WO2018186505A1 - Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation - Google Patents

Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation Download PDF

Info

Publication number
WO2018186505A1
WO2018186505A1 PCT/KR2017/003645 KR2017003645W WO2018186505A1 WO 2018186505 A1 WO2018186505 A1 WO 2018186505A1 KR 2017003645 W KR2017003645 W KR 2017003645W WO 2018186505 A1 WO2018186505 A1 WO 2018186505A1
Authority
WO
WIPO (PCT)
Prior art keywords
centrifugation
exosomes
skin
minutes
recovering
Prior art date
Application number
PCT/KR2017/003645
Other languages
English (en)
Korean (ko)
Inventor
김수
이원종
최은욱
서민구
우은영
박은주
Original Assignee
㈜프로스테믹스
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by ㈜프로스테믹스 filed Critical ㈜프로스테믹스
Priority to PCT/KR2017/003645 priority Critical patent/WO2018186505A1/fr
Publication of WO2018186505A1 publication Critical patent/WO2018186505A1/fr

Links

Images

Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/14—Blood; Artificial blood
    • A61K35/15—Cells of the myeloid line, e.g. granulocytes, basophils, eosinophils, neutrophils, leucocytes, monocytes, macrophages or mast cells; Myeloid precursor cells; Antigen-presenting cells, e.g. dendritic cells
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/30—Nerves; Brain; Eyes; Corneal cells; Cerebrospinal fluid; Neuronal stem cells; Neuronal precursor cells; Glial cells; Oligodendrocytes; Schwann cells; Astroglia; Astrocytes; Choroid plexus; Spinal cord tissue
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K35/00—Medicinal preparations containing materials or reaction products thereof with undetermined constitution
    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/48—Reproductive organs
    • A61K35/51—Umbilical cord; Umbilical cord blood; Umbilical stem cells
    • 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
    • A61K8/96—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution
    • A61K8/98—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin
    • 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
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q7/00—Preparations for affecting hair growth

Definitions

  • the present invention relates to a composition comprising the stem cells or the exo-derived from the culture medium thereof, and having the effect of wound treatment, skin improvement and hair loss prevention or treatment, and a method for producing the same.
  • Stem cells are cells that have been differentiated into specific cells without progress and, if necessary, have the ability to differentiate into all kinds of cells constituting the body such as nerves, blood, and cartilage. There are two ways to obtain such stem cells, firstly from embryos derived from fertilized eggs (embryonic stem cells) and secondly from stem cells (adult stem cells) stored in each part of our adult body. ) Is recovered. Although functionally different, both embryonic and adult stem cells have the ability to differentiate into different cell types. Embryonic stem cells have very good differentiation ability and long telomeres, but they have ethical problems and difficult to obtain large amounts of cells, while adult stem cells can obtain a large number of cells but can be transplanted to others. The risk of infection or differentiation is relatively low.
  • the compounds include cyclosporin derivatives (Korean Patent No. 10-0439467, Korean Patent No. 10-0695611), N-heterocyclic carboxylic acid and carbamate (Korean Patent No. 10-2001-0052502), quina Zolinone derivatives (Korean Patent Laid-Open Publication No. 10-1999-0023754), chrysine 7-O-crotonate (Korean Patent Publication 10-2001-0009474) and 1,2-disubstituted benzenecarboxamide derivatives 1999-0037394) have been reported.
  • such a hair loss treatment or hair growth improving composition is a chemical composition, which can cause fatal side effects for women and infants, and also has side effects such as acceleration of hair loss due to dermatitis in men. Side effects that can be used with prolonged use can also increase patient pain.
  • One object of the present invention is to provide a composition for treating wounds that can stably treat or promote wounds using stem cells or culture-derived exosomes.
  • Still another object of the present invention is to provide a composition for preventing or treating hair loss having a safe but excellent hair growth and hair growth effect using the exosome derived from the stem cells or the culture medium.
  • Wounds are also called wounds, and depending on the cause, they are divided into intestines, jaws, insoles, acne, fissures, insoles, school windows, etc. Symptoms of the wound include pain, bleeding, dysfunction, and when the wound, the living body exhibits a variety of physiological reactions to heal. Normally, injured cells undergo denaturation and death processes, causing flow of cells and tissue fluid to flow out from surrounding tissues, and precipitation of fibrin and granulation tissue are formed to heal wounds.
  • EGF Epidermal Growth Factor
  • FGF Fibroblast Growth Factor
  • TGF- ⁇ Transforming Growth Factor- ⁇
  • TGF- ⁇ TGF- ⁇
  • IGF-I Insulin-like Growth Factor-1
  • the skin consists of the epidermis, the dermis and the subcutaneous tissue.
  • the epidermis the outer layer of skin, consists of the stratified squamous epithelium and acts as a protective barrier for the body to the outside environment.
  • the epidermis is divided from the outside into stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum and stratum basale.
  • the dermis is the layer between the epidermis and the subcutaneous tissue, divided into papillary and reticular dermis, blood vessels, collagen, elastin fibers, pores, hair roots, sebaceous glands, Korean glands, various Sensory nerves, fibroblasts and macrophages are present and occupy the largest part of the skin.
  • the dermis consists primarily of collagen and elastin fibers, which support the skin. Therefore, when such a problem occurs in the dermis, wrinkles occur and skin elasticity is lost, thereby aging the skin.
  • Collagen is known to play the most important role in skin regeneration, skin moisture content, wound healing and wrinkle improvement, and is produced from fibroblasts. Collagen has a function that can contain a large amount of moisture, which serves to supply moisture to the dermis. When aging, the collagen loses its water-containing function and wrinkles increase. Collagen can also heal wounds by filling fibroblasts with sustained collagen production when the wound is injured.
  • Hair loss is caused by a variety of factors, including the slow growth of hair and the lack of growth factors that promote hair root cell growth.
  • This hair loss reduces the quality of life due to mental stress and causes a lot of interpersonal or social life.
  • the image of appearance in social life is very spectacular, and interest in the cause and treatment of hair loss is increasing day by day because the importance of appearance becomes an invisible vitality of self-esteem or social life.
  • the stem cell or its culture-derived exosomes are excellent in skin permeability, and excellent in the effects of wound healing, skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, in addition to hair growth and hair growth effects Discovering outstanding things led to the present invention.
  • compositions for wound treatment comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • the present invention relates to a method for preparing the wound pharmaceutical composition, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  • the present invention can be obtained with a high purity and content of exosomes excellent in wound healing through a five-step centrifugation process.
  • the adult stem cell culture medium of adult stem cells 800 ⁇ 1,200 mg / L D glucose, 500 ⁇ 600 mg / L L- glutamine, 100 ⁇ 200 mg / L sodium pyruvate, 5 ⁇ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • a cosmetic composition for skin improvement comprising the adult stem cells or culture medium derived from the exosome (exosome) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • the present invention relates to a method for preparing the cosmetic composition for skin improvement, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  • the present invention can be obtained with high purity and content exosomes excellent skin improvement effect through a five-step centrifugation process.
  • the adult stem cell culture medium of adult stem cells 800 ⁇ 1,200 mg / L D glucose, 500 ⁇ 600 mg / L L- glutamine, 100 ⁇ 200 mg / L sodium pyruvate, 5 ⁇ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • a pharmaceutical composition for preventing or treating hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • a cosmetic composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • a food composition for preventing or improving hair loss comprising adult stem cells or culture-derived exosomes (exosomes) as an active ingredient.
  • the exosomes are preferably contained in an amount of 1 ⁇ 100 ⁇ g / ml, or 1 ⁇ 50 ⁇ g / ml.
  • the exosomes are included in an amount of less than 1 ⁇ g / ml, the wound healing effect may be insignificant, and when included in excess of 100 ⁇ g / ml, it may be economically problematic.
  • the exosomes may be included in the number of particles of 1 X 10 7 ⁇ 1 X 10 12 / ml. If the particle number of the exosome is less than 1 X 10 7 / ml may be ineffective wound healing, if the particle number exceeds 1 X 10 12 / ml may be an economic problem.
  • the present invention relates to a method for preparing the hair loss prevention or treatment pharmaceutical composition, comprising: centrifuging the adult stem cell culture solution at 100 to 500 g for 5 to 30 minutes; Recovering the supernatant after the first centrifugation and performing secondary centrifugation at 1,000 to 3,000 g for 10 to 30 minutes; Recovering the supernatant after the second centrifugation and performing third centrifugation for 20 to 60 minutes at 5,000 to 20,000 g; Recovering the supernatant after the third centrifugation and performing fourth centrifugation at 100,000 to 120,000 g for 60 to 120 minutes; And recovering the pellet after the fourth centrifugation to recover the pellet by fifth centrifugation for 60 to 120 minutes at 50,000 to 150,000 g.
  • the present invention can be obtained with a high purity and content of exosomes excellent in hair growth and hair growth effect through a five-step centrifugation process.
  • the adult stem cell culture medium of adult stem cells 800 ⁇ 1,200 mg / L D glucose, 500 ⁇ 600 mg / L L- glutamine, 100 ⁇ 200 mg / L sodium pyruvate, 5 ⁇ 15% by volume Inoculated in DMEM medium supplemented with Fetal Bovine Serum (FBS) and 0.5-1.5% by volume of penicillin-streptomycin, followed by 5-10% CO under humidity conditions of 80-95% and 30-40 ° C.
  • FBS Fetal Bovine Serum
  • penicillin-streptomycin penicillin-streptomycin
  • the adult stem cell refers to an undifferentiated cell having a multipotency derived from an adult human cell of a mammal, including a human, for example, bone marrow, blood, brain, skin, fat (ie , Adipose tissue or fat cells), umbilical cord blood, umbilical cord Barton's jelly, and the like.
  • the "adult stem cells” include mesenchymal stem cells derived from adult cells.
  • Adipose-derived stem cells are known from known methods (e.g., WO2000 / 53795 and WO2005 / 042730) from adipose tissue or adipocytes of mammals including humans, preferably humans. It can be obtained through processes such as liposuction and sedimentation, enzymatic treatment of collagenase and the like, and removal of floating cells such as red blood cells by centrifugation.
  • the adipose tissue includes brown or white tissue derived from subcutaneous, retinal, visceral, breast gonad or other adipose tissue sites and can be readily obtained from conventional liposuction.
  • Exosomes in the present invention refers to the small vesicles of the membrane structure secreted from various cells.
  • the diameter of the exosome is approximately 30 to 1,000 nm, which means that the vesicle is released into the extracellular environment due to the fusion of the polycystic body and the plasma membrane.
  • Representative expression markers of exosomes correspond to HSP70, CD63 and CD9.
  • the pharmaceutical composition may further include a suitable carrier, excipient or diluent according to a conventional method.
  • Carriers, excipients and diluents that may be included in the pharmaceutical compositions of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate , Cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, methylhydroxy benzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, and the like, may be used, but is not limited thereto.
  • compositions according to the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, or sterile injectable solutions, respectively, according to a conventional method.
  • when formulated, it may be prepared using diluents or excipients such as commonly used fillers, extenders, binders, wetting agents, disintegrating agents, surfactants, and the like.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and such solid preparations may include at least one excipient such as starch, calcium carbonate, It can be prepared by mixing sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate, talc can also be used.
  • Liquid preparations for oral use include suspensions, solvents, emulsions, and syrups, and include various excipients such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. Can be.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations and suppositories.
  • non-aqueous solvent and suspending agent propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used.
  • base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Routes of administration of the pharmaceutical compositions according to the invention are not limited to these, but are oral, intravenous, intramuscular, intraarterial, intramedullary, intradural, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, Sublingual or rectal. Oral or parenteral release is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intra-articular, intramuscular, intrasternal, intradural, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical compositions of the invention may also be administered in the form of suppositories for rectal administration.
  • the pharmaceutical compositions of the present invention vary depending on a number of factors, including the activity, age, weight, general health, sex, formulation, time of administration, route of administration, rate of release, drug combination and severity of the particular disease to be prevented or treated, of the specific compound employed.
  • the dosage of the pharmaceutical composition may be appropriately selected by those skilled in the art depending on the patient's condition, weight, degree of disease, drug form, route of administration and duration, and 0.0001 to 50 mg / kg or It may be administered at 0.001 to 50 mg / kg. Administration may be administered once a day or may be divided several times. The dosage does not limit the scope of the invention in any aspect.
  • the pharmaceutical compositions according to the invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, suspensions.
  • the food composition may be prepared in the form of various foods, for example, beverages, gums, teas, vitamin complexes, powders, granules, tablets, capsules, sweets, rice cakes, breads, and the like. Since the food composition of the present invention is composed of plant extracts having little toxicity and no side effects, it can be used with confidence even for long-term use for prophylactic purposes.
  • the amount may be added at a ratio of 0.1 to 50% of the total weight.
  • natural carbohydrates include monosaccharides such as glucose, disaccharides such as fructose, sucrose and the like, and common sugars such as polysaccharides, dextrins and cyclodextrins, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • the flavourant include natural flavourants (tautin, stevia extract (for example, rebaudioside A, glycyrrhizin, etc.), synthetic flavors (saccharin, aspartame, etc.).
  • compositions of the present invention include various nutrients, vitamins, minerals (electrolytes), flavors such as synthetic flavors and natural flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloidal thickeners. , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated drinks, and the like.
  • additives can be used independently or in combination.
  • the proportion of such additives is not so critical but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the food composition of the present invention.
  • the cosmetic composition is a lotion, nutrition lotion, nutrition essence, massage cream, beauty bath additives, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen Creams, suntan creams, skin lotions, skin creams, sunscreen cosmetics, cleansing milk, depilatory ⁇ cosmetic ⁇ , face and body lotions, face and body creams, skin whitening creams, hand lotions, hair lotions, cosmetic creams, jasmine Oil, Bath Soap, Water Soap, Beauty Soap, Shampoo, Hand Cleanser (Hand Cleaner), Medicated Soap ⁇ Non-Medical ⁇ , Cream Soap, Facial Wash, Systemic Cleanser, Scalp Cleaner, Hairrin, Cosmetic Soap, Tooth Whitening Gel, Toothpaste Or the like.
  • the composition of the present invention may further comprise a solvent or a suitable carrier, excipient or diluent commonly used in the preparation of the cosmetic composition.
  • the kind of the solvent that can be further added in the cosmetic composition of the present invention is not particularly limited, but, for example, water, saline, DMSO, or a combination thereof may be used, and as the carrier, excipient or diluent, purified water, oil, wax , Fatty acids, fatty acid alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like.
  • a whitening agent, a moisturizing agent, vitamins, sunscreens, perfumes, dyes, antibiotics, antibacterial agents, antifungal agents may be included as necessary.
  • the oil may be hydrogenated vegetable oil, castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, avocado oil, wax, wax, carnauba, candelilla, montan, ceresin, liquid paraffin, lanolin Can be used.
  • Stearic acid, linoleic acid, linolenic acid, oleic acid may be used as fatty acids, cetyl alcohol, octyl dodecanol, oleyl alcohol, pantenol, lanolin alcohol, stearyl alcohol, hexadecanol may be used as fatty acid alcohol.
  • Isopropyl myristate, isopropyl palmitate, butyl stearate may be used as the fatty acid ester.
  • surfactants cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as much as possible.
  • it may include a hygroscopic agent, a thickener, an antioxidant, and the like, which are widely known in the cosmetic field, and their types and amounts are known in the art.
  • Stem cells or the culture-derived exosomes provided by the present invention promote cell migration to the wound site and have excellent wound healing or wound healing promoting activity.
  • the stem cells or the culture-derived exosomes provided by the present invention is excellent in the hair growth and hair growth effect is excellent in the prevention and treatment of hair loss, it is safe without side effects even when ingested or applied to the skin.
  • Figure 1 shows a TEM picture of the exosomes in Experimental Example 1
  • (b) shows a graphical analysis of the size of the particles present in the exosomes.
  • Figure 2 (a) is a photograph confirming the expression of HSP70, CD63 and CD9 markers of exosomes using Western blot in Experimental Example 1, (b) is a CD24 and AQP2 markers of exosomes using real-time PCR The degree of expression is shown in a graph, and (c) and (d) show the expression of CD63, CD9 and CD81 markers of exosomes using FACS.
  • Figure 3 is a photograph showing the change in the movement of human skin fibroblasts to the wound site after treatment of the exo-some obtained in Example 3 in Experimental Example 2 and the fat-derived stem cell culture medium from which the exo-some obtained in Comparative Example 1 is removed.
  • Figure 4 is a graph showing the ratio of human skin fibroblasts moved to the wound site after the treatment of the exo-some obtained in Example 3 in Experimental Example 2 and the fat-derived stem cell culture medium from which the exo-some obtained in Comparative Example 1 is removed .
  • FIG. 5 is a graph showing the expression levels of procollagen, KGF and CD34 after treatment of the exosomes obtained in Example 3 in Experimental Example 3 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed.
  • Figure 6 (a) and (b) is a graph showing the expression level of procollagen and elastin for each concentration after the treatment of the exosomes obtained in Example 3 in Experimental Example 4.
  • Figure 7 (a) to (c) is a graph showing the expression level of KGF, CD34 and VEGF by concentration after the exosome treatment obtained in Example 3 in Experimental Example 5.
  • Example 8 is a graph showing the expression levels of LEF-1, PTC-1 and KGF after treatment of the exosomes obtained in Example 3 and Experimental Example 6 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed. will be.
  • the stem cell or its culture-derived exosomes have excellent skin permeability, and excellent effects such as wound healing, skin moisturizing, skin whitening, wrinkle improvement and anti-aging upon absorption, and also excellent hair growth and hair growth effects.
  • Consent was obtained from a patient who had undergone a subcutaneous fat removal procedure, and the adipose tissue was removed from the patient by liposuction.
  • the extracellular matrix of adipose tissue was treated with 0.075% collagenase for 45 minutes in an incubator at about 37 ° C. and at about 5% CO 2 , and then the resulting adipose tissue was centrifuged at about 1200 g for 5 minutes to form stromal cells containing high density stem cells.
  • Vascular fractions were obtained. The obtained fractions were washed with PBS, other tissues were removed through a 70 ⁇ m nylon cell strainer, and histopaque-1077 (Sigma Co.) was used to separate cell debris and mononuclear cells.
  • the isolated mononuclear cells were cultured using Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin / streptomycin as a medium at about 37 ° C., about 5% CO 2 incubator. After culturing for 24 hours at, the adhering stem cells were isolated by removing non-adherent cells.
  • DMEM Dulbecco's Modified Eagle's Medium
  • FBS fetal bovine serum
  • penicillin / streptomycin penicillin / streptomycin
  • Adipose derived stem cells 1.25 ⁇ 10 5 cells obtained in Example 1 were prepared with 1000 mg / L D glucose, 584 mg / L L-glutamine, 110 mg / L sodium pyruvate, 10% by volume FBS, 1% by volume penicillin -Added to DMEM medium supplemented with streptomycin and passaged for 30 days in a 5% by volume CO 2 incubator under conditions of about 90% humidity and about 37 ° C.
  • the cultures were removed from the passaged culture using a pipette, washed three times with phosphorylation buffer solution, and then supplemented with 365 mg / L L-glutamine, 15 mM HEPES, 55 mg / L sodium pyruvate. Inoculated at a concentration of 1.2 ⁇ 10 6 cells / dish in a 1: 1 (weight ratio) mixed medium of DMEM and Ham's F-12 nutrient mixtures and incubated for 72 hours under hypoxia conditions.
  • Example 2 50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube, and centrifuged at 4 ° C. and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 g for 20 minutes at 4 ° C., and the obtained supernatant was transferred to a tube capable of a new high speed centrifuge, followed by high speed centrifugation at 10,000 g for 30 minutes. .
  • the supernatant was transferred to a new ultra-centrifugal polycarbonate bottle, and the supernatant was removed by ultrafast centrifugation using OPTIMA XE-90 ultracentrifuge from Beckman Coulter for 70 minutes at 110,000 g at 4 ° C. .
  • the settled pellet was resuspended using a micropipette in a tube with 1000 ⁇ l PBS.
  • the resuspended tube was centrifuged for 70 minutes at 100,000 g at 4 ° C. using an OPTIMA MAX-XP ultracentrifuge from Beckman Coulter, and all possible supernatants were removed and the pellet was recovered.
  • a small amount of PBS was added to the recovered pellet, resuspended, separated into 100 ⁇ l, stored at ⁇ 80 ° C., and dissolved if necessary.
  • Example 2 50 ml of the adipose stem cell culture obtained in Example 2 was transferred to a centrifuge tube, and then centrifuged at 4 ° C. and 300 g for 10 minutes. The supernatant was collected and transferred to a new centrifuge tube, which was then centrifuged at 2,000 ° C. for 20 minutes at 4 ° C. The supernatant after 20 min centrifugation at 2,000 ° C. at 4 ° C. was transferred to a new high-speed centrifuge tube, followed by high-speed centrifugation at 10,000 ° C. for 30 minutes at 4 ° C.
  • the supernatant was transferred to a new polycarbonate bottle for ultrafast centrifuge, ultrafast centrifuged using OPTIMA XE-90 ultracentrifuge from Beckman Coulter for 70 minutes at 110,000 g at 4 ° C.
  • the removed fat-derived stem cell culture was prepared.
  • Example 3 The exosomes obtained in Example 3 were observed through a transmission electron microscope (TEM), the photograph is shown in Figure 1 (a), and the particles present in the exosomes obtained as described above through the nanoparticle tracking analysis (NTA) The distribution of each size was analyzed and the results are shown graphically in FIG.
  • TEM transmission electron microscope
  • NTA nanoparticle tracking analysis
  • the exosomes isolated from the adipose-derived stem cell cultures can be confirmed to have a fine spherical structure in nanometers, and as shown in FIG. It can be seen that mainly distributed in 30 ⁇ 150nm.
  • exosomes isolated from the adipose derived stem cell cultures have the characteristics of typical exosomes.
  • Human skin fibroblasts were attached to the culture dish at a concentration of 5 ⁇ 10 4 cells / well and then treated with the exosomes obtained in Example 3 and the adipose derived stem cell culture medium from which the exosomes obtained in Comparative Example 1 were removed. After 48 hours of incubation, RNA was extracted from the cells of each group, and then complementary DNA (cDNA) was synthesized using intron premix and real-time PCR was performed using the same. As genes related to skin anti-aging and regeneration in the cells of each treatment group, the expression of procollagen, KGF and CD34 was confirmed and the results are shown graphically in FIG. 5.
  • cDNA complementary DNA
  • the procollagen maintains skin elasticity and corresponds to a gene related to collagen synthesis necessary for the formation of the main protein of the skin.
  • KGF keratinocyte growth factor
  • CD34 corresponds to a gene involved in skin regeneration as a factor involved in blood vessel regeneration.
  • Human skin fibroblasts were attached to the culture dish at a concentration of 5 ⁇ 10 4 cells / well, and then the exosomes obtained in Example 3 were treated at concentrations of 5 and 10 ⁇ g / ml, respectively. After 48 hours of incubation, RNA was extracted from each group of cells, and cDNA was synthesized using intron premix and real-time PCR was performed using the same. As genes involved in increasing skin anti-aging elasticity in the cells of each treatment group, the expression level of procollagen and elastin was confirmed, and the results are shown graphically in FIG. 6. However, the control group was not treated with exosomes.
  • the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in maintaining skin elasticity and preventing aging.
  • Human skin fibroblasts were attached to the culture dish at a concentration of 5 ⁇ 10 4 cells / well and then the exosomes obtained in Example 3 were treated at concentrations of 5 and 10 ⁇ g / ml, respectively. After 48 hours of incubation, RNA was extracted from each group of cells, and cDNA was synthesized using intron premix and real-time PCR was performed using the same. As genes involved in skin regeneration in the cells of each treatment group, the expression levels of KGF, CD34 and VEGF were confirmed and the results are shown graphically in FIG. 7. However, the control group was not treated with exosomes.
  • the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in skin regeneration.
  • Example 3 After attaching 5 X 10 5 human dermal papilla cells to a 60 mm (SPL, Korea) culture dish, the exosomes obtained in Example 3 and the adipose derived stem cell cultures from which the exosomes obtained in Comparative Example 1 were removed were treated. . After 48 hours of incubation, the cells were recovered, RNA was isolated using RNA isolation kit (Quiagen, USA), and cDNA was synthesized for 1 ug of RNA isolated using cDNA synthesis kit (Intronbio, Korea). Each primer was synthesized in Cosmojintech, primers were used at a concentration of 10 pmol / ul, real-time PCR was detected by the addition of 2X Cyber Green Master Mix (Takara, Japan). As genes involved in hair follicle growth and development in cells of each treatment group, the expression levels of LEF-1, PTC-1 and KGF were confirmed, and the results are shown graphically in FIG. 8.
  • lymphoid enhancer-binding factor-1 (LEF-1) is a gene that promotes the formation and differentiation of hair follicles
  • PTC-1 patched protein, Sonic Hedgehog (Shh) receptor
  • KGF keratinocyte growth factor
  • the exosome obtained from the adipose derived stem cell culture according to the present invention plays an important role in the growth, development and differentiation of hair follicles.
  • the present invention relates to a composition comprising the stem cells or the exo-derived from the culture medium thereof, and having the effect of wound treatment, skin improvement and hair loss prevention or treatment, and a method for producing the same.

Landscapes

  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Animal Behavior & Ethology (AREA)
  • General Health & Medical Sciences (AREA)
  • Veterinary Medicine (AREA)
  • Public Health (AREA)
  • Engineering & Computer Science (AREA)
  • Developmental Biology & Embryology (AREA)
  • Cell Biology (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Chemical & Material Sciences (AREA)
  • Epidemiology (AREA)
  • Pharmacology & Pharmacy (AREA)
  • Virology (AREA)
  • Biotechnology (AREA)
  • Medicinal Chemistry (AREA)
  • Hematology (AREA)
  • Dermatology (AREA)
  • Reproductive Health (AREA)
  • Neurosurgery (AREA)
  • Ophthalmology & Optometry (AREA)
  • Birds (AREA)
  • Neurology (AREA)
  • Mycology (AREA)
  • Nutrition Science (AREA)
  • Food Science & Technology (AREA)
  • Polymers & Plastics (AREA)
  • Medicines Containing Material From Animals Or Micro-Organisms (AREA)

Abstract

La présente invention concerne une composition comprenant un exosome dérivé de cellules souches ou de leur milieu de culture, et présentant les effets de traitement de cicatrices, d'amélioration de la peau, et de prévention ou de traitement de la perte des cheveux. L'exosome dérivé de cellules souches ou du milieu de culture de cellules souches selon la présente invention présente une excellente perméabilité cutanée, et présente également d'excellents effets d'hydratation de la peau, de blanchissement de la peau, anti-rides et anti-vieillissement lorsqu'il est absorbé, et n'irrite pas la peau ou n'a pas d'effets secondaires sur la peau, étant ainsi sûr. De plus, l'exosome dérivé de cellules souches ou du milieu de culture de cellules souches présente de remarquables effets de croissance capillaire et de restauration des cheveux, et présente ainsi des effets remarquables de prévention et de traitement de la perte des cheveux. L'exosome peut être ingéré ou appliqué sur la peau sans aucun effet secondaire, et est ainsi sûr.
PCT/KR2017/003645 2017-04-03 2017-04-03 Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation WO2018186505A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
PCT/KR2017/003645 WO2018186505A1 (fr) 2017-04-03 2017-04-03 Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
PCT/KR2017/003645 WO2018186505A1 (fr) 2017-04-03 2017-04-03 Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation

Publications (1)

Publication Number Publication Date
WO2018186505A1 true WO2018186505A1 (fr) 2018-10-11

Family

ID=63712910

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/KR2017/003645 WO2018186505A1 (fr) 2017-04-03 2017-04-03 Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation

Country Status (1)

Country Link
WO (1) WO2018186505A1 (fr)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110115769A (zh) * 2019-04-08 2019-08-13 浙江大学 一种脑靶向外泌体及其制备方法和应用
CN110559254A (zh) * 2019-10-20 2019-12-13 张新梅 一种外泌体组合物、其制备方法及应用
CN111471647A (zh) * 2020-03-27 2020-07-31 尧舜泽生物医药(南京)有限公司 成体干细胞外泌体的制备方法及其在治疗帕金森疾病中的应用
CN113699104A (zh) * 2021-08-23 2021-11-26 上海太安堂生物医学有限公司 外泌体提取方法、应用该外泌体的精华液及精华液应用
CN115232786A (zh) * 2022-08-02 2022-10-25 莫伊森(厦门)生发有限公司 一种脂肪干细胞外泌体及其应用

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20090119428A (ko) * 2008-05-16 2009-11-19 (주)프로스테믹스 상처 치료 또는 상처 치료 촉진용 약학 조성물
KR20100122087A (ko) * 2008-02-22 2010-11-19 에이전시 포 사이언스, 테크놀로지 앤드 리서치 중간엽 줄기세포 입자
KR101617673B1 (ko) * 2014-10-29 2016-05-03 재단법인대구경북과학기술원 성체줄기세포 및 엘라스틴 유사 폴리펩타이드를 포함하는 상처 치료 촉진용 약학적 조성물
KR20170020245A (ko) * 2015-08-12 2017-02-22 (주)프로스테믹스 성체 줄기세포와 분화된 세포의 혼합 배양액 및 이의 용도

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR20100122087A (ko) * 2008-02-22 2010-11-19 에이전시 포 사이언스, 테크놀로지 앤드 리서치 중간엽 줄기세포 입자
KR20090119428A (ko) * 2008-05-16 2009-11-19 (주)프로스테믹스 상처 치료 또는 상처 치료 촉진용 약학 조성물
KR101617673B1 (ko) * 2014-10-29 2016-05-03 재단법인대구경북과학기술원 성체줄기세포 및 엘라스틴 유사 폴리펩타이드를 포함하는 상처 치료 촉진용 약학적 조성물
KR20170020245A (ko) * 2015-08-12 2017-02-22 (주)프로스테믹스 성체 줄기세포와 분화된 세포의 혼합 배양액 및 이의 용도

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
ZHANG, J. ET AL.: "Exosomes Released from Human Induced Pluripotent Stem Cells-derived M SCs Facilitate Cutaneous Wound Healing by Promoting Collagen Synthesis and Angiogenesis", JOURNAL OF TRANSLATIONAL MEDICINE, vol. 13, no. 1, 1 February 2015 (2015-02-01), pages 1 - 14, XP021218115 *

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN110115769A (zh) * 2019-04-08 2019-08-13 浙江大学 一种脑靶向外泌体及其制备方法和应用
CN110559254A (zh) * 2019-10-20 2019-12-13 张新梅 一种外泌体组合物、其制备方法及应用
CN111471647A (zh) * 2020-03-27 2020-07-31 尧舜泽生物医药(南京)有限公司 成体干细胞外泌体的制备方法及其在治疗帕金森疾病中的应用
CN113699104A (zh) * 2021-08-23 2021-11-26 上海太安堂生物医学有限公司 外泌体提取方法、应用该外泌体的精华液及精华液应用
CN115232786A (zh) * 2022-08-02 2022-10-25 莫伊森(厦门)生发有限公司 一种脂肪干细胞外泌体及其应用

Similar Documents

Publication Publication Date Title
KR20170044999A (ko) 줄기세포 유래 엑소좀을 포함하는 상처 치료, 피부 개선 및 탈모 방지 또는 치료용 조성물 및 그 제조방법
WO2017052267A1 (fr) Composition comprenant des vésicules extracellulaires dérivées d'extrait de plante destinée à l'état de la peau et à prévenir la perte de cheveux
WO2017123022A1 (fr) Exosome dérivé de cellules souches contenant une quantité importante de facteurs de croissance
WO2018186505A1 (fr) Composition destinée au traitement des cicatrices, à l'amélioration de la peau, et à la prévention ou au traitement de la perte de cheveux comprenant un exosome dérivé de cellules souches, et son procédé de préparation
WO2013027984A2 (fr) Composition cosmétique contenant un composant de thé vert
KR101793899B1 (ko) 성체 줄기세포와 분화된 세포의 혼합 배양액 및 이의 용도
KR101894229B1 (ko) 녹용 줄기세포 배양액을 포함하는 탈모 치료용 기능성 조성물
JP2022510151A (ja) エクソソーム及びその多様な用途
KR102311832B1 (ko) 줄기세포 유래 엑소좀을 포함하는 상처 치료, 피부 개선 및 탈모 방지 또는 치료용 조성물 및 그 제조방법
WO2017052271A1 (fr) Composition pour prévenir ou traiter la perte des cheveux
KR20180048431A (ko) 녹용 줄기세포 배양액을 포함하는 피부 재생용 기능성 조성물
WO2020218846A1 (fr) Composition de blanchiment de la peau comprenant un exosome de lait
WO2023027274A1 (fr) Composition cosmétique contenant des exosomes dérivés de racine d'iris germanica en tant que principe actif
WO2019066121A1 (fr) Composition comprenant des vésicules extracellulaires dérivées de plantes
WO2022177217A2 (fr) Composition pour la régénération, le blanchiment, l'antioxydation de la peau, ou le traitement des plaies, comprenant un exosome dérivé de cellules souches utilisé en tant que principe actif, et utilisation associée
WO2017135556A1 (fr) Composition contenant du gdf11 et son utilisation
WO2017146514A1 (fr) Composition cosmétique de soin capillaire contenant un mutant de facteur de croissance des fibroblastes 9 hautement stable en tant que substance active
KR102311835B1 (ko) 줄기세포 유래 엑소좀을 포함하는 상처 치료, 피부 개선 및 탈모 방지 또는 치료용 조성물 및 그 제조방법
KR20220063839A (ko) 상처 치유 또는 피부 재생, 및 탈모 예방 또는 모발 성장 촉진용 중간엽 줄기세포 배양액 및 이의 제조방법
US20200129386A1 (en) Composition for skin whitening or wound treatment, containing liquid plasma
WO2021162206A1 (fr) Composition pour la prévention ou le traitement de la perte de cheveux comprenant un milieu de culture de tissu de follicules pileux
KR101541470B1 (ko) 진세노사이드 ｆ2를 포함하는 탈모방지 또는 발모촉진용 조성물
WO2021096089A1 (fr) Nouvelle composition comprenant des exosomes dérivés de cellules souches et du polydésoxyribonucléotide en tant que principes actifs
WO2021206335A1 (fr) Peptide pour la prévention ou le traitement de la perte des cheveux et utilisation correspondante
KR102124272B1 (ko) Hb-egf를 유효성분으로 포함하는 지방줄기세포의 발모능 증진용 조성물

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 17904395

Country of ref document: EP

Kind code of ref document: A1

NENP Non-entry into the national phase

Ref country code: DE

32PN Ep: public notification in the ep bulletin as address of the adressee cannot be established

Free format text: NOTING OF LOSS OF RIGHTS PURSUANT TO RULE 112(1) EPC (EPO FORM 1205A DATED 22.01.2020)

122 Ep: pct application non-entry in european phase

Ref document number: 17904395

Country of ref document: EP

Kind code of ref document: A1