KR102354916B1 - A novel peptide 3PS_I014 and uses thereof - Google Patents

Abstract

The present invention relates to a novel peptide 3PS_I014 and its use, and more particularly, to a peptide 3PS_I014 consisting of a novel amino acid sequence and an anti-inflammatory use thereof, wherein the novel peptide consisting of the amino acid sequence of SEQ ID NO: 1 is inflammatory in human epidermal cells By decreasing the expression of the cytokines CCL17 and CCL22 that induce

Description

Novel peptide 3PS_I014 and uses thereof

The present invention relates to a novel peptide 3PS_I014 and its use, and more particularly, to a peptide composed of a novel amino acid sequence and its anti-inflammatory use.

As an organ constituting the outside of the body, the skin is an important physical barrier that protects the body from various external physical and chemical stimuli while maintaining internal homeostasis. The skin not only prevents the outflow of moisture and electrolytes, but also maintains body temperature control and homeostasis such as temperature, responds to stimuli such as touch, pressure, pain, and warmth, as well as immune function, vitamin D synthesis, etc. perform the function of

A problem that prevents the skin from performing its functions is called a skin disease. Among them, an inflammatory skin disease in which the skin is inflamed is a disease in which symptoms such as redness, swelling, heat, and pain appear, and by type, contact dermatitis, irritation It is classified as contact dermatitis, allergic contact dermatitis, phototoxic and photoallergic contact dermatitis, contact urticaria syndrome, atopic dermatitis, seborrheic dermatitis, blister eczema, coin eczema, autosensitization dermatitis, housewife eczema, and stasis dermatitis.

Inflammatory skin diseases involve several cells such as T lymphocytes, Langerhans cells, eosinophils and keratinocytes, and are caused by several factors such as cytokines, chemokines, and immunoglobulin molecules. Among them, keratinocytes act as a derivative and target of an immune response occurring in the skin as a component constituting most of the epidermis of the skin.

When skin keratinocytes are exposed to environmental and physiological stress, an inflammatory response is induced, and various inflammatory cytokines such as IL-4, TNF-α, IFN-γ are secreted, and TGF-β, Keratinocyte growth factor (KGF) It also has receptors for these cytokines. In particular, TNF-α, an inflammatory cytokine, affects dermatitis by acting on the production of several cytokines, cell growth and differentiation, and necrosis, and these cytokines reduce the proliferation rate of skin keratinocytes and induce inflammation Increases the secretion of cytokines that exacerbate skin inflammation. That is, inflammatory skin disease is related to the sensitivity of skin keratinocytes to external stimuli and stress, and if factors affecting skin keratinocytes are removed or the response of skin keratinocytes is reduced, inflammatory skin disease can be effective in treating or improving

Since such inflammatory skin diseases not only medically damage the health of the human body, but also damage the appearance, many people have suffered from inflammatory skin diseases until now. For the treatment of inflammatory skin diseases, methods such as applying a moisturizer for maintaining moisture in the skin or administering a steroid for regulating an inflammatory response of the immune system are commonly used. However, these methods only relieved symptoms temporarily. In the case of steroids, when used in excess, blood pressure and blood sugar rise, edema, insomnia, psychiatric problems occur, Cushing's disease, electrolyte abnormalities, menstrual irregularities, increased appetite, weight It has been reported that side effects such as increase, liver function abnormality, bone necrosis, muscle weakness, cataracts, glaucoma, increased intraocular pressure, and delayed wound healing appear. Therefore, in order to treat or improve inflammatory skin diseases, research on the development of new formulations for inflammatory skin diseases that are safe for the skin as well as exhibit anti-inflammatory activity is required.

On the other hand, a peptide preparation is a substance in which 2-50 amino acids, which are protein components, are linked, and is defined as the ‘minimum unit with a protein function’. Peptide formulations select the smallest unit with excellent physiological activity among proteins to control biosignal transduction and function. It can exhibit pharmacological action and activity. In addition, the success rate of new drugs in the clinical stage is more than twice that of low-molecular compounds. Since 2008, clinical trials for new peptide drugs have been increasing, and it has been reported that only 400 new peptide drugs are currently in the clinical stage. Therefore, new peptide formulations suitable for treatment of specific diseases are newly discovered, and research on whether they can be provided as new drugs is continuously being conducted.

The present inventors synthesized a peptide of a new sequence in consideration of this point, and completed the present invention by confirming that the peptide exhibits anti-inflammatory activity in human epithelial cells.

The present invention is to provide a novel peptide 3PS_I014, and to provide an anti-inflammatory use of the novel peptide by confirming the anti-inflammatory activity of the novel peptide 3PS_I014.

The present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

In addition, the present invention provides an anti-inflammatory composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In addition, the present invention provides an anti-inflammatory pharmaceutical composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

According to the present invention, the novel peptide 3PS_I014 consisting of the amino acid sequence of SEQ ID NO: 1 reduces the expression of the cytokines CCL17 and CCL22 that induce inflammation in human epidermal cells, thereby exhibiting anti-inflammatory activity, the novel peptide 3PS_I014 A composition comprising as an active ingredient may be provided as an anti-inflammatory pharmaceutical composition or a cosmetic composition.

1 is an electrophoretic photograph confirming the change in the expression level of the cytokine CCL17 or CCL22 gene when the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to the present invention is treated in a human keratinocyte line (HaCaT cell).

The terms used in this specification have been selected as currently widely used general terms as possible while considering the functions in the present invention, but these may vary depending on the intention or precedent of a person skilled in the art, the emergence of new technology, and the like. In addition, in a specific case, there is a term arbitrarily selected by the applicant, and in this case, the meaning will be described in detail in the description of the corresponding invention. Therefore, the term used in the present invention should be defined based on the meaning of the term and the overall content of the present invention, rather than the name of a simple term.

Unless defined otherwise, all terms used herein, including technical and scientific terms, have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Terms such as those defined in a commonly used dictionary should be interpreted as having a meaning consistent with the meaning in the context of the related art, and should not be interpreted in an ideal or excessively formal meaning unless explicitly defined in the present application. does not

Numerical ranges are inclusive of the values defined in that range. Every maximum numerical limitation given throughout this specification includes all lower numerical limitations as if the lower numerical limitation were expressly written. Every minimum numerical limitation given throughout this specification includes all higher numerical limitations as if the higher numerical limitation were expressly written. Any numerical limitation given throughout this specification shall include all numerical ranges within the broader numerical range, as if the narrower numerical limitation were expressly written down.

Hereinafter, the present invention will be described in more detail.

The present invention provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

amino acid sequence SEQ ID NO: Example 1 KGFIRIHKQV NLTNK One

K of the amino acid sequence is lysine (Lysine, Lys), G is glycine (Glycine, Gly), F is phenylalanine (Phenylalanine, Phe), I is isoleucine (Isoleucine, Ile), R is arginine (Arginine, Arg), H is histidine (His), Q is glutamine (Gln), V is valine (Valine, Val), N is asparagine (Asparagine, Asn), L is leucine (Leucine, Leu), T is Threonine, Thr). The amino acids constituting the peptide include L-form, D-form, and DL-form, and the amino acids constituting the peptide of the present invention include all of them. The peptide includes variants, and includes those in which a part of the structure of the peptide of the present invention is mutated without changing the main activity due to natural or artificial mutation.

In addition, the present invention provides an anti-inflammatory composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient. The peptide 3PS_I014 of the present invention may exhibit an anti-inflammatory effect by suppressing the expression level of the cytokine CCL17 or CCL22 gene in the cells induced in the inflammatory response.

The cell in which the inflammatory response is induced refers to a cell capable of inducing or exacerbating an inflammatory response by stimulation of other cells or their own secreted TNF-α or IFN-γ cytokines. For example, skin cells stimulated by cytokines that induce inflammation, such as TNF-α or IFN-γ, increase the secretion of cytokines such as CCL17 or CCL22, thereby causing skin inflammation. That is, when there is stimulation of cytokines inducing inflammation such as TNF-α or IFN-γ in skin cells, suppression of expression of CCL17 or CCL22, or stimulation of cytokines TNF-α or IFN-γ inducing inflammation It can be said that if the expression is reduced compared to that receiving the treatment, the effect of preventing or inhibiting skin inflammation appears.

Therefore, “inhibiting the expression level of the CCL17 or CCL22 gene” means that the peptide consisting of the amino acid sequence of SEQ ID NO: 1 according to the present invention induces inflammation in skin cells, such as TNF-α or IFN-γ stimulation It may mean a case in which the expression level of the CCL17 or CCL22 gene is reduced compared to that received, or it may mean a case in which the CCL17 or CCL22 gene is not expressed.

The “inflammatory response-induced cell” refers to all cells that have been stimulated by cytokines, chemokines, or immunoglobulins that cause inflammation as cells that have received chemical or immunological stimulation that causes an inflammatory response. For example, it may be skin keratinocytes, and the chemical or immunological stimulation that causes an inflammatory response may be, for example, cytokines secreted by other cells or cells themselves, such as stimulation of TNF-α or IFN-γ. .

In addition, the present invention provides an anti-inflammatory pharmaceutical composition comprising the peptide 3PS_I014 consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The pharmaceutical composition may be for preventing or treating an inflammatory skin disease, and the inflammatory skin disease is one selected from the group consisting of atopic dermatitis, systemic lupus erythematosus, contact dermatitis, allergic skin disease, acne, acne, and urticaria. It may be the above disease, but is not limited thereto.

In the present invention, the term “prevention” refers to any action of inhibiting or delaying inflammatory skin disease by administering, for example, applying the composition according to the present invention to the skin. In the present invention, the term “treatment” refers to any action for improving or beneficially changing the symptoms of inflammatory skin disease by administering, for example, applying the composition according to the present invention to the skin.

The pharmaceutical composition may be formulated in the form of one or more external preparations selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastas, and cataplasmas.

The pharmaceutical composition of the present invention may further include a pharmaceutically acceptable carrier and diluent for formulation. The pharmaceutically acceptable carriers and diluents include starch, sugar, and excipients such as mannitol, fillers and extenders such as calcium phosphate, cellulose derivatives such as carboxymethylcellulose, hydroxypropylcellulose, gelatin, alginate, and polyvinyl blood. binders such as rolidone, lubricants such as talc, calcium stearate, hydrogenated castor oil and polyethylene glycol, disintegrants such as povidone, crospovidone, and surfactants such as polysorbates, cetyl alcohol, and glycerol does not The pharmaceutically acceptable carrier and diluent may be biologically and physiologically compatible with the subject. Examples of diluents include, but are not limited to, saline, aqueous buffers, solvents, and/or dispersion media.

In addition, the present invention provides an anti-inflammatory cosmetic composition comprising the peptide 3PS_I014 consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

The cosmetic composition may be formulated with one or more selected from the group consisting of skins, lotions, creams, serums, emulsions, essences, powders, foundations, sprays, mask packs, sheet packs, sleeping packs, wash-off packs, and peel-off packs. can The cosmetic composition may be formulated by a conventional method.

Specifically, the cosmetic composition can be prepared in general emulsified formulations and solubilized formulations. For example, lotion, such as a softening lotion or a nourishing lotion; emulsions such as facial lotions and body lotions; creams such as nourishing creams, moisturizing creams, and eye creams; essence; makeup ointment; spray; gel; pack; sunscreen; makeup base; foundations such as liquid type, solid type or spray type; powder; makeup removers such as cleansing creams, cleansing lotions, and cleansing oils; Alternatively, it may be formulated as a cleaning agent such as a cleansing foam, soap, body wash, but is not limited thereto. In addition, the cosmetic composition is an external preparation for the skin, and may be formulated as an ointment, patch, gel, cream or spray, but is not limited thereto.

In the cosmetic composition, in addition to the essential components in each formulation, other components may be appropriately blended within the range that does not impair the purpose according to the present invention according to the type or purpose of use, etc. of the formulation.

The cosmetic composition may include a conventionally acceptable carrier, for example, oil, water, surfactant, humectant, lower alcohol, thickener, chelating agent, colorant, preservative, fragrance, etc. may be appropriately formulated, but is limited thereto no.

The acceptable carrier may vary depending on the formulation. For example, when formulated in an ointment, paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide or their Extracts may be used.

When the cosmetic composition is formulated as a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or extracts thereof may be used as a carrier component, and in the case of a spray, chlorofluorohydro It may further contain a propellant such as carbon, propane, butane or dimethyl ether.

When the cosmetic composition is formulated as a solution or emulsion, a solvent, solubilizer, or emulsifier may be used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl benzoate, propylene glycol, 1,3-Butylglycol oil may be used, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan may be used.

When the cosmetic composition is formulated as a suspension, a liquid diluent such as water, ethanol or propylene glycol as a carrier component, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester; Microcrystalline cellulose, aluminum metahydroxide, bentonite, agar or tracanth and the like can be used.

When the cosmetic composition is formulated as a soap, alkali metal salts of fatty acids, fatty acid hemiester salts, fatty acid protein hydrolysates, isethionates, lanolin derivatives, aliphatic alcohols, vegetable oils, glycerol, sugars, etc. may be used as carrier components. can

The cosmetic composition is a fatty substance, organic solvent, solubilizer, thickening agent, gelling agent, softening agent, antioxidant, suspending agent, stabilizing agent, foaming agent, and fragrance commonly used in the industry depending on the quality or function of the final product. , surfactant, water, ionic or nonionic emulsifier, filler, sequestering agent, chelating agent, preservative, blocking agent, wetting agent, essential oil, dye, pigment, hydrophilic or lipophilic active agent, commonly used in cosmetics It may additionally contain adjuvants commonly used in the field of cosmetics or dermatology, such as any other ingredients.

However, the adjuvant and its mixing ratio may be appropriately selected so as not to affect the desirable properties of the cosmetic composition according to the present invention.

Hereinafter, experimental examples and examples will be described in detail to help the understanding of the present invention. However, the following experimental examples and examples are only to illustrate the content of the present invention, the scope of the present invention is not limited to the following experimental examples and examples. Experimental examples and examples of the present invention are provided to more completely explain the present invention to those of ordinary skill in the art.

Hereinafter, reagents used in Examples and the like were commercially available, and the best products were used, and unless otherwise stated, those purchased from Sigma-aldrich were used.

Example 1. Preparation of novel peptides

To synthesize the novel peptide 3PS_I014 consisting of the amino acid sequence of SEQ ID NO: 1, the present inventors used Merrifield's liquid-phase method using a Fmoc amino group protective container. The peptide synthesis method was a solid phase method using Fmoc (9-fluorenylmethoxycarbonyl) as a protecting group of the Nα-amino group of an amino acid.

Specifically, Rink Amide MBHA-Resin was used as a starting material for a peptide having a carboxyl terminus of -NH ₂ , and Fmoc-amino acid-Wang Resin was used as a starting material for a peptide having a carboxyl terminus of -OH. The elongation of the peptide chain by Fmoc-amino acid coupling was performed by DCC (N-hydroxybenzotriazole(HOBt)-dicyclo-hexylcar-bodiimide) method. After coupling the Fmoc-amino acid at the amino terminus of each peptide, the Fmoc group was removed with 20% piperidine/N-methylpyrrolidone (NMP) solution and washed several times with NMP and dichloromethane (DCM), followed by nitrogen dried with gas To this, a solution of TFA (trifluoroacetic acid)-phenol-thioanisole-H2O-triisop- ropylsilane (85: 5: 5: 2.5: 2.5, vol./vol.) was added and reacted for 3 hours to remove the protecting group and remove the peptide from the resin. After separation, the peptide was precipitated with diethyl ether. The crude peptide obtained in this way was purified using an acetonitrile gradient containing 0.1% TFA by a purified reverse phase-HPLC column (Delta Pak, C18 300Å, 15 μm, 19.0 mm x 30 mm). , Waters) was used for purification.

After hydrolysis of the synthetic peptide with 6N-HCl at 110° C. for 24 hours, the obtained residue was concentrated under reduced pressure, dissolved in 0.02 N-HCl, and the amino acid composition was measured with an amino acid analyzer (Hitachi 8500 A). In addition, molecular weight was calculated based on the sequence of the synthesized peptide, and the exact molecular weight was measured using MALDI mass spectrometry (matrixassisted laser desorption ionization mass spectrometer). As a result, since the measured molecular weight and the calculated molecular weight were identical, it was confirmed that a peptide having an accurate amino acid sequence was synthesized.

Example 2. Evaluation of anti-inflammatory activity of peptides

In order to confirm whether the novel peptide 3PS_I014 synthesized in Example 1 exhibits anti-inflammatory activity, the degree of inhibition of cytokines expressed during inflammation in human epidermal cells, human keratinocytes (HaCaT cells), was evaluated.

HaCaT cells were cultured in DMEM (Dulbecco's Modified Eagle's Medium) medium (Gibco, Grand Island, NY, USA) containing 1% penicillin-streptomycin and 10% Fetal bovine serum (FBS) at 37°C and 5% CO ₂ conditions. . DMEM medium containing 10% FBS was dispensed in a 6-well cell culture plate, and the cultured HaCaT cells were cultured at 3 x 10^5/well for 24 hours. After washing with serum-free medium, TNF-α/IFN-γ 10 ng/mL was treated alone (positive control), or TNF-α/IFN-γ 10 ng/mL and the peptide of Example 1 10 μg/mL Simultaneous treatment was followed by incubation for 24 hours. After incubation, the medium was removed from each well, washed with D-PBS, and RNA was isolated using RNA-prep (Bio-Zol, MA500, Biosang) solution according to the manufacturer's method. cDNA was synthesized with 5 μg of isolated RNA, and, as shown in Table 2 below, PCR was performed with each primer targeting the CCL17 or CCL22 gene, and electrophoresis was performed with 1.2% agarose gel to determine the expression of each CCL17 or CCL22 gene. The expression level was confirmed (FIG. 1). GAPDH was used as a house keeping gene, and HaCaT cells that were not treated with TNF-α/IFN-γ or the peptide of Example 1 were used as a negative control.

division (direction) primer sequence SEQ ID NO: CCL17 (forward) actgtctcccgggactacct 2 (reverse) tccctcactgtggctcttct 3 CCL22 (forward) tgccctgggtgaagatgatt 4 (reverse) agaggatgggttagaggggt 5

1, as a result of treating HaCaT cells with TNF-α/IFN-γ, expression of CCL17 and CCL22 genes compared to HaCaT cells not treated with TNF-α/IFN-γ or the peptide of Example 1 at all was increased, which showed that an inflammatory response was induced by treatment of TNF-α/IFN-γ in HaCaT cells.

On the other hand, when HaCaT cells were treated with TNF-α/IFN-γ and the peptide of Example 1 at the same time, the expression of CCL17 and CCL22 genes was reduced compared to the case where only TNF-α/IFN-γ was treated, which was the result of Example 1 It shows that the peptide has the effect of inhibiting the inflammatory response induced by TNF-α/IFN-γ treatment. The above results demonstrate that the peptide of Example 1 exhibits anti-inflammatory activity, suggesting that a composition comprising the peptide of Example 1 can be used as an anti-inflammatory agent.

As described above in detail a specific part of the present invention, for those of ordinary skill in the art, it is clear that this specific description is only a preferred embodiment, and the scope of the present invention is not limited thereby. do. That is, the substantial scope of the present invention is defined by the appended claims and their equivalents.

<110> 3BIGS <120> A novel peptide 3PS_I014 and uses thereof <130> DP-2020-0025 <160> 5 <170> KoPatentIn 3.0 <210> 1 <211> 15 <212> PRT <213> Artificial Sequence <220> <223> 3PS_I014 <400> 1 Lys Gly Phe Ile Arg Ile His Lys Gln Val Asn Leu Thr Asn Lys 1 5 10 15 <210> 2 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL17_f <400> 2 actgtctccc gggactacct 20 <210> 3 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL17_r <400> 3 tccctcactg tggctcttct 20 <210> 4 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL22_f <400> 4 tgccctgggt gaagatgatt 20 <210> 5 <211> 20 <212> DNA <213> Artificial Sequence <220> <223> CCL22_r <400> 5 agaggatggg ttagaggggt 20

Claims (9)

A peptide consisting of the amino acid sequence of SEQ ID NO: 1. An anti-inflammatory composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The inflammation is an anti-inflammatory composition, characterized in that the disease is caused by increased expression of CCL17 or CCL22. The composition for anti-inflammatory according to claim 2, wherein the peptide suppresses the expression level of the cytokine CCL17 or CCL22 gene in the cells induced in the inflammatory response. As an anti-inflammatory pharmaceutical composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The inflammation is a pharmaceutical composition, characterized in that the disease is caused by increased expression of CCL17 or CCL22. delete The pharmaceutical composition according to claim 4, wherein the inflammation is at least one disease selected from the group consisting of atopic dermatitis, systemic lupus erythematosus, contact dermatitis, acne, acne, and urticaria. 5. The pharmaceutical composition according to claim 4, wherein the pharmaceutical composition is formulated in the form of one or more external preparations selected from the group consisting of creams, gels, patches, sprays, ointments, warning agents, lotions, liniments, pastas, and cataplasmas. composition. As an anti-inflammatory cosmetic composition comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient,
The inflammation is a cosmetic composition, characterized in that the disease is caused by increased expression of CCL17 or CCL22. The cosmetic composition of claim 8, wherein the cosmetic composition is selected from the group consisting of skin, lotion, cream, serum, emulsion, essence, powder, foundation, spray, mask pack, sheet pack, sleeping pack, wash-off pack and peel-off pack. A cosmetic composition formulated with one or more.

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