVTRA SEQ ID NO:3 PKVERRPR SEQ ID NO:8 GKRKPVTR SEQ ID NO:4 KVERRPRPR SEQ ID NO:9 KRKPVTRAR SEQ ID NO:5 VERRPRPR SEQ ID NO:10 RKPVTRAR

[0109] Table 1B. M3-M4 peptide variants <![CDATA[ M3 ]]> <![CDATA[ M4 ]]> SEQ ID NO:11 QHQKAKKPWT SEQ ID NO:16 HQKMAKPWTQ SEQ ID NO:12 QHQKAKKPW SEQ ID NO:17 HQKMAKPWT SEQ ID NO:13 QHQKAKKP SEQ ID NO:18 HQKMAKPW SEQ ID NO:14 HQKAKKPWT SEQ ID NO:19 QKMAKPWTQ SEQ ID NO:15 QKAKKPWT SEQ ID NO:20 KMAKPWTQ

[0110] Table 1C. M5-M6 peptide variants <![CDATA[ M5 ]]> <![CDATA[ M6 ]]> SEQ ID NO:21 NTNRPRTKRA SEQ ID NO:26 RPRIPKASAK SEQ ID NO:22 NTNRPRTKR SEQ ID NO:27 RPRIPKASA SEQ ID NO:23 NTNRPRTK SEQ ID NO:28 RPRIPKAS SEQ ID NO:24 TNRPRTKRA SEQ ID NO:29 PRIPKASAK SEQ ID NO:25 NRPRTKRA SEQ ID NO:30 RIPKASAK

[0111] Table 1D. M7-M8 peptide variants <![CDATA[ M7 ]]> <![CDATA[ M8 ]]> SEQ ID NO:31 PVPQKVRPQR SEQ ID NO:36 KTPKAVNKCR SEQ ID NO:32 PVPQKVRPQ SEQ ID NO:37 KTPKAVNKC SEQ ID NO:33 PVPQKVRP SEQ ID NO:38 KTPKAVNK SEQ ID NO:34 VPQKVRPQR SEQ ID NO:39 TPKAVNKCR SEQ ID NO:35 PQKVRPQR SEQ ID NO:40 PKAVNKCR

[0112] Table 1E. M9 peptide variants <![CDATA[ M9 ]]> <![CDATA[ Continued from M9 ]]> SEQ ID NO:41 ATKCKQWQRNMRKVR SEQ ID NO:62 CKQWQRNMRKVR SEQ ID NO:42 ATKCKQWQRNMRKV SEQ ID NO:63 CKQWQRNMRKV SEQ ID NO:43 ATKCKQWQRNMRK SEQ ID NO:64 CKQWQRNMRK SEQ ID NO:44 ATKCKQWQRNMR SEQ ID NO:65 CKQWQRNMR SEQ ID NO:45 ATKCKQWQRNM SEQ ID NO:66 CKQWQRNM SEQ ID NO:46 ATKCKQWQRN SEQ ID NO:47 ATKCKQWQR SEQ ID NO:48 ATKCKQWQ SEQ ID NO:49 TKCKQWQRNMRKVR SEQ ID NO:67 KQWQRNMRKVR SEQ ID NO:50 TKCKQWQRNMRKV SEQ ID NO:68 KQWQRNMRKV SEQ ID NO:51 TKCKQWQRNMRK SEQ ID NO:69 KQWQRNMRK SEQ ID NO:52 TKCKQWQRNMR SEQ ID NO:70 KQWQRNMR SEQ ID NO:53 TKCKQWQRNM SEQ ID NO:54 TKCKQWQRN SEQ ID NO:55 TKCKQWQR SEQ ID NO:56 KCKQWQRNMRKVR SEQ ID NO:71 QWQRNMRKVR SEQ ID NO:57 KCKQWQRNMRKV SEQ ID NO:72 QWQRNMRKV SEQ ID NO:58 KCKQWQRNMRK SEQ ID NO:73 QWQRNMRK SEQ ID NO:59 KCKQWQRNMR SEQ ID NO:60 KCKQWQRNM SEQ ID NO:74 WQRNMRKVR SEQ ID NO:61 KCKQWQRN SEQ ID NO:75 WQRNMRKV

[0113] Table 1F. M10 peptide variants

[0114] Table 1G. M11 peptide variants <![CDATA[ M11 ]]> <![CDATA[ Continued from M11 ]]> SEQ ID NO:106 KTKCKQRQRNMRKVR SEQ ID NO:127 CKQRQRNMRKVR SEQ ID NO:107 KTKCKQRQRNMRKV SEQ ID NO:128 CKQRQRNMRKV SEQ ID NO:108 KTKCKQRQRNMRK SEQ ID NO:129 CKQRQRNMRK SEQ ID NO:109 KTKCKQRQRNMR SEQ ID NO:130 CKQRQRNMR SEQ ID NO:110 KTKCKQRQRNM SEQ ID NO:131 CKQRQRNM SEQ ID NO:111 KTKCKQRQRN SEQ ID NO:112 KTKCKQRQR SEQ ID NO:113 KTKCKQR SEQ ID NO:114 TKCKQRQRNMRKVR SEQ ID NO:132 KQRQRNMRKVR SEQ ID NO:115 TKCKQRQRNMRKV SEQ ID NO:133 KQRQRNMRKV SEQ ID NO:116 TKCKQRQRNMRK SEQ ID NO:134 QRQRNMRK SEQ ID NO:117 TKCKQRQRNMR SEQ ID NO:135 QRQRNMR SEQ ID NO:118 TKCKQRQRNM SEQ ID NO:119 TKCKQRQRN SEQ ID NO:120 TKCKQRQR SEQ ID NO:121 KCKQRQRNMRKVR SEQ ID NO:136 QRQRNMRKVR SEQ ID NO:122 KCKQRQRNMRKV SEQ ID NO:137 QRQRNMRKV SEQ ID NO:123 KCKQRQRNMRK SEQ ID NO:138 QRQRNMRK SEQ ID NO:124 KCKQRQRNMR SEQ ID NO:125 KCKQRQRNM SEQ ID NO:139 RQRNMRK SEQ ID NO:126 KCKQRQ SEQ ID NO:140 RQR

[0115] Table 1H. M12, M13 and M14 peptide variants

[0116] Table 1I. M15 peptide variants

[0117] Table 1J. M16, M17, M18, M20, M22 peptide variants

[0118] Table 1 K.M19 peptide variants

[0119] Table 1 L. M21 peptide variants Example

[0120] The synthetic peptides M1-M16 and the natural peptides M17-M22 correspond to SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, or SEQ ID NO: 291, respectively, and are disclosed in the present invention and are Figure 7 Examples of some mammalian milk protein peptides are listed in. Peptides M1-M22 are all derived from or from camel κ-casein ( Figure 1 ), human milk transferrin ( Figure 2 )、goat αS2-casein ( Figure 3 )、Human lactase-phlorizin hydrolase ( Figure 4 )、goat β-casein ( Figure 5 ), and bovine lysozyme C isozyme ( Figure 6 ).

[0121] As shown in Tables 1A to 1L, the peptides disclosed in the present invention are 8-15 amino acids in length and are cationic in nature, ie, the peptides are positively charged, with charges ranging from 2.1 to up to 7.9.

[0122] For some sequences, the native sequence could not be synthesized, so only modified synthetic peptides were analyzed. For example, the synthesis of the native sequence derived from M14 (SEQ ID NO: 177) failed. Similarly, the synthesis of the native sequences derived from M9 (SEQ ID NO: 41), M10 (SEQ ID NO: 76) and M11 (SEQ ID NO: 106) also failed. The peptides were purchased from Genescript.

[0123] M1-M8 (SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31 and SEQ ID NO: 36) were tested using a direct killing assay. Equal amounts of inocula of G. vaginalis, L. iners and L. crispatus were incubated with water (0.00) or 0.375 mg / ml, 0.75 mg / ml and 1.5 mg / ml of the peptides. After 30 minutes of incubation, aliquots were spotted on HBT bilayer medium, tryptic soy agar (+5% sheep blood) or MRS agar plates, respectively. For these peptides, a concentration of 0.375 mg / ml was sufficient to inactivate the viability of G. vaginalis and L. iners compared to the absence of the peptides.

[0124] like Fig. 8A As shown, M1-M8 (SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31 and SEQ ID NO: 36) were tested using a direct killing assay. Equal amounts of Gardnerella vaginalis, L. iners and L. crispatus inocula were incubated with water (0.00) or 0.375 mg / ml, 0.75 mg / ml and 1.5 mg / ml of the peptide. After 30 minutes of incubation, aliquots were spotted on human blood Tween (HBT) bilayers, tryptic soy agar (+5% sheep blood) or MRS (de Man, Rogosa, and Sharpe) agar plates. For these peptides, a concentration of 0.375 mg / ml was sufficient to inactivate G. vaginalis and L. iners compared to the absence of peptide. Figure 8B The relative minimum bactericidal concentration (MBC) for each of the M1-M8 peptides is provided.

[0125] like Fig. 9As shown, the bacterial pathogens Pseudomonas aeruginosa (P.aeruginosa) and Staphylococcus aureus (S.aureus) associated with skin, burn and wound infections were tested. As shown, Pseudomonas aeruginosa (P.aeruginosa) is more sensitive to the peptides than Staphylococcus aureus (S.aureus), and some peptides are more effective than other peptides in time-killing tests. For example, compared with M1-M5 (SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16 and SEQ ID NO:21), M6-M8 (SEQ ID NO:26, SEQ ID NO:31 and SEQ ID NO:36), M14 (SEQ ID NO:177) and M15 (SEQ ID NO:178) are more effective in reducing the viability of Pseudomonas aeruginosa (P.aeruginosa).

[0126] like Fig. 10A , 10B As shown in Figures 10 and 10C, the indicated peptides were used at a final concentration of 0.5% (5 mg / ml) to determine quantitative colony forming units / ml. For this colony formation assay, the peptides were incubated with the indicated bacterial strains in 0.8% NaCl, and aliquots were taken starting at time T=0 for up to 60 minutes, then serially diluted and plated on tryptic soy agar plates, incubated overnight, and CFU / ml were counted. It was observed that P. aeruginosa was particularly sensitive to peptides M6 (SEQ ID NO: 26), M7 (SEQ ID NO: 31), M14 (SEQ ID NO: 177), and M16 (SEQ ID NO: 213). Similarly, S. aureus and S. epidermidis were also sensitive to these peptides. Specifically, as shown, after 20 minutes, both P. aeruginosa and S. aureus MRSA appeared to be more susceptible to these peptides than to another cationic peptide, Indolicidin.

[0127] like Fig.11A , 11BAs shown in Figures 11 and 11C, the effects of formulations containing 20% ​​propylene glycol and 2% hydroxypropylcellulose and 0.5% by weight of peptides M6 (SEQ ID NO: 26), M7 (SEQ ID NO: 31), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) or M22 (SEQ ID NO: 291) were tested in a colony formation assay. Although the formulation itself (F) (without peptides) did not alter the viability of the cells, the presence of 0.5% final concentration of peptides in the formulation still resulted in the antibacterial activity of the selected peptides. Pseudomonas aeruginosa and S. epidermidis were both more sensitive to activity than S. aureus.

[0128] like Fig. 12A , 12B As shown in 12C and 12D, the effects of formulations containing 20% ​​propylene glycol and 2% hydroxypropylcellulose and 0.5% by weight of peptides M6 (SEQ ID NO: 26), M7 (SEQ ID NO: 31), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) or M22 (SEQ ID NO: 291) were tested in a direct killing assay as described herein. Although the formulation itself (F) (without peptides) did not alter the viability of the cells, the presence of the peptides in the formulations retained the antimicrobial activity of these peptides - for example, Pseudomonas aeruginosa and S. epidermidis were both more sensitive to the activity than S. aureus.

[0129] like Fig.13 As shown, the effects of formulations containing 20% ​​propylene glycol and 2% hydroxypropylcellulose and 0.5 wt% of peptides M6 (SEQ ID NO: 26), M7 (SEQ ID NO: 31), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) or M22 (SEQ ID NO: 291) were tested using a direct killing assay. Although the formulation itself (F) (without peptides) did not alter the viability of Staphylococcus aureus (S. aureus) MRSA, the presence of the peptides in the formulation preserved the antibacterial activity of these peptides, and M22 (SEQ ID NO: 291) was observed to reduce viability starting from 0 minutes.

[0130] Fig.14AThe sensitivity (S) or resistance (R) of 8 peptide-resistant Staphylococcus aureus (S. aureus) strains out of 100 MRSN panel strains (NR-51553, NR-51556, NR-51570, NR51597, NR-51520, NR-51546, NR-51574, and NR-51588) to the listed antibiotics are shown. Fig. 14B The effect of a formulation containing 0.030 mg / ml (30 μg / ml) of peptides M14 (SEQ ID NO: 177) and M16 (SEQ ID NO: 213) in a broth dilution method based on the Clinical and Laboratory Standards Institute (CLSI) of the United States is shown to determine the minimum bactericidal concentration (MBC) of M14 and M16 against each of the eight tested MRSN Staphylococcus aureus (S. aureus) strains in the presence of 0.003% (30 μg / ml) peptides, as shown in the figure. The CLSI-based method is described in CLSI document M07-A10, Vol. 35, No. 2, 2015 (ISBN 1-56238-988-2).

[0131] Fig.15A and Fig. 15B The results of the modified dilution method based on CLSI described above are shown, the test was performed in a 0.01X MH (Mueller Hinton) broth (without carboxymethylcellulose) ( Fig. 15B ) and liquid medium containing 0.5% CMC and 0.01X MH ( Fig. 15B ), the effect of peptide M16 (SEQ ID NO: 213) on the MRSN panel Pseudomonas aeruginosa strain NR-51553 was tested, resulting in a minimum bactericidal concentration (MBC) of 15 μg / ml for this more resistant Pseudomonas aeruginosa strain.

[0132] Fig.16 The results of the improved dilution method based on CLSI as described above are shown for testing the effects of peptides M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177) and M16 (SEQ ID NO: 213) against Staphylococcus aureus (S. aureus) MRSA, respectively, wherein M14 (SEQ ID NO: 177) at a concentration of 250 μg / ml in 0.01X MH liquid culture medium (broth) was more effective at an earlier time (T) of 4 hours, while peptide M16 (SEQ ID NO: 213) was more effective at 24 hours.

[0133] Fig.17Results of a direct killing assay of A. baumannii are shown, where 250 μg / ml M16 (SEQ ID NO: 213) was more effective at an earlier time (T) of 4 hours, while the M14 peptide (SEQ ID NO: 177) was more effective at 24 hours.

[0134] Fig.18A Shown are the results of an ex vivo bactericidal activity assay using vaginal swabs from patients with symptoms of bacterial vaginosis in a direct killing assay disclosed herein, in which M6 (SEQ ID NO:26) and M14 (SEQ ID NO:177) were tested side-by-side on HBT bile agar in the presence of the bacterium Enterococcus faecalis (E. faecalis), along with metronidazole (MET) and clindamycin (CLIN). Fig.18B Shown are the results of this ex vivo activity assay using vaginal swabs from patients testing M6 (SEQ ID NO:26) and M14 (SEQ ID NO:177) side by side with metronidazole (MET) and clindamycin (CLIN) on MRS agar in the presence of Candida albicans. Cytotoxicity assay

[0135] Fig.19 The cytotoxicity results of peptides M6 (SEQ ID NO: 26) and M14 (SEQ ID NO: 177) measured against vaginal epithelial cells (VK2 / E6E7) and cervical cells (HeLa) using a standard calcein dye cytotoxicity assay (e.g., Thermofisher (Invitrogen), catalog number L3224) are shown. The peptides were incubated with cells at peptide concentrations of 1 mg / ml, 5 mg / ml, and 10 mg / ml for 18 hours, and a peptide-free (none) control group and an ethanol positive control group were set up as shown. The peptides M6 and M14 remained viable at concentrations up to 20 mg / ml. Biofilm test

[0136] Fig. 20Results are shown for peptide M6 (SEQ ID NO: 26) in a G. vaginalis biofilm assay, which measures biofilm disruption by the reduction of crystal violet dye as described, for example, in O'Toole, 2011, J. Vis. Exp., 47: 2437. Briefly, disruption of G. vaginalis biofilm was measured as concentrations of peptide M6 increased, i.e., at 0.02 mg / ml, 0.2 mg / ml, and 2 mg / ml of peptide M6, the crystal violet dye was reduced, indicating that the biofilm of G. vaginalis was disrupted. EpiVaginal Model

[0137] Table 2 below shows the results of measuring the effects of peptides M6 (SEQ ID NO: 26) and M14 (SEQ ID NO: 177) on vaginal irritation using the EpiVaginal model to determine the effects of peptides M6 (SEQ ID NO: 26) and M14 (SEQ ID NO: 177) on vaginal irritation in the EpiVaginal model. TM In the tissue model (Mattek), tissue viability was reduced to 50% (ET 50 ) exposure time. Briefly, toxicity was determined by measuring the relative conversion of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) in vaginal tissue samples treated with peptides compared to tissue treated with solvent negative control after exposure to peptides M6 and M14 at a final concentration of 20 mg / ml for different time periods. The positive control was Triton-X-100 at a final concentration of 1%.

[0138] Table 2. Test article Peptide concentration <![CDATA[ET 50 (Hours)]]> M6 2％ >24 M14 2％ >24 Triton-X-100 1％ 1.55 In vivo testing

[0139] The in vivo antimicrobial activity of peptides M6 (SEQ ID NO: 26), M14 (SEQ ID NO: 177), M16 (SEQ ID NO: 213) and / or M22 (SEQ ID NO: 291) at 0.5% (5 mg / ml) in 0.5% carboxymethylcellulose (CMC) was tested in two models: 1) a custom excisional wound infection model using Pseudomonas aeruginosa (P. aeruginosa) strain PAO1 (ATCC 15691), in which the efficacy of the antimicrobial peptides was compared with a control drug (e.g., ciprofloxacin); and 2) an established Staphylococcus aureus (MRSA) USA300 strain ( BAA-1717 TM )) skin infection model (in which the efficacy of antimicrobial peptides (AMPs) was compared with commercially available mupirocin ointment (e.g., or ) for comparison) and Acinetobacter baumannii (A.baumannii) infection model.For these in vivo models, mice are infected with relevant pathogens (e.g., Pseudomonas aeruginosa (P.aeruginosa), Staphylococcus aureus (S.aureus), Acinetobacter baumannii (A.baumannii)). Control mice were killed 1 hour after pathogen infection, and positive control or peptide tested mice were administered twice a day (BID) at intervals of 12 hours within 2.5 days.At the end of each relevant time point (e.g., at 2.5 days), mice were killed, and the number of bacteria was counted from the wound area 11 hours after the last administration. Antiviral test

[0140] In addition to the control group, TZM-bl cells exposed to 5,000 μg / ml, 1,000 μg / ml, 100 μg / ml, 10 μg / ml or 1 μg / ml of peptide M6 (SEQ ID NO: 26) (with or without HIV-1) for 48 hours were used to determine the inhibitory effect on human immunodeficiency virus (HIV). Fig.21A As shown, cells were exposed to HIV alone or to medium alone for co-exposure experiments. Fig. 21B ), cells were exposed to the indicated concentrations (μg / ml) of medium or peptide M6 (SEQ ID NO: 26) for 6 hours (6h), then washed and subsequently exposed to HIV for 36 to 42 hours. In post-exposure experiments ( Fig. 21C ), cells were exposed to HIV for 2 hours, washed, then exposed to HIV and peptide M6 (SEQ ID NO: 26) at the indicated concentrations for 11 hours, washed, and then incubated with culture medium for 36 hours. To assess inhibition, the percentage of HIV infection when cells were exposed to virus and peptide was determined using, for example, a Bright-Glo luciferase assay system (e.g., Britelite, PerkinElmer or Brite-Glo, Promega) as described in Sarzotti-Kelsoe et al., 2014, J. Immunol. Methods, 0: 131-146 or Puertas et al., 2012, J. Clin. Microbiol., 50: 3909-3916. The entire contents of Sarzotti-Kelsoe et al., 2014 and Puertas et al., 2012 are incorporated herein by reference.

[0141] like Fig. 22As shown, HEC1A cells infected with HSV-2G virus at a specific multiplicity of infection (MOI) (as shown) were used to test the inhibitory effect on herpes simplex virus (HSV). Briefly, after 1 hour of exposure, the virus was removed and peptide M6 (SEQ ID NO: 26) was added at a concentration of 5 mg / ml 3 days before qRT-PCR to measure HSV-2 DNA in the cell culture supernatant.

Claims

1. A peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295; or a pharmaceutically or cosmetically acceptable salt thereof.

2. A composition comprising the peptide according to claim 1, or a pharmaceutically or cosmetically acceptable salt thereof, wherein the composition further comprises a viscosity increasing agent.

3. The composition according to claim 2, wherein The viscosity increasing agent is selected from the group consisting of hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone, dextran, and hyaluronic acid.

4. The composition according to claim 2 or 3, wherein The viscosity increasing agent is hydroxypropyl cellulose or carboxymethyl cellulose (CMC).

5. The composition according to any one of claims 2 to 4, wherein The concentration of the viscosity increasing agent ranges from about 0.4% to about 6% based on the total weight or volume of the composition.

6. The composition according to any one of claims 2 to 5, further comprising a solvent.

7. The composition according to claim 6, wherein The solvent is selected from one or more of the group consisting of glycerol, ethanol, methanol, propylene glycol, and isopropanol.

8. The composition according to claim 6, wherein The solvent is selected from one or more of glycerol, propylene glycol, and isopropanol, and its concentration ranges from 0.1% to 30%; or the solvent is selected from methanol and ethanol, and its concentration ranges from 99%.

9. The composition according to any one of claims 6 to 8, wherein The solvent is propylene glycol.

10. The peptide according to claim 1 or the composition according to any one of claims 2 to 9, wherein The peptide or a pharmaceutically or cosmetically acceptable salt thereof has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO:

291.

11. The peptide according to claim 1 or the composition according to any one of claims 2 to 9, wherein The peptide or a pharmaceutically or cosmetically acceptable salt thereof has an amino acid sequence selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 256, and SEQ ID NO:

291.

12. The peptide according to claim 1 or the composition according to any one of claims 2 to 9, wherein The peptide or a pharmaceutically or cosmetically acceptable salt thereof has an amino acid sequence selected from any one of SEQ ID NO:26, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:213 and SEQ ID NO:

291.

13. The composition according to any one of claims 2 to 12, wherein The peptide is present in an amount of about 0.02 mg / mL to 50 mg / mL.

14. The composition according to any one of claims 2 to 13, wherein The peptide is present in an amount of about 0.02 mg / mL to 20 mg / mL.

15. The composition according to any one of claims 2 to 14, wherein The peptide is present in an amount of about 0.375 mg / mL to 10 mg / mL.

16. A method of treating a microbial infection in a subject having a microbial infection, the method comprising administering to the subject a peptide or composition according to any one of claims 1 to 15.

17. The method according to claim 16, wherein: The microbial infection is selected from the group consisting of bacterial infection, fungal infection, and viral infection.

18. The method according to claim 16, wherein: The microbial infection is a bacterial infection selected from the group consisting of Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Gardnerella vaginalis (G. vaginalis), Lactobacillus iners (L. iners), Acinetobacter baumannii (A. baumannii), and Enterococcus faecalis (E. faecalis) infections.

19. The method according to claim 16, wherein: The microbial infection is a Candida albicans (C. albicans) yeast infection.

20. The method according to claim 16, wherein: The microbial infection is a viral infection selected from HIV infection and herpes simplex virus (HSV) infection.

21. A method of treating a surface comprising microorganisms, the method comprising applying to the surface a peptide or composition according to any one of claims 1 to 15.

22. The method according to claim 21, wherein: The surface is a medical device.

23. A composition comprising: a first peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, or a pharmaceutically or cosmetically acceptable salt thereof; and a second peptide having an amino acid sequence selected from any one of SEQ ID NO: 1 to SEQ ID NO: 295, or a pharmaceutically or cosmetically acceptable salt thereof, wherein the first peptide is different from the second peptide.

24. The composition of claim 23, further comprising a viscosity increasing agent.

25. The composition according to claim 24, wherein The viscosity increasing agent is selected from the group consisting of hydroxypropyl cellulose, hydroxypropyl methyl cellulose, carboxymethyl cellulose (CMC), polyvinyl pyrrolidone, dextran, and hyaluronic acid.

26. The composition according to claim 24 or 25, wherein The concentration of the viscosity increasing agent ranges from 0.4% to 6% based on the total weight or volume of the composition.

27. The composition according to any one of claims 23 to 26, further comprising a solvent.

28. The composition according to claim 27, wherein The solvent is selected from the group consisting of glycerol, ethanol, methanol, propylene glycol, and isopropanol.

29. The composition according to claim 27, wherein The solvent is selected from glycerol, propylene glycol, dimethyl sulfoxide (DMSO), and isopropanol, and its concentration ranges from 0.1% to 30%; or the solvent is selected from methanol and ethanol, and its concentration ranges up to 99%.

30. The composition according to any one of claims 23 to 29, wherein The first peptide and the second peptide are respectively selected from any one of the following: SEQ ID NO:1, SEQ ID NO:6, SEQ ID NO:11, SEQ ID NO:16, SEQ ID NO:21, SEQ ID NO:26, SEQ ID NO:31, SEQ ID NO:36, SEQ ID NO:41, SEQ ID NO:76, SEQ ID NO:106, SEQ ID NO:141, SEQ ID NO:142, SEQ ID NO:177, SEQ ID NO:178, SEQ ID NO:213, SEQ ID NO:218, SEQ ID NO:219, SEQ ID NO:220, SEQ ID NO:255, SEQ ID NO:256, and SEQ ID NO:291, and pharmaceutically or cosmetically acceptable salts thereof.

31. The composition according to any one of claims 23 to 29, wherein The first peptide is selected from any one of the following: SEQ ID NO: 1, SEQ ID NO: 6, SEQ ID NO: 11, SEQ ID NO: 16, SEQ ID NO: 21, SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 41, SEQ ID NO: 76, SEQ ID NO: 106, SEQ ID NO: 141, SEQ ID NO: 142, SEQ ID NO: 177, SEQ ID NO: 178, SEQ ID NO: 213, SEQ ID NO: 218, SEQ ID NO: 219, SEQ ID NO: 220, SEQ ID NO: 255, SEQ ID NO: 256, and SEQ ID NO: 291, and the second peptide is selected from any one of the following: SEQ ID NO: 26, SEQ ID NO: 31, SEQ ID NO: 36, SEQ ID NO: 177, SEQ ID NO: 178, and SEQ ID NO: 213, and pharmaceutically or cosmetically acceptable salts thereof.

32. The composition according to any one of claims 23 to 29, wherein The first peptide and the second peptide are respectively selected from any one of the following: SEQ ID NO: 26, SEQ ID NO: 177, SEQ ID NO: 213, and SEQ ID NO: 291, and pharmaceutically or cosmetically acceptable salts thereof.

33. The composition of any one of claims 23-32, further comprising one or more additional peptides selected from the group consisting of: SEQ ID NO: 1 to SEQ ID NO:

295.

34. A method of treating a microbial infection in a subject having a microbial infection, the method comprising administering to the subject a composition according to any one of claims 23-33.

35. The method of claim 34, wherein: The microbial infection is selected from the group consisting of bacterial infection, fungal infection, and viral infection.

36. The method of claim 34, wherein: The microbial infection is a bacterial infection selected from the group consisting of Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), Staphylococcus epidermidis (S. epidermidis), Gardnerella vaginalis (G. vaginalis), Lactobacillus iners (L. iners), Acinetobacter baumannii (A. baumannii), and Enterococcus faecalis (E. faecalis).

37. A method of treating a surface comprising microorganisms, the method comprising applying to the surface a composition according to any one of claims 23 to 33.

38. The method of claim 37, wherein: The surface is a medical device.

39. A cosmetic composition comprising the peptide or composition of any one of claims 1-15 or 23-33, wherein the peptide is optionally a pharmaceutically or cosmetically acceptable salt thereof.

40. The cosmetic composition according to claim 39, wherein The cosmetic composition is selected from the group consisting of creams, lotions, cleansers, solutions, serums, ointments, and make-ups.

41. The cosmetic composition according to claim 39 or 40, wherein The cosmetic composition has an increased shelf life compared to the cosmetic composition without the peptide composition.

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