WO2015160187A1

WO2015160187A1 - COMPOSITION FOR PREVENTING HAIR LOSS, GROWING HAIR, STRENGTHENING HAIR ROOTS, PROMOTING HAIR GROWTH OR INHIBITING GSK-3β PATHWAY COMPRISING HUMAN PLACENTA EXTRACT

Info

Publication number: WO2015160187A1
Application number: PCT/KR2015/003786
Authority: WO
Inventors: Beom Joon Kim; Tae-Rin KWON; Chang Taek Oh; Hae Jung Han; Hye Min Park
Original assignee: JAPAN BIO PRODUCTS Co Ltd; Green Cross Corp Korea; GCJBP Corp
Current assignee: JAPAN BIO PRODUCTS Co Ltd; GC Biopharma Corp; GC Wellbeing Corp
Priority date: 2014-04-15
Filing date: 2015-04-15
Publication date: 2015-10-22
Anticipated expiration: 2016-10-15
Also published as: KR20150118856A

Abstract

The present invention relates to a composition for inhibiting a GSK-3β pathway, which includes a human placenta extract (HPE). The composition according to one exemplary embodiment of the present invention can induce inhibition of a GSK-3β pathway and increase expression of hair growth factors, β- catenin and Wnt3a, by activating a phosphatidylinositide 3-kinase (PI3-K)/protein kinase B (AKT) pathway. Also, the composition according to one exemplary embodiment of the present invention can be useful for patients with hair-loss and treating alopecia by prolonging anagen of hair follicles and activating development of the hair follicles.

Description

COMPOSITION FOR PREVENTING HAIR LOSS, GROWING HAIR, STRENGTHENING HAIR ROOTS, PROMOTING HAIR GROWTH OR INHIBITING GSK-3p PATHWAY COMPRISING HUMAN PLACENTA

EXTRACT

FIELD OF THE INVENTION

The present invention relates to a composition for preventing hair loss, growing hair, strengthening hair roots, promoting hair growth, or inhibiting a GSK-3 pathway, which comprises a human placenta extract as an active ingredient.

BACKGROUND OF THE INVENTION

Hair plays several roles in protecting scalps from external physical stimuli such as sunlight, regulating the body temperature, etc. The hair growth cycle is mainly divided into 3 phases: anagen, catagen, and telogen (Stenn KS, Paus R. Physiol. Rev., vol. 81, (2001), no. 1, pp. 449-494). Dysregulation of the hair growth cycle may induce hair loss or alopecia.

Hair loss is a disease that may accompany physical, psychological, or social problems. Such hair loss may occur in both men and women due to various factors such as aging, a hormone imbalance, stress, nutritional deficiency, etc. In recent years, drugs approved as therapeutic agents for treating male androgenetic alopecia include finasteride for oral administration which is effective for hair growth, and minoxidil (MXD) for topical administration. However, these drugs have disadvantages in that they have an unpredictable effect, and thus show limited and transient efficacy (Rossi A, et al., Recent Pat. Inflamm. Allergy Drug Discov., vol. 6, (2012), no. 2, pp. 130- 136). Also, minoxidil is known to be effective for alleviating hair loss in frontal and vertex areas of the head, and finasteride is known to be effective for alleviating hair loss in a vertex area of the head, but not to be effective for alleviating male pattern baldness, that is, M-shaped hair loss. Further, there are no suitable therapeutic agents for female hair loss. Therefore, development of various alternative therapeutic agents is required.

Glycogen synthase kinase-3 (GSK-3) functions to phosphorylate and inactivate glycogen synthases associated with production of glycogen, and adjusts glucose (sugar) metabolism. Also, GSK-3 is known to regulate many cellular processes through signaling pathways which are important for cell proliferation, stem cell regeneration, apoptosis, and cell development. Accordingly, dysregulation of GSK-3 is known to cause many diseases, including degenerative neuronal diseases (Alzheimer's disease, Parkinson's disease, etc.), strokes, depressive psychosis, type II diabetes mellitus and inflammations.

Particularly, GSK-3 is associated with hair growth by regulating the signal transduction of Wnt/p-catenin to play an important role in morphogenesis and differentiation of hair follicles (Kishimoto J, et al, Genes Dev., vol. 15, (2000), no. 14, pp. 1181-1185; Millar SE. J Invest Dermatol, vol. 118, (2002), no. 2, pp. 215-216). Wnt signals are required for early maturation of hair follicles, and thus GSK-3 structurally regulates a Wnt pathway by inhibiting β-catenin (Andl T, et al, Dev Cell, vol. 2, (2002), no. 5, pp. 643-653). Also, β-catenin may serve as an effector for a keratonin synthesis pathway, and thus stabilization of β-catenin may result in an increase in hair growth.

A Wnt protein is a ligand that may attach (bind) to other proteins referred to as receptors, and free Wnt molecules bind to Wnt receptors in the cell membrane to activate survival/proliferation pathways. Wnt is known to activate β-catenin.

Meanwhile, a human placenta extract (HPE) includes various growth factors, cytokines, and other physiologically active substances, and has been widely used for alleviation of fatigue, antioxidant, etc. (Lee KK, et al., Evid Based Complement Alternat. Med., vol. 2012, (2012) p.130875). In studies using an animal model, HPE is also known to induce improvement of liver function by regenerating the liver and influence wound healing by producing TGF-β and VEGF. However, in spite of the great interest in HPE, the functions of HPE are not fully studied, and no studies on hair growth have been conducted so far.

SUMMARY OF THE INVENTION

Accordingly, it is an object of the present invention to provide a composition for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth.

It is another object of the present invention to provide a composition for inhibiting a GSK-3 P pathway.

In accordance with one aspect of the present invention, there is provided a composition for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth, comprising a human placenta extract (HPE) and minoxidil as active ingredients.

In accordance with another aspect of the present invention, there is provided a composition for inhibiting a glycogen synthase kinase-3 P (GSK-3 P) pathway, comprising HPE and minoxidil as active ingredients.

In accordance with a further aspect of the present invention, there is provided a composition for inhibiting a GSK-3P pathway, comprising HPE as an active ingredient.

In accordance with a still further aspect of the present invention, there is provided a kit for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth, which comprises:

a composition for injections comprising HPE as an active ingredient, and

a composition for skin external application comprising minoxidil as an active ingredient,

whrerein the compositions are administered together.

The composition according to one exemplary embodiment of the present invention can inhibit a GSK-3P pathway and increase expression of β- catenin and Wnt3a, by activating a phosphatidylinositide 3 -kinase (PI 3- kinase)/protein kinase B (AKT) pathway. Also, the composition according to one exemplary embodiment of the present invention can be useful for patients with hair-loss and treating alopecia by prolonging anagen of hair follicles and activating development of the hair follicles.

BRIEF DESCRIPTION OF THE DRAWINGS

The above and other objects and features of the present invention will become apparent from the following description of the invention, when taken in conjunction with the accompanying drawings, which respectively show:

FIGS. 1A and IB: the cell viability in human epidermal keratinocytes (HaCaT) and human dermal papilla cells (hDPCs), respectively, treated with a human placenta extract (HPE) and/or minoxidil (MXD).

FIGS. 2 and 3: visual changes in hair regrowth state and scores of dorsal hair area ratios, respectively, depending on the treatment with HPE and/or MXD in the C57BL/6J mouse model.

FIGS. 4 A and 4B: images of H&E-stained skin tissues and hair anagen/telogen ratios (A/T ratios), respectively, after the treatment with HPE and/or MXD in the C57BL/6J mouse model.

FIGS. 5 A and 5B: the thicknesses of skin and the results obtained by measuring the number of hair follicles in transverse sections, respectively, depending on the treatment with HPE and/or MXD in the C57BL/6J mouse model.

FIGS. 6A and 6B: images of H&E-stained skin tissues, and the results obtained by measuring the number of hair follicles in horizontal sections, respectively, depending on the treatment with HPE and/or MXD in the C57BL/6J mouse model.

FIGS. 7A and 7B: the results obtained by determining expression levels of β-catenin and Wnt3a depending on the treatment with HPE and/or MXD in the C57BL/6J mouse model using an immunohistochemistry assay and an immunofluorescence assay, respectively.

FIG. 8: images of AP-stained skin tissues after the treatment with HPE and/or MXD in hDPCs.

FIGS. 9 and 10: the results obtained by analyzing the expression of a GSK-3 / -catenin signaling factor using a western blotting assay and an immunostaining assay, respectively, after the treatment with HPE and/or MXD in hDPCs.

DETAILED DESCRIPTION OF THE INVENTION The present invention provides a composition for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth, which comprises a human placenta extract (HPE) and minoxidil as active ingredients.

In the present invention, HPE used as one of the active ingredients exhibits an excellent cell proliferation effect (Example 1).

HPE is characterized in that it inhibits a glycogen synthase kinase-3p

(GSK-3 ) pathway. Specifically, the pharmaceutical composition according to one exemplary embodiment of the present invention may inhibit the GSK-3 pathway by activating a phosphatidylinositide 3 -kinase (PI 3-kinase)/protein kinase B (AKT) pathway (Example 6). HPE may have an effect of preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth by increasing expression levels of β-catenin and Wnt3a and activating development of the hair follicles through a Wnt/ -catenin pathway (Example 5). According to one exemplary embodiment of the present invention, it can be seen that HPE induces early anagen in hair follicles and prolongs anagen of hair follicles when HPE is subcutaneously or intradermally injected into a hair-loss region (Example 3), and also stimulates an increase in the number and size of hair follicles (Example 4).

In the present invention, HPE may be administered for 1 to 2 weeks at a dose corresponding to 0.5 to 3 vials (2 mL/vial), preferably 1 to 2 vials.

In the present invention, the term "hair loss" refers to a condition in which the hair is lost or the number of the hair, approximately 100,000 fibers of which are normally present in a human scalp, decreases due to certain factors. The term "prevention of hair loss" refers to a method of preventing a condition of the hair loss.

In the present invention, the term "hair growth" refers to a process where a population of hair germ cells differentiates to grow, which form a cell aggregate in which embryonic cells in the human follicular epidermis are compactly gathered. Specifically, a hair growth process refers to a process in which hair follicles are formed through a hair pre-germ stage, a hair peg stage, and a hair vulvous peg stage; sebaceous glands from which essential natural oily components are secreted are formed when the formed hair follicles develop into a mature state; arrector pili muscles attached to follicular wall and papillae playing a role in supplying nutrients and oxygen to the hair follicles are formed; and hair matrix cells in a region in contact with the papillae receive the nutrients from the papillae and start to differentiate, thereby generating new fibers of hair. The generation of hair is a process in which hair matrix cells differentiate and grow into hair by receiving the nutrients from the papillae.

In the present invention, the term "strengthening hair roots" refers to an increase in the size of dermal papilla cells, an increase in the depth thereof in the scalp, and an increase in depth and thickness of hair, etc. Specifically, a hair root means a living form of hair which is included in hair follicles under the epidermis of the head and has a structure including a hair region, a hair follicle, a papilla, a hair bulb, and a sebaceous gland by formation of a cross- linked cystine between a first region where biosynthesis and tissue formation occur and a second region. A lower end of the hair root is swelled in the form of an onion, the form of which is referred to as a hair bulb. A hair follicle surrounds the hair root in a cylindrical shape by connecting the epidermis and dermis. A sebaceous gland is open toward a direction in which the hair root forms an obtuse angle with a surface of the skin, and a hair muscle composed of smooth muscle next to the sebaceous gland serves to erect a fiber of hair. Papillae are confined in an inner part of the hair bulb surrounded by the dermis. Epithelial cells in the epidermis grow around the papillae, and are gradually keratinized to form fibers of hair. When the hair root starts to grow, the thickness of the skin spanning from a panniculus camosus to an epidermal granular layer of a subcutaneous fat layer increases. Therefore, strengthening hair roots means an increase in the size of dermal papilla cells, an increase in the depth thereof in the scalp, an increase in depth and thickness of hair, etc.

In the present invention, the term "hair growth" refers to a state in which hair grows on the body, that is, generally grows on the head, and the term "promoting hair growth" refers to inducing an increase in the number of hair by stimulating hair growth. Also, the present invention provides a composition for inhibiting a

GSK-3P pathway, which comprises HPE and minoxidil as active ingredients.

The composition for inhibiting a GSK-3 pathway may inhibit the GSK-3 pathway and increase expression levels of β-catenin and Wnt3a by activating a PI 3 -kinase/ AKT pathway.

Wnt/p-catenin signaling promotes the development of new hair follicles and initiates hair morphogenesis (Clevers H. Cell, vol. 127, (2006) no. 3, pp. 469-480; Tsai SY, et al, Dev Biol, vol. 15, (2014) no. 385, pp. 179-188). Wnt cascade signals play a key role in activation of bulge stem cells to perform a hair formation process. Such signals are produced by β-catenin and Lefl (Lowry WE, et al, Genes Dev., vol. 19, (2005) no. 13, pp. 1596-1611). Therefore, the composition for inhibiting a GSK-3 pathway according to one exemplary embodiment of the present invention may promote hair growth.

According to one exemplary embodiment of the present invention, the results of an immunohistochemical examination and immunofluorescence analysis demonstrate that HPE markedly increases expression levels of β- catenin and Wnt3a (Example 5). This is associated with the fact that HPE activates certain functions in anagen of the hair growth cycle, that is, that HPE activates the development of hair follicles by a Wnt/p-catenin pathway. As a result, the hair follicles may be stimulated to enter anagen, and it produces an effect that anagen of the hair follicles may be prolonged.

Also, HPE stimulates an early embryonic development process and plays an important role in the development of the hair follicles. In conclusion, HPE serves to improve hair-promoting activity through mechanisms acting on the Wnt/p-catenin pathway, thereby improving hair growth in the C57BL/6J mouse model.

The composition according to one exemplary embodiment of the present invention may be topically administered to a hair-loss region or a region in which hair growth is required. Specifically, the composition according to one exemplary embodiment of the present invention may be administered using any method of selectively delivering an effective dose of the composition to a region of the skin in which the simulation of hair growth is required. For example, the administration may be achieved by directly topically administering the composition according to one exemplary embodiment of the present invention to a treatment site (for example, rubbing skin of the treatment site with the composition of the present invention in the form of a lotion or gel, or administering the composition of the present invention to the treatment site in the form of a liquid by spraying), or formulating the composition of the present invention into an injectable preparation and injecting the injectable preparation into a treatment site including the scalp (for example, subcutaneously or intradermally injecting the injectable preparation).

According to one exemplary embodiment of the present invention, the administration may be performed using a concomitant administration method of directly topically administering a composition comprising minoxidil to a treatment site and injecting a composition comprising HPE to the treatment site.

Therefore, the present invention provides a pharmaceutical formulation comprising the composition.

The pharmaceutical formulation according to one exemplary embodiment of the present invention may be in the form of an injection or a skin external application, for example, in at least one form selected from the group consisting of an injection, a cream, a gel, a patch, a spray, an ointment, a plaster, a lotion, a microneedle, a roller, and a combination thereof.

According to one exemplary embodiment of the present invention, the composition of the present invention may be formulated into the form of an injection which is subcutaneously injected into a treatment site. In this case, the composition of the present invention may further include an excipient suitable for injection. Preferably, the composition including HPE may be formulated into the form of an injection. Examples of the suitable excipient may include saline, bacteriostatic saline, sterile water, etc., but is not limited thereto. Such an injection formulation may be easily prepared according to methods known in the art.

Also, the composition of the present invention may be formulated into the form of an external preparation for use on the skin which is suitable for being directly administered to a treatment site. In this case, the composition of the present invention may further include a physiologically available carrier or diluent which is suitable for being topically administered to the skin. Preferably, the composition including minoxidil may be formulated into the form of a skin external preparation. Examples of the physiologically available carrier or diluent may include water, physiological saline, a cream, a lotion, various types of gels, and short-chain alcohols and glycols (i.e., ethyl alcohol and propylene glycol), but is not limited thereto.

The preparation according to one exemplary embodiment of the present invention may further include at least one component selected from the group consisting of a sunscreen agent, a skin conditioning agent, a skin protecting agent, a softening agent, a moisturizing agent, a hair conditioning agent, and a mixture thereof when the preparation is in the form of an external preparation for use on the skin.

The sunscreen agent that may be used in the present invention may include at least one component selected from the group consisting of benzophenone-3 (oxybenzone), benzophenone-4(suHsobenzone), benzophenone-8(deoxybenzone), butyl methoxydibenzoylmefhane (avobenzone), DEA-methoxycinnamate(diethanolamine methoxycinnamate), ethyl dihydroxypropyl PABA(ethyl 4-[bis(hydroxypropyl)]aminobenzoate), ethylhexyl dimethyl PABA (padimate O), ethylhexyl methoxycinnamate(octyl methoxycinnamate), ethylhexyl salicylate(octyl salicylate), homosalate, menthyl anthranilate (meradimate), octocrylene, PABA(arninobenzoic acid), phenylbenzimidazole sulfonic acid (ensulizole), TEA-salicylate(trolamine salicylate), titanium dioxide, and zinc oxide, but is not limited thereto.

The skin conditioning agent that may be used in the present invention may include at least one component selected from the group consisting of acetyl cysteine, N-acetyl dihydrosphingosine, an acrylate/behenyl acrylate/dimethicone acrylate copolymer, adenosine, adenosine cyclic phosphate, adenosine phosphate, adenosine triphosphate, alanine, albumen, an algae extract, allantoin and derivatives thereof, an aloe barbadensis extract, aluminum PCA, an amyloglucosidase, arbutin, arginine, azulene, bromelain, buttermilk powder, butylene glycol, caffeine, calcium gluconate, capsaicin, carbocysteine, carnosine, β-carotene, casein, an catalase, cephalin, a ceramide, Chamomilla recutita(Matricaria) flower extract, cholecalciferol, cholesteryl ester, coco-betaine, coenzyme A, modified corn starch, crystallin, cycloethoxymethicone, cysteine DNA, cytochrome C, darutoside, dextran sulfate, a dimethicone copolyol, dimethylsilanol hyaluronate, DNA, elastin, an elastin amino acid, an epidermal growth factor, ergocalciferol, ergosterol, ethylhexyl PCA, fibronectin, folic acid, gelatin, gliadine, β-glucan, glucose, glycine, glycogen, a glycolipid, a glycoprotein, glycoseaminoglycan, glycosphingolipid, horseradish peroxidase, a hydrated protein, a hydrolyzed protein, Jojoba seed oil, keratin, a keratin amino acid, and kinetin, but is not limited thereto.

The skin protecting agent that may be used in the present invention may include at least one component selected from the group consisting of an algae extract, allantoic aluminum hydroxide, aluminum sulfate, betaine, a Cammellia sinensis leaf extract, a cerebroside, dimethicone, glucuronolactone, glycerine, kaolin, lanolin, a malt extract, mineral oil, Vaseline, potassium gluconate, and talc, but is not limited thereto.

The softening agent that may be used in the present invention may include at least one component selected from the group consisting of acetyl arginine, acetylated lanolin, an algae extract, apricot kernel oil PEG- 6 ester, avocado oil PEG- 11 ester, bis-PEG-4 dimethicone, butoxyethyl stearate, CIS- C36 acid glycol ester, C12-C13 alkyl lactate, caprylyl glycol, cetyl ester, cetyl laurate, coconut oil PEG- 10 ester, di-C12-C13 alkyl tartrate, diethyl sebacate, dihydrocholesteryl butyrate, dimethiconol, dimyristyl tartrate, disteareth-5 lauroyl glutamate, ethyl avocadate, ethylhexyl myristate, glyceryl isostearate, glyceryl oleate, hexyldecyl stearate, and hexyl isostearate, but is not limited thereto. The moisturizing agent that may be used in the present invention may include at least one component selected from the group consisting of acetyl arginine, an algae extract, an aloe barbadensis leaf extract, betaine, 2,3- butanediol, chitosan lauroyl glycinate, diglycereth-7 malate, diglycerine, diglycol guanidine succinate, erythritol, fructose, glucose, glycerine, honey, a hydrated wheat protein/PEG-20 acetate copolymer, hydroxypropyltrimonium hyaluronate, inositol, lactitol, maltitol, maltose, mannitol, mannose, methoxy PEG, myristamidobutyl guanidine acetate, polyglyceryl sorbitol, potassium PCA, propyleneglycol, sodium PCA, sorbitol, sucrose, and urea, but the present invention is not limited thereto.

The hair conditioning agent that may be used in the present invention may include at least one component selected from the group consisting of acetylated lanolin, amodimethicone, behenamidopropyl ethyldimonium ethosulfate, behentrimonium chloride, Butyrospermum parkii (shea butter) oil, caprylic acid/capric acid glyceride, ceteareth-20, cetylpyridinum chloride, cocamidopropyl betaine, cocamidopropyl hydroxysultaine, cocodimonium hydroxypropyl hydrolyzed keratin, dicetyldimonium chloride, dimethyl lauramine isostearate, glyceryl stearate SE, guar hydroxypropyltrimonium chloride, hydrolyzed glycosaminoglycan, hydrolyzed keratin, hydroxypropyl guar hydroxypropyltrimonium chloride, isostearyl glyceryl pentaerythrityl ether, laurdimonium hydroxypropyl hydrolyzed wheat protein, linoleamidopropyl dimethylamine dimer dilinoleate, linoleamidopropyl ethyldimonium ethosulfate, linoleamidopropyl PG-dimonium chloride phosphate, methylchloroisothiazolinone, panthenyl hydroxypropyl steardimonium chloride, PEG-40 sorbitan laurate, PET-3 sorbitan stearate, PEG-6 sorbitan stearate, PPG-5-ceteth-20, propylene glycol dicaprylate/dicaprate, an silk amino acid, steapyrium chloride, and stearyl ethylhexyldimonium methosulfate, but is not limited thereto. Therefore, the present invention provides a kit for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth, which comprises a composition for injections including HPE as an active ingredient, and a composition for a skin external application comprising minoxidil as an active ingredient, wherein the compositions are administered together.

The composition according to one exemplary embodiment of the present invention comprising HPE and minoxidil as active ingredients may enhance the efficiency of minoxidil as a hair growth promoter.

According to one exemplary embodiment of the present invention, the composition increases the cell viability in a dose-dependent manner in a HPE plus MXD-treated group, compared to a HPE only- treated group or a MXD only-treated group (see Example 1, and FIGS. 1A and IB). Further, the composition shows a superior activity of converting the hair follicles from early telogen to anagen (see Example 2, and FIGS. 2 and 3), and has a higher hair anagen/telogen ratio (A/T ratio) and an activity of significantly increasing the number of the hair follicles (see Examples 3 and 4, and FIGS. 4A to 6B). Also, it can be seen that the composition of the present invention also has a superior hair follicle-promoting activity by significantly increasing expression levels of β-catenin and Wnt3a (see Example 5, and FIGS. 7 A and 7B).

In the present invention, the term "effective dose" refers to a quantity of the composition which stimulates hair growth associated with hair loss.

Meanwhile, the present invention provides a composition for inhibiting a GSK-3P pathway, which comprises HPE as an active ingredient. The composition may inhibit the GSK-3P pathway by activating a PI 3-kinase/AKT pathway, and thus may increase expression of β-catenin and Wnt3a. Specific descriptions of the HPE and composition for inhibiting a GSK-3P pathway are as described above.

Hereinafter, the present invention will be described in further detail with reference to the following examples. However, it should be understood that the following examples are intended to illustrate the present invention only, and are not intended to limit the scope of the present invention.

Statistical analysis

All the data were expressed as standard errors of samples, and the statistical comparison between treated groups and untreated groups was performed using a one-way ANOVA analysis. Thereafter, the direct comparison among specific groups was performed using post-hoc Tukey. The results are expressed as the mean standard errors of values obtained through at least three independent experiments, and P values of p<0.05: *, p<0.01 : **, and pO.001 : *** were considered to be statistically significant.

Example 1: Analysis of cell proliferation in human dermal papilla cell line and keratinocyte cell line

Isolation and culture of human dermal papilla cells

Human dermal papilla cells (hereinafter referred to as "hDPCs") were purchased from CefoBio (Seoul, Korea) as primary cells, and cultured in Dulbecco's modified Eagle's medium (DMEM; Invitrogen-Gibco-BRL, Grand Island, NY) supplemented with 5% fetal bovine serum (FBS; Invitrogen-Gibco- BRL) and 1% penicillin in a humidified environment. Such cultured hDPCs were re-cultured three or four passages, and then used in this experiment.

Cell proliferation assay

The hDPCs thus obtained were treated with a human placenta extract

(HPE) and/or minoxidil (MXD), and a proliferation level of hDPCs was measured using a CCK-8 assay (Dojindo Molecular Technologies, Inc., Rockville, MD, USA). Specifically, the hDPCs thus obtained were plated on a 96-well plate at a density of 1.5>< 10⁴ cells/well, treated with different concentrations of HPE (0.1%, 0.5%, 1%, 5%, 10%, and 20%) (v/v%) only, and a combination of HPE (5%, 10%, and 20%) (v/v%) and MXD (0.5 μΜ), and continuously incubated for 96 hours. Subsequently, a CCK-8 solution (10 μΐ) was added to the cells in 1 ml of DMEM and incubated at 37°C for 2 hours. The cells were measured for absorbance at 450 nm using a SpectraMax 340 microplate reader (Molecular Devices, LLC, Sunnyvale, CA, USA). When it is assumed that the absorbance of the control (untreated group) was 100%, the cell viability was measured as a ratio with respect to the absorbance of the control. The results are shown in FIG. IB.

Also, the cell viability was measured for an immortalized human keratinocyte cell line (HaCaT) (ATCC, Manassas, VA) in the same manner as described above. The results are shown in FIG. 1A. The results shown in FIGS. 1A and IB are expressed as a fold change in standard deviation with respect to the control (*P < 0.05, **P < 0.01, compared with the control; #P < 0.05, ##P < 0.01, and ###P < 0.001, compared to those of 0.5 μΜ MXD).

As shown in FIG. IB, when the hDPCs were treated with 20% HPE for 96 hours, the cell viability increased by approximately 44% or more. In particular, when the hDPCs were treated with 20% HPE plus 0.5 μΜ MXD, the cell viability was shown to be 142%, a value of which increased by approximately 42%.

When the HaCaT cells were treated with HPE, cell proliferation was promoted, resulting in an increase in the cell viability. When the HaCaT cells were treated with HPE plus MXD, the cell viability significantly increased in a dose-dependent manner (FIG. 1A). Interestingly, it was revealed that, when the cells were treated with 10% HPE plus 0.5 μΜ MXD (130%), the cell viability more significantly increased, compared to when the cells were treated with 10% HPE only (119%). Therefore, it seemed that HPE had a synergistic effect on the proliferation of hDPCs when the cells were treated with HPE plus MXD.

Example 2: In vivo test of hair growth activity of HPE To evaluate an effect of HPE activities on hair growth in vivo, a total of fifty-six (56) female C57BL/6J mice (6 weeks old) were purchased from Japan SLC (Shizuoka, Japan), and randomly divided into seven (7) groups (8 mice/group). The seven groups include an untreated group (Normal); a group (Vehicle) in which ethanol was applied to the skin and saline was then subcutaneously injected into; a group (2% MXD) in which 2% MXD was applied to the skin and saline was then subcutaneously injected into; a group (2% HPE) in which ethanol was applied to the skin and 2% HPE was then subcutaneously injected into; a group (20% HPE) in which ethanol was applied to the skin and 20% HPE was then subcutaneously injected into; a group (100% HPE) in which ethanol was applied to the skin and 100% HPE was then subcutaneously injected into; and a group (2% MXD plus 100% HPE) in which 2% MXD was applied to the skin and 100% HPE was then subcutaneously injected into (hereinafter, referred to as "HPE plus MXD-treated group").

A human placenta hydrolysate of human placenta (Laennec) was purchased from GCJBP Corporation (Yongin, Korea), and used in this experiment. The backs of the C57BL/6 mice whose hair growth cycle was in telogen were shaved using an electric clipper to induce hair growth, as disclosed in the article [Muller-Rover S, et al., J Invest. Dermatol, vol. 1 17, no. 1, pp. 3-15].

From two days after the depilation, four sites on the dorsal skin of each mouse were selected, and 200 μΐ of ethanol or 2% MXD was applied to the skin every third day for 21 days according to the conditions for respective experimental groups. Thereafter, 100 μΐ of saline or HPE (2%, 20%, or 100%) was subcutaneously injected into the same sites. In this case, the control was treated with a vehicle only. All the experiments lasted for 21 days, and the mice were then killed. All the procedures involving the animal experiments were conducted according to the guidelines of the Institutional Animal Care and Use Committee of Chung Ang University in Korea (IRB Number: 13-0002).

Visual change in hair regrowth state

After the depilation, dorsal portions (back skins) of the mice in each group were observed every other day, and photographed at days 0, 8, 15, and 21 using a camera (Canon 3000D). The results are shown in FIG. 2.

As shown in FIG. 2, it was revealed that the skin color of the center regions in the HPE plus MXD-treated group grew dark more rapidly than the same area in the 2% MXD-treated group, indicating that HPE had an effect of promoting hair growth in the HPE plus MXD-treated group more rapidly than in the other groups.

Also, it could be seen that black hair grew over the dorsal portions of all the mice at day 21 , and the hair growth cycle entered mature anagen. From such results, it could be seen that HPE had an influence on the hair growth cycle of the C57BL6/J mice, and showed a superior effect when the mice were treated with HPE plus MXD.

Scores of dorsal hair growth area ratio

To observe a hair growth state after the test started, the dorsal portions of the mice in each group were photographed at days 6, 9, 12, 15, 18, and 21 after the depilation using a digital camera. Evaluations were performed by quantifying the total hair growth rates and the number of hair follicles on digital images photographed using Image-Pro Plus software Version 7.0 (Media

Cybernetics, Inc., Silver Spring, MD, USA). The criteria for evaluation are listed in the following Table 1, and the evaluation was performed based on the hair growth scores. The result are shown in FIG. 3 (n = 8/mice; *p < 0.05, **p < 0.01, and ***P < 0.001, compared to those of the control).

[Table 1]

As shown in FIG. 3, the results obtained at day 21 showed that the hair growth score significantly increased in the HPE plus MXD-treated group (4.88 ± 0.13), compared to that of the 2% MXD-treated group (4.63 ± 0.18).

Also, the weights of newly grown hair in all the test groups were measured, and compared to those of the control (Normal). As a result, it was revealed that newly grown hair had the highest weight in the HPE plus MXD- treated group. From the above-described results, it was confirmed that HPE induced hair growth by promoting the conversion of hair follicles of the mice from early telogen to anagen, and the HPE plus MXD-treated group had a significantly higher hair growth-promoting activity than the 2% MXD-treated group.

Example 3: Quantitative histo morphometry To evaluate an effect of HPE on formation of hair follicles, a hair follicle state and a hair growth level were observed through typical histological examination.

Biological tissues from the dorsal skins of the mice were fixed with 4% paraformaldehyde on day 21 after the depilation, and embedded in paraffin. Thereafter, the tissue was microtomed into transverse and longitudinal segments having a thickness of 5 μιη, and the segments were then transferred onto slide glasses. The skin segments were stained with hematoxylin-eosin (H&E), and observed under an optical microscope. In this case, the individual hair follicles were stagnated in specific hair cycle stages (telogen or anagen I to VI), and classified (Chase HB. Physiol Rev, vol. 34, no. I, pp. 113-126). As shown in FIG. 4A, it could be seen that inner root sheaths in the untreated group were not sufficiently developed, and the hair roots were partially weakly developed. On the other hand, it could be seen that the hair follicles and inner root sheaths were sufficiently developed and hair growth generally increased in the HPE-treated groups.

Also, the number of the hair follicles were counted at the stage of anagen, catagen and telogen of hair, and measured for anagen/telogen (A/T) ratios. The results are shown in FIG. 4B. As a result, it could be seen that the 20% and 100% HPE-treated groups, and the HPE plus MXD-treated group had an increased A/T ratio, compared to the 2% MXD-treated group. The hair cycle stage of the HPE plus MXD-treated group corresponded to anagen VI, but the vehicle group stayed in anagen III. In conclusion, it was confirmed that the A/T ratio (1.53 ± 0.15) of the HPE plus MXD-treated group was higher than that of the 2% MXD-treated group (1.16 ± 0.16).

Meanwhile, the thickness of the skin was defined as a distance from an epidermal granular layer to an upper section of the panniculus camosus of a subcutaneous fat layer, and the thickness of hair was measured as a total length of hair grown on the subcutaneous fat layer during anagen. As the length of hair increased from the hair follicles, the thickness of hair was changed accordingly. The thickness of the skin significantly increased in the HPE plus MXD-treated group (499.25 ± 26.12 μπι), compared to that in the 2% MXD- treated group (443.12 ± 34.66 μιη) (FIG. 5A).

Also, the number of the hair follicles in the transverse skin segment increased in the HPE-treated group, the HPE plus MXD-treated group, and the 2% MXD-treated group, compared to that in the normal group (FIG. 5B)_; Based on the results, it was revealed that the depths of the hair follicles and anagen stage of the hair growth cycle markedly increased in the HPE-treated group, compared to that in the vehicle group. Therefore, it could be seen that HPE promoted hair growth, and effectively prolonged the mature anagen stage. Example 4: Effects of HPE on development of the hair follicles

To measure the number and size of the hair follicles whose hair growth was induced, the skin tissues of the mice were obtained on day 21 after the depilation, fixed with 4% paraformaldehyde, and gradually and horizontally sectioned using a microtome in the direction of the upper and lower dermis. Thereafter, the segments were stained with H&E. The results are shown in FIG. 6A. As shown in FIG. 6A, it was revealed that such segments had the hair follicles spanning from the isthmus to the bulbar portion, thereby enabling the differentiation of vellus, catagen, and telogen hair.

Next, the hair growth area was quantified using Image-Pro Plus software. The results are shown in FIG. 6B. As a result, the number of the hair follicles significantly increased in the HPE plus MXD-treated group (136.00 ± 7.73), compared to that in the 2% MXD-treated group (112.63 ± 6.87). These results showed that HPE stimulated an early embryonic development process, and played an important role in development of the hair follicles.

Example 5: Measurement of expression levels of hair growth- related factor, β-catenin and Wnt3a

To evaluate a mechanism involved in the induction of anagen in the

HPE-treated group and the HPE plus MXD-treated group, the tissue segments of the dorsal skins of the mice were subjected to immunohistochemistry and immunofluorescence analyses using antibodies specific to β-catenin and Wnt3a.

Specifically, the tissue segments of the dorsal skins of the mice obtained in each experimental group were fixed with 4% paraformaldehyde (PFA), embedded in paraffin, and microtomed into longitudinal segments having a thickness of 5 μιη. Thereafter, the longitudinal segments were transferred to probe-on-plus slide glasses (Thermo Fisher Scientific Inc., Pittsburg, PA, USA). Some sections were stained for immunohistochemical markers using monoclonal antibodies against β-catenin (1 :500, 610154, BD Transduction, Lexington, KY, USA) and Wnt3a (1 :500, ab28472, Abeam, Cambridge, MA, USA).

Immunohistochemical staining was performed with a high-temperature antigen unmasking technique. The segments were heated in an unmasking solution (citrate buffer, pH 6.0), washed, and then incubated with anti-mouse primary monoclonal antibodies at room temperature for an hour. Thereafter, the segments were incubated with secondary antibodies (Envision Detection kit K5007, DAKO, Glostrup, Denmark). The reaction product was developed with a diaminobenzidine solution as a chromogen. After the chromogen reaction, the segments were washed, and counterstained with hematoxylin. The stained segments were rinsed, dehydrated, and then covered with Permount (Thermo Fisher Scientific Inc, Fair Lawn, J, USA). A histological change of the hair follicles was evaluated under an optical microscope.

The segments in which anagen of hair was induced by the HPE treatment were stained positive for β-catenin and Wnt3a. The immunohistochemical analysis shown in FIG. 7A showed that the expression levels of β-catenin and Wnt3a were increased by the HPE treatment. Also, the immunofluorescence analysis shown in FIG. 7B showed that β-catenin and Wnt3a were strongly deposited around the hair follicles.

Such immunostaining studies showed that the expression levels of β- catenin and Wnt3a markedly increased in the HPE plus MXD-treated group, compared to that in the HPE-treated group. In particular, putative outer root sheath cells were stained against anti^-catenin antibodies, while potential basophilic hair matrix cells were often stained. Also, Wnt3a staining was also observed in transitional cells and putative hair-like structures surrounding the cells, which could potentially be hair follicle placode formation (Andl T, et al., Dev. Cell, vol. 2, (2002) no. 5, pp. 643-653). These results showed that it was associated with certain functions in anagen, that is, the fact that HPE activated the development of hair follicles through the Wnt/ -catenin pathway (Ridanpaa M, Fodde R, Kielman M. Mech Dev, vol. 109, no. 2, pp. 173-181). Taken together, our findings suggested that HPE played a possible role in hair promoting activity associated with hair morphogenesis. Example 6: GSK-3p pathway inhibitory activity of HPE

AP staining

To determine an effect of HPE on activation of alkaline phosphatase (AP), AP staining was performed using a commercially available AP Live staining kit (Life Technologies) according to the manufacturer's guideline. Specifically, hDPCs were respectively treated with 10% HPE only, 0.5 μΜ MXD only, and 10% HPE plus 0.5 μΜ MXD. After 72 hours, the hDPCs were washed twice with a fresh medium, and then treated with 1 *AP Live staining solution. The hDPCs were reacted at 37 °C for 30 minutes, and washed three or four times again. Images of the AP -positive cells were recorded using an optical microscope with a standard fluorescein isothiocyanate (FITC) filter, and bright field images of the AP-positive cells were also obtained at the same time. The results are shown in FIG. 8.

As shown in FIG. 8, it could be observed that the number of the cells was increased when the cells were treated with 10% HPE plus 0.5 μΜ MXD, compared to when the cells were treated with 10% HPE only, and 0.5 μΜ MXD only, and that the number of the AP positive (AP+) cells increased on the whole. Western blotting analysis

To analyze expression of a GSK-3p/ -catenin signaling factor in the hDPCs using a western blotting assay, first of all, the hDPCs were dissolved in a RIPA buffer (including 50 mM Tris, pH 8.0, 150 mM NaCl, 1% NP-40, 0.1% SDS, 0.5% deoxycholic acid, and 1 mM phenylmethylsulfonyl fluoride (PMSF)). Thereafter, the hDPCs were mixed with a protease inhibitor (Roche Diagnostics, Indianapolis, IN) for 15 minutes in ice, and centrifuged at 20,000g for 10 minutes at 4 °C. The resulting supernatant was re-centrifuged for 10 minutes, and the concentration of proteins was measured using a BCA protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The proteins (20 μg) were assayed by polyacrylamide gel electrophoresis (SDS-PAGE), and adsorbed onto a polyvinylidene fluoride (PVDF) membrane (Millipore Corp, Bedford, MA). The membrane was reacted at room temperature for 2 hours in PBS supplemented with 0.1% Tween-20 and 5% non-fat milk, probed with primary antibodies diluted with PBS/0.2% BSA for an hour, and then washed. Subsequently, the membrane was reacted with secondary HRP-bound anti- rabbit antibodies (Vector Laboratories, Burlingame, CA, USA) and HRP-bound anti-mouse antibodies (Vector Laboratories, Burlingame, CA, USA), and the bands were visualized using an ECL advance kit (Amersham). The bands were standardized using a loading control (Actin) band. The western blotting analysis results are shown in FIG. 9.

The western blotting analysis was performed, as follows. The hDPCs were seeded in a 100 mm culture dish at a density of 5>< 10⁴ cells/ml. After one day, the medium was removed, and a serum-free medium was added.

Then, the hDPCs were incubated again for 24 hours. Thereafter, the hDPCs were respectively treated with 10% HPE only, 0.5 μΜ MXD only, and 10%

HPE plus 0.5 μΜ MXD, and then incubated for 12 hours. After the hDPCs had undergone such a procedure, the proteins were isolated from the cells, and the expression of GSK-3p/p-catenin was determined. As a result, it was revealed that the expression level of GSK-3P (pGSK3 ) in which a serine residue at a 9^th position was phosphorylated remarkably increased when the cells were treated with HPE plus MXD, as shown in FIG. 9. These results demonstrated that HPE had a synergistic effect in inactivating GSK-3P when the cells were treated with HPE plus MXD, compared to when the cells were treated with HPE only, and MXD only. The similar results were obtained after 24 hours and it was found that the expression of β-catenin increased due to inactivation of GSK-3 . Based on these results, it could be seen that the hair growth effect was shown to be synergistic when the cells were treated with HPE plus MXD.

Immunocvtochemistry

To analyze the expression of the GSK-3p/p-catenin signaling factor in the hDPCs using an immunostaining assay, the hDPCs were seeded in a chamber slide glass. After 24 hours, a procedure of removing the medium used, and exchanging the medium with a serum-free medium was performed.

Thereafter, the hDPCs were treated with 5% HPE only, 10% HPE only, 0.5 μΜ

MXD only, and 10% HPE plus 0.5 μΜ MXD, respectively, and then incubated for 48 hours. The obtained cells whose incubation was completed on the chamber slide glass were fixed with 4% paraformaldehyde at room temperature

(RT) for 15 minutes, and then washed with PBS/0.2% BSA. Subsequently, the cells were passed for 15 minutes in PBS/0.2% BSA/0.1% Triton X-100, and then reacted at room temperature for an hour in PBS/0.2% BSA. The cells thus obtained were reacted with β-catenin primary antibodies diluted with

PBS/0.2% BSA at 4°C for 12 hours, and then washed. Then, the cells were reacted at room temperature for an hour using FITC (Novus, Saint Louis, MO,

USA), to which secondary antibodies (Invitrogen) were bound in PBS/0.2%

BSA. The cells were counterstained with 10 μg/ml DAPI. The chamber slide glass was analyzed under an FVlOi-W (2nd generation) confocal laser scanning microscopy (Olympus). The results are shown in FIG. 10.

As shown in FIG. 10, it could be seen that the treatment of HPE plus MXD to the cells increased the dephosphorylation of β-catenin increased to have a synergistic effect in hair growth, compared to when the cells were treated with 10% HPE only.

Claims

WHAT IS CLAIMED IS:

1. A composition for inhibiting a glycogen synthase kinase-3 (GSK-3P) pathway, comprising a human placenta extract (HPE) as an active ingredient.

2. The composition of claim 1, wherein the composition inhibits the GSK-3 pathway by activating a phosphatidylinositide 3 -kinase (PI 3-K)/protein kinase B (AKT) pathway.

3. The composition of claim 1, wherein the composition increases expression of β-catenin and Wnt3a.

4. A composition for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth, comprising HPE and minoxidil as active ingredients.

5. A composition for inhibiting a GSK-3 pathway, comprising HPE and minoxidil as active ingredients.

6. The composition of claim 5, wherein the composition inhibits the GSK-3P pathway by activating a PI 3-K/AKT pathway.

7. The composition of claim 5, wherein the composition increases expression of β-catenin and Wnt3a.

8. A pharmaceutical formulation comprising the composition of any one of claims 1 to 7.

9. The pharmaceutical formulation of claim 8, which is in a form selected from the group consisting of an injection, a cream, a gel, a patch, a spray, an ointment, a plaster, a lotion, a microneedle, a roller, and a combination thereof.

10. A kit for preventing hair loss, growing hair, strengthening hair roots, or promoting hair growth, which comprises:

a composition for an injection comprising HPE as an active ingredient, and

wherein the compositions are administered together.