KR100982179B1 - Peptides Having Activities of Transforming Growth Factor-Beta and Their Uses - Google Patents

Abstract

The present invention provides a method for preventing or treating a TGF-β-marking peptide comprising a specific amino acid sequence and a TGF-β-effective disease or condition comprising the same. It relates to a composition for. The peptide of the present invention is derived from human TGF-β, and has a function or function similar to that of natural human TGF-β. The peptide of the present invention is more stable than natural TGF-β and can improve the problems caused by the large molecular weight of natural TGF-β. The peptide of the present invention can be used for the treatment or amelioration of various diseases or conditions to which TGF-β is applied, and particularly exhibits excellent efficacy on skin whitening and wrinkle improvement.

Transformation growth factor, TGF-β, peptide, stability, whitening, wrinkle improvement

Description

Peptides Having Activities of Transforming Growth Factor-Beta and Their Uses}

The present invention relates to a peptide having the activity of transforming growth factor-beta (TGF-β) and its use.

Transforming growth factor-beta (TGF-β) is a population of polypeptides that regulate cell differentiation and proliferation. Other members of this group include Mullerian inhibitors (Cate et al., Cell , 45: 685-698 (1986)), inhibins (Mason et al., Nature , 318: 659-663). (1985)) and proteins predicted from the transcripts of the Decapentaplage gene complex of Drosophilla (Padgett et al., Nature , 325: 81-84 (1987)). Transformation growth factor-beta (TGF-β) consists of two similar disulfide linked subunits, each having a molecular weight of 13,000 (Assoian et al., J. Biol . Chem ., 258: 7155-7160 (1983); Frolik et al., Proc . Natl . Acad . Sci. USA , 80: 3676-3680 (1983); Frolik et al., J. Biol . Chem ., 260: 10995-11000 (1984)). This is due to several tissues such as placenta (Frolik et al., Nature , 325: 81-84 (1983)), platelets (Childs et al., Proc. Natl . Acad . Sci . USA , 79: 5312-5316 (1982), Assioan et al., J. Biol . Chem ., 258: 7155-7160 (1983), kidneys (Roberts et al., Biochemistry , 22: 5692-5698 (1983)), and bone with reduced mineral content (Seyedin et. al., Proc . Natl . Acad . Sci . USA , 82: 119-123 (1985)). In the presence of 10% serum and epidermal growth factor, TGF-β enhances anchorage independent growth of normal rat kidney fibroblasts (Roberts et al., Proc . Natl . Acad . Sci . USA , 78: 5339-5343 (1981): Roberts et al , Nature, 295:. 417-419 (1982): Twardzik et al, J. Cell Biochem, 28:... man 289-297 (1985)), 10% serum When present, colony formation of AKR-2B fibroblasts can be induced (Tucker et al., Cancer Res ., 43: 1518-1586 (1983). TGF-β has also been shown to cause differentiation of myoblasts and fetal cartilage specific macromolecules in fetal rats (Seyedin et al., J. Biol . Chem ., 261: 5693-5695 (1986)).

In contrast to its effect on cell proliferation, TGF-β purified from human platelets, as well as functional associated proteins isolated from African green monkey cells (BSC-1), has been shown to inhibit the growth of certain cells in culture ( Tucker et al., Science , 226: 705-707 (1984)). TGF-β has also been shown to inhibit the growth of several types of human cancer cell lines (Roberts et., Proc . Natl . Acad . Sci . USA, 82: 119-123 (1985)). This inhibitory / stimulatory effect of TGF-β appears to depend on several factors such as, for example, cell type and cell physiological state (Spon et al., Science , 232: 534 (1986)).

Human TGF-β (Derynck et al., Nature , 316: 701-705 (1985)), Mouse TGF-β (Derynck et al., J. Biol. Chem ., 261: 4377-4379 (1986)) and Simi CDNA clones encoding genes of intra TGF-β (Sharples et. Al., DNA , 6: 239-244 (1987)) were isolated. DNA sequence analysis of these clones revealed that they are synthesized as large precursor polypeptides that produce TGF-β monomers. Very large sequence homology was found between TGF-β precursor proteins from all of the TGF-β sources described above.

It has recently been identified that any protein isolated from bovine bones with reduced mineral content is involved in TGF-β (Seyedin et al., J. Biol . Chem ., 262: 1946-1949 (1987)). This protein is also found in pig platelets (Cheifetz et al., Cell , 48: 409-415 (1987)), human prostate adenocarcinoma cell line PC-3 (Ikeda et al., Biochemistry , 26: 2406-2410 (1987)), And human glioblastoma cell lines (Wrann et al., EMBO J., 6: 1633-1636 (1987)). It was named TGF-β2 because some amino acid sequences of this protein appeared to be homologous to TGF-β. Human TGF-β (Derynck et al., Nature , 316: 701-705 (1985)), mouse TGF-β (Derynck et al., J. Biol . Chem ., 261: 4377-4379 (1986)), described above. And Simian TGF-β (Sharples et al., DNA , 6: 239-244 (1987)) were named TGF-β1.

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.

TGF-β is a very active protein, but its expression is very difficult, expensive, and has a short half-life, thus limiting its smooth use. The inventors of the present invention provide a peptide that can perform functions or actions similar to those of the native human transforming growth factor (TGF-β), but is more stable than the native TGF-β and can improve the problems caused by the large molecular weight of the native TGF-β. To prepare, various kinds of human TGF-β-derived peptides were prepared and screened. As a result, the present invention was completed by selecting a peptide having excellent physiological activity and excellent stability and skin permeability among many peptide candidates.

Accordingly, an object of the present invention is to provide a transforming growth factor-beta (MIG) peptide.

Another object of the present invention is to provide a composition for the prevention or treatment of TGF-β-effective diseases or conditions (TGF-β-effective).

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 an aspect of the present invention, the present invention provides a transforming growth factor-beta (micro) peptide comprising an amino acid sequence represented by SEQ ID NO: 1.

TGF-β is a very active protein, but its expression is very difficult, expensive, and has a short half-life, thus limiting its smooth use. The inventors of the present invention provide a peptide that is capable of functioning or functioning similar to the natural human transforming growth factor (TGF-β) but is more stable than the native TGF-β and can improve the problems caused by the large molecular weight of the native TGF-β. To prepare, various kinds of human TGF-β-derived peptides were prepared and screened. As a result, among the peptide candidates, peptides were selected that were excellent in physiological activity and excellent in stability and skin permeability.

The peptide of the present invention basically comprises an amino acid sequence derived from a natural human TGF-β amino acid sequence.

According to a preferred embodiment of the present invention, the N-terminal or C-terminus of the TGF-β-miking peptide of the present invention is additionally bound to the amino acid sequence attached to the cell, and more preferably to the N-terminus. The cell adhesion amino acid sequence serves to enhance the TGF-β activity of the peptide of the present invention.

The adherent amino acid sequence that may be used in the present invention includes any adherent amino acid sequence known in the art. Preferably, the cell attachment amino acid sequence is RGD (Arg-Gly-Asp), RGDS (Arg-Gly-Asp-Ser), RGDC (Arg-Gly-Asp-Cys), RGDV (Arg-Gly-Asp-Val) , RGES (Arg-Gly-Glu-Ser), RGDSPASSKP (Arg-Gly-Asp-Ser-Pro- Ala-Ser-Ser-Lys-Pro), GRGDS (Gly-Arg-Gly-Asp-Ser), GRADSP ( Gly-Arg-Ala-Asp-Ser-Pro), KGDS (Lys-Gly-Asp-Ser), GRGDSP (Gly-Arg-Gly-Asp-Ser-Pro), GRGDTP (Gly-Arg-Gly-Asp-Thr) -Pro), GRGES (Gly-Arg-Gly-Glu-Ser), GRGDSPC (Gly-Arg-Gly-Asp-Ser-Pro-Cys), GRGESP (Gly-Arg-Gly-Glu-Ser-Pro), SDGR (Ser-Asp-Gly-Arg), YRGDS (Tyr-Arg-Gly-Asp-Ser), GQQHHLGGAKQAGDV (Gly-Gln-Gln-His-His-Leu-Gly-Gly-Ala-Lys-Gln-Ala-Gly -Asp-Val), Gly-Pro-Arg (GPR), Gly-His-Lys (GHK), Tyr-Ile-Gly-Ser-Arg (YIGSR), Pro-Asp-Ser-Gly-Arg (PDSGR), CDPGYIGSR (Cys-Asp-Pro-Gly-Tyr-Ile-Gly-Ser-Arg), LCFR (Leu-Cys-Phe-Arg), EIL (Glu-Ile-Leu), EILDV (Glu-Ile-Leu-Asp Val), EILDVPST (Glu-Ile-Leu-Asp-Val-Pro-Ser-Thr), EILEVPST (Glu-Ile-Leu-Glu-Val-Pro-Ser-Thr), LDV (Leu-Asp-Val) Or LDVPS (Leu-Asp-Val-Pro-Ser), most preferably RGD (Arg-Gly-Asp) . The RGD sequence is selected from fibronectin, an interstitial protein.

While the peptides of the present invention show much better stability than native TGF-β by themselves, the stability can be improved by additional amino acid modifications. According to a preferred embodiment of the present invention, the N-terminus and / or C-terminus of the peptide of the present invention is an acetyl group, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group, polyethylene A protecting group selected from the group consisting of glycol (PEG) and amino acids is further bound. Such protecting groups serve to increase the stability of the peptides of the invention.

More preferably, the protecting group is an amino acid, even more preferably Gly or Ala, most preferably Gly. When amino acids are used as protecting groups, the number of amino acid residues is preferably 1-3, more preferably 1-2, most preferably 1.

The term “stability” herein means not only in vivo stability but also storage stability (eg, room temperature storage stability).

According to a preferred embodiment of the present invention, the peptide of the present invention is a cell attachment amino acid sequence to the N-terminus or C-terminus, preferably the N-terminus of SEQ ID NO: 1, 2 or 3 The protecting group is then bonded to the N-terminus or C-terminus, preferably the N-terminus.

Specific examples of the peptides of the present invention are described in SEQ ID NO: 1.

As used herein, the term "peptide" refers to a linear molecule formed by binding amino acid residues to each other by peptide bonds.

Peptides of the invention can be prepared according to chemical synthesis methods known in the art, in particular solid-phase synthesis techniques (Merrifield,J. Amer . Chem . Soc . 85: 2149-54 (1963); Stewart, et al.,Solid Phase Peptide Synthesis, 2nd. ed., Pierce Chem. Co .: Rockford, 111 (1984)).

The peptide of the present invention not only exhibits excellent physiological activity, but also has excellent thermal stability and stability against physicochemical factors such as acids and alkalis. Therefore, the peptide of the present invention having the excellent long-term preservation can be advantageously applied to products requiring long-term storage, such as pharmaceuticals, quasi-drugs, cosmetics and oral products.

According to another aspect of the present invention, the present invention provides a prevention of diseases or conditions of TGF-β-effective comprising the above-described peptide of the present invention exhibiting TGF-β activity as an active ingredient. Or a therapeutic composition.

Since the composition of the present invention includes the above-described peptide of the present invention as an active ingredient, the overlapping content between the two is omitted in order to avoid excessive complexity of the present specification according to the overlapping description.

The peptide used as an active ingredient in the present invention retains TGF-β activity and exerts the action or function of natural TGF-β when applied to a living body. As used herein, the term "TGF-β activity" refers to all activities conventionally identified with respect to native TGF-β, such as activity of cell proliferation and division. Since the peptide of the present invention is designed to mimic the action of native TGF-β, it can exert all the various in vivo activities of native TGF-β.

Since the peptides used in the present invention not only have the same function or function as natural TGF-β, but also have similar physiological activities, they can be advantageously used for the prevention or treatment of TGF-β-effective diseases or conditions. As used herein, the term “TGF-β-effective disease or condition” means a disease or condition that can be treated or prevented by native TGF-β. TGF-β-effective diseases or conditions are disclosed in US Pat. No. 5,804,363 and US Patent Application Publication No. 20020010134, the teachings of which are incorporated herein by reference.

According to a preferred embodiment of the present invention, the activity exerted by the composition of the present invention, promoting cell proliferation, promoting division, promoting physiological activity of epithelial cells, promoting the formation of blood vessels, promoting nerve regeneration, tissue repair (tissue repair), Healing wounds, treating thrombosis, treating bone defects, treating rheumatoid arthritis, treating uveitis, treating cancer, treating atherosclerosis, promoting collagen, elastin, laminine or hyaluronic acid synthesis, treating periodontal disease or improving skin conditions. Can be expressed.

When the composition of the present invention is applied to periodontal disease, the composition of the present invention may be prepared as a toothpaste, oral cleansing composition or composition for oral care. As used herein, the term “composition for treating periodontal disease” may be replaced with “composition for tooth and mouth care” or “composition for tooth and mouth cleaning”. The peptide of the present invention can promote or prevent the periodontal disease by promoting the physiological activity of epithelial cells of the gum tissue and regenerating damaged gum tissue through rapid healing of gum wounds.

According to a more preferred embodiment, the composition of the present invention has the efficacy or activity of improving the skin condition. In particular, since the peptide used as an active ingredient in the composition of the present invention is much smaller in molecular weight than natural TGF-β, the skin penetration rate is very excellent. Therefore, when the composition of the present invention is applied to the skin topically, it is possible to greatly improve the skin condition. Even more preferably, the improvement of the skin condition by the composition of the present invention is skin whitening, wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturizing improvement, blotch removal, acne treatment, wound removal and skin regeneration effect, Most preferably, skin whitening and wrinkle improvement.

For example, the peptide used as an active ingredient in the present invention exhibits whitening efficacy by effectively blocking melanin formation (eg, inhibiting the activity of tyrosinase) in the skin, and simultaneously removing wrinkles (increase in procollagen or fibronectin). Increase). The whitening composition of the present invention brightens the color of the skin and makes the skin tone uniform, or exhibits effects such as removal of skin pigment and removal of blotch.

Moreover, the peptides of the present invention do not show any toxicity to cells derived from various humans, and thus, it is judged that the utility of the peptides as therapeutic drugs that can be treated very safely is very high.

The compositions of the present invention may be prepared from pharmaceutical and cosmetic compositions.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the above-described peptide of the present invention; 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 peptides described above.

Pharmaceutically acceptable carriers included in the pharmaceutical compositions of the present invention are those commonly used in the preparation, such as lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia rubber, calcium phosphate, alginate, gelatin, Calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrups, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oils, and the like. It is not limited. In addition to the above components, the pharmaceutical composition of the present invention may further include a lubricant, a humectant, a sweetener, a flavoring agent, an emulsifier, a suspending agent, a preservative, and the like. Suitable pharmaceutically acceptable carriers and agents are Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical composition of the present invention may be administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, it may be administered by intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, transdermal administration, or the like. Can be.

Suitable dosages of the pharmaceutical compositions of the present invention may vary depending on factors such as the formulation method, mode of administration, age, weight, sex, morbidity, condition of food, time of administration, route of administration, rate of excretion and response to response of the patient. Can be. On the other hand, the preferred dosage of the pharmaceutical composition of the present invention is 0.0001-100 μg per day.

The pharmaceutical compositions of the present invention may be prepared in unit dose form by formulating with a pharmaceutically acceptable carrier and / or excipient according to methods which can be easily carried out by those skilled in the art. Or may be prepared by incorporation into a multi-dose container. In this case, the formulation may be in the form of a solution, suspension or emulsion in an oil or an aqueous medium, or may be in the form of extracts, powders, granules, tablets or capsules, and may further include a dispersant or stabilizer.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a cosmetically effective amount of the above-described peptide of the present invention; And (b) a cosmetically acceptable carrier.

As used herein, the term “cosmetic effective amount” means an amount sufficient to achieve the skin improving efficacy of the composition 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, animal oils, vegetable oils, waxes, paraffins, starches, trachants, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talc or zinc oxide may be used as carrier components. Can be.

In the case where 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. Especially, in the case of a spray, a mixture of chlorofluorohydrocarbons, propane / Propane or dimethyl ether.

When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as the carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 Fatty acid esters of, 3-butyl glycol oil, glycerol aliphatic ester, polyethylene glycol or sorbitan.

When the formulation of the present invention is a suspension, liquid carrier diluents such as water, ethanol or propylene glycol, suspending agents such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Soluble cellulose, aluminum metahydroxy, bentonite, agar or tracant and the like can be used.

When the formulation of the present invention is a surfactant-containing cleansing, the carrier component is an aliphatic alcohol sulfate, an aliphatic alcohol ether sulfate, a sulfosuccinic acid monoester, isethionate, an imidazolinium derivative, a methyltaurate, a sarcosinate, a fatty acid amide. Ether sulfates, alkylamidobetaines, aliphatic alcohols, fatty acid glycerides, fatty acid diethanolamides, vegetable oils, lanolin derivatives or ethoxylated glycerol fatty acid esters and the like can be used.

The components included in the cosmetic composition of the present invention include components conventionally used in cosmetic compositions, in addition to the peptide and carrier components as active ingredients, and include, for example, conventional agents such as antioxidants, stabilizers, solubilizers, vitamins, pigments and flavorings. Adjuvants may be included.

In summary, the features and advantages of the present invention are as follows:

(Iii) The peptide of the present invention is derived from human TGF-β, and has a function or function similar to that of natural human TGF-β.

(Ii) The peptide of the present invention is more stable than native TGF-β and can improve the problems caused by the large molecular weight of native TGF-β.

(Iii) The peptide of the present invention can be used for the treatment or amelioration of various diseases or conditions to which TGF-β is applied, and particularly exhibits excellent efficacy on skin whitening and wrinkle improvement.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Therefore, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

Example

Example 1 Synthesis of Peptide of SEQ ID NO: 1

500 mg of chloro trityl chloride resin (CTL resin, Novabiochem Cat No. 01-64-0021) was added to a reaction vessel, and 10 ml of methylene chloride (MC) was added thereto, followed by stirring for 3 minutes. The solution was removed, 10 ml of dimethylformamide (DMF) was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of methylene chloride solution was added to the reactor, followed by adding Fmoc-Gln (trt) -OH (Novabiochem) 200 and DIEA (diisopropylethylamine) 400, stirring to dissolve well, and reacting with stirring for 1 hour. After the reaction, the mixture was washed with methanol and DIEA (2: 1) in MC and reacted with resin for 10 minutes, followed by washing with excess MC / DMF (1: 1). The solution was removed, 10 ml of DMF was added thereto, stirred for 3 minutes, and then the solvent was removed again. 10 ml of deprotection solution (20% piperidine / DMF) was added to the reaction vessel and stirred at room temperature for 10 minutes to remove the solution. After the same amount of deprotection solution was added to maintain the reaction again for 10 minutes, the solution was removed, washed twice with DMF, once with MC, once again with DMF for 3 minutes to prepare a Gln (trt) -CTL resin. 10 ml of DMF solution was added to the new reactor, Fmoc-Thr (otbu) -OH (Novabiochem) 200, HoBt (N-Hydroxybenzotriazole) 200 and Bop 200 were added and stirred to dissolve well. 400 DIEA was added twice in fractions into the reactor and stirred for at least 5 minutes until all solids dissolved. The dissolved amino acid mixture solution was placed in a reaction vessel with deprotected resin and reacted with stirring at room temperature for 1 hour. The reaction solution was removed, stirred three times for 5 minutes with DMF solution, and the unreacted solvent was removed. A small amount of the reaction resin was taken and the degree of reaction was checked using a Ninhydrine test. Thr (otbu) -Gln (trt) -CTL resin was prepared by two deprotection reactions similarly as above with the deprotection solution. After washing sufficiently with DMF and MC and once again Kaiser test, the following amino acid attachment experiment was performed similarly to the above. That is, Fmoc-Asp (otbu), Fmoc-Leu, Fmoc-Ser (tBu), Fmoc-Trp (boc), Fmoc-Ile, Fmoc-Thr (tBu), Fmoc based on the amino acid sequence selected as shown in FIG. Chain reaction was performed in order of -Asp (otbu), Fmoc-Gly, Fmoc-Arg (pbf), and Fmoc-Gly. The prepared peptidyl resin was deprotected by the above-described method to remove Fmoc, and then washed three times with DMF, MC, and methanol, dried with flowing nitrogen air slowly, and dried completely under reduced pressure under vacuum under P ₂ O ₅ . Peptitile resin was prepared. 30 ml of fugitive solution (containing 81.5% of Trifluroacetic acid (TFA), 5% of distilled water, 5% of thioanisole, 5% of phenol, 2.5% of EDT, and 1% of TIS) was prepared in the prepared resin. After the addition, the reaction was kept shaking at room temperature for 2 hours. The resin was filtered by filtration and the resin was washed with a small amount of TFA solution and then combined with the mother liquor. Distillation was carried out using the reduced pressure to half the total volume and 50 ml of cold ether was added to induce precipitation, followed by centrifugation to collect the precipitate and washing with two more cold ethers. The mother liquor was removed and dried sufficiently under nitrogen to yield 130 mg of peptide (11 residues) of NH2-Ile-Leu-Tyr-Tyr-Val-Gly-Arg-Thr-Pro-Lys-Val-OH SEQ ID NO: 1 before purification. The molecular weight 1308.7 (theoretical value 1308.62) was obtained using a molecular weight meter (Perseptive Pioneer DE-STR ABI, USA).

delete

2 is a high performance liquid phase chromatography on the peptide which has not been synthesized and purified.

Example 2: Evaluation of Stability of Peptides

To confirm the stability of the purified SEQ ID NO: 1 peptide, the peptide was dissolved in 50 mM Tris-HCl (pH 8.0) buffer to 10 μg / ml. As a control, recombinant TGF-β1 (Sigma) produced from Escherichia coli at a concentration of 1 μg / ml was prepared in the buffer solution. The prepared solution was placed in a glass vial and then left at 40 ° C. MTT assay (Scudiero, DA, et al. Cancer Res., 48) for NIH-3T3 cells (Bank of Korea Cell Line) was sampled on days 0, 1, 10, 25, 50, 75 and 100 by standing at 40 ° C. : 4827-4833 (1988)) to measure the residual amount of the remaining peptide (Fig. 3).

As can be seen in Figure 3, in the case of recombinant TGF-β1 it can be seen that the activity is rapidly reduced over time, the peptide of the present invention was confirmed that the activity lasts for a long time.

Example  3: Nanoization Peptide  Produce

50 mg of the peptide obtained in the above example was accurately weighed, and then dissolved by sufficiently stirring with 500 ml of distilled water. Peptide solution is mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol and a little oil phase, and the amount is adjusted with distilled water so that the total amount is 1 L, followed by high pressure with a microfluidizer. To emulsify using to prepare a nanosome of about 100 nm size. The prepared nanosomes were used for cosmetic production with a final concentration of about 50 ppm.

Formulation example  1: flexible cosmetics

Including the peptide nanosomes prepared in Example 1, the flexible cosmetics consisting of the following composition was prepared according to a general lotion manufacturing method.

ingredient Content (% by weight) Peptide of SEQ ID NO: 1 0.001 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 Quantity Benzophenone-9 0.05 incense a very small amount Purified water Balance Sum 100

Formulation example  2: nutrition cream

Peptide nanosomes prepared in Example 1, the nutrition cream consisting of the following composition was prepared according to the general nutrition cream manufacturing method.

ingredient Content (% by weight) Peptide of SEQ ID NO: 1 0.001 Meadowfoam oil 3.0 Cetearyl Alcohol 1.5 Stearic acid 1.5 Glyceryl Stearate 1.5 Floating paraffin 10.0 Beeswax 2.0 Polysorbate 60 0.6 Solbitan Sesquioleate 2.5 Squalane 3.0 1,3-butylene glycol 3.0 glycerin 5.0 Triethanolamine 0.5 Tocopheryl acetate 0.5 Preservative, pigment Quantity incense Quantity Purified water Balance Sum 100

Formulation example  3: nutrition cosmetics

Including the peptide nanosomes prepared in Example 1, nutrient lotion consisting of the following composition was prepared according to a general lotion manufacturing method.

ingredient Content (% by weight) Peptide of SEQ ID NO: 1 0.002 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 Carboxy Vinyl Polymer 1.0 Preservative, pigment Quantity incense Quantity Purified water Balance Sum 100

Formulation example  4: essence

Including the peptide nanosomes prepared in Example 1, an essence consisting of the following composition was prepared according to a general essence preparation method.

ingredient Content (% by weight) Peptide of SEQ ID NO: 1 0.005 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 Carboxy Vinyl Polymer 0.2 Triethanolamine 0.18 Octyldodec-16 0.4 ethanol 6.0 Incense, preservative, coloring Quantity Purified water Balance Sum 100

Example 4 Reduction of Melanin Pigment of Peptide

In order to measure the melanin reduction effect of the peptide synthesized in Example 1, melanocytes were cultured in C57BL / 6 mice, and then melanin production was induced with α-MSH (α-melanocyte stimulating hormone, Sigma). After treatment for each concentration was measured melanin production inhibitory effect. Melanosite of mice was incubated at 37 ° C. and 5% CO ₂ with medium containing 10% fetal bovine serum (Sigma) in DMEM (Dulbecco's modified Eagle's media, Sigma). After incubating the cells at a concentration of 1 X 10 ⁵ cells / well in a 24-well plate and confirming cell adhesion, the control group was treated with nothing but only the solvent, and the positive control group received α-MSH 200. μg / ml and the rest of the dishes were treated with a concentration of 1 μg / ml and 10 μg / ml. Each dish was incubated for 3 days after adding test substance. In this case, the test substance is dissolved in each of the components in a medium solvent, and then dissolved in a mixed solvent having a propylene glycol: ethanol: purified water ratio of 5: 3: 2, diluted to a test concentration, and mixed in the same ratio. After removing the culture medium by centrifugation, the melanin production of the cells could be visually confirmed, and the results for the treatment group of 10 μg / ml concentration are shown in FIG. 4. As can be seen in Figure 4, the melanin production was rapidly increased in the group containing the α-MSH, the group treated with the peptide showed a similar melanin production when not treated with the α-MSH. This suggests that when a factor that causes melanin production is applied to the skin, the peptide can inhibit the activity and brighten the tone of the skin.

For more precise experiments, the cells were washed with phosphate buffered saline (PBS), dissolved in 1 N sodium hydroxide, the absorbance was measured at 400 nm, and the inhibition of melanogenesis was calculated (Dooley's method). Shown in The result of FIG. 5 is consistent with the result of FIG. 4.

Example 5 Reduction of Tyrosinase Activity of Peptides

In order to verify the whitening activity of the peptide of SEQ ID NO: 1 synthesized in Example 1 and the transforming growth factor, the melanin production inhibitory ability after treatment with α-MSH, a melanogenesis factor, was investigated. B16F10 cells (Korea Cell Line Bank) were inoculated with 1 x 10 ⁵ cells and cultured for 3 days. After the cells were settled, the medium was replaced with 2% serum, and the negative control group was not treated. The positive control group was treated with α-MSH at a concentration of 200 ng / ml. The other dishes were treated with the peptide of SEQ ID NO: 1 prepared in Example together with 200 μg / ml of α-MSH at concentrations of 1 μg / ml and 10 μg / ml, respectively. After culturing for 4 days, the cells were collected and the cells were collected and treated with lysis buffer to extract proteins. The extracted protein was quantified by BCA (bicinchonic acid) method, and 10 L-DOPA (L-3,4-dihydroxyphenylalanine, Sigma) was added to 90 protein solutions for each group, and reacted at 37 ℃ for 30 minutes. The absorbance was measured on a spectrophotometer to determine the activity of tyrosinase.

As can be seen in Figure 6, in the group containing only α-MSH, the activity of tyrosinase is enhanced, but by the additional peptide The activity was reduced by more than 30%, which is shown as a level of conformity with respect to peptide amount. This suggests that the peptide of the present invention can lighten the tone of the skin by inhibiting the activity when the factor of increasing the activity of tyrosinase is applied to the skin and the like, and the peptide of the present invention shows an active mechanism such as transformation growth factor. I think it is.

Example 6: Inhibition of melanogenesis markers of SEQ ID NO: 1 peptide

In order to more reliably verify the whitening activity of TGF-like peptides in mouse melanoma cells induced with melanin by treating with α-MSH, tyrosinase-related protein-1 (TRP1), TRP2 and microphthalmia-associated transcription factor (MITF) RNA production was observed by reverse transcriptase polymerase chain reaction.

First, 3 days of culture was performed by inoculating 1 × 10 ⁵ B16F10 cells in a 6-well cell culture dish. After the cells had settled, the medium was exchanged with 2% serum, and the control group added solvent only and the positive control group contained 200 μg / ml of α-MSH, while the remaining dishes contained 200 μg of α-MSH and 10 μg of the peptide of SEQ ID NO: 1. Treated at / ml concentration. The cells were cultured for 4 days and the morphology of the cells was observed through a microscope. As shown in FIG. 7, the melanosomes induced by α-MSH disappeared by the peptide of SEQ ID NO: 1.

Subsequently, mRNA was extracted from the cultured cells and subjected to reverse transcriptase polymerase chain reaction using primers of TRP1, TRP2 and MITF. The sequence of the primers used in the polymerization reaction is as follows: TRP1; F-primer, 5′-GACAGACCGCTGTGGCTCAT-3 ′; R-primer, 5′-CTCCAGACGCAGGAGGTGGTA-3 ′; TRP2; F-primer, 5′-GCATGACGGTGGACAGCCTAGT-3 ′; R-primer, 5′-GTGTGGTGATCACGTAGTCGG-3 ′; MITF; F-primer, 5′-CCAGCCTGGCGATCATGTCATGC-3 ′; R-primer; 5`-GGTTGGCTGGACAGGAGTTGCTG-3`.

As a result, the peptide of the present invention was found to inhibit the expression of TRP1, TRP2 and MITF genes related to melanogenesis (Fig. 8).

Example 7 Procollagen and Fibronectin Production-Promoting Effects of Sequence 1 Peptides

Incubated for 48 hours The peptide of SEQ ID NO: 1 synthesized in NIH3T3 cells was treated at a concentration of 1 μg / ml and 10 μg / ml, and after 72 hours, the concentrations of procollagen and fibronectin, which are markers of skin wrinkle improvement, were measured. Concentration measurements were performed using a procollagen ELISA kit (Takara, Japan) and a fibronectin ELISA kit (Chemicon, USA). As can be seen in Figure 9, the peptide of the present invention increased the procollagen production of fibroblasts. In addition, as can be seen in Figure 10, the peptide of the present invention increased the fibronectin production of fibroblasts. Through this, it can be seen that the peptide of SEQ ID NO: 1 exhibits very good wrinkle improvement effect upon treatment on the skin.

Example 8 Cytotoxicity Test of Sequence 1 Peptide

To determine whether there is intracellular toxicity of skin cells to the peptide of the present invention, refer to the method of Rizino et al. (Rizzino, et al. Cancer Res. , 48: 4266 (1988)), HaCat cells ( It was measured by colorimetric method of SRB (Sulforhodamine B, Sigma) using the Korea Cell Line Bank), NIH3T3 cells (Korea Cell Line Bank) and B16F10 cell line (Korea Cell Line Bank). Each cell line was cultured using a 250 ml tissue culture flask containing Eagle's minimal essential media (EMEM) containing 10% FBS (fetal bovine serum). The cultured cell line was removed from the bottom of the culture vessel with 0.25% trypsin solution and centrifuged to collect only cell precipitates. It was resuspended in EMEM medium containing no FBS, and then placed in a 96-well tissue culture plate to be 1 x 10 ⁵ cells per well, and incubated at 37 ° C. and 7% CO ₂ conditions for 24 hours. After 24 hours, the medium was changed to the same culture except for the serum completely, and the blank sample and the peptide were sterilely dissolved in water and 10% dimethyl sulfoxide (DMSO) to prepare a standard, followed by 10 ng / ml, 100 ng / ml, 72 hours were incubated at the concentration of 1 μg / ml, 10 μg / ml, and 100 μg / ml under the same conditions as above. After the incubation was completed, the culture supernatant was removed and washed once with PBS. After removing the washing solution, treated with colorimetric SRB solution, washed sufficiently with PBS, and observed the state of the living cells by observing each cell under a microscope, and measured the absorbance at ultraviolet light 590 nm to measure the survival state of each cell. 11c.

As shown in Figures 11a-11c, for HaCat cells, a type of keratinocyte line, NIH3T3 cells, a type of fibroblast, and B16F10 cell line, a type of melanocytes, there was no decrease in the number of cells by peptides administered from low to high concentrations. No change was observed in the microscopic observation of the cells. Through this, the peptide of the present invention can be seen that there is no toxicity of the skin-related cells, which can be seen that even if the peptide is treated on the skin will not have a significant effect on the skin cells.

Having described the specific part of the present invention in detail, it is apparent to those skilled in the art that the specific technology is merely a preferred embodiment, and the scope of the present invention is not limited thereto. Thus, the substantial scope of the present invention will be defined by the appended claims and equivalents thereof.

1 is amino acid sequence of human transforming growth factor-beta (TGF-β) and the sequence information of the peptide of the present invention.

Figure 2 is a graph showing the results of high performance liquid chromatography analysis of the peptide prepared according to the embodiment of the present invention.

3 is a graph showing the results of measuring the stability of the peptide prepared according to the embodiment of the present invention.

Figure 4 is an experimental result comparing the melanin production reduction effect of melanocytes after treatment of the peptide prepared according to the embodiment of the present invention in B16 melanoma cells treated with α-MSH.

FIG. 5 shows the results of absorbance analysis of melanin production after treatment of the peptide prepared according to the embodiment of the present invention in B16 melanoma cells treated with α-MSH.

Figure 6 is a figure measuring the activity of tyrosinase after treatment of the peptide prepared according to the embodiment of the present invention in B16 melanoma cells treated with α-MSH.

Figure 7 is a photograph observing the melanoma changes of melanoma cells after treatment of the peptide prepared according to the embodiment of the present invention in B16 melanoma cells treated with α-MSH.

8 is a diagram of reverse transcriptase polymerase chain reaction demonstrating the effect of reducing the melanogenesis marker after treatment of the peptide prepared according to the embodiment of the present invention in B16 melanoma cells treated with α-MSH.

9 is cultured NIH3T3 Figure shows the increased production of procollagen after treatment of the peptide prepared according to the embodiment of the present invention in fibroblasts.

10 is cultured NIH3T3 Figure shows the increase in the production of fibronectin after treatment of the peptide prepared according to an embodiment of the present invention in fibroblasts.

11A-11C show the results of cytotoxicity experiments on keratinocytes, fibroblasts and B16 melanoma cells using the peptides prepared according to the Examples of the present invention.

<110> Caregen. Co. Ltd. <120> Peptides Having Activities of Transforming Growth Factor-Beta and          Its Uses <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 11 <212> PRT <213> Artificial Sequence <220> <223> TGF-Beta Mimicking Peptide 1 <400> 1 Ile Leu Tyr Tyr Val Gly Arg Thr Pro Lys Val   1 5 10  

Claims (10)

TGF-β (transforming growth factor-beta) activity showing the TGF-β (transforming growth factor-beta) -mimicking peptide consisting of the amino acid sequence represented by the first sequence sequence showing the activity, arteriosclerosis, A pharmaceutical composition for preventing or treating a disease or conditions of TGF-β-effective selected from the group consisting of wound, rheumatoid arthritis, uveitis and cancer. delete delete delete delete delete delete delete Wrinkle improvement or skin comprising TGF-β (transforming growth factor-beta) -mimicking peptide as an active ingredient consisting of an amino acid sequence represented by the first sequence of the sequence listing showing TGF-β (transforming growth factor-beta) activity Whitening cosmetic composition. delete

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