KR101104223B1 - Interleukin 10-Derived Peptides and Uses Thereof - Google Patents

Abstract

The present invention relates to IL-10-derived peptides and compositions comprising them. The peptide of the present invention has the same function as that of human IL-10 and is very excellent in stability and skin permeability compared to natural IL-10. According to the invention, the composition comprising the peptide of the invention exerts very good efficacy in the prevention, treatment or amelioration of Th1-mediated immune diseases, in particular inflammation. In addition, the compositions of the present invention have excellent efficacy in improving skin conditions such as atopic dermatitis, atopic dermatitis, psoriasis or acne treatment. Therefore, the excellent activity and stability of the peptide of the present invention described above, it can be very advantageously applied to medicines, quasi-drugs and cosmetics.

Interleukin-10, Th1-mediated immune disorders, atopy, psoriasis

Description

Interleukin 10-Derived Peptides and Uses Thereof}

The present invention relates to an IL 10-derived peptide and a composition comprising the same.

Typical atopic or allergic dermatitis, including atopic dermatitis, psoriasis, seborrheic dermatitis, and the like, have been well studied as immune related diseases. The term "dermatitis" as used herein is generally defined as inflammation of the skin (Stedman's Medical Dictionary, 27th edition, Lippincott Williams & Wilkins (2000)). As used herein, the term "contact dermatitis" is an inflammatory response of the skin to an antigen (or allergen) or stimulant (Stedman's Medical Dictionary, supra). Stimulants directly affect the skin, causing direct tissue damage, while allergens induce an immune response that causes inflammation and tissue damage. Some common stimulants are materials such as wool and synthetic fibers, soaps and bleaches, perfumes and cosmetics, dust and sand, tobacco smoke and chlorine, petroleum or solvents and the like. Typically allergens are substances from food, plants, or animals that irritate the skin and elicit an immune response. If contact with the allergen is repeated, then contact dermatitis then develops into eczema, which is accompanied by the selection and infiltration of the cells. "Atopic dermatitis" and "atopic eczema" or "eczema" and related terms may be used interchangeably and refer to a complex disease primarily caused by cellular immune deficiency and increased immunoglobulin antibody E (IgE). . In addition, allergens, which are irritants to the skin, each develop dermatitis more often than mere exposure to allergic triggers. Anxiety, stress and depression can all play a role in the exacerbation of eczema. In addition, people with atopic eczema may turn out to have an increased number of eosinophils. Atopic dermatitis (AD) is a universal multifactorial disease affected by both genetic predisposition and environmental factors. Strong genetic predisposition, which determines the risk of AD, has been established through twin studies. The pair-wise concordance rate for identical twins was 0.72 compared to 0.23 for fraternal twins. The magnitude of this concordance suggests that genetic predisposition is critical for the development of atopic dermatitis (Schultz Larsen F. Atopic dermatitis: a genetic-epidemiologic study in a population-based twin sample. J Am Acad Dermatol , 28: 719-23 (1993)). The risk of children getting AD is 50% if one parent has atopic dermatitis (AD, asthma or allergic rhinitis) and 75% if both parents have it. It is therefore believed that genetic predisposition plays a predominant role in the predisposition to atopic disease. As already mentioned above, atopy has been found to be an imbalance between Th2- and Th1-type immune responses, at least partially mediated by Th2 cytokines (IL-4, IL-12 and IL-5) (Mosmann TR, Sad S. The expanding universe of T-cell subsets: Th1, Th2 and more.Immunol Today , 17: 138-46 (1996); Grewe M, Bruijnzeel-Koomen CA, Schopf E, Thepen T, Langeveld-Wildschut AG, Ruzicka T, et al. , A role for Th1 and Th2 cells in the immunopathogenesis of atopic dermatitis.Imunmun Today , 19: 359-61 (1998)). However, certain papers have shown that Th1-type cytokines (IFNGs) do not down-regulate allergic immune responses, but rather exacerbate the inflammatory process (Hansen G, Berry G, DeKruyff RH, Umetsu DT.Allergen-specific). Th1 cells fail to counterbalance Th2 cell-induced airway hyperreactivity but cause severe airway inflammation.J Clin Invest , 103: 175-83 (1999)). Thus, the dermatological response in AD can be seen to be affected by both Th2- and Th1-type cytokines.

On the other hand, cytokines comprising the transforming growth factor β1 (TGF-β1) and interleukin 10 have been shown to have a general immunosuppressive activity that blocks both Th1- and Th2-type cytokine activity, similar to IL-10. It is possible to control dermatitis or atopic dermatitis caused by rapid immunosensitization agents if peptide fragments are performed (Letterio JJ, Roberts AB. Regulation of immune responses by TGF beta.Annu Rev Immunol , 16: 137-61 (1998). Hobbs K, Negri J, Klinnert M, Rosenwasser LJ, Borish L. Interleukin-10 and transforming growth factor-beta promoter polymorphisms in allergies and asthma.Am J Respir Crit Care Med , 158: 1958-62 (1998)). In addition, it has been suggested that genes that help determine the production or activity of numerous candidate genes, especially Th1 and Th2 cytokines, in several gene clusters (5q, 6p, 11q13, 12q15-24.1 and 14q) are prone to atopic disease. (Anderson GG, Morrison JF. Molecular biology and genetics of allergy and asthma.Arch Dis Child , 78: 488-96 (1998); Forrest S, Dunn K, Elliott K, Fitzpatrick E, Fullerton J, McCarthy M, et al. ., Identifying genes predisposing to atopic eczema J Allergy Clin Immunol, 104: 1066-70 (1999); Ono SJ Molecular genetics of allergic diseases Annu Rev Immunol, 18:... 347-66 (2000)).

IL-10 is a cytokine that can mediate many actions or effects. IL-10 inhibits macrophage / monocite and T-cell lymphocyte replication and secretion of inflammatory cytokines (IL-1, TNF-α, TGF-β, IL-6, IL-8 and IL-12) It is known to be a potent immunosuppressive Th-2 cell cytokine produced by functional lymphocyte cells (Redpath S, Ghazal P, Gascoigne NR. Hijacking and exploitation of IL-10 by intracellular pathogens. Trends Microbiol , 9:86 -92 (2001)). Several reports have demonstrated that quantitative differences in IL-10 transcription and / or expression contribute to potent immunosuppression, which is associated with the mediated alternative allele or haploid type. IL-10 has been isolated in both mouse and human cells and is involved in regulating the immune response of different classes or subsets of T-helper (Th) cells. Th cells can be classified into different subsets distinguished by their cytokine production properties. In general, after being activated by antigen or mitotic ratin, Th1 T cell clones produce interleukin-2 (IL-2) and IFN-γ, and Th2 cell clones produce IL-10, IL-4 and IL-5. Secrete. Two classes of Th cell clones produce cytokines such as tumor necrosis factor-α (TNF-α), IL-3 and granulocyte-macrophage colony stimulating factor (GM-CSF). The third class of Th cells (Th0) produces IL-2, IFN-γ, IL-4, IL-5, TNF-α, IL-3 and GM-CSF simultaneously. In addition, different cytokine production patterns of Th1 and Th2 cells reflect their helper function. Th1 cells are mainly involved in delayed hypersensitivity and Th2 cells are involved in antibody production. Since antibody (Th2 pathway) and delayed hypersensitivity (Th1 pathway) responses are often independent of each other, Th1 and Th2 cells are thought to have a cross-regulatory effect. IFN- [gamma] produced by Th1 cells inhibits proliferation of Th2 cells, and IL-10 produced by Th2 cells inhibits cytokine synthesis, in particular IFN- [gamma] and IL-2 production by Th1 cell clones.

On the other hand, delayed hypersensitivity reactions are generally cell-mediated immune responses characterized by edema and cellular infiltration of tissue by T cells and monocytes and / or macrophages. Some cytokine sets are separately involved in delayed hypersensitivity and humoral immune responses (Cher et al. , J. Immunol. , 138: 3688 (1987); and mosmann et al. , 1987 and 1989, supra). ). Diseases associated with these classes of responses can be caused by improper production of a related set of cytokines to search for peptides attributable to IL-10, leading to several factors caused by expressed delayed hypersensitivity, especially TNF-α. , IFN-γ and the like to inhibit the growth of tumor mononuclear cells, etc. was able to explore the function.

The present inventors have prepared and screened various kinds of human IL-10-derived peptides in order to produce peptides having the same or similar functions or functions as natural growth factor IL (interleukin) -10, but having better stability than native IL-10. It was. As a result, the present invention has been completed by selecting peptides having excellent physiological activity (for example, promoting immune cell differentiation and pro-inflammatory cytokine inhibition) as well as stability and skin permeability among many peptide candidates.

It is therefore an object of the present invention to provide a peptide exhibiting growth factor activity.

Another object of the present invention is to provide a composition for preventing or treating a Th1-mediated immune disease.

Still another object of the present invention is to provide a composition for improving skin conditions.

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 peptide exhibiting growth factor activity having the amino acid sequence of SEQ ID NO: 1.

According to another aspect of the invention, the present invention provides a composition for the prevention or treatment of Th1-mediated immune disease comprising the peptide as an active ingredient.

The present inventors have prepared and screened various kinds of human IL-10-derived peptides in order to produce peptides having the same or similar functions or functions as natural growth factor IL (interleukin) -10, but having better stability than native IL-10. It was. As a result, among peptide candidates, peptides were selected that were not only excellent in physiological activity (eg, promoting immune cell differentiation, inhibiting pro-inflammatory cytokines), but also having excellent stability and skin permeability.

In the present invention, the peptide of the present invention is represented by the following general formula (1):

Formula 1

Glu-Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn

The present inventors have synthesized a number of cytokine related peptides exhibiting the above-described properties based on sites related to the function of IL-10 and the like. In more detail, the peptide of the present invention is prepared by predicting a binding site for a cytokine receptor protein and optimizing the amino acid sequence of the predicted site. Peptides of the present invention are provided by screening for a peptide capable of producing candidate peptides by screening for a predictable site of receptor binding and screening the peptides with the highest anti-inflammatory activity among these posterior peptides.

According to a preferred embodiment of the invention, the peptide of the invention is derived from human IL-10, which is illustrated in Table 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 peptides of the present invention perform the functions of natural human IL-10, thereby lowering the functions of various cytokines attributable to Th1, macrophage / monocite, T-cell lymphocyte replication and pro-inflammatory cytokines (TNF-α). , INF-γ, IL-6, IL-8 and IL-12).

According to a preferred embodiment of the present invention, the peptide of the present invention has the ability to promote the growth of immune cells.

According to a preferred embodiment of the present invention, cells in which cell growth or differentiation is promoted by the peptides of the present invention are hematopoietic stem cells or immune cells, more preferably B lymphocytes, monocytes, neutrophils, Eosinophils, basophils or macrophages, most preferably B lymphocytes.

According to a preferred embodiment of the present invention, the peptide of the present invention inhibits the production of pro-inflammatory cytokines (see Figure 4).

The peptide of the present invention is superior in stability to natural cytokines by itself, but may be further improved by modification of amino acids. According to a preferred embodiment of the present invention, the C-terminus of the peptide is modified with a hydroxyl group (-OH), an amino group (-NH ₂ ), azide (-NHNH ₂ ) and the like.

According to a preferred embodiment of the invention, the N-terminus of the peptide is from the group consisting of acetyl group, fluorenyl methoxy carbonyl group, formyl group, palmitoyl group, myristyl group, stearyl group and polyethylene glycol (PEG) The protecting group selected is combined.

Modification of the above-mentioned amino acid serves to greatly improve the stability of the peptide of the present invention. The term “stability” herein means not only in vivo stability but also storage stability (eg, room temperature storage stability). The above protecting groups serve to protect the peptides of the present invention from the attack of protein cleavage enzymes in vivo.

On the other hand, the present invention provides a composition for the prevention, treatment or improvement of Th1-mediated immune disease comprising the above-mentioned peptide as an active ingredient.

As demonstrated in the examples below, the cytokine-related peptides of the invention are immunorelevant substances (eg, TNF-α, INF-γ, IL) overexpressed by lipopolysaccharide (LPS) treatment similar to native IL-10. -1β, IL-6 and IL-12) have the ability to inhibit and reduce the expression or activity (see Figures 4 and 5). In addition, in the present invention, the expression or activity of the immune-related substance causes delayed-type hypersensitivity and humoral immune response.

The Th1-mediated immune disease to which the composition of the present invention is applied is not particularly limited and is preferably applied to inflammatory disease, autoimmune disease or transplant rejection. More preferably, the Th1-mediated immune disease to which the composition of the present invention is applied is an autoimmune disease or inflammatory disease, and even more preferably, atopic dermatitis, psoriasis, acne, atopic rhinitis (hay fever), allergic dermatitis ( Eczema), chronic sinusitis or seborrheic dermatitis.

Th1-mediated immune diseases to which the composition of the present invention is applied include atopic dermatitis, psoriasis, acne, atopic rhinitis (hay fever), allergic dermatitis (eczema), chronic sinusitis, seborrheic dermatitis, colitis, inflammatory bowel disease , Type 1 diabetes mellitus, type 2 diabetes mellitus, rheumatoid arthritis, reactive arthritis, osteoarthritis, psoriasis, scleroderma, porosity, atherosclerosis, myocarditis, endocarditis, pericarditis, cystic fibrosis, Hashimoto's thyroiditis, Graves' disease, leprosy, Syphilis, Lyme, Borreliosis, Neuro-Borelliosis, Tuberculosis, Sarcoidosis, Lupus, Discus lupus, Alumni Lupus, Lupus Nephritis, Systemic Lupus Erythematosus, Asthma, Macular Degeneration , Uveitis, irritable bowel syndrome, croissant disease, shogran syndrome, fibromyalgia, chronic fatigue syndrome, chronic fatigue immunodeficiency syndrome, myalgia encephalomyelitis, muscular dystrophy Isaac sclerosis, Parkinson's disease, multiple sclerosis, autism spectrum disorders, including, attention deficit disorder and attention deficit hyperactivity disorder, but is not limited thereto.

According to another aspect of the present invention, the present invention provides a composition for improving skin conditions comprising the above-described peptide of the present invention as an active ingredient.

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

Since the peptide of the present invention has a much lower molecular weight than natural human growth hormones, the skin penetration rate is excellent. Therefore, when the composition of the present invention is applied to the skin topically, the skin condition can be effectively improved.

According to a preferred embodiment of the present invention, the skin condition improvement by the composition of the present invention is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, mushroom removal, acne treatment, wound removal and skin regeneration effect, more preferred It is wrinkle improvement, skin elasticity improvement, skin aging prevention, wound removal and skin regeneration effect. For example, the peptide used as an active ingredient in the present invention promotes the proliferation of fibroblasts, induces increased synthesis of collagen, hyaluronic acid or fibronectin from these cells, and regenerates the keratinocyte layer, epithelial layer and dermal layer of the skin. Or it grows, eventually exerts the effects of wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, wound removal, skin regeneration.

According to the present invention, the peptide of the present invention has a very excellent thermal stability compared to the natural growth hormone (see Fig. 6). Existing growth hormones have the following problems: (a) low synthesis, (b) difficulty in manufacturing, (c) high cost and (d) low stability at temperature and long term storage. However, since the peptide of the present invention can be synthesized at a very low cost and is physicochemically stable at high temperature, it can prevent the degradation of physiological activity to the maximum and have a more improved therapeutic effect by increasing the remaining period in the body. Therefore, the peptide of the present invention can be used in a form suitable for the use of medicines and cosmetics.

According to a preferred embodiment of the present invention, the composition of the present invention comprises (a) a pharmaceutically effective amount of the cytokine-related peptide of the present 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 aforementioned cytokine-related peptide.

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, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil, and the like It doesn't happen. 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 formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

The pharmaceutical compositions of the present invention may be prepared in unit dosage 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 incorporating 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 aqueous medium, or may be in the form of extracts, powders, granules, tablets, capsules, or gels (eg, hydrogels), and may further include dispersants or stabilizers. .

In addition, the composition of the present invention comprises (a) a cosmetically effective amount of the cytokine-related peptide of the present invention described above; 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 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.

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, an 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.

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

(a) The cytokine-related peptide of the present invention may function the same as that of human IL-10.

(b) The peptide of the present invention is very excellent in stability and skin permeability compared to natural IL-10.

(c) Compositions comprising the peptides of the invention exert very good efficacy in preventing, treating or ameliorating Th1-mediated immune diseases, in particular inflammation.

(d) According to the present invention, the composition of the present invention has excellent efficacy in improving skin conditions such as atopic dermatitis, atopic dermatitis, psoriasis or acne treatment.

(e) The excellent activity and stability of the peptides of the present invention make it possible to be very advantageously applied to medicines, quasi-drugs and cosmetics.

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

Synthesis Example 1 Synthesis of EAYMTMKIRN (SEQ ID NO: 1)

700 mg of chloro trityl chloride resin (CTL resin, Nova biochem 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 dichloromethane solution was added to the reactor, 200 mmole of Fmoc-Asn (trt) -OH (Bachem, Swiss) and 400 mmole of diisopropyl ethylamine (DIEA) were added to the mixture, followed by stirring to dissolve well. I was. After the reaction, the mixture was washed with methanol and DIEA (2: 1) in dichloromethane (DCM) for 10 minutes and washed with excess DCM / DMF (1: 1). 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 deprotection solution (20% of piperidine / DMF) was added to the reaction vessel and stirred at room temperature for 10 minutes to remove the solution. After adding the same amount of deprotection solution and maintaining the reaction again for 10 minutes, the solution was removed and washed 3 times with DMF, once with MC, once with DMF once to prepare Asn (trt) -CTL resin. 10 ml of DMF solution was added to a new reactor, 200 mmole of Fmoc-Arg (tBu) -OH (Bachem, Swiss), 200 mmole of HoBt, and 200 mmole of Bop were stirred and dissolved. 400 mmole DIEA was added to the reactor twice in fractions 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 and stirred with a DMF solution three times for 5 minutes and then removed. A small amount of the reaction resin was taken and the degree of reaction was checked using a Nihydrin Test. The deprotection reaction was performed twice as described above with the deprotection solution to prepare Arg (tBu) -Asn (trt) -CTL resin. After sufficient washing with DMF and MC, once again Kaiser test was performed, and the following amino acid attachment experiment was performed as above. Fmoc-Ile, Fmoc-Lys (Boc), Fmoc-Met, Fmoc-Thr (tBu), Fmoc-Met, Fmoc-Tyr (tBu), Fmoc-Ala and Fmoc-Glu (tBu) based on selected amino acid sequences The chain reaction was carried out in the order of. The Fmoc-protector was reacted twice with a deprotection solution twice for 10 minutes and then washed well. Acetylation of acetic anhydride, DIEA, and HoBt was performed for one hour, and then the prepared peptidyl resin was washed three times with DMF, MC, and methanol, and dried slowly by flowing nitrogen air. After drying, a fugitive solution (Trifluroacetic acid) 81.5%, distilled water 5%, Thioanisole (5%) was added thereto, and then shaken occasionally at room temperature to maintain a reaction for 2 hours. The resin was filtered off 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, and then the precipitate was collected by centrifugation and washed twice with cold ether. The mother liquor was removed and dried sufficiently under nitrogen to synthesize 1.0 g of NH2-Glu-Ala-Tyr-Met-Thr-Met-Lys-Ile-Arg-Asn-OH peptide 1 before purification (yield: 87.0%). The molecular weight of 1256.9 (theoretical value: 1256.5) was obtained when measured using a molecular weight meter.

Sequence number Amino acid sequence Analytical Values (Mass Spectrometer) Analytical value Theoretical value One NH2-EAYMTMKIRN-OH 1256.9 1256.5

Test Example 1 Inhibitory Effect of Synthetic Peptides on Growth of Lymphocytes

The growth effect of B lymphocytes on the peptides of the present invention was verified. Cultivated cells from Ramos B lymphocytes (Korea Bank of Korea, Korea) were cultured using a 250 ml tissue culture flask containing DMEM (Dulbecco's minimal essential media, Gibco) containing 10% FBS (fetal bovine serum) It was. 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 DMEM culture medium containing 10% FBS, and then placed in a 24-well tissue culture plate to be 4 X 10 ⁴ cells per well, and cultured under 37 ° C and 7% CO2 conditions for 48 hours. After incubation, washed twice with DMEM medium containing no FBS, replaced, and then the blank sample and the peptide of the present invention were dissolved sterilely in water and 10% DMSO for standard preparation, and then 1 ng / ml, 50 ng / Cultured under the same conditions as above for 3 days at concentrations of ml, 100 ng / ml and 1 μg / ml. After the incubation was completed, the culture supernatant was removed and the cells were fixed with ethanol. After washing with PBS, the washing solution was removed and treated with a colorless SRB solution. After washing sufficiently with PBS, the absorbance was measured at 590 nm, and the survival state of the cells was shown in FIG. 3. As can be seen in Figure 3, it was confirmed that the growth of B lymphocytes changed by LPS concentration-dependently recovered by the peptide of the present invention.

Test Example 2: Validation of Cytotoxicity of Synthetic Peptides

To determine whether the skin cells are cytotoxic, refer to the method of Rigino et al. (Rizzino, et al. Cancer Res. , 48: 4266 (1988)), HaCat cells (Korea Cell Line Bank), NIH3T3 It was measured by the SRB colorimetric method using cells (Korea Cell Line Bank) and B16F10 cell line (Korea Cell Line Bank). Cell lines were cultured using a 250 ml tissue culture flask containing DMEM containing 10% FBS. 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. This was resuspended in DMEM 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 for 24 hours. After 24 hours, the medium was exchanged with the same culture solution except for serum, and the blank sample and the peptide of the present invention were sterilely dissolved in water and 10% DMSO for standard preparation, and then 10 ng / ml, 100 ng / ml, 1 72 hours were incubated under the same conditions as above at concentrations of μg / ml, 10 μg / ml and 100 μg / ml. After the incubation was completed, the culture supernatant was removed and the cells were fixed with ethanol. After washing with PBS, the washing solution was removed and treated with a colorless SRB solution. After washing sufficiently with PBS, the absorbance was measured at 590 nm of ultraviolet light to confirm the survival state of each cell. For HaCat cells, a type of keratinocytes, NIH3T3 cells, a type of fibroblasts, and B16F10, a type of melanocytes, no decrease in cell numbers was observed by peptides administered from low to high concentrations. No change was observed in the observations (no results were shown). 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.

Test Example 3: Measurement of Inflammatory Cytokine Reduction of Synthetic Peptides Using Reverse Transcriptase Polymerase Chain Reaction

In order to more directly confirm that the inflammation-induced cytokine is reduced by treating the synthetic peptides, HaCat cells were treated with the synthetic peptides, and then mRNA production of TNF-α was observed by reverse transcriptase polymerase chain reaction.

First, induction experiments of TNF-α, IL-6, IL-12, IL-1β and INF-γ were inoculated with 1 × 10 ⁵ Hacat cells in a 6-well cell culture dish and cultured for 3 days. After the cells had settled, the medium was exchanged with 2% serum, where only the solvent was used as a control, 10 μg / ml LPS (lipopolysaccharide, Sigma, USA) as a control, and the synthetic peptide was treated at the same concentration as the positive control. After injecting μg / ml and 10 μg / ml, 6 hours of incubation was performed. Subsequently, mRNA was extracted from cultured cells treated with the control and the trigger, and the trigger and the peptide, and reverse transcription polymerase chain reaction was performed using primers of TNF-α. The nucleotide sequences of TNF-α, INF-γ, IL-6, IL-12, IL-1β and Actin used as primers are as follows: TNF-α, 5′-GATCTCAAAGACAACCAACTAGTG-3 ′ (forward) and 5′- CTCCAGCTGGAAGACTCCTCCCAG-3` (reverse); INF-γ, 5′-TGCATCTTGGCTTTGCAGCTCTTCCTCATGGC-3 ′ (forward) and 5′-TGGACCTGTGGGTTGTTGACCTCAAACTTGGC-3 ′ (reverse); IL-6, 5′-AGAGGAGACTTCACAGAGGA-3 ′ (forward) and 5′-ATCTCTCTGAAGGACTCTGG-3 ′ (reverse); IL-12, 5′-GGAAGCACGGCAGCAGAATA-3 ′ (forward) and 5′-AACTTGAGGGAGAAGTAGGAATGG-3 ′ (reverse); IL-1β, 5′-GGAAGCACGGCAGCAGAATA-3 ′ (forward) and 5′-AACTTGAGGGAGAAGTAGGAATGG-3 ′ (reverse); And actin, 5′-GATCTCAAAGACAACCAACTAGTG-3 ′ (forward) and 5′-CTCCAGCTGAAGACTCCTCCCAG −3 ′ (reverse).

Experimental results show that the synthetic peptides inhibit the expression of TNF-α, IL-6, IL-12, IL-1β and INF-γ, which play a major role in the immune response as IL-10. 4). In addition, Western blotting experiments using an antibody of TNF-α (Santa Cruz, USA) confirmed that the degree of protein expression in the cells of the same experiment was reduced (Fig. 5), which means that the synthetic peptide functions similar to IL-10. Means to do.

Test Example 4: Thermal Stability of Prepared Peptides

The peptide prepared through the synthesis example was dissolved in PBS at a concentration of 0.11 mg / ml with the standard form cytokine (IL-13) purchased from R & D (USA). The prepared solution was placed in a glass vial of 1 ml each and left at 37 ° C. The solution, which was left at 37 ° C, was sampled at 0, 5, 10, 20, 30, 40, and 70 days, centrifuged by date to remove denatured peptides or proteins, and the supernatant was taken and quantified using HPLC (Fig. 6). In the case of synthetic peptides, the residual amount was higher than that of natural growth factors, and the samples were treated with cells in the same manner as in Test Example 1, and the remaining activity was measured to measure the thermal stability of the peptides. It was found that activity was higher than self-flow (FIG. 6).

Example 1: Preparation of Nanonized Peptides

50 mg of each of the peptides obtained in the above synthesis example was accurately weighed, and then sufficiently dissolved in 500 ml of distilled water and dissolved. The mixture solution is mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol and a little oil phase, and then 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 preparation alone or in combination with a final concentration of about 1,000 ppm.

Formulation Example 1: Softening Longevity

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 Nanosomes 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: Moisturizing Cream

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

ingredient Content (% by weight) Peptide Nanosomes 0.001 Meadowfoam oil 3.0 Cetearyl alcohol 1.5 Stearic acid 1.5 Glyceryl Stearate 1.5 Liquid 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: Moisturizing Cream

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

ingredient Content (% by weight) Peptide Nanosomes 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

At least one kind of nanosomes of the four peptide nanosomes prepared in Example 1 was prepared, and an essence consisting of the following composition was prepared according to a general essence preparation method.

ingredient Content (% by weight) Peptide Nanosomes 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

1 is a graph showing the results of high performance liquid chromatography analysis of the peptides prepared by the synthesis examples of the present invention.

Figure 2 is a graph showing the growth inhibition of LPS stimulated active lymphocytes by synthetic peptides.

3 is a micrograph showing growth inhibition of LPS stimulated active lymphocytes by synthetic peptides.

Figure 4 is a result showing the reduction of inflammation-induced cytokines of synthetic peptides. Inflammation was induced by LPS treatment to observe the expression levels of inflammatory cytokines TNF-α, IL-6, IL-12, IL-1β and INF-γ. The peptide of the present invention significantly reduced the mRNA levels of TNF-α, IL-6, IL-12, IL-1β and INF-γ.

5 is a result showing the reduction of TNF-α by the synthetic peptide using Western blot.

Figure 6 is a graph comparing the thermal stability of the control and the peptide of the present invention.

<110> Caregen <120> Interleukin 10-Derived Peptides and Uses Thereof <160> 1 <170> KopatentIn 1.71 <210> 1 <211> 10 <212> PRT <213> Homo sapiens <400> 1 Glu Ala Tyr Met Thr Met Lys Ile Arg Asn   1 5 10  

Claims (10)

Peptide showing growth factor activity having the amino acid sequence of SEQ ID NO: 1. The peptide of claim 1, wherein the peptide is derived from IL-10. According to claim 1, wherein the peptide is a peptide, characterized in that to restore the growth of immune cells activated by lipopolysaccharide (LPS). The peptide of claim 3, wherein the immune cells are B lymphocytes, neutrophils, eosinophils, basophils, monocytes or macrophages. The peptide of claim 1, wherein the peptide inhibits the production of pro-inflammatory cytokines. 6. The peptide of claim 5, wherein the pro-inflammatory cytokine is TNF-α, INF-γ, IL-1β, IL-6 or IL-12. A composition for preventing or treating a Th1-mediated immune disease, comprising the peptide of any one of claims 1 to 6 as an active ingredient. 8. The composition of claim 7, wherein the Th1-mediated immune disease is an inflammatory disease, an autoimmune disease or transplant rejection. A composition for improving skin conditions comprising the peptide of any one of claims 1 to 6 exhibiting growth factor activity as an active ingredient. 10. The composition according to claim 9, wherein the improvement of the skin condition is wrinkle improvement, skin elasticity improvement, skin aging prevention, skin moisturization improvement, blotch removal, acne treatment, wound removal and skin regeneration.

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