WO2019117577A1 - 키스펩틴을 포함하는 항노화 또는 항염증 조성물 - Google Patents

키스펩틴을 포함하는 항노화 또는 항염증 조성물 Download PDF

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Publication number
WO2019117577A1
WO2019117577A1 PCT/KR2018/015653 KR2018015653W WO2019117577A1 WO 2019117577 A1 WO2019117577 A1 WO 2019117577A1 KR 2018015653 W KR2018015653 W KR 2018015653W WO 2019117577 A1 WO2019117577 A1 WO 2019117577A1
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WIPO (PCT)
Prior art keywords
skin
kisspeptin
kispeptin
active fragment
preventing
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PCT/KR2018/015653
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English (en)
French (fr)
Korean (ko)
Inventor
정수경
모상현
윤석균
이경은
경서연
강승현
김연준
박명삼
이정훈
서효현
Original Assignee
코스맥스 주식회사
주식회사 바이오에프디엔씨
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Priority claimed from KR1020180125412A external-priority patent/KR102209869B1/ko
Application filed by 코스맥스 주식회사, 주식회사 바이오에프디엔씨 filed Critical 코스맥스 주식회사
Priority to CN201880004016.6A priority Critical patent/CN110167522B/zh
Publication of WO2019117577A1 publication Critical patent/WO2019117577A1/ko

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    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
    • A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
    • A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
    • A23L33/17—Amino acids, peptides or proteins
    • A23L33/18—Peptides; Protein hydrolysates
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/1703—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates
    • A61K38/1709—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans from vertebrates from mammals
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • A61Q19/08—Anti-ageing preparations
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
    • A—HUMAN NECESSITIES
    • A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
    • A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
    • A23V2200/00—Function of food ingredients
    • A23V2200/30—Foods, ingredients or supplements having a functional effect on health
    • A23V2200/318—Foods, ingredients or supplements having a functional effect on health having an effect on skin health and hair or coat

Definitions

  • Functional cosmetics may include products that delay skin aging and help to improve wrinkles of the skin, products that protect the skin from ultraviolet rays and the external environment, or products that help to whiten skin.
  • functional cosmetics it is necessary to develop functional ingredients that can exhibit physiological efficacy in the skin.
  • the human skin undergoes changes due to various internal and external factors as it gets older.
  • the secretion of various hormones that regulate metabolism is reduced internally, and the function of immune cells and the activity of cells are lowered, so that the biosynthesis of immune proteins and biocompatible proteins necessary for the living body is reduced.
  • the amount of ultraviolet rays reaching the surface of the sunlight increases and the environmental pollution is further increased.
  • free radicals and active noxious oxygen are increased and the thickness of the skin decreases, Not only the elasticity is reduced but also the skin color is gray, the skin trouble frequently occurs, and the spots, the freckles and the black spots are also increased.
  • Detailed mechanism of skin aging phenomenon is as follows: reduction of keratinocyte and fibroblast cleavage, reduction of collagen synthesis, increase of MMP (Matrix metalloproteinase) generation, increase of signaling of melanin generation, The elasticity of the skin decreases, and the number of spots, freckles, and black spots increases. In particular, the balance of hormones in the forties is broken, and cell regeneration ability, collagen synthesis ability, and skin lipid production ability, which plays an important role in skin moisturization, are greatly reduced.
  • MMP Microx metalloproteinase
  • UV light activates stress hormones repeatedly and damages the skin. This can lead to skin side effects such as the collapse of dermal fibrous tissue, just as overdose of steroids on skin.
  • UV stimulation is a major cause of glucocorticoid activity in human skin known as stress hormones such as cortisol. It also increases the expression of 11? -Hydroxysteroid dehydrogenase type 1 (11? -HSD1), an enzyme in the body that activates cortisol.
  • 11? -HSD1 11? -Hydroxysteroid dehydrogenase type 1
  • the increase in the activity and expression of 11? -HSD1 by ultraviolet rays is closely related to skin aging, and thus has been attracting attention as a new target for inhibiting skin aging.
  • Inflammation is a physiological response that protects the body from harmful environmental conditions, such as the ingress of external foreign matter, such as bacteria, and mechanical damage. Excessive inflammation is also a major cause of skin damage. Inflammation causes an increase in several types of polynuclear leukocytes (PMNs) and immunological substances, and these cells contain various types of proteolytic enzymes, such as inflammatory cell products (such as elastase, hyalurinidase, and lipoxygenase ) And cytokine secretion. These actions may then cause harmful damage to adjacent tissue and non-tissue cell components and may lead to severe tissue damage of chronic inflammation. In addition, the damage of the connective tissues not only causes a decrease in the elasticity of the skin to cause wrinkles but also adversely affects the regeneration and proliferation of the cells, resulting in quick skin aging.
  • PMNs polynuclear leukocytes
  • proteolytic enzymes such as inflammatory cell products (such as elastase, hy
  • One aspect is to provide a cosmetic composition comprising kisspeptin or an active fragment thereof as an active ingredient.
  • Another aspect is to provide a use for the use of kispeptin or an active fragment thereof in the preparation of a cosmetic composition.
  • Another aspect is to provide a composition for external application for skin for preventing, ameliorating or treating a skin condition comprising kisspeptin or an active fragment thereof as an active ingredient, or preventing or ameliorating skin aging.
  • Another aspect is to provide a pharmaceutical composition for preventing or ameliorating skin aging comprising kispeptin or an active fragment thereof as an active ingredient, and for the prophylaxis and treatment of inflammatory diseases.
  • Another aspect is to provide a method of preventing, ameliorating or treating skin conditions comprising administering kisspeptin or an active fragment thereof to an individual in need thereof.
  • Another aspect is to provide a use of kisspeptin or an active fragment thereof for use in the inhibition or amelioration of skin aging, in the preparation of a pharmaceutical composition for the prophylaxis and treatment of inflammatory diseases.
  • Yet another aspect is to provide a health functional food for the prevention or treatment of skin conditions comprising kispeptin or an active fragment thereof, or for preventing or ameliorating skin aging.
  • Another aspect is the use of kisspeptin or active fragments thereof for use in the manufacture of a composition for preventing, ameliorating or treating skin conditions.
  • One aspect provides a composition comprising kisspeptin or an active fragment thereof as an active ingredient.
  • Kisspeptin is used interchangeably with metastin or KISS-1 peptides and can refer to a protein encoded by the KISS1 gene.
  • Kisspeptin is a G-protein coupled receptor ligand for GPR54 and is known as a human metastasis inhibitory gene with the ability to inhibit melanoma and breast cancer metastasis.
  • GpR54 signaling plays an important role in initiating the secretion of gonadotropin releasing hormone (GnRH) in puberty.
  • Gonadotropin releasing hormone is released from the hypothalamus and acts on the anterior pituitary gland to produce luteinizing hormone (LH) and follicle It causes the release of stimulating hormone (FSH).
  • kispeptin can directly stimulate GnRH release, it transmits negative and positive feedback signals of steroid hormones to GnRH neurons, acts as a gatekeeper to the onset of puberty, ) And photoperiodic information.
  • the kisspeptin is at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% Or may have sequence identity.
  • the active fragment may be composed of the 27th to 54th, 45th to 49th, or 45th to 54th amino acid sequences from the N-terminal of the amino acid sequence of SEQ ID NO: 1.
  • the C-terminus of the active fragment may be amidated.
  • the active fragment has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96% , 97% or more, 98% or more, 99% or more, or 100% sequence identity.
  • the effective amount may be from 0.0001 mg to 10,000 mg, 0.001 mg to 1000 mg, 1.0 mg to 100 mg, 0.01 mg to 1000 mg, 0.01 mg to 100 mg, 0.01 mg to 10 mg, or 0.01 mg to 1 mg.
  • the kisspeptin or active fragment thereof may be present in an amount of from 0.001% to 80%, such as 0.01% to 60%, 0.01% to 40%, 0.01% to 30%, 0.01% From 0.05 wt% to 40 wt%, from 0.05 wt% to 30 wt%, from 0.05 wt% to 20 wt%, from 0.01 wt% to 10 wt%, from 0.01 wt% to 5 wt% From 0.05% to 10%, from 0.05% to 5%, from 0.1% to 60%, from 0.1% to 40%, from 0.1% to 30%, from 0.1% to 20% By weight, 0.1% by weight to 10% by weight, or 0.1% by weight to 5% by weight.
  • the kispeptin or active fragment thereof may be one that has activity to inhibit MMP (Matrix metalloproteinase), ll [beta] -HSDl or an inflammatory factor such as inflammatory cytokine, IL-6, or IL-8 have. Therefore, the kispeptin or an active fragment thereof is effective for prevention, improvement, or treatment of skin aging, skin damage, skin condition or inflammatory disease.
  • MMP Microx metalloproteinase
  • ll [beta] -HSDl or an inflammatory factor such as inflammatory cytokine, IL-6, or IL-8 have. Therefore, the kispeptin or an active fragment thereof is effective for prevention, improvement, or treatment of skin aging, skin damage, skin condition or inflammatory disease.
  • composition may be one for the prevention, amelioration or treatment of aging, for example, skin aging, skin damage, skin conditions or inflammatory diseases.
  • skin aging refers to the type and intangible changes that appear on the skin with age, such as skin thinning, the number of dermal cells or blood vessels, DNA damage recovery, Refers to a reduction in wound healing, skin barrier function, epidermal water retention, sweat secretion, sebum secretion, vitamin D production, physical damage protection, chemical abilities, immune response, sensory function, and body temperature control.
  • the kisspeptin or active fragment thereof may be for improving skin aging induced by an exogenous factor or endogenous factor.
  • the extrinsic factor refers to various external factors such as ultraviolet (light), and the endogenous factor is also referred to as a chronological factor, which is mainly caused by the passage of time.
  • the skin aging specifically includes not only early symptoms of aging induced by external stimuli such as ultraviolet rays, pollution, smoke, chemicals, etc., but also natural aging phenomena caused by aging of skin cells And is a concept that includes both wrinkles, elasticity reduction, skin burning and drying phenomenon. Also, wrinkles include those in which irritation caused by changes in internal and external factors changes the constituents of the skin tissue to cause wrinkles.
  • Examples of such skin conditions or conditions associated with the skin include skin aging, skin photoaging, scarring, dermatitis, atopic dermatitis, pruritus, eczematous skin disease, dry eczema, erythema, urticaria, psoriasis, drug rash or acne, , Insect and parasitic mediated diseases, superficial dermatophytosis, bacterial infectious diseases, viral diseases, adult diseases, autoimmune vesicular diseases, connective tissue diseases, dyspepsia, pigmented sclerosis and the like.
  • the skin damage is caused by external physical damage, penetration of chemicals, bacteria, fungi, viruses, exposure to ultraviolet rays, water loss of skin, wrinkles due to aging, oxidation by free radicals (active oxygen) May include damage from a clinical and cosmetic point of view of the skin tissue or cells including skin inflammation, seborrheic dermatitis, redness (redness, erythema), edema, burning, eczema, pruritus (itching), and atopic dermatitis .
  • the skin includes all parts of the body including the face, hands, arms, legs, feet, chest, belly, back, buttocks, and scalp.
  • conditions associated with the inflammatory disease or inflammation include dermatitis, allergy, atopy, conjunctivitis, periodontitis, rhinitis, otitis, sore throat, tonsillitis, pneumonia, gastric ulcer, gastritis, Crohn's disease, colitis, gout, ankylosing spondylitis, rheumatic fever, lupus, Inflammatory bowel disease, fibromyalgia, psoriatic arthritis, osteoarthritis, rheumatoid arthritis, periarthritis, tendonitis, hay fever, tendinitis, myositis, hepatitis, cystitis, nephritis, sjogren's syndrome, multiple sclerosis and acute and chronic inflammatory diseases have.
  • the composition may be a cosmetic composition.
  • the cosmetic composition may be one for prevention or improvement of skin aging, for skin moisturizing, for strengthening skin barrier, for preventing or improving skin wrinkles, for preventing or improving skin damage, or for preventing or improving skin inflammation .
  • the cosmetic composition may be, for example, a cosmetic formulation of softening agent, nutritional lotion, massage cream, nutritional cream, essence, pack, gel, ampoule or skin sticking type.
  • the ingredients contained in the cosmetic composition may contain, in addition to the above-mentioned composition, the components conventionally used in cosmetic compositions, for example, conventional auxiliary agents such as stabilizers, solubilizers, vitamins, . ≪ / RTI >
  • composition may be a composition for external application to the skin.
  • the external preparation for skin may be a cream, a gel, an ointment, a skin emulsifier, a skin suspension, a transdermal patch, a drug-containing bandage, lotion, or a combination thereof.
  • the external preparation for skin may be used in the form of an ointment, a lotion, a spray, a patch, a cream, a powder, a suspension, a gel or a gel.
  • the external preparation for skin is usually used as a component used in external skin preparations such as cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, or a combination thereof, and may be suitably blended as necessary.
  • cosmetics or medicines such as an aqueous component, an oily component, a powder component, an alcohol, a moisturizer, a thickener, an ultraviolet absorber, a whitening agent, an antiseptic, , Coloring agents, various skin nutrients, or a combination thereof, and may be suitably blended as necessary.
  • the external preparation for skin may be a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid, glucose, fructose, fructose, fructose and other herbal medicines, various herbal medicines, tocopherol acetate, glycyrrhizic acid, Sugars such as trehalose and the like can also be appropriately compounded.
  • a metal blocker such as sodium edetate, sodium edetate, sodium citrate, sodium polyphosphate, sodium metaphosphate or gluconic acid, caffeine, tannin, bellapamil, licorice extract, glabridine, Vitamin C, ascorbic acid magnesium phosphate, ascorbic acid glucoside, arbutin, kojic acid
  • the composition may also be a quasi-drug composition.
  • quadsi-quasi-drugs means fibers, rubber products or the like, which are used for the purpose of treating, alleviating, treating or preventing diseases of humans or animals, weak acting on the human body or acting directly on the human body, Products similar to or similar to those used in the manufacture of a medicinal product for use in the prevention or treatment of an infectious disease, for use in the preparation of a medicament for the prevention of infection, for use in the diagnosis, treatment, alleviation, treatment or prevention of a human or animal condition or disease, Machinery, or apparatus, and that is not an apparatus, machine, or apparatus of an article used for the purpose of imparting pharmacological effects to the structure or function of a person or animal. .
  • the geraniol may be added as it is or may be used in combination with other quasi-drugs, and may be suitably used according to a conventional method.
  • the amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).
  • the quasi-drug composition includes, but is not limited to, personal hygiene products, external preparation for skin, disinfectant cleaner, shower foam, gagrin, wet tissue, detergent soap, hand wash, humidifier filler, mask, ointment or filter filler.
  • the personal care product may be a soap, a wet tissue, a tissue paper, a shampoo, a toothpaste, a hair care product, an air freshner gel or a cleaning gel.
  • the composition may be a pharmaceutical composition.
  • the pharmaceutical composition may additionally comprise a pharmaceutically acceptable diluent or carrier.
  • the diluent may be lactose, corn starch, soybean oil, microcrystalline cellulose, or mannitol
  • the lubricant may be magnesium stearate, talc, or combinations thereof.
  • the carrier may be an excipient, a disintegrant, a binder, a lubricant, or a combination thereof.
  • the excipient may be microcrystalline cellulose, lactose, low substituted hydroxy cellulose, or a combination thereof.
  • the disintegrant may be carboxymethylcellulose calcium, starch glycolate sodium, calcium monohydrogen phosphate anhydrate, or a combination thereof.
  • the binder may be polyvinylpyrrolidone, low-substituted hydroxypropylcellulose, hydroxypropylcellulose, or combinations thereof.
  • the lubricant may be magnesium stearate, silicon dioxide, talc, or a combination thereof.
  • the pharmaceutical composition may be formulated into an oral or parenteral dosage form.
  • Oral administration formulations may be granules, powders, solutions, tablets, capsules, dry syrups, or combinations thereof.
  • the parenteral dosage form may be an injectable.
  • the composition may be a health functional food composition.
  • the health functional food composition may be used together with kispeptin or an active fragment thereof alone or another food or food ingredient, and may be suitably used according to a conventional method.
  • the amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment).
  • the composition of the present specification may be added in an amount of not more than 15 parts by weight based on the raw material in the production of food or beverage.
  • the beverage composition may contain various flavors or natural carbohydrates as additional components such as ordinary beverages.
  • the natural carbohydrates are sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol.
  • sugar saccharides such as monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, polysaccharides such as dextrin and cyclodextrin, xylitol, sorbitol and erythritol.
  • sweeteners include natural sweeteners such as tau martin and stevia extract, synthetic sweeteners such as saccharin and aspartame, and the like.
  • the health food composition may further comprise at least one selected from the group consisting of nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and its salts, organic acids, protective colloid thickeners, pH adjusting agents, stabilizers, preservatives, glycerin, The carbonating agent used, or a combination thereof.
  • the health functional food composition may also contain natural fruit juice, fruit juice beverage, flesh for the manufacture of vegetable drinks, or combinations thereof.
  • another aspect provides a method of preventing, ameliorating, or treating the condition of an individual comprising treating or administering an effective amount of the above composition to a subject in need thereof.
  • the state or composition of the individual is the same as described above.
  • the subject may be a mammal, such as a person, a cow, a horse, a pig, a dog, a sheep, a goat, or a cat.
  • the kisspeptin or active fragment thereof has an inhibitory activity of MMP-1 or an inflammatory cytokine and is useful for preventing, improving or treating skin aging, skin damage, skin condition, or inflammatory disease It is effective.
  • 1 is a graph showing the results of HPLC analysis of active fragments of kisspeptin according to one embodiment.
  • FIG. 2 is a diagram showing the chemical structure and the three-dimensional predicted structural model for active fragments of kispeptin according to one embodiment
  • FIG. (A) chemical structure of kisspeptin-1 (C 63 H 83 N 17 O 14 ); (b) Three-dimensional predictive structural model; (C) chemical structure of kisspeptin-E (C 31 H 39 N 9 O 9 ); (d) Three-dimensional expected structure.
  • FIG. 3 is a graph showing that kisspeptin-1 and kispeptin-E according to one embodiment inhibit the expression of MMP-1.
  • FIG. 4 is a graph showing that kisspeptin-1 and kispeptin-E according to one embodiment inhibit the expression of IL-6.
  • FIG. 5 is a graph showing that kisspeptin-1 and kispeptin-E according to one embodiment inhibit the expression of IL-8.
  • FIG. 6 is a graph showing that kisspeptin-1 and kispeptin-E according to one embodiment inhibit the expression of 11? -HSD1.
  • Kisspeptin-1 (the 45th to 54th amino acid sequence (SEQ ID NO: 3) from the N-terminus of the amino acid sequence of the entire kispeptin) and SEQ ID NOs: 2 and 4 of the kisspeptin active fragment from the kisspeptin represented by SEQ ID NO: .
  • SEQ ID NO: 2 was synthesized by a conventional solid phase peptide synthesis method using 9-fluorenylmethoxycarbonyl (Fmoc) as an amino acid protecting group, and the resin was Fmoc-conjugated with phenyl- Wang resin was used.
  • Fmoc 9-fluorenylmethoxycarbonyl
  • the extension of the amino acid residues was carried out by using N-hydroxybenzotriazole (HOBt) and N, N'-Diisopropyl carbodiimide (DIC) as active agents.
  • Amino acids, HOBt, and DIC used in each step were reacted for 5 hours at room temperature for 2 hours. After completion of the reaction, the resultant was washed six times with dimethylformamide (DMF), and then washed six times with DCM (dichloromethane), followed by drying of the resin.
  • DMF dimethylformamide
  • DCM dichloromethane
  • the dried peptide of SEQ ID NO: 1-Resin was reacted with a mixed solution of trifluoroacetic acid: triisopropylsilane: water (90: 5: 5 (v / v)) at room temperature for 2 hours to isolate the peptide from the resin, The peptide was precipitated with ether and crude peptide was obtained using a centrifuge.
  • the obtained peptide of SEQ ID NO: 1 was purified by reversed phase high performance liquid chromatography (Kromasil, C18, 5 mu, 110 ⁇ ) using acetonitrile and water containing 0.1% trifluoroacetic acid as a solvent. 250 * 21.2 mm) (yield: 34.8%).
  • Sequence Nos. 3, 4 and 5 were also synthesized in the same manner as above, and the yields were 32.4%, 38.0%, and 35%, respectively.
  • the kisspeptin is Kisspeptin 1 (SEQ ID NO: 3) capped with NH2 in the 45th to 54th amino acid sequence from the N-terminus of the entire kisspeptin amino acid sequence (SEQ ID NO: 1) and Kisspeptin E (SEQ ID NO: 5) capped with NH2 in the 45th to 49th amino acid sequence from the N-terminus of the amino acid sequence of the peptin (SEQ ID NO: 1)) was used to confirm the inhibition of MMP-1.
  • Kisspeptin 1 SEQ ID NO: 3
  • Kisspeptin E SEQ ID NO: 5
  • MMP-1 collagenase
  • Hs68 human fibroblast cell line Hs68 after a busy as a 6-well plate the number of 4x10 5, and incubated for 24 hours at 37 °C, an incubator of 5% CO 2 condition. After the medium was removed, DPBS was added and UVB of 12 mJ / cm 2 was irradiated to the remaining cells except the non-UVB group. Next, ascorbic acid (AA) as a positive control group, and kisspeptin mimetic peptide as an experimental group were added at different concentrations and further cultured for 24 hours.
  • AA ascorbic acid
  • AA kisspeptin mimetic peptide
  • Real-time PCR is performed in a real-time PCR machine using a mixture of a target protein, MMP-1 primer, and a cyanine dye, SYBR Green supermis (Applied Biosystems, USA) And the degree of expression of the MMP-1 gene was finally evaluated.
  • the sequences of the primers for PCR and the reaction conditions are shown in Table 1 below.
  • MMP-1 forward primer SEQ ID NO: 8 After activation of the polymerase at 94 ° C for 5 minutes, polymerization was carried out at 40 ° C for 30 seconds at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 seconds MMP-1 reverse primer SEQ ID NO: 9 ⁇ -actin forward primer SEQ ID NO: 10 ⁇ -actin reverse primer SEQ ID NO: 11
  • 1 is a graph showing that kisspeptin according to one embodiment inhibits the expression of MMP-1.
  • the fibroblast cell line Hs68 the cells were cultured in 6 well plates and then dispensed as the number of 4x10 5, in 37 °C, an incubator of 5% CO 2 condition for 24 hours. Subsequently, the medium was removed and DPBS was added. After UVB irradiation of 15 mJ / cm 2 to the remaining cells except for the UVB non-control group, kisspeptin was added by concentration (1 nM, 10 nM, 100 nM, and 1000 nM) Lt; / RTI > for 24 hours. Then, the expression levels of IL-6 and IL-8 overexpressed by UVB were evaluated in the same manner as in Experimental Example 1, except that the primers shown in Table 2 were used. The results are shown in FIGS. 2 and Respectively.
  • IL-6 forward primer SEQ ID NO: 12 After activation of the polymerase at 94 ° C for 5 minutes, polymerization was carried out at 40 ° C for 30 seconds at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 seconds IL-6 reverse primer SEQ ID NO: 13 IL-8 forward primer SEQ ID NO: 14 IL-8 reverse primer SEQ ID NO: 15
  • Figure 2 is a graph showing that kisspeptin according to one embodiment inhibits the expression of IL-6.
  • Figure 3 is a graph showing that kisspeptin according to one embodiment inhibits the expression of IL-8.
  • kisspeptin As shown in FIG. 2 and FIG. 3, the expression of LPS-induced IL-6 and IL-8 in the kispeptin-treated group was significantly reduced as compared with the untreated control group. In particular, the expression level of IL-6 was decreased to a level similar to that of the normal control.
  • kisspeptin according to one embodiment has an anti-inflammatory activity and can be usefully used as a composition for preventing, ameliorating, or treating inflammation, for example, skin inflammation or skin damage.
  • the kisspeptin mimetic peptide inhibits mRNA expression of 11? -HSD1 induced by UVB.
  • human fibroblasts were inoculated in a 6-well cell culture dish at a density of 4 ⁇ 10 5 , and cultured in a 5% CO 2 incubator at 37 ° C. for 24 hours.
  • UVB was irradiated at 12 mJ, followed by ascorbic acid as a positive control group and kisspeptin mimetic peptide as an experimental group, followed by culturing for 24 hours.
  • the cells were then recovered and RNA was isolated by adding 1 ml of trizol (RNA iso, DAKARA, Japan). Then, the expression level of 11? -HSD1 overexpressed by UVB was evaluated in the same manner as in Experimental Example 1, except that the primer shown in Table 3 below was used. The results are shown in FIG.
  • primer order Reaction conditions 11? -HSD1 forward primer SEQ ID NO: 16 Polymerase activation at 94 ° C for 5 minutes, polymerization at 95 ° C for 30 seconds, 50 ° C for 1 minute, and 72 ° C for 1 minute 40 cycles 11 [beta] -HSD1 reverse primer SEQ ID NO: 17
  • FIG. 4 is a graph showing the inhibition of 11? -HSD1 expression of kisspeptin according to one embodiment.
  • the expression of 11 ⁇ -HSD1, a stress hormone converting enzyme, in the kispeptin-1 and kispeptin-E treated groups was significantly reduced compared to the untreated control group.
  • the expression of 11? -HSD1 could be remarkably suppressed compared with that of ascorbic acid, which is a positive control.
  • kisspeptin according to one embodiment can effectively inhibit skin aging which can be caused by external stimuli by inhibiting the anti-inflammatory activity as well as skin stress hormone-inducing enzyme expression.

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PCT/KR2018/015653 2017-12-12 2018-12-11 키스펩틴을 포함하는 항노화 또는 항염증 조성물 WO2019117577A1 (ko)

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Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020065510A (ko) * 1999-12-17 2002-08-13 다케다 야쿠힌 고교 가부시키가이샤 ＫｉＳＳ-1 펩티드의 제조 방법
JP2003300906A (ja) * 2002-04-12 2003-10-21 Daiichi Fine Chemical Co Ltd がん転移抑制因子の安定化
WO2007084211A2 (en) * 2005-11-11 2007-07-26 The General Hospital Corporation Use of gpr54 ligands for treatment of reproductive disorders, proliferative disorders, and for contraception
WO2009046858A2 (en) * 2007-09-11 2009-04-16 Mondobiotech Laboratories Ag Therapeutic uses of kisspeptin 13 and compositions thereof
US20160074320A1 (en) * 2013-04-12 2016-03-17 Universidade De Sao Paulo - Usp Pharmaceutical compostions comprising kisspeptin or derivatives thereof

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GB0719592D0 (en) * 2007-10-08 2007-11-14 Medical Res Council Compound, use and method
CN106544322B (zh) * 2016-12-06 2019-10-08 东华大学 一种用于研究Kiss1基因表达调控的报告系统及其构建方法

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20020065510A (ko) * 1999-12-17 2002-08-13 다케다 야쿠힌 고교 가부시키가이샤 ＫｉＳＳ-1 펩티드의 제조 방법
JP2003300906A (ja) * 2002-04-12 2003-10-21 Daiichi Fine Chemical Co Ltd がん転移抑制因子の安定化
WO2007084211A2 (en) * 2005-11-11 2007-07-26 The General Hospital Corporation Use of gpr54 ligands for treatment of reproductive disorders, proliferative disorders, and for contraception
WO2009046858A2 (en) * 2007-09-11 2009-04-16 Mondobiotech Laboratories Ag Therapeutic uses of kisspeptin 13 and compositions thereof
US20160074320A1 (en) * 2013-04-12 2016-03-17 Universidade De Sao Paulo - Usp Pharmaceutical compostions comprising kisspeptin or derivatives thereof

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