Composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, comprising a double-stranded miRNA as an active ingredient

Abstract

The present invention relates to a composition for improving gray hair, promoting hair growth and / or preventing or improving hair loss, comprising a double-stranded miRNA as an active ingredient, more specifically, to a pharmaceutical or cosmetic composition for improving gray hair, promoting hair growth and / or preventing or improving hair loss, comprising a double-stranded miRNA miR-3139, miR-3189, miR-3199 or miR-8485, a double-stranded oligonucleotide structure comprising the same, or nanoparticles comprising the structure as an active ingredient. The composition according to the present invention can prevent gray hair and slow down the progression of gray hair by activating melanocytes and promoting melanin production, and can improve hair that has already progressed to gray hair to the hair state before gray hair. In addition to the activity of melanocytes, it can promote the proliferation of hair papilla cells and keratinocytes present in hair follicles, induce the development of the outer root sheath of hair and hair growth, and exert the effect of promoting hair growth and / or preventing hair loss.

Description

Technical Field

[0001] The present invention relates to a composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, containing double-stranded miRNA as an active ingredient. More specifically, the present invention relates to a pharmaceutical or cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, containing miR-3139, miR-3189, miR-3199 or miR-8485, which is double-stranded miRNA, a double-stranded oligonucleotide structure containing the same, or nanoparticles containing the structure as an active ingredient.

Background Art

[0002] Human hair greying is a phenomenon of physiological aging and is known to be caused by a decrease in melanin pigment due to the decline and depletion of the functions of melanocytes or melanocyte stem cells in hair follicles. Melanin pigment is an important factor that determines hair color. Melanin is synthesized and secreted from melanosomes present in melanocytes, and after being transmitted to adjacent keratinocytes, fibroblasts, etc., it moves along with hair growth to maintain hair color. Currently, simple hair dyeing procedures are used as a solution for hair greying. However, dyeing is a temporary method, and re-dyeing is essential due to the growth of white hair. Components contained in hair dye dyes have various side effects such as irritating the scalp and hair follicles, causing allergies and dermatitis. Currently, research related to the regulation of melanin pigment production is limited to the development of substances that suppress symptoms such as pigmentation caused by excessive induction of skin melanin due to various factors such as genetic factors, aging, hormones, growth factors, and ultraviolet rays, and the elucidation of their mechanisms of action, which show skin whitening effects. Conversely, research on the mechanisms of action and the development of substances related to promoting melanin production to overcome hair greying or reduced melanin synthesis has hardly progressed. In addition, melanogenesis is regulated by the interaction of various signals (SCF-KIT-RAS-ERK signaling pathway, MC1R-cAMP-PKA signaling pathway, Wnt-β-catenin signaling pathway). Due to such complex mechanisms of action, it is actually not easy to elucidate the mechanisms of action and develop new substances to prevent or improve hair greying.

[0003] Recently, androgenetic alopecia (AGA), which mainly occurs in middle-aged men and has been regarded as a genetic disease in men, has become prevalent not only in young men but also in women due to serious environmental pollution, stress, changes in diet, and the rapid aging of the population. As a result, the interest in hair loss has increased, and the hair loss market has been growing rapidly. It is known that hair loss is induced by various factors such as genetic factors, male hormones, aging, and blood circulation disorders. Currently, only finasteride, which suppresses the production of dihydrotestosterone (DHT), a male hormone (androgen), and minoxidil, which improves vasodilation and blood circulation, are approved therapeutic drugs. However, these drugs have temporary and limited efficacy depending on the administration method and dosage, and are accompanied by various side effects. Therefore, the development of new substances that can induce the activation and proliferation of hair follicle cells to promote hair growth without side effects is required.

[0004] The hair growth cycle consists of three stages: anagen, in which hair grows most actively; catagen, in which growth stops and hair degradation begins; and telogen, in which the activity of hair follicles stops. Hair loss refers to the natural shedding of hair that has stopped growing due to the hair growth cycle, and it is known that various factors can cause hair loss. Although drug development for alleviating hair loss symptoms has been carried out for a long time, currently, there are no drugs other than finasteride, an oral hair loss treatment agent, and minoxidil, a topical hair loss treatment agent. However, inducing the alleviation of hair loss symptoms through the mechanism of suppressing male hormone production has limitations in efficacy and is accompanied by side effects. Therefore, it is necessary to develop substances with new mechanisms and strategies that show efficacy without side effects. Promoting the activation and proliferation of hair follicle cells to quickly induce the conversion of telogen hair to anagen is one of the important strategies for developing hair loss treatment agents. Therefore, the development of new substances that can induce the activation and proliferation of hair follicle cells to promote hair growth is required.

[0005] Techniques for suppressing gene expression are important tools in the development of therapeutic drugs and target validation for disease treatment, and are being developed in various aspects to overcome the limitations of existing traditional drug treatment methods. One of them is the use of interfering RNA (RNA interference, hereinafter referred to as RNAi) (Iorns, E., Lord, C.J., Turner, N. & Ashworth, A., Nat Rev Drug Discov 6, 556-68. 2007.). RNAi is a method used to suppress gene expression, and because of its significant gene suppression effect with simplicity and low cost, the application fields of this technology are diversifying. siRNA is a single-stranded RNA composed of 16 to 27 nucleotides and acts as a component of a ribonucleoprotein complex called RNA Induced Silencing Complex (RISC) in cells. RISC functions as an RNA enzyme scissors, cleaving messenger RNA (hereinafter referred to as mRNA) to inhibit the production of proteins from mRNA. The siRNA contained in RISC binds to mRNA with a sequence complementary to the siRNA sequence to form double-stranded RNA, and RISC acts as an RNA enzyme scissors to cleave the target mRNA, preventing mRNA from performing its function as a template for continuously producing proteins.

[0006] Thus, RNAi-based therapeutic agents are evaluated as a technology that is more advanced than the therapeutic agents of the single-molecule substances in terms of blocking mRNA before the protein production stage and utilizing the RNA and the intracellular RISC system. However, there are also side effects that cannot be solved by siRNA-based technologies, which is a phenomenon called off-target effect. As described above, it degrades mRNA that binds complementarily to the siRNA sequence, but also binds to and induces degradation of mRNA that is only partially complementary rather than complementary to the entire siRNA sequence, thus inducing the degradation of non-target mRNA, which is called off-target effect.

[0007] To overcome the technical difficulties of the aforementioned RNAi-based therapeutic agents, research has been actively conducted to use microRNA (hereinafter referred to as miRNA) as a therapeutic agent (Agostini, M. & Knight, R.A., Oncotarget 5, 872-81, 2014; Dangwal, S. & Thum, T., Annu Rev Pharmacol Toxicol 54, 185-203, 2014.). miRNA is an RNA composed of 16 to 27 nucleotides and is classified as a protein non-coding RNA with respect to messenger RNA that is translated into protein (Carthew, R.W. & Sontheimer, E.J., Cell 136, 642-55, 2009; MacFarlane, L.-A. & Murphy, P.R., Current Genomics 11, 537-561, 2010; Bartel, D.P., Cell 136, 215-33, 2009). miRNA is recorded in the genomes of higher plant and animal cells and is known to play a central role in regulating cell metabolism and functions including cell generation, growth, differentiation, and death. To date, more than 2,000 miRNAs have been known in the human genome, and the functions of a considerable number of these miRNAs are still unknown.

[0008] miRNAs are transcribed from the genome into RNA by an RNA polymerase called Pol II, but their initial lengths are so diverse that they cannot be specified (Carthew, R.W. & Sontheimer, E.J., Cell 136, 642-55, 2009; Brodersen, P. & Voinnet, O., Nat Rev Mol Cell Biol 10, 141-148, 2009.). This is due to the diversity of the positions where miRNAs are contained in the genome. They are located in introns, which are parts of mRNA that do not participate in protein production, and are transcribed simultaneously with the production of mRNA. They are also produced in various ways, such as when they are located in the inter-genic region between genes on the genome and are transcribed independently (Malone, C.D. & Hannon, G.J., Cell 136, 656-68, 2009.). The initial miRNA precursors thus generated are called primary microRNAs (primary miRNAs). Primary miRNAs are edited into precursor miRNAs (precursor miRNAs, hereinafter referred to as pre-miRs) by an RNA-cleaving enzyme called Drosha in the nucleus (Bartel, D.P., Cell 136, 215-33, 2009.). Pre-miRs form an RNA hairpin structure and are composed of approximately 70-80 nucleotides. Pre-miRs inside the cell nucleus are transported from the nucleus to the cytoplasm by exportin proteins and are further processed in the cytoplasm by another RNA-cleaving enzyme called Dicer to generate double-stranded mature miRNAs (mature microRNAs, hereinafter referred to as mature miRs when described as miR without other modifiers) composed of 16-27 nucleotides. One strand of the double-stranded miR is selectively selected and binds to the ribonucleoprotein complex RISC to become active, and binds to the target mRNA using the miR sequence.

[0009] Generally, mRNA can be broadly divided into three parts based on its involvement in protein production, namely the coding region containing protein translation information, the 5'-UTR (Un-Translated Region) and 3'-UTR that do not have protein translation information as the 5' and 3' parts of the coding region respectively. siRNA, which causes the degradation of target mRNA by utilizing a complementary sequence to mRNA, acts regardless of whether it is related to the 5'-UTR, 3'-UTR, or the coding region of mRNA, while miR mainly binds to the 3'-UTR (Carthew, R.W. & Sontheimer, E.J., Cell 136, 642-55, 2009; Bartel, D.P., Cell 136, 215-33, 2009.). In addition to the difference in the binding position to mRNA, as a unique feature of siRNA and other miRNAs, siRNA mainly binds to mRNA containing a sequence complementary to the entire siRNA sequence, whereas for miR, a limited-size seed region sequence located at the 2-8 nucleotide position from the 5' end of miRNA is mainly used for target mRNA recognition. Therefore, even if the entire miRNA sequence does not have a completely complementary sequence to the target gene and contains a certain non-complementary sequence, it does not affect miRNA activity (Bartel, D.P., Cell 136, 215-33, 2009.). Since the sequence size of the seed region is 6-8 nucleotides, there are various types of mRNAs with a sequence complementary to this in the 3' UTR. For this reason, one type of miRNA can simultaneously control multiple types of mRNAs. Such properties of miRNA enable it to function as an efficient regulator involved in the control of many aspects of cell physiology leading to cell division, growth, differentiation, and death. Also, the function of miRNA as a regulator has the advantage that it can be effectively applied to various diseases. In the case of siRNA, it aims to suppress the expression of a single gene, while miRNA can simultaneously inhibit the expression of multiple genes involved in various signaling pathways.Most mRNAs contain regions in their 3' UTRs where one or more miRNAs may bind, and according to certain bioinformatics calculations, it is known that protein production of approximately 30% of all mRNAs is regulated by miRNAs.

[0010] Despite the excellent effects and diverse applications of miRNAs or siRNAs, for miRNAs / siRNAs to be developed as therapeutic agents, they must be effectively delivered to target cells through improving the stability of miRNAs / siRNAs in the body and enhancing the cell delivery efficiency (FY Xie., Drug Discov. Today. 2006 Jan; 11(1-2):67-73). To improve in vivo stability and solve the problem of non-specific innate immune stimulation of miRNAs / siRNAs, studies have been actively conducted to modify some nucleotides or the backbone of miRNAs / siRNAs to be resistant to nucleases, or to utilize delivery carriers such as viral vectors, liposomes, or nanoparticles. Delivery systems using viral vectors such as adenoviruses and retroviruses have high transfection efficacy but high immunogenicity and oncogenicity. On the other hand, non-viral delivery systems containing nanoparticles have lower cell delivery efficiency compared to viral delivery systems, but have high stability in vivo, can be specifically delivered to the target, and have the advantages of absorbing (uptake) and internalizing the encapsulated RNAi oligonucleotides into cells or tissues with little cytotoxicity and natural immune induction, and are currently evaluated as a more promising delivery method compared to viral delivery systems (Akhtar S, J Clin Invest. 2007 December 3; 117(12):3623-3632).

[0011] Methods of using nanocarriers in non-viral delivery systems involve forming nanoparticles using various polymers such as liposomes and cationic polymer complexes, and carrying miRNA / siRNA on these nanoparticles, i.e., nanocarriers, and delivering them to cells. Among the methods of using nanocarriers, the mainly utilized methods include polymeric nanoparticles, polymer micelles, lipoplexes, etc. Among these, the method using lipoplexes is composed of cationic lipids and interacts with the anionic lipids of the cell endosome to induce the destabilization effect of the endosome and play a role in intracellular delivery. Also, it is known that by linking a chemical substance or the like to the terminal site of the miRNA / siRNA sense (passenger) strand to give enhanced pharmacokinetic characteristics, high efficiency can be induced in vivo (J Soutschek, Nature 11; 432(7014):173-8, 2004). At this time, the stability of miRNA / siRNA changes depending on the nature of the chemical substance bound to the end of the miRNA / siRNA sense or antisense (guide) strand.

[0012] For example, siRNA conjugated with a polymer compound such as polyethylene glycol (PEG) becomes a delivery agent with improved miRNA / siRNA stability by interacting with the anionic phosphate groups of miRNA / siRNA to form a complex under conditions where a cationic substance is present (SH Kim, J Control Release 129(2):107-16, 2008). In particular, micelles composed of polymer complexes are extremely small in size compared to other systems used as drug delivery carriers, such as microspheres or nanoparticles, have a very uniform distribution, and are self-assembled structures, so they have the advantage of being easy to ensure the quality control and reproducibility of the formulation. To improve the intracellular delivery efficiency of miRNA / siRNA, technologies for ensuring stability and efficient cell membrane permeability have been developed through miRNA / siRNA conjugates in which a hydrophilic substance, a biocompatible polymer (e.g., polyethylene glycol) is conjugated to miRNA / siRNA by simple covalent bonding or linker-mediated covalent bonding (Korean Registered Patent No. 883471).

[0013] However, simply chemically modifying miRNA / siRNA and conjugating polyethylene glycol (PEGylation) has the drawback of low stability in vivo and poor delivery to target organs. To solve such drawbacks, a double-stranded oligonucleotide structure in which hydrophilic and hydrophobic substances are conjugated to a double-stranded oligonucleotide, particularly a double-stranded oligoribonucleotide such as miRNA / siRNA, has been developed. The structure forms self-assembled nanoparticles named SAMiRNA (self-assembled micelle inhibitory RNA) due to the hydrophobic interaction of the hydrophobic substance (Korean Registered Patent No. 1224828). TM (self-assembled micelle inhibitory RNA), but the SAMiRNA TM technology has the advantage of being able to obtain nanoparticles that are very small in size and homogeneous compared to conventional delivery technologies.

[0014] SAMiRNA TM As a specific example of the technology, PEG or HEG (Hexaethylenglycol) is used as a hydrophilic substance. PEG, a synthetic polymer, is often used to increase the solubility of pharmaceuticals, especially proteins, and to regulate pharmacokinetics. PEG is a polydisperse substance. A batch of polymer is composed of the sum of different numbers of monomers, and its molecular weight shows a Gaussian curve shape. The polydisperse value (Mw / Mn) represents the degree of homogeneity of the substance. That is, when PEG has a low molecular weight (3 - 5 kDa), it shows a polydispersity index of about 1.01, and when it has a high molecular weight (20 kDa), it shows a high polydispersity index of about 1.2, indicating that the higher the molecular weight, the relatively lower the homogeneity of the substance. Therefore, when PEG is conjugated to a pharmaceutical, there is a drawback that the polydisperse characteristics of PEG are reflected in the conjugate and it is not easy to verify a single substance. Although there is a tendency to produce substances with a low polydispersity index through the improvement of the synthesis and purification process of PEG, especially when PEG is conjugated to a substance with a small molecular weight, there are inconvenient points such as it is not easy to confirm whether the conjugation has been easily carried out. There are problems due to the polydispersity characteristics of the substance (Francesco M. VDRUG DISCOVERY TODAY (2005) 10(21):1451 - 1458). Along with this, recently, in an improved form of the existing self - assembling nanoparticle SAMiRNA TM technology, SAMiRNA TM is formed by blocking the hydrophilic substances of the double - stranded oligonucleotide structure that constitutes SAMiRNA with uniform 1 - 15 monomers having a certain molecular weight and basic units containing a linker as needed. By using an appropriate number of these as needed, a new form of delivery technology with a smaller size and a dramatically improved polydispersity compared to the conventional SAMiRNA TM has been developed.

[0015] Therefore, as a result of the inventors' efforts to find miRNAs that can improve the graying of hair, it was confirmed that miR-3139, miR-3189, miR-3199, or miR-8485 can activate melanocytes, respectively, promote the production of melanin, and improve the graying of hair. In addition to the activation of melanocytes, the promotion of the proliferation of hair follicle dermal papilla cells, keratinocytes, etc. present in the hair follicle was confirmed, and the present invention was completed by confirming the increase in the outer root sheath (ORS) of the hair follicle and the hair length.

Prior Art Documents

Patent Documents

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Patent Document 1

Patent Document 2

Patent Document 3

Non-Patent Documents

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Summary of the Invention

[0018] The present invention provides a novel composition that can improve the graying of hair by activating melanogenic cells in hair follicles and promoting melanogenesis, induce the promotion of hair follicle cell proliferation to prevent or improve hair loss, and promote hair growth.

[0019] To achieve the above object, the present invention provides (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1): A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199 or miR-8485. Or (iii) Provided is a pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, comprising, as an active ingredient, nanoparticles containing the double-stranded oligonucleotide structure.

[0020] The present invention also provides to a subject in need of improving graying of hair, promoting hair growth and / or preventing or improving hair loss (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1): A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199 or miR-8485. Or (iii) Provided is a cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, comprising, as an active ingredient, nanoparticles containing the double-stranded oligonucleotide structure.

[0021] The present invention provides to a subject in need of improving graying of hair, promoting hair growth and / or preventing or improving hair loss (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1): A-X-R-Y-B Structural formula (1) In the above structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) A method for improving hair graying, promoting hair growth and / or preventing or improving hair loss, comprising the step of administering nanoparticles containing the double-stranded oligonucleotide structure is provided.

[0022] The present invention is for improving hair graying, promoting hair growth and / or preventing or improving hair loss (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y- Structural formula (1) In the above structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) The use of nanoparticles containing the double-stranded oligonucleotide structure is provided.

[0023] The present invention is for the manufacture of a pharmaceutical or cosmetic for improving hair graying, promoting hair growth and / or preventing or improving hair loss (i) miR-3139, miR-3189, miR-3199, or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y- Structural formula (1) In the above structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199 or miR-8485. Or (iii) The use of nanoparticles containing the double-stranded oligonucleotide structure is provided.

Brief Description of the Drawings

[0024] Figure 1 shows the results of a primary screening for measuring the melanin production amount in human skin cancer cell line (M21) using 1728 types of human miRNAs carried on SAMiRNA. TM

[0025] Figure 2 shows the results of a secondary screening of 18 types with high melanin production amount selected through screening in two types of human skin cancer cell lines (M21, SK-MEL28).

[0026] Figure 3 shows the results of evaluating the reproducibility of the melanin production amount and color change efficacy in human skin cancer cell line (SK-MEL-28) for 9 types of miRNAs with the highest melanin production amount through the first and second screenings.

[0027] Figure 4 shows the results of analyzing the changes in melanin production signal transduction through RT-qPCR reaction to analyze the mechanism of action of 9 types of miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199, miR-933 with the highest melanin production amount in SK-MEL-28 cell line.

[0028] Figure 5 shows the results of analyzing the changes in melanin production signal transduction of 9 types of miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199, miR-933 selected from human epidermal melanocytes cell line through RT-qPCR reaction.

[0029] ​Figure 6 shows the results of confirming changes in melanogenesis signal transduction by Western blot analysis for the mechanism of action analysis of nine types of miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199, and miR-933 selected from the SK-MEL-28 cell line.

[0030] Figure 7 shows the results of observing the promotion of outer root sheath (ORS) and hair follicle cell proliferation in human white hair for the four finally selected miR-3139, miR-3189, miR-3199, and miR-8485.

[0031] Figure 8 shows the results of observing the efficacy (color change) in human white hair for the four finally selected miR-3139, miR-3189, miR-3199, and miR-8485.

[0032] Figure 9a shows the results of analyzing cell color change and melanin production by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line.

[0033] Figure 9b shows the results of confirming changes in melanogenesis signal transduction by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line by RT-qPCR.

[0034] Figure 9c shows the results of confirming changes in melanogenesis signal transduction by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line by immunoblot analysis.

[0035] Figure 9d shows the results of confirming the efficacy of changes in melanogenesis signal transduction by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line through immunocytochemistry analysis.

[0036] Figure 10a shows the results of selecting GSK3β as the target gene of miR-3199.

[0037] Figure 10b shows the results of confirming the ability to inhibit GSK3β by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line using RT-qPCR analysis.

[0038] Figure 10c shows the results of confirming the ability to inhibit GSK3β by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line using immunoblot analysis.

[0039] Figure 10d shows the results of confirming the ability to inhibit GSK3β by SAMiRNA-miR-3199 treatment in the SK-MEL-28 cell line using immunocytochemistry analysis.

[0040] Figure 10e shows the results of confirming that miR-3199 directly binds to the 3' UTR of GSK3β mRNA and acts through luciferase reporter assay analysis.

[0041] Figure 11a shows the analysis results of cell color change and melanin production by SAMiRNA-miR-3199 treatment in human epidermal melanocytes.

[0042] Figure 11b shows the results of confirming the change in melanin production signal transduction by SAMiRNA-miR-3199 treatment in human epidermal melanocytes using RT-qPCR analysis.

[0043] Figure 11c shows the results of confirming the signal transduction change in melanin production by SAMiRNA-miR-3199 treatment in human epidermal melanocytes using immunoblot analysis.

[0044] Figure 12a shows the results of evaluating the cytotoxicity and innate immune toxicity of SAMiRNA-miR-3199 in human dermal papilla cells, keratinocytes, and human melanocytes.

[0045] Figure 12b shows the results of evaluating the cytotoxicity and innate immunotoxicity of SAMiRNA-miR-3199 by checking for the presence or absence of an increase in inflammatory cytokines.

Best Mode for Carrying Out the Invention

[0046] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. In general, the nomenclature used herein is well known and commonly used in the technical field.

[0047] In the present invention, due to the need to prevent, inhibit or improve the graying and hair loss of hair, a screening library for 1728 human miRNAs was synthesized (Table 2), and this was treated with M21 and SK-MEL-28 cells, which are human skin cancer cell lines. By comprehensively analyzing the measurement of melanin production and the change in cell color, four types of miRNAs with good effects, such as miR-3139, miR-3189, miR-3199, and miR-8485, were discovered. In addition, it was confirmed that the above miRNAs can improve melanocyte activity, promote melanin production, and improve the graying of hair, the mechanism of action of promoting melanin production was elucidated, and it was confirmed that melanin production in hair can be promoted without side effects through cytotoxicity and innate immunotoxicity evaluation. In addition, it was confirmed that the cell proliferation of dermal papilla cells and keratinocytes present in the hair follicle, together with melanin-producing cell activity, is promoted, and the development of the outer root sheath and the length of the hair increase.

[0048] Therefore, from one aspect, the present invention (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) a double-stranded oligonucleotide construct containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) A pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, comprising as an active ingredient nanoparticles containing the double-stranded oligonucleotide structure.

[0049] From another aspect, the present invention (i) miR-3139, miR-3189, miR-3199, or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) A cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, comprising as an active ingredient nanoparticles containing the double-stranded oligonucleotide structure.

[0050] In the present invention, the composition is characterized by improving graying of hair, promoting hair growth and / or preventing or improving hair loss by melanocyte activity and hair follicle cell proliferation, but is not limited thereto.

[0051] In the present invention, the miR-3139 is characterized by containing the base sequence represented by SEQ ID NO: 9 and the base sequence represented by SEQ ID NO: 10, and preferably, it is a DNA / DNA, RNA / RNA, or DNA / RNA hybrid composed of the base sequence represented by SEQ ID NO: 9 and the base sequence represented by SEQ ID NO: 10. miR-3139 5'-CAGGCAUCUGUUGAGCUCCUAUU-3' (SEQ ID NO: 9) 5'-UAGGAGCUCAACAGAUGCCUGUU-3' (SEQ ID NO: 10)

[0052] In the present invention, the miR-3189 is characterized by comprising the nucleotide sequence represented by SEQ ID NO: 5 and the nucleotide sequence represented by SEQ ID NO: 6, and preferably is a DNA / DNA, RNA / RNA, or DNA / RNA hybrid composed of the nucleotide sequence represented by SEQ ID NO: 5 and the nucleotide sequence represented by SEQ ID NO: 6. miR-3189 5'-UGCCCCAUCUGUGCCCUGGGUAGGA-3' (SEQ ID NO: 5) 5'-CCCUUGGGUCUGAUGGGGUAG-3' (SEQ ID NO: 6)

[0053] In the present invention, the miR-3199 is characterized by comprising the nucleotide sequence represented by SEQ ID NO: 15 and the nucleotide sequence represented by SEQ ID NO: 16, and preferably is a DNA / DNA, RNA / RNA, or DNA / RNA hybrid composed of the nucleotide sequence represented by SEQ ID NO: 15 and the nucleotide sequence represented by SEQ ID NO: 16. miR-3199 5'-CUUUCUCCUAAGGCAGUCCCUUU-3' (SEQ ID NO: 15) 5'-AGGGACUGCCUUAGGAGAAAGUU-3' (SEQ ID NO: 16)

[0054] In the present invention, the miR-8485 is characterized by comprising the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2, and preferably is a DNA / DNA, RNA / RNA, or DNA / RNA hybrid composed of the nucleotide sequence represented by SEQ ID NO: 1 and the nucleotide sequence represented by SEQ ID NO: 2. miR-8485 5'-ACGUGUGUGUGUGUGUGUGUU-3' (SEQ ID NO: 1) 5'-CACACACACACACACACGUAU-3' (SEQ ID NO: 2)

[0055] The miR-3189, miR-3199, and miR-8485 of the present invention were confirmed to increase the expression of microphthalmia-associated transcription factor (MITF), tyrosinase (TYR), tyrosinase-related protein 1 (TYRP1), and tyrosinase-related protein 2 (TYRP2), and miR-3139 was confirmed to increase the expression of TYR and TYRP2.

[0056] The genes whose expression is increased by the miRNA of the present invention are known to have the following functions.

[0057] MITF is a transcription factor that regulates the expression of the TYR, TYRP1, and TYRP2 genes, which are enzymes directly involved in melanin pigment synthesis, and is known as an important transcription factor for the development and differentiation of melanocytes (D'Mello, S. A., Finlay, G. J., Baguley, B. C. & Askarian-Amiri, M. E. Signaling Pathways in Melanogenesis. Int J Mol Sci. 17 (2016); Kawakami A, Fisher DE. The master role of microphthalmia-associated transcription factor in melanocyte and melanoma biology. Lab Investig. (2017)).

[0058] TYR, TYRP1, and TYRP2 are mainly involved in converting tyrosine into melanin pigment. In particular, TYR and TYRP2 are known as important enzymes that affect the quantity and quality of melanin (NIU, C., AISA, H.A..、2017: Upregulation of melanogenesis and tyrosinase activity: potential agents for vitiligo. Molecules 22, E1303. (2017); D'Mello, S. A., Finlay, G. J., Baguley, B. C. & Askarian-Amiri, M. E. Signaling Pathways in Melanogenesis.Int J Mol Sci. 17 (2016)).

[0059] As described in the background art above, the seed region corresponding to the second base to the eighth or ninth base of the miRNA active sequence is the main factor of activity. However, when producing double-stranded oligonucleotides, long double-stranded oligonucleotides containing this can also be produced and used.

[0060] Also, the miRNA may be double-stranded and, in other embodiments, may contain a single-molecule polynucleotide. For example, it may be an antisense oligonucleotide or miRNA, but is not limited thereto.

[0061] On the other hand, miR-3139, miR-3189, miR-3199, or miR-8485 can contain a sequence in which one or more bases are substituted, deleted, or inserted in the sense strand constituting it or the antisense strand complementary thereto.

[0062] The double-stranded oligonucleotide according to the present invention can include an overhang, which is a structure including one or more unpaired nucleotides at the 3'-end of one or both strands. Further, the sense strand or the antisense strand is preferably composed of 19 to 31 nucleotides, but is not limited thereto.

[0063] In the present invention, in the case of an oligo conjugate in which a hydrophilic substance and a hydrophobic substance are bound to the RNA or DNA oligonucleotide, not only can the oligonucleotide be efficiently delivered in vivo through a conjugate in which the hydrophilic substance and the hydrophobic substance are bound to both ends of the RNA or DNA oligonucleotide, but also the stability can be improved.

[0064] In addition, self-assembled nanoparticles are formed by the hydrophobic interaction of the hydrophobic substance. Such nanoparticles not only have extremely excellent delivery efficiency in the body and stability in the body, but also have a very uniform particle size due to the improvement of the structure and are easy to perform QC (Quality control). Therefore, there is an advantage that the manufacturing process as a drug is simple.

[0065] In one aspect, the hydrophilic substance is (P) n , (P m -J) n or (J-P m ) n wherein P is a hydrophilic substance monomer, n is 1 to 200, m is 1 to 15, and J is a linker that links m hydrophilic substance monomers to each other or m hydrophilic substance monomers and the oligonucleotide.

[0066] In the present invention, the hydrophilic substance is characterized by having a molecular weight of 200 to 10,000.

[0067] In the present invention, the hydrophilic substance is any one selected from the group consisting of polyethylene glycol, polyvinyl pyrrolidone, and polyoxazoline, but is not limited thereto.

[0068] In the present invention, the hydrophilic substance monomer (P) is characterized by having the structure of the following compound (1).

Chemical formula

[0069] In the present invention, the linker (J) is characterized by being selected from the group consisting of PO3 - , SO3, and CO2.

[0070] In the present invention, the molecular weight of the hydrophobic substance is characterized by being 250 to 1,000, and the hydrophobic substance is a steroid derivative, glyceride derivative, glycerol ether, polypropylene glycol, C 12 to C 50 unsaturated or saturated hydrocarbon, diacylphosphatidylcholine, fatty acid, phospholipid, and lipopolyamine, but is not limited thereto.

[0071] In the present invention, the steroid derivative is characterized by being selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholestanylamine, but is not limited thereto.

[0072] In the present invention, the glyceride derivative is selected from, but not limited to, mono-, di- and tri-glycerides.

[0073] In the present invention, the covalent bond represented by X and Y is characterized by being a non-degradable bond or a degradable bond. The non-degradable bond is characterized by being an amide bond or a phosphorylation bond, and the degradable bond is characterized by being a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzyme-degradable bond, but is not limited thereto.

[0074] When the hydrophilic substance is A, the double-stranded oligonucleotide structure according to the present invention can have the structure of the following structural formula (1').

Chemical formula

[0075] As one aspect, the double-stranded oligonucleotide structure containing miRNA according to the present invention is a double-stranded oligonucleotide structure containing the structure of the following structural formula (2). A-X-5' R 3' Y-B Structural formula (2) In the structural formula (2), A, B, X, Y and R have the same definitions as in structural formula 1.

[0076] More preferably, the double-stranded oligonucleotide structure has the structure of the following structural formula (2').

Chemical formula

[0077] In one aspect, the hydrophilic substance may be a cationic or non-ionic polymer substance having a molecular weight of 200 to 10,000, preferably a non-ionic polymer substance having a molecular weight of 1,000 to 2,000. In the case of the hydrophilic substance, as the non-ionic hydrophilic polymer compound, for example, it is characterized by being any one selected from the group consisting of polyethylene glycol, polyvinylpyrrolidone, and polyoxazoline, but is not limited thereto.

[0078] In another aspect, when the hydrophilic substance is (P m -J) n or (J-P m ) n the double-stranded oligonucleotide structure according to the present invention has the structure of the following structural formula (3) or structural formula (4). (P m -J) n -X-R-Y-B Structural formula (3) (J-P m ) n -X-R-Y-B Structural formula (4) In the structural formula (3) and structural formula (4), P is a hydrophilic substance monomer, n is 1 to 200, m is 1 to 15, J is a linker that connects between m hydrophilic substance monomers or between m hydrophilic substance monomers and the oligonucleotide to each other, X and Y are each independently a simple covalent bond or a covalent bond via a linker, and R is the specific miRNAs of the present invention. More preferably, the double-stranded oligonucleotide structure containing miRNA according to the present invention has the structure of the following structural formula (3').

Chemical formula

[0079] More preferably, the double-stranded oligonucleotide structure containing miRNA according to the present invention has the structure of the following structural formula (4').

Chemical formula

[0080] The hydrophilic substance monomer (P) in the structural formula (3) and the structural formula (4) can be used without limitation as long as it is a monomer of a nonionic hydrophilic polymer that meets the purpose of the present invention. Preferably, it is a monomer selected from the compounds (1) to (3) described in Table 1, more preferably the monomer of the compound (1). G in the compound (1) can preferably be selected from CH2, O, S and NH.

[0081] In particular, among the hydrophilic substance monomers, the monomer represented by the compound (1) can introduce various functional groups, has good affinity in vivo, and not only has excellent biocompatibility such as inducing few immune reactions, but also increases the in vivo stability of the oligonucleotide contained in the structure according to the structural formula (3) and the structural formula (4), and can increase the delivery efficiency. It is very suitable for the production of the structure according to the present invention.

[0082]

Table 1

[0083] The hydrophilic substance in the structural formula (3) and the structural formula (4) preferably has a total molecular weight in the range of 1,000 to 2,000. Therefore, for example, when hexaethylene glycol by the compound (1), that is, a substance in which G in the structural formula (3) and the structural formula (4) is O and m is 6 is used, since the molecular weight of the hexaethylene glycol spacer is 344, the number of repeating times (n) is preferably 3 to 5. The present invention, if necessary, in the structural formula (3) and the structural formula (4) (P m -J) or (J-Pm It is characterized in that the repeating unit of the hydrophilic group represented by ( )), that is, the hydrophilic substance block, can be used in an appropriate number represented by n. The hydrophilic substance monomer P and the linker J contained in each hydrophilic substance block may be the same or different independently between each hydrophilic substance block. That is, when three hydrophilic substance blocks are used (n = 3), the hydrophilic substance monomer by compound (1) is used in the first block, the hydrophilic substance monomer by compound (2) is used in the second block, and the hydrophilic substance monomer by compound (3) is used in the third block. Thus, different hydrophilic substance monomers may be used for each hydrophilic substance block, or any one hydrophilic substance monomer selected from the hydrophilic substance monomers by compound (1) to compound (3) may be used in the same way for all hydrophilic substance blocks. Similarly, for the linker that mediates the bonding of the hydrophilic substance monomers, the same linker may be used for each hydrophilic substance block, or different linkers may be used for each hydrophilic substance block. Also, m, which is the number of hydrophilic substance monomers, may be the same or different between each hydrophilic substance block. That is, three hydrophilic substance monomers are linked (m = 3) in the first hydrophilic substance block, five (m = 5) in the second hydrophilic substance block, and four (m = 4) in the third hydrophilic substance block. Thus, different numbers of hydrophilic substance monomers may be used, or the same number of hydrophilic substance monomers may be used in all hydrophilic substance blocks.

[0084] In the present invention, the linker (J) is preferably selected from the group consisting of PO3 - , SO3 and CO2, but is not limited thereto. It is obvious to those skilled in the art that any linker can be used as long as it meets the object of the present invention depending on the monomer of the hydrophilic substance used and the like.

[0085] All or part of the hydrophilic substance monomer may be modified, if necessary, to have functional groups necessary for binding to other substances such as target-specific ligands.

[0086] In some cases, one to three phosphate groups may be bonded to the 5'-end of the antisense strand of the double-stranded oligonucleotide structure containing miRNA specific to the gene.

[0087] For example, the double-stranded oligonucleotide structure containing miRNA has the structure of the following structural formula (3'') or structural formula (4''). [Chemical formula] The hydrophobic substance (B) plays a role in forming nanoparticles composed of the oligonucleotide structure according to structural formula (1) through hydrophobic interaction.

[0088] The hydrophobic substance preferably has a molecular weight of 250 to 1,000, and steroid derivatives, glyceride derivatives, glycerol ether, polypropylene glycol, C 12 to C 50 unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, lipopolyamines, etc. can be used, but are not limited thereto. It is obvious to those having ordinary knowledge in the technical field to which the present invention pertains that any hydrophobic substance can be used as long as it meets the object of the present invention.

[0089] The steroid derivative can be selected from the group consisting of cholesterol, cholestanol, cholanic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine, and the glyceride derivative can be selected from mono-, di-, and tri-glycerides, etc. At this time, the fatty acid of the glyceride is preferably an unsaturated or saturated fatty acid having C 12 to C 50 .

[0090] In particular, among the hydrophobic substances, saturated or unsaturated hydrocarbons and cholesterol are preferable in that they have the advantage of being easily bound at the synthesis stage of the oligonucleotide structure according to the present invention.

[0091] The hydrophobic substance binds to the distal end on the opposite side of the hydrophilic substance and may bind to any position of the sense strand or antisense strand of miRNA.

[0092] In the present invention, the hydrophilic substance, hydrophilic substance block, or hydrophobic substance and the oligonucleotide are bound by a simple covalent bond or a covalent bond (X or Y) via a linker. The covalent bond may be either a non-degradable bond or a degradable bond. At this time, examples of the non-degradable bond include an amide bond or a phosphorylated bond, and examples of the degradable bond include a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-degradable bond, but are not limited thereto.

[0093] The miRNA sequence that can be used as an active ingredient of a composition for improving graying of hair, preventing or improving hair loss, promoting hair follicle cell proliferation, and / or promoting hair growth by promoting melanocyte activity and melanin production provided by the present invention is a sequence derived from a human gene, but the gene from which the miRNA is derived is not limited to a human gene, and miRNA sequences obtained from genes derived from other animals can also be used.

[0094] The miRNA can be used in the form of various miRNA derivatives (miRNA mimics) that generate the biological equivalent effects of miRNA, and modified miRNAs containing miRNA sequences with the same seed region can be used. At this time, the lengths of the miRNA sense and antisense sequences can be reduced, and short derivatives composed of 15 nucleotides in length can also be used.

[0095] As miRNA derivatives for the miRNA, they can partially contain a phosphorothiolate structure in which the phosphate backbone structure of RNA is substituted with other elements such as sulfur, and can be used in a form that is entirely or partially substituted with DNA, PNA (peptide nucleic acids), and LNA (locked nucleic acid) molecules instead of RNA. Also, they can be used in a form in which the 2'-hydroxyl group of the RNA sugar is substituted with various functional structures, which includes but is not limited to methylation, methoxylation, fluorination, etc.

[0096] The miRNA is not limited to the double-stranded RNA of the mature miRNA and the miRNA derivative derived therefrom, and can be used in the form of a miRNA precursor. The miRNA precursor can also be partially or entirely substituted with the RNA phosphate backbone structure, DNA, PNA, and LNA of the RNA nucleic acid, and the 2'-hydroxyl group of the RNA sugar molecule can be modified.

[0097] The miRNA can be used in the form of a miRNA precursor or primary miRNA (pre-miRNA), which can be synthesized by chemical methods or transmitted to cells in the form of a plasmid for expression.

[0098] In the present invention, methods for transmitting miRNA to cells cultured in a culture dish can include methods using a mixture with a cationic lipid, methods of transmission by electrical stimulation, and methods using a virus, etc. However, those skilled in the art can easily apply and use various methods known in the art for transmitting miRNA, and are not limited to the above methods.

[0099] From another aspect, the present invention relates to a double-stranded oligonucleotide structure and nanoparticles containing the miRNA of miR-3139, miR-3189, miR-3199, or miR-8485.

[0100] As described above, the double-stranded oligonucleotide structure containing the miRNA is amphiphilic, containing both hydrophobic and hydrophilic substances. The hydrophilic part has an affinity through interactions such as hydrogen bonding with water molecules present in the body, so it tends to face outward. The hydrophobic substances tend to face inward through hydrophobic interactions between them, forming thermodynamically stable nanoparticles. That is, hydrophobic substances are located at the center of the nanoparticles, and hydrophilic substances are located in the outer direction of the double-stranded oligonucleotide containing the miRNA, forming nanoparticles in a form that protects the miRNA sequence.

[0101] The nanoparticles according to the present invention are characterized in that they may be formed only of double-stranded oligonucleotide structures having the same sequence, or may be composed of double-stranded oligonucleotide structures having different sequences. Double-stranded oligonucleotide structures containing different miRNA that promote melanogenesis and promote hair follicle cell proliferation may be included in the nanoparticles according to the present invention. For example, the nanoparticles may be composed of a mixture of a double-stranded oligonucleotide structure containing miR-3139 and a double-stranded oligonucleotide structure containing miR-8485.

[0102] In addition, a double-stranded oligonucleotide containing a miRNA that promotes melanogenesis and promotes hair follicle cell proliferation through other melanocyte activities other than the double-stranded oligonucleotide structure containing the miRNA, or a double-stranded oligonucleotide structure containing the same may be further included in the composition according to the present invention.

[0103] In the present invention, the double-stranded oligonucleotide structure (SAMiRNA TM ) containing the miRNA and the melanocyte activity and melanogenesis promoting effect of the nanoparticles and the resulting improvement effect on graying were confirmed. In addition, the promoting effect on the proliferation of dermal papilla cells and keratinocytes present in the hair follicles was confirmed together with the activity of melanogenic cells.

[0104] Therefore, the present invention can utilize the composition for pharmaceutical use and cosmetics. The pharmaceutical composition is characterized in that it is used in a dosage form selected from ointments, pastes, gels, jellies, serums, aerosol sprays, non-aerosol sprays, foams, creams, lotions, solutions or suspensions, but is not limited thereto.

[0105] When the composition is used as a cosmetic, although not limited thereto, hair tonic, hair conditioner, hair essence, hair lotion, hair nourishing lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nourishing cream, hair moisturizing cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nourishing pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservation treatment agent, hair dye, hair waving agent, hair bleaching agent, hair gel, hair glaze, hair dressing agent, hair lacquer, hair moisturizer, hair mousse or hair spray. It is characterized by being used in a dosage form selected from the dosage forms.

[0106] In addition to the above-mentioned active ingredient, the composition of the present invention can be produced by further containing one or more pharmaceutically acceptable carriers. The pharmaceutically acceptable carrier must be compatible with the active ingredient of the present invention, and physiological saline, sterilized water, Ringer's solution, buffered physiological saline, dextrose solution, maltodextrin solution, glycerol, ethanol, and one or a mixture of two or more of these components can be used, and other ordinary additives such as antioxidants, buffers, bacteriostatic agents can be added as necessary. Further, a diluent, a dispersant, a surfactant, a binder, and a lubricant can be additionally added to formulate into injection dosage forms such as aqueous solutions, suspensions, emulsions, etc. In particular, it is desirable to formulate and provide in the form of a freeze-dried (lyophilized) dosage form. For the production of the freeze-dried dosage form, methods commonly known in the technical field to which the present invention belongs can be used, and a stabilizer for freeze-drying may be added. Furthermore, appropriate methods in the art or methods disclosed in Remington's pharmaceutical Science (Mack Publishing company, Easton PA) can be utilized to preferably formulate according to each disease or component.

[0107] The content and administration method of the active ingredient and the like contained in the composition of the present invention can be determined by an ordinary expert in the technical field based on the number of melanocytes and the degree of functional decline of an ordinary individual, and the severity of the symptoms and sites of graying. Also, it can be formulated in various forms such as acids, tablets, injections, ointments, functional cosmetics, etc., and can also be provided in unit-dose or multi-dose containers, such as sealed ampoules and bottles.

[0108] When the double-stranded oligonucleotide structure containing the miRNA according to the present invention, the composition or nanoparticles containing the same are used in the production of functional cosmetics or topical skin agents, the dosage form of the functional cosmetics or topical skin agents is characterized by being selected from the group consisting of creams, lotions, gels, water-soluble liquids, and essences, but is not limited thereto.

[0109] From another aspect, the present invention relates to a subject in need of improving graying of hair, promoting hair growth and / or preventing or improving hair loss (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) a double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) A method for improving hair whitening, promoting hair growth and / or preventing or improving hair loss, comprising the step of administering nanoparticles containing the double-stranded oligonucleotide structure is provided.

[0110] From another aspect, the present invention relates to (i) miR-3139, miR-3189, miR-3199 or miR-8485 for improving graying of hair, promoting hair growth and / or preventing or improving hair loss (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) a double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) Use of nanoparticles containing the double-stranded oligonucleotide structure is provided.

[0111] From another aspect, the present invention relates to (i) miR-3139, miR-3189, miR-3199, or miR-8485 for the manufacture of a pharmaceutical or cosmetic for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1): A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance, B is a hydrophobic substance, X and Y each independently represent a simple covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199 or miR-8485. Or (iii) Provide the use of nanoparticles containing the double-stranded oligonucleotide structure.

Example

[0112] Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art that these examples are for illustrative purposes only and should not be construed as limiting the scope of the present invention.

[0113] Example 1: Screening for miRNAs that promote melanogenesis 1-1: Promoting Melanin Production Human miRNA Candidate Group As the 21st version of the human miRNA sequence provided by the miRNA database miRBase (www.mirbase.org), 1728 miRNAs were synthesized in the form of being incorporated into a double-stranded oligonucleotide structure (SAMiRNA TM ) according to the stem-loop structure criterion (Table 2). The synthesis of all the synthesized miRNA strands was discriminated and confirmed for the presence or absence of the intended sequence using a mass spectrometer of the MALDI-TOF method. Using 1728 miRNAs as targets for human skin cancer cell lines, screening was performed to find the miRNA with the highest melanin production. The sequences of the 1728 human miRNA sense strands experimented in the present invention are known in miRbase (http: / / www.mirbase.org). When the seed region is present only in one strand, a complementary sequence was synthesized for transmission to cells in the form of a double strand.

[0114] For example, the miRNA of miR-3139 was produced as follows. SEQ ID NO: 9: 5'-CAGGCAUCUGUUGAGCUCCUAUU-3' SEQ ID NO: 10: 5'-UAGGAGCUCAACAGAUGCCUGUU-3'

[0115] Also, when the seed sequence is present in both strands, each strand was synthesized as the sense and antisense strands. For example, the miRNA of miR-3189 was produced as follows. SEQ ID NO: 5: 5'-UGCCCCAUCUGUGCCCUGGGUAGGA-3' (sense) SEQ ID NO: 6: 5'-CCCUUGGGUCUGAUGGGGUAG-3' (antisense)

[0116]

Table 2

[0117] 1-2: Cell Culture and miRNA Transfection To discover miRNAs that effectively increase melanin production, the human melanoma M21 (Korean cell line bank, KR), a human-derived skin cancer cell line, was used. The M21 cell line was cultured at 37°C / 5% CO2 using DMEM medium (Hyclone, US) containing 10% Fetal Bovine Serum (FBS, Hyclone, US) and 1% Penicillin-Streptomycin (Hyclone, US). M21 cells were dispensed into a 12 well plate (Falcon, US) at a condition of 4×10 4 cells / well, and the next day, miRNAs were transfected at 40 nM according to the manufacturer's protocol using Lipofectamine RNAiMAX (Invitrogen, US).

[0118] 1-3: Primary Screening and Reproducibility Evaluation of 1728 miRNAs by Measuring Melanin Production 1728 kinds of miRNAs were repeatedly transfected into M21 cells three times by the method of Example 1-2. After culturing for 72 hours, all the cells were collected, reacted with 1M NaOH (Bioneer, KR) at 37°C for 2 hours according to the melanin extraction protocol, and the absorbance was measured at a wavelength of 405 mm to compare the color change and the amount of melanin production. After culturing for 72 hours, all the cells were collected, reacted with 1M NaOH (Bioneer, KR) at 37°C for 2 hours according to the melanin extraction protocol, and the absorbance was measured at a wavelength of 405 mm to analyze the amount of melanin production. For reproducibility evaluation, the top 9 kinds of miRNAs were used to treat the M21 cell line by the method of Example 1-2. After culturing for 72 hours, all the cells were collected, reacted with 1M NaOH (Bioneer, KR) at 37°C for 2 hours according to the melanin extraction protocol, and the absorbance was measured at a wavelength of 405 mm to analyze the amount of melanin production.

[0119] As a result, nine miRNAs that showed a melanin production level of 13.0 μg / ml or more (1.88-fold or more compared to the negative control group) in the M21 cell line, which had processed a total of 1,728 types, and showed a melanin production level similar to that of the positive control group, IBMX (Sigma, US) / CuSO4 (Sigma, US) treatment group, were selected (Figure 1). Even in the repeated experiments for reproducibility evaluation, higher color change and melanin production were confirmed in the M21 cell line by the top nine selected miRNAs compared to the negative control group (Figure 2, Table 3).

[0120]

Table 3

[0121] Example 2: Screening of the final sequences through reproducibility evaluation of the nine selected miRNAs and analysis of the melanin production mechanism 2-1: Cell Culture and miRNA Transfection For the reproducibility evaluation of the top nine in melanin production, SK-MEL-28 (Korean cell line bank, KR), a human-derived skin cancer cell line, was used. The SK-MEL-28 cell line was cultured at 37°C / 5% CO2 using MEM medium (Hyclone, US) containing 10% FBS (Hyclone, US) and 1% Penicillin-Streptomycin (Hyclone, US). SK-MEL-28 cells were dispensed into a 12-well plate (Falcon, US) at a condition of 4×10 4 cells / well, and the next day, miRNAs were transfected at 40 nM according to the manufacturer's protocol using Lipofectamine RNAiMAX (Invitrogen, US).

[0122] 2-2: Reproducibility Evaluation of the Top 9 miRNAs through Melanin Production Analysis For the reproducibility evaluation of the top 9 selected miRNAs, color change and melanin production were measured in SK-MEL-28 cells. miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199 or miR-933 was transfected into SK-MEL-28 cells 3 times each according to the method of Example 2-1. After culturing for 72 hours, all cells were collected to observe color change. For measuring melanin production, after reacting with 1 M NaOH (Bioneer, KR) at 37°C for 2 hours according to the melanin extraction protocol, absorbance was measured at a wavelength of 405 nm to analyze melanin production.

[0123] As a result, significant color change and increased melanin production were observed in the groups treated with miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199 or miR-933 compared to the negative control group. When compared with the melanin inducer as the positive control group, significantly better color change and increased melanin production were confirmed (Figure 3).

[0124] 2-3: RT-qPCR Analysis for Analyzing the Mechanism of Promoting Melanin Production in SK-MEL-28 Cells To analyze the mechanism of action of the top 9 selected miRNAs in promoting melanin production, RT-qPCR analysis was performed for gene analysis of important factors in the melanin production process, such as MITF, TYR, TYRP1, and TYRP2. The SK-MEL-28 cell line was seeded at 4×10 4After dispensing at cells / well, the cells were cultured under the conditions of 37°C / 5% CO2. On the next day, miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199 or miR-933 was transfected into SK-MEL-28 cells three times repeatedly by the method of Example 2-1. After culturing for 96 hours, total RNA was extracted from the cell lysate using Universal RNA extraction kit (Bioneer, KR), and using this RNA as a template, AccuPower GreenStar TM RT-qPCR Master Mix (Bioneer, KR) was used to analyze the mRNA expression levels of the said gene (Human qPCR panel kit, Bioneer, KR) and RPL13A (Human reference qPCR primer set, Bioneer, KR) by qRT-PCR according to the manufacturer's protocol. The Ct values of the two genes derived after the qPCR array were used to calculate the relative amount (fold change) of each gene mRNA in the control group compared to the experimental group by relative quantitative analysis through the 2(- Delta Delta C(T)) Method [Livak KJ, Schmittgen TD. 2001. Methods. Dec;25(4):402-8]. The primer sequences for each gene are as follows (Table 4).

[0125]

Table 4

[0126] As a result, it was confirmed that the gene expressions of TYR, TYRP1 and TYRP2, which are enzymes directly involved in melanin synthesis in melanosomes, were increased by the nine miRNAs (Figure 4). In the case of MITF, a transcription factor, it was confirmed that there was a weak increase or no change, which is interpreted as being due to the mechanism that the regulation of MITF is regulated not only by gene expression but also by protein stability and activation (activity).

[0127] 2-4: RT-qPCR Analysis for Analyzing the Mechanism of Promoting Melanin Production in Human Melanocyte-forming Cells We analyzed the gene expression of MITF, TYR, TYRP1, and TYRP2 by nine miRNAs using human epidermal melanocytes obtained from normal human skin tissue. Human epidermal melanocytes (Invitrogen, US) cells were cultured at 1 × 10 5 After dispensing at 1000 cells / well, the cells were cultured in mammalian cell culture / primary cell culture (Gibco, US) containing human melanocyte growth supplement-2 (Gibco, US) at 37°C / 5% CO2. The next day, the cells were treated with 9 types of miR-8485, miR-7978, miR-6074, miR-3132, miR-4644, miR-3139, miR-3189, miR-3199, or miR-933 at 5μM each. After 96 hours of culture, total RNA was extracted from the cell lysate using a Universal RNA extraction kit (Bioneer, KR), and this RNA was used as a template in AccuPower GreenStar TM The mRNA expression levels of the genes (Human qPCR panel kit, Bioneer, KR) and RPL13A (Human reference qPCR primer set, Bioneer, KR) were analyzed by qRT-PCR using RT-qPCR Master Mix (Bioneer, KR) according to the manufacturer's protocol.

[0128] As a result, in human normal melanocyte-forming cells, unlike the results of melanoma cells M21, it was confirmed that the gene expression of MITF increased by about 1.2 to 1.7 times compared to the negative control group by miR-8485, miR-7978, miR-3139, miR-3189, miR-3199, and miR-933. Also, in the case of TYR, it was confirmed that it increased by about 1.2 to 3.1 times by 9 kinds of miRNAs. In the case of TYRP1 and TYRP2, differences in expression patterns were observed for each of the 9 miRNA sequences. However, in the case of miR-8485, miR-7978, miR-3189, miR-3199, and miR-933, both TYRP1 and TYRP2 showed a tendency to increase. In the case of miR-6074, miR-3139, and miR-4644, it was confirmed that only TYRP2 increased (Figure 5).

[0129] 2-5: Western Blot Analysis for Analyzing the Mechanism of Promoting Melanin Production Western blot analysis was performed to analyze the expression of MITF and TYR proteins, which are important factors in the melanin production process. The SK-MEL-28 cell line was dispensed into a 6-well plate (Falcon, US) at 1×10 5 cells / well and then cultured under the conditions of 37°C / 5% CO2. The next day, miR-3139, miR-3189, miR-3199, or miR-8485 was transfected into SK-MEL-28 cells by the method of Example 2-1. After culturing for 96 hours, analysis was performed according to the western blot protocol. As the primary antibodies, MITF (Santa Cruz Biotechnology Inc., US) and Tyrosinase (Santa Cruz Biotechnology Inc., US) were used. As the secondary antibodies, HRP-linked-anti-mouse IgG (Cell Signaling Technology, US) and HRP-linked-anti-rabbit IgG (Cell Signaling Technology, US) were used.

[0130] As a result, it was confirmed that MITF and TYR proteins were increased by nine miRNAs, similar to the above RT-qPCR results (Figure 6). By comprehensively analyzing the cell color changes, melanin synthesis amount, and the expression results of major genes and proteins in the melanin production process after treatment with the top nine selected miRNAs, four miRNAs, miR-3139, miR-3189, miR-3199, and miR-8485, with the highest melanin synthesis amount and obvious gene and protein expression of MITF, TYR, and TYRP1 / 2, were finally selected (Table 5).

[0131]

Table 5

[0132] Example 3: Efficacy evaluation of the four finally selected miRNAs on human hair Using the four finally selected miRNAs, an efficacy evaluation was carried out on human white hair. On the experimental day, the hair was collected by plucking the tip of the white hair, cut about 1 - 2 cm from the hair root, and then cultured in 200 μL of DMEM / F12 medium (Gibco, US) containing 10% FBS (Hyclone, US) and 1% Penicillin-Streptomycin (Hyclone, US) in a 24-well plate (Falcon, US). The growth of white hair root cells, hair color changes, and properties were observed while treating the four miRNAs at a concentration of 5 μM at two-day intervals for about 35 days.

[0133] As a result, there were no significant changes during the treatment period in the negative control group. However, in the groups treated with miR-3139, miR-3189, miR-3199, or miR-8485, with the passage of time, the outer root sheath part of the hair follicle expanded, an increase in the proliferation or migration of hair follicle cells and keratinocytes was observed, and an increase in the length of the hair follicle was also observed (Figure 7). In addition, it was confirmed that the overall graying was improved including the hair shaft (Figure 8).

[0134] Example 4: Evaluation of the Efficacy of SAMiRNA-miR-3199 in Promoting Melanogenesis in SK-MEL-28 Cells 4-1: Cell Culture and Treatment with SAMiRNA-miR-3199 For the evaluation of the efficacy of SAMiRNA-miR-3199 in promoting melanogenesis, the human-derived skin cancer cell line SK-MEL-28 (Korean cell line bank, KR) was used. The SK-MEL-28 cell line was cultured at 37 °C / 5% CO2 using MEM medium (Hyclone, US) containing 10% FBS (Hyclone, US) and 1% Penicillin-Streptomycin (Hyclone, US). SK-MEL-28 cells were dispensed into a 12-well plate (Falcon, US) at a condition of 4×10 4 cells / well and treated at a concentration of 5 μM the next day.

[0135] 4-2: Melanin Production Analysis by Treatment with SAMiRNA-miR-3199 SK-MEL-28 cells were treated with SAMiRNA-miR-3199 by the method of Example 4-1. After culturing for 72 hours, all the cells were collected to observe the color change. For the measurement of the melanin production amount, after reacting with 1 M NaOH (Bioneer, KR) at 37 °C for 2 hours according to the melanin extraction protocol, the absorbance was measured at a wavelength of 405 mm to analyze the melanin production amount.

[0136] As a result, when treated with SAMiRNA-miR-3199, a significant color change and an increase in the amount of melanin were observed compared to the negative control group (Figure 9a).

[0137] 4-3: Expression Analysis of Melanin Production-related Genes by Treatment with SAMiRNA-miR-3199 RT-qPCR analysis was performed for the gene analysis of melanogenesis-related genes MITF, TYR, TYRP1, and TYRP2 by SAMiRNA-miR-3199 treatment. SK-MEL-28 cell line was treated with SAMiRNA-miR-3199 by the method of Example 4-1. After culturing for 96 hours, total RNA was extracted from the cell lysate using the Universal RNA extraction kit (Bioneer, KR), and this RNA was used as a template for AccuPower GreenStarTM The mRNA expression levels of the gene (Human qPCR panel kit, Bioneer, KR) and RPL13A (Human reference qPCR primer set, Bioneer, KR) were analyzed by qRT-PCR using RT-qPCR Master Mix (Bioneer, KR) according to the manufacturer's protocol. The Ct values of the two genes derived after qPCR array were used to calculate the relative amount (fold change) of each gene mRNA in the experimental group compared with the control group through relative quantitative analysis using the 2(-Delta Delta C(T)) Method [Livak KJ, Schmittgen TD. 2001. Methods. Dec;25(4):402-8].

[0138] As a result, it was confirmed that MITF, a core transcription factor for melanin synthesis, was significantly increased by SAMiRNA-miR-3199 treatment, and the gene expressions of TYR, TYRP1, and TYRP2, which are core enzymes for melanin synthesis, were greatly increased (Figure 9b).

[0139] 4-4: Analysis of the Expression Level of Melanin Production-related Proteins by Treatment with SAMiRNA-miR-3199 Immunoblot was performed for the analysis of MITF and TYR protein expression, which are core factors in the melanin production process. The SK-MEL-28 cell line was seeded at 1×10 in a 6-well plate (Falcon, US). 5After dispensing at cells / well, it was cultured under the conditions of 37 °C / 5% CO2. The next day, the SK-MEL-28 cell line was treated with 5 μM SAMiRNA-miR-3199. After culturing for 96 hours, analysis was performed according to the immunoblot protocol. MITF (Santa Cruz Biotechnology Inc., US), Tyrosinase (Santa Cruz Biotechnology Inc., US), and α-Tubulin (Cell Signaling Technology, US) were used as primary antibodies, and HRP-linked-anti-mouse IgG (Cell Signaling Technology, US) and HRP-linked-anti-rabbit IgG (Cell Signaling Technology, US) were used as secondary antibodies.

[0140] As a result, similar to the gene expression analysis results by RT-qPCR, it was confirmed that the MITF and TYR proteins were significantly increased by the treatment with SAMiRNA-miR-3199 (Figure 9c).

[0141] 4-5: Intracellular Expression Analysis of Melanin Production-related Proteins by Treatment with SAMiRNA-miR-3199 For the analysis of the intracellular expression patterns of MITF and TYR proteins, which are the core factors of melanogenesis, immunocytochemistry analysis was performed. Before cell dispensing, cover glasses were placed in a 12-well plate (Falcon, US), coated with Poly-L-lysine for 1 hour, and then the SK-MEL-28 cell line was seeded at 2×10 4Dispensed at cells / well and cultured under the conditions of 37 °C / 5% CO2. The next day, the SK-MEL-28 cell line was treated with SAMiRNA-miR-3199 by the method of Example 4-1. After culturing for 96 hours, it was reacted with a 0.1% Triton-X100 solution for 10 minutes for cell permeabilization, then blocked with a solution containing 1% BSA for 1 hour. For the primary antibodies, MITF (abcam, UK) and Tyrosinase (Santa Cruz Biotechnology Inc., US) were used, and for the secondary antibodies, HRP-linked-anti-mouse IgG (Cell Signaling Technology, US) and HRP-linked-anti-rabbit IgG (Cell Signaling Technology, US) were used. Images were analyzed using a spinning disk confocal (Dragonfly high-speed confocal image platform, Andor) for fluorescence analysis of the stained cells.

[0142] As a result, in the case of MITF, a transcription factor, the nuclear expression increased significantly by SAMiRNA-miR-3199 treatment, and in the case of TYR, a melanin synthase, the expression in the cytoplasm with melanosomes increased significantly (Figure 9d).

[0143] Example 5: Target gene analysis and mechanism analysis of SAMiRNA-miR-3199 5-1: miR-3199 Target Gene Analysis Using the miRNA target gene prediction program (TargetScan, http: / / www / targetscan.org / ), among many predicted genes capable of binding to the seed sequence of miR-3199, the GSK3β gene, which is a key regulator of the main signaling pathway of the melanogenesis mechanism, was selected. Binding of miR-3199 to 4153-4159 of the 3'UTR of GSK3β was predicted (Figure 10a).

[0144] 5-2: Analysis of the Ability of SAMiRNA-miR-3199 to Inhibit the mRNA of Target Gene GSK3β RT-qPCR analysis was performed to analyze the inhibitory ability of SAMiRNA-miR-3199 on the target gene GSK3β mRNA. The SK-MEL-28 cell line was dispensed into a 12-well plate (Falcon, US) at 4×10 4 cells / well and then cultured under the conditions of 37 °C / 5% CO2. The next day, the SK-MEL-28 cell line was treated with SAMiRNA-miR-3199 by the method of Example 4-1. After culturing for 24 hours, total RNA was extracted from the cell lysate using the Universal RNA extraction kit (Bioneer, KR). Using this RNA as a template, the mRNA expression levels of GSK3β (Bioneer, KR) and RPL13A (Human reference qPCR primer set, Bioneer, KR) were analyzed by qRT-PCR according to the manufacturer's protocol using the AccuPower GreenStar TM RT-qPCR Master Mix (Bioneer, KR). The Ct values of the two genes derived after the qPCR array were used to calculate the relative amount (fold change) of each gene mRNA in the control group compared to the experimental group by relative quantitative analysis through the 2(- Delta Delta C(T)) Method [Livak KJ, Schmittgen TD. 2001. Methods. Dec;25(4):4 02-8]. The primer sequences for GSK3β are as follows (Table 6).

[0145]

Table 6

[0146] As a result, it was confirmed that the target gene GSK3β was significantly decreased by the treatment with SAMiRNA-miR-3199 (Figure 10b).

[0147] 5-3: Analysis of the Expression Level of GSK3β Protein by Treatment with SAMiRNA-miR-3199 An immunoblot was performed to analyze the ability of SAMiRNA-miR-3199 to inhibit GSK3β protein. The SK-MEL-28 cell line was dispensed into a 6-well plate (Falcon, US) at 1×10 5 cells / well and then cultured under conditions of 37°C / 5% CO2. The SK-MEL-28 cell line was treated with SAMiRNA-miR-3199 by the method of Example 4-1. After culturing for 96 hours, analysis was performed according to the immunoblot protocol. The primary antibodies used were GSK3β (Cell Signaling Technology Inc., US), β-catenin (Becton, Dickinson and Company Inc, US), and α-Tubulin (Cell Signaling Technology, US), and the secondary antibodies used were HRP-linked-anti-mouse IgG (Cell Signaling Technology, US) and HRP-linked-anti-rabbit IgG (Cell Signaling Technology, US).

[0148] As a result, similar to the results of GSK3β gene expression analysis by RT-qPCR, it was confirmed that the GSK3β protein decreased by SAMiRNA-miR-3199 treatment. GSK3β phosphorylates β-catenin to promote proteolysis and reduce protein stability. When the expression of GSK3β is inhibited, the protein stability of β-catenin increases and the protein increases. Therefore, it was confirmed that the β-catenin protein increased by SAMiRNA-miR-3199 treatment (Figure 10c).

[0149] 5-4: Analysis of the Expression Pattern of Intracellular GSK3β Protein by Treatment with SAMiRNA-miR-3199 Immunocytochemistry was performed to analyze the intracellular GSK3β protein expression pattern by SAMiRNA-miR-3199 treatment. Before cell dispensing, cover glasses were placed in a 12-well plate (Falcon, US) and coated with Poly-L-lysine for 1 hour. Then, the SK-MEL-28 cell line was at 2×104 They were dispensed at cells / well and cultured under the conditions of 37 °C / 5% CO2. The next day, the SK-MEL-28 cell line was treated with SAMiRNA-miR-3199 by the method of Example 4-1. After culturing for 48 hours, they were reacted with a 0.1% Triton-X100 solution for 10 minutes for cell permeabilization, then blocked with a solution containing 1% BSA for 1 hour. For the primary antibodies, GSK3β (Cell Signaling Technology Inc., US) and β-catenin (Becton, Dickinson and Company Inc, US) were used, and for the secondary antibodies, HRP-linked-anti-mouse IgG (Cell Signaling Technology, US) and HRP-linked-anti-rabbit IgG (Cell Signaling Technology, US) were used. They were analyzed using a spinning disk confocal (Dragonfly high-speed confocal image platform, Andor) for fluorescence analysis of the stained cells.

[0150] As a result, it was confirmed that the expression of GSK3β, the target gene, decreased by the treatment with SAMiRNA-miR-3199, and it was confirmed that the β-catenin protein increased significantly in the nucleus and cytoplasm (Figure 10d).

[0151] 5-5: Analysis of the Direct Binding of SAMiRNA-miR-3199 to the 3'UTR of GSK3β mRNA A luciferase reporter assay was performed to analyze the direct binding of miR-3199 to the 3'UTR of GSK3β mRNA. The SK-MEL-28 cell line was seeded in a 12 well plate (Falcon, US) at 3×10 4After dispensing at cells / well, they were cultured under the conditions of 37 °C / 5% CO2. On the next day, the SK-MEL-28 cell line was transfected with a vector inserted with GSK3β-wild type (WT) with a normal GSK3β binding site or GSK3β-mutant (MT) with a mutated GSK3β binding site (in a non-binding form) using Lipofectamine RNAiMAX (Invitrogen, US). At the same time, 5 μM of SAMiRNA-miR-3199 was treated respectively. After culturing for 96 hours, an experiment was conducted and analyzed according to the manufacturer's protocol using the Dual-Luciferase® Reporter Assay System (PromegaTM Corporation, US) for luciferase activity measurement.

[0152] As a result, when SAMiRNA-miR-3199 was treated on cells expressing GSK3β-WT with a normal binding site, the luciferase activity significantly decreased, and it was confirmed that there was no change in luciferase activity by SAMiRNA-miR-3199 treatment in cells expressing GSK3β-MT with a mutated binding site (Figure 10e). Thereby, it was confirmed that miR-3199 directly binds to the 3' UTR (4153-4159) of GSK3β mRNA and inhibits GSK3β.

[0153] Example 6: Evaluation of the Efficacy of SAMiRNA-miR-3199 in Promoting Melanin Production in Human Melanocytes 6-1: Cell Culture and Treatment with SAMiRNA-miR-3199 For the evaluation of the melanogenesis-promoting efficacy of SAMiRNA-miR-3199, Human Epidermal Melanocytes (HEMs, Invitrogen, US) obtained from normal human skin tissue were used. HEMs were cultured in mammalian cell culture / primary cell culture medium (Gibco, US) containing melanocyte growth supplement-2 (Gibco, US) at 37 °C / 5% CO2 conditions. HEMs were dispensed into a 12 well plate (Falcon, US) at a condition of 1×10 5 cells / well, and the next day, SAMiRNA-miR-3199 was treated at a concentration of 5 μM.

[0154] 6-2: Melanin Production Analysis by Treatment with SAMiRNA-miR-3199 For the evaluation of the efficacy of SAMiRNA-miR-3199, the color change and the amount of melanin production were measured for HEMs. HEMs were treated with SAMiRNA-miR-3199 at a concentration of 5 μM by the method of Example 6-1. After culturing for 72 hours, all the cells were collected to observe the color change. For the measurement of the amount of melanin production, after reacting with 1M NaOH (Bioneer, KR) at 37 °C for 2 hours according to the melanin extraction protocol, the absorbance was measured at a wavelength of 405 mm to analyze the amount of melanin production.

[0155] As a result, when treated with SAMiRNA-miR-3199, a significant color change and an increase in the amount of melanin were observed compared to the negative control group (Figure 11a).

[0156] 6-3: Expression Analysis of Melanin Production-related Genes by Treatment with SAMiRNA-miR-3199 After the treatment with SAMiRNA-miR-3199, RT-qPCR analysis was performed for gene analysis such as MITF, TYR, TYRP1, TYRP2, GSK3β, etc. HEMs were treated with SAMiRNA-miR-3199 by the method of Example 6-1. After culturing for 96 hours, total RNA was extracted from the cell lysate using a Universal RNA extraction kit (Bioneer, KR), and using this RNA as a template, AccuPower GreenStar TMThe mRNA expression levels of the said gene (Human qPCR panel kit, Bioneer, KR) and RPL13A (Human reference qPCR primer set, Bioneer, KR) were analyzed by qRT-PCR using RT-qPCR Master Mix (Bioneer, KR) according to the manufacturer's protocol. The Ct values of the two genes derived after qPCR array were used to calculate the relative amount (fold change) of each gene mRNA in the experimental group compared to the control group through relative quantitative analysis by the 2(-Delta Delta C(T)) Method [Livak KJ, Schmittgen TD. 2001. Methods. Dec;25(4):402-8].

[0157] As a result, it was confirmed that the gene expressions of MITF, TYR, TYRP1, and TYRP2 increased significantly by SAMiRNA-miR-3199 treatment (Figure 11b). Also, it was confirmed that GSK3β, which is the target gene of miR-3199, decreased significantly (Figure 11b).

[0158] 6-4: Analysis of the Expression Level of Melanin Production Core Proteins by Treatment with SAMiRNA-miR-3199 Immunoblot was performed to analyze the changes in MITF and TYR protein expressions by SAMiRNA-miR-3199 treatment. HEMs were dispensed into a 6-well plate (Falcon, US) at 2×10 5 cells / well and then cultured under the conditions of 37°C / 5% CO2. HEMs were treated with SAMiRNA-miR-3199 by the method of Example 6-1. After culturing for 96 hours, analysis was performed according to the immunoblot protocol. The primary antibodies used were MITF (Santa Cruz Biotechnology Inc., US), Tyrosinase (Santa Cruz Biotechnology Inc., US), and α-Tubulin (Cell Signaling Technology, US), and the secondary antibody used was HRP-linked-anti-mouse IgG (Cell Signaling Technology, US).

[0159] As a result, similar to the gene expression analysis results by RT-qPCR, it was confirmed that MITF and TYR proteins increased by SAMiRNA-miR-3199 treatment (Figure 11c).

[0160] Example 7: Evaluation of cytotoxicity and innate immunotoxicity of SAMiRNA-miR-3199 7-1: Cytotoxicity Evaluation of SAMiRNA-miR-3199 For the cytotoxicity evaluation of SAMiRNA-miR-3199, Human follicle dermal papilla cell (HFDPC, Promocell, DE), which is a human dermal papilla cell, HaCaT (ATCC, US), which is a keratinocyte, and Human Epidermal Melanocytes (Invitrogen, US) were used. HFDPC 3×10 3 cells / well, HaCaT 4×10 3 cells / well, and HEMs 5×10 3 cells / well were dispensed and then cultured under the conditions of 37°C / 5% CO2. The next day, SAMiRNA-miR-3199 was treated to each cell at different concentrations (0, 1, 5, 10, 20 μM). After culturing for 72 hours, for cytotoxicity measurement, an experiment was conducted and analyzed according to the manufacturer's protocol using a WST assay kit (DOGEN, KR).

[0161] As a result, no cytotoxicity was observed even at a high concentration of 20 μM of SAMiRNA-miR-3199 in all cell lines (Figure 12a).

[0162] 7-2: Natural Immune Toxicity Evaluation of SAMiRNA-miR-3199 For the evaluation of the innate immune toxicity of SAMiRNA-miR-3199, human peripheral blood mononuclear cells (PBMCs) (Cellular Technology Limited, US) were used. After dispensing at 5×105 cells / well in a 12-well plate (Falcon, US), the cells were cultured under conditions of 37°C / 5% CO2. The next day, SAMiRNA-miR-3199 was treated at different concentrations (0, 5, 10, 20 μM). After culturing for 6 hours, total RNA was extracted from the cell lysates using the Universal RNA extraction kit (Bioneer, KR), and using this RNA as a template, AccuPower GreenStar TM RT-qPCR Master Mix (Bioneer, KR) was used to analyze the mRNA expression levels of inflammatory cytokine genes (Human Immune qPCR panel kit, Bioneer, KR) and RPL13A (Human reference qPCR primer set, Bioneer, KR) by qRT-PCR according to the manufacturer's protocol. The primer sequences for each gene are as follows (Table 7).

[0163]

Table 7

[0164] As a result, even at the high concentration of 20 μM of SAMiRNA-miR-3199, no increase in inflammatory cytokines (such as IL-1β, IL-6, IL-12β, TNF-α, INF-γ, etc.) was observed, and it was confirmed that no innate immune toxicity appeared (Figure 12b).

[0165] Example 8: Double-stranded oligonucleotide structure (SAMiRNA) The double-stranded oligonucleotide structure produced in the present invention has a structure as shown in the following structural formula (5). C 18 -S-S-C6- 5' S 3' - polyethylene glycol 2000 Structural formula (5) 3' AS 5'-PO4 In the structural formula (5), S is the sense strand of miRNA, AS is the antisense strand of miRNA, PO4 is a phosphate group, polyethylene glycol is a hydrophilic substance monomer, and polyethylene glycol 2000 is a linker, and a phosphate group (PO3 - ) is bonded through, and C 24 is a hydrophobic substance and contains a disulfide bond, and 5' and 3' mean the terminal directions of the double-stranded oligo RNA.

[0166] For the sense strand of miRNA of the structural formula (5), using DMT-polyethylene glycol 2000-CPG as a support and connecting phosphodiester bonds that form an RNA backbone structure by using β-cyanoethyl phosphoramidite, after synthesizing an oligo RNA hydrophilic substance structure containing a sense strand with polyethylene glycol bonded to the 3'-terminal site, a hydrophobic C 24 containing a disulfide bond was bonded to the 5'-terminal to produce a sense strand of a desired RNA polymer structure. In the case of the antisense strand for annealing with the sense strand, an antisense strand having a sequence complementary to the sense strand was produced by using a method of connecting phosphodiester bonds constituting the RNA backbone structure by using β-cyanoethyl phosphoramidite, and then an antisense strand with a phosphate group bonded to the 5'-terminal was produced.

Industrial Applicability

[0167] The composition according to the present invention can activate melanocyte-forming cells, promote the production of melanin, prevent the graying of hair, slow down its progression rate, improve the hair that has already undergone graying to the hair state before graying, and has no side effects differentiated from the simple dyes that have been repeatedly used to conceal the conventional graying of hair, and can be usefully utilized as a pharmaceutical use and in cosmetics and hair dyeing compositions. Further, it can promote the proliferation of dermal papilla cells and keratinocytes present in the hair follicle together with the activity of melanin-producing cells, and can be usefully utilized as a pharmaceutical use and a cosmetic composition that can induce the alleviation of alopecia and hair growth promotion without side effects.

[0168] As described above, specific parts of the content of the present invention have been described in detail. However, it will be apparent to those skilled in the art that these specific techniques are merely preferred embodiments, and the scope of the present invention is not limited thereby. Therefore, it can be said that the substantial scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) a double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the above structural formula (1), A is a hydrophilic substance selected from any one of the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone, and polyoxazoline, or a hydrophilic substance having the following structural formula (5) or structural formula (6): (A’ m -J) n Structural formula (5) (J-A’ m ) n Structural formula (6) In the above structural formula (5) and structural formula (6), A’ is a hydrophilic substance monomer, m is 1 to 15, n is 1 to 10, J is a linker that interconnects m hydrophilic substance monomers or m hydrophilic substance monomers and oligonucleotides, The hydrophilic substance monomer A’ is any one selected from the following compounds (1) to (3), PO 3 - 、SO 3 and CO 2 The linker (J) selected from the group consisting of, B is a hydrophobic substance selected from the group consisting of steroid derivatives, glyceride derivatives, glycerol ethers, polypropylene glycols, C １２ to C ５０ unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, and lipopolyamines, The steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine, The glyceride derivative is selected from mono-, di-, and tri-glycerides, X and Y each independently mean a single covalent bond or a covalent bond via a linker, and R means miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) nanoparticles containing the double-stranded oligonucleotide structure A pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss, containing the same as an active ingredient.

2. The miR-3139 contains the base sequence represented by SEQ ID NO: 9 and the base sequence represented by SEQ ID NO: 10, and is the pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 1.

3. The miR-3189 contains the base sequence represented by SEQ ID NO: 5 and the base sequence represented by SEQ ID NO: 6, and is the pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 1.

4. The miR-3199 contains the base sequence represented by SEQ ID NO: 15 and the base sequence represented by SEQ ID NO: 16, and is the pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 1.

5. The miR-8485 contains the base sequence represented by SEQ ID NO: 1 and the base sequence represented by SEQ ID NO: 2, and is the pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 1.

6. The hydrophilic substance has a molecular weight of 200 to 10,000, and is the pharmaceutical composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 1.

7. The pharmaceutical composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 1, wherein the molecular weight of the hydrophobic substance is 250 to 1,000.

8. The pharmaceutical composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 1, wherein the covalent bond represented by X and Y is a non-degradable bond or a degradable bond.

9. The pharmaceutical composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 8, wherein the non-degradable bond is an amide bond or a phosphorylation bond.

10. The pharmaceutical composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 8, wherein the degradable bond is a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond, or an enzyme-degradable bond.

11. (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance that is polyethylene glycol (PEG), B is a hydrophobic substance that is C6-S-S-C18, X and Y each independently mean a simple covalent bond or a covalent bond via a linker, and R means miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) A nanoparticle containing the double-stranded oligonucleotide structure The pharmaceutical composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss, which contains the above as an active ingredient.

12. (i) miR-3139, miR-3189, miR-3199 or miR-8485, (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1): A-X-R-Y-B Structural formula (1) In the above structural formula (1), A is a hydrophilic substance selected from any one of the group consisting of polyethylene glycol (PEG), polyvinylpyrrolidone, and polyoxazoline, or a hydrophilic substance having the following structural formula (5) or structural formula (6): (A’ m -J) n Structural formula (5) (J-A’ m ) n Structural formula (6) In the above structural formula (5) and structural formula (6), A’ is a hydrophilic substance monomer, m is 1 to 15, n is 1 to 10, and J is a linker that interconnects m hydrophilic substance monomers or m hydrophilic substance monomers and oligonucleotides, The hydrophilic substance monomer A’ is any one selected from the following compounds (1) to (3), and the linker (J) is selected from the group consisting of PO 3 - , SO 3 and CO 2 ; B is a hydrophobic substance selected from the group consisting of steroid derivatives, glyceride derivatives, glycerol ethers, polypropylene glycols, C １２ to C ５０ unsaturated or saturated hydrocarbons, diacylphosphatidylcholine, fatty acids, phospholipids, and lipopolyamines, The steroid derivative is selected from the group consisting of cholesterol, cholestanol, cholic acid, cholesteryl formate, cholestanyl formate, and cholesteryl amine, The glyceride derivative is selected from mono-, di-, and tri-glycerides, X and Y each independently represent a single covalent bond or a covalent bond via a linker, and R represents miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) nanoparticles containing the double-stranded oligonucleotide structure A cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss, containing the same as an active ingredient.

13. The miR-3139 contains the base sequence represented by SEQ ID NO: 9 and the base sequence represented by SEQ ID NO: 10, and the cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 12.

14. The miR-3189 contains the base sequence represented by SEQ ID NO: 5 and the base sequence represented by SEQ ID NO: 6, and the cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 12.

15. The miR-3199 contains the base sequence represented by SEQ ID NO: 15 and the base sequence represented by SEQ ID NO: 16, and the cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 12.

16. The miR-8485 contains the base sequence represented by SEQ ID NO: 1 and the base sequence represented by SEQ ID NO: 2, and the cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 12.

17. The hydrophilic substance has a molecular weight of 200 to 10,000, and the cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 12.

18. The molecular weight of the hydrophobic substance is 250 to 1,000, and the cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 12.

19. The covalent bond represented by X and Y is a non-degradable bond or a degradable bond, and the cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 12.

20. The non-degradable bond is an amide bond or a phosphorylation bond, and the cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 19.

21. The degradable bond is a disulfide bond, an acid-degradable bond, an ester bond, an anhydride bond, a biodegradable bond or an enzyme-degradable bond, and the cosmetic composition for improving graying of hair, promoting hair growth and / or preventing or improving hair loss according to claim 19.

22. (ii) A double-stranded oligonucleotide structure containing the structure of the following structural formula (1), A-X-R-Y-B Structural formula (1) In the structural formula (1), A is a hydrophilic substance which is polyethylene glycol (PEG), B is a hydrophobic substance which is C6-S-S-C18, X and Y each independently mean a simple covalent bond or a covalent bond via a linker, and R means miR-3139, miR-3189, miR-3199, or miR-8485. Or (iii) Nanoparticles containing the double-stranded oligonucleotide structure A cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss, containing [[ID=]] as an active ingredient.

23. The cosmetic composition is selected from the group consisting of hair tonic, hair conditioner, hair essence, hair lotion, hair nutrient lotion, hair shampoo, hair rinse, hair treatment, hair cream, hair nutrient cream, hair moisture cream, hair massage cream, hair wax, hair aerosol, hair pack, hair nutrient pack, hair soap, hair cleansing foam, hair oil, hair dryer, hair preservation treatment agent, hair dye, hair waving agent for hair, hair bleaching agent, hair gel, hair glaze, hair dressing agent, hair lacquer, hair moisturizer, hair mousse, and hair spray. The cosmetic composition for improving graying of hair, promoting hair growth, and / or preventing or improving hair loss according to claim 12, characterized in that.

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