KR20200071043A - Composition for preventing hair loss or promoting hair growth comprising epireguline or epiregulin-derived peptide as an active ingredient - Google Patents

Abstract

The present invention relates to a composition for preventing hair loss or promoting hair growth comprising epiregulin or a fragment thereof as an active ingredient and, more particularly, to a composition for preventing hair loss or promoting hair growth comprising epiregulin or a fragment thereof as an active ingredient, and also a composition for preventing hair loss or promoting hair growth comprising adipose stem cells treated with epiregulin as an active ingredient. It has been confirmed that epiregulin according to the present invention or a fragment thereof promotes the induction of hair growth in a telogen phase of hair roots, and also remarkably increases an effect of hair growth in a mouse, and thus epiregulin according to the present invention or a fragment thereof may be easily used for the purpose of preventing hair loss or promoting hair growth. In addition, the adipose stem cell treated with epiregulin according to the present invention shows an enhancement in proliferation ability and migration ability and shows an inhibition of aging, and thus is useful as a therapeutic agent for hair loss or cosmetics for preventing or ameliorating hair loss. According to the present invention, the adipose stem cell treated with epiregulin shows a remarkable increase in proliferation ability and migration ability and shows a remarkable inhibition of aging, and it has been confirmed that there is a remarkable increase in an effect of inducing anagen hairs and hair growth compared to a control group when the adipose stem cell is administered into an animal model.

Description

Composition for preventing hair loss or promoting hair growth comprising epireguline or epiregulin-derived peptide as an active ingredient}

The present invention relates to a composition for preventing hair loss or promoting hair growth comprising epiregulin or a fragment thereof as an active ingredient, and more specifically, a composition for preventing hair loss or promoting hair growth comprising epiregulin or a fragment thereof as an active ingredient, It relates to a composition for preventing hair loss or promoting hair growth, which contains adipose stem cells treated with epiregulin as an active ingredient.

Recently, as interest in beauty has increased, so has interest in the treatment of alopecia. Alopecia refers to a condition in which there is no hair where the hair should normally be. Hair does not have an important physiological function that is directly related to life, but it has a very large role from a cosmetic point of view. In addition, it has functions such as UV protection and hair protection. In the case of severe hair loss, there may be problems in social life, and it can have a serious psychological effect, which is important in terms of quality of life.

Hair loss can be clinically divided into scar-induced scarring hair loss and non-scarring hair loss only hair. In the case of scarring hair loss, the hair follicles are destroyed, so the hair does not come back. Hair is made in what is called a hair follicle, and each hair follicle periodically undergoes phases of activity and arrest. The time interval of these hair cycles varies depending on the body part, but in the case of hair, it enters the telogen for about 3 to 4 months through the anagen of 2 to 6 years and the catagen of 2 to 4 weeks. do. Each hair follicle has 10 to 20 hair follicle growth cycles over its lifetime.

The most widely used surgical method for the treatment of hair loss is autologous hair transplantation, and drug treatment using propesia and minoxidil is widely performed. In the case of drug treatment, the hair growth effect is shown at the time of administration, but when the treatment is stopped, hair loss is resumed, especially in the case of minoxidil, there are side effects such as sexual dysfunction.

On the other hand, Epiregulin (Epiregulin; EPR) is a protein encoded by the EREG gene in humans, Epiregulin is known as a ligand of the epidermal growth factor receptor (EGFR), but the contents related to hair growth are It is not known.

In addition, a method for treating hair loss using stem cells has recently been spotlighted, and a recent study confirmed that mice with a hair cycle of telogen are converted to anagen by fat stem cells pretreated with hypoxia or vitamin C. (Won et al., J Dermatol Sci 57:134-137,2010; Kim et al., Stem Cells Dev 23:1364-1376, 2014), which can be seen that this is due to the improvement of the hair regeneration ability of adipocytes. . In addition, it is known that PDGF-A, a growth factor expressed and secreted from adipose stem cells, regulates the activity of follicle stem cells and induces anagen in the hair cycle (Festa et al., Cell 146:761-771, 2011, Tomita et al., J Dermatol Sci 43:105-115, 2006). However, the function and effect of epiregulin on fat stem cells have not been confirmed.

Thus, the present inventors not only confirmed that the induction of hair growth is promoted by epiregulin or a fragment thereof after extensive research to develop a method for improving the production yield of the adipose stem cells and enhancing the hair growth effect, as well as epiregulin The proliferation and migration of adipocytes are increased, aging is suppressed, and the hair regenerative capacity of epiregulin-treated adipocytes is improved, and the present invention has been completed.

An object of the present invention is to provide a composition for preventing hair loss or promoting hair growth, which contains Epiregulin or a fragment thereof as an active ingredient.

Another object of the present invention is to provide a proliferation method capable of enhancing the ability to induce hair growth of adipose stem cells.

Another object of the present invention is to provide a pharmaceutical composition or a cosmetic composition for preventing hair loss or promoting hair growth, which contains adipose stem cells or culture medium having enhanced hair growth inducing ability as an active ingredient.

Another object of the present invention is to provide a composition capable of enhancing the proliferation, migration, and aging inhibition of adipocytes.

In order to achieve the above object,

The present invention provides a composition for preventing hair loss or promoting hair growth, comprising Epiregulin (EPR) or a fragment thereof.

In one preferred embodiment of the present invention, the epiregulin fragment may be represented by the amino acid sequence of SEQ ID NO: 1.

According to another preferred embodiment of the present invention, the composition may be a pharmaceutical composition, quasi-drug composition or cosmetic composition.

The present invention also provides a proliferation method capable of enhancing the ability to induce hair growth of adipose stem cells, comprising culturing adipose derived stem cells in a medium to which epiregulin is added.

The present invention also provides a pharmaceutical composition for preventing hair loss or promoting hair growth, which contains adipose stem cells treated with epiregulin or a culture medium thereof as an active ingredient.

The present invention also provides a cosmetic composition for preventing hair loss or promoting hair growth, which contains adipose stem cells cultured by treating epiregulin or a culture medium thereof as an active ingredient.

The present invention also provides a composition for promoting proliferation or migration of adipocytes containing epiregulin as an active ingredient.

The present invention also provides a composition for inhibiting aging of adipose stem cells containing epiregulin as an active ingredient.

The present invention also provides a pharmaceutical composition for preventing hair loss or promoting hair growth, which contains adipose stem cells treated with epiregulin or a culture medium thereof as an active ingredient.

The present invention also provides a cosmetic composition for preventing hair loss or promoting hair growth, which contains adipose stem cells cultured by treating epiregulin or a culture medium thereof as an active ingredient.

Epiregulin according to the present invention or fragments thereof not only promotes hair growth induction in the telogen of the hair root, but also confirms that the hair growth effect is significantly increased in the mouse, so the epiregulin of the present invention or its The fragment can be easily utilized for the purpose of preventing hair loss or promoting hair growth.

In addition, the adipose stem cells treated with epiregulin according to the present invention have improved proliferation capacity, mobility, and suppressed aging, and are useful as a hair loss treatment agent or a cosmetic for preventing or improving hair loss.

Adipose stem cells treated with epiregulin according to the present invention have the advantage of significantly increasing the proliferative capacity during cultivation, thereby significantly reducing the cultivation period.

According to the present invention, adipose stem cells treated with epiregulin have a markedly increased proliferation capacity and mobility, and aging is significantly suppressed, and when administered to an animal model, a significant increase in hair growth induction and hair growth effects compared to the control group Was confirmed.

1 is a view confirming the hair growth effect of Epiregulin in experimental animals.
2 is a view confirming an increase in the length of a growing mustache of mice by treating epiregulin.
Figure 3 shows the results of confirming the degree of cell proliferation by staining and direct counting with trypan blue after epiregulin treatment and culture in papillary cells (hDPC).
4 is a view confirming the hair growth effect (A) and the degree of proliferation of dermal papilla cells (B) in Epiregulin fragments in experimental animals.
Figure 5 shows the effect of improving the proliferation capacity of fat stem cells according to epiregulin treatment.
Figure 6 shows the effect of improving the mobility of fat stem cells according to epiregulin treatment through scratch analysis.
7 shows the effect of improving the mobility of adipose stem cells according to epiregulin treatment using transwell.
Figure 8 shows the effect of improving the regenerative capacity of adipose stem cells according to epiregulin treatment through colony assay.
Figure 9 shows the anti-aging effect of fat stem cells according to epiregulin treatment through beta-galactocidase staining.
10 is a view confirming the hair growth effect of adipose cells pre-treated with Epiregulin in experimental animals.

Hereinafter, the present invention will be described in detail.

In one aspect, the present invention relates to a composition for preventing hair loss or promoting hair growth, comprising Epiregulin (EPR) or a fragment thereof.

The epiregulin is a family of epidermal growth factors and plays an important role in skin cell differentiation and growth. The epiregulin fragment may include GQCIYLSGDRC (SEQ ID NO: 1), and may be applied by modifying an amino acid sequence as needed to increase hair loss prevention or hair growth promoting efficacy. The modified epiregulin fragment may preferably include GQCIYLNNNNN (SEQ ID NO: 2), but the amino acid sequence is 90% or more, 93% or more, 95% or more, 96% or more, 97% or more, 98% or more, or 99% or more identical sequences.

"Hair loss", which can be prevented or treated by the composition of the present invention, is a phenomenon in which hair completely comes out of the scalp, and can prevent or treat male pattern hair loss and female pattern hair loss, and prevent or treat male pattern hair loss caused by hair follicle damage. Is preferred, but is not limited thereto.

The term "hair growth" of the present invention means that hair is generated from the scalp, and specifically means to induce hair loss by forming hair follicles in a hair loss area or a hairless area (hairless area). It is used in the art in the same sense as "hair growth" or "wool" which means that the length of the hair is increased (ie, hair growth).

In one embodiment of the present invention, as a result of confirming the hair-generating effect by treating epiregulin on the back of the hair-removed mouse, the group treated with epiregulin is faster and richer in hair than the control group. It was found that there is a better hair growth effect (FIG. 1). In addition, as a result of tissue culture by treating Epiregulin, it was confirmed that the length of the mouse mustache significantly increased in the culture medium treated with Epiregulin (FIG. 2). Furthermore, when epiregulin was cultured by treating the papillary cells, it was confirmed that the proliferation capacity of the papilloma cells in the culture medium treated with epiregulin was significantly improved (FIG. 3).

In another embodiment of the present invention, as a result of injecting epiregulin fragments into a hair removal mouse or the like, it was found that, as shown in FIG. 4A, hair growth phase induction was accelerated in mice treated with epiregulin.

In addition, as a result of confirming the proliferation effect of the dermal papilla cells by epiregulin fragments, as shown in FIG. 4B, it was confirmed that the proliferation of the dermal papilla cells was significantly increased in a concentration-dependent manner.

Therefore, since it was confirmed that the epiregulin (Epiregulin; EPR) or a fragment thereof of the present invention effectively induces hair growth, it can be easily utilized for the purpose of preventing hair loss or promoting hair growth.

The composition of the present invention may be prepared as a pharmaceutical composition, quasi-drug composition or cosmetic composition.

The composition to which the pharmaceutical composition, quasi-drug composition or cosmetic composition containing the composition of the present invention can be applied can be applied to any part of the body that requires hair growth as well as the scalp. For example, it can be used to improve the condition of damaged hair or hair due to trauma or wide forehead or M-type forehead, eyelashes or eyebrows and alopecia for simple cosmetic effect.

When the composition of the present invention is prepared as a pharmaceutical composition, the pharmaceutical composition of the present invention includes a pharmaceutically acceptable carrier. The pharmaceutically acceptable carrier included in the pharmaceutical composition of the present invention is commonly used in formulation, 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, but is not limited thereto Does not work. The pharmaceutical composition of the present invention may further include a lubricant, a wetting agent, a sweetener, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, and in the case of parenteral administration, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, transdermal administration, mucosal administration, and eye drop administration may be administered.

Suitable dosages of the pharmaceutical compositions of the invention are variously prescribed by factors such as formulation method, mode of administration, patient's age, weight, sex, morbidity, food, time of administration, route of administration, rate of excretion, and response sensitivity. Can be. Preferably, the dosage of the pharmaceutical composition of the present invention is 0.0001-100 mg/kg (body weight) on an adult basis.

The pharmaceutical composition of the present invention is prepared in a unit dose form by formulating using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily carried out by those skilled in the art to which the present invention pertains. Or it can be manufactured by incorporating into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension, syrup or emulsion in an oil or aqueous medium, or may be in the form of ex-agent, powder, granule, tablet or capsule, and may further include a dispersant or stabilizer.

When the composition of the present invention is prepared as a quasi-drug composition, the fat-derived stem cells showing the effect of preventing hair loss or promoting hair growth can be added as it is, or used with other quasi-drugs or quasi-drug components, and can be suitably used according to a conventional method. have. The mixing amount of the active ingredient can be appropriately determined depending on the intended use.

The quasi-drug composition for preventing hair loss or promoting hair growth of the present invention is not particularly limited in its formulation, and can be variously formulated in the form of quasi-drugs known in the art as exhibiting the effect of preventing hair loss or promoting hair growth. The formulated quasi-drugs are hair tonic, hair lotion, hair cream, hair spray, hair mousse, hair gel, hair conditioner, hair shampoo, hair rinse, hair pack, hair treatment, eyebrow hair growth agent, eyelash hair growth agent, eyelash nutrition agent, pet Shampoo for pets, rinse for pets, hand sanitizer, detergent soap, soap, antiseptic cleaner, wipes, mask, ointment, patch or filter filler, etc., and includes all quasi-drugs in the usual sense.

In addition, in each formulation, the quasi-drug composition for preventing hair loss or promoting hair growth may be arbitrarily selected and blended with other ingredients according to the formulation or purpose of use. The mixing amount of the active ingredient may be appropriately determined depending on the intended use, and may include, for example, thickening agents, stabilizers, solubilizing agents, conventional adjuvants such as vitamins, pigments and fragrances, and carriers.

When the composition of the present invention is prepared as a cosmetic composition, the ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions other than the adipose-derived stem cells as an active ingredient, for example, antioxidants, stabilizers, Solubilizers, conventional adjuvants such as vitamins, pigments and fragrances, and carriers.

The cosmetic composition of the present invention can be prepared in any formulation conventionally prepared in the art, for example, solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleaning , Oil, powder foundation, emulsion foundation, wax foundation, and spray, etc., but is not limited thereto.

When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, trakant, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide, etc. may be used as a carrier component. Can be.

When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder can be used as a carrier component, and in the case of a spray, additionally chloro fluorohydrocarbon, propane /Propellant such as butane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizing agent or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 , 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan fatty acid ester.

When the formulation of the present invention is a suspension, liquid diluents such as water, ethanol or propylene glycol as carrier components, ethoxylated isostearyl alcohol, suspensions such as polyoxyethylene sorbitol esters and polyoxyethylene sorbitan esters, microcrystals Sex cellulose, aluminum metahydroxide, bentonite, agar or trakant, etc. can be used.

When the formulation of the present invention is a surfactant-containing cleansing, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivatives, methyltaurate, sarcosinate, fatty acid amide as a carrier component Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

In another aspect, the present invention relates to a proliferation method capable of enhancing hair growth inducing ability of adipose stem cells, including the step of culturing by treating epiregulin with adipose stem cell culture medium.

Epiregulin contained in the culture medium of the present invention may be added at a concentration of 5 to 50 ng/ml as described above.

The culture step of the present invention may be to inoculate the adipose stem cells in a medium containing epiregulin and culture for 48 to 72 hours.

In the present invention, the culture may be performed according to a suitable medium and culture conditions known in the art. Such a culture process can be easily adjusted and used by those skilled in the art. These various culturing methods have been disclosed in various literatures (eg, James et al., Biochemical Engineering , Prentice-Hall International Editions). According to the cell growth method, suspension culture and adhesion culture are divided into batch, fed-batch, and continuous culture according to the culture method.

The medium used for cultivation must adequately satisfy the culture requirements of adipose stem cells. The medium includes various carbon sources, nitrogen sources, trace element components and growth factors. Examples of carbon sources that can be used are glucose, sucrose, lactose, fructose, carbohydrates such as maltose, starch, cellulose, fats such as soybean oil, sunflower oil, castor oil, coconut oil, palmitic acid, stearic acid, linoleic acid Fatty acids such as, glycerol and alcohols such as ethanol, and organic acids such as acetic acid. These carbon sources can be used alone or in combination. Examples of nitrogen sources that can be used include peptone, yeast extract, gravy, malt extract, corn steep liquor (CSL), and organic nitrogen sources and urea such as soybean wheat, ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate and ammonium nitrate. Inorganic nitrogen sources are included. These nitrogen sources may be used alone or in combination. As the personnel, the medium may include potassium dihydrogen phosphate, dipotassium hydrogen phosphate and the corresponding sodium-containing salt. It may also include metal salts such as magnesium sulfate or iron sulfate. In addition, amino acids and suitable precursors may be included. During the culture, compounds such as ammonium hydroxide, potassium hydroxide, ammonia, phosphoric acid and sulfuric acid can be added in an appropriate manner to adjust the pH. In addition, during the culture, an antifoaming agent such as a fatty acid polyglycol ester can be used to suppress bubble formation. Preferably, the culture medium in the present invention may use a medium commonly used in the field of animal cell culture technology, commercially available Dulbecco's Modified Eagle Medium (DMEM), DMEM-F12 (mix of DMEM and Ham's F-12 medium) , MEM (Minimum Essential Medium), IMDM (Iscove's Modified Dulbecco's Medium), α-MEM (MEM Alpha Modification), RPMI 1640 (Roswell Park Memorial Institute 1640), M199/F12 (Medium 199 and Ham's F-12 medium), IMEM (Improved MEM), MCDB 131 medium, etc. may be used, and the medium may be used as an additional addition of epiregulin or HB-EG as described above. In addition, if necessary, fetal bovine serum (FBS), growth factor, or differentiation-related factors may be additionally included, and antibiotics commonly used for stem cell culture, for example, penicillin and strepto Antibiotics, such as streptomycin, kanamycin, ampicillin, or amphotericin B, can be added alone or in combination. When cultured, CO ₂ at a constant concentration (for example, 5%) can be maintained as in normal animal cell culture conditions. The temperature at the time of incubation is usually 20°C to 45°C, preferably 25°C to 40°C.

After the end of the culture, a centrifugation or filtration process may be performed to separate the culture medium and the "fat" stem cells, and these steps may be performed by a person skilled in the art as necessary.

In one embodiment of the present invention, it was confirmed that the proliferation and migration (migration) of adipocytes after epiregulin treatment was increased to confirm the activation of adipocytes by epiregulin (FIGS. 5 to 7 ).

In another embodiment of the present invention, the regenerative ability and anti-aging ability of epiregulin-treated fat stem cells were confirmed (FIGS. 8 and 9 ).

In another embodiment of the present invention, the effect of hair generation was confirmed by transplanting 　 adipose stem cells treated with epiregulin on the back of a mouse from which hair was removed. As a result, it was found that the group transplanted with epiregulin-activated “fat stem cells” had faster and more abundant hair than the control group, and thus had better hair growth effect (FIG. 10 ).

Therefore, in another aspect, the present invention relates to a pharmaceutical composition for preventing, treating or promoting alopecia, comprising adipose stem cells cultured by treating epiregulin or a culture medium thereof as an active ingredient.

The pharmaceutical composition can be used for cell transplantation.

In another aspect, the present invention relates to a quasi-drug composition for preventing hair loss or improving hair growth, which contains epiregulin-treated fat stem cells or a culture medium thereof as an active ingredient.

In another aspect, the present invention relates to a cosmetic composition for preventing hair loss or improving hair growth, which contains epiregulin-treated fat stem cells or a culture medium thereof as an active ingredient.

For the pharmaceutical composition, quasi-drug composition and cosmetic composition, the contents described above may be applied in the same way.

The present invention will be described in more detail through examples. These examples are only for illustrating the present invention, it will be apparent to those skilled in the art that the scope of the present invention is not to be construed as limited by these examples.

Confirm the hair growth effect of Epiregulin

After removing hair from the back of a 6-week-old male C3H rat using a pet hair depilator and a hair removal cream, 100 μl of Epiregulin (Peprotech, Cat No. 100-04) at 100 ng/ml for 12 days on rats During the subcutaneous injection. On the 16th day, we examined the degree of hair regeneration by pushing the newly grown hair on a rat's back using a razor and weighing it.

As a result, as shown in Figure 1, it was found that the hair growth phase induction was accelerated in mice treated with Epiregulin.

Confirming the effect of tissue culture on Epiregulin-treated media

The growth mouse mustache (anagen vibrissal) of normal mice was isolated using a scalpel and tweezers. The isolated mouse mustache was cultured by treating 5-20 ng epiregulin in a quantitative medium (2 mM L-glutamine, 10 μg/ml insulin, 10 ng/ml hydrocortisone, penicillin and serum-free Williams E medium). . Individual hair follicles were photographed 72 hours after initiation (Edmund Optics Ltd, UK). Changes in hair length were calculated from the pictures and expressed as the mean±SE of 8-10 hair follicles.

As a result, as shown in FIG. 2, in the group treated with epiregulin than the control group, the length of the mustache of the separated mice increased.

Confirmation of proliferation effect of papillary cells by epiregulin treatment

Follicle Dermal Papilla cell growth media (Promocell, Heidelberg, Germany) medium containing 1% Antibiotic and Antimyotic Hyclone sv30079.91 for human dermal papilla cells (hDPC) The cells were prepared by inoculation to and cultured in a 5% carbon dioxide incubator at 37°C. The prepared hDPC in 12-well was inoculated into the culture medium at a concentration of 5×10 ³ /well. After incubation overnight, 5-20 ng of Epiregulin was treated to further incubate for 48 hours. Then, add 500 μl of trypsin, float cells in each well, neutralize by adding the same amount of medium, and collect the medium mixed with the cells in a 1.5 ml tube. Here, only 50 μl was taken, 50 μl of trypan blue was stained, and the number of cells was calculated using the formula to confirm the degree of cell proliferation.

As a result, as shown in FIG. 3, when epiregulin was treated, it was confirmed that proliferation of breast papilla cells increased.

Confirmation of hair growth effect and proliferation effect of dermal papilla cells by Epiregulin fragment

4-1: Confirmation of hair growth effect

In the present invention, in order to confirm the hair growth effect by epiregulin fragments, 11 amino acids represented by GQCIYLSGDRC (SEQ ID NO: 1) were synthesized by requesting peptron, and then experiments were performed in the same manner as in Example 1.

As a result, as shown in Figure 4A, it was found that the hair growth phase induction was faster in mice treated with Epiregulin.

4-2: Breast nipples Check the proliferation effect of cells

In the present invention, in order to confirm the hair growth effect by epiregulin fragments, experiments were performed in the same manner as in Example 3.

As a result, as shown in Figure 4B, it was confirmed that the proliferation of breast papilla cells is significantly increased in a concentration-dependent manner.

Separation and culture of adipose-derived stem cells from adipose tissue

Human adipose-derived stem cells were obtained by liposuction of a known method from the subcutaneous fat (Kim et al. J Dermatol Sci 53:96-102, 2009). Specifically, the human adipose tissue supplied from the hospital was washed twice with PBS (a commonly used buffer), and then 0.075% collagenase was added thereto, followed by incubation for 50 minutes in a 37°C incubator. Next, a medium in which PBS and α-MEM medium were mixed in half was added to the culture medium, and then centrifuged at 1200 rpm, the supernatant was removed, and only the material settled into pellets was used. PBS was added to the separated pellets, mixed thoroughly, filtered with a mesh of 100 μm Nile, centrifuged, and only pellets were obtained and mixed and cultured in the medium. The obtained adipose stem cells were cultured at 37° C., 5% CO ₂ in α-MEM medium (Hyclone, Thermo Scientific) containing 10% FBS (GIBCO, Invitrogen), 1% penicillin/streptomycin (GIBCO), 7 Cells of ~9 passages were used. Flow cytometry confirmed the positive expression of CD44, CD73, CD90, CD105, HLA-1 and PODXL and the negative expression characteristics of CD34 and CD45 (Kim et al. Cell Death Dis 4:e588, 2013; Kim et al., Cell Biol Int 38:32-40, 2014), the ability to differentiate into adipocytes, osteoblasts and chondrocytes was confirmed (Song et al. Stem Cells Dev 17:451-461, 2008).

Confirmation of the effect of improving the proliferation capacity of fat stem cells by epiregulin treatment

The adipose stem cells obtained in Example 1 were observed under a microscope, and the cells were floated with trypsin and centrifuged. The supernatant was discarded, leaving only the cells in the lower layer. The cells were well released by adding 1 X PBS and stained with trypan blue. The number of cells was calculated using the formula to confirm the degree of cell proliferation.

As a result, as shown in Figure 5, it was confirmed that the proliferation of fat stem cells significantly increased compared to the control group in all cases cultured by treatment with Epiregulin.

Confirmation of the effect of improving the migration ability of adipose stem cells by epiregulin treatment

In order to confirm the improvement of the migration capacity of the adipose stem cells according to the epiregulin treatment, a mobility analysis test using scratch analysis and transwell was performed.

Specifically, for scratch analysis, the same number of cells were incubated until they were completely filled in a 6-well dish, and then scratches were generated using a suitable tool, and Fng-free medium and 5ng, 20ng concentration of Epiregulin were used. The medium was added, and observed for a certain period of time and photographed to analyze the results.

As a result, as shown in FIG. 6, it was confirmed that the number of cell migration increased after treatment of epiregulin with adipose stem cells.

On the other hand, in the transfer ability test using a transwell, the same number of cells were planted in a 10 mm plate, and the original medium was discarded when it was grown by 40-50% the next day. After that, the medium containing FBS and the composition medium to which 5 ng and 20 ng of epiregulin were added respectively are added again and cultured for 2-3 days. This is a step of changing the cells to cells with growth and mobility by giving a molecular mechanism by epiregulin. After that, the composition medium was discarded, and cultured again for 16 hours with a medium without FBS, and then the same number of cells were seeded in a transwell. At this time, the medium entering the transwell should always be free of FBS. In the lower chamber part, 700 µl of normal medium containing FBS was added, and after 24 hours of incubation, staining with a crystal dye was performed to analyze the number of cells that moved the transwell membrane.

As a result, as shown in FIG. 7, it was confirmed that after the epiregulin treatment, the ability of fat stem cells to migrate was improved depending on the concentration.

Confirmation of self-renewal effect of fat stem cells by epiregulin treatment

Seeds 1 x 10 ⁴ adipose stem cells, treated with epiregulin (5,20ng) after 24 hours, and stained with crystal violet reagent after 10 days. Epiregulin was totaled 3 times every 3 days for 10 days. It was mixed with a new medium and replaced. Thereafter, a photograph was taken and the number of colonies having 50 or more cells was counted and shown in FIG. 8.

As a result, as shown in FIG. 8, after treatment with epiregulin, the self-renewal effect was confirmed from the sudden increase in the colony of fat stem cells compared to the control group.

Confirmation of the anti-aging effect of fat stem cells by epiregulin treatment

6 passage cultured 6 passage fat stem cells were inoculated at a concentration of 1 X 10 ⁵ . After 24 hours of incubation, Epiregulin was treated with 5 and 20 ng, respectively, and after 4 days of incubation, the passage was increased again, and Epiregulin was continuously treated to 12 passages. After 12 passages, cells were stained and analyzed using an X-Gal kit (Sigma-Aldrich, MO, USA).

As a result, as shown in FIG. 9, it was confirmed that aging of the fat stem cells cultured by treating epiregulin was suppressed.

Confirmation of the hair growth effect of adipose stem cells pre-treated with epiregulin in experimental animals

After removing the hair on the back of a 6-week-old male C3H rat using a pet hair depilator and a hair removal cream, 3 x 10 ⁴ fat stem cells treated with 20 ng of epiregulin for 2 days were injected subcutaneously into the rat. After 12 to 16 days, we looked at the degree of hair regeneration by pushing the newly grown hair on the rat back and weighing it.

As a result, as shown in FIG. 10, it was found that hair growth induction was accelerated in rats injected subcutaneously with adipose stem cells pretreated with epiregulin.

<110> Industry-Academic Cooperation Foundation, Yonsei University <120> Composition for preventing hair loss or promoting hair growth comprising epireguline or epiregulin-derived peptide as an active ingredient <130> 1066820 <150> KR 10-2018-0158063 <151> 2018-12-10 <160> 2 <170> KoPatentIn 3.0 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> epiregulin-derived peptide <400> 1 Gly Gln Cys Ile Tyr Leu Ser Gly Asp Arg Cys 1 5 10 <210> 2 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> epiregulin-derived peptide <400> 2 Gly Gln Cys Ile Tyr Leu Asn Asn Asn Asn Asn 1 5 10

Claims (8)

Epiregulin (Epiregulin; EPR) or a pharmaceutical composition for preventing hair loss or hair growth comprising a fragment thereof.
According to claim 1, wherein the epiregulin fragment is a pharmaceutical composition for preventing hair loss or promoting hair growth, comprising the amino acid sequence of SEQ ID NO: 1.
Epiregulin (Epiregulin; EPR) or a quasi-drug composition for preventing hair loss or hair growth comprising a fragment thereof.
Epiregulin (Epiregulin; EPR) or a cosmetic composition for promoting hair loss comprising a fragment thereof.
A pharmaceutical composition for preventing hair loss or promoting hair growth, which contains Epiregulin-treated fat stem cells or a culture medium thereof as an active ingredient.
The pharmaceutical composition for preventing hair loss or promoting hair growth according to claim 5, further comprising a pharmaceutically acceptable carrier.
[6] The pharmaceutical composition for preventing hair loss or promoting hair growth according to claim 5, wherein the composition is used for cell transplantation.
A cosmetic composition for preventing hair loss or promoting hair growth, which contains fat stem cells cultured by treating Epiregulin or a culture medium thereof as an active ingredient.

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