WO2016085250A1 - 발모촉진용 조성물 - Google Patents

발모촉진용 조성물 Download PDF

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Publication number
WO2016085250A1
WO2016085250A1 PCT/KR2015/012708 KR2015012708W WO2016085250A1 WO 2016085250 A1 WO2016085250 A1 WO 2016085250A1 KR 2015012708 W KR2015012708 W KR 2015012708W WO 2016085250 A1 WO2016085250 A1 WO 2016085250A1
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Prior art keywords
hair growth
culture medium
cell culture
promoting
parts
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PCT/KR2015/012708
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English (en)
French (fr)
Inventor
안동현
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안동현
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.)
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Publication date
Priority claimed from KR1020140168236A external-priority patent/KR101570004B1/ko
Priority claimed from KR1020150126824A external-priority patent/KR20160064954A/ko
Application filed by 안동현 filed Critical 안동현
Priority to US15/531,340 priority Critical patent/US10842735B2/en
Publication of WO2016085250A1 publication Critical patent/WO2016085250A1/ko
Priority to US16/389,572 priority patent/US20190240142A1/en

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    • 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
    • A61K8/981—Cosmetics or similar toiletry preparations characterised by the composition containing materials, or derivatives thereof of undetermined constitution of animal origin of mammals or bird
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/16—Amides, e.g. hydroxamic acids
    • A61K31/164—Amides, e.g. hydroxamic acids of a carboxylic acid with an aminoalcohol, e.g. ceramides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/41—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having five-membered rings with two or more ring hetero atoms, at least one of which being nitrogen, e.g. tetrazole
    • A61K31/4164—1,3-Diazoles
    • A61K31/4188—1,3-Diazoles condensed with other heterocyclic ring systems, e.g. biotin, sorbinil
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K33/00—Medicinal preparations containing inorganic active ingredients
    • A61K33/24—Heavy metals; Compounds thereof
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • 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
    • 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
    • A61K38/18—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1808—Epidermal growth factor [EGF] urogastrone
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1825—Fibroblast growth factor [FGF]
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    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/18—Growth factors; Growth regulators
    • A61K38/1858—Platelet-derived growth factor [PDGF]
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    • 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
    • A61K8/19—Cosmetics or similar toiletry preparations characterised by the composition containing inorganic ingredients
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    • A—HUMAN NECESSITIES
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    • 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
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    • A61K8/673—Vitamin B group
    • A—HUMAN NECESSITIES
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    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
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    • 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
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0653—Adipocytes; Adipose tissue
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    • 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
    • C12N5/0652—Cells of skeletal and connective tissues; Mesenchyme
    • C12N5/0662—Stem cells
    • C12N5/0663—Bone marrow mesenchymal stem cells (BM-MSC)
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/74—Biological properties of particular ingredients
    • A61K2800/78—Enzyme modulators, e.g. Enzyme agonists
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    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
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    • A61K2800/00—Properties of cosmetic compositions or active ingredients thereof or formulation aids used therein and process related aspects
    • A61K2800/80—Process related aspects concerning the preparation of the cosmetic composition or the storage or application thereof
    • A61K2800/85—Products or compounds obtained by fermentation, e.g. yoghurt, beer, wine
    • A—HUMAN NECESSITIES
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    • A61K35/12—Materials from mammals; Compositions comprising non-specified tissues or cells; Compositions comprising non-embryonic stem cells; Genetically modified cells
    • A61K35/28—Bone marrow; Haematopoietic stem cells; Mesenchymal stem cells of any origin, e.g. adipose-derived stem cells
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    • C12N2500/00—Specific components of cell culture medium
    • C12N2500/05—Inorganic components
    • C12N2500/10—Metals; Metal chelators
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    • C12N2500/22—Zinc; Zn chelators
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    • C12N2501/10—Growth factors
    • C12N2501/11—Epidermal growth factor [EGF]
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    • C12N2533/50—Proteins
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Definitions

  • This technique is a technique for the treatment of hair loss, and more specifically, a composition for promoting hair growth which has an excellent hair growth effect and can greatly simplify the procedure.
  • Hair has no direct physiological function that is important to life, but it protects the body from external stratification, buffering, ultraviolet blocking, and external stimuli, and absorbs unnecessary metals such as arsenic, mercury, and zinc from the body. Hair is divided into anagen in which hair grows, catagen in which growth stops and hairball shrinks, and telogen in which hair follicles are pushed upwards and hair falls out as hair follicles contract. In the same place, new hairs are created, causing the old hair to fall out and regenerate into the growth phase, repeating growth and dropout throughout life.
  • the causes include decreased follicular function by male hormone involvement, decreased scalp physiological function, Korean small blood flow disorder to scalp tension, malnutrition, stress, side effects caused by drugs, genetic factors, chemicals, leukemia, tuberculosis, malignant lymphoma, etc.
  • severe hair loss can cause problems in social life and can also have a severe impact on psychology, which is very important in terms of quality of life.
  • the effects of hair loss prevention are greater than those of hair loss, and can cause side effects such as sexual dysfunction in Propecia and malformed childbirth in pregnant women.
  • the genes associated with hair loss are delivered to hair follicles or blocked by gene expression.
  • gene therapy has been developed, there is a problem that the efficacy and safety of the treatment are uncertain, the cost of the treatment, and therefore the clinical application is not available.
  • hair loss treatment methods using stem cells have recently been shown, but hair loss treatment methods using stem cells are currently used to treat hair loss of stem cells. In most cases, it is induced to differentiate into hair follicle cells by directly injecting it into the indicated area, and this method is not autologous stem cells, which is impossible to treat, and the therapeutic effect is not sustained, and it takes long time and expensive cost. In order to remedy this problem, attempts have been made to treat hair loss using culture medium produced during stem cell culture, not stem cells, but the level of commercialization has not yet been achieved. Detailed description of the invention
  • the purpose of the present invention is to provide a composition for promoting hair growth having a very high hair growth promoting effect and a very simple human delivery method of the active ingredient, with a very high utilization value of the composition.
  • the hair growth promoting composition according to an embodiment of the present invention is 100 parts by weight of stem cell culture medium obtained from the culture medium of mesenchymal stem cells cultured for 2 generations or more, 100 to 300 parts solvent, 0.001 to 0.1 parts by weight zinc. 5 to 50 parts by weight of the panthenol compound and 0.0001 to 0.1 parts by weight of water-soluble vitamins.
  • composition is delivered to the human body by spraying (spraying) or spreading on the scalp to promote hair growth.
  • the stem cell culture medium includes the steps of (a) culturing the mesenchymal stem cells, and (b) three-dimensional culture of the mesenchymal stem cells cultured in two or more passages with a biocompatible scaffold in a serum-free medium. Stem cell culture fluid obtained from coarse culture medium.
  • the mesenchymal stem cells may be human adipocyte derived stem cells.
  • the stem cell culture medium contains VEGF, HGF, FCF, TFG- ⁇ , and EGF as growth factors, and may include collagen, fibronectin, laminin, and hyaluronic acid as extracellular matrix proteins.
  • panthenol-based compound examples include D-panthenol
  • water-soluble vitamins examples include D-biotin
  • the hair growth promoting composition according to the present invention can utilize the culture medium, a by-product of stem cell culture, to achieve economic efficiency of enjoyment cell culture.
  • the hair growth promoting effect of the hair growth promoting composition according to the present invention is excellent, and it is possible to achieve a certain hair growth effect only by administering the composition for about several months.
  • composition for promoting hair growth is simply sprayed, sprayed or sprayed on the scalp or hair.
  • the application of active ingredients can be carried out just by applying, which promotes hair growth
  • the procedure with the composition can be greatly simplified.
  • FIG. 1 shows hair loss using a composition for promoting hair growth according to an embodiment of the present invention.
  • This picture shows the hair changes over time after treatment.
  • the hair growth promoting composition is mesenchymal stem cells cultured at least two passages
  • the stem cell culture fluid obtained from the culture medium.
  • the stem cell culture fluid includes not only the culture medium containing various additives, but also metabolites generated during the culture process of the stem cells.
  • the process of obtaining the stem cell culture medium comprises (a) mesenchymal stem cells.
  • Three-dimensional culture with biocompatible scaffolds in serum-free medium and (c) collecting culture medium.
  • the mesenchymal stem cells are preferably cultured in substrate and propagation medium and then subcultured, and (b) mesenchymal stem cells are cultured at 3 days intervals. It is desirable to obtain enjoyed cell culture fluid at least three times while exchanging.
  • the serum-free medium can be cultured by adding at least one of basic fibroblast growth factor (bFGF) and epidermal growth factor (EGF).
  • biocompatible scaffolds are cell supports with cell-adhesive surfaces, which may be natural or synthetic polymers, for example, alginate, protein, collagen, fibrin, hyaluronic acid, salose, poly (alpha-hydroxy). Acid) series, poly (vinyl alcohol), polyanhydride and the like.
  • the mesenchymal stem cell culture composition of the present invention for achieving the above-mentioned objectives is prepared according to the above method, which contains a large amount of growth factors and cytokines, and in particular, a high concentration of VEGF and EGF.
  • the mesenchymal stem cell culture medium composition of the present invention contains 4 times or more of VEGF and 17 times or more of EGF, compared to the conventional culture medium composition.
  • the present invention relates to mesenchymal stem cells in serum-free medium.
  • Stem cell culture medium containing a large amount of physiologically active substance obtained by effective culture is included as a component of the hair growth promoting composition.
  • Mesenchymal stem cells used in the culture can be obtained from bone marrow, umbilical cord blood, and adipose tissue. Stem cells have the advantage of being relatively accessible, simple to collect, and capable of obtaining large quantities from an individual.
  • a large amount of mesenchymal stem cells can be grown in a short period of time by appropriately using basal medium and propagation medium in culturing mesenchymal stem cells.
  • a three-dimensional culture using a biocompatible scaffold in serum-free medium in the process of obtaining a culture solution can be used to stably maintain cells for a long time, thereby producing a large amount of cell culture solution.
  • the stem cell culture medium produced according to the method of the present invention is characterized by significantly higher growth factors such as VEGF and EGF compared to the culture medium produced in conventional two-dimensional culture.
  • bovine-derived serum is required to culture and maintain mesenchymal stem cells, and in order to use the cell culture for clinical purposes, it is desirable that the culture fluid does not contain any serum derived from mesenchymal stem cells. If the medium does not contain bovine serum, it is difficult to keep cells stable.
  • the mesenchyme in serum-free medium does not contain bovine serum.
  • a method for stably culturing stem cells is provided.
  • the culture medium obtained according to this method contains a much higher amount of growth factors than the culture medium produced by conventional methods.
  • the vascular endothelial growth factor (VEGF) content in the stem cell culture medium is increased by about four times that of the conventional culture method, providing a significantly improved effect, and thus can be used clinically.
  • VEGF vascular endothelial growth factor
  • the culture can be carried out in a simple form of bar without using the plate commonly used for cell culture.
  • the number of cells can be adjusted.
  • mesenchymal enjoyment cells are not stably maintained when plate culture is performed in serum-free medium and are eliminated from the cell culture surface.
  • the mesenchymal stem cells are cultured together with biocompatible scaffolds.
  • the mesenchymal stem cells with adherent properties can be stably maintained within the three-dimensional scaffold.
  • fibringlu is used as an example of a biocompatible scaffold
  • the mesenchymal stem cells are at least 80% for 14 days. Survival was shown and high levels of VEGF were secreted continuously compared to two-dimensional culture.
  • the stability of cells and the amount of growth factors secreted in the culture medium when cultured by adding bFGF (basic fibroblast growth factor) and / or EGF (epidermal growth factor) to serum-free medium Can increase.
  • meenchy stem cells are capable of self-proliferation and multipotency, and express the CD73 +, CD90 +, CD105 +, CD14-, CD20-, CD34-, and CD45-phosphorus phenotypes. These cells represent, but are not limited to, bone marrow, adipose tissue, umbilical cord blood, etc. However, fat-derived stem cells are preferred in terms of the amount of active ingredient in the culture medium.
  • physiologically active substance refers to cytokines, cell growth factors, etc., which may affect the function of a cell or body.
  • stem cell culture fluid refers to the cell culture supernatant that cultured the fungal cells. The stem cell culture fluid contains various physiologically active substances secreted from the cells during the fungal cell culture.
  • Biocompatibility scaffolds are made of a material that has affinity with cells and is called “cell adhesion” and means a support that can attach and culture cells three-dimensionally. Examples include, but are not limited to, alginates, proteins, collagen, fibrin, hyaluronic acid, salose, etc. Examples of synthetic polymers include poly (alpha-hydroxy acid) series, poly (vinyl alcohol), polyanhydride It has rounds and is not limited to this.
  • three-dimensional cultures can be performed with biocompatible scaffolds such as fibrin glue.
  • three-dimensional cultures with fibrin glue when culturing mesenchymal enjoyment cells in serum-free medium are performed.
  • the content of human-derived growth factors, especially VEGF and EGF can be dramatically increased.
  • stem cells are stably maintained and secreted a high concentration of cell growth factors, so the culture medium can be obtained at least three times by changing the culture medium every three days.
  • the three-dimensional culture method of it is possible to cultivate in the bar, unlike the conventional plate culture, so that a large capacity production is possible.
  • the culture of the mesenchymal stem cells can be carried out according to the following procedure.
  • the medium used for cell culture is not limited to this. '
  • Proliferation medium was 10% bovine serum, EGF (epidermal growth factor) at Ol-lOOng / i concentration. And / or DMEM or DMEM F12 containing a basic fibroblast growth factor (bFGF) at a concentration of 0.1-100 ng / i, rapidly growing adherent mesenchymal stem cells and acting to increase the amount of cells in a large amount in a short period of time. .
  • bFGF basic fibroblast growth factor
  • the growth medium is removed and the cells are trypsinized to remove the cells from the culture vessel.
  • the cultured cells are washed three times with PBS to remove FBS, and cultured in serum-free medium as adhered to a biocompatible scaffold.
  • Cell culture in scaffolds does not require conventional cell culture containers. Therefore, it is possible to cultivate a large amount in a sterile bottle or a culture bag, which has the advantage of being easily cultivated at a lower cost.
  • the stem cell culture medium contains VEGF, HGF, FCF, TFG- ⁇ , and EGF as growth factors, and may include collagen, fibronectin, laminin, and hyaluronic acid as extracellular matrix proteins.
  • the hair growth promoting composition comprises zinc having 0.001 to 0.1 parts by weight relative to 100 parts by weight of the stem cell culture solution.
  • the hair growth promoting composition further comprises 5 to 5 parts by weight of 100 parts by weight of the stem cell culture solution.
  • a panthenol compound having 50 parts by weight can be used.
  • the substrate tenol compound include D-pannetol compounds, and other panthenol derivatives may be used.
  • composition for promoting hair growth comprises water-soluble vitamins having 0.0001 to 0.1 parts by weight relative to 100 parts by weight of the enjoyment cell culture medium.
  • vitamin D-biotin and the like can be used, and various types of water-soluble vitamins can be additionally used.
  • the hair growth promoting composition contains a water-soluble solvent, and may contain 100 to 300 parts by weight of an aqueous solvent with respect to 100 parts by weight of the stem cell culture liquid. Saline water is preferred ⁇
  • composition for promoting hair growth can exhibit a hair growth effect by spraying or spraying on the scalp or simply spreading it evenly on the scalp. That is, it is not necessary to treat the scalp by injection. Can be simplified.
  • composition for promoting hair growth was prepared in a composition as shown in Table 1 below.
  • FIG. 1 is a photograph showing hair changes over time after treatment to a baldness patient using a hair growth promoting composition according to an embodiment of the present invention.
  • the subject patient was treated with the composition for promoting hair growth prepared in the above example once a week, by spraying 7cc each.

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Abstract

본 발명은 2계대 이상 배양한 중간엽 줄기세포의 배양배지로부터 수득된 줄기세포 배양액 100중량부; 아연 0.001 내지 0.1 중량부; 판테놀계 화합물 5 내지 50중량부; 및 수용성 비타민 0.0001 내지 0.1 중량부를 포함하고, 두피에 분사 또는 도포되는 방식에 의하여 발모를 촉진하는 발모촉진용 조성물을 제공한다. 상기 발모촉진용 조성물은 효과가 우수할 뿐만 아니라 조성물의 전달 방식이 단순하여 치료 방식의 경제성을 도모할 수 있다.

Description

명세서
발명의명칭:발모촉진용조성물
기술분야
[1] 본기술은탈모치료분야의기술로서,보다구체적으로는우수한발모효과를 갖고시술을매우간소화할수있는발모촉진용조성물에관한것이다.
배경기술
[2] 최근미용에관심이높아지면서탈모치로에관한관심또한높아지고있다. 모발은생명에직접적인중요한생리적기능은가지고있지않지만외부의 층격으로부터완충과자외선차단，외부자극으로부터인체를보호하며,신체에 불필요한비소，수은，아연등의중금속을흡수하여체외로배출하는기능을 가지고있다.모발은모발이성장하는성장기 (anagen),성장이멈추고모구부가 축소되면서대사과정이늦어지는퇴행기 (catagen)，모낭이수축되면서모근이 위쪽으로밀려올라가모발이빠지게되는휴지기 (telogen)로구분되며，같은 장소에서새로운모 ^이생성됨으로인해오래된모발이탈락하고성장기로 전환되는모주기 (hair cycle)을가지고일생동안성장과탈락을반복한다.
[3] 이러한모발이정상적으로있어야할곳에없는상태를탈모라하며,그
원인으로남성호르몬관여에의한모포기능의저하，두피생리기능의저하，두피 긴장에의한국소혈류장애，영양불량，스트레스,약물에의한부작용,유전적 요인,화학물질，백혈병，결핵，악성임파종등의질병을들고있다.위와같은 기능외에탈모가심한경우사회생활을하는데문제가있을수있으며 심리적으로도위축되어심각한영향을미칠수있어삶의질측면에서탈모 치료는매우중요하다.
[4] 현재탈모치료법으로가장많이쓰이고있는방법은자기자신의머리털을 이용하여이식하는자가모발이식수술법과미녹시딜과프로페시아를사용하는 약물치료가널리사용되고있다.미녹시딜의경우혈관확장을통한모세포에 영양공급증가및칼륨채널개방효과 (potassium channel opening effect)등이모발 성장올유도하며，프로페시아의경우 DHT(dihydrotestosterone)의생성을 저해하는효과로모발성장을유도하지만이두약물모두치료할당시에는발모 효능이나타나나,치료가중단되면탈모가다시진행되고모발성장의
효과보다는탈모방지의효과가더욱크며，프로페시아의경우성기능장애， 임신한여성의경우기형아출산과같은부작용이나타날수있다.최근,탈모와 관련된유전자를모낭에전달하거나유전자발현을차단하는방법으로 시술되는유전자치료법이개발되었으나,치료의효능및안전성이불확실하고, 치료비용이고가이어서임상적용이용이하지않다는문제점이있다.
[5] 한편，최근에는줄기세포를이용한탈모치료방법등도시도되고있으나.현재 줄기세포를이용한탈모치료방법은줄기세포를탈모나무모의증상을 나타내는부위에직접주입하여모낭세포로분화하도록유도하는방법이 대부분인데,이러한방법은자가줄기세포가아니면치료가불가능하며, 치료효과가지속적으로유지되지못할뿐만아니라,오랜시간과고가의비용이 소요된다는단점이있다.이러한문제점을개선하기위하여줄기세포가아닌 줄기세포배양시생산되는배양액을사용하여탈모를치료하고자하는시도가 이루어지고있으나，아직상업화수준의실효성올거두고있지못한실정이다. 발명의상세한설명
기술적과제
[6] 본발명의목적은모발의성장촉진효과가매우우수하고，유효성분의인체 전달방식이매우간단하여조성물의활용가치가매우높은발모촉진용 조성물을제공하는것이다.
과제해결수단
[7] 본발명의일실시예에따른발모촉진용조성물은 2계대이상배양한중간엽 줄기세포의배양배지로부터수득된줄기세포배양액 100중량부,용매 100내지 300증량부，아연 0.001내지 0.1증량부,판테놀계화합물 5내지 50중량부및 수용성비타민 0.0001내지 0.1중량부를포함한다.
[8] 두피에분사또는도포되는방식에의하여발모를촉진하는발모촉진용
조성물.상기조성물은두피에분사 (분무)또는도포되는방식에의하여인체에 전달되어발모를촉진한다.
[9] 상기줄기세포배양액은， (a)중간엽줄기세포를배양하는단계,및 (b) 2계대 이상배양한중간엽줄기세포를무혈청배지에서생체적합성스캐폴드와함께 3차원배양하는단계를거친배양배지로부터수득된줄기세포배양액일수 있다.
[10] 상기중간엽줄기세포는인간지방세포유래줄기세포일수있다.
[11] 상기줄기세포배양액은성장인자로서, VEGF, HGF, FCF, TFG-βΙ및 EGF를 포함하고,세포외기질단백질로서,콜라겐,피브로넥틴，라미닌및히알루론산을 포함할수있다.
[12] 상기판테놀계화합물의예로서는 D-판테놀을들수있으며，상기수용성 비타민의예로서는 D-바이오틴을들수있다.
발명의효과
[13] 본발명에따른발모촉진용조성물은줄기세포배양의부산물인배양액을 활용함으로써，즐기세포배양의경제적효율을도모할수있다ᅳ
[14] 무엇보다중요한점은,본발명에따른발모촉진용조성물의발모촉진효과의 우수성이며,대략수개월의조성물투여만으로도확실한발모효과가발휘될수 있다.
[15] 한편,상기발모촉진용조성물은두피나두발에단순히분사，분무또는
도포하는것만으로도유효성분의전달이이루어질수있어상기발모촉진용 조성물을이용한시술이매우간소화될수있다.
도면의간단한설명
[16] 도 1은본발명의일실시예에따른발모촉진용조성물을이용하여탈모
환자에게치료한후의시간경과에따른모발변화를보여주는사진이다.
발명의실시를위한형태
[17] 이하본발명의일실시예에따른발모촉진용조성물을자세하게설명하도록 한다.
[18] 상기발모촉진용조성물은 2계대이상배양한중간엽줄기세포
배양배지로부터수득된줄기세포배양액을포함한다.상기줄기세포배양액은 다양한첨가제를포함한배양배지뿐만아니라줄기세포의배양과정중에 발생된대사산물을포함한다.
[19] 구체적으로，상기줄기세포배양액의수득과정은 (a)중간엽줄기세포를
배양하는단계; (b) 2계대이상배양한중간엽줄기세포를수집하여
무혈청배지에서생체적합성스캐폴드와 3차원배양하는단계;및 (c)배양 배지를수집하는단계를포함할수있다.
[20] 여기에서 , (a)중간엽줄기세포의배양은기질배지및증식배지에서배양한후 계대배양하는것이바람직하고, (b)중간엽줄기세포의 3차원배양은 3일 간격으로배지를교환하면서적어도 3회이상즐기세포배양액을얻는것이 바람직하다.그리고，상기무혈청배지에 bFGF(basic fibroblast growth factor)및 EGF(epidermal growth factor)증적어도하나를첨가하여배양할수있다.
[21] 또한，생체적합성스캐폴드는세포접착성인표면을갖는세포지지체로서 천연및합성고분자일수있으며,예를들어알지네이트,단백질，콜라젠， 피브린,히알루론산,샐를로오스，폴리 (알파-하이드록시산)계열,폴리 (비닐 알콜),폴리안하이드라이드등이포함된다.
[22] 상기다른목적을달성하기위한본발명의중간엽줄기세포배양액조성물은, 상기방법에따라제조되어다량의성장인자와사이토카인을함유하며，특히 VEGF및 EGF를고농도로함유하는것올특징으로한다.바람직하게는본 발명의중간엽줄기세포배양액조성물은종래의배양액조성물에비하여 VEGF는 4배이상， EGF는 17배이상함유한다.
[23] 본발명은무혈청배지에서중간엽줄기세포 (mesenchymal stem cell)를
효과적으로배양하여얻어지는다량의생리활성물질을함유하는줄기세포 배양액을발모촉진용조성물의성분으로포함하는것이다.배양에사용하는 중간엽줄기세포는골수，제대혈，지방조직둥으로부터얻을수있다.특히지방 유래줄기세포는비교적접근이수월하고채취가간단하며한개체로부터 다량을얻을수있다는장점이있다.
[24] 본발명에서는중간엽줄기세포를배양하는데있어서기저배지，증식배지를 적절히사용하여배양함으로써，다량의중간엽줄기세포를단기간내에 효과적으로얻을수있다.또한，본발명의방법에서는배양액을얻는과정에서 무혈청배지중생체적합성스캐폴드를이용하여 3차원배양을실시함으로써， 장기간동안세포를안정하게유지할수있어다량의세포배양액을생산할수 있다.이처럼본발명의방법에따라생산된줄기세포배양액은종래의 세포배양법인 2차원배양으로생산된배양액에비해 VEGF, EGF와같은 성장인자의함량이월등히높은것을특징으로한다.중간엽줄기세포는인간의 골수，지방조직，제대혈등에서얻게되므로채취가능한양이제한적이고，이들 조직으로부터얻어지는중간엽줄기세포의양또한매우한정적이다.줄기세포 배양액을임상적용도로사용하기위해서는,한정된수의세포에서다량의 줄기세포배양액을얻는것과배양액중에포함되어있는생리활성물질의 함량이높은것이바람직하다.
[25] 일반적으로중간엽줄기세포를배양하고유지하기위해서는소유래혈청이 필요한데，세포배양액을임상적용도로사용하기위해서는배양액증에소유래 혈청이포함되지않는것이바람직하다.중간엽줄기세포의배양배지에소유래 혈청이포함되지않을경우세포를안정적으로유지하기어렵다.
[26] 본발명에서는소유래혈청이포함되지않은무혈청배지에서중간엽
줄기세포를안정적으로배양할수있는방법을제공한다.이방법에따라얻어진 배양액은기존의방법으로생산된배양액에비해매우고함량의성장인자를 포함한다.예를들어，본발명의배양방법에따라얻어진줄기세포배양액중 포함된 VEGF(vascular endothelial growth factor)함량은종래의배양방법에비해 약 4배증가하여현저히개선된효과를제공하며，따라서임상적으로매우 유용하게사용할수있다ᅳ
[27] 본발명의방법에따라，즐기세포를피브린글루와같은생체적합성
스캐폴드와함께 3차원배양을실시할경우,세포배양을위해일반적으로 사용하는플레이트를사용하지않고간단한형태의바를에서배양이
가능하므로매우경제적이고대용량의배양액을손쉽게얻을수있다.뿐만 아니라， 2차원배양에서는세포배양면의크기에따라세포수가제한되므로 줄기세포에서분비되는생리활성물질의함량도제한적인반면,본발명에따른
3차원배양을실시할경우세포수를조정할수있으므로배양액중의
생리활성물질의농도를필요에따라증가시키는것이가능하다.
[28] 일반적으로중간엽즐기세포는무혈청배지에서플레이트배양을할경우 안정적으로유지되지않고세포배양면으로부터탈락되어사멸하게된다.본 발명에서는중간엽줄기세포를생체적합성스캐폴드와함께배양함으로써， 부착하여자라는성질을가진중간엽줄기세포가 3차원스캐폴드내에서 안정적으로유지될수있도록하였다.생체적합성스캐폴드의한예로서 피브린글루를사용할경우,중간엽줄기세포는 14일동안적어도 80%이상의 생존율을나타냈으며, 2차원배양에비해고함량의 VEGF를지속적으로 분비하였다. [29] 또한，본발명의방법에따르면，무혈청배지에 bFGF(basic fibroblast growth factor)및 /또는 EGF(epidermal growth factor)를첨가하여배양할경우세포의 안정성및배양액중분비되는성장인자의함량을증가시킬수있다.
[30] 본명세서에서 "중간엽줄기세포 (mesenchy stem cells)"는자기증식이가능하고 다분화능을가지고있으며， CD73+, CD90+, CD105+, CD14-, CD20-, CD34-, CD45-인세포표현형을나타내는세포를의미하고,골수，지방조직，제대혈 등에서분리될수있으며，이에한정되지는않는다.다만,배양액의유효성분의 양적인측면에서지방유래줄기세포가바람직하다.
[31] 본발명에서 "생리활성물질"은세포나신체의기능에영향을미칠수있는 사이토카인，세포성장인자등을통칭하여나타낸다. "줄기세포배양액 "은 즐기세포를배양한세포배양상등액을지칭한다.줄기세포배양액은즐기세포 배양과정에서세포로부터분비되는여러가지생리활성물질을함유하고있다.
[32] "생체적합성스캐폴드"는세포와친화성을가지며이른바 "세포접착성 "인 표면을지닌재료로만들어지며세포를 3차원적으로부착하고배양시킬수있는 지지체를의미한다.천연유래지지체의예로는알지네이트,단백질，콜라젠， 피브린,히알루론산,샐를로오스등이있고이에한정되지는않는다ᅳ합성 고분자의예를들면폴리 (알파-하이드록시산)계열,폴리 (비닐알콜)， 폴리안하이드라이드둥이있으며이에한정되지는않는다.
[33] 본발명에서는피브린글루와같은생체적합성스캐폴드와함께 3차원배양을 수행할수있다.본발명의방법에따르면,중간엽즐기세포를무혈청배지에서 배양할때피브린글루와함께 3차원배양을실시함으로써，인간유래성장인자， 특히 VEGF, EGF의함량올획기적으로증가시킬수있다.즉 2차원배양에서는 저산소조건을주더라도배양액증함유되는 VEGF등의함량을높이는데 한계가있으며，줄기세포배양액을 1희밖에얻을수없지만, 3차원배양에서는 줄기세포가안정적으로유지되면서고농도의세포성장인자를지속적으로 분비하므로 3일에한번씩배양배지를교환하면서최소 3회이상배양배지를 얻을수있다.또한，본발명의 3차원배양방법에따르면，종래의플레이트 배양과달리,바를에서배양이가능하므로대용량생산이가능하다.
[34] 상기중간엽줄기세포의배양은다음과정에따라실시될수있으며，
세포배양에사용되는배지는이에한정되지않는다. '
[35] (1)중간엽줄기세포의배양
[36] 해당조직에서얻은중간엽줄기세포를기질배지에현탁시켜 10,000~40，000 cells/crf의농도로배양용기에접종한후배양한다.기질배지는 10%우혈청이 포함되어있는 DMEM또는 VlEM F12(Dulbecco's Modified Eagle Medium/Ham's F-12 Nutrient Broth)배지로，약 24시간배양한다.
[37] (2)증식배지 (expansion media)에서의배양.
[38] 기질배지를제거한후，증식배지에서배양하여부착성세포를증식시킨다. 증식배지는 10%우혈청， O.l-lOO ng/i 농도의 EGF(epidermal growth factor) 및 /또는 0.1~100 ng/ i농도의 bFGF(basic fibroblast growth factor)를포함하는 DMEM또는 DMEM F12로，부착성인중간엽줄기세포를신속하게증식시켜 세포양을단기간에대량으로증가시키는작용을한다.
[39] (3)계대배양
[40] 세포가배양용기바닥의 80내지 90%를채우면증식배지를제거하고트립신 처리를통해세포를배양용기에서떼어낸다.계대배양을위해서는세포를
1 :3-1:4로희석하여새로운배양용기에서증식배지와함께배양한다.이와같은 방법으로추가적인계대배양이가능하다.
[41] (4)생체작합성스캐폴드와함께배양
[42] 배양한세포는 PBS로 3회이상세척하여 FBS를제거해주고,무혈청배지에서 생체적합성스캐폴드에부착된형태로배양한다.스캐폴드내에서의세포 배양은통상적인세포배양용기를필요로하지않으므로,무균바틀또는 culture bag에서다량으로배양이가능하여보다낮은비용으로편리하게배양할수있는 장점이있다.
[43] 상기줄기세포배양액은성장인자로서， VEGF, HGF, FCF, TFG-βΙ및 EGF를 포함하고,세포외기질단백질로서,콜라겐，피브로넥틴，라미닌및히알루론산을 포함할수있다.
[44] 상기발모촉진용조성물은상기줄기세포배양액 100중량부에대하여 , 0.001 내지 0.1중량부를갖는아연을포함한다.또한,상기발모촉진용조성물은상기 줄기세포배양액 100중량부에대하여， 5내지 50중량부를갖는판테놀계 화합물을포함한다.기판테놀계화합물의예로서는, D-판네틀둥의화합물올들 수있으며，기타판테놀유도체등이사용될수있다.
[45] 상기발모촉진용조성물은상기즐기세포배양액 100중량부에대하여 , 0.0001 내지 0.1중량부를갖는수용성비타민을포함한다.상기수용성
비타민으로서는, D-바이오틴등이사용될수있고기타다양한형태의수용성 비타민이추가적으로사용될수있다.
[46] 상기각각의성분，아연,판테놀계화합물,수용성비타민등은수용액
상태로서，상기줄기세포배양액과흔합된다.결국,상기발모촉진용조성물에는 수용성용매가포함되어，상기줄기세포배양액 100중량부에대하여 100내지 300증량부의수용성용매가포함될수있다ᅳ상기수용성용매로서는식염수 둥의물성분이바람직하다ᅳ
[47] 상기발모촉진용조성물은두피에분무,또는분사방식에의하거나단순히 두피에균일하게도포하는것만으로도모발증식효과를나타낼수있다.즉， 주사제에의한두피투여방식등에의하지않아도되므로，치료의간소화가 가능할수있다.
[48] 이하에서는본발명의일실시예에따른발모촉진용조성물의발모효과를 입증하기위한실험결과를설명하도록한다ᅳ
[49] 심시예:심험용밤모촉 ᅵ ^조성물의제조 [50] 하기표 1과같은조성으로발모촉진용조성물을제조하였다.
[51] [표 1]
Figure imgf000008_0001
[52]
[53] 심험용발모촉 ᅵ용조성물밤모특성 가
[54] 도 1은본발명의일실시예에따른발모촉진용조성물을이용하여탈모 환자에게치료한후의시간경과에따른모발변화를보여주는사진이다.
[55] 대상환자에게상기와같은실시예에의하여제조된발모촉진용조성물을 일주일에한번씩, 7cc씩분무방법으로치료하였다.
[56] 도 1을참조하면,관리전상태에대비하여볼때，시간경과에따라서모발의 증식이확연하게이루어진것을확인할수있었다.특히，단 2개월의 지료만으로도유효성있는모발의증식이일어날수있음을확인하였다.탈모 환자의유형에따라다소의효과차가발생할것으로사료되나효과의 현저성이나유효성은층분히확인되었다고판단하였다.

Claims

청구범위
2계대이상배양한중간엽줄기세포의배양배지로부터수득된줄기세포 배양액 100중량부;
용매 100내지 300증량부;
아연 0.001내지 αι중량부;
판테놀계화합물 5내지 50중량부;및
수용성비타민 0.0001내지 0.1중량부를포함하고，
두피에분사또는도포되는방식에의하여발모를촉진하는발모촉진용 조성물ᅳ
제 1항에있어서,
상기줄기세포배양액은， (a)중간엽줄기세포를배양하는단계,및 (b) 2계대이상배양한중간염즐기세포를무혈청배지에서생체적합성 스캐폴드와함께 3차원배양하는단계를거친배양배지로부터수득된 줄기세포배양액인것을특징으로하는발모촉진용조성물.
제 1항에있어서,
상기증간엽줄기세포는인간지방세포유래줄기세포인것을특징으로 하는발모촉진용조성물.
제 1항에있어서,
상기줄기세포배양액은성장인자로서, VEGF, HGF, FCF, TFG-βΙ및 EGF를포함하고，세포외기질단백질로서，콜라겐，피브로넥틴，라미닌및 히알루론산을포함하는것을특징으로하는발모촉진용조성물.
제 1항에있어서，
상기판테놀계화합물은 D-판테놀을포함하는것을특징으로하는 발모촉진용조성물ᅳ
제 1항에있어서，
상기수용성비타민은 D-바이오틴을포함하는것올특징으로하는 발모촉진용조성물.
2계대이상배양한인간지방세포유래중간엽줄기세포를소유래혈청이 포함되지않은무혈청배지에서성장인자인 bFGF또는 EGF를별도로 첨가하여배양하는단계를거친배양배지로부터,줄기세포배양액
100중량부를수득하는단계;
물 100내지 300주량부에，아연으 001내지 0.1중량부， D-판테놀 5내지 50 중량부및 D-바이오틴 0.0001내지 0.1중량부를흔합하여흔합용액을 준비하는단계;
상기줄기세포배양액에상기흔합용액을흔할한이후,그혼할물을 두피에분사또는도포되는방식에의하여발모를촉진하는발모촉진 방법. [청구항 8] 제 7항에있어서，
상기줄기세포배양액은， (a)중간엽줄기세포를배양하는단계,및 (b) 2계대이상배양한중간엽즐기세포를무혈청배지에서생체적합성 스캐폴드와함께 3차원배양하는단계를거친배양배지로부터수득된 줄기세포배양액인것을특징으로하는발모촉진방법
[청구항 9] 제 1항에있어서，
상기중간엽즐기세포는인간지방세포유래줄기세포인것을특징으로 하는발모촉진방법
[청구항 10] 2계대이상배양한인간지방세포유래중간엽줄기세포를소유래혈청이 포함되지않은무혈청배지에서성장인자인 bFGF또는 EGF를별도로 첨가하여배양하는단계를거친배양배지로부터수득된줄기세포 배양액 2nrf;
아연수용액 lm (아연 lmg);
D-판테놀수용액 lm^D-판테놀 250mg);및
D-바이오틴수용액 lmf(D-바이오틴 0.12mg);를포함하고, 두피에분사또는도포되는방식에의하여발모를촉진하는발모촉진용 조성물.
PCT/KR2015/012708 2014-11-28 2015-11-25 발모촉진용 조성물 WO2016085250A1 (ko)

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