KR101809375B1 - Pharmaceutical Composition for Improving Vitiligo or Treating Gray Hair Comprising a Src Kinase Inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for treating amelanosis containing Src kinase inhibitor, and a cosmetic composition. The Src kinase inhibitor of the present invention increases synthesis of melanin in cells, and also promotes expression of genes encoding factors involved in melanin production. Accordingly, the composition containing the Src kinase inhibitor can be suitable to be used in drugs, quasi-drugs, and cosmetic products.

Description

FIELD OF THE INVENTION [0001] The present invention relates to a pharmaceutical composition for improving vitiligo or treating white moth with Src kinase inhibitor,

The present invention relates to a pharmaceutical composition comprising a Src kinase inhibitor.

In vivo, there is a rule (that is, self tolerance) that does not cause an immune response to the self body, and in contrast, it is an autoimmune disease.

Vitiligo is an autoimmune disease caused by an autoimmune reaction to melanocytes. It is a skin disease in which a discolored half-skin appears on the skin. It occurs in about 0.1-2% of the world's population. Pathologically, melanocytes, which produce melanin, are destroyed by autoimmune reactions, resulting in the deficiency of melanin pigment that makes up the skin color. It usually appears as white spots on the face and extremities, underarms and groin, and can spread around. In severe cases, hair follicle elanocytes are destroyed, and white discolored hairs appear on lesion sites. Untreated patients require continuous therapy with steroids or ultraviolet light because the lesion worsens in most patients. However, since the conventional treatment method often fails to provide adequate treatment, a more effective treatment method is urgently required.

On the other hand, Src family kinases (SFK) are composed of eight members as non-receptor tyrosine kinases, but mainly three members (SRC, FYN and YES) are widely expressed. These SFKs are activated by various external stimuli, such as growth factor and extracellular matrix components. Oncogenic cytoplasmic tyrosine kinases in "oncogenes and tumor surpressors" Oxford New York Tokyo: Oxford University (Oxford, 1986). Press; 1997; pp: 97-130). Thus, inhibitors of SFK have been studied and applied as various cancer therapies (Hollande F, Pannquin J, Joubert D. The long road to colorectal cancer therapy: searching for the right signals. Drug Resist Updat 2010; 13: 44- 56; Korean Patent Publication No. 10-2016-0089378).

However, the efficacy of SFK in melanin-related diseases such as vitiligo is unclear. Therefore, in the present invention, SFK inhibitor is required to improve vitiligo or to treat white moth disease.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have sought to find substances for effective improvement or treatment against vitiligo or white mite. As a result, the present inventors have found that treatment of siRNA with Src kinase inhibitors and Src kinase widely used as therapeutic agents for various cancers promotes melanin synthesis in cells (for example, melanoma cells) and expression of melanin- , Thereby completing the present invention.

It is therefore an object of the present invention to provide a pharmaceutical composition for the treatment of melanin deficiency diseases.

Another object of the present invention is to provide a cosmetic composition.

It is another object of the present invention to provide a method for promoting melanin synthesis.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition for treating a melanin deficiency disease comprising a Src kinase inhibitor.

According to another aspect of the present invention, there is provided a cosmetic composition comprising a Src kinase inhibitor.

The present inventors have sought to find substances for effective improvement or treatment against vitiligo or white mite. As a result, the present inventors have found that treatment of siRNA with Src kinase inhibitors and Src kinase widely used as therapeutic agents for various cancers promotes melanin synthesis in cells (for example, melanoma cells) and expression of melanin- .

The Src family kinase is a tyrosine kinase present in the cytoplasm involved in many intracellular mechanisms. It has been reported that the abnormal regulation of Src kinase shows significant carcinogenic activity, and its overexpression has been measured in tumors, and its inhibitor has been used as a cancer treatment agent.

Interestingly, the present invention provides a pharmaceutical composition for the treatment of melanin deficiency diseases comprising a Src kinase inhibitor as an initial report showing that Src kinase inhibitors are effective for the improvement of vitiligo or white moth.

In some embodiments of the invention, the Src kinase inhibitor promotes the synthesis of melanin.

In some embodiments of the present invention, the pharmaceutical composition of the present invention increases the expression of melanin-producing markers, and more particularly, melanogenesis markers such as microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 ), Increases the expression of TRP2 or tyrosinase (see Figures 1 to 3).

Src kinase inhibitors that may be used in the present invention can be any known substance known in the art if they are inhibitors that increase melanin synthesis, such as PP1 (CAS 172889-26-8), PP2 ((CAS 172889- 27-9), dasatinib (CAS 302962-49-8), su6656 (CAS 330161-87-0), bosutinib (CAS 380843-75-4), saracatinib (CAS 379231 (CAS 179248-59-0), A 419259 trihydrochloride (CAS 364042-47-7), AG 538 (CAS 133550), KX2-391 (CAS 897016-82-9), Src kinase inhibitor I -18-2), AGL 2263, Bcr-abl inhibitor II (CAS 607702-99-8), alteucine (CAS 31186-12-6), herbimycin A (CAS 70563-58-5), PD 166285 PDGFR tyrosine kinase inhibitor IV (CAS 627518-40-5), calpostatin C (CAS 121263-19-2), EGF / FGF / PDGF receptor tyrosine kinase inhibitors (CAS 1135256-66-4), stearosporin (CAS 62996-74-1), lavendustin A (CAS 125697-92-9), luteolin (CAS 491 -70-3), TX-1918 (CAS 503473-32-3), geldanamycin (CAS 30562-34-6), MNS (CAS 1485-00-3), TX- 1123 (CAS 157397-06-3 ), GW5074 (CAS 220904-83-6), erlotinib HCl (CAS 183319-69-9), NVP-BHG712 (CAS 940310-85-0), GW2580 (CAS 870483-87-7), AEE788 But are not limited to, TAK-901 (CAS 934541-31-8), midosutaurin (CAS 120685-11-2), PD173074 (CAS 219580-11-7) no.

In some embodiments of the invention, the Src kinase inhibitor of the present invention comprises PP1, PP2, su6656, dasatinib, conservinib, saracatibib and KX2-391, more particularly PP2, su6656, dasatinib , Conservative tinib, and saracatinib.

In addition, Src kinase inhibitors that may be used in the present invention may include siRNA, shRNA, miRNA, antisense oligonucleotides, aptamers and ribozymes against Src kinase.

The term "siRNA" as used herein means a nucleic acid molecule capable of mediating RNA interference or gene silencing (see WO 00/44895, WO 01/36646, WO 99/32619, WO 01/29058, WO 99/07409 and WO 00/44914). Since siRNA can inhibit the expression of a target gene, it is provided as an efficient gene knockdown method or as a gene therapy method. siRNAs were first discovered in plants, insects, fruit flies and parasites, but recently they have been applied to mammalian cell research to develop / use siRNAs (Degot S, et al., 2002; Degot S, et al., 2004; Ballut L , et al., 2005).

An siRNA molecule that may be used in the present invention is a siRNA molecule that has a sense strand (e. G., A sequence corresponding to a Src kinase gene mRNA sequence) and an antisense strand (e. G., A sequence complementary to a Src kinase gene mRNA sequence) They may be arranged on the opposite sides of each other to form a double-stranded structure. In addition, siRNA molecules that may be used in the present invention may have a single stranded structure with self-complementary sense and antisense strands.

The siRNA is not limited to a complete pair of double-stranded RNA portions that are paired with each other, but is paired by a mismatch (the corresponding base is not complementary), a bulge (no base corresponding to one chain) May be included. Specifically, the total length is 10 to 100 bases, more specifically 15 to 80 bases, and even more specifically 20 to 70 bases.

As used herein, the term "shRNA (small hairpin RNA or short hairpin RNA)" refers to an RNA sequence that produces a robust hairpin turn, which can be used to silence gene expression through RNA interference. shRNA uses a vector for introducing cells and mainly uses a U6 promoter capable of expressing shRNA. These vectors are always transferred to daughter cells, allowing genetic silencing to be inherited. The shRNA hairpin structure is degraded into an intracellular machinery siRNA and bound to an RNA-induced silencing complex. The above-described complex binds to and degrades mRNA matched to the siRNA bound thereto. shRNAs are transcribed by RNA polymerase III, and production of shRNAs in mammalian cells may cause interferon responses as cells recognize shRNA as a viral attack and find defensive means. In addition, shRNAs can also be used in plants and other systems, and the U6 promoter is not necessary. In the case of plants, the CaMV (cauliflower mosaic virus) 35S promoter, which is a conventional promoter having a very strong continuous expression ability, can be used.

As used herein, the term "microRNA (miRNA)" refers to a material that binds to the 3'-UTR of mRNA (messenger RNA) as a single strand RNA molecule of 21-25 nucleotides and controls gene expression of eukaryotes (Bartel DP, et al., Cell, 23: 116 (2): 281-297 (2004)). The production of miRNA is made into a stem-loop structure precursor miRNA (pre-miRNA) by Drosha (RNase III type enzyme), and it is transferred to the cytoplasm and cleaved by Dicer to make mature miRNA [Kim VN, et al., Nat Rev Mol Cell Biol., 6 (5): 376-385 (2005)]. MiRNAs prepared as described above are involved in development, cell proliferation and death, lipid metabolism, tumor formation, etc. by controlling the expression of target proteins [Wienholds E, et al., Science, 309 (5732): 310-311 ); Nelson P, et al., Trends Biochem Sci., 28: 534-540 (2003); Lee RC, et al., Cell, 75: 843-854 (1993); And Esquela-Kerscher A, et al., Nat Rev Cancer, 6: 259-269 (2006).

The term "antisense oligonucleotide " as used herein refers to DNA or RNA or a derivative thereof containing a nucleic acid sequence complementary to the sequence of a specific mRNA, and is capable of binding to a complementary sequence in mRNA and translating mRNA into a protein . ≪ / RTI > For example, the antisense sequence of the present invention means a DNA or RNA sequence complementary to a Src kinase and capable of binding to a Src kinase mRNA, and is capable of translating Src kinase mRNA, translocating it into the cytoplasm, And may inhibit essential activities for all other overall biological functions. The length of the antisense nucleic acid is 6 to 100 bases, specifically 8 to 60 bases, more specifically 40 bases in 10.

As used herein, the term "ribozyme" is a type of RNA. It is an RNA that has the same function as an enzyme that recognizes the base sequence of a specific RNA and cleaves itself. The ribozyme consists of a region that specifically binds with a complementary base sequence of the target messenger RNA strand and a region that cleaves the target RNA. As used herein, the term "aptamer" refers to an oligonucleotide (generally an RNA molecule) that binds to a particular target. Specifically, the term " aptamer "as used herein refers to an oligonucleotide For example, an RNA aptamer). The general contents of the Aptamer are described in Bock LC et al., Nature 355 (6360): 564-6 (1992); Hoppe-Seyler F, Butz K "Peptide aptamers: powerful new tools for molecular medicine". J Mol Med. 78 (8): 426-30 (2000); Cohen BA, Colas P, Brent R. "An artificial cell-cycle inhibitor isolated from a combinatorial library". Proc Natl Acad Sci USA. 95 (24): 14272-7 (1998), the disclosures of which are incorporated herein by reference.

The term "complementary ", as used herein, refers to the ability of a targeting moiety in a nucleic acid molecule to be selectively (e.g., Src kinase gene) targeted under certain hybridization or annealing conditions, specifically under physiological conditions Quot; is meant to include one or more mismatch nucleotide sequences that are sufficiently complementary to hybridize to < RTI ID = 0.0 > a " complementary " More specifically, it is completely complementary.

In some embodiments of the invention, the compositions of the present invention comprise (a) a pharmaceutically effective amount of a Src kinase inhibitor of the invention as described above; And (b) a pharmaceutically acceptable carrier.

As used herein, the term "pharmaceutically effective amount" means an amount sufficient to achieve the efficacy or activity of the Src kinase inhibitor described above.

In certain embodiments of the invention, the diseases to which the compositions of the invention may be applied are selected from the group consisting of leukoderma, vitiligo, quadrichrome vitiligo, vitiligo ponctue, syndromic syndrome, (For example, Alezzandrini syndrome, Hermansky-Pudlak syndrome, ChediakHigashi syndrome, Griscelli syndrome (Elejalde syndrome, syndrome, Griscelli syndrome type 2 and Griscelli syndrome type 3), Waardenburg syndrome, Tietz syndrome, Cross-syndrome type 2, Syndrome, ABCD syndrome, albinism-deafness syndrome, and Vogt-Koyanagi-Harada syndrome), as well as a variety of other disorders, Oculocutaneous albinism, white moth, hypomelanosis [idiopathic guttate hypomelanosis, phylloid hypomelanosis, progressive macular hypomelanosis], stain albinism Peybaldism, nevus depigmentosus, postinflammatory hypopigmentation, pityriasis alba, Vagabond's leukomelanoderma, Yemenite deaf-blind (Yemenite deaf-blind hypopigmentation syndrome, Wende-Bauckus syndrome, Woronoff's ring, melanin deficiency (amelanism), leucism, and skin decolorization-related diseases.

The pharmaceutically acceptable carriers to be contained in the pharmaceutical composition of the present invention are those conventionally used in the present invention and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, But are not limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methylcellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. It is not. The pharmaceutical composition of the present invention may further contain a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc., in addition to the above components. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington ' s Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally. For example, the oral administration can be carried out in the form of tablets, pills, granules, capsules, powders, granules, powders, emulsions, syrups and pellets. In the case of parenteral administration, , Transdermal administration, subcutaneous injection, and the like.

 The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on such factors as formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, route of administration, excretion rate, . On the other hand, the preferred dosage of the pharmaceutical composition of the present invention may be 0.01-400 mg per day, more specifically 0.5-300 mg per day, and more particularly 1.0-200 mg per day, It is not.

In some embodiments of the invention, the compositions of the present invention may be applied to mammals, more particularly humans, mice, rats, guinea pigs, rabbits, monkeys, pigs, horses, cows, sheep, However, the present invention is not limited thereto.

The pharmaceutical composition of the present invention may be formulated into a unit dosage form by using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by those having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions or emulsions in oils or aqueous media, or in the form of excipients, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

In addition, the composition of the present invention can also be manufactured into a cosmetic composition.

In some embodiments of the invention, the compositions of the present invention comprise (a) a cosmetically effective amount of a Src kinase inhibitor of the present invention as described above; And (b) an cosmetically acceptable carrier.

As used herein, the term "a cosmetically effective amount" means an amount sufficient to achieve the melanin synthesis promoting effect by the Src kinase inhibitor of the present invention described above.

The cosmetic composition of the present invention may be prepared in any form conventionally produced in the art and may be in the form of a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant- , Oil, powder foundation, emulsion foundation, wax foundation and spray, but is not limited thereto. More specifically, it can be manufactured in the form of a soft lotion, a nutritional lotion, a nutritional cream, a massage cream, an essence, an eye cream, a cleansing cream, a cleansing foam, a cleansing water, a pack, a spray or a powder.

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

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

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

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

When the formulation of the present invention is an interfacial active agent-containing cleansing, the carrier component may include aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyltaurate, sarcosinate, fatty acid amide Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters.

The ingredients contained in the cosmetic composition of the present invention include components commonly used in cosmetic compositions, in addition to Src kinase inhibition and carrier components as an active ingredient, such as antioxidants, stabilizers, solubilizers, vitamins, And may include conventional adjuvants.

According to another aspect of the present invention, there is provided a method of promoting melanin synthesis comprising administering to a subject a Src kinase inhibitor.

Since the method of the present invention includes the Src kinase inhibitor of the present invention described above as an active ingredient, the overlapping contents between the two are omitted in order to avoid the excessive complexity of the present specification according to the overlapping description.

According to the present invention, the Src inhibitor of the present invention promotes melanin synthesis and increases gene expression of related factors (see Figures 1 to 4).

In some embodiments of the invention, the expression of the melanin-producing marker may be detected through a PCR amplification reaction. The amplification of the present invention is performed according to a polymerase chain reaction (PCR). More specifically, the primers of the present invention are used for amplification reactions. In some embodiments of the invention, the amplification of the invention is carried out according to RT-PCR.

The term "amplification reaction" as used herein refers to a reaction to amplify a nucleic acid molecule. A variety of amplification reactions have been reported in the art, including polymerase chain reaction (PCR) (US Pat. Nos. 4,683,195, 4,683,202 and 4,800,159), reverse-transcription polymerase chain reaction (RT-PCR) (Sambrook et al., Molecular Cloning. (McPherson and Moller, 2000), the method of Davey, C. et al (EP 329,822), the method of Miller, HI (WO 89/06700) Ligase chain reaction (LCR), Gap-LCR (WO 90/01069), repair chain reaction (EP 439,182), transcription-mediated amplification (TMA) 88,135), self sustained sequence replication (20) (WO 90/06995), selective amplification of target polynucleotide sequences (U.S. Patent No. 6,410,276), selective amplification of target polynucleotide sequences Consensus sequence primed polymerase chain reaction (CP- PCR (US Patent No. 4,437,975), arbitrarily primed polymerase chain reaction (AP-PCR) (U.S. Patent Nos. 5,413,909 and 5,861,245), nucleic acid sequence based amplification ; NASBA (U.S. Patent Nos. 5,130,238; 5,409,818; 5,554,517; and 6,063,603) and strand displacement amplification. Other amplification methods that may be used are described in U.S. Patent Nos. 5,242,794, 5,494,810, 4,988,617, and U.S. Patent No. 09 / 854,317.

As used herein, the term "primer " means an oligonucleotide in which the synthesis of a primer extension product complementary to a nucleic acid chain (template) is induced, that is, the presence of a polymerizing agent such as a nucleotide and a DNA polymerase, It can act as a starting point for synthesis at suitable temperature and pH conditions. Specifically, the primer is a deoxyribonucleotide and a single strand. The primers used in the present invention may include naturally occurring dNMPs (i.e., dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. In addition, the primers may also include ribonucleotides.

The primer should be long enough to be able to prime the synthesis of the extension product in the presence of the polymerizing agent. The appropriate length of the primer is determined by a number of factors, such as temperature, application, and the source of the primer. The term "annealing" or "priming ", as used herein, refers to oligodeoxynucleotides or nucleic acids apposition to a template nucleic acid, wherein the polymerase is capable of polymerizing a nucleotide to form a template nucleic acid, To form nucleic acid molecules.

When the method of the present invention is carried out using a primer, target genes (for example, MITF, TRP1, TRP2 or tyrosinase) can be simultaneously detected in an analysis target (for example, melanocytes) by performing a gene amplification reaction. Accordingly, the present invention provides a gene amplification reaction using a primer that binds mRNA extracted from an analyte to cDNA synthesized as a template.

The primer used in the present invention is hybridized or annealed at one site of the template to form a double-stranded structure. Nucleic acid hybridization conditions suitable for forming such a double-stranded structure can be found in Nucleic Acid Hybridization < RTI ID = 0.0 > (" , A Practical Approach, IRL Press, Washington, DC (1985).

A variety of DNA polymerases can be used in the amplification of the present invention, including the "Clenow" fragment of E. coli DNA polymerase I, the thermostable DNA polymerase and the bacteriophage T7 DNA polymerase. Specifically, the polymerase is a thermostable DNA polymerase from a variety of bacterial species, including Thermus aquaticus (Taq), Thermus thermophilus (Tth), Thermus filiformis, Thermis flavus, Thermococcus literalis, and Pyrococcus furiosus .

When performing the polymerization reaction, it is preferable to provide the reaction vessel with an excessive amount of the components necessary for the reaction. The excess amount of the components required for the amplification reaction means an amount such that the amplification reaction is not substantially restricted to the concentration of the component. It is desirable to provide the reaction mixture with such joins as Mg ^{2 +} , dATP, dCTP, dGTP and dTTP to such an extent that the desired degree of amplification can be achieved. All enzymes used in the amplification reaction may be active under the same reaction conditions. In fact, buffers make all enzymes close to optimal reaction conditions. Thus, the amplification process of the present invention can be carried out in a single reaction without changing conditions such as the addition of reactants.

In the present invention, annealing is carried out under stringent conditions that allow specific binding between the target nucleotide sequence and the primer. The stringent conditions for annealing are sequence-dependent and vary with environmental variables. The amplified target gene (specifically, MITF, TRP1, TRP2 or tyrosinase) is subjected to gel electrophoresis by a suitable method, for example, the above-mentioned amplification reaction product, and the resultant band is observed and analyzed, Can be detected.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to pharmaceutical compositions and cosmetic compositions for the treatment of melanin deficiency diseases comprising a Src kinase inhibitor.

(b) The Src kinase inhibitor of the present invention not only increased intracellular melanin synthesis but also promoted gene expression of melanin production-related factors.

(c) Therefore, the composition containing the Src kinase inhibitor of the present invention can be very advantageously applied to medicines, quasi-drugs and cosmetics.

FIG. 1A shows the results of promoting melanogenesis in G316 cells according to the treatment concentration of su6656. The upper panel is a photograph taken with an optical microscope and the lower panel is a G361 cell line obtained by centrifuging it. su6656 treatment concentration: (a), 0 M; (b), 1 nM; (c), 10 nM; (d), 100 nM; (e), 1 [mu] M; And (f), 10 [mu] M.
FIG. 1B shows PCR results showing melanin production-related gene expression according to the treatment concentration of su6656. GADPH is a loading control.
Figure 2a shows the results of promoting melanogenesis in G316 cells according to the treatment concentration of PP2. The upper panel is a photograph taken with an optical microscope and the lower panel is a G361 cell line obtained by centrifuging it. PP2 treatment concentration: (a), 0 M; (b), 1 nM; (c), 10 nM; (d), 100 nM; (e), 1 [mu] M; And (f), 10 [mu] M.
FIG. 2B is a PCR showing the expression of a melanin-related gene according to the treatment concentration of PP2. GADPH is a loading control.
FIG. 3 shows the results of (a) promoting melanogenesis in G316 cells according to the treatment concentration of dasatinib and (b) expressing a related gene.
FIG. 4 shows the results of stimulating melanin production (a) and expression of related genes (b) in G316 cells according to treatment of siRNA against Src kinase.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Example 1: Analysis of melanin synthesis by treatment with Src kinase inhibitor su6656

To determine melanin content, human melanoma cell line G361 was cultured and then treated with untreated (negative control), alpha-MSH (a-melanocyte stimulating hormone; positive control) and su6656 (Src kinase inhibitor) The melanin stimulating effect was measured. G361 cells were cultured in low glucose DMEM (Dulbecco's modified Eagle's media) supplemented with 10% fetal bovine serum and 1% penicillin / streptomycin at 37 ° C and 5% CO ₂ . Cells were cultured in a 6-well plate at a concentration of 0.8 × 10 ⁴ cells / well (cells / well), and cells were adhered. After that, the control group was treated with 100 nM α-MSH wells were treated with 1 nM, 10 nM, 100 nM, 1 μM and 10 μM su6656. In each well, medium was changed every 3 days after addition of test substance, and su6656 and α-MSH were also treated in the medium. Wherein the treatment material was added to the treatment concentration after each component was dissolved in the medium. After the cells were treated with the test material for 6-9 days, the number and shape of G361 cells were observed with a microscope. Cells were removed using trypsin-EDTA and the culture medium was removed by centrifugation. After washing with 1XPBS, PBS was removed by centrifugation and the degree of melanogenesis was observed by pellet. As shown in Fig. 1A, the production of melanin was increased in the su6656-treated cell group.

In addition, total mRNA of G361 treated with α-MSH and su6656 was extracted to confirm the increase of melanin production at the gene level. To extract total mRNA, total mRNA was obtained by dissolving the cells with the reagent provided in the kit using an RNA extraction kit. CDNA was synthesized with 500ng of mRNA obtained using 10,000 U reverse transcriptase and 50ng / ul oligo (dT) primer. Expression of MITF, TRP1, TRP2, and tyrosinase genes related to melanin production was confirmed by PCR using cDNA synthesized by RT-PCR. The base sequence of MITF is 5'-caaactggaagacatcctga-3 'right 5'-cttccatgctcatactgctc-3' and the base sequence of TRP1 is 5'-gctgaggagatacaatgctg-3 'right 5'-atttctgtgttggtgactgg-3'. The base sequence of TRP2 is 5'-gtggacagcctagtgaacaa-3 'right 5'-cgtaggatgtagggaccact-3' and the nucleotide sequence of tyrosinase is left 5'-gacataaccgggaatcctac-3 'right 5'-tgaaaagagtctgggtctga-3'. PCR was performed with primers of melanin production related genes at 95 ° C for 2 minutes, 95 ° C for 20 seconds, 58 ° C for 40 seconds, 72 ° C for 1 minute, and 72 ° C for 5 minutes. The product of the PCR was loaded on 1.5% agarose gel to confirm the expression pattern of the gene.

As shown in FIG. 1B, the su6656-treated cells showed similar levels of gene expression to the α-MSH-treated group, which is a positive control.

Example 2 Analysis of Melanin Synthesis Following Treatment of Other Src Kinase Inhibitors, PP2 or Dasatinib

Was carried out in the same manner as described in Example 2, except that the other Src kinase inhibitors, PP2 or dasatinib, were treated.

As shown in Figs. 2A-2B and Fig. 3, the treatment of the Src kinase inhibitor showed a similar level of gene expression as that of the group treated with the positive control group of [alpha] -MSH, in addition to the increase of melanin synthesis amount.

Example 3: Analysis of melanin synthesis by treatment with Src siRNA

Small interfering RNA (siRNA, IDT, USA) was used to suppress the expression of Scr. After replacing the medium of the cultured cells for 24 hours with the medium containing no serum and antibiotics, cultivate for 2 hours. After 2 hours, 20 nM of Src siRNA is transfected with RNAiMAX and stored at room temperature for 30 minutes. After 5 hours, replace with medium containing serum and antibiotics, transfection siRNA in the same manner every 3-4 days, and incubate for 6-9 days.

As shown in Fig. 4, treatment of Src siRNA not only increased the synthesis amount of melanin, but also increased gene expression of melanin synthesis-related factors.

Example 4: Preparation of nanosomal formulations containing a Src kinase inhibitor

The Src kinase inhibitor was mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol and a small amount of oil, and the mixture was adjusted to a total volume of 1 L with distilled water. Then, The nanosomes of about 100 nm in size were prepared by emulsification using high pressure. The prepared nanosomes were used alone or in combination for cosmetic preparation at a final concentration of about 10 nM to about 10 μM.

Formulation Example 1: Softening Age

The softening water containing the nano-sized formulation prepared in Example 4 and having the following composition was prepared according to the general method of producing lotion.

ingredient Content (% by weight) Nanosome 0.01-20 1,3-butylene glycol 6.0 glycerin 4.0 PEG 1500 1.0 Sodium hyaluronate 1.0 Polysorbate 20 0.5 ethanol 8.0 Preservative, pigment Suitable amount Benzophenone-9 0.05 incense a very small amount Purified water Balance Sum 100

Formulation Example 2: Moisturizing cream

A nutritional cream comprising the nano-zose formulation prepared in Example 4 and having the following composition was prepared according to the general method of producing a moisturizing cream.

ingredient Content (% by weight) Nanosome 0.01-20 Meadow Foam Oil 3.0 Cetearyl alcohol 1.5 Stearic acid 1.5 Glyceryl stearate 1.5 Liquid paraffin 10.0 Wax 2.0 Polysorbate 60 0.6 Sorbitan sesquioleate 2.5 Squalane 3.0 1,3-butylene glycol 3.0 glycerin 5.0 Triethanolamine 0.5 Tocopheryl acetate 0.5 Preservative, pigment Suitable amount incense Suitable amount Purified water Balance Sum 100

Formulation Example 3: Moisturizing lotion

The nutritional lotion comprising the nano-zose formulation prepared in Example 4 and having the following composition was prepared according to the general method of producing lotion.

ingredient Content (% by weight) Nanosome 0.02-20 1,3-butylene glycol 4.0 glycerin 4.0 Cetearyl alcohol 0.8 Glyceryl stearate 1.0 Triethanolamine 0.13 Tocopheryl acetate 0.3 Liquid paraffin 5.0 Squalane 3.0 Macadamia nut oil 2.0 Polysorbate 60 1.5 Sorbitan sesquioleate 0.5 Carboxyvinyl polymer 1.0 Preservative, pigment Suitable amount incense Suitable amount Purified water Balance Sum 100

Formulation Example 4: Essence

An essence comprising the nano-sized preparation prepared in Example 4 and having the following composition was prepared according to a general essence production method.

ingredient Content (% by weight) Nanosome 0.05-20 glycerin 10.0 1,3-butylene glycol 5.0 PEG 1500 2.0 Allantoin 0.1 DL-Panthenol 0.3 EDTA-2Na 0.02 Hydroxyethylcellulose 0.1 Sodium hyaluronate 8.0 Carboxyvinyl polymer 0.2 Triethanolamine 0.18 Octyldodec-16 0.4 ethanol 6.0 Fragrance, preservative, pigment Suitable amount Purified water Balance Sum 100

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is obvious that the same is by way of illustration and example only and is not to be construed as limiting the scope of the invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims (15)

SiRNA kinase inhibitors selected from the group consisting of PP2 (CAS 172889-27-9), su6656 (CAS 330161-87-0), siRNA against dasatinib and Src kinase, Chromosomal aberrations, hypochromatosis, hypochromatosis, Crohn ' s disease, Crohn ' s disease, Crohn ' s disease, Wherein the melanin deficiency is selected from the group consisting of Huntington's disease, Huntington's disease, Huntington's disease, Huntington's disease, Huntington's disease, Huntington's disease, Huntington's disease, The pharmaceutical composition according to claim 1, wherein the composition promotes the synthesis of melanin. The pharmaceutical composition for treating melanin deficiency diseases according to claim 1, wherein the composition increases the expression of a melanin-producing marker. 4. The method according to claim 3, wherein the melanin-producing marker gene is a microphthalmia-associated transcription factor (MITF), tyrosinase-related protein 1 (TRP1), tyrosinase-related protein 2 (TRP2) or tyrosinase A pharmaceutical composition. The method according to claim 1, wherein the Src kinase inhibitor further comprises PP1 (CAS 172889-26-8), conservedinib, saracatinib or KX2-391 (CAS 897016-82-9) A pharmaceutical composition for therapeutic use. The pharmaceutical composition of claim 1, wherein the Src kinase inhibitor further comprises a shRNA, miRNA, antisense oligonucleotide, aptamer or ribozyme for Src kinase. delete Wherein the at least one Src kinase inhibitor is selected from the group consisting of PP2 (CAS 172889-27-9), su6656 (CAS 330161-87-0), dasatinib and siRNA against Src kinase. delete 9. The cosmetic composition according to claim 8, wherein the Src kinase inhibitor further comprises PP1 (CAS 172889-26-8), conservedinib, saracatinib or KX2-391 (CAS 897016-82-9). 9. The cosmetic composition of claim 8, wherein the Src kinase inhibitor further comprises an shRNA, miRNA, antisense oligonucleotide, aptamer or ribozyme for the Src kinase. 9. The cosmetic composition according to claim 8, wherein the cosmetic composition has an improvement of vitiligo or an effect of improving white hair. One or more Src kinase inhibitors selected from the group consisting of PP2 (CAS 172889-27-9), su6656 (CAS 330161-87-0), siRNA against dasatinib and Src kinase are administered to subjects other than humans Lt; RTI ID = 0.0 > melanin < / RTI > synthesis. 14. The method of enhancing melanin synthesis according to claim 13, wherein the Src kinase inhibitor promotes the synthesis of melanin. 14. The method of promoting melanin synthesis according to claim 13, wherein said subject comprises a mouse, rat, guinea pig, rabbit, monkey, pig, horse, cattle, sheep, nutrition, dog or cat.

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