Cecropin peptide isolated from trichoplusia ni larvae and use thereof for treating sepsis, bacterial infection, or inflammatory diseases

Abstract

The present invention relates to a composition for preventing or treating gram-negative bacterial infections, the composition comprising T. ni cecropin. According to an aspect of the present invention, the peptide T. ni cecropin has the excellent effects of reducing liver and kidney toxicity values, inflammatory cytokine production, and endotoxins and inhibiting inflammatory cell infiltration in animal models infected with LPS-infected endotoxemia and Carbapenem-resistant Acinetobacter baumannii and can thus be effectively used for preventing or treating bacterial infections (e.g., gram-negative bacterial infections), sepsis, or inflammatory diseases.

Description

Cecropin peptide isolated from cabbage silver moth larvae and its use for treating sepsis, bacterial infection, or inflammatory diseases

[0001] The present invention relates to a cecropin peptide isolated from the larvae of the cabbage moth (Trichoplusia ni) and its use in the treatment of sepsis, bacterial infection, or inflammatory disease.

[0002] Inflammation is a defense response of biological tissues to a specific stimulus. It refers to a complex lesion that causes three things: tissue degeneration, circulatory disturbance, exudation, and tissue proliferation. Its causes include physical factors such as mechanical injury, temperature, and radiation, chemical factors such as toxins, and parasitic infections, with bacteria being the most common. Beyond these primary causes, its pathogenesis is complex, influenced by various secondary factors, individual predispositions, and immunity. Furthermore, the inflammatory response is one of the most important pathological responses, a host defense mechanism against wounds, microbial infections, and other factors.

[0003] Antibiotic resistance is a growing threat worldwide. Polymyxins, such as colistin and polymyxin B, are effective in treating Gram-negative bacterial infections, but overuse has led to widespread resistance to colistin. Carbapenems, such as imipenem and meropenem, are effective in treating Gram-negative bacterial infections, but overuse has also led to widespread resistance to carbapenems. The World Health Organization (WHO) has classified carbapenem-resistant Acinetobacter baumannii as the group most in need of new antibiotics (J. Med. Soc. 2018, 32, 76). However, the existing antibiotics used to treat colistin- and carbapenem-resistant Gram-negative bacterial infections have limited use due to toxic effects, and because it takes time and money to appropriately optimize the antibiotic dosage and evaluate toxicity, therapeutic options are still limited and new antibiotics are urgently needed.

[0004] Broadly speaking, the antigen-antibody response, also known as the immune response, plays a key role in biological defense in invertebrates, including humans. However, insects protect themselves by strengthening their innate immune system, which differs from the acquired immune system, such as the antigen-antibody response. Thus, innate immunity serves as a crucial defense system against initial infections not only in insects but also in vertebrates, including humans. A crucial element of the insect innate immune system is the acquisition of substances with bactericidal and inactivating properties against various microorganisms. Among these, peptides and proteins with antimicrobial properties, particularly antimicrobial peptides, have high potential for disease control in humans and livestock. These insect-derived antipathogenic peptides are expected to be useful not only for human and livestock disease prevention but also for preventing and treating crop damage caused by various pathogenic bacteria.

[0005] These antimicrobial peptides can be divided into three groups based on their structure. The first group is cysteine-rich β-sheet peptides, the second group is α-helical amphipathic peptide molecules, and the third group is proline-rich (Trp-rich) peptides, such as proline or tryptophan (Mayasaki et al., Int. J. Antimicrob. Agents, 9, 269-280, 1998; Andreu, D.; Rivas, L. Biopolymers 1998, 47, 415-433). These antimicrobial peptides are known to play important roles in host defense and the innate immune system. In addition, the above antimicrobial peptides have various structures depending on the amino acid sequence, and the most common of these structures is a structure that forms an amphiphilic α-helix without a cysteine ​​residue, such as melittin or cecropin, which are antimicrobial peptides found in insects.

[0006] About 200 types of insect antimicrobial proteins and peptides have been isolated from insects since cecropin was first reported by Dr. Boman's research team from the Cecropia moth. These can be broadly divided into cecropins, defensins, and peptides containing a lot of florine and glycine amino acids according to their structure and size. Among these, cecropins are basic proteins composed of 31 to 39 amino acids, have an α-helix-hinge-α-helix structure including an amphipathic helix structure at the N-terminus, and are heat-stable and exhibit a wide range of antimicrobial activity (Boman and Hultmark, Annu. Rev. Micrbiol., 41: 1003-126, 1987).

[0007] T. nicecropin, an antimicrobial peptide isolated from the larvae of the cabbage moth (Trichoplusia ni, T. ni), a member of the order Lepidoptera, is characterized by the presence of an additional Phe (phenylalanine, F) at the N-terminus, unlike the known insect cecropin (Non-patent Document 1). However, there have been no reports on the in vivo antimicrobial activity of T. nicecropin or its therapeutic effect on infections such as colistin-resistant Escherichia coli (ColREC) and carbapenem-resistant Acinetobacter baumannii (CRAB) infections.

[0008] Accordingly, the inventors of the present invention completed the present invention by confirming the in vivo antibacterial activity and anti-inflammatory effect of T. nicecropin through cell experiments and animal experiments obtained by stimulation with LPS, colistin-resistant Escherichia coli (ColREC), or carbapenem-resistant Acinetobacter baumannii (CRAB).

[0009] One aspect provides a pharmaceutical composition for preventing or treating sepsis or septic shock, bacterial infection, or inflammatory disease, comprising a peptide represented by the amino acid sequence of sequence number 1 as an active ingredient.

[0010] Another aspect is to provide a pharmaceutical composition or health functional food for preventing, improving or treating sepsis or septic shock, bacterial infection or inflammatory disease, containing the above peptide as an active ingredient.

[0011] Another aspect provides a method for inhibiting bacterial growth, a method for preventing or treating sepsis or septic shock, a method for preventing or treating bacterial infections, or a method for preventing or treating inflammatory diseases, comprising administering the peptide to a subject or treating a sample.

[0012] Another aspect is to provide an antimicrobial composition (e.g., a pharmaceutical composition, a food additive, a feed additive, a preservative composition, and a quasi-drug composition) comprising the peptide.

[0013] Another aspect provides the use of the peptide for the manufacture of an antibacterial composition, a pharmaceutical composition or a health functional food for preventing, treating or ameliorating sepsis or septic shock, bacterial infection or inflammatory disease.

[0014] The terminology used in this specification is intended to appropriately express preferred embodiments of the present invention, and may vary depending on the intent of the user or operator, or the customary practices in the field to which the present invention pertains. Therefore, the definitions of these terms should be determined based on the contents of this specification as a whole. Throughout this specification, when a part is said to "include" a certain component, unless otherwise specifically stated, this does not mean that other components are excluded, but rather that other components may be included.

[0015] Unless otherwise defined, all technical terms used in this invention may be used with the same meanings as commonly understood by those skilled in the art in the relevant field of the present invention. Furthermore, while preferred methods and samples are described herein, similar or equivalent methods may also be included within the scope of the present invention. The contents of all publications cited as references herein may be incorporated into the present invention.

[0016] One aspect provides a peptide represented by the amino acid sequence of sequence number 1.

[0017] In one specific example, the peptide may be a peptide derived from the cabbage moth (Trichoplusia ni). The peptide may be isolated from, but is not limited to, a larva, a pupa, or the moth itself of the cabbage moth.

[0018] In one specific example, the peptide may be cecropin. The cecropin may comprise the amino acid sequence of SEQ ID NO: 1 (RWKFFKKIEKVGQNIRDGIIKAGPAVAVVGQAASITGK-NH2). The peptide may have an amino group (-NH2) naturally or naturally modified at the C-terminus.

[0019] In addition, the peptides of the present invention can be synthesized according to their length by methods well known in the art, for example, by an automated peptide synthesizer, and can also be produced by genetic engineering techniques. Specifically, the peptides of the present invention can be prepared by standard synthetic methods, recombinant expression systems, or any other method known in the art. Thus, the peptides according to the present invention can be synthesized by a number of methods, including, for example, methods including: (a) synthesizing the peptide stepwise or by fragment assembly by means of solid-phase or liquid-phase methods, and isolating and purifying the final peptide product; or (b) expressing a nucleic acid construct encoding the peptide in a host cell and recovering the expression product from the host cell culture; or (c) performing cell-free in vitro expression of a nucleic acid construct encoding the peptide and recovering the expression product; Or a method of obtaining fragments of a peptide by any combination of (a), (b) and (c), and then linking the fragments to obtain a peptide, and recovering the peptide.

[0020] Another aspect provides a health functional food for preventing, treating or improving sepsis or septic shock, bacterial infection or inflammatory disease, containing the above peptide as an active ingredient.

[0021] Another aspect provides a method for inhibiting bacterial growth, a method for preventing or treating sepsis or septic shock, a method for preventing or treating bacterial infections, or a method for preventing or treating inflammatory diseases, comprising administering the peptide to a subject or treating a sample.

[0022] Another aspect provides an antimicrobial composition (e.g., a pharmaceutical composition, a food additive, a feed additive, a preservative composition, and a quasi-drug composition) comprising the peptide.

[0023] Another aspect provides the use of the peptide for the manufacture of an antibacterial composition, a pharmaceutical composition or a health functional food for preventing, treating or ameliorating sepsis or septic shock, bacterial infection or inflammatory disease.

[0024] As used herein, the term "antibacterial" means inhibiting, reducing or preventing the growth of bacteria, inhibiting or reducing the ability of bacteria to cause infection in a subject, or inhibiting or reducing the ability of bacteria to multiply or remain infectious in the environment.

[0025] The above peptide may exhibit significant antibacterial activity even at low concentrations (e.g., 1 μM, 2 μM, 3 μM, 4 μM, 5 μM, 6 μM, 7 μM, 8 μM, 9 μM, or 10 μM).

[0026] In one specific example, the peptide may have antibacterial activity against Gram-positive or Gram-negative bacteria. Preferably, the peptide may have antibacterial activity against Gram-negative bacteria.

[0027] The above gram-positive bacteria (gram-negative bacteria) may be at least one selected from the group consisting of Bacillus subtilis, Staphylococcus aureus, and Staphylococcus epidermidis, but is not limited thereto.

[0028] The above gram-negative bacteria (gram-negative bacteria) may be at least one selected from the group consisting of Enterobacter sp. bacteria, Salmonella sp. bacteria, Vibrio sp. bacteria, Mycobacterium sp. bacteria, Shigella sp. bacteria, Psedomonas sp. bacteria, Acinetobacter sp. bacteria, and Escherichia sp. bacteria, but is not limited thereto.

[0029] The above-mentioned E. coli bacteria may be, but are not limited to, Escherichia coli, Escherichia albertii, Escherichia blattae, Escherichia fergusonii, Escherichia hermannii, or Escherichia vulneris.

[0030] The above Salmonella bacteria may be, but are not limited to, Salmonella typhimurium, Salmonella enterica, Salmonella typhi, Salmonella paratyphi, Salmonella enteritidis, or Salmonella cholerasuis.

[0031] The above Pseudomonas genus bacteria may be, but are not limited to, Pseudomonas aeruginosa or Pseudomonas stutzeri.

[0032] The above Vibrio genus bacteria may be, but are not limited to, Vibrio vulnificus, Vibrio cholerae, Vibrio parahemolyticus, Vibrio alginolyticus, Vibrio mimicus, Vibrio hollisae, Vibrio fluvialis, Vibrio metchnikovii, Vibrio damsela, or Vibrio furnisii.

[0033] The above Mycobacterium genus bacteria may be, but are not limited to, Mycobacterium leprae, Mycobacterium intracellulare, Mycobacterium avium, Mycobacterium bovis, or Mycobacterium marinum.

[0034] The above Shigella genus bacteria may be, but are not limited to, Shigella dysenteriae, Shigella flexneri, Shigella boydii, or Shigella sonnei.

[0035] The above Acinetobacter genus bacteria are Acinetobacter baumannii, Acinetobacter calcoaceticus, Acinetobacter haemolyticus, Acinetobacter junii, Acinetobacter johnsonii, Acinetobacter lwoffii, Acinetobacter radioresistens, Acinetobacter ursingii, Acinetobacter schindleri, Acinetobacter parvus, Acinetobacter baylyi, Acinetobacter bouvetii, Acinetobacter It may be, but is not limited to, Acinetobacter towneri, Acinetobacter tandoii, Acinetobacter grimontii, Acinetobacter tjernbergiae or Acinetobacter gerneri.

[0036] The above Enterobacter genus bacteria may be, but are not limited to, Enterobacter aerogenes, Enterobacter agglomerans or Enterobacter cloacae.

[0037] Preferably, the Gram-negative bacteria may be Acinetobacter sp. bacteria or Escherichia sp. bacteria.

[0038] In one specific example, the Gram-negative bacteria may be multidrug-resistant Gram-negative bacteria.

[0039] The above multidrug-resistant gram-negative bacteria may be, but are not limited to, carbapenem or colistin resistance.

[0040] The above carbapenem-resistant gram-negative bacteria may be, but are not limited to, carbapenem-resistant Acinetobacter baumannii (CRAB).

[0041] The above colistin-resistant gram-negative bacteria may be, but are not limited to, colistin-resistant Escherichia coli (ColREC).

[0042] In one specific example, it may have the endotoxin neutralizing ability or endotoxin removing ability.

[0043] In one specific example, the peptide may alleviate inflammation in a subject infected with endotoxin (LPS) or Gram-negative bacteria. Preferably, the peptide may inhibit the expression or production of any one or more selected from the group consisting of nitric oxide (NO), tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), or interleukin-1β (IL-1β).

[0044] In one specific example, the peptide may reduce the level of endotoxin (LPS) or toxicity in the liver or kidney of a subject infected with Gram-negative bacteria. Preferably, the peptide may inhibit the production of one or more of the following enzymes: alanine aminotransferase (AST), aspartate aminotransferase (ALT), and blood urea nitrogen (BUN).

[0045] In one specific example, the peptide may increase the permeability of the bacterial cell membrane, thereby damaging the cell membrane.

[0046] In one specific example, the peptide may inhibit inflammatory cell infiltration into body tissues.

[0047] The term "sepsis" or "septic shock" in this specification refers to systemic inflammatory response syndrome (SIRS) caused by rapid excessive secretion of inflammatory cytokines, also called a "cytokine storm," which is an immune response of the human body to an invading pathogen.

[0048] The above inflammatory diseases include edema, dermatitis, allergies, atopy, asthma, conjunctivitis, periodontitis, allergic and non-allergic rhinitis, otitis media, pharyngitis, tonsillitis, pneumonia, gastric ulcers, chronic and acute gastritis, Crohn's disease, colitis, chronic and acute enteritis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis of the shoulder joint, tendinitis, tenosynovitis, myositis, acute and chronic hepatitis, cystitis, acute and chronic nephritis, Sjogren's syndrome, multiple sclerosis, diabetes, chronic and acute rhinitis, chronic obstructive pulmonary disease, pulmonary fibrosis, irritable bowel syndrome, migraine, headache, back pain, fibromyalgia, myofascial disease, viral infection, bacterial infection, fungal infection, burns, wounds due to surgical or dental operation, prostaglandin E hyperactivity syndrome, atherosclerosis It may be any one or more selected from the group consisting of, but not limited to, sclerosis, arthritis, Hodgkin's disease, pancreatitis, iritis, scleritis, uveitis, and eczema.

[0049] In one specific example, the sepsis, septic shock or inflammatory disease may be caused by, but is not limited to, carbapenem-resistant Acinetobacter baumannii (CRAB) or colistin-resistant Escherichia coli (ColREC).

[0050] The term “prevention” in this specification means any act of inhibiting or delaying the occurrence, spread and recurrence of sepsis, septic shock or inflammatory disease by administering a pharmaceutical composition according to the present invention.

[0051] As used herein, "treatment" refers to any action that improves or beneficially alters the symptoms of sepsis, septic shock, or an inflammatory disease by administering the composition. Those skilled in the art to which the present invention pertains will be able to reference materials provided by the Korean Medical Association and other sources to determine the precise criteria for diseases for which the composition of the present invention is effective, and to determine the degree of improvement, enhancement, and treatment.

[0052] The above pharmaceutical composition can be administered in a pharmaceutically effective amount.

[0053] The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing any side effects, and the effective dosage level can be determined based on factors including the patient's health condition, cause of the disease, severity, activity of the drug, sensitivity to the drug, method of administration, time of administration, route of administration and excretion rate, duration of treatment, drugs used in combination or concurrently, and other factors well known in the medical field.

[0054] The above pharmaceutical composition may be administered as an individual treatment or in combination with other treatments, sequentially or simultaneously with conventional treatments, or in single or multiple doses. Considering all of the above factors, it is important to administer the amount that achieves maximum effect with the minimum amount possible without causing side effects, a determination readily available to those skilled in the art.

[0055] The composition of the present invention can be administered parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) or orally, depending on the intended method, and the dosage range varies depending on the patient's weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity of the disease. The daily dosage of the composition according to the present invention is 0.0001 to 10 mg / ml, preferably 0.0001 to 5 mg / ml, and it is more preferable to administer it once or several times a day.

[0056] The pharmaceutical composition may include a carrier, diluent, excipient, or a combination of two or more thereof commonly used in biological preparations. The term "pharmaceutically acceptable" as used herein means that the composition exhibits a property of not being toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for delivering the composition in vivo, and for example, compounds described in Merck Index, 13th ed., Merck & Co. Inc., saline solution, sterile water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or more of these components may be mixed and used, and other common additives such as antioxidants, buffers, and bacteriostatic agents may be added as necessary. In addition, diluents, dispersants, surfactants, binders, and lubricants may be additionally added to formulate the composition into a main use form such as an aqueous solution, suspension, or emulsion, pills, capsules, granules, or tablets. Furthermore, it can be preferably formulated according to each disease or ingredient using an appropriate method in the field or the method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

[0057] The pharmaceutical composition may be in one or more dosage forms selected from the group consisting of oral dosage forms, topical preparations, suppositories, sterile injectable solutions and sprays, with oral or injectable dosage forms being more preferred.

[0058] The term "administration" herein means providing a given substance to a subject or patient by any appropriate method, and may be administered parenterally (for example, intravenously, subcutaneously, intraperitoneally, or locally in the form of an injection) or orally, depending on the intended method. The dosage may vary depending on the patient's weight, age, sex, health, diet, administration time, administration method, excretion rate, and disease severity. Liquid preparations for oral administration of the composition of the present invention include suspensions, oral solutions, emulsions, syrups, etc., and may include various excipients such as wetting agents, sweeteners, fragrances, preservatives, etc. in addition to commonly used simple diluents such as water and liquid paraffin. Preparations for parenteral administration may include, but are not limited to, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, etc. The pharmaceutical composition of the present invention may be administered by any device that allows the active substance to move to target cells. Preferred administration methods and formulations include intravenous injections, subcutaneous injections, intradermal injections, intramuscular injections, and drip injections. The injections can be manufactured using aqueous solvents such as saline solution and Ringer's solution, non-aqueous solvents such as vegetable oils, higher fatty acid esters (e.g., ethyl oleate, etc.), alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.), and pharmaceutical carriers such as stabilizers to prevent deterioration (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers to adjust pH, and preservatives to inhibit microbial growth (e.g., phenylmercuric nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

[0059] The pharmaceutical composition may further comprise a pharmaceutically acceptable additive. At this time, the pharmaceutically acceptable additive may be starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate, lactose, mannitol, maltose, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, Opadry, sodium starch glycolate, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, calcium stearate, sucrose, dextrose, sorbitol, and talc. The pharmaceutically acceptable additive according to the present invention is preferably included in the composition in an amount of 0.1 to 90 parts by weight, but is not limited thereto.

[0060] The above pharmaceutical composition may be administered in conjunction with an existing therapeutic agent. The concurrent administration of the pharmaceutical composition may be administered before, simultaneously with, or after the administration of the existing therapeutic agent.

[0061] The above food composition may be used by adding the above antimicrobial composition as is, or in combination with other foods or food ingredients, and may be used appropriately according to conventional methods. The above composition may include food-related and scientifically acceptable food additives in addition to the active ingredient, and the amount of the active ingredient mixed may be appropriately determined depending on the intended use (prevention, health, or therapeutic treatment).

[0062] The term "food supplement additive" used in the present invention means a component that can be added to food as an auxiliary, and can be appropriately selected and used by those skilled in the art as added in the manufacture of health functional foods of each formulation. Examples of food supplement additives include various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic flavoring agents and natural flavoring agents, coloring agents and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, etc., but the types of food supplement additives of the present invention are not limited by the above examples.

[0063] The food composition of the present invention may include a health functional food.

[0064] As used herein, the term "health functional food" refers to a food manufactured and processed in the form of tablets, capsules, powders, granules, liquids, pills, etc. using raw materials or ingredients with beneficial functional properties for the human body. "Functionality" here refers to regulating nutrients for the structure and functions of the human body or achieving beneficial health effects, such as physiological effects.

[0065] The above health functional food can be manufactured by methods commonly used in the art, and during the manufacturing process, raw materials and ingredients commonly added in the art can be added. In addition, the formulation of the health functional food can be manufactured without limitation as long as it is a formulation recognized as a health functional food. The food composition of the present invention can be manufactured in various forms, and the health functional food of the present invention can be consumed as a supplement to enhance the effect of improving gram-negative bacterial infections.

[0066] In addition, there is no limitation on the type of health food in which the composition of the present invention can be used, and it can be manufactured by mixing other appropriate auxiliary ingredients and known additives that can be included in health functional foods, depending on the selection.

[0067] Examples of foods that can be manufactured include dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, and vitamin complexes, and the extract according to the present invention can be manufactured by adding it to juice, tea, jelly, and juice manufactured using the extract as a main ingredient.

[0068] When used as a feed composition, the composition may be a highly concentrated solution of 20 to 90% or may be manufactured in the form of powder or granules. The feed composition may further include one or more of organic acids such as citric acid, fumaric acid, adipic acid, lactic acid, and malic acid; phosphates such as sodium phosphate, potassium phosphate, acid pyrophosphate, and polyphosphate (polyphosphate); and natural antioxidants such as polyphenol, catechin, alpha-tocopherol, rosemary extract, vitamin C, green tea extract, licorice extract, chitosan, tannic acid, and phytic acid. The composition may be formulated in the form of a conventional feed and may include conventional feed ingredients together.

[0069] The feed may further include grains such as ground or shredded wheat, oats, barley, corn and rice; plant-based protein feeds such as feeds mainly composed of rapeseed, soybeans and sunflower; animal-based protein feeds such as blood meal, meat meal, bone meal and fish meal; dry ingredients composed of sugars and dairy products such as various types of milk powder and whey powder, and may further include nutritional supplements, digestion and absorption enhancers, growth promoters and the like.

[0070] The feed composition may be administered to animals alone or in combination with other feed additives in an edible carrier. Furthermore, the feed additives may be readily administered to animals as a top dressing, by mixing them directly into animal feed, or in an oral formulation separate from the feed. When the feed additives are administered separately from animal feed, they may be prepared as immediate-release or sustained-release formulations by combining them with food-grade edible carriers, as is well known in the art. Such edible carriers may be solid or liquid, such as cornstarch, lactose, sucrose, soybean flakes, peanut oil, olive oil, sesame oil, and propylene glycol. When a solid carrier is used, the feed additive may be in the form of a tablet, capsule, powder, troche, or saccharide tablet, or a top dressing in a microdispersible form. When a liquid carrier is used, the feed additive may be in the form of a gelatin soft capsule, or a syrup, suspension, emulsion, or solution.

[0071] Additionally, the feed may contain additives such as preservatives, stabilizers, wetting or emulsifying agents, and solution accelerators. The feed additives may be added to animal feed by injection, spraying, or mixing.

[0072] The feed or feed additive of the present invention can be applied to a number of animal diets including mammals, poultry, and fish.

[0073] As the mammals, it can be used for pigs, cows, sheep, goats, laboratory rodents, and laboratory rodents, as well as pets (e.g., dogs, cats), and as the poultry, it can be used for chickens, turkeys, ducks, geese, pheasants, and quail, and as the fish, it can be used for trout, but is not limited thereto.

[0074] According to one aspect, T. nicecropin has excellent in vivo antibacterial and anti-inflammatory activities, including reducing hepatic and renal toxicity levels, inflammatory cytokine production, bacterial counts, and endotoxins in animal models infected with LPS-induced endotoxemia or CRAB-induced endotoxemia, and inhibiting inflammatory cell infiltration. Therefore, T. nicecropin has the effect of being useful for the prevention or treatment of bacterial infections (e.g., Gram-negative bacterial infections), sepsis, or inflammatory diseases.

[0075] Figure 1 shows the in vitro cytotoxicity of T. nicecropin antimicrobial peptides; (a) shows cell viability (%) in RAW 264.7 murine macrophage cells for 24 hours; (b) shows cell viability (%) in L-929 cells.

[0076] Figure 2 is a graph showing the concentration-dependent (0 to 100 μM) toxicity effect induced by antimicrobial peptides on sheep red blood cells.

[0077] Figure 3 shows the biofilm formation inhibition activity of T. nicecropin antimicrobial peptides; (a) shows the antibiofilm activity of colistin-resistant Escherichia coli (ColREC) 1557 by an antimicrobial peptide; (b) shows the antibiofilm activity of ColREC 12 by an antimicrobial peptide.

[0078] Figure 4 shows the results of an LAL (Limulus Amebocyte Lysate) analysis showing the LPS neutralizing ability of the peptide.

[0079] Figure 5 shows the displacement results of BODIPY-TR-cadaverine from LPS after peptide treatment.

[0080] Figure 6 shows the results of isothermal titration calorimetry showing the binding affinity of T. nicecropin (0.1 mM) and 0.025 mM LPS.

[0081] Figure 7 shows the results of measuring the fluorescence intensity induced by bacterial membrane disruption by peptides; (a) shows the results in E. coli; (b) shows the results in ColREC 1557; (c) shows the results in ColREC 12.

[0082] Figure 8 is a graph showing the time-dependent killing activity according to T. nicecropin treatment in E. coli.

[0083] Figure 9 shows the results of coating a dehydrated sample with platinum and imaging it with a scanning electron microscope; (a) is a scanning electron microscope (SEM) image of untreated E. coli; (b) is a SEM image of E. coli after treatment with T. nicecropin.

[0084] Figure 10 shows the reference one-dimensional nuclear magnetic resonance (NMR) spectrum of T. nicecropin (top) and the saturation shift difference (STD) NMR spectrum (bottom) obtained through interaction with LPS (bottom) dissolved in 20 mM sodium phosphate buffer (pH 5.9).

[0085] Figure 11 shows the results of circular dichroism spectroscopy measurements for observing the secondary structure of T. nicecropin in a membrane-mimicking environment.

[0086] Figure 12 shows the concentration-dependent inhibitory effect of T. nicecropin on (a) nitrite production and (b) IL-6 production, an inflammatory cytokine, in RAW 264.7 cells stimulated with LPS for 16 hours.

[0087] Figure 13 shows the concentration-dependent inhibitory effect of T. nicecropin on (a) nitrite production and (b) IL-6 production, an inflammatory cytokine, in RAW 264.7 cells stimulated with ColREC for 16 hours.

[0088] Figure 14 shows the results of measuring the effect of T. nicecropin on reducing serum endotoxin (LPS) concentration in a mouse model of LPS infection endotoxemia using LAL (Limulus Amebocyte Lysate) analysis.

[0089] Figure 15 shows the results of measuring the effect of T. nicecropin treatment on the suppression of the expression of inflammatory cytokines (IL-6) in the blood in a mouse model of LPS infection.

[0090] Figure 16 shows the results of measuring the effects of T. nicecropin treatment on improving liver damage (AST and ALT) and kidney damage (BUN) in a mouse model of LPS infection; (a) shows the effect of reducing AST; (b) shows the effect of reducing ALT; (c) shows the effect of reducing BUN.

[0091] Figure 17 shows the results of measuring cell infiltration by T. nicecropin treatment in a mouse model of LPS infection using the hematoxylin & eosin method.

[0092] Figure 18 shows changes in the total number of CRAB bacteria by T. nicecropin treatment in a mouse model of CRAB infection; (a) shows changes in the total number of CRAB bacteria in the lungs; (B) shows changes in the total number of CRAB bacteria in the liver; (c) shows changes in the total number of bacteria in the kidneys.

[0093] Figure 19 shows the results of measuring the effect of T. nicecropin treatment on reducing serum endotoxin (LPS) concentration in a CRAB infection mouse model using LAL (Limulus Amebocyte Lysate) analysis.

[0094] Figure 20 shows the results of measuring the effect of suppressing the expression of inflammatory cytokines in the blood by treatment with T. nicecropin in a mouse model of CRAB infection; (a) shows the effect of suppressing the expression of TNF-α; (b) shows the effect of suppressing the expression of IL-6; (c) shows the effect of suppressing the expression of IL-1β.

[0095] Figure 21 shows the results of measuring the effect of T. nicecropin treatment on improving liver damage (AST and ALT) and kidney damage (BUN) in a mouse model of CRAB infection; (a) shows the effect of reducing AST; (b) shows the effect of reducing ALT; (c) shows the effect of reducing BUN.

[0096] Figure 22 shows the results of measuring cell infiltration by T. nicecropin treatment in a CRAB infection mouse model using the hematoxylin & eosin method.

[0097] Figure 23 shows the results of measuring the survival rate by the treatment effect of CRAB infection by T. nicecropin treatment in a CRAB infection mouse model.

[0098] Hereinafter, preferred embodiments are presented to aid understanding of the present invention. However, the following embodiments are provided solely to facilitate a better understanding of the present invention and are not intended to limit the scope of the present invention. The embodiments are susceptible to various modifications, and thus the embodiments are not limited to the embodiments disclosed below and may be implemented in various forms.

[0099]

[0100] Example 1. Confirmation of antibacterial activity of peptides

[0101] To confirm the antibacterial activity of peptides against various Gram-negative bacteria, the minimum inhibitory concentration (MIC) was measured. Specifically, peptides for confirming antibacterial activity were Cecropin A from Hyalophora cecropia, Cecropin A from Trichoplusia ni, Polymyxin B, Colistin, and Melittin. The cecropin peptide was synthesized in the solid phase using Fmoc (fluorenylmethoxycarbonyl) by request from Anygen Co., Ltd. (Gwangju, Korea). Specifically, it was purified to a purity of 95% or higher using high-performance liquid chromatography using a C18 column, and characterized using matrix-assisted laser-desorption ionization-time-of-flight mass spectrometry (MALDI-TOF). Escherichia coli (KCTC 1682), used as a standard Gram-negative bacteria, was purchased from the Korea Center for Microbiology (KCTC, Jeongeup, Republic of Korea). Acinetobacter baumannii (KCCM 40203) and Pseudomonas aeruginosa (KCCM 11328) were purchased from the Korea Center for Microbiology (KCCM, Seoul, Republic of Korea). Klebsiella pneumoniae (NCCP 16054) was purchased from the National Institute of Health's Multidrug-Resistant Bacteria and Pathogen Resource Bank (Osong, Republic of Korea). In addition, colistin-resistant bacteria, colistin-resistant E. coli NMA 1557 (ColREC 1557) and NMS 12 (ColREC 12), colistin-resistant A. baumannii NMS 1915 (ColRAB 1915), and colistin-resistant K. pneumoniae NMS 139 (ColRKP 139) and carbapenem-resistant A. baumannii NMS 1915 (CRAB 1915), carbapenem-resistant A. baumannii NMS 1919 (CRAB 1919), carbapenem-resistant A.A. baumannii NMS 1707 (CRAB 1707), carbapenem-resistant A. baumannii NMS 1693 (CRAB 1693), and carbapenem-resistant A. baumannii NMS 1695 (CRAB 1695) were purchased from the National Institute of Health's Multidrug-Resistant Bacteria Specialized Pathogen Resource Bank (Osong, Republic of Korea) and used. The minimum growth inhibitory concentration of the peptide against each strain was measured, and the geometric mean (GM) value, which is the mean minimum inhibitory concentration (MIC) value of all the bacterial strains used, was confirmed. Additionally, the peptide concentration that induces 10% hemolysis of heparin-treated red blood cells in vitro, HC.And the relative selective index, which is the minimum peptide concentration that causes 10% hemolysis, was also confirmed. If no detectable hemolysis was observed at 100 μM, the value of 200 μM was used to calculate the relative selective index. The selective index was calculated as HC 10 / MIC (μM), and the larger the value, the greater the cell selectivity for bacteria.

[0102] The antibacterial activities of the antibiotic peptides against standard Gram-negative bacteria and colistin-resistant Escherichia coli are shown in Table 1 below.

[0103]

[0104] MicroorganismMinimum growth inhibitory concentration (μM)Cecropin AT. niCecropinPolymyxin BColistinMelittinE. coli220.250.258A. baumannii210.50.254P. aeruginosa842132K. pneumoniae110.250.2532ColREC 155711882ColREC 1211442ColRAB 19151164>642ColRKP <1392264>6432GM2.251.6317.8833.7214.25HC 10 ​2002002002003.1 Relative Selection Index 88.89123.0811.195.930.22

[0105]

[0106] Table 1 shows the antibacterial activity of antibiotic peptides against various Gram-negative standard bacteria and colistin-resistant Escherichia coli.

[0107] As shown in Table 1, polymyxin B and colistin inhibited the antibacterial activity against colistin-resistant strains, whereas T. nicecropin exhibited antibacterial activity similar to that of the reference strain even in resistant strains. Furthermore, the average minimum inhibitory concentration (GM) for the reference strain and colistin-resistant strains was the lowest for T. nicecropin at 1.63, indicating the highest antibacterial activity.

[0108] Additionally, the antibacterial activity of antibiotic peptides against carbapenem-resistant strains was measured, and the carbapenem-based antibacterial agents Imipenem and Meropenem were used together in the experiment. The results are shown in Table 2.

[0109]

[0110] Microorganism Minimum Growth Inhibitory Concentration (μM) Cecropin AT. nicecropin Polymyxin BColistin Melittin Imipenem Meropenem CRAB 169311140.5> 256128 CRAB 169542480.5> 256256 CRAB 19151164> 2562> 256128 CRAB 170710.50.50.52> 256128 CRAB 191910.564 2561> 256128 GM1.601.00 26.7104.91.20 256153.6 HC 10 2002002002003.1200200Relative Selection Index1252007.491.912.580.781.30

[0111]

[0112] Table 2 shows the antibacterial activity of antibiotic peptides against various carbapenem-resistant Acinetobacter bacteria.

[0113] As shown in Table 2, Imipenem and Meropenem, which are carbapenem antibiotics, were confirmed to have no antibacterial activity because they showed resistance to carbapenem. In addition, these antibiotics are low-molecular-weight antibiotics for which hemolytic activity cannot be measured, and therefore relative selectivity indices could not be calculated, and thus were not applied. The CRAB 1915 strain is a bacterium that is simultaneously resistant to carbapenem and colistin, and neither colistin nor polymyxin B showed antibacterial activity, but T. nicecropin showed excellent antibacterial activity in all strains.

[0114] From the above results, it was confirmed that T. nicecropin has excellent antibacterial activity and cell selectivity against colistin-resistant Escherichia coli and carbapenem-resistant Acinetobacter bacteria compared to representative antibiotics.

[0115]

[0116] Example 2. Confirmation of peptide toxicity

[0117] To investigate the toxic effects of the peptides on mammalian cells, cell viability was determined. Specifically, RAW 264.7 mouse macrophage cells and human embryonic kidney (HEK) cells used in the experiment were obtained from the Korean Cell Line Bank (Seoul, Korea). The cells were maintained in DMEM culture medium (Thermo Fischer Scientific Inc., MA, USA) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin in a humidified 5% CO2 incubator at 37°C. The cytotoxicity of the peptides was analyzed using the WST-8 cell proliferation assay (Biomax Co., Ltd, Seoul, Korea), and the experiment was performed according to the kit protocol. The peptide samples used in Example 1 were then dissolved in dimethyl sulfoxide (DMSO) to prepare a 10 mg / mL stock solution. Cells (1 10 4 ) were seeded in 96-well plates, and treated with peptides at concentrations of 0 to 100 μM at 80% confluency, and then cultured for 24 hours. After culture, WST-8 reagent was added, and the change in absorbance at 450 nm with respect to the reagent blank was read, and the value was expressed as percent cell survival. Statistical analysis was performed using two-way ANOVA with Dunnett's comparison test. Each value is expressed as the mean ± SEM of three independent experiments, and *P < 0.05, **P < 0.01, ***P < 0.001 indicate statistical significance, and ns indicates no significance. The results are shown in Fig. 1.

[0118] As shown in Fig. 1, T. nicecropin was confirmed to not exhibit toxicity up to a concentration of 100 μM in RAW 264.7 mouse macrophages. In contrast, melittin exhibited 96.9% toxicity at a concentration of 12.5 μM in RAW 264.7 mouse macrophages. In L-929 cells, T. nicecropin showed a viability rate of over 95%, whereas melittin showed a viability rate of less than 5% at 1.6 μM.

[0119] Additionally, hemolytic activity was measured in sheep red blood cells, and the results are shown in Fig. 2.

[0120] As shown in Fig. 2, when hemolytic activity was measured in sheep red blood cells, melittin exhibited 84.3% hemolytic activity at a concentration of 12.5 μM, whereas T. nicecropin did not exhibit hemolytic activity even at a concentration of 100 μM. From the above results, it was confirmed that T. nicecropin does not exhibit toxicity to animal cells.

[0121]

[0122] Example 3. Peptide effect on inhibition of biofilm formation

[0123] Gram-negative bacteria form biofilms as a self-defense mechanism to survive in unfavorable environments, thereby increasing antibiotic resistance. Microbial interactions with surface materials mediate primary adhesion and development as a matrix-enclosed structure. Therefore, a crystal violet-based assay was performed to investigate the biofilm formation inhibition ability of peptides against colistin-resistant strains (ColREC 1557 and ColREC 12). Specifically, bacterial cells cultured in MH medium containing 0.2% (w / v) glucose were plated at a density of 2 × 10 5After seeding at a concentration of 1 CFU / mL, the cells were exposed to various concentrations (0 to 32 μM) of the peptides at 37°C for 16 h. The bacterial cells were then fixed with 100% methanol for 15 min and stained with crystal violet for 1 h. The plates were then washed with distilled water, dried, and dissolved in 100% ethanol (v / v). The color development, which indicates the degree of biofilm formation, was measured at 595 nm and expressed as the percentage of biofilm formation. The results are shown in Figure 3.

[0124] As shown in Fig. 3, in ColREC 1557, T. nicecropin showed 75.3% inhibition even at a concentration of 2 μM, and more than 90% inhibition at 4 μM. In contrast, colistin was confirmed to inhibit only 58.6% of biofilm formation in ColREC 1557 at 16 μM. In addition, while 2 μM colistin inhibited 28.9% of biofilm formation in ColREC 12, the same concentrations of T. nicecropin and cecropin A showed 82.5% and 80.8% inhibition rates, respectively, confirming excellent inhibitory efficacy.

[0125]

[0126] Example 4. Antibacterial activity mechanism of peptides

[0127] LPS is a major component of the outer membrane of Gram-negative bacteria and induces TLR4-mediated inflammatory signals. There is growing interest in peptides with excellent antiendotoxin activity capable of removing bacterial LPS for the treatment of Gram-negative infections. To confirm peptide-mediated LPS-neutralizing activity, experiments were conducted using the Limulus Amebocyte Lysate (LAL) chromogenic endotoxin assay kit (GenScript, New Jersey, USA). Specifically, peptides at concentrations of 1.6 to 50 μM were allowed to interact with 2 ng / mL LPS for 10 minutes at 37°C. LL-37, known to have high LPS-neutralizing activity, was used as a control. LAL enzyme (10 μM) was added to the peptide-LPS complex and incubated for 10 minutes before adding a chromogenic substrate. Endotoxin levels were expressed as endotoxin units (EU) per milliliter by measuring absorbance at 545 nm three times. The results are shown in Fig. 4.

[0128] As shown in Figure 4, all peptides were confirmed to neutralize LPS in a concentration-dependent manner.

[0129]

[0130] Additionally, the binding ability of LPS and peptides was investigated by the BODIPY-TR cadaverine (BC, Thermo Fisher Scientific, MA, USA) displacement assay. This assay was conducted based on the principle that BC fluorescent dye quenches upon binding to cell-free LPS and emits fluorescence upon binding of LPS to the peptide. Specifically, the probe complex was prepared by mixing 5 μg / mL BC and 5 μg / mL LPS (E. coli O111:B4, Sigma-Aldrich, St. Louis, MO, USA) in 50 mM Tris buffer (pH 7.4) at room temperature for 6 h. Peptides, including the LL-37 control (1.6–50 μM), known to have high LPS binding ability, were allowed to interact with the LPS-BC mixture in a black 96-well plate for 30 min. Relative fluorescence intensities were recorded at an excitation wavelength of 580 nm and an emission wavelength of 620 nm using a fluorescence microplate reader (Molecular Devices, San Jose, USA). The results are shown in Fig. 5.

[0131] Figure 5 shows the displacement results of BODIPY-TR-cadaverine from LPS after peptide treatment.

[0132] As shown in Figure 5, T. nicecropin induced BC displacement in a concentration-dependent manner. Furthermore, compared to the 100% displacement rate of the well-known LPS-binding peptide LL-37, both T. nicecropin and cecropin A were found to increase BC displacement by 79.7% and 71.3%, respectively. This confirmed that these peptides exhibit LPS neutralizing and binding interactions similar to LL-37.

[0133] Additionally, an isothermal titration calorimetry (ITC) experiment was performed to measure the binding affinity of the peptide to LPS, and the results are shown in Fig. 6.

[0134] As shown in Fig. 6, the excellent binding affinity of T. nicecropin for LPS was confirmed, indicating that this was an exothermic process caused by strong electrostatic interactions.

[0135] From the above results, it was confirmed that T. nicecropin exhibits antibacterial activity against Gram-negative bacteria by interacting with LPS and neutralizing LPS.

[0136]

[0137] Example 5. Membrane depolarizing ability of peptides

[0138] To determine the ability of the peptide to penetrate the bacterial outer membrane, 1-N-phenylnaphthylamine (NPN) was used for the assay. Specifically, the strain was washed with washing buffer (5 mM HEPES, 20 mM glucose, pH 7.4) and then resuspended in dilution buffer (5 mM HEPES, 20 mM glucose, 0.1 M KCl, pH 7.4). Fluorescence changes in the negative control (cells with dye) and cells treated with various concentrations of the peptide (1 to 8 μM) were recorded using a fluorescence spectrophotometer. The results are shown in Figure 7.

[0139] As shown in Figure 7, a dose-dependent increase in fluorescence intensity was observed upon treatment of each bacterium with various concentrations of peptide. In E. coli, colistin and T. nicecropin exhibited similar permeability, whereas in colistin-resistant bacteria, T. nicecropin permeated the membrane more efficiently than colistin.

[0140] From the above results, it was confirmed that T. nicecropin can overcome the resistance mechanism of existing antibiotics by destroying the outer membrane of colistin-resistant bacteria.

[0141]

[0142] Example 6. Ability of peptides to damage E. coli membranes

[0143] The optimal duration of peptide exposure for E. coli was determined by considering the bacterial mortality rate over time. To measure the antibacterial activity of the peptide over time, 2 mL of bacterial suspension was treated with various concentrations (2 μM, 4 μM) of T. nicecropin and incubated at 37°C. At each time point (5, 10, 15, 30, 45, 60, and 120 min), 100 μL of culture medium was plated onto LB agar plates. Colonies were counted after 12 h of incubation at 37°C. The results are shown in Figure 8.

[0144] As shown in Fig. 8, for 2 μM and 4 μM T. nicecropin, live bacteria were completely eradicated after 2 hours of incubation.

[0145]

[0146] Next, changes in the biofilm morphology were analyzed using field emission-scanning electron microscopy (SU8020; Hitachi, Tokyo, Japan). Cultured bacteria were treated with 4 μM T. nicecropin for 4 h, washed with PBS, and fixed with 2.5% (w / v) glutaraldehyde overnight at 4°C. They were then further fixed with 1% osmium tetroxide and dehydrated using gradually increasing concentrations of 50, 60, 70, 80, 90, and 100% ethanol solutions. The dehydration process was then prolonged by varying the volume ratio of ethanol to isoamyl acetate to 2:1, 1:1, and 1:2, followed by incubation in hexamethyldisilane for an additional 30 min. The dehydrated samples were coated with platinum and imaged using a scanning electron microscope. The results are shown in Figure 9.

[0147] As shown in Figure 9, bacterial membrane damage was observed after exposure of E. coli to 4 μM T. nicecropin for 4 h. Control bacteria maintained a smooth, intact appearance, whereas bacteria exposed to the peptide exhibited shrinkage and membrane surface damage.

[0148] These results confirm that the peptide targets the membrane, causing damage and causing leakage of internal substances. Furthermore, the peptide induced cytoplasmic leakage through cell membrane disruption, ultimately leading to cell death. This demonstrates that T. nicecropin exerts its antibacterial activity through a bacterial membrane disruption mechanism.

[0149]

[0150] Example 7. Confirmation of peptide-LPS interaction using saturation transfer difference (STD) method.

[0151] To investigate the interaction between the peptide and LPS (E. coli O111:B4, 25 μM), saturation transfer difference (STD) NMR spectra were obtained. NMR experiments were performed using a Bruker 700 MHz spectrometer. Specifically, a 0.5 mM nicecropin sample was dissolved in 20 mM sodium phosphate buffer, pH 5.9, and 15 μM LPS was added. The spectra were selectively saturated at -3.0 ppm for the LPS resonance, and a reference spectrum was observed at 40 ppm. STD-NMR data were obtained by subtracting the off-resonance spectrum from the on-resonance spectrum using 40 Gaussian pulses with a duration of 50 ms and a total saturation time of 2 s. The results are shown in Figure 10.

[0152] As shown in Fig. 10, the upper spectrum shows the one-dimensional proton spectrum of the peptide alone, whereas the lower spectrum shows the saturation transfer difference (STD) effect of the peptide bound to LPS. A strong STD effect was observed due to the hydrophobic interactions of three aromatic hydrophobic residues characteristic of T. nicecropin with the lipids of LPS. In addition, amide protons and aliphatic protons also interacted with LPS and exhibited STD effects. In particular, the strong STD effect in the 7.0–7.7 ppm region corresponding to the aromatic ring protons of Trp2, Phe4, and Phe5 shown below confirmed that these aromatic residues are important for the interaction with LPS. Therefore, it was predicted that T. nicecropin has antibacterial activity by destroying the cell membrane of Gram-negative bacteria through this strong interaction with LPS.

[0153]

[0154] Example 8. Comparison of secondary structures of peptides

[0155] Circular dichroism (CD) spectroscopy was performed to investigate the secondary structural changes of the peptides. All CD experiments on the peptides were performed using a J-810 spectropolarimeter (Jasco, Tokyo, Japan) with a 1-mm path length cell at 25°C. CD spectra of the peptides were recorded from 190 to 250 nm at 0.1 nm intervals at 50 μM. CD experiments were performed in aqueous solution or in a membrane-like environment of 50 mM DPC micelles or 100 mM SDS micelles. The CD spectral data obtained from 10 scans were averaged and smoothed using the J-810. CD data were expressed in deg cm 2 dmole -1 The mean residue ellipticity (θ) of the unit is expressed. The results are shown in Fig. 11.

[0156] As shown in Fig. 11, the T. nicecropin peptide exhibited a random coil structure in an aqueous environment, but was confirmed to exhibit a complete helical structure in the DPC micelle state mimicking a zwitterionic cell membrane or in the SDS micelle state mimicking a negatively charged bacterial cell. The double negative maxima at 205 nm and 220 nm showed the characteristic of an α-helical structure, which confirmed that this is a structural characteristic of antimicrobial peptides with an amphipathic α-helical structure for destroying bacterial cell membranes.

[0157]

[0158] Example 9. Confirmation of the anti-inflammatory effect of T. nicecropin

[0159] Example 9.1. Anti-inflammatory effect of rhamnetin in LPS-stimulated RAW264.7 cells.

[0160] Nitric oxide (NO) overproduction is known to be associated with acute and / or chronic inflammation in response to bacterial lipopolysaccharide (LPS). In Example 4, we confirmed that T. nicecropin exhibited antimicrobial activity through effective interaction with LPS, and thus we hypothesized that it could modulate LPS-induced inflammatory signaling. Therefore, the inhibitory effect of the peptide on nitrite production was evaluated using the Griess assay. RAW264.7 cells (1 х 10 5) were pretreated with T. nicecropin at concentrations of 0.6 to 10 μM for 1 hour, and then stimulated with 20 ng / mL LPS for 16 hours. Afterwards, the supernatant and Griess reagent were added in equal proportions and reacted. The color change was measured at 540 nm. The concentration of nitrite content was evaluated using a standard curve of sodium nitrite. In addition, the production of the inflammatory cytokine IL-6 in the culture medium was quantified using an enzyme-linked immunosorbent assay kit (ELISA; R&D Systems, Minneapolis, MN, USA) and analyzed according to the kit protocol. All assays were performed in triplicate, and the concentration of IL-6 was measured by measuring the absorbance at 450 nm using a microplate reader. The results are shown in Fig. 12.

[0161] As shown in Figure 12, the T. nicecropin peptide inhibited NO secretion by 80.1% at 5 μM. In the case of IL-6 (interleukin-6), ELISA-based analysis confirmed that T. nicecropin inhibited by 86.5% at a concentration of 5 μM, confirming a high inhibition rate.

[0162]

[0163] Example 9.2. Anti-inflammatory effect of T. nicecropin peptide in RAW264.7 cells stimulated with colistin-resistant E. coli (ColREC).

[0164] From the above Example 9.1, the anti-inflammatory activity of T. nicecropin peptide against LPS-induced inflammatory response, which is the main cause of immune response in Gram-negative bacteria infection, was confirmed, and it was assumed that it could regulate inflammatory signal induced by colistin-resistant E. coli. Therefore, RAW 264.7 cells (1 Х 10 5) were pretreated with T. nicecropin peptide at concentrations of 0.6 to 10 μM for 1 h, and then treated with 1 Х 10 5 The culture was stimulated with CFU of colistin-resistant E. coli. The results are shown in Fig. 13.

[0165] As shown in Figure 13, it was confirmed that the concentrations of nitrite and IL-6 were significantly increased by colistin-resistant E. coli, and a concentration-dependent inhibitory effect by T. nicecropin peptide was confirmed. That is, it was confirmed that when T. nicecropin was treated, NO and IL-6 were suppressed at a higher level than when colistin was treated.

[0166] From the above results, it was confirmed that the nitrite and inflammatory cytokine suppression ability of the T. nicecropin peptide was excellent not only for LPS-induced inflammatory response but also for inflammatory response induced by colistin-resistant bacteria, thereby confirming the excellent anti-inflammatory effect of the T. nicecropin of the present invention.

[0167]

[0168] Example 10. Effect of T. nicecropin peptide in an LPS-infected animal model of endotoxemia.

[0169] Example 10.1. Confirmation of the effect of T. nicecropin peptide on reducing serum endotoxin.

[0170] To determine whether T. nicecropin peptides reduce endotoxin LPS in a mouse model of endotoxemia infected with LPS, a Limulus amebocyte lysate (LAL) assay was performed. Specifically, the LAL assay was performed using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's recommendations. The results are shown in Figure 14.

[0171] As shown in Figure 14, the LPS level in the LPS-treated group was significantly higher than that in the T. nicecropin-treated group. This confirmed that the LPS-neutralizing ability confirmed through the in vitro LAL assay was maintained in vivo.

[0172]

[0173] Example 10.2. Measurement of cytokine expression levels

[0174] To confirm the inhibitory effect of T. nicecropin on in vivo inflammatory cytokines in the LPS-infected endotoxemia mouse model, the expression level of IL-6, an inflammatory cytokine, was determined using ELISA kits (R&D system). The results are shown in Figure 15.

[0175] As shown in Figure 15, cytokine secretion in the blood increased after LPS infection, and a significant decrease in cytokine secretion was observed in the group treated with T. nicecropin. This confirms that T. nicecropin suppresses the production of inflammatory cytokines in vivo.

[0176]

[0177] Example 10.3. Measurement of liver damage (AST and ALT) and kidney damage (BUN) in blood

[0178] To determine the effects of T. nicecropin peptide on in vivo liver and kidney damage in the LPS-infected endotoxemia mouse model, AST, ALT, and BUN concentrations in mouse blood were measured. Specifically, a standard kit provided by Asan Pharmaceutical was used, and AST, ALT, and BUN were detected in serum according to the manufacturer's instructions. PBS was used as a negative control, and a standard solution was used as a positive control. The results are shown in Figure 16.

[0179] As shown in Figure 16, the hepatotoxicity and nephrotoxicity levels were significantly increased in the LPS-injected group, whereas the AST, ALT, and BUN levels were reduced by 49.5, 54.2, and 66.7%, respectively, in the T. nicecropin peptide-treated group. This confirmed that the T. nicecropin peptide has a hepatoprotective and renal protective effect in the mouse model.

[0180] In addition, in a non-LPS infected mouse model, the AST, ALT, and BUN levels were not significantly different from those of the control group after T. nicecropin peptide treatment, confirming the stability of the T. nicecropin peptide.

[0181]

[0182] Example 10.4. Identification of lung infiltrating inflammatory cells

[0183] To determine the degree of cellular infiltration in the lung tissue of the mouse model induced by T. nicecropin peptide, pulmonary infiltrating inflammatory cells were identified. Specifically, lungs were removed from the LPS-infected endotoxemia mouse model, each sample fixed in 10% formalin buffer, and then dehydrated with alcohol. The dehydrated tissue was then embedded in wax, and coronal sections were dewaxed, stained with hematoxylin & eosin (H&E), and examined for cellular infiltration under an optical microscope. The results are shown in Figure 17.

[0184] As shown in Figure 17, treatment with T. nicecropin confirmed a reduction in inflammatory cells infiltrating the lungs. This confirmed the excellent effect of T. nicecropin in suppressing inflammatory cell infiltration of lung tissue caused by LPS infection.

[0185]

[0186] Example 11. Therapeutic effect of T. nicecropin peptide in a CRAB-infected sepsis animal model

[0187] Example 11.1. Antibacterial activity in a mouse model of sepsis infected with carbapenem-resistant Acinetobacter bacteria.

[0188] The antibacterial activity of T. nicecropin as a peptide antibiotic was confirmed using a sepsis mouse model (CRAB infection mouse model) established using carbapenem-resistant Acinetobacter. The sepsis model was prepared using carbapenem-resistant Acinetobacter as the infectious strain (Jang M. et al., Int. J. Mol. Sci., 20:4895, 2019), and the mice were randomly selected and divided into four groups of five each. As a control group, normal mice were intraperitoneally injected with PBS (pH 7.4), and T. nicecropin was intraperitoneally injected at a dose of 1 mg / kg. To measure the antibacterial activity, the experimental group mice were pretreated with T. nicecropin for 1 hour, and then challenged with carbapenem-resistant Acinetobacter (5 х 10 6 CFU / mouse, IP) were infected. As a sepsis control group, carbapenem-resistant Acinetobacter strains (5 Х 10 6 Mice infected with 100 CFU / mouse, IP) were used. After 16 hours, the mice were euthanized with an overdose of chloroform, and lung, liver, and kidney tissues were collected and subjected to biochemical and histological analyses. Specifically, lysates of the lung, liver, and kidney tissues collected above in cold PBS were evaluated for bacterial colony counts. The results are shown in Figure 18.

[0189] As shown in Figure 18, T. nicecropin was confirmed to reduce bacterial counts in the mouse lungs, liver, and kidneys by approximately 87.6%, 84.7%, and 92.3%, respectively. This confirmed the excellent antibacterial effect of T. nicecropin against sepsis-causing bacteria in a septic shock mouse model.

[0190]

[0191] Example 11.2. Confirmation of the effect of T. nicecropin peptide on reducing serum endotoxin.

[0192] To determine whether the T. nicecropin peptide reduces endotoxin LPS in the serum of the CRAB infection mouse model, a Limulus amebocyte lysate (LAL) assay was performed. Specifically, the LAL assay was performed using the Pierce LAL Chromogenic Endotoxin Quantification Kit (Thermo Scientific, Rockford, IL, USA) according to the manufacturer's recommendations. The results are shown in Figure 19.

[0193] As shown in Figure 19, the LPS level in the carbapenem-resistant Acinetobacter treatment group was significantly higher than that in the T. nicecropin treatment group. Therefore, it was confirmed that the LPS neutralizing ability confirmed through the in vitro LAL assay was maintained in vivo.

[0194]

[0195] Example 11.3. Measurement of cytokine production inhibition ability

[0196] To confirm the inhibitory effect of T. nicecropin on in vivo inflammatory cytokines in the CRAB infection mouse model, the expression levels of inflammatory cytokines TNF-α, IL-6, and IL-1β were determined using ELISA kits (R&D system). The results are shown in Figure 20.

[0197] As shown in Figure 20, after infection with carbapenem-resistant Acinetobacter, the secretion of inflammatory cytokines in the blood increased, and in the group treated with T. nicecropin, the increased secretion of inflammatory cytokines was significantly reduced. Therefore, it was confirmed that T. nicecropin suppresses the production of inflammatory cytokines in vivo.

[0198]

[0199] Example 11.4. Measurement of liver damage (AST and ALT) and kidney damage (BUN) in blood

[0200] To determine the effects of T. nicecropin peptide on in vivo liver and kidney damage in the CRAB infection mouse model, blood AST, ALT, and BUN concentrations were measured. Specifically, a standard kit provided by Asan Pharmaceutical was used, and AST, ALT, and BUN were detected in serum according to the manufacturer's instructions. PBS was used as a negative control, and a standard solution was used as a positive control. The results are shown in Figure 21.

[0201] As shown in Figure 21, in the group injected with carbapenem-resistant Acinetobacter, hepatotoxicity and nephrotoxicity levels significantly increased, whereas in the group treated with T. nicecropin peptide, AST, ALT, and BUN levels decreased by 71.9, 71.2, and 63.9%, respectively. Therefore, it was confirmed that the T. nicecropin peptide has a protective effect on the liver and kidney in the mouse model.

[0202] In addition, when T. nicecropin peptide was treated in a mouse model not infected with carbapenem-resistant Acinetobacter, the AST, ALT, and BUN levels were not significantly different from those of the control group, confirming the stability of the T. nicecropin peptide.

[0203]

[0204] Example 11.5. Confirmation of the effect of reducing lung infiltrating inflammatory cells.

[0205] To determine the degree of cellular infiltration in the lung tissue of the CRAB infection mouse model induced by T. nicecropin peptide, pulmonary infiltrating inflammatory cells were identified. Specifically, lungs were removed from the mouse model, each sample fixed in 10% formalin buffer, and then dehydrated with alcohol. The dehydrated tissue was then embedded in wax, and coronal sections were dewaxed. Next, the tissue was stained with hematoxylin & eosin (H&E), and cellular infiltration was examined under a light microscope. The results are shown in Figure 22.

[0206] As shown in Figure 22, a decrease in inflammatory cells infiltrating the lungs was observed in the group treated with T. nicecropin. This confirms the excellent effect of T. nicecropin in suppressing inflammatory cell infiltration of lung tissue caused by carbapenem-resistant Acinetobacter infection.

[0207]

[0208] Example 11.7. Survival rate measurement results

[0209] To confirm the therapeutic effect of T. nicecropin peptide in sepsis caused by Gram-negative bacteria, the survival ability was evaluated in a septic shock mouse model caused by carbapenem-resistant Acinetobacter infection. Specifically, the experimental group was treated with carbapenem-resistant Acinetobacter (1 Х 10 7 The group injected intraperitoneally with 10 CFU / mouse was used, and the control group was injected with only PBS (pH7.4) or T. nicecropin (10 mg / kg). The experimental group was pretreated with T. nicecropin (10 mg / kg) for 1 hour and then injected with carbapenem-resistant Acinetobacter (1 Х 10 7CFU / mouse, IP) and injected intraperitoneally with T. nicecropin (10 mg / kg) three times at 6-hour intervals. Ten ICR (6-week-old) mice were used for each group. The number of surviving mice was checked at 1-hour intervals for up to 30 hours after intraperitoneal injection of carbapenem-resistant Acinetobacter, and then checked at 6-hour intervals thereafter for up to 96 hours, and the number of surviving mice was expressed as a percentage (%). The results are shown in Fig. 23.

[0210] As shown in Figure 23, all 10 mice in the control group infected with carbapenem-resistant Acinetobacter died within 21 hours, whereas all 10 mice in the experimental group co-injected with T. nicecropin showed a 100% survival rate up to 96 hours. This confirmed that T. nicecropin has an excellent therapeutic effect on septic shock caused by carbapenem-resistant Acinetobacter infection.

Claims

1. A pharmaceutical composition for preventing or treating sepsis or septic shock, comprising a peptide represented by the amino acid sequence of sequence number 1 as an active ingredient.

2. In claim 1, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the peptide is a peptide derived from the cabbage moth (Trichoplusia ni).

3. In claim 1, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the sepsis is sepsis caused by gram-negative bacteria or antibiotic-resistant gram-negative bacteria.

4. In claim 3, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the gram-negative bacteria are Acinetobacter sp. bacteria or Escherichia sp. bacteria.

5. In claim 3, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the antibiotic-resistant gram-negative bacteria are carbapenem- or colistin-resistant bacteria.

6. In claim 5, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the carbapenem- or colistin-resistant gram-negative bacteria is carbapenem-resistant Acinetobacter baumannii (CRAB) or colistin-resistant Escherichia coli (ColREC).

7. In claim 7, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the sepsis or septic shock is caused by endotoxemia or bacterial infection.

8. In claim 1, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the peptide has endotoxin neutralizing or endotoxin removing ability.

9. In claim 1, A pharmaceutical composition for preventing or treating sepsis or septic shock, wherein the peptide increases the permeability of a bacterial cell membrane and damages the cell membrane.

10. A pharmaceutical composition for preventing or treating bacterial infection, comprising a peptide represented by the amino acid sequence of sequence number 1 as an active ingredient.

11. In claim 10, A pharmaceutical composition for preventing or treating a bacterial infection disease, wherein the bacteria are gram-negative bacteria or antibiotic-resistant gram-negative bacteria.

12. In claim 11, A pharmaceutical composition for preventing or treating bacterial infection, wherein the antibiotic-resistant gram-negative bacteria are carbapenem-resistant Acinetobacter baumannii (CRAB) or colistin-resistant Escherichia coli (ColREC).

13. A pharmaceutical composition for preventing or treating an inflammatory disease, comprising a peptide represented by the amino acid sequence of sequence number 1 as an active ingredient.

14. An antibacterial composition comprising a peptide represented by the amino acid sequence of sequence number 1 as an active ingredient.

15. An antimicrobial composition according to claim 14, wherein the antimicrobial composition is any one selected from the group consisting of a pharmaceutical composition, a food additive, a feed additive, a preservative composition, and a quasi-drug composition.

16. A method for inhibiting the growth of bacteria, comprising the step of treating a sample in vitro or administering to an animal other than a human a peptide represented by the amino acid sequence of sequence number 1.

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