KR102086229B1 - Use of papiliocin derivatives - Google Patents

Abstract

The present invention relates to the use or prevention of inflammatory diseases, particularly sepsis or septic shock, of the papiliosin derivatized derivative peptides. Nontoxic, selective antimicrobial activity against gram negative / positive bacteria, multidrug resistant strains, inhibits the production of inflammatory cytokines induced by LPS stimulation, improves liver and kidney damage in septic shock model, inflammatory cytokine expression It improves survival through inhibition, antibacterial action against sepsis-inducing bacteria in lung, liver and kidney, and reduction of lung cell infiltration, and thus can be usefully used as a composition for preventing or treating inflammatory diseases, especially sepsis or sepsis shock. have.

Description

Papillosin derivatives and uses thereof {USE OF PAPILIOCIN DERIVATIVES}

FIELD OF THE INVENTION The present invention relates to the use of antimicrobial papiliosin derivative peptides, and in particular to the use of the 12-mer papiliosin derivative peptides for the prevention or treatment of inflammatory diseases, in particular sepsis or septic shock.

In a broad sense, antigenic antibody responses, called immune responses, have played a major role in biological defense in invertebrates, including humans. However, insects and amphibians protect themselves by strengthening innate immune mechanisms, which are different from acquired immune mechanisms such as antigenic antibody responses. Thus, innate immunity plays an important role as an important defense against early infections not only in insects and amphibians but also in vertebrates including humans. A very important means of the innate immune system is to obtain antimicrobial peptides with bactericidal and inactivating effects on various microorganisms. Recently, as the problem of antibiotic resistance becomes serious, interest in new drug candidates using such antibiotic peptides is increasing. Antibiotic peptides have antimicrobial activity by targeting bacterial membranes, and thus, have the characteristics of maintaining activity against strains resistant to conventional antibiotics. Therefore, various naturally occurring antibiotic peptides have been spotlighted as important biological resources in the search for a new concept of antibiotics that will replace existing antibiotics threatened by the appearance of resistant strains.

The antimicrobial proteins and peptides of insects discovered to date have been described by Boman's team. About 200 species have been isolated from insects since they were first reported in the cecropia moth, which can be largely divided into peptides containing many amino acids, such as cecropins, defensins, florins and glycine, depending on their structure and size. Secropins are basic proteins consisting of 31 to 39 amino acids, for example, an isolated antimicrobial peptide papiliosin from a larva butterfly. Papiliosine has low hemolyticability, very low toxicity to mammalian cells, and has antimicrobial activity targeting bacterial cell membranes, and α-helix- including N-terminal amphiphilic helix structure and C-terminal hydrophobic helix It has a hinge-α-helix structure and has excellent antibacterial and anti-inflammatory activity against various pathogens and resistant strains. In particular, Gram-negative bacteria selectively have excellent antimicrobial activity and low toxicity. In addition, the N-terminal helix structure of papillosin plays an important role in antimicrobial activity, and the second residue, tryptophan, has been found to be important for antimicrobial and anti-inflammatory activity. Also, by removing two residues from the N-terminal helix structure region of papyliocin, the antimicrobial activity of papyliocin derivatives having N-terminal sequences up to 22mer, 20mer, 18mer, 16mer, 14mer, 12mer, and 10mer was measured. As a result, up to 12-mer peptides were found in standard strains of E. coli, A.baumannii, P. aeruginosa, S.aureus, and B. Subtilis . Very low antimicrobial activity (Dasom Jeon, Binu Jacob, Chulhee Kwak, and Yangmee Kim "Short Antimicrobial Peptides Exhibiting Antibacterial and Anti-inflammatory Activities Derived from the N-terminal Helix of Papiliocin" Bulletin of Korean Chemical Society, 38, 1260-1268 (2017)). From this, the N-terminal 12-mer peptide sequence is considered to be the minimum length that can achieve a three-turn helix, it was confirmed that the derivative of the shortest length to maintain the antimicrobial activity, on the basis of this in the present invention papillosin N By designing a derivative of the terminal 12-mer peptide, the peptide was invented to improve economic efficiency, low toxicity and high antimicrobial activity due to short residue sequence.

On the other hand, the inflammatory response is a biological defense response caused by various factors, such as infection by the pathogen or tissue damage, and performs an initial protective action to limit the damage only to the site of infection or damage. In most cases, this inflammatory response leads to the elimination of pathogenic factors using components of intrinsic immunity and the induction of specific adaptive immunity. Redness, swelling, fever, and pain known to be associated with inflammation include increased local blood flow, decreased local blood flow rate, and increased blood permeability due to the expansion of blood vessels by the action of inflammatory mediators and cytokines at the site of inflammation. Results of continuous immune responses such as increased extravasation of plasma components, increased extravasation of immune cells due to increased adhesion of vascular endothelial cells to circulating immune cells, and increased migration to the site of infection by chemotaxis. to be. Sepsis is defined as a situation in which an infection is identified or suspected along with a systemic inflammatory response. Mainly caused by the penetration of bacteria into the blood vessels or cells due to the administration of therapeutic agents that induce immunosuppression and burns or visceral rupture (Cohen J. The immunopathogenesis of sepsis.Nature. 2002; 420: 885-91.) Can be induced by bacteria and Gram-negative bacteria. In the case of Gram-positive sepsis, bacteria continue to survive and multiply inside the cell, whereas Gram-negative sepsis is a circulatory system in which gram-negative bacteria or endotoxins, one of its cell wall components, excessively invade the blood in the primary infectious place. It is caused by spreading through the whole body. When bacterial infection occurs, macrophages at the site of infection are activated to secrete tumor necrosis factor (TNF) -α and interleukin-1 (IL), thereby increasing the release of plasma proteins into tissues, and increasing the release of phagocytes and lymphocytes into tissues. Leads to increased migration and increased adhesion of platelets to the vessel wall. In this way, local blood vessels are blocked and pathogens are concentrated at the site of infection. In particular, sepsis develops systemic infections and is accompanied by severe vascular occlusion induced by TNF-α. Systemic release of TNF-α also results in loss of plasma volume due to vasodilation and increased permeability of blood vessels, causing shock.

Septic shock is defined as systemic inflammatory response syndrome with infection (Bone RC. The pathogenesis of sepsis. Ann. Intern. Med. 1991; 115: 457.). In septic shock, TNF-α further triggers disseminated intravascular coagulation (blood coagulation) resulting in clot production and large consumption of blood clotting proteins in small blood vessels. Because the patient's ability to coagulate blood is lost, vital organs such as the kidneys, liver, heart, and lungs are damaged due to normal perfusion failure, resulting in fever, hypotension, myocardial suppression, dehydration, acute renal failure, and respiratory arrest. Accompany you. Sepsis shock is the most common cause of life-threatening inpatients in the intensive care unit with a 45% mortality rate. The high morbidity and mortality associated with sepsis is due in part to bacterial endotoxins that stimulate proinflammatory cytokines factor secretion from macrophages and monocytes. Lipopolysaccharide (LPS) in the Gram-negative bacterial outer membrane activates macrophages and produces a series of significant amounts of proinflammatory cytokines and nitric oxide (NO) free radicals during endotoxic shock. Promotes secretion Sepsis is a serious disease with a mortality rate of 23% to 46%, depending on when it is found, and is a growing health burden and is known as a health policy challenge (Dellinger RP: Cardiovascular management of septic shock. Crit Care Med 2003 ; 31: 946-55; OsbornTM, Tracy JK, Dunne JR, Pasquale \ M, Napolitano LM: Crit Care Med 2004; 32: 2234-40; Angus DC, Linde-Zwirble WT. Lidicker J, Clermont G, Carcillo J , Pinsky MR: Crit Care Med 2001; 29: 1303-10). Sepsis is the most common cause of non-cardiovascular intensive care death, with at least 750,000 new cases annually in the United States, 50% of which progress to septic shock and about half of 200,000 deaths (Martin GS, Mannino DM, Eaton S, Moss M: N Engl J Med 2003; 348: 1546-54.).

So far, no fundamental treatment for the treatment of sepsis has been identified. Sepsis is not better with conventional anti-inflammatory treatments, and even the only FDA-approved drotrecogin alfa, Xigris ^® , Engl. J. Med. 2012, 366, 2055-2064 is sepsis in the clinical stage. The effect of treatment on the drug is not clear, and research on this is being stopped. Currently, sepsis is usually treated with antibiotics or antifungal injections, and the medication and duration of treatment depends on the type of microorganism. Hemodialysis or transfusion may also be given depending on the patient's condition. If antibiotics and antifungal drugs are heard well, sepsis can be cured, but it can be difficult to treat in people who are infected with drug-resistant microorganisms, in patients with weak immunity, or when treatment is started too late. As such, in relation to the gradual increase in pathogens resistant to various antibiotics, treatment of sepsis has emerged as a very important problem. In particular, as bacterial infections resistant to antibiotics have soared, it is urgent to develop new treatments for sepsis because antibiotics alone cannot effectively treat them. The inventors have identified that the N-terminal sequence 12-mer of papyliocin, a natural antimicrobial peptide, and the new 12-mer peptides developed therefrom can treat or prevent sepsis caused by bacterial infection. The present invention has been completed.

The present inventors intended to design a 12-mer peptide having high economical efficiency, low toxicity and high antimicrobial activity by reducing the residue sequence from the sequence of papyliocin, and thus, papillocin N-terminal sequence 12-mer having antimicrobial activity. And the novel 12-mer peptides developed therefrom have completed the present invention by confirming the prophylactic and therapeutic effects in inflammation-related sepsis animal models.

Accordingly, it is an object of the present invention to provide antimicrobial papyliocin derivative peptides.

It is also an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of inflammatory diseases, sepsis or septic shock, which comprises a papiliosin derivative peptide as an active ingredient.

It is also an object of the present invention to provide a cosmetic composition for improving an inflammatory disease comprising a papiliosin derivative peptide as an active ingredient.

In addition, an object of the present invention is to provide a food composition for improving an inflammatory disease comprising a papillosin derivative peptide as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for the prevention or treatment of inflammatory diseases, sepsis or septic shock comprising the papillosin derivative peptides and the active ingredient thereof.

In another aspect, the present invention provides a cosmetic composition for improving inflammatory disease comprising a papillosin derivative peptide as an active ingredient.

In addition, the present invention provides a food composition for improving inflammatory disease, sepsis or septic shock, which comprises a papiliosin derivative peptide as an active ingredient.

Papiliosin derivative peptides according to the present invention have excellent economic efficiency as a 12-mer, have selective antimicrobial activity against Gram-negative / positive bacteria, multidrug-resistant strains without toxicity to humans, and inflammatory cytokines caused by LPS stimulation Inflammatory diseases because they improve the survival rate by inhibiting the production of, and improving the liver and kidney damage in the sepsis shock model, inhibiting the expression of inflammatory cytokines, antibacterial action against sepsis-inducing bacteria in the lung, liver and kidney, and reducing lung cell infiltration. In particular, it can be usefully used as a composition for preventing or treating sepsis or septic shock.

1 is a diagram showing cytotoxicity through human erythrocyte hemolytic activity of the peptides represented by SEQ ID NOs: 1-7.
2 is a diagram confirming the secondary structure of the peptides represented by SEQ ID NOS: 1 to 7 by circular dichroism spectroscopy.
Figure 3 is a diagram confirming the inhibitory effect of the production amount of the inflammation-induced cytokines TNF-α and IL-6 induced by LPS stimulation of the peptides represented by SEQ ID NOS: 1-7.
4 is a diagram confirming the change in survival rate in the E. Coli K1 infected sepsis shock mouse model by the treatment of the derivative peptide Pap12-6 of SEQ ID NO: 6.
FIG. 5 shows liver damage indices AST and ALT) and kidney damage indices BUN in an E. Coli K1 infected sepsis shock mouse model by treatment of the derivative peptide Pap12-6 of SEQ ID NO: 6
Figure 6 is a diagram confirming the effect of inhibiting the expression of inflammatory cytokines in vivo in the E. Coli K1 infected sepsis shock mouse model by the treatment of the derivative peptide Pap12-6 of SEQ ID NO:
A: Expression levels of inflammatory cytokines TNF-a and IL-1β following peptide administration in sepsis shock mouse model blood; And
B: Inflammatory cytokine expression according to peptide administration in homogenate of sepsis shock mouse model lung.
FIG. 7 is a diagram showing changes in total bacterial bacterial strain in organs in the E. coli infected sepsis shock mouse model treated with the derivative peptide Pap12-6 of SEQ ID NO: 6.
FIG. 8 is a diagram showing cell invasion in lung tissue of the E. coli K1 infected sepsis shock mouse model treated with the derivative peptide Pap12-6 of SEQ ID NO: 6.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings. However, the following embodiments are presented by way of illustration of the present invention, when it is determined that the detailed description of the well-known technology or construction known to those skilled in the art may unnecessarily obscure the subject matter of the present invention, the detailed description may be omitted. However, the present invention is not limited thereto. The invention is susceptible to various modifications and applications within the scope of the following claims and the equivalent scope thereof.

In addition, terms used in the present specification (terminology) are terms used to properly express the preferred embodiment of the present invention, which may vary depending on the intention of the user, the operator, or customs in the field to which the present invention belongs. Therefore, the definitions of the terms should be made based on the contents throughout the specification. Throughout the specification, when a part is said to "include" a certain component, it means that it may further include other components, except to exclude other components unless specifically stated otherwise.

All technical terms used in the present invention, unless defined otherwise, are used in the meaning as commonly understood by those skilled in the art in the related field of the present invention. Also described herein are preferred methods or samples, but similar or equivalent ones are within the scope of the present invention. The contents of all publications described herein by reference are incorporated into the present invention.

In one aspect, the invention relates to RWKIFKKX ₁ X ₂ X ₃ X ₄ X ₅ (SEQ ID NO: 9), wherein X ₁ is I, A or V, X ₂ is E, A or V, X ₃ is K, X ₄ is V or K and X ₅ is G, A, V or W.

In one embodiment, the papiliosin derivative peptide is RWKIFKKIEKVG (SEQ ID NO: 1), RWKIFKKAAKKG (SEQ ID NO: 2), RWKIFKKVVKKG (SEQ ID NO: 3), RWKIFKKAAKKA (SEQ ID NO: 4), RWKIFKKVVKKA (SEQ ID NO: 5), RVKKVV (SEQ ID NO: 5) 6) or the amino acid sequence of RWKIFKKVVKKW (SEQ ID NO: 7).

In one embodiment, the papiliosin derivative peptide is derived from the peptide of SEQ ID NO: 1 with alanine or valine as the eighth (isoleucine) and ninth (glutamate) residues in order to enhance amphotericity to increase antimicrobial activity and reduce toxicity. It may be a 12-mer peptide consisting of SEQ ID NO: 2 to SEQ ID NO: 7 consisting of the amino acid sequence substituted, and substituted with the 11th (Val) residues with lysine, and the 12th (Gly) residues with alanine, valine or tryptophan.

In one embodiment, the papiliosin derivative peptides may have antimicrobial activity against gram negative / positive bacteria and multidrug resistant bacteria.

In one aspect, the present invention relates to a pharmaceutical composition for the prevention or treatment of inflammatory diseases comprising a papillosin derivative peptide as an active ingredient.

In one embodiment, the papyliocin derivative peptide is RWKIFKKIEKVG (SEQ ID NO: 1), RWKIFKKAAKKG (SEQ ID NO: 2), RWKIFKKVVKKG (SEQ ID NO: 3), RWKIFKKAAKKA (SEQ ID NO: 4), RWKIFKKVVKKA (SEQ ID NO: 5), RVKKVK (VWKKVK) Number 6) or the amino acid sequence of RWKIFKKVVKKW (SEQ ID NO: 7).

In one embodiment, the pharmaceutical composition of the present invention comprises a papillocin derivative peptide of RWKIFKKVVKKA (SEQ ID NO: 5), RWKIFKKVVKKV (SEQ ID NO: 6) or RWKIFKKVVKKW (SEQ ID NO: 7), which is particularly low in toxicity and has excellent antibacterial and anti-inflammatory activity. It may be included as an active ingredient, and it is most preferable to include the papiliosin derivative peptide of RWKIFKKVVKKV (SEQ ID NO: 6) as an active ingredient.

In one embodiment, the pharmaceutical composition of the present invention may include one or more papyliocin derivative peptides of SEQ ID NO: 1 to 7 as an active ingredient in combination.

In one embodiment, the inflammatory disease is edema, dermatitis, allergy, atopic, asthma, conjunctivitis, periodontitis, allergic and non-allergic rhinitis, otitis media, sore throat, tonsillitis, pneumonia, gastric ulcer, chronic and acute gastritis, Crohn's disease, colitis , Chronic and acute enteritis, hemorrhoids, gout, ankylosing spondylitis, rheumatic fever, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay salt, 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, fascia disease, viral infection, bacterial infection, fungal infection, burns, surgical Or wounds caused by dental surgery, prostaglandin E excess syndrome, atherosclerosis, arthritis, Hodgkin's disease, pancreatitis, iris, scleritis, It may be any one selected from the group consisting of uveitis, eczema, sepsis or septic shock, more preferably sepsis or septic shock.

In one embodiment, sepsis or septic shock can be caused by infection of Gram negative bacteria, Gram negative bacteria are Enterobacter sp. Bacteria, Salmonella sp. Bacteria, Vibrio sp.) bacteria, Mycobacterium sp. bacteria, Shigella sp. bacteria, Acinetobacter sp. bacteria, Pseedomonas sp. bacteria and E. coli Escherichia sp.) May be any one or more selected from the group consisting of bacteria, and may be Acinetobacter baumannii , Psedomonas aeruginosa or Escherichia coli .

In one embodiment, sepsis or septic shock can be caused by infection with Gram-positive bacteria, and Gram-positive bacteria are Bacillus sp. Bacteria, Staphylococcus sp. Bacteria, Enterococcus spp. ( Enterococcus sp.) Bacteria and Streptococcus sp. Bacteria may be any one or more selected from the group consisting of, Bacillus subtilis (B acillus subtilis ), Staphylococcus aureus ( Staphylococcus aureus ) Staphylococcus epidermidis .

In one embodiment, sepsis or septic shock can be caused by infection with multidrug-resistant bacteria, and the multidrug-resistant bacteria are multidrug resistance- Escherichia coli (MDREC), multidrug resistance- Pseudomonas aeruginosa , MDRPA), multidrug resistance-Acinetobacter baumannii (MDRAB) and methicillin-resistant Staphylococcus aureus (MRSA) have.

In one embodiment, each or a combination of the papiliosin derivative peptides can prevent or treat sepsis or septic shock by having antibacterial activity against sepsis-causing bacteria in the body.

In one embodiment, each or a combination of the papiliosin derivative peptides can prevent or treat sepsis or septic shock by inhibiting organ damage due to sepsis.

In one embodiment, each or a combination of papyliocin derivative peptides may be administered before / simultaneously / after sepsis or septic shock induction by bacteria.

The pharmaceutical composition of the present invention may further include a known inflammatory disease, sepsis or septic shock therapeutic agent in addition to each or a combination of papyliocin derivative peptides as an active ingredient, and may be used in combination with other known treatments. .

In the present invention, the term "prevention" means any action that inhibits or delays the development, spread and recurrence of an inflammatory disease, sepsis or septic shock by administration of a pharmaceutical composition according to the invention, the term "treatment" By the administration of the papyliosin derivative peptide of the invention, or a pharmaceutically acceptable salt thereof, or a composition comprising the same, it means any action that improves or advantageously alters the symptoms of an inflammatory disease, sepsis or septic shock. Those skilled in the art to which the present invention pertains can refer to the data presented by the Korean Medical Association and the like to know the exact criteria of the disease for which the composition is effective, and to determine the extent of improvement, improvement and treatment. will be.

The term "therapeutically effective amount" as used in combination with an active ingredient in the present invention means an amount effective for preventing or treating a target disease, and the therapeutically effective amount of the composition of the present invention may be various factors, for example, a method of administration. This may vary depending on the patient's condition, destination and patient's condition. Therefore, when used in humans, the dosage should be determined in an appropriate amount in consideration of both safety and efficiency. It is also possible to estimate the amount used in humans from an effective amount determined through animal testing. Such considerations when determining the effective amount include, for example, Hardman and Limbird, eds., Goodman and Gilman's The Pharmacological Basis of Therapeutics, 10th ed. (2001), Pergamon Press; And E.W. Martin ed., Remington's Pharmaceutical Sciences, 18th ed. (1990), Mack Publishing Co.

The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. As used herein, the term "pharmaceutically effective amount" refers to an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and that does not cause side effects, and an effective dose level refers to Factors including health condition, inflammatory disease, sepsis or cause of septic shock, severity, drug activity, drug sensitivity, method of administration, time of administration, route and rate of release, duration of treatment, combination or combination of drugs And other factors well known in the medical arts. The compositions of the present invention may be administered as individual therapeutic agents or in combination with other therapeutic agents, may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered in single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can achieve the maximum effect with a minimum amount without side effects, which can be readily determined by one skilled in the art.

The pharmaceutical compositions of the present invention may comprise carriers, diluents, excipients or combinations of two or more thereof conventionally used in biological preparations. As used herein, the term "pharmaceutically acceptable" means to exhibit properties that are not toxic to cells or humans exposed to the composition. The carrier is not particularly limited as long as it is suitable for in vivo delivery of the composition, for example, Merck Index, 13th ed., Merck & Co. Inc. Compounds, saline solution, sterile water, Ringer's solution, buffered saline solution, dextrose solution, maltodextrin solution, glycerol, ethanol and one or more of these components can be mixed and used as needed. Conventional additives may be added. In addition, diluents, dispersants, surfactants, binders and lubricants may be additionally added to formulate into main dosage forms, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like. Furthermore, it may be preferably formulated according to each disease or component by a suitable method in the art or using a method disclosed in Remington's Pharmaceutical Science (Mack Publishing Company, Easton PA, 18th, 1990).

In one embodiment, the pharmaceutical composition may be one or more formulations selected from the group consisting of oral formulations, external preparations, suppositories, sterile injectable solutions and sprays, with oral or injectable formulations being more preferred.

As used herein, the term "administration" means providing a substance or substance to an individual or patient in any suitable manner, and according to the method desired, non-oral administration (eg, intravenous, subcutaneous, intraperitoneal). Or topically applied as an injection formulation) or orally, and the dosage varies depending on the weight, age, sex, health condition, diet, time of administration, method of administration, rate of excretion and severity of the patient. Liquid preparations for oral administration of the compositions of the present invention include suspensions, solvents, emulsions, syrups, and the like, and various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. And the like may be included together. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized formulations, suppositories, and the like. The pharmaceutical composition of the invention may be administered by any device in which the active substance may migrate to the target cell. Preferred modes of administration and preparations are intravenous, subcutaneous, intradermal, intramuscular, injectable and the like. Injections include non-aqueous solvents such as aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g. ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH control, to prevent microbial growth Pharmaceutical carriers such as preservatives (eg, mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like).

As used herein, the term "individual" means monkeys, cows, horses, sheep, pigs, chickens, turkeys, quails, cats, and dogs, including humans who may or may develop the inflammatory disease, sepsis or septic shock. All animals, including mice, rats, rabbits or guinea pigs, can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to an individual. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable additive, wherein the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, calcium hydrogen phosphate , Lactose, mannitol, syrup, gum arabic, pregelatinized starch, corn starch, powdered cellulose, hydroxypropyl cellulose, oppadry, sodium starch glycolate, carnauba lead, synthetic aluminum silicate, stearic acid, magnesium stearate, aluminum stearate, Calcium stearate, sucrose, dextrose, sorbitol, talc and the like can be used. The pharmaceutically acceptable additive according to the present invention is preferably included 0.1 parts by weight to 90 parts by weight based on the composition, but is not limited thereto.

In one aspect, the present invention relates to a cosmetic composition for improving an inflammatory disease comprising a papillosin derivative peptide as an active ingredient.

In one embodiment, the cosmetic composition of the present invention may be a cosmetic composition for anti-inflammatory use.

The cosmetic composition of the present invention may be prepared by including a cosmetically effective amount and a cosmetically acceptable carrier of each of the above-described papyliosin derivative peptides of the present invention (SEQ ID NOs: 1 to 7) or a combination thereof. have.

As used herein, the term “cosmetic effective amount” means an amount sufficient to achieve the anti-inflammatory efficacy of the composition of the invention described above.

The appearance of the cosmetic composition contains a cosmetically or dermatologically acceptable medium or base. These are all formulations suitable for topical application, for example emulsions, suspensions, microemulsions, microcapsules, microgranules or ionic (liposomes) obtained by dispersing an oil phase in solutions, gels, solids, pasty anhydrous products, aqueous phases and It may be provided in the form of a nonionic vesicle dispersant or in the form of a cream, skin, lotion, powder, ointment, spray or cone stick. These compositions can be prepared according to conventional methods in the art. The composition according to the invention can also be used in the form of a foam or in the form of an aerosol composition further containing a compressed propellant.

The cosmetic composition according to an embodiment of the present invention is not particularly limited in the formulation, for example, supple cosmetics, astringent cosmetics, nourishing cosmetics, nutrition cream, massage cream, essence, eye cream, eye essence, cleansing It may be formulated into cosmetics such as creams, cleansing foams, cleansing water, packs, powders, body lotions, body creams, body oils and body essences.

When the formulation of the present invention is a paste, cream or gel, animal carriers, plant fibers, waxes, paraffins, starches, tracantes, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide, etc. may be used as carrier components. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used, in particular, in the case of spray, additionally chlorofluorohydrocarbon, propane Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solvating agent or emulsifying agent is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butylglycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystalline Cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, linolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The cosmetic composition of the present invention is a skin, lotion, cream, essence, pack, foundation, color cosmetics, sun cream, two-way cake, face powder, compact, makeup base, skin cover, eye shadow, lipstick, lip gloss, lip fix, eyebrow pencil It may be applied to cosmetics such as lotion, and detergents such as shampoo and soap.

The cosmetic composition according to an embodiment of the present invention may further include a functional additive and a component included in the general cosmetic composition in addition to the extract containing the active ingredient. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, polymer peptides, polymer polysaccharides, sphingolipids and seaweed extract.

In addition to the said functional additive, you may mix | blend the component contained in a general cosmetic composition with the cosmetic composition of this invention as needed. In addition to the other components included, oils and fats, moisturizers, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, fungicides, antioxidants, plant extracts, pH adjusters, alcohols, pigments, flavorings, blood circulation Accelerators, cooling agents, limiting agents, purified water, and the like.

In one aspect, the present invention relates to a food composition for improving an inflammatory disease comprising a papiliosin derivative peptide as an active ingredient.

Papiliosin derivative peptides of the present invention have little toxicity to animals, and thus can be used as food compositions.

When the composition of the present invention is used as a food composition, each or a combination of the papillosin derivative peptides may be added as it is, or used with other foods or food ingredients, and may be appropriately used according to a conventional method. The composition may include a food acceptable additive in addition to the active ingredient, the amount of the active ingredient may be appropriately determined according to the purpose of use (prevention, health or therapeutic treatment).

As used herein, the term "food supplement" refers to a component that can be added to food supplements, and can be appropriately selected and used by those skilled in the art as being added to prepare health functional foods of each formulation. Examples of food additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic and natural flavors, colorants and fillers, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH regulators, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like, but is not limited by the examples of the food supplement additive of the present invention.

The food composition of the present invention may include a health functional food. As used herein, the term "health functional food" refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and pills using raw materials or ingredients having useful functions for the human body. Here, 'functional' means to obtain a useful effect for health purposes such as nutrient control or physiological action on the structure and function of the human body. The health functional food of the present invention can be prepared by a method commonly used in the art, and the preparation can be prepared by adding raw materials and ingredients commonly added in the art. In addition, the formulation of the health functional food can also be prepared without limitation as long as the formulation is recognized as a health functional food. The food composition of the present invention can be prepared in various forms of formulation, the health functional food of the present invention can be ingested as an adjuvant for improving the improvement effect of the inflammatory disease.

In addition, there is no restriction on the kind of health foods in which the composition of the present invention can be used. In addition, the composition comprising each or a combination of the papiliosin derivative peptides of the present invention as an active ingredient may be prepared by mixing known additives with other appropriate auxiliary ingredients that may be contained in the health functional food according to the choice of those skilled in the art. Examples of foods that can be added include meat, sausages, breads, chocolates, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products, including ice cream, various soups, beverages, teas, drinks, alcoholic beverages and Vitamin complexes and the like, can be prepared by adding the extract according to the present invention as a main ingredient juice, tea, jelly and juice.

The present invention will be described in more detail with reference to the following examples. However, the following examples are only intended to embody the contents of the present invention, and the present invention is not limited thereto.

Example 1 Synthesis of Antibiotic Peptides

To determine the antimicrobial activity of papyliocin derivatives having N-terminal sequences up to 22mer, 20mer, 18mer, 16mer, 14mer, 12mer or 10mer by removing two residues from the N-terminal helix structure region of papyliocin As a result, up to 12-mer peptides were detected in standard strains of E. coli, A.baumannii, P. aeruginosa, S.aureus and B. Subtilis . Has very low antimicrobial activity (data not shown). Accordingly, in order to prepare a 12-mer peptide (Pap12-1) and a single antibiotic peptide derivative derived from papyliocin having antimicrobial activity, N- (9-fluorenyl) methoxycarbonyl (N- (9-fluorenyl) Antibiotic peptides were synthesized and purified by solid phase synthesis using methoxycarbonyl (Fmoc) synthesis ( Biochimica et Biophysica Acta (BBA) -Biomembranes 1798.10 (2010): 1913-1925). Specifically, from the peptide consisting of SEQ ID NO: 1 (Pap12-1) consisting of the 12 residue sequences of the N-terminal of papyliocin, the eighth (isorucin) to enhance the antimicrobial activity to increase the antimicrobial activity and lower the toxicity 12-dimer of Table 1 by substituting the 9th (glutamate) residue with alanine or valine, the 11th (Val) residue with lysine, and the 12th (Gly) residue with alanine, valine or tryptophan. New peptides (Pap12-2 to Pap12-7) were designed and synthesized. Thereafter, the concentrations of the synthesized peptides were quantified using a UV spectrophotometer, and the purity of the final peptides (> 98%) was analyzed by reverse-phase high performance liquid chromatography (Reverse-phase HPLC).

Peptide
designation Amino acid sequence (-NH2) Majesty Mean amphipathic moment <μH> SEQ ID NO: Pap12-1 RWKIFKKIEKVG +4 0.486 One Pap12-2 RWKIFKKAAKKG +6 0.459 2 Pap12-3 RWKIFKKVVKKG +6 0.555 3 Pap12-4 RWKIFKKAAKKA +6 0.483 4 Pap12-5 RWKIFKKVVKKA +6 0.580 5 Pap12-6 RWKIFKKVVKKV +6 0.653 6 Pap12-7 RWKIFKKVVKKW +6 0.736 7 Melittin
(Control peptide) GIGAVLKVLTTGLPALISWIKRKRQQ 8

As a result, antimicrobial peptides composed of amino acid sequences of SEQ ID NOs: 1 to 7 as shown in Table 1 were synthesized.

Example 2. Antimicrobial Activity of Antibiotic Peptides

2-1. Antimicrobial Activity of Antibiotic Peptide against Gram Positive / Negative Strains

In order to compare the antimicrobial activity of the antibiotic peptides synthesized in Example 1, the antimicrobial activity of the antibiotic peptides against Gram-negative standard strains and Gram-positive standard strains was previously known as melittin (control peptide). ) And comparative measurements. Antimicrobial activity was determined using minimum inhibitory concentration (MIC) of peptides that did not divide cells in nutrient-rich MH medium. Specifically, the strains described in Table 2 below were purchased, diluted with MH medium so that the number of bacteria was 2Х10 ⁶ CFUs (colony-forming units) per ml, and 100 μl were dispensed into 96-well micro titration plates, followed by MH medium. 100 μl of the peptide solutions (2: 1 stepwise diluted solutions) of SEQ ID NOS: 1 to 7 of the present invention diluted with. The MIC of the peptides was determined by incubating the plates at 37 ° C. for 16 hours and measuring the absorbance of each well at 620 nm using an ELISA reader (Bio-Tek Instruments). The HC ₁₀ value means a concentration indicating 10% hemolytic activity in the hemolytic activity performed in Example 3 below, and the Therapeutic index, which means pharmacological utility, was calculated as HC ₁₀ / mean MIC.

division Strain name Deposit number source Gram-
Positive bacteria Bacillus subtilis
( Bacillus subtilis ) KCTC 3068 Korea Cell Line Bank
(Korean Collection for Type Cultures) Staphylococcus aureus
( Staphylococcus aureus ) KCTC 1621 Staphylococcus epidermidis KCTC 1917 Gram-
Negative bacteria Escherichia coli
( Escherichia coli ) KCTC 1682 Korea Cell Line Bank
(Korean Collection for Type Cultures) Pseudomonas Eruginosa
( Psedomonas aeruginosa ) KCTC 1637 Acinetobacter Baumani
( Acinetobacter baumannii ) KCTC 2508 Antibiotic
Resistant bacteria MDREC CCARM 1229 Antibiotic Resistant Bacterial Bank
(Culture collection of antimicrobial resistant microbe) CCARM 1238 MDRAB CCARM 12010 CCARM 12220 MDRPA CCARM 2002 CCARM 2003 MRSA CCARM 3114 CCARM 3090 CCARM 3126

bacteria Minimal inhibitory concentration (MIC) (μM) Pap12-1 Pap12-2 Pap12-3 Pap12-4 Pap12-5 Pap12-6 Pap12-7 Melittin Gram-negative bacteria E.coli 16 16 16 8 8 4 4 4 A. baumannii 16 16 8 16 4 2 2 2 P. aeruginosa 16 16 16 16 8 4 4 4 Mean antimicrobial activity 16 16 13.3 13.3 6.6 3.3 3.3 3.3 HC ₁₀ (μM) 800 800 800 800 800 800 172 0.76 Therapeutic index 50 50 60.2 60.2 121.2 242.4 53.0 0.23 Gram-positive bacteria S.aureus 64 16 8 8 8 4 4 4 B.Subtilis 32 16 16 16 16 8 8 2 S.epidermidis 32 32 16 16 16 8 8 2 Mean antimicrobial activity 42.7 21.3 13.3 13.3 13.3 6.6 6.6 2.6 Therapeutic index 18.7 37.6 60.2 60.2 60.2 121.2 26.5 0.29

As a result, as shown in Table 3, it can be seen that the six derivative peptides of Pap12-2 to Pap12-7 of the present invention have superior antimicrobial activity compared to the parent peptide Pap12-1. In particular, Pap12-6 and Pap12-7 had an antimicrobial activity equivalent to melittin, a positive control known to have high antimicrobial activity in Gram-negative bacteria. In addition, Pap12-6 and Pap12-7 showed the highest antimicrobial activity against Gram-positive bacteria, and similar to melittin against S. aureus .

2-2. Confirmation of Antimicrobial Activity of Antibiotic Peptide against Multidrug-resistant Bacteria

In order to confirm the antimicrobial activity against the multidrug-resistant bacteria of the antibiotic peptides synthesized in Example 1, the antimicrobial activity against six multidrug-resistant Gram-negative bacteria and three multidrug-resistant Gram-positive bacteria (antibiotic resistant bacteria) was measured. Specifically, the multidrug-resistant strains described in Table 2 were purchased, diluted with MH medium so that the number of bacteria was 2Х10 ⁶ CFUs / ml, and 100 μl were dispensed into 96-well microtiter plates, followed by MH medium. Dilute Pap12-1, Pap12-2, Pap12-3, Pap12-4, Pap12-5, Pap12-6 and Pap12-7 antibiotic peptide solution (2: 1 (V / V) stepwise diluted solution) and Mellie Tin (positive control) was added to 100 μl in each well. The MIC of each peptide was determined by incubating the plate for 16 hours at 37 ° C. and then measuring the absorbance of each well at 620 nm using an ELISA reader (Bio-Tek Instruments).

Microorganisms Minimal inhibitory concentration (MIC) (μM) Pap12-1 Pap12-2 Pap12-3 Pap12-4 Pap12-5 Pap12-6 Pap12-7 Melittin Gram-negative bacteria MDREC 1229 16 32 16 16 16 8 8 4 MDREC 1238 16 32 16 16 16 8 8 8 MDRPA 2002 32 > 32 32 32 32 8 8 8 MDRPA 2003 16 > 32 32 32 32 8 8 4 MDRAB 12010 16 > 32 16 32 32 8 8 4 MDRAB 12220 16 32 16 16 32 8 8 4 Gram-positive bacteria MRSA 3090 > 32 > 32 16 32 32 16 16 8 MRSA 3114 > 32 > 32 16 32 32 16 16 8 MRSA 3126 > 32 > 32 16 32 32 16 16 8

As a result, as shown in Table 4, all derivative peptides except Pap12-2 was superior to the antimicrobial activity against multidrug-resistant bacteria compared to Pap12-1. In particular, Pap12-6 and Pap12-7 are papillocin derivative peptides that have a high degree of antimicrobial activity against melittin (positive control) and Gram-negative multidrug resistant bacteria which are known to have high antimicrobial activity. Could. In addition, Pap12-6 and Pap12-7 were found to have the highest antimicrobial activity against Gram-positive multidrug-resistant bacteria. Therefore, the derivative peptides Pap12-6 and Pap12-7 were found to be excellent peptide antibiotic candidates.

Example 3. Toxicity Verification of Antibiotic Peptides

In order to confirm the cytotoxicity of the antibiotic peptides represented by SEQ ID NOs: 1 to 7 prepared in Example 1, human erythrocyte hemolytic activity was confirmed. Human erythrocytes were diluted with PBS (phosphate-buffered saline: 35 mM phosphate buffer / 150 mM NaCl mixed solution, pH 7.4), and then washed three times by centrifugation at 1000 g for 10 minutes. 100 μl of the 8.0% erythrocyte solution diluted with PBS was loaded into a 96-well microtiter plate, and then mixed with 100 μl of the antibiotic peptide solution represented by SEQ ID NOS: 1 to 7, respectively, at 37 ° C. Incubate for 1 hour. The incubated 96-well microtiter plates were centrifuged at 1000 g for 5 minutes, and then 100 μl of the supernatant was transferred to another 96-well microtiter plate and the absorbance at 405 nm was measured. At this time, melittin was used as a positive control antimicrobial peptide showing strong hemolytic activity. The measurement of hemolytic activity was calculated as 100% hemolysis when treated with 0.1% Triton X-100, and the hemolytic activity (degree of cell destruction) of antibiotic peptides was calculated as% hemolysis (Equation 1).

A: 405 nm absorbance in peptide solution;

B: 405 nm absorbance at 0.1% Triton X-100; And

C: 405 nm absorbance in PBS solution.

As a result, melittin, a positive control, showed high toxicity at low concentrations of 10 μM or less, showing high toxicity, while peptide Pap12-7 represented by SEQ ID NO: 7 showed hemolytic activity of 21% at 100 μM, but toxicity up to 25 μM. It did not show, it was found that there is no toxicity in the concentration range having antibacterial activity (Fig. 1). In addition, all six peptides (SEQ ID NOS: 1 to 6) except the peptide Pap12-7 represented by SEQ ID NO: 7 did not show hemolytic activity even at a high concentration of 400 μM and were marked as 0, indicating that they were selective for bacteria. In addition, HC ₁₀ value means a concentration showing a hemolytic activity of 10%, peptides of SEQ ID NO: 1 to 6 did not show hemolytic activity even at 400μM was estimated to 800μM, Pap12-7 of SEQ ID NO: 7 was calculated as 172μM .

Example 4. Secondary Structure Determination of Antibiotic Peptides

In order to measure the secondary structure of the antibiotic peptides represented by SEQ ID NOs: 1 to 7, prepared in Example 1, the secondary structure was measured using a circular dichroism spectrometer. The spectra of the aqueous solution, SDS micelles (micelle) and DPC micelles showed no secondary structure in the aqueous solution, but it was found that all antibiotic peptides had an alpha helix structure in micelles, which are similar to the biofilm (FIG. 2). Therefore, it was found that the length of the 12-mer residue sequence of the papyliocin derivative peptides of the present invention was an efficient three-turn alpha-helix structure. In particular, Pap12-6 and Pap12-7 peptides with high antimicrobial activity and the highest amphotericity were found to have the highest alpha helix structure, indicating that the secondary structure is important for antimicrobial activity.

Example 5 Inhibitory Effects of Antibiotic Peptides on Induced Inflammatory Cytokine Production

The effects of antibiotic peptides represented by SEQ ID NOS: 1 to 7 on the production of inflammatory cytokines TNF-α and IL-6, which are caused by LPS stimulation known as pathogens, were confirmed. Specifically, macrophage Raw264.7 from mice was present in 5% CO ₂ in RPMI1640 medium containing 10% fetal bovine serum (FBS) and 1% antibiotics (100 U / mL penicillin, 100 ug / mL streptomycin). Incubated at 37 ° C. Each well of a 96-well microtiter plate was aliquoted to 200 μl to give a 1Х10 ⁵ cell count. After 96-well microtiter plates were incubated overnight at 37 ° C. in a 5% CO ₂ incubator, the peptides of SEQ ID NOS: 1 to 7 were reacted at 10 or 20 μM concentration for 1 hour, followed by 20ng of LPS. / ml treatment and incubate for 18-24 hours. At this time, only a culture medium was used as a negative control, and a culture solution treated with only LPS was used as a positive control. Subsequently, the immune plates were coated and washed overnight with the primary antibodies, anti-mTNF-α antibody and anti-mIl-6 antibody, respectively, followed by blocking buffer (5% BSA in PBS) to stop non-specific reactions. Blocking at 4 ° C. In a plate coated with the primary antibody, 50 μl of the cell culture supernatant after the LPS treatment 18 to 24 hours was applied to the plate and incubated for 2 hours. Then, two hours after treatment with each of the colorable secondary antibodies that recognize mTNF-α and mIl-1β, TMB (3,3`, 5,5`-) was added after 40 minutes with streptavidin HRP. tetramethybensidine) solution. After 10-15 minutes of color development, the color was stopped by H ₂ SO ₄ , and the amount of mTNF-α and mIl-6 dissolved in the culture supernatant was measured at 540 nm using an ELISA reader (Molecular Devices, Sunnyvale, CA). The amounts of mTNF-α and mIl-6 were quantified in comparison with the absorbance values of the standard.

As a result, all peptide derivatives reduced the production of inflammatory-induced cytokines TNF-α and IL-6 (FIG. 3). In particular, the activity of Pap12-5, Pap12-6 and Pap12-7 was excellent. Specifically, the amount of mTNF-α expressed when Pap12-5 was treated with 10 μM and 20 μM was 53.4% and 38.9% of the control group, respectively, and the amounts of mIL-6 were 61.5% and 44.7% of the control group, respectively. In addition, when PapRW12-7 was treated with 10 μM and 20 μM, the amount of mTNF-α decreased to 37.4% and 34.0% of the control, respectively, and the amount of mIL-6 decreased to 60.3% and 40.4% of the control, respectively. In particular, when Pap12-6 was treated with 10 μM and 20 μM, the amount of mTNF-α decreased by 29.2% and 25.1% in control, respectively, and the amount of mIL-6 decreased by 58.7% and 27.3% in control, respectively. Through this, it was found that all derivative peptides of the present invention are very effective in reducing the amount of inflammation-inducing cytokines TNF-α and IL-6, and among the seven peptides, Pap12-6 has the most significant effect. there was.

Pap12-6 did not show hemolytic activity even at 400μM when hemolytic activity was measured, and showed very low toxicity, and antimicrobial activity was highest when measuring antimicrobial activity. Among the 7 antibiotic peptides of the present invention, the peptide with the highest bacterial selectivity At the same time, the inhibitory ability of TNF-α and IL-6, which are inflammation-inducing cytokines, is high, and thus, it was confirmed that it is the most useful peptide antibiotic candidate. Accordingly, Pap12-6 peptide was selected as the best peptide antibiotic candidate among the seven derivative peptides, and the experiment was performed in the following sepsis shock mouse model.

Example 6 Effect of Improving Survival Rate of Pap12-6 in Sepsis Shock Mouse Model

Survival changes were confirmed in the E. Coli K1 infected sepsis shock mouse model by treatment of the peptide peptide Pap12-6 of SEQ ID NO: 6 selected above. Specifically, 10 mg / kg of Pap12-6 peptide was injected into the abdominal cavity of 5 ICR (6 week old) mice in each group, and 1 hour later, Escherichia coli E. coli K1 (1 × 10 ⁷ CFU /) diluted with LB (Lysogeny broth) medium. mice were injected intraperitoneally, and the number of surviving animals was checked every 0, 6, 12, 18, 24, 30, 36, 48, 60, 72, 84 and 96 hours and expressed in terms of percent (control). Injects only E. coli K1 (1 × 10 ⁷ CFU / mouse).

As a result, the control group injected with nothing and the mouse group injected with only Pap12-6 peptide (10 mg / kg) survived for 96 hours, but the group of mice induced with sepsis shock by injection of E. coli K1 in 18 hours. All died (FIG. 4). On the other hand, mice injected with Pap12-6 peptide (10 mg / kg) and E. coli K1 survived all 5 mice up to 36 hours, and 3 survived up to 96 hours, significantly improving survival for sepsis shock ( 4).

Example 7 Effects of Pap12-6 on Impairing Liver and Kidney Damage in Sepsis Shock Mouse Model

Liver damage indices AST and ALT and kidney damage indices BUN were confirmed in the E. Coli K1 infected sepsis shock mouse model by treatment of the derivative peptide Pap12-6 of SEQ ID NO: 6. Specifically, Pap12-6 peptide (1 mg / day 1 hour prior to intraperitoneal injection of E. coli (1 × 10 ⁶ CFU / mouse) using three Balb / c (6 weeks old, E. coli model) in each group. kg) were intraperitoneally injected. After overnight injection, the blood of the mice was collected, left at room temperature for 30 minutes, and centrifuged at 4 ° C. and 13000 rpm for 15 minutes to separate serum. Thereafter, 10 µl of substrate solution was added to a 96-well plate to measure AST, and 2 µl of the separated serum was added and incubated at 37 ° C. for 60 minutes. Thereafter, 10 µl of the coloring solution was added and incubated for 20 minutes at room temperature. Then, 100 µl of 0.4N NaOH solution was added thereto, and the absorbance was measured at 505 nm after 10 minutes. In addition, 10 μl of substrate solution was added to a 96 well plate to measure ALT, and then 2 μl of the separated serum was added and incubated at 37 ° C. for 30 minutes. Thereafter, 10 µl of the coloring solution was added and incubated at room temperature for 20 minutes, and 100 µl of 0.4N NaOH solution was added thereto, and then 10 minutes later, the absorbance was measured at 505 nm. In addition, 100 μl of enzyme solution was added to the 96-well plate to measure BUN, and then 1 μl of the separated serum, standard solution and purified water were added thereto, and then incubated at 37 ° C. for 5 minutes. Thereafter, 100 µl of the coloring solution was added and incubated at 37 ° C. for 5 minutes, and then absorbance was measured at 580 nm.

As a result, when E. coli was injected, hepatotoxicity and nephrotoxicity were significantly increased compared with the control group, whereas when treated with Pap12-6 peptide, hepatotoxicity levels of AST and ALT were reduced to 43.8% and 41.6%, respectively. The BUN value was reduced to 61.4%, it was confirmed that the toxicity level was lowered (Fig. 5).

Example 8 Inhibitory Effect of Pap12-6 Expression on Inflammatory Cytokines in Blood and Lung of Sepsis Shock Mouse Model

Inhibition of the expression of inflammatory cytokines in vivo in E. coli K1 infected sepsis shock mouse model by treatment with the derivative peptide Pap12-6 of SEQ ID NO: 6 was confirmed. Specifically, Pap12-6 peptide (1 week before E. coli K1 (1 × 10 ⁶ CFU / mouse) was intraperitoneally injected using 3 Balb / c (6 weeks old, E. coli model) in each group. 1 mg / kg) was intraperitoneally injected. After 2 hours based on bacterial intraperitoneal injection, mouse blood of each group was collected and incubated at room temperature for 30 minutes, and then serum was separated by centrifugation at 4 ° C. and 13000 rpm for 15 minutes. Mouse lung homogenates from each group were taken overnight overnight based on bacterial intraperitoneal injection and homogenized in crushing buffer. Subsequently, the immunoplates were coated and washed overnight with primary antibodies, anti-mTNF-α and anti-mIL-1β antibodies, followed by blocking buffer (2% BSA in PBS) to stop non-specific reactions. Blocking for 1 hour. The serum and lung homogenate prepared above were diluted 10-fold in the plate coated with the primary antibody, respectively, and treated with 100 μl of the plate and incubated at room temperature for 2 hours. After 1 and a half hours after treatment with the developable secondary antibody that recognizes mTNF-α and mIL-1β, TMB (3,3`, 5,5`-tetramethybensidine was added after 30 minutes with streptavidin HRP). ) Color solution. Color of 10 to 15 minutes from the time of color development was stopped by H ₂ SO ₄ was measured at 540nm using a (Molecular Devices, Sunnyvale, CA) mTNF-α and the amount of mIL-1β ELISA reader dissolved in the culture supernatant. The mTNF-α and mIL-1β amounts were quantified in comparison with the standard absorbance values.

As a result, by treatment with Pap12-6, the expression levels of inflammatory cytokines TNF-α and IL-1β decreased to 63.2% and 48.0% in the blood of sepsis shock mouse model (FIG. 6A), 51.0% in the lung and It was found to decrease to 49.0% (FIG. 6B).

Example 9 Antibacterial Effect of Pap12-6 in Lung, Liver and Kidney of Sepsis Shock Mouse Model

In order to identify the effect of inhibiting bacterial growth in the organ (or antimicrobial effect) in the E. Coli K1 infected sepsis shock mouse model by treatment of the derivative peptide Pap12-6 of SEQ ID NO: 6, three Balb / c (6 At week 1, Pap12-6 peptide (1 mg / kg) was intraperitoneally injected 1 hour prior to E. coli (1 × 10 ⁶ CFU / mouse) intraperitoneal injection using the E. coli model. Sixteen hours after injection, lungs, livers and kidneys were harvested from each group of mice, sterilized and placed in 1 ml of sterile PBS, followed by homogenization of each tissue on ice under aeration hood. Each lung, liver and kidney homogenate was diluted 1: 1000 and 10 μl of each dilution was plated on LB agar plates. The plated plates were incubated at 37 ° C. for 24 hours and then colony production was measured.

As a result, the total bacterial count in lung, liver and kidney of the sepsis shock mouse model was significantly reduced to 25.8%, 30.2% and 17.3%, respectively, according to the Pap12-6 peptide administration (FIG. 7). Through this, Pap12-6 was found to have an excellent antimicrobial effect against sepsis-inducing bacteria in the sepsis shock mouse model.

Example 10 Effect of Pap12-6 on Pulmonary Cell Infiltration Reduction in Sepsis Shock Mouse Model

To determine whether the degree of inflammatory cells infiltrating into the lung of the E. coli infected sepsis shock mouse model by treatment with the derivative peptide Pap12-6 of SEQ ID NO: 6 was administered, Pap12-6 peptide (1 mg / kg) was administered. After 1 hour, E. coli was administered, and after 24 hours, the lungs of each mouse were removed. Tissue blocks were collected from the extracted lungs, fixed in 4% paraformaldehyde for 12 hours and embedded in wax. The coronal wax was removed, stained with hematoxylin & eosin (H & E), and cell infiltration was examined by an optical microscope, and digital images were obtained.

As a result, it was confirmed that the treatment of Pap12-6 reduced the inflammatory cells infiltrating into the lung by the normal state (not sepsis-induced) in the sepsis shock mouse model (FIG. 8).

<110> Konkuk University Industrial Cooperation Corp <120> USE OF PAPILIOCIN DERIVATIVES <130> PN1806-192 <160> 9 <170> KoPatentIn 3.0 <210> 1 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-1 <400> 1 Arg Trp Lys Ile Phe Lys Lys Ile Glu Lys Val Gly   1 5 10 <210> 2 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-2 <400> 2 Arg Trp Lys Ile Phe Lys Lys Ala Ala Lys Lys Gly   1 5 10 <210> 3 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-3 <400> 3 Arg Trp Lys Ile Phe Lys Lys Val Val Lys Lys Gly   1 5 10 <210> 4 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-4 <400> 4 Arg Trp Lys Ile Phe Lys Lys Ala Ala Lys Lys Ala   1 5 10 <210> 5 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-5 <400> 5 Arg Trp Lys Ile Phe Lys Lys Val Val Lys Lys Ala   1 5 10 <210> 6 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-6 <400> 6 Arg Trp Lys Ile Phe Lys Lys Val Val Lys Lys Val   1 5 10 <210> 7 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> Pap12-7 <400> 7 Arg Trp Lys Ile Phe Lys Lys Val Val Lys Lys Trp   1 5 10 <210> 8 <211> 26 <212> PRT <213> Artificial Sequence <220> <223> melittin <400> 8 Gly Ile Gly Ala Val Leu Lys Val Leu Thr Thr Gly Leu Pro Ala Leu   1 5 10 15 Ile Ser Trp Ile Lys Arg Lys Arg Gln Gln              20 25 <210> 9 <211> 12 <212> PRT <213> Artificial Sequence <220> <223> papiliocin derivative <400> 9 Arg Trp Lys Ile Phe Lys Lys Xaa Xaa Xaa Xaa Xaa   1 5 10

Claims (13)

An antimicrobial papiliosin derivative peptide comprising the amino acid sequence of RWKIFKKVVKKV (SEQ ID NO: 6) or RWKIFKKVVKKW (SEQ ID NO: 7). delete A pharmaceutical composition for the prophylaxis or treatment of an inflammatory disease comprising the papiliosin derivative peptide of claim 1 as an active ingredient. delete The pharmaceutical composition for preventing or treating inflammatory disease according to claim 3, wherein the inflammatory disease is sepsis or septic shock. The pharmaceutical composition for preventing or treating inflammatory disease according to claim 5, wherein the sepsis or septic shock is caused by an infection of Gram-negative bacteria. According to claim 6, Gram-negative bacteria are Enterobacter sp. Bacteria, Salmonella sp. Bacteria, Vibrio sp. Bacteria, Mycobacterium sp. Bacteria, Inflammatory disease, any one or more selected from the group consisting of Shigella sp. Bacteria, Acinetobacter sp. Bacteria, Psedomonas sp. Bacteria and Escherichia sp. Bacteria Pharmaceutical composition for the prophylaxis or treatment of. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 5, wherein the sepsis or the septic shock is caused by an infection of Gram-positive bacteria. According to claim 8, Gram-positive bacteria are Bacillus sp. Bacteria, Staphylococcus sp. Bacteria, Enterococcus sp. Bacteria and Streptococcus sp. At least one selected from the group consisting of, a pharmaceutical composition for preventing or treating inflammatory diseases. The pharmaceutical composition for preventing or treating an inflammatory disease according to claim 5, wherein the sepsis or the septic shock is caused by an infection of the multidrug-resistant bacterium. The multidrug resistant bacterium is multidrug resistance- Escherichia coli (MDREC), multidrug resistant Pseudomonas aeruginosa (MDRPA), multidrug resistant acinetobacter Baumani (multidrug resistance-Acinetobacter). baumannii, MDRAB) and methicillin-resistant Staphylococcus aureus (MRSA), any one or more selected from the group consisting of, a pharmaceutical composition for the prevention or treatment of inflammatory diseases. Claim 1, a cosmetic composition for improving inflammatory disease comprising the papiliosin derivative peptide as an active ingredient. A food composition for improving inflammatory disease comprising the papiliosin derivative peptide of claim 1 as an active ingredient.

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