WO2021162206A1 - Composition pour la prévention ou le traitement de la perte de cheveux comprenant un milieu de culture de tissu de follicules pileux - Google Patents

Composition pour la prévention ou le traitement de la perte de cheveux comprenant un milieu de culture de tissu de follicules pileux Download PDF

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WO2021162206A1
WO2021162206A1 PCT/KR2020/016026 KR2020016026W WO2021162206A1 WO 2021162206 A1 WO2021162206 A1 WO 2021162206A1 KR 2020016026 W KR2020016026 W KR 2020016026W WO 2021162206 A1 WO2021162206 A1 WO 2021162206A1
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container
hair follicle
composition
medium
culturing
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Korean (ko)
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용동은
박철순
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주식회사 마이크로바이오틱스
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • C12N5/0602—Vertebrate cells
    • C12N5/0625—Epidermal cells, skin cells; Cells of the oral mucosa
    • C12N5/0627—Hair cells
    • C12N5/0628—Hair stem cells; Hair progenitors
    • 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
    • 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
    • A61P17/14—Drugs for dermatological disorders for baldness or alopecia
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/12—Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M25/00—Means for supporting, enclosing or fixing the microorganisms, e.g. immunocoatings
    • C12M25/06—Plates; Walls; Drawers; Multilayer plates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/113—Acidic fibroblast growth factor (aFGF, FGF-1)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2501/00—Active agents used in cell culture processes, e.g. differentation
    • C12N2501/10—Growth factors
    • C12N2501/115—Basic fibroblast growth factor (bFGF, FGF-2)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/50—Proteins
    • C12N2533/52—Fibronectin; Laminin
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2533/00—Supports or coatings for cell culture, characterised by material
    • C12N2533/90—Substrates of biological origin, e.g. extracellular matrix, decellularised tissue

Definitions

  • the present invention relates to a composition for preventing or treating hair loss comprising a hair follicle tissue culture solution and various uses thereof.
  • the hair follicle refers to the sac-shaped epithelial tissue surrounding the hair root, and the hair root refers to the part where the hair is embedded in the skin. Hair grows by active cell division near the hair follicle. If this cell division capacity is reduced, the hair follicles atrophy and migrate to a shallower part of the skin. Normally, the cell division ability is reacquired, and the generated new cell group moves to the depths of the skin, where new hair follicles are formed, and the hair grows and pushes out the old hair. .
  • Minoxidil an external drug that is commercially available with FDA approval
  • Propecia which is an oral drug, finasteride, as the main ingredient. may slow down progress.
  • in vitro experiments have difficulties in long-term experiments because nutrition or oxygen supply through blood vessels is blocked during cell culture.
  • the media for culturing hair follicle organs that have been commonly used since 2000 are Williams' medium E, 10 ⁇ g/ml insulin, 10 ⁇ g/ml transferrin, and 10 ⁇ g/ml sodium selenite. (sodium selenite) and 10 ⁇ g / ml hydrocortisone (hydrocortisone) has a medium composition is added.
  • One object of the present invention is to add a ROCK inhibitor and a basement membrane protein to a first container, and then (a) attaching hair follicle cells to the first container; (b) culturing in a cell culture medium after treating the medium in the first vessel; (c) step of attaching the hair follicle cells to the second container and passage; and (d) culturing after adding the medium to the second container; to a composition for promoting hair follicle formation comprising a culture solution of hair follicle cells cultured by.
  • the hair follicle cells after treating the ROCK inhibitor and basement membrane protein in the first container, (a) attaching the hair follicle cells to the first container; (b) culturing after treating the medium in the first container; (c) step of attaching the hair follicle cells to the second container and passage; and (d) culturing after treating the medium in the second container; to a composition for promoting hair follicle formation comprising a culture solution of hair follicle cells cultured by.
  • ROCK inhibitor refers to a substance capable of inhibiting a signal mediated by Rho kinase.
  • ROCK is a smooth muscle contraction, actin cytoskeleton organization (actin cytoskeleton organization), platelet activation, myosin phosphatase cell adhesion, downregulation of migration, proliferation and survival (downregulation) of aortic smooth muscle cells (aortic smooth muscle cells) thrombin-induced response, Hypertrophy of cardiomyocytes, bronchial smooth muscle contraction, smooth muscle contraction and cytoskeletal reorganization of non-muscle cells, dose-regulated anion channel activation, neurite retraction, wound healing, It plays an important role in various cellular functions, such as cell transformation and gene expression.
  • the ROCK inhibitor is 4-[(1R)-1-aminoethyl]-N-pyridin-4-ylcyclohexane-1-carboxamide (Y-27632), the group consisting of HA-1077, Y-39983 and Wf-536 It may be at least one selected from, but preferably Y-27632, but is not limited thereto.
  • the basement membrane in the bonding of the dermis and the epidermis, the basement membrane (BM) has various functions, and the most obvious function is to close the epidermis and the dermis, and in this way, mechanically stable cohesion of the two layers of tissues ) to ensure
  • the basement membrane also influences the "polarity" and composition of the epidermis while at the same time becoming a clear boundary between the epidermis and the dermis. It is assumed that the basement membrane initiates epidermal differentiation of keratinocytes and maintains the proliferative state of the basal cell layer. Under normal conditions, the basement membrane also prevents direct contact of epidermal cells with the dermis. However, when a wound in which the basement membrane is damaged occurs, direct contact with the dermis is made, and then the cells change their behavior to initiate the wound healing process.
  • basement membrane protein refers to a protein constituting the basement membrane.
  • the protein is a laminin or synthetic laminin 511 E8 fragment.
  • the shared laminin 511 E8 fragment is known as a laminin variant having improved cell adhesion activity.
  • the basement membrane protein may be laminin, but is not limited thereto.
  • the laminin may be a synthetic laminin 511 E8 fragment, but is not limited thereto.
  • hair follicle refers to a pouch-shaped epithelial tissue surrounding the hair root
  • the hair root refers to the part where the hair is embedded in the skin
  • hair follicle refers to cells that can be collected from the hair follicle tissue.
  • Follicle associated stem cells Human Hair Follicle Dermal Papilla Cells, Human Hair Follicle Germinal Matrix Cells, Human umbilical cells. It may be at least one selected from the group consisting of vein endothelial cells).
  • the first container and the second container are multi-plates or incubators such as 24-well, 96-well, and 384-well commonly used for cell culture, but are not limited thereto.
  • the hair follicle cells may be cells derived from hair follicle tissue.
  • the hair follicle tissue may have a hair shaft attached thereto.
  • the hair shaft is a portion exposed on the scalp surface, and occupies most of the hair, and when the cross section is cut and enlarged under a microscope, the outer layer is divided into three layers: cuticle, cortex, and medulla. lose
  • (e) removing the hair follicle cells in the present invention may further include.
  • the term "culture medium” means obtained by culturing cells in a culture medium.
  • the culture medium may or may not contain cells.
  • a method for preparing a cell-free culture medium may be performed using a known method. For example, after culturing the cells, the cell culture solution is recovered, and the recovered culture solution is filtered using a micro syringe filter or centrifuged to obtain a cell-free culture solution from which cells are removed.
  • the pore size of the micro filter may be appropriately selected within a size capable of removing cells, and is preferably 0.1 to 10 ⁇ m.
  • composition of the present invention may further include epidermal growth factor (EGF) and fibroblast growth factor (FGF) in the culture medium, but is not limited thereto.
  • EGF epidermal growth factor
  • FGF fibroblast growth factor
  • epidermal growth factor binds to the receptor EGFR to promote cell growth, proliferation, and differentiation
  • human EGF consists of 53 amino acids (Exp Cell Res., 284(1):2- 13, 2003). It is also reported that such EGF plays an important role in skin and hair regeneration (J Dermatol Sci., 72(2):81-86, 2013).
  • the fibroblast growth factor is FGF1 (aFGF), FGF2 (bFGF), FGF3 (int-2), FGF4 (HST), FGF5, FGF6 (HST2), FGF7 (KGF), It may be one or more selected from the group consisting of FGF9, FGF10, FGF11, FGF12, FGF12B, FGF14, FGF16, FGF17, FGF19, FGF20, FGF21 and FGF23, but is not limited thereto.
  • the concentration ratio of the EGF and the FGF4 may be 1:10 to 10:1.
  • the concentration of EGF may be 10ng/ml to 500ng/ml
  • the concentration of FGF4 may be 10ng/ml to 500ng/ml.
  • the medium may further include Dulbecco Modified Eagle Medium (DMEM), antibiotics and Fetal bovine serum (FBS).
  • DMEM Dulbecco Modified Eagle Medium
  • antibiotics antibiotics
  • Fetal bovine serum FBS
  • the antibiotic may be 0.1% to 5% penicillin, but is not limited thereto.
  • the medium may further include bFGF.
  • the bFGF may be included in an amount of 0.0001 wt% to 0.005 wt%, but is not limited thereto.
  • the medium may further include b27.
  • the b27 may be included in 0.01 wt% to 0.5 wt%, but is not limited thereto.
  • composition ratio of bFGF and b27 may be 1:10 to 1:1000, preferably 1:100, but is not limited thereto.
  • b27 is a chemically defined, medium supplement mainly used for the growth of neurons.
  • the step (c) may be to coat the hair follicle cells with an extracellular matrix and then attach to the second container and passaging.
  • extracellular matrix refers to a three-dimensional molecular complex with various compositions, and is involved in morphogenesis, proliferation, cell differentiation and tissue repair due to growth factor and cytokine depot properties.
  • the matrix may be at least one selected from Matrigel, NuFF feeder cells, and Geltrax.
  • the period of culturing in steps (b) and (d) may be 20 to 40 days, preferably 25 to 35 days.
  • the hair follicle cells after treating the ROCK inhibitor and the basement membrane protein in the first container, (a) attaching the hair follicle cells to the first container; (b) culturing after treating the medium in the first container; (c) step of attaching the hair follicle cells to the second container and passage; and (d) culturing after treating the medium in the second container; Or it relates to a composition for promoting hair growth or hair growth.
  • hair loss refers to a state in which hair is lost in a region where hair should normally exist, and generally refers to the loss of hair on the scalp. Unlike soft hair, which is colorless and thin, hair loss can cause cosmetic problems.
  • the hair loss can be clinically divided into two types of scarring and non-scarring.
  • scarring scarring
  • hair follicles are destroyed, so hair is not regenerated, whereas scars are not formed.
  • Hair follicles are maintained in hair loss (non-scarring), so the hair is regenerated after the symptomatic site disappears.
  • the hair loss is alopecia areata, alopecia totalis, alopecia universalis, androgenic alopecia, telogen effluvium, anagen effluvium, or It may be, but is not limited to, chemotherapy-induced alopecia.
  • composition for preventing, alleviating, treating or promoting hair growth or hair growth of the present invention may be provided as a pharmaceutical composition, a cosmetic composition or a food composition.
  • a method for preventing, alleviating, and treating hair loss comprising administering to an individual an effective amount of a composition comprising the hair follicle cell culture medium according to the present invention; Or it relates to a method for promoting hair growth or hair growth.
  • the "individual” refers to an individual in need of hair loss, prevention or treatment of a wound, or an individual in need of skin improvement, and may include both mammals and non-mammals.
  • mammals include humans, non-human primates such as chimpanzees, other apes or monkey species; livestock animals such as cattle, horses, sheep, goats, pigs; domestic animals such as rabbits, dogs or cats; laboratory animals such as rodents such as rats, mice or guinea pigs, but are not limited thereto.
  • the "administration" refers to the process of introducing the active ingredient of the present invention to an individual by any suitable method, and the preparation of the composition administered as described above is not particularly limited, and solid form preparation, liquid It may be administered as a preparation in the form of an aerosol preparation for inhalation, or in a solid form preparation which is intended to be converted immediately before use into a liquid form preparation for oral or parenteral administration, for example, powders, granules, capsules. , tablets, oral dosage forms such as aqueous suspensions, external preparations, suppositories, and sterile injection solutions may be formulated and administered, but the present invention is not limited thereto.
  • a pharmaceutically acceptable carrier may be additionally administered together with the composition of the present invention.
  • the pharmaceutically acceptable carrier may include a binder, a lubricant, a disintegrant, an excipient, a solubilizer, a dispersing agent, a stabilizer, a suspending agent, a dye, a fragrance, etc.
  • a buffer Preservatives, analgesics, solubilizers, isotonic agents, stabilizers, etc. can be mixed and used.
  • bases, excipients, lubricants, preservatives, etc. can be used for topical administration.
  • the dosage form of the composition of the present invention can be prepared in various ways by mixing with a pharmaceutically acceptable carrier as described above.
  • a pharmaceutically acceptable carrier for example, in the case of oral administration, it can be prepared in the form of tablets, troches, capsules, elixirs, suspensions, syrups, wafers, etc., and in the case of injections, it can be prepared in the form of unit dose ampoules or multiple doses. have.
  • it can be formulated as a solution, suspension, tablet, capsule, sustained release formulation, and the like.
  • examples of carriers, excipients and diluents suitable for formulation include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, malditol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate or mineral oil may be used.
  • fillers, anti-agglomeration agents, lubricants, wetting agents, fragrances, emulsifiers, preservatives and the like may be further included.
  • the route of administration of the composition according to the present invention is, but not limited to, oral, intravenous, intramuscular, intraarterial, intramedullary, intrathecal, intracardiac, transdermal, subcutaneous, intraperitoneal, intranasal, intestinal, topical, sublingual or work. Oral or parenteral administration is preferred.
  • parenteral includes subcutaneous, intradermal, intravenous, intramuscular, intraarticular, intrasynovial, intrasternal, intrathecal, intralesional and intracranial injection or infusion techniques.
  • the pharmaceutical composition of the present invention may also be administered in the form of a suppository for rectal administration.
  • a "pharmaceutically effective amount” refers to an amount sufficient of an agent to provide a desired biological result. The result may be reduction and/or alleviation of the signs, symptoms or causes of a disease, or any other desirable change in the biological system.
  • an “effective amount” for therapeutic use is the amount of a composition disclosed herein required to provide a clinically significant reduction in disease.
  • An appropriate “effective” amount in any individual case can be determined by one of ordinary skill in the art using routine experimentation. Accordingly, the expression “effective amount” generally refers to the amount in which the active substance has a therapeutic effect. In the case of the present invention, the active substance is a preventive, ameliorating or therapeutic agent for cancer.
  • the composition of the present invention may vary depending on several factors including the activity of the specific composition used, age, weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
  • the dosage of the composition may vary depending on the patient's condition, body weight, degree of disease, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and 0.0001 to 100 mg/kg or 0.001 to 0.001 to daily It can be administered at 100 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
  • the composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
  • the hair follicle cells after treating the ROCK inhibitor and the basement membrane protein in the first container, (a) attaching the hair follicle cells to the first container; (b) culturing after treating the medium in the first container; (c) step of attaching the hair follicle cells to the second container and passage; and (d) culturing after treating the medium in the second container; it may be a composition for preventing, treating or alleviating a wound comprising the cultured hair follicle cell culture solution.
  • composition of the present invention promotes the formation of fibroblasts, known as important regulators in the wound healing process (Wnt3a Induces Myofibroblast Differentiation by Upregulating TGF-beta Signaling Through SMAD2 in a beta-Catenin-Dependent Manner. Jon MC, et al. (2011) ) Plos ONE.
  • composition for preventing, treating or alleviating wounds of the present invention may be provided as a pharmaceutical composition, a cosmetic composition, or a food composition.
  • the present invention in another embodiment, it relates to a method for preventing, treating or alleviating a wound, comprising administering to a subject an effective amount of the composition according to the present invention.
  • the hair follicle cells after treating the ROCK inhibitor and the basement membrane protein in the first container, (a) attaching the hair follicle cells to the first container; (b) culturing after treating the medium in the first container; (c) step of attaching the hair follicle cells to the second container and passage; and (d) culturing the medium after treating the medium in the second container;
  • skin improvement refers to a process of treating, alleviating, or alleviating skin damage caused by intrinsic or extrinsic factors of the skin, or its effects, and more specifically, skin whitening. , wrinkle improvement, elasticity enhancement, skin regeneration, skin moisturizing, anti-aging, skin irritation alleviation, acne prevention or improvement, and/or atopic prevention or improvement effect, but is not limited thereto.
  • the composition of the present invention has a very good stem cell proliferation promoting ability, when it is applied to the human body, the skin condition can be improved by promoting or activating the proliferation of adult stem cells present in the skin.
  • Adult stem cells present in the skin are, for example, stem cells present in the basal layer of the epidermis (interfollicular epidermis) between hair follicles and hair follicles, stem cells present in hair follicles, or adipose-derived stems present in the subcutaneous fat layer. is a cell
  • composition for improving skin of the present invention may be provided as a cosmetic composition, a food composition, or a pharmaceutical composition.
  • it in another embodiment, relates to a method for improving skin, comprising administering to a subject an effective amount of the composition according to the present invention.
  • the hair follicle cell culture solution according to the present invention has excellent wound healing activity by promoting cell migration to the wound site. In addition, it has excellent skin improvement effects such as skin moisturizing, skin whitening, wrinkle improvement and anti-aging by increasing the activity of fibroblasts. In addition, the hair follicle cell culture solution according to the present invention has excellent hair growth and hair growth effects, so it is excellent in preventing and treating hair loss, and it is safe without side effects even when ingested or applied to the skin.
  • prevention may include, without limitation, any act of blocking or suppressing or delaying symptoms of various diseases using the composition of the present invention.
  • treatment may include, without limitation, any action in which symptoms of various diseases are improved or beneficial by using the composition of the present invention.
  • the composition of the present invention may vary depending on various factors including the activity of the specific composition used, age, weight, general health, sex, diet, administration time, administration route, excretion rate, drug formulation, and the severity of the specific disease to be prevented or treated.
  • the dosage of the pharmaceutical composition may vary depending on the patient's condition, weight, disease severity, drug form, administration route and period, but may be appropriately selected by those skilled in the art, and may be 0.0001 to 50 mg/kg per day or It may be administered at 0.001 to 50 mg/kg. Administration may be administered once a day, or may be administered in several divided doses. The above dosage does not limit the scope of the present invention in any way.
  • the pharmaceutical composition according to the present invention may be formulated as pills, dragees, capsules, solutions, gels, syrups, slurries, and suspensions.
  • the cosmetic composition includes lotion, nutritional lotion, nutritional essence, massage cream, cosmetic bath water additive, body lotion, body milk, bath oil, baby oil, baby powder, shower gel, shower cream, sunscreen lotion, sunscreen cream, Suntan cream, skin lotion, skin cream, sunscreen cosmetics, cleansing milk, depilatory ⁇ cosmetic ⁇ , face and body lotion, face and body cream, skin whitening cream, hand lotion, hair lotion, cosmetic cream, jasmine oil, Bath soap, water soap, beauty soap, shampoo, hand sanitizer (hand cleaner), medicinal soap (for non-medical use), cream soap, facial wash, whole body cleaner, scalp cleaner, hair conditioner, makeup soap, tooth whitening gel, toothpaste, etc. form can be prepared.
  • the composition of the present invention may further include a solvent or a suitable carrier, excipient or diluent commonly used in the preparation of cosmetic compositions.
  • the type of solvent that can be further added to 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 a carrier, excipient or diluent, purified water, oil, wax , fatty acids, fatty alcohols, fatty acid esters, surfactants, humectants, thickeners, antioxidants, viscosity stabilizers, chelating agents, buffers, lower alcohols, and the like.
  • it may include a whitening agent, a moisturizer, a vitamin, a sunscreen, a perfume, a dye, an antibiotic, an antibacterial agent, an antifungal agent.
  • Hydrogenated vegetable oil castor oil, cottonseed oil, olive oil, palm oil, jojoba oil, and avocado oil may be used as the oil.
  • the wax beeswax, spermaceti, carnauba, candelilla, montan, ceresin, liquid paraffin, and lanolin may be used. can be used
  • fatty acid stearic acid, linoleic acid, linolenic acid, and oleic acid
  • fatty acid alcohol cetyl alcohol, octyl dodecanol, oleyl alcohol, panthenol, lanolin alcohol, stearyl alcohol, and hexadecanol
  • fatty acid ester isopropyl myristate, isopropyl palmitate, and butyl stearate may be used.
  • surfactant cationic surfactants, anionic surfactants and nonionic surfactants known in the art can be used, and surfactants derived from natural products are preferred as far as possible.
  • it may include a desiccant, a thickener, an antioxidant, etc. widely known in the cosmetic field, and the types and amounts thereof are as known in the art.
  • the food composition of the present invention may be prepared in the form of various foods, for example, beverages, gums, tea, vitamin complexes, powders, granules, tablets, capsules, confectionery, rice cakes, bread, and the like.
  • the amount may be added in a proportion of 0.1 to 50% of the total weight.
  • the food composition when the food composition is prepared in the form of a beverage, there is no particular limitation other than containing the food composition in the indicated ratio, and it may contain various flavoring agents or natural carbohydrates as additional ingredients, like a conventional beverage. That is, as natural carbohydrates, monosaccharides such as glucose, disaccharides such as fructose, and polysaccharides such as sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol are included. can do.
  • natural carbohydrates monosaccharides such as glucose, disaccharides such as fructose, and polysaccharides such as sucrose, common sugars such as dextrin and cyclodextrin, and sugar alcohols such as xylitol, sorbitol and erythritol are included. can do.
  • flavoring agent examples include natural flavoring agents (taumartin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.) and synthetic flavoring agents (saccharin, aspartame, etc.).
  • the food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), synthetic flavoring agents and flavoring agents such as natural flavoring agents, coloring agents, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickeners , pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonation agents used in carbonated beverages, and the like.
  • These components may be used independently or in combination.
  • the proportion of these additives is not critical, but is generally selected in the range of 0.1 to about 50 parts by weight per 100 parts by weight of the composition of the present invention.
  • composition for promoting hair follicle formation comprising a hair follicle cell culture solution.
  • FIG. 1 shows a method of culturing hair follicle tissue for each time.
  • Figure 2 shows the transfer of the hair follicle tissue collected in the well.
  • Figure 3 shows a photograph of each step of culturing the hair follicle tissue.
  • 5 is a culture of the hair follicle tissue attached to the geltrax and Y-27632.
  • FIG. 8 is a view showing the measurement of mobility by treating the fibroblasts with a composition containing a hair follicle cell culture solution.
  • FIG. 9 is a graph showing the collagen gene expression level when fibroblasts are treated with a composition containing a hair follicle cell culture medium.
  • FIG. 10 is a graph showing the ELN gene expression level when fibroblasts are treated with a composition containing a hair follicle cell culture medium.
  • FIG. 11 is a graph showing the KGF gene expression level when fibroblasts are treated with a composition containing a hair follicle cell culture medium.
  • FIG. 12 shows the results of calculating the amount of fibroblasts when the fibroblasts are treated with a composition containing a hair follicle cell culture medium.
  • FIG. 13 is a graph showing the expression level of FGF-10 gene when dermal papilla cells are treated with a composition containing a hair follicle cell culture medium.
  • FIG. 14 is a graph showing the expression levels of FGF-7 and vEGF genes when dermal papilla cells are treated with a composition containing a hair follicle cell culture medium.
  • One object of the present invention is to add a ROCK inhibitor and a basement membrane protein to a first container, and then (a) attaching hair follicle cells to the first container; (b) culturing in a cell culture medium after treating the medium in the first vessel; (c) step of attaching the hair follicle cells to the second container and passage; and (d) culturing after adding the medium to the second container; to a composition for promoting hair follicle formation comprising a culture solution of hair follicle cells cultured by.
  • the hair follicle cells of Preparation Example 1 were cultured. As shown in Figures 2 and 4, the hair follicle tissue was placed in a 6-well plate with Dulbecco Modified Eagle Medium (DMEM) + Y-27632 (10 ⁇ M) + I-matrix (recombinant laminin E8 fragment) (5 ⁇ l/ 2ml) + 1% penicillin was added to adhere, and then placed in a cell incubator. When the medium was insufficient without changing the medium for about 30 days, an appropriate amount was filled.
  • DMEM Dulbecco Modified Eagle Medium
  • Y-27632 10 ⁇ M
  • I-matrix recombinant laminin E8 fragment
  • Y-27632 (10 ⁇ M) was added and the cells were attached. After that, after coating with Gel Trax every 30 days, Y-27632 (10 ⁇ M) was added and the cells were adhered and cultured.
  • DMEM Dulbecco Modified Eagle Medium
  • penicillin + 10% FBS penicillin + 10% FBS was used, and an appropriate amount was filled when the medium was insufficient.
  • the hair follicle cells of Preparation Examples 2 to 4 were cultured. Add follicle cells to Dulbecco Modified Eagle Medium (DMEM) + Y-27632 (10 ⁇ M) + I-matrix (recombinant laminin E8 fragment) (5 ⁇ l/2ml) + 1% penicillin in 6 well plates After attachment, the cells were placed in a cell incubator. After that, when the medium was insufficient for about 30 days, an appropriate amount was filled. The composition ratio of the medium used at this time is shown in Table 1 below.
  • DMEM Dulbecco Modified Eagle Medium
  • Y-27632 10 ⁇ M
  • I-matrix recombinant laminin E8 fragment
  • the hair follicle cells of Comparative Examples 1 to 3 were cultured. Hair follicle cells were added to Dulbecco Modified Eagle Medium (DMEM) + I-matrix (recombinant laminin E8 fragment) (5 ⁇ l/2ml) + 1% penicillin in a 6-well plate to attach, and then put in a cell incubator. put After that, when the medium was insufficient for about 30 days, an appropriate amount was filled.
  • DMEM Dulbecco Modified Eagle Medium
  • I-matrix synthetic laminin E8 fragment
  • the culture solution of Comparative Example 1 is a culture solution having the same concentration except for Y-27632 in the culture solution of Preparation Example 2
  • the culture solution of Comparative Example 2 is Y-27632 in the culture solution of Preparation Example 3
  • the culture solution of Comparative Example 3 corresponds to a culture solution having the same concentration except for Y-27632 in the culture solution of Preparation Example 4.
  • a culture solution was obtained in the process of subculturing the hair follicle cells cultured in the process of Preparation Example 1, centrifuged, and the supernatant was filtered to extract the hair follicle cell culture solution. Specifically, cells were first detached with trypsin-EDTA and then centrifuged at 3000 RPM for 5 minutes. Next, only the supernatant was collected and centrifuged again at 3000 RPM for 5 minutes. Next, only the supernatant was removed and foreign substances including cells were removed using a 0.22 ⁇ M filter system.
  • EGF + FGF4 + hair follicle cell culture composition was treated as compared to the control group (CTRL) not treated with the composition.
  • the concentration of EGF was 200 ng/ml, and the concentration of FGF4 was 100 ng/ml.
  • Figure 10 shows the expression level of elastin protein gene (ELN) when fibroblasts were treated with EGF + FGF4 and EGF + FGF4 + hair follicle cell culture composition, respectively. Compared to EGF + FGF4, ELN in EGF + FGF4 + hair follicle cell culture composition. expression was increased.
  • EGF elastin protein gene
  • KGF Keratinocyte Growth Factor
  • FIG. 12 shows the amount of proliferation of fibroblasts when the composition containing the hair follicle cell culture solution is treated.
  • EGF+FGF4+hair follicle cell culture medium composition EGF+FGF4, EGF, and FGF4 has increased.
  • the concentration of EGF was 200ng/ml
  • the concentration of FGF4 was 100ng/ml.
  • the expression level of FGF-10 was checked to determine whether there was a difference in the cell activation effect depending on whether Y-27632 was included in the hair follicle cell culture medium.
  • the hair follicle cell culture solution prepared in Preparation Examples 2 to 4 and the hair follicle cell culture solution prepared in Comparative Examples 1 to 3 were treated with dermal papilla cells, the expression level of FGF-10-related mRNA was compared in Figure 13. shown in
  • the expression levels of FGF-7 and vEGF were checked to determine whether there was a difference in cell activation effect depending on whether Y-27632 was included in the hair follicle cell culture medium.
  • the expression levels of FGF-7 and vEGF-related mRNA were compared when the dermal papilla cells were treated with the hair follicle cell culture medium prepared in Preparation Example 4 and the hair follicle cell culture medium prepared in Comparative Example 3, and it is shown in FIG. 14 . It was.
  • the present invention relates to a composition for preventing or treating hair loss comprising a hair follicle tissue culture solution and various uses thereof.

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Abstract

La présente invention concerne une composition pour favoriser la formation de follicules pileux, comprenant un milieu de culture de cellules de follicules pileux, et diverses utilisations associées.
PCT/KR2020/016026 2020-02-10 2020-11-13 Composition pour la prévention ou le traitement de la perte de cheveux comprenant un milieu de culture de tissu de follicules pileux WO2021162206A1 (fr)

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CN115786247B (zh) * 2021-12-29 2023-10-20 朗肽生物制药股份有限公司 一种无血清培养基及其在维持毛囊活性、毛发养护及移植方面的应用

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