JP7350652B2 - Composition for promoting melanin containing specific siRNA - Google Patents

Description

The present specification relates to a composition for darkening hair, preventing a hypomelanin disease, or improving a hypomelanin disease, which contains a specific siRNA.

Melanin is a phenolic biopolymer substance that has a complex form of black pigment and protein, and is responsible for the browning or browning that occurs when the cut surfaces of apples, potatoes, and bananas are exposed to the air. It is observed in the fur, skin, hair, and eyes of animals. When melanin is overproduced and deposited on the skin, it causes spots and freckles, and is directly linked to darkening of the hair. Melanin also accelerates skin and hair aging, and can even induce skin cancer.

Melanocyte stimulating hormone (MSH) is secreted by ultraviolet rays, inflammation, hormones, etc., and MSH reacts with receptors to increase cAMP in melanin cells and synthesize melanin. is secreted outside of melanin cells and plays a role in protecting the skin or hair from ultraviolet rays. Melanin synthesis is mainly regulated by α-MSH, and known proteins involved in melanin synthesis include MITF, TYR, TRP1, and TRP2.

Melanin in hair is an important factor that determines the color of hair.Melanin is produced in melanocytes in the hair follicle and is transferred to keratinocytes in the hair cortex, then moves upward as the hair grows. do. Hair whitening, which is recognized as a phenomenon of physiological aging, is induced by a decrease in melanin production due to a numerical decrease in hair melanin-forming cells and a decline in the function of melanin-forming cells. Currently, hair dyeing is used as a solution to hair graying, but hair dyeing is a temporary method and it is necessary to dye the hair again as the gray hair grows. The ingredients present in these products become irritating ingredients, causing contact dermatitis and skin allergies, which has become a problem.

Furthermore, when an abnormality occurs in melanin biosynthesis, abnormal whitening of the skin appears due to abnormal coloring, which may lead to diseases such as albinism and vitiligo.

On the other hand, siRNA (small interfering RNA), which is an interfering RNA composed of about 10 to 60 nucleic acids, binds to RISC (RNA-induced silencing complex) etc. in the cell and combines with complementary base sequences in the cell. It is well known that it has an RNA interference effect that specifically acts on mRNA that has a target mRNA, etc., and suppresses target mRNA. siRNA can inhibit gene expression even in doses 4 to 10 times lower than antisense oligonucleotides, has low cytotoxicity and is excellent in selectively inhibiting gene expression, and can be used to treat cancer and genetic diseases. If the gene that induces protein expression is inhibited, the cause of the disease can be eliminated, so it is attracting a lot of interest as a new and effective disease treatment agent.

Korean Publication Patent No. 10-2016-0066986 Korean Published Patent No. 10-2011-0130282

ALLOUCHE, J. et al., “In Vitro Modeling of Hyperpigmentation Associated to Neurofibromatosis Type 1 Using Melanocytes Derived from Human Embryonic Stem Cells”, Preceedings of the National Academy of Sciences, 2015, vol. 112, no. 29, pages 9034- 9039 ROY, R. et al., “Association between Risk of Oral Precancer and Genetic Variations in MicroRNA and Related Processing Genes”, Journal of Biomedical Science, 2014, vol. 21, thesis no. 48, internal pages 1-7 LI, Y. et al., “VIT1/FBOX11 Knockdown Induces Morphological Alterations and Apoptosis in B10BR Mouse Melanocytes”, International Journal of Melecular Medicine, 2009, Vol. 23, pages 673-678

In one aspect, the present invention provides a method for blackening hair, preventing hair whitening, preventing hypomelanin diseases, and hypomelanin diseases, which contains siRNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. Its purpose is to provide a composition for the treatment of hypomelanin diseases or for the improvement of hypomelanin diseases, to develop the field of gray hair care and fields related to hypomelanin diseases, and to promote the welfare of related consumers and patients. do.

In order to solve the above-mentioned problems, the present invention, in one aspect, provides a method for hair darkening, hair whitening prevention, and treatment of melanin-lowering diseases, which contains siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. Provided are compositions for prevention, treatment of hypomelanin diseases, or amelioration of hypomelanin diseases.

In other aspects, the present invention provides a method for blackening hair, a method for preventing hair whitening, and a method for treating hypomelanin diseases, which comprises administering to an individual a composition comprising siRNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2. Provided are methods for preventing, treating, or ameliorating hypomelanin-related diseases.

In another aspect, the present invention provides an effective method for use in hair darkening, hair whitening prevention, prevention of melanin-lowering diseases, treatment of melanin-lowering diseases, or improvement of melanin-lowering diseases. The present invention provides siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as a component, and a composition containing the same.

In another aspect, the present invention provides SEQ ID NO. Alternatively, the present invention provides non-therapeutic cosmetic uses of siRNA consisting of the base sequence of SEQ ID NO: 2 and compositions containing the same. In other aspects, the present invention provides a composition for darkening hair, a composition for preventing hair whitening, a composition for preventing hypomelanin diseases, a composition for treating hypomelanin diseases, or a composition for treating hypomelanin diseases. Provided is a use of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 for use in producing a composition for improving.

In another aspect of the invention, the composition may be a pharmaceutical composition or a cosmetic composition.

When using the composition containing siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient according to one aspect of the present invention, it is possible to prevent hair darkening, hair whitening, prevention of hypomelanin diseases, or It is possible to effectively treat and improve melanin-lowering diseases.

FIG. 2 is a diagram showing that melanin content is significantly increased by siRNA consisting of the base sequence of SEQ ID NO: 1 or 2 in Melan-a cells. These are Western blot data showing that tyrosinase and TRP1 are significantly increased by siRNA consisting of the base sequence of SEQ ID NO: 1 or 2 in Melan-a cells. FIG. 2 is a diagram showing that melanin content is significantly increased by siRNA consisting of the base sequence of SEQ ID NO: 1 or 2 in B16F1 cells. These are Western blot data showing that tyrosinase and TRP1 are significantly increased by siRNA consisting of the base sequence of SEQ ID NO: 1 or 2 in B16F1 cells.

The present specification will be explained in detail below.

As used herein, "hair" refers to a thread-like structure made of keratinized epithelial cells that appears on the surface of the skin, and specifically refers to body hair, including hair and body hair. It is a broad concept.

As used herein, the term "skin" refers to the tissue that covers the body surface of an animal, and is defined in the broadest sense to include not only the tissue that covers the body surface such as the face or body, but also the scalp and hair.

As used herein, "blackening" means an increase in the amount of colored pigment in the skin or hair, and in particular refers to a phenomenon in which the amount of melanin pigment increases, causing the skin or hair to take on a dark color. can mean For example, hair darkening includes the phenomenon of hair turning black, and also includes the prevention and prevention of hair whitening and gray hair.

As used herein, "whitening" refers to a decrease in the amount of colored pigment in the skin or hair, and in particular refers to a phenomenon in which the amount of melanin pigment decreases and the skin or hair becomes pale in color. It can mean something. For example, hair albinism or gray hair includes the phenomenon of hair turning white.

In this specification, "hair blackening effect substance" or "hair whitening prevention effect effect substance" is a general term for substances that darken the brightness of hair or prevent further progress of hair whitening. It's good. It may also be a general term for substances that act to induce, improve, and promote the deposition of colored pigments, especially melanin, and may include a single compound, polymer, DNA, RNA, siRNA, miRNA, peptide, It may include, but is not limited to, proteins.

As used herein, "hypomelanin disease" refers to a disease in which melanin deposition is abnormally suppressed or melanin production is reduced due to an abnormality in the synthesis or decomposition of melanin pigment, or a disease in which melanin is excessively removed. include. For example, albinism, leukoderma, partial albinism, albinism, vitiligo, quadrichrome vitiligo, vitiligo punctue, idiopathic eczema. Syndromic Albinism (e.g., Alezandrini syndrome, Hermansky-Pudlak syndrome, Chediak-Pudlak syndrome) Shi syndrome prince group (Chediak-Higashi syndrome), Griscelli syndrome (Elejalde syndrome), Griscelli syndrome type 2, and Griscelli syndrome 3 (Griscelli syndrome type 3)), Waardenburg syndrome Waardenburg syndrome, Tietz syndrome, Cross-MuKusick-Breen syndrome, ABCD syndrome (A BCD syndrome), albinism-deafness syndrome (Albinism- deafness syndrome) and Vogt-Koyanagi-Harada syndrome), oculocutaneous albinism, and hypomelanosis (idiopathic idiopathic guttate hypomelanosis ), phylloid hypomelanosis, progressive macular hypomelanosis), patchy scleroderma, piebaldism, nevus depigment osus), post-inflammatory hypopigmentation (postinflammatory hypopigmentation), pityriasis alba, Vagabond's leukomelanoderma, Yemeni hypopigmentation syndrome te deaf-blind hypopigmentation syndrome), Wende - Includes Wende-Bauckus syndrome, Woronoff's ring, amelanism, and leucism.

As used herein, "substances that are effective in preventing, improving, and treating hypomelanin diseases" refer to substances that prevent or suppress the onset of hypomelanin diseases, substances that reduce or alleviate the symptoms of hypomelanin diseases, Substances that eliminate the causes of hypomelanin diseases, substances that promote melanin synthesis, substances that prevent the suppression of melanin synthesis, and substances that suppress the decomposition of melanin, including the entire skin, the entire hair, and specific hair. A general term for substances that have the effect of darkening the brightness of a specific skin area or inducing, improving, and increasing the deposition of melanin pigment, and is a single compound, polymer, DNA, RNA, It may include, but is not limited to, siRNA, miRNA, peptide, protein, etc.

In one aspect, the present invention provides a method for blackening hair, preventing hair whitening, preventing hypomelanin diseases, and hypomelanin diseases, which contains siRNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient. The present invention provides a composition for treating or ameliorating hypomelanin diseases.

According to one embodiment, siRNA including the base sequence of SEQ ID NO: 1 may be represented by "#1", and siRNA including the base sequence of SEQ ID NO: 2 may be represented by "#2", and each The siRNA sequence may include the following base sequence.
Base sequence of #1 (SEQ ID NO: 1): 5'-CCAAACCAGAACAAGCAGAACA-3'
Base sequence of #2 (SEQ ID NO: 2): 5'-GAACACGAAGGCUGUAAACAAA-3'

The siRNA may include a base sequence having 80%, preferably 90%, more preferably 100% homology with the base sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2.

The siRNA may be an independent single-stranded RNA containing the above-mentioned sequence or a sequence having 80 to 100% homology to the above-mentioned sequence, or may be a double-stranded RNA. The siRNA includes a sense strand RNA and an antisense strand RNA complementary thereto, or is a single strand RNA with a stem-loop structure in which the sense strand RNA and the antisense strand RNA are connected by a loop. There may be. Note that the double strand or stem region may include an unpaired portion due to mismatch (corresponding bases are not complementary), bulge (corresponding base is missing on one strand), or the like. The total length of the siRNA is 10 to 60 bases, preferably 15 to 40 bases, more preferably 18 to 30 bases. Further, the loop region has no special meaning in sequence, and it is sufficient to have about 3 to 10 bases simply to connect the sequences at appropriate intervals. The structure of the siRNA end can be either a blunt end or a cohesive end. The structure of the sticky end can be either a protruding structure in which the 3' end protrudes and/or a structure in which the 5' end protrudes, and the number of protruding bases is not limited. In addition, siRNA can contain, for example, a low molecular RNA molecule (for example, a natural RNA molecule such as tRNA, rRNA, or viral RNA, or an artificial RNA molecule) in the protruding part of one end, within a range that can maintain the effect of suppressing the expression of the target gene. ) may be included. The structure of the siRNA end does not need to have a cleavage structure on both sides, and one end of the siRNA may be connected by a linker RNA. The length of the linker is not particularly limited as long as it does not cause any problem when pairing the stem portions.

Furthermore, in the siRNA according to one aspect of the present invention, the RNA strand can include at least one chemical modification within its sugar moiety, nucleobase moiety, or internucleotide structure. Such modifications may make it possible to inhibit destruction of siRNA by nucleases in vivo. Any chemical modification that can improve the stability and biocompatibility of siRNA according to an aspect of the invention in vivo is within the scope of the invention. Among the preferred variants on the sugar moiety, mention may be made of the 2'-position of the ribose, such as 2'-deoxy, 2'-fluoro, 2'-amino, 2'-thio, or 2'-O-alkyl. , and preferably a 2'-O-methyl replacing the normal 2'-OH group on the ribonucleotide or a modification that occurs in the presence of a methylene bridge between the 2' and 4' positions of the LNA. For nucleobases, 5-bromo-uridine, 5-iodo-uridine, N3-methyl-uridine, 2,6-diaminopurine (DAP, 5-methyl-2'-deoxycytidine, 5-(1-propynyl)- It is possible to use modified bases such as 2'-deoxy-uridine (pdU), 5-(1-propynyl)-2'-deoxycytidine (pdC) or bases linked to cholesterol.Internucleotide backbone Preferred variations of include the substitution of phosphodiester groups in the backbone by phosphorothioate, methylphosphonate, phosphorodiamidate groups, or N-(2-aminoethyl)-glycine ( PNA, peptide nucleic acids). Various modified bases, sugars, and backbones are used in modified nucleic acids of the morpholino type (fixed to a morpholine ring and phosphorodiamidate). (bases linked by groups) or PNA (bases fixed to N-(2-aminoethyl)-glycine units linked by peptide bonds).

The siRNA according to the present invention also includes "isolated" and "bound" to other delivery and delivery means, which means that it is not in its natural state, but in its natural state. It includes what is sought and may be obtained by any means involving human intervention. In particular, siRNA according to the invention can be produced using chemical synthesis, enzymatic methods, polymerase chain reaction (PCR), amplification of a specific nucleotide sequence, or recombinant synthesis of a variety of already existing siRNAs. It may be obtained by purifying a nucleic acid structure or siRNA.

According to another embodiment, the siRNA may enhance melanin production or deposition.

According to another embodiment, the siRNA may enhance the expression of one or more of tyrosinase and TRP1 (Tyrosinase-Related Protein 1).

The siRNA according to one aspect of the present invention promotes the production or deposition of melanin in melanin-a cells and B16F1 cells, and targets tyrosinase, an enzyme that produces melanin, and TRP1 protein, which is involved in the formation of melanin. It was confirmed that it promotes the expression of Furthermore, when treated with the siRNA, the number of cells in which tyrosinase or TRP1 is expressed may be increased. Tyrosinase is an enzyme involved in the formation of melanin, and when the expression of tyrosinase is enhanced, the production or deposition of melanin increases. Furthermore, TRP1 is a protein involved in the synthesis of melanin, and like tyrosinase, when the expression of TRP1 is enhanced, the production or deposition of melanin increases. Melanin in hair is an important factor that determines the color of hair.Melanin is produced in melanin cells in the hair follicle and is transferred to keratinocytes in the hair cortex, then moves upward as the hair grows. Therefore, hair whitening is induced by a decrease in melanin production due to a decrease in the number of hair melanin cells and a decline in the function of melanin cells. Through the siRNA, it is possible to promote the production of melanin and the expression of enzymes and proteins involved in this, thereby making it possible to darken the skin and hair and prevent the whitening of the skin and hair. can do.

Further, in one aspect, the present invention can promote melanin production and enhance the expression of enzymes and proteins involved in this through the siRNA, so it can also prevent and prevent melanin-lowering diseases and symptoms. It has also been discovered that it can be treated and improved.

According to another aspect of the invention, the composition may further comprise any carrier that allows for improved biocompatibility and transmissibility of the siRNA according to the invention. In particular, vehicles that can be used with siRNA include liposomes, exosomes, peptides (eg, cell-penetrating peptides), and a variety of nanoparticles. Furthermore, recombinant vectors, plasmids, etc. containing the siRNA can also be used as means for transmission or delivery. Various techniques and methods known in the art can be applied to the transmission or transportation means, and modifications and improvements can be made depending on the dosage form.

The siRNA is preferably, but not limited to, operably linked to at least a promoter for proper transcription in cells to which it is transferred. The promoter may be of any type as long as it can function in cells. The siRNA according to one aspect of the present invention includes a leader sequence, a polyadenylation sequence, a promoter, an enhancer, an upstream activation sequence, a signal peptide sequence, and a transcription termination factor as necessary for efficient action. It may further include regulatory sequences.

According to another aspect of the invention, the content of siRNA in the composition may range from 0.000000001% to 20% by weight based on the total weight of the composition. Further, the content is 0.000000001% by mass or more, 0.000000005% by mass or more, 0.00000001% by mass or more, 0.00000005% by mass or more, 0.0000001% by mass or more, based on the total mass of the composition. 0.0000005 mass% or more, 0.000001 mass% or more, 0.000005 mass% or more, 0.00001 mass% or more, 0.00005 mass% or more, 0.0001 mass% or more, 0.0005 mass% or more, 0 .001 mass% or more, 0.005 mass% or more, 0.01 mass% or more, 0.05 mass% or more, 0.1 mass% or more, 0.3 mass% or more, 0.5 mass% or more, 0. It may be 8% by mass or more, 1% by mass or more, 3% by mass or more, 5% by mass or more, 8% by mass or more, 10% by mass or more, 12% by mass or more, 15% by mass or more, or 18% by mass or more. Further, the content is 20% by mass or less, 18% by mass or less, 15% by mass or less, 12% by mass or less, 10% by mass or less, 8% by mass or less, 5% by mass or less, based on the total mass of the composition. 3% by mass or less, 1% by mass or less, 0.8% by mass or less, 0.5% by mass or less, 0.3% by mass or less, 0.1% by mass or less, 0.05% by mass or less, 0.01% by mass 0.005% by mass or less, 0.001% by mass or less, 0.0005% by mass or less, 0.0001% by mass or less, 0.00005% by mass or less, 0.00001% by mass or less, 0.000005% by mass or less , 0.000001 mass% or less, 0.0000005 mass% or less, 0.0000001 mass% or less, 0.00000005 mass% or less, 0.00000001 mass% or less, or 0.000000005 mass% or less.

According to yet another aspect of the invention, the dosage of the siRNA may range from 0.000001 mg/kg/day to 20 mg/kg/day. Further, the dosage is 0.000001 mg/kg/day or more, 0.000005 mg/kg/day or more, 0.00001 mg/kg/day or more, 0.00005 mg/kg/day or more, 0.00001 mg/kg/day. 0.0005mg/kg/day or more, 0.0001mg/kg/day or more, 0.0005mg/kg/day or more, 0.001mg/kg/day or more, 0.005mg/kg/day or more, 0.01mg /kg/day or more, 0.05mg/kg/day or more, 0.1mg/kg/day or more, 0.5mg/kg/day or more, 0.8mg/kg/day or more, 1mg/kg/day or more, 2mg /kg/day or more, 3mg/kg/day or more, 5mg/kg/day or more, 8mg/kg/day or more, 10mg/kg/day or more, 12mg/kg/day or more, 15mg/kg/day or more, or 18mg /kg/day or more. Further, the dosage is 20 mg/kg/day or less, 18 mg/kg/day or less, 15 mg/kg/day or less, 12 mg/kg/day or less, 10 mg/kg/day or less, 8 mg/kg/day or less, and 5 mg/kg/day or less. /kg/day or less, 3mg/kg/day or less, 2mg/kg/day or less, 1mg/kg/day or less, 0.8mg/kg/day or less, 0.5mg/kg/day or less, 0.1mg/kg /day or less, 0.05mg/kg/day or less, 0.01mg/kg/day or less, 0.005mg/kg/day or less, 0.001mg/kg/day or less, 0.0005mg/kg/day or less, 0 It may be less than .00001 mg/kg/day, less than 0.00005 mg/kg/day, less than 0.00001 mg/kg/day, or less than 0.000005 mg/kg/day. Determination of the dosage of the active ingredient is within the level of ordinary skill, and the daily dosage of the drug depends on various factors such as the degree of progression of the drug, time of onset, age, health condition, and complications of the subject to be administered. may vary depending on various factors.

In another aspect, the present invention may be a composition for preventing, treating, and improving melanin-lowering diseases, which contains siRNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient.

According to one embodiment, the hypomelanotic disease includes vitiligo, depigmented nevus, pityria alba, albinism, post-inflammatory hypopigmentation, patchy scleroderma, partial albinism, idiopathic guttate hypomelanosis. It may be one or more selected from the group consisting of melanosis and punctate albinism. Melanin is observed in the outer fur, hair, skin, head, eyes, etc. of animals, and when melanin production is insufficient, deposition is suppressed, or excessive decomposition occurs, hypomelanin diseases develop. There are things to do. In one aspect, the present invention enhances the production or deposition of melanin, and also enhances the expression of tyrosinase and TRP1, and thus can be used for the prevention, improvement, and treatment of the hypomelanin diseases.

According to one aspect of the invention, the composition may be a pharmaceutical composition.

The pharmaceutical composition may contain pharmaceutical auxiliaries such as carriers, excipients, preservatives, preservatives, stabilizers, wetting agents or emulsification promoters, salts and/or buffers for adjusting osmotic pressure, The composition may further contain other therapeutically useful substances, and may be formulated into various oral or parenteral dosage forms by conventional methods.

Examples of the oral preparations include tablets, pills, hard and soft capsules, solutions, suspensions, emulsifiers, syrups, powders, powders, fine granules, granules, and pellets. In addition to the active ingredient, it contains surfactants, diluents (e.g. lactose, dextrose, sucrose, mannitol, sorbitol, cellulose and glycine), lubricants (e.g. silica, talc, stearic acid and its magnesium or calcium salts). , and polyethylene glycol). The tablets may further contain binders such as magnesium aluminum silicate, starch paste, gelatin, tragacanth, methylcellulose, sodium carboxymethylcellulose and polyvinylpyrrolidine, and optionally disintegrants such as starch, agar, alginic acid or its sodium salts. It may contain pharmaceutical additives such as absorbents, coloring agents, flavoring agents, and sweetening agents. The tablets may be manufactured by conventional mixing, granulating or coating methods.

The form for parenteral administration may be a transdermal dosage form, such as an injection, a drop, an ointment, a lotion, a gel, a cream, a spray, a suspension, an emulsion, a suppository, or a patch. However, the dosage form is not limited to these.

The pharmaceutical composition may be administered orally or parenterally, and when administered parenterally, the injection method may be selected from external skin, intraperitoneal, rectal, intravenous, intramuscular, subcutaneous, intradermal, intrathoracic or intracerebrovascular injection. It's okay to do so.

The pharmaceutical composition may be a skin external preparation, and the skin external preparation is a general term for any composition that can be applied to the skin, and includes pharmaceuticals or quasi-drugs in various dosage forms. may be included in this.

According to another aspect of the present invention, the composition may be a cosmetic composition for hair darkening, hair whitening prevention, and prevention and improvement of melanin-lowering diseases.

The cosmetic composition may further include functional additives and ingredients included in common cosmetic compositions. The functional additive may include a component selected from the group consisting of water-soluble vitamins, oil-soluble vitamins, high-molecular peptides, high-molecular polysaccharides, sphingolipids, and seaweed extracts. Other ingredients include oils and fats, humectants, emollients, surfactants, organic and inorganic pigments, organic powders, ultraviolet absorbers, preservatives, bactericides, antioxidants, plant extracts, and pH adjustment. agents, alcohol, pigments, fragrances, blood circulation promoters, cooling agents, antiperspirants, purified water, etc.

The dosage form of the cosmetic composition is not particularly limited, and may be appropriately selected depending on the intended purpose. For example, skin lotion, skin softener, skin toner, astringent, lotion, milk lotion, moisture lotion, nourishing lotion, massage cream, nourishing cream, moisture cream, hand cream, foundation, essence, nourishing essence, pack, soap, cleansing foam, It may be manufactured in one or more dosage forms selected from the group consisting of cleansing lotion, cleansing cream, body lotion, and body cleanser, but is not limited thereto.

When the dosage form according to one aspect of the present invention is a paste, cream, or gel, carrier components include animal fibers, vegetable fibers, wax, paraffin, starch, tragacanth, cellulose derivatives, polyethylene glycol, silicone, bentonite, silica, Talc or zinc oxide may be used.

When the dosage form according to one aspect of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, or polyamide powder may be used as carrier components, especially when the dosage form is a spray. may further include propellants such as chlorofluorohydrocarbons, propane/butane, or dimethyl ether.

When the dosage form according to one aspect of the present invention is a solution or emulsion, a solvent, solvating agent, or emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, These include ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol aliphatic esters, polyethylene glycol, or fatty acid esters of sorbitan.

When the dosage form according to one aspect of the invention is a suspension, carrier components include a liquid diluent such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitol ester. Suspending agents such as oxyethylene sorbitan esters, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tragacanth may be used.

When the dosage form according to one aspect of the present invention is a surfactant-containing cleansing, carrier components include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinate monoester, isethionic acid, imidazolium derivative, Methyl taurates, sarcosinates, fatty acid amide ether sulfates, alkylamido betaines, fatty alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives, or ethoxylated glycerol fatty acid esters may be used.

According to another aspect of the invention, the composition may be a food or health food composition.

The food or health food composition may be in a liquid or solid dosage form, and includes, for example, various foods, beverages, gums, teas, vitamin complexes, functional health foods, and health supplements. It may be used in the form of powder, granules, tablets, capsules, or drinks. In addition to the active ingredients, the food composition of each dosage form may be formulated with ingredients commonly used in the field, selected without difficulty by those skilled in the art, depending on the dosage form or purpose of use. Synergistic effects may occur when applied simultaneously with raw materials.

There are no particular restrictions on the liquid components that may be contained in addition to the active ingredients disclosed herein, and the liquid components may contain various flavoring agents, natural carbohydrates, etc. as additional components, similar to ordinary beverages. Examples of said natural carbohydrates include monosaccharides such as glucose, fructose, disaccharides such as maltose, sucrose, and common sugars such as dextrins, cyclodextrins, and sugar alcohols such as xylitol, sorbitol, erythritol. As said flavoring agents, natural flavors (thaumatin, stevia extract (eg rebaudioside A, glycyrrhizin, etc.)) and synthetic flavors (saccharin, aspartame, etc.) may be advantageously used. The proportion of said natural carbohydrates may generally be about 1 to 20 g, and in one aspect about 5 to 12 g per 100 ml of the compositions disclosed herein.

In one aspect, the food composition includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavors, colorants and enhancers (cheese, chocolate, etc.), pectic acid and its like. It may contain salts, alginic acid and its salts, organic acids, protective colloid thickeners, pH regulators, stabilizers, preservatives, glycerin, alcohol, carbonating agents used in carbonated beverages, and the like. In other aspects, it may include fruit pulp for the production of natural fruit juices and vegetable beverages. These components may be used independently or in combination. The proportions of such additives may vary, but are generally selected in the range of 0.001 to about 20 parts by weight per 100 parts by weight of the compositions disclosed herein.

Hereinafter, the configuration and effects of one aspect of the present invention will be explained in more detail through Examples and Experimental Examples. Note that the following examples are merely illustrative for the purpose of helping understanding of the present invention, and the scope and scope of the present invention are not limited thereto.

[Example 1]
Cell and skin model preparation
Normal human epidermal melanocytes (NHEMs, Cascade Biologics, Portland, OR, USA) were obtained and melan-A, which is melanocytes derived from C57BL/6 J (black, a/a) mice, was obtained. ( Melan-A) cell line was obtained from Dr. Dorothy C. Bennett (St. George's Hospital Medical School, London, UK). B16F1 mouse melanoma cell line was obtained from ATCC (Manassas, VA, USA).

Normal human epidermal melanocytes were maintained under Human Melanocyte Growth Supplements (HMGS) (Cascade Biologics, Inc., Mansfield, UK) in M-254 medium. Melan-A cells were cultured in RPMI 1640 medium with 10% (v/v) fetal bovine serum, 1% (v/v) penicillin-streptomycin, and 0.2 μM phorbol 12-myristate 13-acetate. acetate). B16F1 mouse melanoma cell line was cultured in DMEM supplemented with 10% (v/v) fetal calf serum and 1% (v/v) penicillin-streptomycin.

Reagents and siRNA transfection
α-MSH, arbutin, and kojic acid were obtained from Sigma-Aldrich (St. Louis, MO, USA). siRNA #1 (5'-CCAACCAGAACAAGCAGAACA-3'), #2 (5'-GAACACGAAGGCUGUAAACAAA-3'), and negative control group siRNA (scrambled control siRNA, siNC) (5'-CCUACGC CACCAAUUUCGU-3') is Jeno It was obtained from Genolution (Seoul, Republic of Korea). Melan-a and B16F1 cells cultured in a 60 mm dish were transfected with siRNA using Lipofectamine® RNAi MAX (Invitrogen, Carlsbad, CA, USA). After culturing with siRNA for 24 hours, the cells were treated with each compound (reagent) and maintained for 48 hours.

Melanin Assay Cells were treated with trypsin/EDTA, centrifuged at 1000xg for 5 minutes, and washed twice with PBS. The final cell pellet in the tube was photographed and the cell pellet was dissolved in 1N NaOH. The homogenized cell extract was transferred to a 96-well plate, and the relative amount of melanin at absorbance at 405 nm was measured using an ELISA plate reader.

Western blot analysis All lysates were prepared in 2x Laemmli sample buffer (2x Laemmli sample buffer, 62.5mM Tris-HCl, pH 6.8, 25% [v/v] glycerol, 2% [w/v] SDS, 5 % [v/v] β-mercaptoethanol, and 0.01% [w/v] bromophenol blue) (Bio-Rad, Hercules, CA, USA). All cellular proteins were measured using Bradford solution (Bio-Rad). The samples were then separated by SDS-PAGE and transferred to a PVDF membrane (Bio-Rad). After blocking with 4% (w/v) skim milk in TBST (25mM Tris, 140mM NaCl, and 0.05% [v/v] Tween® 20), the membrane was The cells were incubated overnight with the following antibodies.

Anti-actin antibody (MAB1501, 1:10,000) was obtained from Millipore (Temecula, CA, USA), anti-αPEP7 (tyrosinase) antibody (anti-αPEP7 (tyrosinase) antibody, 1:10,000) :1000) and anti-αPEP1 (TRP1) antibody (1:1000) were obtained from V. J. Hearing (NIH, Bethesda, MD). For protein detection, the membrane was incubated with HRP-conjugated secondary antibodies and signal was detected using the SuperSignal® West Dura HRP Detection Kit (Pierce, Rockford, IL, USA). It was measured using

Statistical analysis Each data was obtained from at least three independent experiments and is expressed as mean±SE (standard error). Statistical evaluation of the results was performed using one-way ANOVA (*: p<0.05, **: p<0.01).

[Experimental Example 1] Regulation of melanin production and related enzymes and proteins by siRNA Melan-a cells were transfected with siRNA (#1, #2) and scrambled control siRNA (scrambled control siRNA, siNC). After 48 hours, the cells were treated with a specific substance D (0.5 μM) that suppresses melanin production or cultured for 2 days under serum-free serum conditions, and then the melanin content was measured. As can be seen from Figure 1, treatment with specific substance D that inhibits melanin production or melan-a cell culture under serum-free conditions inhibited melanin production in the control group (siNC), but stimulation (specific Melanin production, which was inhibited by substance D or serum starvation), was significantly enhanced by siRNA (#1, #2) (Figure 1).

We also conducted experiments regarding the expression of melanin-forming enzymes. As a result of Western blot analysis, when Melan-A cells were transfected with siRNA (#1, #2), the levels of tyrosinase and TRP1 proteins were significantly increased compared to wild-type cells (Figure 2).

B16F1 cells were also transfected with siNC and siRNA (#1, #2), and one day later, treated with a specific substance D (0.5 μM) that suppresses melanin formation or treated with serum-free medium. After a day, the degree of melanin production was confirmed, and tyrosinase and TRP1 were analyzed by Western blotting.

As a result, melanin production, which was suppressed by specific substance D or serum starvation in B16F1 cells, was significantly increased by siRNA (#1, #2) transfection, as in melan-a cells (FIG. 3). Furthermore, the expressions of tyrosinase and TRP1, which were suppressed by specific substance D or serum starvation, were significantly increased by siRNA (#1, #2) transfection (FIG. 4).

Taking these results together, the siRNAs (#1, #2) can promote melanin synthesis, as well as the expression of melanin-producing enzymes and proteins involved in melanin production. Since it is possible to prevent hair blackening and hair whitening, the present invention, in one aspect, can be used for the care of hair blackening and gray hair. Further, since melanin production can be promoted in a specific area of the skin or in the entire skin using such features according to one aspect of the present invention, one aspect of the present invention can be used to treat hypomelanin-related diseases. It is possible to prevent, treat, and improve symptoms.

Examples of dosage forms of the composition according to one aspect of the present invention will be described below, but application to various other dosage forms is also possible, and these are not intended to limit the present invention. This is for specific explanation.

[Dosage Form Example 1] Soft capsule 10 mg of liposome containing 2 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 80 to 140 mg of L-carnitine, 180 mg of soybean oil, 2 mg of palm oil, 8 mg of hydrogenated vegetable oil, 4 mg of yellow wax and 6 mg of lecithin were mixed and filled into one capsule according to a conventional method to produce a soft capsule.

[Dosage Form Example 2] Tablet 10 mg of liposome containing 2 mg of siRNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 200 mg of galacto-oligosaccharides, 60 mg of lactose, and 140 mg of maltose were mixed and granulated using a fluidized bed dryer. , 6 mg of sugar ester was added thereto, and the mixture was compressed using a tablet machine to produce tablets.

[Dosage Form Example 3] Granules 10 mg of liposome containing 2 mg of siRNA having the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 250 mg of anhydrous crystalline glucose, and 550 mg of starch are mixed and formed into granules using a fluidized bed granulator. After that, the mixture was filled into packages to produce granules.

[Dosage Form Example 4] Drink After mixing 10 mg of liposome containing 2 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 10 g of glucose, 0.6 g of citric acid, and 25 g of liquid oligosaccharide, 300 ml of purified water was mixed. Additionally, fill each bottle with 200ml. After filling bottles, the mixture was sterilized at 130°C for 4 to 5 seconds to produce a drink.

[Dosage Form Example 5] Injection Prepare an injection according to the usual method using 20 mg of liposome containing 5 mg of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, an appropriate amount of sterile distilled water for injection, and an appropriate amount of a pH adjuster. Manufactured.

[Dosage form example 6] Soft lotion (skin lotion)
10% by mass of liposomes containing 1.0% by mass of siRNA having the base sequence of SEQ ID NO: 1 or 2, 1.00% by mass of L-ascorbic acid-2-phosphate magnesium salt, water-soluble collagen (1% aqueous solution) ) 1.00% by mass, sodium citrate 0.10% by mass, citric acid 0.05% by mass, licorice extract 0.20% by mass, 1,3-butylene glycol 3.00% by mass, purified water as the remainder. A softening lotion (skin lotion) was produced using this method.

[Dosage Form Example 7] Cream 10% by mass of liposomes containing 1.00% by mass of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 2.00% by mass of polyethylene glycol monostearate, self-emulsifying monostearic acid Glycerin 5.00% by mass, propylene glycol 4.00% by mass, squalene 6.00% by mass, tri2-ethylhexane glyceryl 6.00% by mass, glycosphingolipid 1.00% by mass, 1,3-butylene glycol 7 00% by mass, 5.00% by mass of beeswax, and purified water as the remaining amount to produce a creamy preparation.

[Dosage Form Example 8] Pack 10% by mass of liposomes containing 1.00% by mass of siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2, 13.00% by mass of polyvinyl alcohol, L-ascorbic acid-2-phosphate Magnesium salt 1.00% by mass, lauroyl hydroxyproline 1.00% by mass, water-soluble collagen (1% aqueous solution) 2.00% by mass, 1,3-butylene glycol 3.00% by mass, ethanol 5.00% by mass, A pack was manufactured using purified water as the remaining amount.

[Dosage Form Example 9] Health Food A health food was manufactured according to a conventional method using the composition shown in the table below.

The composition ratio of the vitamin and inorganic mixture was made using ingredients that are relatively suitable for health foods as an example, but the composition ratio may be modified as desired and may be carried out according to the usual manufacturing method of health foods. After mixing the above components, the mixture may be used to produce a health food composition according to a conventional method.

[Dosage form example 10] Health drink

As shown in Table 2, add the remaining amount of purified water to a total volume of 900 ml, mix the ingredients according to the usual health drink manufacturing method, and stir and heat at 85 ° C. for about 1 hour. The resulting solution was filtered, and the resulting filtrate was placed in a sterilized 2 liter container, sealed and sterilized, and then stored in a refrigerator to produce a health drink.

Although specific parts of the content of the present invention have been described in detail above, those with ordinary knowledge in the art will understand that such specific descriptions are merely preferred embodiments, and that the scope of the present invention will be limited by this. It is clear that there are no restrictions. It is therefore intended that the substantial scope of the invention be defined by the appended claims and their equivalents.

Claims (8)

A composition for hair darkening or hair whitening prevention comprising siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient ,
The content of siRNA in the composition is in the range of 0.000000001% by mass to 20% by mass, based on the total mass of the composition.
composition . A composition for the prevention and treatment of hypomelanin diseases containing siRNA consisting of the base sequence of SEQ ID NO: 1 or SEQ ID NO: 2 as an active ingredient ,
The content of siRNA in the composition is in the range of 0.000000001% by mass to 20% by mass, based on the total mass of the composition.
composition . The composition according to claim 1 or 2, wherein the siRNA promotes melanin production or deposition. The composition according to claim 1 or 2, wherein the siRNA enhances the expression of one or more of tyrosinase and TRP1. The composition according to claim 1 or 2, wherein the dosage of the siRNA is in the range of 0.000001 mg/kg/day to 20 mg/kg/day. The hypomelanin diseases include albinism, vitiligo, depigmented nevus, pityriasis alba, albinism, post-inflammatory hypopigmentation, patchy scleroderma, partial albinism, idiopathic guttate hypomelanosis, and The composition according to claim 2, which is one or more selected from the group consisting of punctate albinism. 3. The composition according to claim 1 or 2, wherein the composition is a pharmaceutical composition. 3. The composition according to claim 1, wherein the composition is a cosmetic composition for darkening hair, preventing hair whitening, and preventing and improving melanin-lowering diseases.

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