WO2011136433A1 - Milieu conditionné de cellules souches humaines dérivées de l'adipose possédant un effet de pousse de cheveux et son utilisation - Google Patents

Milieu conditionné de cellules souches humaines dérivées de l'adipose possédant un effet de pousse de cheveux et son utilisation Download PDF

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Publication number
WO2011136433A1
WO2011136433A1 PCT/KR2010/003551 KR2010003551W WO2011136433A1 WO 2011136433 A1 WO2011136433 A1 WO 2011136433A1 KR 2010003551 W KR2010003551 W KR 2010003551W WO 2011136433 A1 WO2011136433 A1 WO 2011136433A1
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derived stem
medium
human adipose
hair
stem cells
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PCT/KR2010/003551
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English (en)
Korean (ko)
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김동수
이원종
박병순
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(주)프로스테믹스
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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
    • 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

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  • the present invention relates to a culture medium of human fat-derived stem cells having a wool effect and its use, and more particularly, to cultivate human fat-derived stem cells in a serum-free medium, and then, containing the conditioned medium obtained by filtration as an active ingredient.
  • a pharmaceutical composition for preventing or preventing hair loss, a method of inducing wool by topically applying or intradermal injection of the pharmaceutical composition to a hair loss site or a hair loss site, and a hair loss treatment agent and alopecia treatment agent comprising the pharmaceutical composition as an active ingredient will be.
  • Alopecia refers to abnormal hair loss, and usually hair loss. Hair has a period of development, growth, degeneration and resting periods, and it is known that about 50 to 100 hairs fall out normally in the resting period.
  • alopecia There are many causes of alopecia: maternal sequelae, stress, hyperthyroidism or hypothyroidism, excessive diet, iron deficiency, chemo sequelae, and male-hormonal alopecia. skin diseases such as androgenetic alopecia) and scalp ringworm are known.
  • Propecia is a synthetic anti-androgen that inhibits 5-alpha reductase, an enzyme that converts testosterone to dihydrotestosterone (DHT).
  • DHT dihydrotestosterone
  • continuous doses of propecia may have side effects such as erectile dysfunction and sexual dysfunction in men, and may be caused by male genital malformation of the fetus when absorbed into the skin of pregnant women and women of childbearing age.
  • Minoxidil is used for the treatment of vasodilators and alopecia, and side effects include scalp itching.
  • the treatment with the pharmacotherapy was effective at the time of administration, but it was found that hair loss proceeds again when the treatment is stopped.
  • Self hair transplantation also has the advantage of being semi-permanent, but has the disadvantage of relatively high cost.
  • Korean Patent Laid-Open Publication No. 2008-0097593 discloses a cell therapeutic agent containing adult stem cells and hair follicle cells derived from human adipose tissue
  • Korean Patent No. 10-0771171 discloses a composition for treating baldness using hair follicle stem cells as an active ingredient. Is disclosed.
  • the present invention is derived from the above requirements, the present inventors incubated in human serum-derived stem cells in serum-free medium, and then the conditioned medium obtained through filtration is immortalized keratin forming cells and constituent cells of the epidermis and dermis; The present invention has been found to effectively propagate human dermal papilla cells, thus completing the present invention.
  • the present invention provides a pharmaceutical composition for hair loss prevention or wool induction containing the culture medium obtained by filtration after culturing human adipose derived stem cells in serum-free medium.
  • the present invention also provides a method for inducing wool, wherein the pharmaceutical composition is topically applied or intradermal injection to the hair loss site or hair loss site.
  • the present invention also provides a hair loss treatment agent comprising the pharmaceutical composition as an active ingredient.
  • the present invention provides a therapeutic agent for alopecia comprising the pharmaceutical composition as an active ingredient.
  • Conditional medium of the human adipose derived stem cells of the present invention is expected to be effectively used for the treatment and prevention of alopecia because it has the activity of inducing regeneration and wool of skin tissue.
  • Figure 1 shows the results of measurement of cell proliferation by MTT assay after treatment of human dermal papilla cells with human adipose derived stem cell-condition medium and adipocyte-condition medium.
  • Isolated human adipose tissue-derived stem cells (ADSCs) were maintained in ⁇ -MEM for 9, 12 and 15 days.
  • Differentiation of human adipose derived stem cells into adipocytes was carried out for 9, 12 and 15 days by adipocyte differentiation media (insulin, dexamethasone, isobutyl-methyl-xanthine and indomesacin). Induced.
  • Human adipocyte-derived stem cell-condition medium and differentiated adipocyte-condition medium were collected after 48 hours of incubation in serum-free ⁇ -MEM, respectively.
  • Human dermal papilla cells were cultured in human adipose derived stem cell-condition medium and adipocyte-condition medium, and cell proliferation was measured by MTT assay after 24 and 48 hours. Proliferation of human dermal papilla cells was significantly increased in the group treated with human adipose derived stem cell-conditioned medium for 48 hours, whereas treatment with adipocyte-conditioned medium inhibited proliferation. Student's T test was used for statistical analysis and all results were averaged over at least three independent experiments. In addition, different sources of human adipose derived stem cells were used for each independent experiment. *: significance test for negative control at p ⁇ 0.05, d: duration of treatment in each medium.
  • Figure 2 shows the expression changes of signaling proteins in human dermal papilla cells after human adipose derived stem cell-condition medium treatment. After treatment with human adipose derived stem cell-condition medium, the expression of phosphorylated Akt was slightly increased and the expression of phosphorylated Erk was clearly increased.
  • Figure 3 shows the results of analyzing the cell cycle and cell cycle-related proteins of human dermal papilla cells after treatment with human adipose derived stem cell-condition medium.
  • Human adipose derived stem cell-conditioning medium increased the proliferation of human dermal papilla cells through regulation of the cell cycle.
  • Treatment with 48 hours of human adipose derived stem cell-conditioned media resulted in an increase in S phase and a decrease in G1 arrest compared to the control (top).
  • CNT control
  • ADSC-CM human adipose derived stem cell-conditioned medium treated with human dermal papilla cells.
  • Figure 4 shows the results of measuring the proliferation of immortalized keratin forming cells by MTT assay after treatment of immortalized keratin forming cells with human adipose derived stem cell-condition medium.
  • Immortalized keratinocytes are starved for 24 hours and then incubated in human adipose derived stem cell-conditioned medium for 48 hours. The proliferation of the cells was then measured by MTT assay.
  • Treatment of human adipose derived stem cell-conditioned media significantly increased proliferation of immortalized keratin forming cells at concentrations of 10-30%. All results are averaged over at least three independent experiments.
  • different sources of human adipose derived stem cells were used for each independent experiment. Student's T test was used for statistical analysis. *: Significance test for negative control at p ⁇ 0.05.
  • the resulting values were expressed as mean ⁇ standard error, measured over 6 days of cumulative growth of at least 15 hair follicles for each growth condition. Student's T test was used for statistical analysis. Five human adipose derived stem cell-conditioned media were randomly applied to five male scalp samples, respectively. *: Significance test for negative control at p ⁇ 0.05.
  • FIG. 6 shows hair growth stimulation by human adipose derived stem cells in C3H / HeN nude mice. Induction of hair growth was measured using 7-week-old C3H / HeN nude mice and hair follicles were synchronized during the resting phase.
  • Human adipose derived stem cells (5 ⁇ 10 5 cells) and a control group, PBS (phosphate buffered saline), were injected intradermally into the dorsal skin of mice every three days for 9 days. Hair growth was measured 12 weeks after the first injection. As a result, it was observed that the hair growth was increased in the group treated with human adipose derived stem cells compared to the control group treated with PBS.
  • 0.2 mm mesoroller (Moohan, Korea) was used 10 times before treatment to increase skin absorption. Hair growth stimulating effects were measured by darkening of the skin. After 12 weeks, hair growth was increased in the group to which the human adipose derived stem cell-condition medium was applied topically compared to the control group.
  • FIG. 7 shows H & E staining results on the dorsal skin of C3H / HeN nude mice treated with control and human adipose derived stem cell-conditioned media. Histological evaluation was performed 2 and 4 weeks after the first topical application of human adipose derived stem cell-condition medium. As a result, it was observed that the number of hair follicles increased after 4 weeks of human adipose derived stem cell-condition medium treatment.
  • the present invention is a pharmaceutical composition for preventing hair loss or induction of wool containing a conditioned medium obtained by culturing human adipose derived stem cells in a serum-free medium, followed by filtration.
  • a conditioned medium obtained by culturing human adipose derived stem cells in a serum-free medium, followed by filtration.
  • the condition medium is preferably cultured in human adipose derived stem cells in serum-free modified eagle medium ( ⁇ -MEM) for 40 to 60 hours, centrifuged and then 0.2 to 0.25 ⁇ m filter It can be obtained by filtration, more preferably, human adipose derived stem cells can be obtained by incubating for 48 hours in serum-free ⁇ -MEM (modified Eagle medium), followed by centrifugation and filtration with a 0.22 ⁇ m filter.
  • serum-free modified eagle medium ⁇ -MEM
  • the condition medium may promote scalp and hair growth by increasing the proliferation of human dermal papilla cells and human keratinocytes.
  • the stem cells refer to undifferentiated cells having the ability to differentiate into various types of tissue cells, and are separated from embryonic stem cells and adult tissues separated from the inner cell mass of the blastocyst. It can be broadly classified into adult stem cells.
  • Adult stem cells refer to undifferentiated cells having mutipotency derived from adult tissues of mammals including humans, preferably humans. For example, various stem cells such as bone marrow, blood, brain, skin, fat, umbilical cord blood, etc. Can be derived from adult cells.
  • the human adipose derived stem cells are a kind of adult stem cells isolated from human adipose tissue. Acquisition of adipose tissue can be obtained incidentally in the process of liposuction, which is conventionally performed, and thus has an advantage of easily obtaining and culturing a sufficient amount of stem cells, and is also superior in safety to bone marrow harvesting.
  • the human adipose derived stem cells may be cultured in a conventional manner using a stem cell culture medium, for example, a serum medium.
  • a stem cell culture medium for example, a serum medium.
  • the human adipose derived stem cells may be cultured in an ⁇ -modified Eagle medium containing 10% fetal bovine serum, and then finally cultured in a serum-free ⁇ -modified Eagle medium to increase the protein content in the obtained culture.
  • the serum-free medium step minimizes the differentiation by exposing the stem cells isolated from adipose tissue to a specific extreme environment, and enables the recovery of as much protein as possible from the culture medium.
  • compositions comprising the conditional medium of the present invention may further include suitable carriers, excipients and diluents commonly used in the preparation of pharmaceutical compositions.
  • the pharmaceutical dosage forms of the conditioned medium of the present invention may be used in the form of their pharmaceutically acceptable salts, and may be used alone or in combination with other pharmaceutically active compounds as well as in a suitable collection.
  • compositions comprising the conditioned medium according to the present invention may be in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories, and sterile injectable solutions, respectively, according to a conventional method. Can be formulated and used.
  • Carriers, excipients and diluents that may be included in the composition comprising the conditioned medium include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium Various compounds or mixtures including silicates, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.
  • diluents or excipients such as fillers, extenders, binders, wetting agents, disintegrating agents, and surfactants are usually used.
  • Solid preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, and the solid preparations include at least one excipient such as starch, calcium carbonate, sucrose in the extract. ) Or lactose, gelatin and the like are mixed.
  • lubricants such as magnesium stearate and talc are also used.
  • Oral liquid preparations include suspensions, solvents, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin.
  • Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories.
  • the 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.
  • As the base of the suppository witepsol, macrogol, tween 61, cacao butter, laurin butter, glycerogelatin and the like can be used.
  • Preferred dosages of the conditioned medium of the present invention vary depending on the condition and weight of the patient, the extent of the disease, the form of the drug, the route of administration and the duration, and may be appropriately selected by those skilled in the art.
  • the conditioned medium of the present invention is preferably administered at 0.0001 to 100 mg / kg, preferably at 0.001 to 100 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.
  • Conditional media of the invention can be administered to mammals, such as mice, mice, livestock, humans, and the like by a variety of routes. All modes of administration can be expected, for example by oral, rectal or intravenous, intramuscular, subcutaneous, topical application or intradermal injection.
  • the present invention also provides a method for inducing wool, wherein the pharmaceutical composition is topically applied or intradermal injection to the hair loss site or hair loss site.
  • the dosage form may preferably be topical application or intradermal injection, but is not limited thereto.
  • the present invention provides a hair loss treatment and alopecia treatment comprising the pharmaceutical composition as an active ingredient.
  • the hair loss treatment agent and alopecia treatment agent of the present invention can be applied not only to the scalp for the treatment of androgenetic alopecia and gynecologic alopecia that may occur after menopause or ovarian removal surgery, but also to any part of the body that needs hair growth. have.
  • Pharmaceutically acceptable carriers included in the hair loss treatment and hair loss treatment of the present invention are conventionally used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, Gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, It is not limited to this.
  • the hair loss treatment agent and the alopecia treatment agent of the present invention are in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient, according to methods which can be easily carried out by those skilled in the art. It may be prepared by or incorporated into a multi-dose container, and may further include a dispersant or stabilizer.
  • the hair loss treatment agent and the alopecia treatment agent of the present invention may be used as a single therapy, but may also be used in combination with other conventional hair growth-inducing drug therapy or surgical therapy, and when combined with such a therapy, may exhibit an maximal effect.
  • Human adipose derived stem cells were cultured as previously described (Lee et al., 2004, Cell Physiol biochem 14, 311-324). Human adipose derived stem cells were collected from subcutaneous adipose tissue obtained by liposuction from five women of average age 43.8 years. Human adipose derived stem cells were treated with 0.075% of collagen type I (collagenase type I, Sigma-Aldrich, St. Louis, MO, USA) for 45 minutes at 37 ° C.
  • collagen type I collagen type I (collagenase type I, Sigma-Aldrich, St. Louis, MO, USA) for 45 minutes at 37 ° C.
  • CM conditioned media
  • human adipose derived stem cells (5 ⁇ 10 5 cells) and mature adipocytes (adipocytes) were cultured in serum-free ⁇ -MEM.
  • the periodic change of hair involves rapid remodeling of epidermal and dermal components, thus proliferation of cultured human dermal papilla cells (hDPCs) and immortalized keratinocytes (HaCaT cells).
  • Human dermal papilla cells and immortalized keratinocytes were cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco-BRL) containing 10% fetal bovine serum.
  • DMEM Dulbecco's modified Eagle's medium
  • MTT assay 3- (4,5-dimethyl-2-thiazolyl) -2,5-diphenyl-2H-tetrazolium bromide.
  • human adipose derived stem cell-conditioning medium increases the proliferation of human dermal papilla cells through regulation of the cell cycle.
  • human adipose derived stem cell-conditioning media have been shown to promote dermal papilla cells survival and proliferation by activating Erk and Akt signaling pathways. It is reported that dermal papilla size is closely related to hair growth cycle, and dermal papilla cell numbers are reported to increase in the anagen phase (Elliott et al., 1999, J Invest Dermatol 113, 873-877).
  • human adipose derived stem cell-conditioned media significantly promoted proliferation of immortalized keratinocytes at concentrations of 10-30% (FIG. 4).
  • Human adipose derived stem cell-condition medium was added to Williams E medium at 12.5%, 25%, 37.5% or 50% levels and then cultured for 6 days.
  • the length of hair follicles in the 12.5% treatment group significantly increased to 40% compared to the control group, which was higher than the positive control group treated with 1 mM of minoxidil (FIG. 5).
  • results of the present invention indicate that human adipose derived stem cells promote hair growth by increasing the proliferation of human dermal papilla cells, and are also possible in epidermal cells by regulating the cell cycle and activating the anagen phase of the hair cycle. . Therefore, rational manipulation of human adipose derived stem cells is considered as a good means for promoting hair growth.

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  • Animal Behavior & Ethology (AREA)
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Abstract

La présente invention concerne une composition médicamenteuse pour empêcher la perte de cheveux ou induire la pousse de cheveux, qui comprend un principe actif sous la forme d'un milieu conditionné obtenu grâce à la culture de cellules souches humaines dérivées de l'adipose dans un milieu sans sérum et la filtration de la culture résultante, un procédé pour induire la pousse de cheveux dans lequel la composition médicamenteuse est injectée de manière intradermique ou déposée de manière topique sur une zone de perte de cheveux ou une zone chauve, et un agent de traitement de la perte de cheveux et un agent de traitement de l'atrichie comprenant la composition médicamenteuse en tant que principe actif.
PCT/KR2010/003551 2010-04-26 2010-06-03 Milieu conditionné de cellules souches humaines dérivées de l'adipose possédant un effet de pousse de cheveux et son utilisation WO2011136433A1 (fr)

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KR1020100038540A KR101749218B1 (ko) 2010-04-26 2010-04-26 양모 효과를 가지는 인간 지방유래 줄기세포의 조건 배지 및 이의 용도
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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US20140205563A1 (en) * 2011-05-06 2014-07-24 BioRegenerative Sciences, Inc. Compositions derived from stem cell released molecules & methods for formulation thereof
US9545370B2 (en) 2012-05-08 2017-01-17 BioRegenerative Sciences, Inc. Bioactive compositions and methods for their preparation and use
CN106554941A (zh) * 2016-12-01 2017-04-05 济南万泉生物技术有限公司 一种治疗脱发的脂肪间充质干细胞
US9730964B2 (en) 2011-03-15 2017-08-15 Cell Ideas Pty Ltd. Pharmaceutical compositions and topical use thereof
EP3148556A4 (fr) * 2014-04-21 2017-11-08 Sanjay Dhar Préparations de traitement de la peau

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
KR101869850B1 (ko) * 2016-11-04 2018-06-21 주식회사 자올 두피 침투력 효과가 증진된 탈모 방지 또는 모발성장 촉진 조성물을 포함하는 헤어팩
EP3692998A4 (fr) 2017-10-02 2021-05-26 Hirotaro Fukuoka Composition pharmaceutique destinée à être utilisée pour la modification du cuir chevelu ou de la peau, la cicatrisation d'une plaie ou la modification des cheveux

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Cited By (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9730964B2 (en) 2011-03-15 2017-08-15 Cell Ideas Pty Ltd. Pharmaceutical compositions and topical use thereof
US10967008B2 (en) 2011-03-15 2021-04-06 Cell Ideas Pty Ltd Pharmaceutical compositions and topical use thereof
US20140205563A1 (en) * 2011-05-06 2014-07-24 BioRegenerative Sciences, Inc. Compositions derived from stem cell released molecules & methods for formulation thereof
US9446075B2 (en) * 2011-05-06 2016-09-20 Bioregenerative Sciences Compositions derived from stem cell released molecules and methods for formulation thereof
US9545370B2 (en) 2012-05-08 2017-01-17 BioRegenerative Sciences, Inc. Bioactive compositions and methods for their preparation and use
EP3148556A4 (fr) * 2014-04-21 2017-11-08 Sanjay Dhar Préparations de traitement de la peau
CN106554941A (zh) * 2016-12-01 2017-04-05 济南万泉生物技术有限公司 一种治疗脱发的脂肪间充质干细胞

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