Composition for promoting hair growth containing guanidine derivative

Abstract

The present invention relates to a composition for improving hair and, more specifically, to a composition for promoting hair growth or preventing and treating alopecia, comprising a non-toxic guanidine derivative as an active ingredient. The composition has an excellent effect of promoting hair growth and hair follicle regeneration, and can be effectively applied to various fields of medicines, foods, feeds, cosmetic compositions and the like for promoting hair growth or preventing, treating and improving alopecia.

Description

[0001] This application is a divisional application of a patent application with application number 201980075425.X, application date November 14, 2019, and invention name “Composition containing guanidine derivatives for promoting hair growth”. Technical Field

[0002] The present invention relates to a composition for promoting hair growth, in particular to a pharmaceutical composition for promoting hair growth, comprising a guanidine derivative as an effective ingredient. Background Art

[0003] Hair repeats a three-stage cycle, namely the growth phase (anagen), the regression phase (catagen) connected to hair extinction, and the resting phase (telogen) when growth stops. If the number of hairs in the growth phase decreases and the ratio of the regression phase to the resting phase increases, the hair will fall out abnormally, leading to hair loss. As a method for treating hair loss, there are many attempts such as surgical methods such as hair transplantation or drug therapy. Although surgical methods are very effective, the disadvantages are that they are very expensive, the operation process is difficult, and the financial and mental burden on patients is very heavy. In addition, this is only a temporary prescription and cannot be a fundamental treatment method.

[0004] In addition, minoxidil, which is widely used in drug therapy and is described in US Patent No. 4596812, was originally used as a therapeutic agent for hypertension, but it has begun to be used as a hair growth promoter as reports of hair growth phenomena due to side effects have emerged. However, minoxidil has many side effects, such as poor effect, scalp loosening, erythema, contact dermatitis such as keratin, etc. (Journal of the Korea Medical Association, 56 (1), 45). Therefore, it is now necessary to develop a safe therapeutic agent that has excellent anti-hair loss and hair growth promoting effects and no side effects. Summary of the invention

[0005] [Technical issues]

[0006] The present inventors have established a composition for promoting hair growth or preventing, treating and ameliorating hair loss, which can promote hair growth and regeneration of hair follicles, by using a non-toxic guanidine derivative as an active ingredient, thereby completing the present invention.

[0007] [Technical solution]

[0008] One object of the present invention is to provide a pharmaceutical composition comprising a guanidine derivative or a pharmaceutically acceptable salt thereof as an effective ingredient for promoting hair growth or preventing or treating hair loss.

[0009] Another object of the present invention is to provide a cosmetic composition comprising a guanidine derivative or a cosmetically acceptable salt thereof as an active ingredient for promoting hair growth or preventing or improving hair loss.

[0010] Another object of the present invention is to provide a food composition comprising a guanidine derivative or a nutritionally acceptable salt thereof as an effective ingredient for promoting hair growth or preventing or improving hair loss.

[0011] Another object of the present invention is to provide a feed composition comprising a guanidine derivative or a nutritionally acceptable salt thereof as an effective ingredient for promoting hair growth or preventing or improving hair loss.

[0012] Another object of the present invention is to provide a method for promoting hair growth or preventing or treating hair loss, comprising administering a pharmaceutical composition containing a guanidine derivative or a pharmaceutically acceptable salt thereof as an active ingredient to a subject in need thereof.

[0013] Another object of the present invention is to provide a guanidine derivative or a pharmaceutically acceptable salt thereof or a composition containing the guanidine derivative as an active ingredient for promoting hair growth or preventing or treating hair loss.

[0014] Another object of the present invention is to provide a use of a guanidine derivative or a pharmaceutically acceptable salt thereof or a composition containing the guanidine derivative as an active ingredient for preparing a drug for promoting hair growth or preventing or treating hair loss.

[0015] [Beneficial Effects]

[0016] When the composition provided by the present invention is used, it provides a better effect of promoting hair growth and hair follicle regeneration than commercial minoxidil. In addition, compared with existing guanidine derivatives, the composition provided by the present invention has a better effect of promoting hair growth, and therefore can be applied to various fields such as medicines, foods, feeds and cosmetic compositions for promoting hair growth, preventing, treating and improving hair loss. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The chemical formula of the guanidine derivative is shown.

[0018] Figure 2 The graph shows the promoting effect of a series of guanidine derivative compounds on the reprogramming of mouse embryonic fibroblasts into iPSCs.

[0019] Figure 3 The graph shows the promoting effects of a series of guanidine derivative compounds on the reprogramming of human foreskin fibroblasts into iPSCs.

[0020] Figure 4 This is a graph showing the effects of a series of guanidine derivative compounds on promoting the maintenance of the stemness of human embryonic stem cells.

[0021] Figure 5a The graph shows the hair growth effect of IM compound treatment in mice confirmed with the naked eye.

[0022] Figure 5b It is a graph showing the hair growth effect of mice upon IM compound treatment.

[0023] Figure 6a The results of confirming the effect of IM compound treatment on the hair follicle cycle by H&E staining on the longitudinal section of mouse skin are shown.

[0024] Figure 6b Shown are changes in the hair follicle cycle upon IM compound treatment.

[0025] Figure 6c The results of confirming the effect of IM compound treatment on hair follicle cycle by H&E staining on mouse skin cross-sections are shown.

[0026] Figure 6d The effect of increasing the number of hair follicles upon IM compound treatment is shown.

[0027] Figure 7a Shown are the expression levels of K15, β-catenin, and Ki67 at day 7 of IM compound treatment.

[0028] Figure 7b Shown are the results of FACS analysis of K15 / β-catenin and Ki67 / Shh at day 7 of IM compound treatment.

[0029] Figure 7c Shown are the results of histochemical analysis of K15, β-catenin, and Shh at day 20 of IM compound treatment.

[0030] Figure 7d Shown are the results of FACS analysis of K15 / β-catenin and Ki67 / Shh at day 20 of IM compound treatment.

[0031] Figure 8a Shown are the results comparing histochemical analysis for K15, PDK, Ki67 and PDH at day 7 of IM compound and minoxidil treatment.

[0032] Figure 8b Shown are the results comparing histochemical analysis for K15, PDK, Ki67 and PDH at day 20 of IM compound and minoxidil treatment.

[0033] Figure 8c Shown are the results comparing FACS analysis for K15 / PDK and Ki67 / PDH at day 7 of IM compound and minoxidil treatment.

[0034] Figure 8d The results of comparing the expression levels of OXPHOS-related enzymes (NDUS 3 and ATP5B), glycolysis-related enzymes (HK2 and LDHA), and cell stemness-related genes (Wnt1a, Lef-1, Gli-1, and Versican) when treated with IM compound and minoxidil are shown.

[0035] Fig. 9 The graph shows the hair growth effect of IM compound treatment in mice confirmed with the naked eye.

[0036] Fig.10 The graph shows the hair growth effect of female mice upon IM compound treatment confirmed with the naked eye.

[0037] Fig.11a The graph shows the hair growth effect of male mice by IM compound treatment confirmed with the naked eye.

[0038] Fig.11b The graph shows the hair growth effect of male mice by IM compound treatment confirmed with the naked eye.

[0039] Fig.12a Shown are the results of histochemical analysis of K15, PDK, Ki67, and PDH at day 20 of IM compound treatment.

[0040] Figure 12b Shown are the results comparing FACS analysis for K15 / PDH and Ki67 / PDK at day 20 of IM compound and minoxidil treatment.

[0041] Fig.13a The graph shows the hair growth effects of the IM compound and metformin treatment in mice compared with the naked eye.

[0042] Fig.13b This is a graph showing a comparison of the hair growth effects of IM compound and metformin treatment on mice.

[0043] Fig.14a It is a graph showing the repair effect of auricle tissue based on IM compound treatment.

[0044] Fig.14b is a graph showing the repair effect of auricle tissue based on IM compound treatment. DETAILED DESCRIPTION

[0045] According to one aspect of the present invention, there is provided a pharmaceutical composition for promoting hair growth or preventing or treating hair loss, comprising a compound of the following Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an effective ingredient:

[0046] [Chemical formula 1]

[0047]

[0048] Wherein, in the above chemical formula 1,

[0049] A is a single bond or -C(NH)-NH-,

[0050] R1 is -(CH2) n- , where n is an integer from 1 to 4,

[0051] R2 is unsubstituted or substituted by halogen or one or more substituents selected from C1-C4 alkyl. 10 Aryl.

[0052] For example, the compound may be any one of the compounds of the following Chemical Formulae 2 to 4:

[0053] [Chemical formula 2]

[0054]

[0055] [Chemical formula 3]

[0056]

[0057] [Chemical formula 4]

[0058]

[0059] The compounds of Chemical Formulae 2 to 4 are hydrophobic cationic small molecules containing a guanidine or biguanide core, and their chemical names are (N-1-(2-methyl)phenethylbiguanide, N-1-(3,4-dichloro)phenethylbiguanide and 1-(3,4-dimethylphenethyl)guanidine, respectively. In the following examples, these compounds are named "IM176OUT05" (or "IM"), "IM176OUT04" and "IM177OUT02", respectively.

[0060] The compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof provided by the present invention can promote the reprogramming of animal somatic cells including humans, maintain cell stemness, and further promote the progress of the hair follicle cycle, accelerate the regeneration of hair follicles, and increase the number of hair follicles, thereby bringing about the effect of promoting hair growth, preventing or treating hair loss.

[0061] For example, it is known that the stem cells of cells can play an important role in the formation of hair growth and / or hair follicles, especially through the β-catenin signal transduction process, thereby conferring stem cell properties and maintaining this state, which is a factor regulating hair growth, hair regeneration and / or hair follicle formation (Nature Cell Biology, 2017, 19: 1017-1027; In vivo, 2019, 33: 1209-1220). In this regard, in a specific embodiment of the present invention, it has been confirmed that guanidine derivative compounds can promote the reprogramming of somatic cells and maintain the cell stemness of stem cells, that is, these compounds can promote hair growth, prevent or treat hair loss by regulating hair growth, hair regeneration and / or the formation of hair follicles. Furthermore, in order to confirm these more specifically, by applying these compounds to the cell level and animal models, it was confirmed that the expression of β-catenin in hair follicles was increased, the number of hair follicles was increased, and the effect of actually promoting hair growth and / or hair growth was confirmed.

[0062] The term "promoting hair growth" used in this specification refers to hair growth, and is used to express an increase in the speed and amount of hair growth. In addition, it means that the hair root function is strengthened, or the hair shedding and generation cycle is shortened, thereby increasing the number of hair growing in the hair follicles and thus increasing the number of hair follicles.

[0063] Compared with minoxidil, which is currently used as a hair growth promoter, the pharmaceutical composition can shorten the resting phase in the hair growth and shedding cycle, promote the entry into the growth phase, and has an excellent effect on promoting hair growth. In addition, the composition has no side effects, including skin problems.

[0064] In one embodiment of the present invention, the pharmaceutical composition may contain the compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof at a concentration of 1 to 5%, particularly at a concentration of 1 to 4% or 1 to 3%.

[0065] In one embodiment of the present invention, the pharmaceutical composition can promote cell reprogramming or maintain cell stemness. In addition, the pharmaceutical composition can promote hair follicle regeneration and promote hair growth. In one embodiment of the present invention, the composition increases the expression of one or more of the group consisting of β-catenin, Shh (sonic hedgehog), HK2 (hexokinase 2), LDHA (lactate dehydrogenase A), NDUS3 (NADH dehydrogenase iron-sulfur protein 3), ATP5B (ATP synthase subunit β), Wnt1a, Lef-1 (lymphoid enhancer binding factor 1), Gli-1 (GLI family zinc finger 1), versican, PDH (pyruvate dehydrogenase) and PDK (pyruvate dehydrogenase kinase).

[0066] Specifically, the pharmaceutical composition can increase the expression of β-catenin and Shh, glycolysis-related enzymes HK2 and LDHA, OXPHOS-related enzymes NDUS 3 and ATP5B, and cell stemness-related genes Wnt1a, Lef-1, Gli-1 and versican involved in hair follicle development, which promote hair follicle regeneration and development, and can increase the expression of enzyme PDK that promotes glycolysis conversion and enzyme PDH for mitochondrial OXPHOS reaction. Compared with minoxidil, which is currently used as a hair growth promoter, it can promote the expression of the above factors with better effects, thereby bringing excellent efficacy in promoting hair growth.

[0067] The pharmaceutical composition can promote the expression of genes related to hair follicle development and activate various signals for hair regeneration. In addition, by directly stimulating stem cell metabolism and promoting glycolysis, OXPHOS can be further promoted. Thus, the pharmaceutical composition can contribute to the active energy metabolism required for hair growth.

[0068] The pharmaceutical composition can be administered in a pharmaceutically effective amount, and the above-mentioned "pharmaceutically effective amount" refers to a dose sufficient to treat the disease at a reasonable benefit / risk ratio applicable to medical treatment. This can be determined based on factors such as the type of disease, severity, age, gender, drug activity, sensitivity to the drug, administration time, administration route and excretion rate, treatment time, concurrent drugs used, and other factors well known in the medical field.

[0069] The pharmaceutical composition can be used alone or in combination with other therapeutic agents, and can be administered sequentially or simultaneously with existing therapeutic agents. Considering the above factors, it is important to administer the dose that achieves the maximum effect at the lowest dose without side effects, which can be easily determined by those skilled in the art. For example, it can be increased or decreased according to the administration route, severity of the disease, gender, weight, age, etc. Therefore, the above-mentioned administration dose does not limit the scope of the present invention in any way.

[0070] The term "pharmaceutically acceptable salt" used in this specification refers to retaining the desired biological and / or physiological activity in the above-mentioned compounds and all salts with the least undesirable toxicological effect. It can be an addition salt formed using an organic acid or an inorganic acid, and the above-mentioned organic acid can, for example, include formic acid, acetic acid, propionic acid, lactic acid, butyric acid, isobutyric acid, trifluoroacetic acid, malic acid, maleic acid, malonic acid, fumaric acid, succinic acid, succinic acid monoamide, glutamic acid, tartaric acid, oxalic acid, citric acid, glycolic acid, glucuronic acid, vitamin C, benzoic acid, phthalic acid, salicylic acid, anthranilic acid, dichloroacetic acid, aminoglycolic acid, benzenesulfonic acid, p-toluenesulfonic acid and methanesulfonic acid salts. In addition, the above-mentioned inorganic acid can, for example, include hydrochloric acid, bromic acid, sulfonic acid, phosphoric acid, nitric acid, carbonic acid and boric acid salts, preferably hydrochloride or acetate form, more preferably hydrochloride form.

[0071] For example, the compositions provided according to the present invention may also include suitable carriers, excipients and diluents that are commonly used to make pharmaceutical compositions. The compositions are sterile or aseptic, and such solutions are sterile or aseptic, and when suitable for use in water, buffers, isotonic agents or animals or humans, may include other ingredients that do not cause allergic or other adverse reactions and are well known to those skilled in the art.

[0072] The term "pharmaceutically acceptable carrier" as used in this specification includes any and all solvents, dispersion media, coverings, antibacterial agents, antifungal agents and isotonic agents, etc. The use of the above media and preparations as pharmaceutically active substances is well known in the art. In addition to active ingredients and non-mixable conventional media or preparations, it is also considered to be used in therapeutic compositions. In addition, supplementary active ingredients can also be mixed into the composition.

[0073] The composition can be prepared in the form of liquid, emulsion, suspension or cream, and can be used for parenteral administration. The dosage of the composition can be used according to the conventional dosage for preventing and treating hair loss, and it is best to use it appropriately according to the patient's age, gender, condition, absorption of active ingredients in the body, inactivity rate and concomitant drugs.

[0074] In one embodiment of the present invention, the pharmaceutical composition is preferably prepared as a non-oral preparation, and can be prepared as a skin external preparation or a subcutaneous injection for local use at the desired hair growth site.

[0075] In one embodiment of the present invention, the above-mentioned external skin preparation can be prepared as any one of the preparations selected from the group consisting of creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes and poultices.

[0076] In addition, the pharmaceutical composition can be used as a subcutaneous injection, which can be injected subcutaneously using a conventional injection needle. In addition, a microneedle array in which a plurality of microneedles with a diameter of tens to hundreds of μm and a length of tens to thousands of μm are arranged together can be used to directly inject into the desired hair growth site.

[0077] According to another aspect of the present invention, there is provided a cosmetic composition comprising the compound of Chemical Formula 1 or a cosmetically acceptable salt thereof as an active ingredient for promoting hair growth or preventing or improving hair loss.

[0078] The term "cosmetically acceptable salt" used in this specification is the same as the definition of "pharmaceutically acceptable salt" mentioned above.

[0079] The above-mentioned cosmetic composition can be prepared into hair growth oil, hair milk, hair care lotion, shampoo, hair conditioner, hair essence, hair gel, hair mask, hair cream, soap, eyebrow hair growth agent, eyebrow nutrient, eyelash hair growth agent and eyelash nutrient, etc. In addition, the above-mentioned cosmetic composition can contain various ingredients of conventional cosmetic compositions such as surfactants, vitamins, preservatives, viscosity enhancers, pH adjusters, fragrances, pigments, etc. according to each dosage form.

[0080] According to another aspect of the present invention, there is provided a food composition comprising the compound of Chemical Formula 1 or a nutritionally acceptable salt thereof as an effective ingredient for promoting hair growth or preventing or improving hair loss.

[0081] The term "nutritionally acceptable salt" used in this specification is the same as the definition of "pharmaceutically acceptable salt" mentioned above.

[0082] The food composition provided by the present invention can be used as a health functional food. The above-mentioned "health functional food" refers to a food manufactured and processed using functional raw materials or ingredients useful to the human body in accordance with the relevant laws on health functional foods, and "functional" means that the nutrients are taken for the purpose of regulating the structure and function of the human body, or obtaining useful effects for health purposes such as physiological effects.

[0083] The food composition provided by the present invention may include conventional food additives. The suitability of the above-mentioned "food additives" shall be determined based on the specifications and standards related to the variety according to the general principles and general experimental methods of the food additive guidelines approved by the Food and Drug Safety Administration, unless otherwise specified.

[0084] The varieties listed in the above-mentioned "Food Additive Guidelines" include: chemical synthetics such as ketones, glycerin, potassium citrate, niacin, cinnamic acid, natural additives such as pigments, licorice extract, crystalline cellulose, sorghum pigment, guar gum, sodium L-glutamate preparations, flour-added alkali agents, preservative preparations, tar pigment preparations and other mixed preparations.

[0085] The food composition provided by the present invention may contain 0.01 to 95 weight percent, preferably 1 to 80 weight percent, of a guanidine derivative or a nutritionally acceptable salt thereof relative to the total weight of the composition. Furthermore, the guanidine derivative or a nutritionally acceptable salt thereof contained in the food composition provided by the present invention may be obtained by a method identical or similar to the extraction method mentioned in the preparation of the pharmaceutical composition, but is not limited thereto.

[0086] In addition, the food composition provided by the present invention can be prepared and processed into tablets, capsules, powders, granules, liquids, pills, etc. for the purpose of promoting hair growth and / or improving hair loss.

[0087] For example, the health functional food in the form of tablets can be prepared by granulating a mixture of a guanidine derivative or a nutritionally acceptable salt thereof, an excipient, a binder, a disintegrant and other additives by conventional methods, and then adding a lubricant and the like for compression molding, or directly compressing the mixture. In addition, the health functional food in the form of tablets can contain flavoring agents and the like as needed, and can also be coated with an appropriate coating agent as needed.

[0088] In the health functional food in capsule form, hard capsules can be prepared by filling a conventional hard capsule with a mixture of powdered guanidine derivatives or their nutritionally acceptable salts, and additives such as excipients, or their granules or coated granules, and soft capsules can be prepared by filling a capsule base such as gelatin with a mixture of guanidine derivatives or their nutritionally acceptable salts, and additives such as excipients. The soft capsules can contain plasticizers such as glycerin or sorbitol, colorants, preservatives, etc. as needed.

[0089] The pelleted health functional food can be formed by a mixture of guanidine derivatives or their food acceptable salts, excipients, binders, disintegrants, etc. by appropriate methods, and can be coated with sugar or other appropriate coating agents, or coated with starch, talcum powder or appropriate substances as needed.

[0090] Granular health functional foods can be prepared into granules by a mixture of guanidine derivatives or their nutritionally acceptable salts, excipients, binders, disintegrants, etc. in an appropriate manner, and may contain spices, flavoring agents, etc. as needed. Granular health functional foods can be made to pass through the No. 12 sieve (1680 μm), No. 14 (1410 μm) and No. 45 (350 μm) sieves, and in the next particle analysis experiment, all of them pass through the No. 12 sieve, and the amount of particles remaining on the No. 14 sieve accounts for less than 5.0% of the total amount, and the amount of particles passing through the No. 45 sieve accounts for less than 15.0% of the total amount.

[0091] The definitions of the above-mentioned excipients, binders, disintegrants, lubricants, flavoring agents, fragrances and other related terms may include those recorded in the well-known literature in the technical field and having the same or similar functions (Explanation of the Korean Pharmacopoeia, Moonsung Publishing House, Korean Pharmaceutical University Association, 5th revised edition, p33-48, 1989).

[0092] The types of the above-mentioned food are not particularly limited. The food to which the extract provided by the present invention can be added includes: beverages, chewing gum, vitamin complexes, beverages, etc., and generally includes all health functional foods.

[0093] According to another aspect of the present invention, there is provided a feed composition comprising the above-mentioned compound or a nutritionally acceptable salt thereof as an effective ingredient for promoting hair growth or preventing or improving hair loss.

[0094] The term "feed" used in this specification refers to food ingested by animals, specifically means a substance that sustains the life of animals or supplies organic or inorganic nutrients required for producing meat, milk, etc. The above-mentioned materials may include feed additives and may be prepared in various forms well known in the art.

[0095] The type of the above-mentioned feed is not particularly limited, and feed commonly used in the art can be used. Non-limiting examples of the above-mentioned feed include: plant feeds such as cereals, roots and tubers, food processing by-products, poultry, fibers, oils and fats, starches, dregs, or cereal by-products; and animal feeds such as proteins, inorganic substances, oils and fats, minerals, oils and fats, single-cell proteins, animal plankton, or food. These can be used alone or in combination of two or more.

[0096] In addition, the compound provided by the present invention or its nutritionally acceptable salt can be used as a feed additive added to a feed composition. The feed additive can be used to improve the productivity or health of the target animal, but is not limited to this. The feed additive can belong to the supplementary feed in the feed management method.

[0097] The feed additives in this specification can be mixed with one or more ingredients of organic acids such as citric acid, fumaric acid, fatty acids, lactic acid, or natural antioxidants such as polyphenols, catechol, vitamin E, vitamin C, green tea extract, chitosan, tannic acid, etc., and other conventional additives such as buffers and antibacterial agents can be added as needed. In addition, it can be prepared in liquid, capsule, granule or tablet form as needed.

[0098] The above feed or feed additives may also be supplemented with substances that have various effects, such as supplementing nutrients and preventing weight loss, improving the digestibility and utilization of cellulose in feed, improving oil quality, preventing reproductive disorders and increasing pregnancy rates, and preventing high temperature stress in summer. For example, various auxiliary agents such as amino acids, inorganic salts, vitamins, antioxidants, antimycins, microbial preparations, and crushed or broken wheat, barley, corn and other plant protein feeds, blood meal, meat meal, fish meal and other animal protein feeds, animal fats and plant fats, and other main ingredients can also be used together with nutritional supplements, growth promoters, digestion and absorption promoters, and disease preventive agents.

[0099] The feed and feed additives of this specification can reach the level of most animals including mammals and poultry. For example, in order to maintain or improve the appearance, it can be used for livestock such as cattle and goats; pets such as dogs and cats, but it is not limited to this.

[0100] According to another aspect of the present invention, there is provided a method for promoting hair growth or preventing or treating hair loss, comprising administering a pharmaceutical composition comprising the compound of Chemical Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient to a subject in need thereof.

[0101] The term "prevention" in the present invention refers to all actions that inhibit or delay the occurrence, spread and recurrence of hair loss by administering the composition provided by the present invention, and "treatment" refers to all actions that lead to improvement or favorable changes of the above symptoms by administering the composition provided by the present invention.

[0102] The term "subject" in the present invention refers to all animals including monkeys, cattle, horses, sheep, pigs, chickens, turkeys, quails, cats, dogs, mice, rats, rabbits or guinea pigs, including humans who have or may suffer from the above-mentioned hair loss. By administering the pharmaceutical composition provided by the present invention to the individual, the above-mentioned disease can be effectively prevented or treated. The pharmaceutical composition provided by the present invention can be administered in parallel with existing therapeutic agents.

[0103] The term "administer" in the present invention refers to providing a predetermined substance to a patient by any appropriate method. The administration route of the composition provided by the present invention can be administered by any conventional route as long as it can reach the target tissue. Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intravascular administration, oral administration, topical administration, intrasplenic administration, intrapulmonary injection, and intrarectal administration, but are not limited thereto. In addition, the pharmaceutical composition provided by the present invention can also be administered by any device for moving the active substance to the target cell.

[0104] According to another aspect of the present invention, there is provided a use of the compound of the above Chemical Formula 1, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound as an active ingredient for promoting hair growth or preventing or treating hair loss.

[0105] Another aspect of the present invention provides a use of the compound of the above Chemical Formula 1, a pharmaceutically acceptable salt thereof, or a pharmaceutical composition containing the compound as an active ingredient for preparing a drug for promoting hair growth or preventing or treating hair loss.

[0106] Hereinafter, the present invention will be further described in detail by the following examples. However, these examples are only for illustrative purposes, and the scope of the present invention is not intended to be limited by these examples.

[0107] Reagent preparation

[0108] As guanidine derivatives, (N-1-(2-methyl)phenethyl biguanide (hereinafter referred to as "IM176OUT05" or "IM"), N-1-(3,4-dichloro)phenethyl biguanide (hereinafter referred to as "IM176OUT04"), and 1-(3,4-dimethylphenethyl)guanidine (hereinafter referred to as "IM177OUT02") were synthesized. The syntheses are respectively the same as Example 22, Example 1, and Example 133 of Korean Patent Publication No. 10-2016-0146852 (2016.12.21). In addition, minoxidil was purchased from Hyundai Pharm (Seoul, Republic of Korea), and metformin was purchased from Cayman Chemical (Ann Arbor, MI, USA). The chemical structural formulas of the guanidine derivatives, minoxidil, and metformin of the present invention are as follows Figure 1 shown.

[0109] Example 1. Reprogramming effect of guanidine derivatives

[0110] Example 1.1. Comparison of the reprogramming effects of guanidine derivatives in mouse cells

[0111] It was confirmed that rotenone (ETC complex I inhibitor) promoted efficient somatic cell reprogramming at a subtoxic dose (10 nM), and the effects of rotenone and the guanidine derivatives of the present invention on generating iPSCs were compared as follows ( Figure 2 ).

[0112] OSKM reprogramming factors were introduced into mouse embryonic fibroblasts (MEFs), and during reprogramming into iPSCs, rotenone, the guanidine derivative of the present invention, and phenformin were treated at the indicated concentrations. After 14 days, alkaline phosphatase (AP) staining was performed ( Figure 2 A), and quantified the number of AP+ colonies ( Figure 2B), IM 156 and phenformin did not show the effect of promoting reprogramming, on the contrary, the number of AP+ colonies increased due to the treatment of IM176OUT 05, IM176OUT 04 and IM177OUT 02. In particular, when IM176OUT 05 (hereinafter referred to as IM) was treated at concentrations of 10nM, 20nM and 50nM, it showed the effect of improving reprogramming efficiency by 16.8 times, 13.9 times and 13.7 times, respectively. After that, the experiment only used the above three compounds that effectively improved the reprogramming effect, and conducted additional efficacy evaluation.

[0113] Example 1.2. Confirmation of the reprogramming effect of guanidine derivatives in human cells

[0114] OSKM reprogramming factors were introduced into human foreskin fibroblasts (HFF) to reprogram them into iPSCs. IM176OUT 05, IM176OUT 04, and IM177OUT 02, which had been confirmed to have reprogramming effects on mouse cells in Example 1.1. above, were treated with the concentrations indicated. AP staining was performed 28 days later ( Figure 3 A), and quantified the number of AP+ colonies ( Figure 3 B) All three compounds showed the best reprogramming effect when treated at a concentration of 10 nM. In particular, it was confirmed that IM176OUT 05 showed an effect of increasing the reprogramming efficiency by more than 2.4 times when treated at concentrations of 10 nM, 100 nM, and 500 nM.

[0115] Example 1.3. Confirmation of the Cell Stemness-Maintaining Effect of Guanidine Derivatives

[0116] In addition, the compounds provided by the present invention have been confirmed to have the effect of maintaining the stemness of human embryonic stem cells (hESC) ( Figure 4 Specifically, if hESCs are cultured in unconditioned medium (UM), they lose their stemness and weaken their AP activity. Figure 4 A), IM176OUT 05, IM176OUT 04, and IM177OUT 02 were used to increase AP staining from a low concentration. In particular, it was confirmed that when IM176OUT 05 was used at a concentration of 10 nM and 100 nM, the cell stemness maintenance effect was increased by 7.6 times and 8.2 times, respectively ( Figure 4 B).

[0117] The results of Examples 1.1 to 1.3 above show that, in the iPSC generation process of mice and humans, 10 nM IM176OUT 05 not only most effectively improves the reprogramming efficiency, but also shows the effect of maintaining the stemness of human embryonic stem cells. The following examples use the above conditions for subsequent research.

[0118] Example 2. Hair regeneration effect experiment of IM176OUT 05

[0119] Example 2.1. Hair regeneration model

[0120] The back skin hair of 7-week-old C57BL / 6 mice (Dae han BioLink, Chungbuk, Republic of Korea) was depilated using animal clippers and hair wax (Veet, Oxy Recrit Benckiser, Seoul, Republic of Korea) during the telogen phase. From the second day, 200 μl of placebo (control group), 1% IM, 1% minoxidil or 1% metformin were applied to the depilated area every day using a sterilized cotton swab. The image of each animal was captured every day, and the darkness of the back color was quantified in the same area (1.6 x 3 cm) using the Image J program. The mice were sacrificed and skin tissues were obtained on days 0, 7, 14 and 20. Half of the tissue was used for RNA isolation, and the remaining half was fixed overnight with 4% paraformaldehyde for histochemical analysis.

[0121] Example 2.2. Histological analysis

[0122] The fixed tissues in Example 1.1. above were deposited in 30% sucrose and embedded in OCT compound (Sakura Finetek USA Inc, Torrance, CA, USA). Frozen sections were obtained using a cryostatsection (Leica, Wetzlar, Germany) and stained with hematoxylin (Sigma) and eosin (Sigma) (H&E) or the respective antibodies listed in Table 1 below. Immunohistochemical analysis was performed and fluorescent images were taken with an Olympus microscope (Olympus).

[0123] Table 1

[0124] Antibody Catalog Number Company Name Dilution ratio Anti-keratin 15 ab52816 abcam 1:100 Anti-β-catenin c ab32572 abcam 1:200 Anti-β-catenin (FACS) ab22656 abcam 1:500 Anti-Ki67 ab15580 abcam 1:200 Anti-Shh (IHC) sc-9024 Santacruz 1:80 Anti-Shh (FACS) ab135240 abcam 1:300 Anti-PDK sc-7140 Santacruz 1:50 Anti-PDH (rabbit IgG) sc-292543 Santacruz 1:50 Anti-PDH (mouse IgG) sc-377092 Santacruz 1:50 Normal rabbit IgG #2729 Cell signaling technology 1:500 Anti-Histone Antibody H3 #4620 Cell signaling technology 1:50 Anti-trimethyl histone H3(Lys4) 04-745 Millipore 1:200 Anti-trimethyl histone H3 (Lys27) 07-449 Millipore 1:200 Anti-Dimethyl Histone H3(Lys9) 07-441 Millipore 1:200 Anti-NANOG A300-397A Bethyl Lab 1:250 Anti-SSEA1 MAB2155 R&D Systems 1:200

[0125] Example 2.3. Quantitative testing of tissue morphology

[0126] According to the principle of correctly classifying the hair cycle stage, each hair follicle was classified through H&E stained microscopic photos on the longitudinal section of each mouse (n>10) to quantify the hair cycle. The percentage of hair growth phase (anagen) of each group was calculated. The hair follicles in the same area (1300μm width) were counted using H&E stained microscopic photos on the cross section of each mouse (n>10).

[0127] Example 2.4. FACS analysis

[0128] PBS was injected into the mice, and after removing the blood, the skin tissue was obtained and cut into small pieces. The tissue was digested with trypsin at 37°C for 150 minutes, filtered through a 75μm nylon mesh, and then filtered through a 30μm mesh (BD Biosciences). Single cells were fixed, dialyzed, and blocked according to the immunostaining protocol. The cells were stained with specific antibodies (Table 1 above) and stained with BD Accuri TM C6 (BD Biosciences) was used for analysis.

[0129] Example 2.5. Analysis of auricular punch

[0130] For the pinna tissue repair analysis, three 2 mm diameter holes were punched in each ear using a punching device (dissecting needle (Fine Science Tools, British Columbia, Canada). The indicated concentration of IM was applied daily with a sterilized cotton swab. The hole area was measured with digital calipers (VWR, Radnor, PA, USA).

[0131] Statistical analysis

[0132] All data represent more than 3 independent biological data of repeated experiments. For all animal experiments, the experiments were repeated 6 times in n>5 ​​treatment groups. Student's t-test was used to compare between groups, and p<0.05 indicated statistical significance.

[0133] Example 3. Confirmation of the hair regeneration effect of IM176OUT 05

[0134] Example 3.1. Hair growth promoting effect of IM176OUT 05 in mice

[0135] Through mouse experiments, we confirmed whether IM compounds can promote hair growth without showing toxicity or other side effects. 7-week-old C57BL / 6 mice were synchronized by removing hair in the telogen phase, and various concentrations of IM were applied to the back skin of the mice every day. On the 9th day, black pigmentation and hair growth were significantly confirmed in the area where 1% IM was used ( Fig. 9 ).

[0136] In addition, the results of comparing the efficacy of IM and the existing hair growth promoter minoxidil showed that hair growth can be promoted in both male and female mice. In particular, IM showed a strong promoting effect on hair growth in female mice. Specifically, on the 8th day of IM treatment, the skin color of IM-treated mice became significantly darker than that of the control group or minoxidil-treated mice. In addition, on the 10th day, hair growth was significantly promoted by IM treatment (Figure 5). In contrast, the results of additional independent experiments showed that for female mice, IM was more effective than minoxidil ( Fig.10 In contrast, in male mice, IM had an effect similar to that of minoxidil ( Fig.11a and Fig.11b ).

[0137] In addition, the results of comparing IM and other biguanide derivatives such as metformin with regard to hair growth showed that from day 8, the dark intensity of the back skin of metformin-treated mice increased compared with the control group, but no significant effect was observed ( Fig.13a and Fig.13b ).

[0138] During the experiment, no side effects including skin problems were observed in the IM-treated mice.

[0139] Example 3.2. Effect of promoting hair follicle regeneration in mice by IM176OUT05

[0140] The results of tissue morphometric analysis showed that IM can stimulate the hair follicle cycle ( Figure 6a On day 20, in longitudinal sections, in the control group of mice, most of the hair follicles were quantitatively present in the growth phase III, whereas in the IM-treated mice, most of the hair follicles were mainly present in the growth phase V and VI stages in the late growth phase ( Figure 6b In addition, on day 7, the number of hair follicles in the IM-treated mice was significantly increased compared with the control group in cross-section ( Figure 6c ), on day 20, the number of hair follicles in IM-treated mice or minoxidil-treated mice was significantly greater than that in the control group ( Figure 6dFurthermore, on day 7, keratin 15 (K15), a marker of hair follicle stem cells, was more strongly expressed in the hair follicle bulge area of ​​IM-treated mice compared to the control group or minoxidil-treated mice ( Figure 7a ).

[0141] In addition, on day 7, the expression of β-catenin, which mediates hair follicle regeneration, was increased in IM-treated mice. At this time, Ki 67-positive proliferating cells were found in IM-treated mice, indicating that the hair follicle cycle and the expansion of proliferative precursor cells were enhanced by IM ( Figure 7a ). The results of quantification of the K15+ / β-catenin+ population by FACS analysis showed that it occupied 3.2%, 11.7%, and 6.0% of the cell population in the single cells of the skin of the control group, IM-treated mice, and minoxidil-treated mice, respectively ( Figure 7b This showed a 3.7-fold increase in IM-treated mice compared to the control group ( Figure 7b ).

[0142] By day 20, K15 and β-catenin were clearly detected in all mouse groups ( Figure 7c ), K15+ / β-catenin+ group was detected in more than 24% of all groups ( Figure 7d Another essential factor for hair follicle development, Shh (Sonic Hedgehog), was clearly detected in IM-treated mice or minoxidil-treated mice ( Figure 7c On day 7, the Ki67+ / Shh+ group accounted for less than 5% of all mouse groups ( Figure 7b ), but by day 20, the percentage of single cells in the skin of control, IM-treated or minoxidil-treated mice increased to 13.8%, 36.7% and 34%, respectively ( Figure 7d On day 20, the Ki67+ / Shh+ group increased 2.7-fold in IM-treated mice compared with the control group ( Figure 7d ).

[0143] Example 3.3. Effect of IM176OUT 05 on stem cell metabolism and proliferation activity of proliferative progenitor cells in the early stage of hair follicle regeneration

[0144] It is well known that K15-positive hair follicle stem cells express pyruvate dehydrogenase kinase (PDK), an enzyme that promotes the switch to glycolysis, while Ki 67-positive proliferating cells strongly express cellulose dehydrogenase (PDH), an enzyme that responds to mitochondrial OXPHOS. On day 7, PDK expression was detected in K15-positive stem cells from IM-treated mice, but not in control or minoxidil-treated mice ( Figure 8aOn day 7, the results of FACS analysis showed that although the K15+ / PDK+ population was not detected in the control group or minoxidil-treated mice, it was confirmed that it occupied 5.9% of the cell population in single cells of the IM-treated mouse skin ( Figure 8c ). Thus, it was confirmed that the glycolytic stem cell population (K15+ / PDK+) of IM-treated mice increased by at least 6.6 times compared to the control group.

[0145] On day 7, PDH expression was clearly confirmed in Ki 67-positive proliferating cells of IM-treated mice. Figure 8a ), and on day 20, high levels were detected in proliferating cells of all populations ( Figure 8b In addition, on day 7, the Ki67+ / PDH+ population increased by 13.7% in IM-treated mice, whereas it only increased by 5.0% and 8.3% in the control group and minoxidil-treated mice, respectively ( Figure 8c ). Furthermore, the results of the analysis of the mutual expression pattern of each marker showed that on day 20, the expression of K15 / PDH and Ki67 / PDK were independently detected in IM-treated mice ( Fig.12a ), indicating that PDK-expressing glycolytic K15-positive hair follicle stem cells and PDH-expressing OXPHOS-dependent Ki 67-positive proliferative cells are clearly distinguished from each other. Furthermore, FACS analysis confirmed that the PDK+ cell population and the Ki67+ cell population were clearly separated, and each population was increased by IM-treatment or minoxidil-treatment on day 20 ( Figure 12b In addition, it was also detected that the populations separated from PDH+ cells and K15+ cells were increased by IM-treatment or minoxidil-treatment on day 20 ( Figure 12b ).

[0146] Example 3.4. Effect of IM176OUT 05 on the expression of genes related to metabolism and hair regeneration

[0147] The expression levels of metabolism and hair regeneration-related genes were analyzed in the skin tissues of each mouse group. The expression of glycolysis-related enzymes (HK2 and LDHA) was significantly induced in IM-treated mice on day 7, and significantly increased in both IM-treated and minoxidil-treated mice compared to the control group on day 20. At this stage, the expression of OXPHOS-related enzymes (NDUS 3 and ATP5B) increased in both IM-treated and minoxidil-treated mice compared to the control group. This suggests that IM can promote glycolysis in the early stages of hair follicle regeneration and further promote OXPHOS in the later stages, thereby contributing to the active energy metabolism required for hair growth. The expression of stemness-related genes involved in hair follicle development (Wnt1a, Lef-1, Gli-1, and versican) was significantly increased in the skin of IM-treated mice on day 7, and minoxidil-treated mice also increased their expression compared with the control group (Wnt1a, Lef-1, and Gli-1). On day 20, both IM-treated and minoxidil-treated mice significantly induced the expression of the above stemness-related genes ( Figure 8d ).

[0148] Example 3.5. Tissue regeneration effect based on IM176OUT 05

[0149] The effect of IM on tissue regeneration was observed through the repair of auricular tissue. The results showed that IM reduced the wound area of ​​the auricular tissue in a volume-dependent manner. Therefore, in addition to activating hair growth, IM can also activate tissue regeneration (Figure 14). Based on these results, IM can repair hair follicle tissue damaged by hair loss, thereby preventing possible damage in advance.

[0150] Those skilled in the art will recognize that the present invention may be implemented in other specific forms without departing from the spirit or essential features of the present invention. The described embodiments should be considered in all respects only as illustrative and not restrictive. Therefore, the scope of the present invention is indicated by the appended claims rather than the preceding description. All changes falling within the equivalent meanings and scope of the claims should be included within the scope of the present invention.

Claims

1. Use of a pharmaceutical composition comprising a compound represented by the following chemical formulae 2 to 4 or a pharmaceutically acceptable salt thereof as an active ingredient in the preparation of a drug for regenerating tissue: [Chemical formula 2] [Chemical formula 3] [Chemical formula 4] 2. The use according to claim 1, in, The pharmaceutical composition comprises the compounds represented by the Chemical Formulas 2 to 4 or pharmaceutically acceptable salts thereof at a concentration of 1% to 5%.

3. The use according to claim 1, in, The pharmaceutical composition promotes cell reprogramming or maintains cell stemness.

4. The use according to claim 1, in, The pharmaceutical composition enhances hair follicle regeneration.

5. The use according to claim 1, in, The pharmaceutical composition increases the expression of one or more of the group consisting of β-catenin, Shh (sonic hedgehog), HK2 (hexokinase 2), LDHA (lactate dehydrogenase A), NDUS3 (NADH dehydrogenase iron-sulfur protein 3), ATP5B (ATP synthase subunit β), Wnt1a, Lef-1 (lymphoid enhancer binding factor 1), Gli-1 (GLI family zinc finger 1), versican, PDH (pyruvate dehydrogenase) and PDK (pyruvate dehydrogenase kinase).

6. The use according to claim 1, in, The pharmaceutical composition is prepared as a formulation for external application to the skin.

7. The use according to claim 6, in, The preparation is any one selected from the group consisting of creams, gels, patches, sprays, ointments, plasters, lotions, liniments, pastes and poultices.

8. The use according to claim 1, in, The tissue is a hair follicle or skin.

9. Use of a cosmetic composition comprising the compound according to any one of claims 1 to 2 or a cosmetically acceptable salt thereof as an active ingredient in the preparation of a cosmetic for regenerating tissue.

10. The use according to claim 9, in, The tissue is a hair follicle or skin.

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