KR20140131193A - Method and Composition for regulating hair growth targeting placental growth factor - Google Patents

Abstract

Disclosed are a pharmaceutical composition and a cosmetic composition comprising placental growth factors for increasing a growth period of hair, and a method for screening a compound for increasing a growth period of hair. According to the present invention, the composition and the method increase a growth period during periods of hair by regulating placental growth factors, thereby being used for alleviating, relieving, and treating hair loss.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a hair growth regulating method and a composition for targeting a placental growth factor,

The present invention relates to compositions and methods for preventing or ameliorating alopecia.

Currently, developed countries are rapidly entering an aging society, and alopecia is one of the typical aging phenomena, occurring in more than 50% of males in their 50s or older. Alopecia are the most common problem of elderly people, and the treatment method is urgently required along with the improvement of quality of life. Alopecia refers to the disappearance of hair on the head or body. Currently, there are many ways to treat alopecia, but there are many limitations. For example, minoxidil treats hair loss through vasodilatation, inhibition of male hormones, but such a drug is not a fundamental treatment based on the hair growth cycle, and drug treatment can prevent hair loss However, if treatment is discontinued, hair loss may occur more rapidly, and hair transplantation is difficult to treat due to the limitation of the number of hair that can be implanted.

U.S. Patent No. 6,773,881 discloses a method for controlling hair growth, and discloses a method for screening hair growth promoting materials through VEGF regulation.

U.S. Patent No. 6844326 discloses a method of treating alopecia, which discloses a method of treating hair loss using p21, a cyclin-dependent carcinostatic agent.

Therefore, it is necessary to develop a therapeutic agent for promoting hair growth based on understanding of hair growth cycle and hair loss mechanism, or for a substance which keeps hair follicle in hair growth period long in hair cycle.

Throughout this specification, the patent literature is referred to and its citations are indicated. The disclosures of the cited patent documents are hereby incorporated by reference herein in their entirety to better illustrate the state of the art to which the invention pertains and the teachings of the present invention.

The present invention is to provide a method or composition capable of promoting hair growth based on understanding of hair growth cycle and hair loss mechanism or maintaining long hair follicle growth period in hair cycle.

 In one aspect, the present invention provides a pharmaceutical composition for increasing hair growth comprising a placental growth factor.

As the placental growth factor used herein, various types and forms may be used as long as they exhibit the activity according to the present invention, for example, an electric field, an active fragment, a placental growth factor receptor binding site, or a placental growth factor peptide mimetic. In another preferred embodiment according to the present application, the placental growth factor is one or more of the placental growth factors 1 to 4, in particular the placental growth factor 1, 2 or 4, more particularly the placental growth factor 2 or 4.

The hair according to the present invention is present in various parts of the body, including but not limited to scalp, face, hair of the body part such as legs, chest, armpit, etc., except for the scalp and face .

The composition for increasing the growth of plants according to the present invention can be used as a variety of diseases, conditions, or hair growth requiring growth of hair follicles, and can be used, for example, for alleviation, improvement, prevention or treatment of alopecia. But are not limited to, for example, alopecia areata, alopecia areata, alopecia areata, resting alopecia, growing alopecia, chemotherapy-induced alopecia, drug-induced alopecia, hair growth.

In another aspect, the invention also provides a method of screening for a substance that modulates hair growth. In one embodiment according to the present application, the method comprises the steps of: providing a test substance; Providing a hair follicle or hair follicle-derived cell; Treating the hair follicle or hair follicle-derived cells with the test substance; And evaluating whether or not the test substance modulates PLGF of the hair follicle or hair follicle-derived cell, wherein the step of evaluating is to select a candidate substance as a candidate substance that up-regulates the expression or activity of PLGF as compared with the control .

The material selected by the screening method according to the present invention is effective on hair in various parts of the body such as hair on the scalp, face, scalp and face, such as legs, chest, and armpit.

In the method according to the present invention, the upregulation of expression of the PLGF may include modulation in the transcription step, translation step, or transcription and translation step, or increasing the activity of PLGF.

In another aspect, the present invention also includes a cosmetic composition for increasing hair growth, comprising a composition according to the present invention or a placenta growth factor or an analogue thereof, and the cosmetic composition may be manufactured into various formulations depending on the mode of use thereof.

The composition and method for increasing hair growth through regulation of PLGF according to the present invention can promote hair growth based on the understanding of hair growth cycle and hair loss mechanism or can maintain long hair follicle during hair growth period, , Can be effectively used for alleviation or treatment.

FIG. 1A shows the effect of PLGF on hair growth in the cultivation of xylanated follicular organs. The length of the hair follicle was measured by using a stereo microscope on days 4 and 8 of culturing (a) and the graph (b). Results were expressed as mean ± SE (Standard Error). * P is <0.05 compared to the control. The numbers on each line of the graph are the percentages relative to the control.
Fig. 1B shows the induction of proliferation of mosquito-keratinocytes by PLGF, which is the result of immunofluorescence analysis of keratinocytes in hair follicles derived from three subjects. Ki67 positive is red, and DAPI stained nuclei are blue. For quantitative analysis, the number of Ki67 positive cells was counted and normalized to the number of DAPI stained cells. Results were expressed as mean ± SE. * P is <0.05 compared to the control. The scale bar is 100 mu m and the magnification of the microscope is x200.
FIG. 2A shows the increase of mRNA expression of various genes related to hDPC proliferation and hair growth induction by PLGF. After hDPC was treated with 50 ng / mL PLGF, 30 minutes (a) and 24 hours (b) RNA was extracted and quantitatively analyzed by real-time PCR (polymerase chain reaction).
FIG. 2B shows the increase in mRNA expression of various genes related to hDPC proliferation and hair growth induction by PLGF. After hDPC was treated with 50 ng / mL PLGF for 24 hours, total RNA was extracted and analyzed by reverse transcription PCR .
FIG. 3A shows the increase in hDPC survival time by PINGS, which is the result of Western blot analysis for 24 hours (a) and 72 hours (b) after treating hDPC with the indicated amount of PLGF.
Figure 3b shows the increase in survival time of hDPC by PINGS. HDPC was incubated with lμM LY294002 (Akt-Protein Kinase B-inhibitor, a) or PD98059 (ERK-Extracellular signal-regulated kinase-inhibitor, b) After 24 hours of treatment, total RNA was extracted and analyzed by real-time quantitative PCR. * P is <0.05 compared to the control.
FIG. 4A shows the effect of increasing PLGF-induced hair follicle growth and period of growth period. FIG. 4A shows the results of tissue analysis (a) and hair follicle length (b) and hair follicle length of the skin samples obtained on day 5 after treatment with PLGF or minoxidil, And skin thickness (c). The numbers displayed in each bar graph are percentages of the percent change compared to the control, the results being expressed as mean ± SE, and * P being <0.01 compared to the control. The scale bar is 100 mu m and the magnification of the microscope is x200.
FIG. 4B shows the effect of increasing PLGF-induced hair follicle growth and period of growth, showing tissue analysis (a) and hair follicle diameter (b) and hair follicle size (D) by H & E staining of skin samples obtained on day 21 after treatment with control, PLGF or minoxidil, Cell death (c). The diameter of the hair follicle was measured as the thickest part called the "Auber" line, and the cell death was measured by the TUNEL method using keratinocytes of the hair follicle, and the TUNEL positive cells were shown as green fluorescence. The results are expressed as mean ± SE, and * P is <0.01 compared to the control. The scale bar is 100 μm and the microscope magnification is x 200.
FIG. 5 shows the effect of PLGF on the growth of hair growth during hair growth. FIG. 5 (a) shows the effect of hair loss on C57BL / 6 mice treated with control, PLGF or minoxidil b) and hair cycle score (c) and skin thickness (d). The hair cycle score was measured using the assigned values (telogen = 1, anagen I to VI = 2 to 7) and the skin thickness was the thickness from the epidermis to the subcutaneous fat. The results showed as mean ± SE, * P is the control group compared to <0.05, * * P is <0.01 compared to control group. The scale bar is 100 mu m and the magnification of the microscope is x200.

The present invention is based on the finding that placental growth factor reduction is associated with hair loss, and it has been found that the treatment of placental growth factor promotes hair growth, increases the duration of the growing period and hair thickness in the hair cycle.

Thus, in one aspect, this disclosure relates to methods of modulating hair growth and hair thickness targeting placental growth factors. The present methods are directed to controlling the gene expression of placental growth factors and / or the production / concentration increase and / or activity of placental growth factor proteins.

As used herein, modulation refers to an increase in the transcription of a gene into mRNA, or the translation of the transcribed mRNA into a protein, and / or the modulation of the activity of a biological function of the protein, in one embodiment, upregulation. Further, the adjustment includes both the adjustment in the in-vivo state, the adjustment in the in-vivo state, and the adjustment in the x-ray state.

Hair follicles have very complex anatomical structures and differentiation systems. The hair follicles are divided into the infundibulum, the isthmus and the inferior segment based on the area where the ducts of the arrector pili and the sebaceous gland are attached. There is a dermal papilla (DP) and a hair matrix part in the hair roots located at the bottom part. The inferior segment consists of hair shaft, inner root sheath (IRS), outer root sheath (ORS), and dermal sheath from the back. During the growing season, hair matrix cells actively divide and produce hairs. At this time, the dermal papilla protruding toward the mosquito beneath the hair follicle is very important for the hair follicle formation, and if the dermal papilla is removed, the hair follicle loses its ability to produce the hair follicle.

Growing hair has a growth cycle and consists largely of the anagen phase, the catagen phase and the telogen phase. Growth period is the period of hair growth, regressive period is the period of slow growth of hair, and rest period is when hair growth is stopped. In the human scalp, 85-90% of the hair is in the growth phase, less than 1% is in the retrograde phase, and 14% is in the rest phase. When the regenerating period begins, the cell division and melanogenesis in the mosquitoes are stopped, the hair bulb is contracted, and the surface envelope is separated from the dermal papilla. As the hair moves upwards, the lower part of the hairy paw is withdrawn, leaving the strand of epithelial cells of the epithelium surrounded by the fibrous membrane, the growth of the inner root of the root, and the lower part of the hairy stem It is surrounded by the formed keratin, which is called club hair. This periodic change in hair growth leads to rapid changes and reconstruction of epidermal and dermal components of the hair follicles.

Regulation of hair growth and hair thickness through modulation of PLGF according to the present invention increases the duration of the hair follicle growth cycle, especially the growth period, in particular. Accordingly, in another aspect, the present invention provides a pharmaceutical composition for increasing hair growth comprising placental growth factors.

The increase is, for example, an increase of at least about 5%, especially at least about 10%, more particularly at least about 20%, of the growth period compared to the case where PLGF is not regulated, but is not limited thereto. It would be possible to set an appropriate range. Or from an increase in the number of hairs.

The compositions according to the present invention are effective in alleviating, improving, preventing, or treating hair loss. The term " treatment ", &quot; alleviation &quot; or &quot; improvement &quot;, as used herein, refers to any act that improves or alleviates symptoms of a related disorder by administration of the composition. Those skilled in the art will be able to ascertain the precise criteria of the disease by referring to the data provided by the Korean Medical Association, and to judge the degree of improvement, improvement, and treatment of the disease.

As used herein, the term "subject" is an animal, particularly a mammal, more particularly a human being, who is the subject of prevention, treatment,

The term "prophylactic, ameliorative or therapeutically effective amount ", as used herein, is an amount sufficient to exhibit the desired effect, and may vary depending on the degree of hair loss and / or other drugs used concomitantly, The amount of a pharmaceutical composition comprising an active ingredient that elicits a biological or medicinal response in a subject that can be determined by the researcher, physician or other clinician, including promoting hair growth, alopecia relief, inhibition of progression, .

The placental growth factor is a member of the VEGF (vascular endothelial growth factor) subfamily and is 42% homologous to VEGF and plays an important role in angiogenesis in development. PLGF is mostly expressed in the placental tropoblast during pregnancy, but it is also expressed in other tissues such as myeloid cells, smooth muscle cells, endothelial progenitor cells, and exists in the cell membrane, cytoplasm or secreted form. PLGF is expressed in three different isoforms of PLGF-1 (131 amino acids aa), 2 (152 aa), 3 (203 aa) and 4 by alternative splicing . Of these, PLGF-2 and 4 contain 21 amino acids with a very basic heparin binding capacity at the C-terminal side. In the case of mice, only one type of PLGF corresponding to PLGF-2 is present. PlGF related matters are described, for example, in Bate, Biochem Soc Trans. December 2011; 39 (6): 1576-1582.

The compositions herein may include a variety of natural or synthetic placental growth factors, or equivalents thereof, as long as they exhibit the effect according to the invention. The equivalents include variants, variants, analogs or derivatives having the activity of a placental growth factor, and may be natural or synthetic. For example, the PLGF of the full length includes not only its active fragment, its binding site to its receptor, or other active site, or a part of nucleic acid or amino acid, and includes a peptide mimetic as well as a recombinant protein . Peptide mimetics refers to a material that mimics a naturally occurring peptide in terms of structural and / or activity, and includes non-natural chemical structures and bonds that are not found in one or more non-peptides or peptides.

Various origins and forms of PLGF may be used herein, particularly those derived from mammals, particularly those derived from humans. Human placental growth factors are known, for example, the gene sequence is NM_002632 (PlGF-2) and the protein sequence is (NP_002623.2). PLGF 1 to 4 can also be used, and in one embodiment according to the present invention PLGF 1 or 2 or a mixture thereof, in particular PLGF-2, in particular human-derived PLGF-2, is used.

This may include various substances that increase the expression of placental growth factors at the mRNA and / or protein level and / or increase the activity of placental growth factors.

In another aspect, the invention provides a pharmaceutical composition for increasing hair growth comprising a placental growth factor. The composition according to the present invention can be used for accelerating hair growth through increase of hair growth period, alleviation, improvement, prevention or treatment of various alopecia, and the range to which the composition can be applied is not particularly limited as long as hair loss occurs, Can be used in the scalp, face or other body parts such as the armpit, chest and the like and can be administered by various routes of administration such as systemic or topical, especially topical, oral, transdermal, or injection, especially percutaneous. In addition, the composition according to the present invention promotes the growth of hair follicles by inducing an increase or activation of PLGF to increase the growth of hair follicles, and can be used as various diseases, conditions, or hair growth promoting agents required for increasing the growth of hair follicles. For example, But are not limited to, alopecia areata, alopecia areata, alopecia areata, growth alopecia, chemotherapy-induced alopecia, drug-induced alopecia, hair growth.

The therapeutic agent or pharmaceutical composition according to the present invention may be formulated in a suitable form together with a commonly used pharmaceutically acceptable carrier. "Pharmaceutically acceptable" refers to compositions which are physiologically tolerated and which, when administered to humans, do not normally cause allergic reactions such as gastrointestinal disorders, dizziness, or the like. The pharmaceutically acceptable carrier may be a mixture of saline, sterilized water, Ringer's solution, buffered saline, dextrose solution, maltodextrin solution, glycerol, ethanol, liposome and at least one of these components. , A buffer solution, a bacteriostatic agent, and the like may be added. In addition, it can be formulated into injection formulations, pills, capsules, granules or tablets such as aqueous solutions, suspensions, emulsions and the like by additionally adding diluents, dispersants, surfactants, binders and lubricants, Specific antibody or other ligand can be used in combination with the carrier. Can further be suitably formulated according to the respective disease or ingredient according to the appropriate method in the art or using the methods disclosed in Remington's Pharmaceutical Science (recent edition), Mack Publishing Company, Easton PA have.

The method of administering the composition of the present invention is not particularly limited thereto, and it is possible to apply the known method of administering the inhibitor, and the composition can be administered parenterally (for example, locally) or orally, Oral administration is preferable, and in the case of parenteral administration, it can be administered in the form of a patch applied or adhered to the skin, and other routes and means are not excluded. In the case of typical drugs, the dosage unit comprises, for example, from about 0.001 mg to about 100 mg, but does not exclude the following ranges and above. The daily dosage may be about 0.1 μg to 10 g, and may be administered once a day or divided into several times a day.

The dosage ranges vary widely depending on the patient's body weight, age, sex, health condition, diet, administration time, administration method, excretion rate, and severity, and known doses can be applied.

A therapeutically effective amount of a pharmaceutical composition according to the present application may be used in an amount of about 0.1 to 500 μg / Kg, especially about 10 to about 400 μg / Kg.

The dosage will vary depending on the conditions of the subject, such as age, body weight and diet, strength of the formulation, progression of the disease state, and manner and time of administration and the specific PLGF modulator. The optimal dosage to be administered can be readily determined by one of ordinary skill in the art.

The pharmacological properties of the present invention may further contain, in addition to the above-mentioned active ingredients, one or more active ingredients exhibiting the same or similar functions, or a compound that maintains / increases the solubility and / or absorbency of the active ingredients. In addition, the composition of the present invention can be used alone or in combination with methods of using surgery, drug treatment and biological response modifiers for preventing and / or improving hair loss.

In another aspect, the present invention discloses a cosmetic composition for increasing the hair growth period comprising the composition according to the present invention or a placenta growth factor as described above, and the content may vary depending on the specific use or formulation of the desired product, (W / v), from about 5 to 20% (w / v), or from about 100 μg to 500 μg / kg (w / v) , Especially about 300 μg.

The cosmetic composition of the present invention may contain, in addition to the above-mentioned active ingredients, conventional additives and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances.

The cosmetic compositions of the present invention may also be formulated into a wide variety of suitable formulations conventionally manufactured in the art, including, for example, solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, But are not limited to, cleansing, oil, spray, and the like. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, a hair tonic, a shampoo, a rinse, a cleansing cream, a cleansing foam, a cleansing water, a hair pack, a spray or a powder.

When the composition of the present invention is a paste, cream or gel, an animal oil, a vegetable oil, a wax, a paraffin, a starch, a tracer, a cellulose derivative, polyethylene glycol, silicon, bentonite, silica, talc or zinc oxide may be used as a carrier component .

When the composition of the present composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component. In particular, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the composition is a solution or an emulsion, a solvent, a solubilizing agent or an emulsifying agent is used as a carrier component, and examples thereof include water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, , 3-butyl glycol, glycerol aliphatic ester, polyethylene glycol or fatty acid esters of sorbitan can be used.

When the formulation of the present composition is a suspension, a carrier such as water, a liquid diluent such as ethanol or propylene glycol, a suspension such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, Cellulose, aluminum metahydroxide, bentonite, agar or tracant, etc. may be used.

When the formulation of the composition of the present invention is an interface-active agent-containing cleansing, the carrier component is selected from the group consisting of aliphatic alcohol sulfates, aliphatic alcohol ether sulfates, sulfosuccinic acid monoesters, isethionates, imidazolium derivatives, methyltaurates, sarcosinates, fatty acid amide ethers Alkylamido betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester. According to a preferred mode of administration, the compositions of the present invention may comprise at least one pharmaceutically acceptable excipient, especially a dermatologically acceptable excipient.

Further, in the composition for dermatological preparation of each of the formulations, components other than the above-mentioned essential components may be selected and mixed without difficulty by those skilled in the art depending on other compositions for dermal use or the purpose of use.

The compositions according to the invention may also comprise at least one adjuvant selected from auxiliaries known to the person skilled in the art, thickeners, preservatives, flavors, colorants, chemical or mineral salts filters, moisturizers, hot spring water and the like. Preservatives commonly used in cosmetics include, for example, molecules having antimicrobial activity, such as capryl derivatives (capryloylglycine and glyceryl caprylate), such as hexanediol and sodium levorinate, zinc and copper derivatives (Gluconate and PCA), phytosphingosine and derivatives thereof, benzoyl peroxide, pyrrothonol amine, zinc pyrocite and selenium sulfide, econazole, ketoconazole, or topical antibiotics such as erythromycin and clindamycin .

In another aspect, the invention also provides a method of screening for a substance that modulates hair growth. In one embodiment according to the present application, the method comprises the steps of: providing a test substance; Providing hair follicle cells; Treating the hair follicle cell with a test substance; And evaluating whether or not the test substance regulates PLGF of the hair follicular keratinocyte, wherein the evaluating step comprises the step of selecting as a candidate substance a substance which upregulates the expression or activity of PLGF as compared with the control group . &Lt; / RTI &gt;

As used herein, "test substance" means a substance that is expected to increase the expression or activity at the gene and / or protein level of PLGF, such as a low molecular weight compound, a high molecular weight compound, a mixture of compounds Or a cell or tissue culture), or biopharmaceuticals (e.g., proteins, antibodies, peptides, DNA, RNA, PNAs) or sugars and lipids.

The test materials can be obtained from libraries of synthetic or natural compounds, and methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the desired synthetic library method, &quot; 1-bead 1-compound &quot; library method, and by synthetic library methods using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc. Natl. Acad. Sci. U.S.A. 90, 6909, 1993; Erb et al. Proc. Natl. Acad. Sci. U.S.A. 91, 11422, 1994; Zuckermann et al., J. Med. Chem. 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew. Chem. Int. Ed. Engl. 33,2059,1994; Carell et al., Angew. Chem. Int. Ed. Engl. 33, 2061; Gallop et al., J. Med. Chem. 37, 1233, 1994, and the like.

For example, biologics can also be used for screening. Biologics refers to a cell or a biomolecule, which refers to a substance produced using biomolecules, proteins, nucleic acids, carbohydrates, lipids, or in vivo and in vitro cell systems. Biomolecules may be provided alone or in combination with other biomolecules or cells. Biomolecules include, for example, proteins or biological organic materials found in polynucleotides, peptides, antibodies, or other plasma.

The hair follicle or hair follicle derived cells used in the method of the present invention may be selected from the group consisting of a hair follicle organ itself, a hair follicle cell derived from a hair follicle, a keratinocyte, in particular a hair matrix keratinocyte and a cell line established from the cell, for example, an outer root sheath But is not limited thereto.

 When treated with the test substance, when the expression and / or activity of PLGF is increased as compared to the untreated control, the test substance is selected as a candidate substance to regulate hair growth. About 10%, about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 90% 100% or more, or more can be selected as candidates. These candidate substances may be developed as a hair growth regulator through clinical experiments or the like, or may be included as an ingredient in a cosmetic composition.

Also, when treated with the above test substance, the increase in the expression and / or activity of PLGF as compared to the untreated control group may be measured directly or indirectly.

Upregulation of the expression of PLGF includes, for example, modulation at the mRNA and / or cDNA and protein levels, such as in the transcriptional, translational, or transcriptional and translation stages, and the expression or activity of PLGF And can be measured by methods known in the art. For example, mRNA / cDNA can be measured by Northern blotting, reverse transcription PCR, quantitative PCR, and the protein can be measured through immunofluorescence analysis using a tissue, Western blotting, etc. The activity can be measured, for example, Whether or not to bind to a receptor and to determine the expression or activity of a known downstream protein by binding. For example, PLGF binds to VEGFR1 (Flt-1) or neurofilin 1 to activate VEGFR1-positive cells, followed by activation of Akt pathway related to survival, proliferation-related MAPK signaling pathway, protein kinase C pathway It is known.

It can also be measured using a variety of methods known in the art including, for example, hair follicle length, skin thickness, hair thickness, or other methods described herein.

Hereinafter, embodiments are provided to facilitate understanding of the present invention. However, the following examples are provided only for the purpose of easier understanding of the present invention, and the present invention is not limited to the following examples.

Example

Experimental Method

1. Follicular organ and dermal papilla cell culture:

Hair follicles were isolated from volunteer scalp tissue and cultured in Williams' E medium (Invitrogen, USA) at 37 ° C in a 5% CO ₂ incubator.

The dermal papilla cells were cultured in a DMEM medium (Invitrogen) containing 10% fetal bovine serum at 37 ° C in a 5% CO ₂ incubator. The dermal papilla cells regulate the matrix keratinocytes of the hair follicles, and matrix keratinocytes of the hair follicles are known to participate in hair follicle growth.

(10 ng / mL or 50 ng / mL) (R & D Systems, USA) or minoxidil (0.1% bovine serum albumin in PBS) to measure the effect of PLGF on the growth of hair follicle MXN, 2 mg / ml, positive control, Sigma-Aldrich, USA) for 8 days.

The length of the hair follicles was photographed on the 4th and 8th days after the addition of the substance using a stereo microscope (Olympus stereo microscope).

2. Analysis of mRNA expression : Total RNA was extracted from the dermal papilla cells cultured in the above manner after placenta growth factor (50 ng / mL) was treated for 30 minutes or 24 hours. RevertAid First Strand cDNA synthesis kit (Thermo Scientific, USA) CDNA was synthesized using the manufacturer's method, and mRNA expression of IGF-1, HGF, and VEGF genes related to hair cell proliferation and versican and alkaline phosphatase genes related to hair growth activity were examined using real-time quantitative PCR . Reverse transcription PCR was performed using the SYBR Green RT-PCR reagent kit according to the manufacturer's instructions. Quantitative real time PCR was performed using the SYBR PCR Master Mix according to the manufacturer's instructions in a quantitative PCR machine (ABI 7500, Applied Biosystems, USA). The PCR conditions were 95 ℃ for 10 min, 95 ℃ for 15 sec, and 50 ℃ (annealing and elongation) for 1 min. The primers used were as follows.

[Table 1]

3. Protein analysis : After treatment of placental growth factor (10 ng / mL or 50 ng / mL) for 3 hours, 24 hours, or 72 hours in the cultured dermal papilla cells, Expression of P-ERK, P-Akt, Bcl2 and Cyclin D1 protein was measured by Western blot using the following antibodies: anti-total ERK, anti-phosphorylated ERK, anti-total Akt, anti-phosphorylated Akt, , anti-cyclin D1 (Cell Signaling Technology, USA), anti-ß-actin (Santa Cruz Biotechnology, USA)

4. Animal test : 7-week-old female mice (C57BL / 6 mice, body weight 18-20 g) were purchased from Koatech (Korea). Mice were fed four to five cages per cage under general breeding conditions, and tap water and feed were freely consumed. In the first animal experiment, in order to investigate the hair growth promoting effect in C57BL / 6 mice in which a total of 32 hair cell cycles were in the resting period, hair on the back part of the resting mouse was pulled as described previously (Paus R et al., The American Journal of pathology 144: 719-341, 1994). In summary, the wax / rosin mixture is melted and then applied to the back, peeled off, and then removed to remove hair from the resting period, and the removed hair is synchronized to the hair follicle. From the day after hair removal, minocystil (2 mg / ml) was injected intradermally with mouse placenta growth factor (10 μg / mL) (R & D systems) or positive control twice a day.

After the injection of placental growth factor, the hair growth promoting effect was observed by measuring the length of the hair follicle by measuring the thickness of the skin by conducting histological examination at the 5th day. On day 21, histological examination was performed to determine the thickness of the hair bulb, and TdT-mediated dUTP nick end labeling (TUNEL) analysis described below was performed to determine the degree of apoptosis. Placental growth factors were used to determine whether the growth period of hair follicles was maintained for a long time.

In the second animal experiment, in order to examine whether placental growth factor induces hair follicle growth in 8-week-old C57BL / 6 mice in which 14 hair cell cycles were in the resting period, hair of the dorsal part of the resting mouse was shaved To remove hair. The area of the removed hair was maintained at the resting period for about 4 weeks. From the day after removing the hair, minoxidil (2 mg / ml) was injected intradermally with placenta growth factor (10 μg / mL) or positive control twice a day. All of the mice were examined for histologic staining by hematoxylin and eosin staining using an image analysis program (Image J, PowerShot S51S, Japan) The tissue morphology was observed by performing hematoxylin and eosin (H & E) staining, and the hair cycle score (Hair Cycle Score) , HCS) were measured as previously described (Maurer et al., The American Journal of pathology 150: 1433-41, 1997). In summary, hair cycles for randomly extracted hair follicles (telogen = 1, anagen I to VI = 2 to 7), and the score was given HCS per mouse group means the average value obtained by adding the hair points of the selected hair follicles per group divided by the number of selected follicles. To evaluate the hair follicles.

5. Immunofluorescent staining : To observe cell proliferation, cells were stained with Ki67 antibody (DAKO, Carpinteria, USA) as a cell proliferation marker and immunofluorescent staining was performed as described below.

In addition, TUNEL staining (In Situch death detection kit, fluorescein, Roche Diagnostics, Germany) was performed as previously described (Yoon SY et al., PloS one 6: e28474, 2011) . Nuclei were counter-stained with DAPI mounting kit (Vector Laboratories, USA). Images of TUNEL stained cells were obtained using the Leica Application Suite Advanced Fluorescence Software (LAS AF, Leica Microsystems, Heerbrugg, Switzerland). For quantitative analysis of cell death, the number of TUNEL positive cells in hair follicles was measured and 80 hair follicles per mouse group were observed.

Example 1 Increase in Follicle Length by PLGF Treatment

As described above, when human-derived hair follicles were cultured and treated with PLGF, the hair follicle length was significantly increased in the group treated with placenta growth factor as compared with the negative control group (see FIG. 1A). In addition, Ki67 antibody staining as a marker of cell proliferation markedly increased the proliferation of hair follicles in the group treated with PLGF as compared with the control group (see Fig. 1B).

This indicates that the placental growth factor has an effect of directly promoting hair growth even when the vascular system is not present.

Example 2 Expression of various genes related to hDPC proliferation and hair growth induction by PLGF

In order to observe the increase of mRNA expression of various genes related to the proliferation of hDPC (human dermal papilla cell) and induction of hair growth by PLGF, hair follicular cells cultured as described above were treated with 50 ng / mL PLGF, ) And 24 hours (b) total RNA was extracted and analyzed by quantitative real time PCR and reverse transcription PCR. As a result, mRNA expression of insulin growth factor 1 (IGF-1), human growth factor (HGF) and vascular endothelial growth factor (VEGF), which are genes involved in hair cell proliferation, Versican, extracellular matrix proteoglycan) and alkaline phosphatase mRNA expression was increased (see Figures 2a and 2b).

As shown in FIGS. 3A and 3B, PLGF treatment significantly increased the survival time of hDPC. After treatment of hDPC with the indicated amount of PLGF, placental growth factor (10 ng / mL or 50 ng / mL) was treated with dermal papilla cells for 24 hours (a) and 72 hours The protein expression of P-ERK, Cyclin D1, which promotes proliferation, was increased. Furthermore, hDPC was treated with 1 μM of LY294002 (Akt inhibitor, Invitrogen) or PD98059 (ERK inhibitor, Sigma-Aldrich) for 48 hours and 24 hours, respectively. Total RNA was extracted and analyzed by real-time quantitative PCR. VEGF Receptor 1 coding gene) or PLGF mRNA expression was significantly reduced.

This indicates that the placental growth factor inhibits apoptosis and induces cell proliferation.

Example 3 Enhancement of Follicular Growth and Growth Period by PlGF

In order to measure the effect of PLGF on hair follicle growth and growth period, hair was removed from the resting period of mice using wax and the removed hair was synchronized to the hair follicle growth period, . On the next day after hair removal, tissue analysis and hair follicle length and skin thickness were measured by H & E staining of skin samples obtained on days 5 and 21 after treatment with mouse PLGF or minoxidil, respectively. As a result, in the group injected with placenta growth factor 5 days after placenta growth factor injection, the hair follicle length was significantly increased compared with the control group, skin thickness was also increased, and skin thickness was increased in the growing period, The growth of hair is promoted in the group and it is induced to the growth period (see Fig. 4A). In addition, as a result of histological examination at 21 days, in the group injected with placenta growth factor, the thickness of hair bulb was increased and the number of positive cells of TUNEL indicating cell death was decreased compared with the control group. (See Fig. 4B).

Example 4: Promotion of hair growth by PLGF

In order to examine whether PLGF induces hair to the growth period, hair on the back skin of a resting mouse was removed using a razor. From the day following removal of hair, C57BL / 6 mice were treated with PLGF or minoxidil in the back control of C57BL / 6 mice and then analyzed by H & E staining for skin tissue on day 41 And hair cycle score and skin thickness were measured.

As a result, as shown in Fig. 5, in the group treated with PLGF, the growth period of the hair follicle was significantly induced and skin thickness became thicker than the negative control group. This indicates that placental growth factors induce hair follicles at the time of growth.

While the present invention has been described in connection with what is presently considered to be the preferred embodiments, it is to be understood that the invention is not limited to the disclosed embodiments, but, on the contrary, .

All technical terms used in the present invention are used in the sense that they are generally understood by those of ordinary skill in the relevant field of the present invention unless otherwise defined. The contents of all publications referred to herein are incorporated herein by reference.

Claims (10)

A pharmaceutical composition for increasing hair growth, comprising a placental growth factor.
The pharmaceutical composition according to claim 1, wherein the placental growth factor is at least one of placental growth factors 1 to 4 derived from a human.
The pharmaceutical composition according to claim 1, wherein the placenta growth factor is an electric field, an active fragment, a placental growth factor receptor binding site or a placental growth factor peptide mimetic.
The pharmaceutical composition according to claim 1, wherein the hair is hair of a body part such as a leg, a chest or an armpit, excluding the scalp, face, scalp and face.
The composition according to claim 1, wherein the composition for increasing growth is used for alleviation, improvement, prevention or treatment of hair growth stimulation or hair loss.
A method of screening a substance that regulates hair growth,
Providing a test substance;
Providing a hair follicle or hair follicle-derived cell;
Treating the hair follicle or hair follicle-derived cells with the test substance; And
And evaluating whether or not the test substance regulates PLGF of the hair follicle or hair follicle-derived cell, wherein the evaluating step comprises selecting a candidate substance as a substance that upregulates the expression or activity of PLGF as compared with the control group A method for screening hair growth regulators.
The method according to claim 6, wherein the hair follicle is a hair follicular organ, and the hair follicle-derived cell is a dermal papilla cell or keratinocyte.
The pharmaceutical composition according to claim 6, wherein the hair is hair of a body part such as a leg, a chest or an armpit, excluding scalp, face, scalp and face. 7. The method of claim 6, wherein the up-regulation of the expression of PLGF is regulated in a transcription step, a translation step, or a transcription and translation step.
A cosmetic composition for increasing hair growth, comprising the composition according to claim 1.

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