Peptide mimetic with excellent effect in controlling itch-inducing cytokines and cosmetic composition containing the same

Abstract

The present invention relates to a peptidomimetic and a composition containing the same that exhibits excellent inhibitory effects on itch-inducing cytokines (TNF-α, IL-4, IL-6, and IL-8), and more specifically, to a peptidomimetic and a composition containing the same that include a histidine derivative capable of effective control of itch-inducing cytokines. The tetrapeptide containing the histidine derivative of the present invention exhibits excellent inhibitory effects on the inflammatory cytokines TNF-α, IL-4, IL-6, and IL-8, and also exhibits excellent stability in the human body when applied, making it extremely useful in industrial fields such as cosmetics and pharmaceuticals.

Description

Technical Field

[0001] The present invention relates to a peptidomimetic having an excellent effect on controlling itch-inducing cytokines (TNF-α, IL-4, IL-6, and IL-8) and a composition containing the same. More specifically, the present invention relates to a peptidomimetic containing a histidine derivative capable of effectively controlling itch-inducing cytokines and a composition containing the same.

Background Art

[0002] Peptides are the core materials of biology, the functional minimum units of proteins, and are composed of 2 to 50 or fewer amino acids. They not only exhibit excellent efficacy in small amounts but also have no toxicity, so they are widely used as the main raw materials for pharmaceuticals, foods, and cosmetics. In particular, peptide cosmetic materials that are safe and effective for the human body are not only increasing in usage day by day, but the development of new materials is very important in the industry due to effects such as import substitution.

[0003] In addition, peptidomimetic is an analog of a peptide and is a core next-generation bio-material that can epochally overcome the disadvantage of poor in vivo stability while maintaining the high efficacy that is the advantage of the peptide.

[0004] Atopy can be said to be "chronic skin inflammation due to immune imbalance", and the immune imbalance is particularly caused by Th1 and Th2 immune imbalances. It is known that Th2 immunity is increased in most patients with skin pruritus.

[0005] Atopic dermatitis is a chronic, intractable inflammatory skin disease characterized by itching (pruritus), skin redness, lichenification, and skin infections. Its causes can be divided into congenital (genetic) and acquired factors. Genetic factors include filaggrin gene mutations, while acquired factors include stimulation by various triggering substances and immunological factors. These factors and damage to the skin barrier are caused by complex interactions between them.

[0006] Cytokines secreted by immune cells (TNF-α, IL-4, IL-6, and IL-8) mediate immune responses, perpetuate inflammation, and worsen skin barrier dysfunction, leading to pruritus.

[0007] In this regard, Patent Document 1: Korean Published Patent No. 2022-0086511 discloses an anti-inflammatory peptide for the prevention or treatment of atopic dermatitis, and Patent Document 2: Korean Published Patent No. 2021-0154536 discloses an anti-allergic or atopic dermatitis improving composition containing a pentapeptide as an active ingredient, but both had the problem of low stability in the body.

[0008] Therefore, in the process of researching and developing various peptomimetic compounds containing histidine derivatives that can effectively control inflammatory cytokines (TNF-α, IL-4, IL-6, and IL-8), which are the main causes of skin itching, the present inventors confirmed that tetrapeptides containing histidine derivatives, which are peptomimetic compounds having a specific sequence, show excellent effects in controlling itch-inducing cytokines (TNF-α, IL-4, IL-6, and IL-8), and thus completed the present invention. [Prior art documents] [Patent Documents]

[0009] [Patent Document 1] Republic of Korea Published Patent No. 2022-0086511 [Patent Document 2] Republic of Korea Published Patent No. 2021-0154536 [Non-patent literature]

[0010] [Non-Patent Document 1] Biochemistry, 11:1726-1732(1972); Pure & Appl. Chem., Vol.56, No.5, pp.595-624, 1984 [Overview of the Initiative] [Problems that the invention aims to solve]

[0011] The object of the present invention is to provide a tetrapeptide containing a histidine derivative that can effectively control inflammatory cytokines (TNF-α, IL-4, IL-6, and IL-8), and a composition containing the same. Another object of the present invention is to provide a method for producing tetrapeptides (peptidomimetic) containing histidine derivatives that can effectively control inflammatory cytokines (TNF-α, IL-4, IL-6, and IL-8). [Means for solving the problem]

[0012] To achieve the above objective, the present invention provides a tetrapeptide comprising a histidine derivative having the structure of the following chemical formula I.

[0013] [ka]

[0014] Furthermore, the present invention provides a cosmetic composition containing a tetrapeptide as an active ingredient, which includes a histidine derivative having the structure of the chemical formula I.

[0015] In the present invention, the cosmetic composition is characterized by having an effect of improving pruritus or skin inflammation.

[0016] In the present invention, the cosmetic composition is characterized by being in a dosage form selected from the group consisting of lotion, nourishing cream, eye cream, essence, pack, lotion and gel.

[0017] The present invention also provides a method for producing a tetrapeptide containing a histidine derivative, comprising: (a) obtaining Fmoc-His(Bis-cyclohexylpropyl)-NH-resin represented by Chemical Formula 1 by a solid-phase synthesis method; (b) sequentially bonding Fmoc-His(Bis-cyclohexylpropyl)-OH and Fmoc-Arg(protecting group)-OH to the resin obtained in step (a) to obtain a resin to which a protected peptide represented by Chemical Formula 2 is bonded; (c) simultaneously removing the resin and the protecting groups of the amino acids from the resin to which the protected peptide obtained in step (b) is bonded to obtain an unpurified tetrapeptide represented by Chemical Formula 3; and (d) purifying the unpurified tetrapeptide obtained in step (c) to obtain a tetrapeptide represented by Chemical Formula I.

[0018]

Chem.

[0019]

Chem.

[0020] [[ID=2十七]]

Chem.

[0021]

Chem.

[0022] In the present invention, the resin is selected from the group consisting of Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin.

[0023] In the present invention, the acidic solutions that simultaneously remove the protecting groups of resin and amino acids are TFA / phenol / water / TIPS (88 / 5 / 5 / 2), TFA / phenol / water / thioanisole / EDT (82.5 / 5 / 5 / 5 / 2.5), TFA / phenol / water / thioanisole / 1-decanethiol (82.5 / 5 / 5 / 5 / 2.5), TFA / DTT / water / TIPS (88 / 5 / 5 / 2), TFA / phenol (95 / 5), and TFA / phenol / Methanesulfonic The product is characterized by being selected from the group consisting of acid (95 / 2.5 / 2.5), TFA / thioanisole / EDT / anisole (90 / 5 / 3 / 2), TFA / TES (95 / 5), TFA / water (95 / 5), TFA / DCM / indole (70 / 28 / 2), and TFA / TIPS / water (95 / 2.5 / 2.5). [Effects of the Invention]

[0024] The histidine derivative-containing peptidomimetic of the present invention is highly effective in controlling inflammatory cytokines such as TNF-α, IL-4, IL-6, and IL-8, and exhibits excellent stability in the human body, making it extremely useful in industrial fields such as cosmetics. [Brief explanation of the drawing]

[0025] [Figure 1] This is a step diagram illustrating the synthesis of a tetrapeptide containing a histidine derivative according to one embodiment of the present invention. [Figure 2] This is the molecular weight measurement (MALDI-TOF MS) result of a tetrapeptide containing a histidine derivative according to the present invention. [Figure 3] This is an HPLC chromatogram showing the purity of a tetrapeptide containing a histidine derivative according to the present invention. [Figure 4] This figure shows the results of evaluating the cytotoxicity of tetrapeptides containing histidine derivatives according to the present invention in HaCaT cells. [Figure 5a] The figure shows the results of evaluating the cytokine regulatory activity of a tetrapeptide containing a histidine derivative according to the present invention in HaCaT cells. [Figure 5b] The figure shows the results of evaluating the cytokine regulatory activity of a tetrapeptide containing a histidine derivative according to the present invention in HaCaT cells. [Figure 5c] The figure shows the results of evaluating the cytokine regulatory activity of a tetrapeptide containing a histidine derivative according to the present invention in HaCaT cells. [Figure 5d] The figure shows the results of evaluating the cytokine regulatory activity of a tetrapeptide containing a histidine derivative according to the present invention in HaCaT cells.

[0026] [Best mode for carrying out the invention] Unless otherwise specified herein, abbreviations used to designate amino acids and protecting groups are based on the terminology recommended by the IUPAC-IUB Commission of Biochemical Nomenclature (Non-Patent Literature 1: Biochemistry, 11:1726-1732 (1972); Pure & Appl. Chem., Vol.56, No.5, pp.595-624, 1984).

[0027] In this specification, the term "peptide" means a linear molecule formed by the bonding of amino acid residues to each other via peptide bonds.

[0028] The abbreviations used herein for solvents, reagents, protecting groups, and amino acids are as follows: ACN: Acetonitrile Arg: Arginine DCM: Dichloromethane DMSO: Dimethyl sulfoxide DMF: Dimethylformamide (N,N-Dimethylformamide) DTT: Dithiolthreitol EDT: 1,2-Ethanedithiol Fmoc: 9-Fluorenyloxycarbonyl

[0029] His: Histidine HPLC: High Performance Liquid Chromatography IL: Interleukin Mtr: 4-Methoxy-2,3,6-trimethylphenyl-sulfonyl Trt: Triphenylmethyl (or Trityl) Pbf: 2,2,4,6,7-Pentamethyl-dihydrobenzofuran-5-sulfonyl

[0030] Pmc: 2,2,5,7,8-Pentamethylchroman-6-sulfonyl Tos: para-toluenesulfonyl TES: Triethylsilane TNF-α: Tumor necrosis factor-alpha TFA: Trifluoroacetic acid TIPS: Triisopropylsilane

[0031] In the process of researching and developing various antimicrobial peptide mimetic compounds containing histidine derivatives, this invention produced a tetrapeptide containing a histidine derivative, which is a peptide mimetic compound having a specific sequence. It was confirmed that this compound exhibits not only excellent antimicrobial effects but also superior anti-inflammatory effects.

[0032] Therefore, the present invention relates, from one perspective, to a tetrapeptide (peptidomimetic) comprising a histidine derivative having the structure of the following chemical formula I.

[0033] [ka]

[0034] Furthermore, the present invention relates to a cosmetic composition containing a tetrapeptide as an active ingredient, which includes a histidine derivative having the structure of the chemical formula I.

[0035] The aforementioned cosmetic composition is effective in inhibiting inflammation-inducing cytokines such as TNF-α, IL-4, IL-6, and IL-8, but is not limited thereto.

[0036] The content of the tetrapeptide containing a histidine derivative having the structure of chemical formula I in the cosmetic composition of the present invention can be appropriately adjusted depending on the application, application form, intended use, and expected effect, and may be, for example, within 0.0001 to 99.9% by weight of the total composition weight, taking into consideration the content versus effect.

[0037] When the cosmetic composition of the present invention is used as a pharmaceutical or quasi-pharmaceutical, it can be applied by topical application via transdermal coating.

[0038] The aforementioned composition means a composition that is applied transdermally to the skin and scalp and can be used in the manufacture of all cosmetic products, including basic cosmetics, body cosmetics, makeup cosmetics, and hair products. It may be in the form of an emulsion, cream, gel, liquid, etc., but there are no special restrictions on its form.

[0039] Furthermore, the cosmetic composition of the present invention may further contain, in addition to the active ingredient, all types of ingredients that can be used in normal product manufacturing and dosage formulation, such as fragrances, pigments, antioxidants, preservatives, humectants, thickeners, softeners (skin conditioning agents), chelating agents, surfactants, stabilizers, synthetic polymers, etc., and the types and content thereof can be appropriately adjusted according to the use and purpose of the final product.

[0040] Furthermore, the compositions of the present invention may contain solvents that are commonly included in the applicable form. Suitable solvents may include one or more selected from purified water, ethanol, and polyols. When producing the compositions of the present invention using such solvents, the solubility of the compounds in the solvent may vary slightly depending on the type of compound or the mixing ratio of the solvents. However, those skilled in the art in which the present invention belongs can appropriately select and apply the type and amount of solvent according to the characteristics of the product.

[0041] The present invention also relates to a method for producing a tetrapeptide containing a histidine derivative, comprising: (a) obtaining an Fmoc-His(Bis-cyclohexylpropyl)-NH-resin represented by chemical formula 1 using a solid-phase synthesis method; (b) sequentially attaching Fmoc-His(Bis-cyclohexylpropyl)-OH and Fmoc-Arg(protecting group)-OH to the resin obtained in step (a) to obtain a resin to which a protected peptide represented by chemical formula 2 is attached; (c) simultaneously removing the protecting groups of the resin and amino acids from the resin to which the protected peptide is attached obtained in step (b) to obtain an unpurified tetrapeptide represented by chemical formula 3; and (d) purifying the unpurified tetrapeptide obtained in step (c) to obtain a tetrapeptide represented by chemical formula I.

[0042] [ka]

[0043] [ka]

[0044] [ka]

[0045] [ka]

[0046] The method for producing a tetrapeptide containing the histidine derivative of the present invention will be described below, step by step.

[0047] (a) Step: A solid-phase synthesis method is used to obtain Fmoc-His(Bis-cyclohexylpropyl)-NH-resin represented by chemical formula 1.

[0048] Examples of suitable resins include rink amide resin, rink amide MBHA resin, rink amide AM resin, and rink amide RAM resin.

[0049] Examples of solvents used in the present invention include, but are not limited to, dichloromethane, N,N-Dimethylformamide, N,N-Dimethylacetamide, N-Methylpyrrolidone, chloroform, 1,2-Dichloroethane, tetrahydrofuran, 1,4-Dioxane, methanol, ethanol, isopropanol, ethylene glycol, methyl acetate, and ethyl acetate.

[0050] (b) Step: Fmoc-His(Bis-cyclohexylpropyl)-OH and Fmoc-Arg(protecting group)-OH are sequentially attached to the resin obtained in step (a) to obtain a resin to which a protected peptide represented by chemical formula 2 is attached.

[0051] Step (b) again includes (i) removing the amino acid protecting group (N-terminal amino group) bound to the resin by treating it with a base and washing the resin, and (ii) reacting and binding a newly bound protected amino acid to the washed resin.

[0052] The protecting groups in Chemical Formula 2 of the present invention are: Methoxymethyl group, Benzyloxymethyl group, Tripheylmethyl group, tert-Butyldimethylsilyl group, tert-Butyldiphenylsilyl group, Phenyldimethylsilyl group, Triphenylsilyl group, Triethylsilyl group, Triisopropysilyl group, para-Methyloxybenzyl group, Tetrahydropyran group, Tetrahydrofuran group, tert-Butyl group, Diphenylmethyl group, 2-Chlorotrityl group, Benzyl group, o-Nitrobenzyl o-Nitrobenzyl group, p-Nitrobenzyl group, 9-Phenylxanthyl group, Methoxytrityl group, Allyl group, Methyl group, Methoxyethoxymethyl group, Ethoxyethyl group, Methylthiomethyl group, 2-Naphthylmethyl group, tert-butyl tert-Butyloxycarbonyl group, benzyloxycarbonyl group, acetyl group, benzoyl group, para-Toluenesulfonyl group, 2,4,6-Trimethylphenylsulfonyl (mesityl-2-sulfonyl) group, 4-Methoxy-2,3,6-Trimethylphenylsulfonyl (4-Methoxy-2,3Examples include the 6-trimethylphenylsulfonyl (6-trimethylphenylsulfonyl) group, Pmc(2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS(1,2-dimethylindole-3-sulfonyl) group, Mtr(4-methyoxy-2,3,5-trimethylphenylsulfonyl) group, and NO2(Nitro) group. It can include a para-toluenesulfonyl group, a 2,4,6-trimethylphenylsulfonyl (mesityl-2-sulfonyl) group, a 4-methoxy-2,3,6-trimethylphenylsulfonyl group, a Pmc(2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, It is preferable to use the Pbf(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl) group, MIS(1,2-dimethylindole-3-sulfonyl) group, Mtr(4-methyoxy-2,3,5-trimethylphenylsulfonyl) group, NO2(Nitro) group, Pmc(2,2,5,7,8-pentamethylchroman-6-sulfonyl) group, Pbf(2,2,4,6,7-pentameth It is more preferable to use the yldihydrobenzofuran-5-sulfonyl) group, MIS(1,2-dimethylindole-3-sulfonyl) group, Mtr(4-methoxy-2,3,5-trimethylphenylsulfonyl) group, or NO2(Nitro) group, and it is most preferable, but not limited to, that the amide protecting group be Pbf(2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl).

[0053] Examples of bases that remove the amino acid protecting group (the N-terminal amide group) include piperidine, 4-methylpiperidine, pyrrolidine, piperazine, hydrazine hydrate, DBU (1,8-Diazabicyclo[5.4.0]undec-7-ene), 4-methylpiperidine, and 1-methyl-3-butylimidazolithium tetrafluoroborane. Organic bases such as BF4, ethanolamine, cyclohexylamine, dicyclohexylamine, tris(2-aminoethyl)amine, 1,3-dicyclohexanebis-(methylamine), 1,4-bis-(3-aminopropyl)piperazine, diethylamine, and 4-dimethylaminopyridine can be used, or inorganic bases such as lithium hydroxide, sodium hydroxide, calcium hydroxide, and potassium hydroxide can be used.

[0054] In the present invention, the reagents used when reacting and binding the protected amino acid to the washed resin include DCC (N,N-Dicyclohexylcarbodiimide), DIC (N,N-Diisopropylcarbodiimide), BOP (Benzotriazole-1-yl-oxy-tris-(dimethylamino)-phosphonium hexafluorophosphate), PyBOP (Benzotriazol-1-yl-oxytripyrrolidinophosphoniumhexafluorophosphate), PyBrOP (Bromo-tripyrrolidino-phosphonium hexafluorophosphate), PyAOP (7-Aza-benzotriazol-1-yloxy-tripyrrolidino-phosphonium hexafluorophosphate), and PyOxim (Ethyl cyano(hydroxyimino)acetato-O2)-tri-(1-pyrrolidinyl)-phosphonium hexafluorophosphate), HBTU(O-Benzotriazole-N,N,N',N'-tetramethyluroniumhexafluorophosphate), HCTU(2-(6-Chloro-1H-benzotriazol-1-yl)-N,N,N',N'-tetramethylaminium hexafluorophosphate), HDMC(N-[(5-chloro-1H-benzotriazol-1-yl)-dimethylamino-morpholino]-uronium hexafluorophosphate N-oxide), TBTU(O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluroniumtetrafluoroborate), HATU(2-(1H-7-Azabenzotriaz TATU(2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluroniumtetrafluoroboratemethanaminium), COMU(1-[1-(Cyano-2-ethoxy-2-oxoethylidene-aminooxy)-dimethylamino-morpholino]-uronium hexafluorophosphate), TOTT(2-(1-Oxy-pyridin-2-yl)-1,1,3,3-tetramethyl-isothiouronium tetrafluoroborate), EDC·HCl (N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide hydrochloride), TFFH (Tetramethylfluoroformamidinium hexafluorophosphate), EEDQ (N-Ethoxycarbony-2-ethoxy-1,2-dihydro-quinoline), T3P (2-Propanephosphonic acid) anhydride), DEPBT(3-(Diethoxyphosphoryloxy)-1,2,3-benzotriazin-4(3H)-one), Oxyma(Ethyl Examples include cyanohydroxyiminoacetate, HOBt (1-Hydroxybenzotriazole), HOOBT (HODhbt, Hydroxy-3,4-dihydro-4-ox-1,2,3-benzo-trazine), BTC (bis-Trichloromethylcarbonate or Triphosgene), CDI (1,1'-Carbonyldiimidazole), 6-ClHOBt (1-Hydroxy-6-chloro-benzotriazole), HOAt (1-Hydroxyazabenzotriazole), and HOSu (N-Hydroxysuccinimide).

[0055] (c) Step: The protecting groups of the resin and amino acids are simultaneously removed from the resin to which the protected peptide obtained in step (b) is bound, to obtain an unpurified tetrapeptide represented by chemical formula 3.

[0056] The process of simultaneously removing the protecting groups of the resin and amino acids is carried out in the presence of an acidic solution, which may be TFA / phenol / water / TIPS(88 / 5 / 5 / 2), TFA / phenol / water / thioanisole / EDT(82.5 / 5 / 5 / 5 / 2.5), TFA / phenol / water / thioanisole / l-decanethiol(82.5 / 5 / 5 / 5 / 5 / 2.5), TFA / DTT / water / TIPS(88 / 5 / 5 / 2), TFA / phenol(95 / 5), or TFA / phenol / Methanesulfonic Examples of solutions include acid (95 / 2.5 / 2.5), TFA / thioanisole / EDT / anisole (90 / 5 / 3 / 2), TFA / TES (95 / 5), TFA / water (95 / 5), TFA / DCM / indole (70 / 28 / 2), and TFA / TIPS / water (95 / 2.5 / 2.5).

[0057] (d) Step: The unpurified tetrapeptide obtained in step (c) is purified to obtain the tetrapeptide represented by chemical formula I.

[0058] The aforementioned purification can be carried out using methods commonly used for peptide purification, and normal-phase or reverse-phase high-performance liquid chromatography (HPLC) can be used. The solvent used in the purification can be selected from the group consisting of methanol, ethanol, isopropanol, acetonitrile, purified water, and mixed solutions thereof, and acids such as trifluoroacetic acid, acetic acid, and formic acid can be used together. [Modes for carrying out the invention]

[0059] [Examples] Throughout this specification, the percentages used to indicate the concentration of a particular substance, unless otherwise specified, refer to (weight / weight)% for solid / solid, (weight / volume)% for solid / liquid, and (volume / volume)% for liquid / liquid.

[0060] Example 1: Preparation of H-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM)

[0061] [ka]

[0062] Linkamide AM resin (substitution rate = 0.70 mmol / g, 100 mmol) and N,N-dimethylformamide (1000 ml, hereinafter referred to as "DMF") were placed in a solid-phase synthesis reactor with a filtration membrane. After expanding the resin for 10 minutes, the solvent was removed through the filtration membrane under reduced pressure. Fmoc-His(Bis-cyclohexylpropyl)-OH (molecular weight = 626.85 g / mol) (188.06 g, 300 mmol, 3.0 equivalents) and Oxyma (molecular weight = 142.11 g / mol, 46.90 g, 330 mmol) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g / mol, 41.7 g, 330 mmol) was added dropwise to the mixed reaction mixture, and the mixture was slowly stirred at room temperature for 5 hours. The resin was washed twice with 1000 ml of DMF, then 1000 ml of 20% piperidine / DMF solution was added and stirred for 15 minutes. After that, the reaction mixture was removed by vacuum filtration, and the resin was treated once more in the same manner. After washing the resin a total of six times with 1000 ml of DMF, H-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) was obtained (substitution rate: 0.7 mmol / g, yield >99%).

[0063] Example 2: Preparation of H-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM)

[0064] [ka]

[0065] In a reactor containing H-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) (100 mmol), Fmoc-Arg(Pbf)-OH (molecular weight = 648.8 g / mol) (194.64 g, 300 mmol, 3.0 equivalents) and Oxyma (molecular weight = 142.11 g / mol, 46.90 g, 330 mmol) were dissolved in 1000 ml of DMF and then added. DIC (molecular weight = 126.2 g / mol, 41.7 g, 330 mmol) was added dropwise to the mixed reaction mixture, and the mixture was slowly stirred at room temperature for 5 hours. After washing the resin twice with 1000 ml of DMF, 1000 ml of 20% piperidine / DMF solution was added and the mixture was stirred for 15 minutes. The reaction mixture was removed by vacuum filtration, the resin was treated once more in the same manner, and after washing the resin a total of six times with 1000 ml of DMF, H-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) was obtained (yield > 99%).

[0066] Example 3: Preparation of H-Arg(Pbf)-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM)

[0067] [ka]

[0068] In a reactor containing H-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) (100 mmol), Fmoc-Arg(Pbf)-OH (molecular weight = 648.8 g / mol, 194.64 g, 300 mmol) and Oxyma (molecular weight = 142.11 g / mol, 46.90 g, 330 mmol) were dissolved in 1000 ml of DMF and added. DIC (molecular weight = 126.2 g / mol, 41.7 g, 330 mmol) was added dropwise to the mixed reaction mixture, and the mixture was slowly stirred at room temperature for 5 hours. The resin was washed twice with 1000 ml of DMF, then 1000 ml of 20% piperidine / DMF solution was added and the mixture was stirred for 15 minutes. The reaction mixture was removed by vacuum filtration, and the resin was treated again in the same manner. After washing the resin a total of six times with 1000 ml of DMF, H-Arg(Pbf)-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) was obtained (yield > 99%).

[0069] Example 4: Preparation of H-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM)

[0070] [ka]

[0071] In a reactor containing H-Arg(Pbf)-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) (100 mmol), Fmoc-Arg(Pbf)-OH (molecular weight = 648.8 g / mol, 194.64 g, 300 mmol) and Oxyma (molecular weight = 142.11 g / mol, 46.90 g, 330 mmol) were dissolved in 1000 ml of DMF and added. DIC (molecular weight = 126.2 g / mol, 41.7 g, 330 mmol) was added dropwise to the mixed reaction mixture, and the mixture was slowly stirred at room temperature for 5 hours. The resin was washed twice with 1000 ml of DMF, then 1000 ml of 20% piperidine / DMF solution was added and the mixture was stirred for 15 minutes. The reaction mixture was removed by vacuum filtration, and the resin was treated again in the same manner. After washing the resin a total of six times with 1000 ml of DMF, H-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) was obtained (yield > 99%).

[0072] Example 5: Preparation of Crude H-Arg-Arg-Arg-His(Bis-cyclohexylpropyl)-NH2

[0073] [ka]

[0074] H-Arg(Pbf)-Arg(Pbf)-Arg(Pbf)-His(Bis-cyclohexylpropyl)-NH-resin (Rink Amide AM) was placed in a reactor, and 3 L of cooled TFA / TIPS / water (95 / 2.5 / 2.5) solution was gradually poured in. The mixture was then stirred at room temperature for 3 hours. The reaction mixture was slowly added dropwise to 12 L of cooled diethyl ether to precipitate the peptide. After stirring at room temperature for 30 minutes, the peptide was collected by filtration, and the solvent was removed under reduced pressure. The mixture was dried in a vacuum dryer for 5 hours to obtain 77.7 g of white Crude H-Arg-Arg-Arg-His(Bis-cyclohexylpropyl)-NH2. (Molecular weight 844.17 g / mol, Crude peptide purity 46.9%, yield 92.0%)

[0075] Example 6: Preparation of H-Arg-Arg-Arg-His(Bis-cyclohexylpropyl)-NH2

[0076] [ka]

[0077] 0.03 g of Crude H-Arg-Arg-Arg-His(Bis-cyclohexylpropyl)-NH2 obtained in Example 5 was dissolved in purified water and filtered through a 0.46 μm membrane. The filtrate was repeatedly injected using industrial HPLC (230 nm, 500 ml / min, 10 micron C18 column, increasing the initial acetonitrile concentration in 0.1% TFA from 27% to 68% within 20 minutes), and the main fraction was fractionated to obtain 29.45 g of the tripeptide represented by chemical formula I (SEQ ID NO: 1) (yield: 36.8%, purity 99.21%). For reference, in the sequence RRRHX of SEQ ID NO: 1, X represents Bis-cyclohexylpropyl bonded to H.

[0078] The molecular weight and purity of the obtained histidine derivative-containing peptidomimetic were confirmed, and the results are shown in Figures 2 and 3.

[0079] Experimental Example 1: Evaluation of Peptidomimetic Toxicity in Keratin Cells (HaCaT)

[0080] A cytotoxicity test was performed on the peptide mimetic prepared in Example 6.

[0081] HaCaT cells were placed in a 96-well plate, 1 x 10 cells per well. 3 After seeding the cells, five candidate peptidomimetic substances were treated at concentrations of 5 μM, 10 μM, 20 μM, and 40 μM, respectively, for 24 hours. After 24 hours, 20 μl of MTT reagent (50 mg / ml) was dispensed into each well. After 4 hours, formazan formation was confirmed under a microscope, the medium was removed, and then 100 μl of DMSO was dispensed into each well. After ensuring that the formazan and DMSO were thoroughly mixed with a pipette, the mixture was incubated at 37°C for 10 minutes, and then the absorbance at 540 nm was measured to calculate the cell viability (%) of the experimental group compared to the control group.

[0082] Statistical analysis was performed using SPSS 18.0 (SPSS Inc. Chicago, IL, USA) to calculate the mean (M) and standard deviation (SD). One-way ANOVA validation and Scheffe post-hoc validation were performed to compare the control group and the experimental group. The statistical significance level was set at p < 0.05.

[0083] When the cytotoxicity of HaCaT against the peptidomimetic was confirmed using the MTT assay, cell viability was over 80% up to a concentration of 40 μM (Figure 4).

[0084] Experimental Example 2: Evaluation of the anti-inflammatory efficacy of peptide mimetic cells in keratinocytes (HaCaT) (Verification of the inhibitory effect on TNF-α, IL-4, IL-6, and IL-8 mRNA expression)

[0085] The anti-inflammatory efficacy of the peptidomimetic produced in Example 6 was evaluated, confirming its ability to regulate inflammatory cytokines (TNF-α, IL-4, IL-6, and IL-8).

[0086] HaCaT cells were placed in a 12-well plate, 1 x 10 per well. 5 After seeding the cells, they were treated with peptidomimetic at concentrations of 5 μM, 10 μM, and 20 μM 24 hours later, and then treated with TNF-α (10 ng / ml) and INF-γ (10 ng / ml) 30 minutes later. Cytotoxicity was confirmed by microscopy after 6 hours. Subsequently, the cells were washed three times with PBS, and after removing the PBS, 500 μl of TRIzol was dispensed into each well. The TRIzol was then transferred to an e-tube, and RNA extraction using TRIzol was performed.

[0087] After treating the solution with TRIzol to extract RNA, it was transferred to a microcentrifuge tube. Chloroform was added to the microcentrifuge tube, and vortexing was performed for 20-30 seconds. The solution was then centrifuged at 13,000 rpm at 4°C for 15 minutes. The solution separated into three parts. The layer containing the supernatant RNA was separated using a pipette, and isopropyl alcohol was added. After being left at room temperature for 10 minutes, the solution was centrifuged at 13,000 rpm at 4°C for 20 minutes.

[0088] The supernatant was discarded, the mixture was washed with 75% EtOH (in DEPC water), and the mixture was centrifuged at 13,000 rpm at 4°C for 5 minutes. The supernatant was then discarded, the microtube was inverted and dried for 1 hour, and then the RNA was dissolved in DEPC water. The extracted RNA was stored at -80°C, and the RNA concentration was calculated by measuring the absorbance at 260 nm / 280 nm using a UV spectro-photometer.

[0089] The synthesized cDNA was amplified using Nuna® Universal qPCR Master Mix (New England Biolabs, MA, USA) via a Mic qPCR Cycler (Bio Molecular Systems, NSW, Austria) to amplify cytokines. The primers used in this study were: TNF-α sense: 5'-CCT ACC AGA CCA AGG TAC AC-3' (SEQ ID NO: 2), TNF-α anti-sense: 5'-AGG GGG TAA TAA AGG GAT TG-3' (SEQ ID NO: 3), IL-4 sense: 5'-ATG GGT CTC ACC TCC CAA CTG CT-3' (SEQ ID NO: 4), IL-4 anti-sense: 5'-CAG CTC GAA GAC TTT GAA TAT TTC TCT CTC-3 (SEQ ID NO: 5), IL-6 sense: 5'-AAA GAG GCA CTG CCA GAA AA-3' (SEQ ID NO: 6), and IL-6 anti-sense: 5'-ATC TGA GG TGC CCA TGC TAC-3 (SEQ ID NO: 7). The anti-inflammatory effects of peptidomimetic were evaluated by calculating the relative mRNA expression levels of cytokines (TNF-α, IL-4, IL-6, and IL-8) using the Ct (threshold cycle: the number of cycles at which a constant amount of amplified product is reached in a region where amplification occurs exponentially) of the target gene and the Ct of the reference gene.

[0090] Statistical analysis was performed using SPSS 18.0 (SPSS Inc., Chicago, IL, USA) to calculate the mean (M) and standard deviation (SD). One-way ANOVA validation and Scheffe post-hoc validation were performed to compare the control group and the experimental group. The statistical significance level was set at p<0.05. After treating with peptidomimetic at different concentrations, cytokine expression was confirmed, and the results are shown in Figure 5. Starting from a concentration of 5 μM, TNF-α mRNA expression was significantly suppressed in a concentration-dependent manner, and even higher levels of TNF-α mRNA expression suppression were observed.

[0091] IL-4 mRNA expression was also suppressed in a concentration-dependent manner starting from a concentration of 5 μM.

[0092] Upon checking IL-6 mRNA expression, it was found to be significantly reduced from a 10 μM concentration.

[0093] IL-8 mRNA expression was inhibited in a concentration-dependent manner starting from 5 μM.

[0094] Having described in detail certain aspects of the present invention, such specific techniques are merely preferred embodiments and do not limit the scope of the invention to those with ordinary skill in the art. Therefore, the substantial scope of the invention is defined by the appended claims and their equivalents.

Claims

1. Chemical formula I below: 【Chemistry 1】 It has a structure A tetrapeptide containing a histidine derivative characterized by the following:

2. The active ingredient is a tetrapeptide containing a histidine derivative having the structure of chemical formula I as described in claim 1. A cosmetic composition characterized by the following features.

3. The aforementioned cosmetic composition has an effect of improving pruritus or skin inflammation. The cosmetic composition according to claim 2.

4. The cosmetic composition is a dosage form selected from the group consisting of lotion, nourishing cream, eye cream, essence, pack, and gel. The cosmetic composition according to claim 2.

5. (a) A solid-phase synthesis method to obtain Fmoc-His(Bis-cyclohexylpropyl)-NH-resin represented by the following chemical formula 1, (b) The step of sequentially attaching Fmoc-His(Bis-cyclohexylpropyl)-OH and Fmoc-Arg(protecting group)-OH to the resin obtained in step (a) above to obtain a resin to which a protected peptide represented by the following chemical formula 2 is attached, (c) The step of simultaneously removing the protecting groups of the resin and amino acids from the resin to which the protected peptide obtained in step (b) is bound, in order to obtain an unpurified tetrapeptide represented by the following chemical formula 3, (d) The step of purifying the unpurified tetrapeptide obtained in step (c) above to obtain a tetrapeptide represented by the following chemical formula I, 【Chemistry 2】 【Transformation 3】 【Chemistry 4】 【Transformation 5】 A method for producing a tetrapeptide containing a histidine derivative, characterized by the above.

6. The resin is selected from the group consisting of Rink Amide Resin, Rink Amide MBHA Resin, Rink Amide AM Resin, and Rink Amide RAM Resin. A method for producing a tetrapeptide according to claim 5.

7. Acidic solutions that simultaneously remove protecting groups from resins and amino acids include TFA / phenylol / water / TIPS (88 / 5 / 5 / 2), TFA / phenylol / water / thioanisole / EDT (82.5 / 5 / 5 / 5 / 2.5), TFA / phenylol / water / thioanisole / 1-decanethiol (82.5 / 5 / 5 / 5 / 2.5), TFA / DTT / water / TIPS (88 / 5 / 5 / 2), TFA / phenylol (95 / 5), and TFA / phenylol / Methanesulfonic Selected from the group consisting of acid (95 / 2.5 / 2.5), TFA / thioanisole / EDT / anisole (90 / 5 / 3 / 2), TFA / TES (95 / 5), TFA / water (95 / 5), TFA / DCM / indole (70 / 28 / 2), and TFA / TIPS / water (95 / 2.5 / 2.5). A method for producing a tetrapeptide according to claim 5.

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