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WO2021230393A1 - Peptide for treating inflammatory skin diseases and use thereof - Google Patents

Peptide for treating inflammatory skin diseases and use thereof Download PDF

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Publication number
WO2021230393A1
WO2021230393A1 PCT/KR2020/006233 KR2020006233W WO2021230393A1 WO 2021230393 A1 WO2021230393 A1 WO 2021230393A1 KR 2020006233 W KR2020006233 W KR 2020006233W WO 2021230393 A1 WO2021230393 A1 WO 2021230393A1
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WIPO (PCT)
Prior art keywords
peptide
inflammatory skin
present
seq
skin diseases
Prior art date
Application number
PCT/KR2020/006233
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French (fr)
Korean (ko)
Inventor
조대호
Original Assignee
주식회사 카인사이언스
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Priority to PCT/KR2020/006233 priority Critical patent/WO2021230393A1/en
Publication of WO2021230393A1 publication Critical patent/WO2021230393A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K8/00—Cosmetics or similar toiletry preparations
    • A61K8/18—Cosmetics or similar toiletry preparations characterised by the composition
    • A61K8/30—Cosmetics or similar toiletry preparations characterised by the composition containing organic compounds
    • A61K8/64—Proteins; Peptides; Derivatives or degradation products thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61Q—SPECIFIC USE OF COSMETICS OR SIMILAR TOILETRY PREPARATIONS
    • A61Q19/00—Preparations for care of the skin
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids

Definitions

  • the present invention relates to a peptide for the treatment of inflammatory skin diseases and uses thereof.
  • Psoriasis is an immune-mediated autoimmune skin disease induced by chronic activation of inflammatory cell infiltration and dysregulation of epidermal keratinocytes in the skin.
  • IL-17 which is secreted and stimulated by IL-23, is known to be importantly involved in inducing psoriasis.
  • Topical administration is a treatment including steroids, coal tar, anthralin, vitamin D3 and its analogues, retinoids and sunburn, etc., and such topical administration has side effects of skin thinning, stretch marks, burns, irritation, and photosensitivity.
  • steroids may induce tolerance, which may affect subsequent treatment.
  • Phototherapy which involves the administration of psoralen along with ultraviolet B or ultraviolet A, has the disadvantage of promoting skin aging and increasing the incidence of skin cancer.
  • oral administration applied to psoriasis with severe symptoms is currently widely used as administration of an immunomodulatory agent such as cyclosporine, but there is a risk of nephrotoxicity or hypertension due to prolonged use.
  • the present invention was devised to solve the above problems, and as a result of intensive research on the preparation for the treatment of inflammatory skin diseases including psoriasis, the present inventors prepared a small-sized peptide consisting of 9 amino acids, and the peptide was It was confirmed that there was an excellent effect in the treatment of inflammatory skin diseases, and based on this, the present invention was completed.
  • a peptide for treating inflammatory skin diseases which consists of an amino acid represented by SEQ ID NO: 1.
  • Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising the peptide or a polynucleotide encoding the same as an active ingredient.
  • Another object of the present invention is to provide a health functional food / cosmetic composition for preventing or improving inflammatory skin disease, comprising the peptide as an active ingredient.
  • another object of the present invention is to provide a method for preventing, treating or improving inflammatory skin diseases, which includes administering to an individual a pharmaceutically effective amount of the peptide or a polynucleotide encoding the same.
  • Another object of the present invention is to provide the use of the peptide or a polynucleotide encoding the same for use as a composition for the prevention, treatment or improvement of inflammatory skin diseases.
  • the present invention provides a peptide consisting of amino acids represented by SEQ ID NO: 1.
  • the present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising a peptide or a polynucleotide encoding the same as an active ingredient, consisting of the amino acid represented by SEQ ID NO: 1.
  • the inflammatory skin disease is psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratoses ), pemphigus, corns, warts, and lichen planus.
  • the present invention provides a cosmetic composition for preventing or improving inflammatory skin disease, comprising a peptide as an active ingredient, consisting of an amino acid represented by SEQ ID NO: 1.
  • composition may be in the form of a suspension, emulsion, paste, gel, cream, lotion, powder, wax or spray.
  • the present invention provides a method for preventing or improving inflammatory skin disease comprising administering the peptide or a polynucleotide encoding the same to an individual.
  • the present invention provides a method for treating inflammatory skin disease, comprising administering the peptide or a polynucleotide encoding the same to an individual.
  • the present invention provides the use of the peptide for treating inflammatory skin diseases.
  • the present invention provides the use of the peptide or a polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating inflammatory skin diseases.
  • the present invention provides the use of the peptide for use as a cosmetic composition for preventing or improving inflammatory skin disease.
  • the novel synthetic peptide according to the present invention has not only an excellent therapeutic effect on inflammatory skin diseases, but also a very small-scale peptide consisting of 9 amino acids, low molecular weight and biodegradable, so it is less likely to cause hypersensitivity reaction, so side effects caused by the administration of external substances can be minimized. As such, it is expected that it can be used as an effective substance that can replace the existing agents for treating inflammatory skin diseases.
  • Figure 2 shows the results of scoring the degree of reduction of erythema (Erythema) according to the peptide treatment of the present invention.
  • the present invention provides a peptide, consisting of amino acids represented by SEQ ID NO: 1.
  • peptide refers to a polymer consisting of two or more amino acids linked by an amide bond (or peptide bond), and for the purpose of the present invention, inhibiting, improving and improving the occurrence of dry skin diseases including psoriasis and It refers to a peptide having a therapeutic activity.
  • amide bond or peptide bond
  • the peptide itself is too large to effectively enter the target tissue or cell, or has a short half-life, which reveals disadvantages that it disappears in the body in a short period of time.
  • the present invention has an effective therapeutic effect and It has technical significance in that it developed a peptide consisting of 9 amino acids at the same time.
  • the peptide of the present invention may consist of the amino acid represented by SEQ ID NO: 1, and each of the amino acid sequence represented by SEQ ID NO: 1 and at least 75%, preferably at least 80%, more preferably at least 90%, most preferably may include an amino acid sequence having 95% or more sequence homology, and may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability. .
  • peptides of the present invention may be included.
  • the functional variants include biological equivalents of the peptide sequences described herein (SEQ ID NO: 1).
  • additional changes may be made to the amino acid or polynucleotide sequence of the peptide to further improve the binding affinity and/or other biological properties of the peptide.
  • Such modifications are deletions of amino acid sequence residues in the peptide. insertions, and/or substitutions, and may be made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge magnitude, and the like.
  • the peptide of the present invention can be obtained by various methods well known in the art. As an example, it may be prepared using polynucleotide recombination and protein expression systems, or synthesized in vitro through chemical synthesis such as peptide synthesis, and cell-free protein synthesis.
  • the protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but the modification of the peptide, particularly the stability of the peptide As long as it is a possible component, it may be included without limitation.
  • the term “stability” refers to storage stability (eg, room temperature storage stability) as well as in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo.
  • polynucleotide polynucleotide
  • polynucleotide is a polymer to which nucleotides are bound, and serves to transmit genetic information.
  • encoding the peptide of SEQ ID NO: 1 encoding the peptide of SEQ ID NO: 1, and each of the polynucleotide sequence encoding the peptide at least 75%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% It may include a sequence having aberrant sequence homology.
  • the "homology” is intended to indicate a degree of similarity with a wild-type amino acid sequence or a polynucleotide sequence, and the comparison of such homology can be performed using a comparison program well known in the art, and homology between two or more sequences can be calculated as a percentage (%).
  • the present invention is a pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising as an active ingredient a peptide or a polynucleotide encoding the same, consisting of amino acids represented by SEQ ID NO: 1; Use of the peptide or polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating inflammatory skin diseases; And it provides a method for treating inflammatory skin disease, comprising administering to an individual a therapeutically effective amount of the peptide or a polynucleotide encoding the same.
  • treatment refers to any action in which the symptoms of a wound are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.
  • “individual” refers to a subject in need of treatment of a wound, and more specifically, refers to mammals such as human or non-human primates, mice, dogs, cats, horses and cattle.
  • the inflammatory skin disease is psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratosis ( seborrheic keratoses), pemphigus, corns, warts, and may be at least one selected from the group consisting of lichen planus, preferably psoriasis, but is not limited thereto.
  • the peptide of the present invention or a polynucleotide encoding the same may be delivered in a colloidal suspension, powder, saline, lipid, liposome, microsphere microspheres), or a pharmaceutically acceptable carrier such as nano spherical particles. They may form complexes with, or be associated with, vehicles and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.
  • pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, which are commonly used in the formulation.
  • it may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
  • the pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically applied) according to a desired method, and the dosage may be determined by the patient. Although it varies depending on the condition and weight, the degree of disease, the drug form, the administration route and time, it may be appropriately selected by those skilled in the art.
  • a fatty substance an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance , surfactant, water, ionic emulsifier, non-ionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocker, wetting agent, essential oil, dye, pigment, hydrophilic active agent, lipophilic active agent or lipid vesicle It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations for skin.
  • the above ingredients may be introduced in an amount generally used in the field of dermatology.
  • the pharmaceutical composition when provided as an external preparation for skin, it may be in the form of an ointment, patch, gel, cream or spray, but is not limited thereto.
  • the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount.
  • pharmaceutically effective amount means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined.
  • the pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered simultaneously with, separately, or sequentially from a conventional therapeutic agent, and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
  • the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate, excretion rate, disease type, and drugs used in combination, the route of administration, It can increase or decrease according to the severity of obesity, sex, weight, age, etc.
  • the present invention is a health functional food / cosmetic composition for preventing or improving inflammatory skin disease, comprising a peptide or a polynucleotide encoding the same as an active ingredient, consisting of an amino acid represented by SEQ ID NO: 1; Use of the peptide or polynucleotide encoding the same for use as a health functional food / cosmetic composition for preventing or improving inflammatory skin disease; And it provides a method for preventing or improving inflammatory skin disease, comprising administering to an individual a therapeutically effective amount of the peptide or a polynucleotide encoding the same.
  • the term “improvement” refers to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms.
  • the health functional food/cosmetics composition may be used simultaneously with or separately from a drug for treatment before or after the onset of a related disease in order to prevent or improve inflammatory skin disease.
  • the active ingredient may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method.
  • the mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement).
  • the composition of the present invention may be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw material.
  • the amount may be less than or equal to the above range.
  • the health functional food composition of the present invention may contain other ingredients as essential ingredients without any particular limitation in addition to containing the active ingredient.
  • various flavoring agents or natural carbohydrates may be contained as additional ingredients.
  • natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol.
  • natural flavoring agents taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)
  • synthetic flavoring agents sacharin, aspartame, etc.
  • the ratio of the natural carbohydrate may be appropriately determined by the selection of those skilled in the art.
  • the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.
  • the cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing agent , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing 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.
  • a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing agent , oil, powder foundation, emulsion foundation, wax foundation, spray, etc. but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing cream, a massage cream, an essence, an eye cream, a cleansing cream,
  • the effective carrier contained in the cosmetic composition of the present invention may be a carrier commonly used in the art, depending on the formulation.
  • the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component.
  • lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.
  • a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.
  • the formulation of the present invention is a suspension
  • a liquid diluent such as water, ethanol or propylene glycol
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals
  • cellulose aluminum metahydroxide, bentonite, agar or tracanth
  • a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester
  • microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.
  • the formulation of the present invention is a surfactant-containing cleansing agent
  • Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.
  • ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the active ingredient and carrier ingredient, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. may include
  • a peptide consisting of the amino acid sequence (RDGRDGPSP) represented by SEQ ID NO: 1 was prepared. Thereafter, the synthesized peptide was purely separated using high performance liquid chromatography (SHIMADZU Prominence HPLC), and a Shiseido capcell pak C18 column (4.6 x 50 mm) was used as the column. In addition, the mass of the synthesized peptide was confirmed using a mass spectrometer (HP 1100 series LC/MSD).
  • Imiquimod was used to induce a skin inflammatory response similar to psoriasis (Journal of Investigative Dermatology 2015;135:2764-2774.).
  • IMQ Imiquimod
  • IMQ 5% IMQ
  • Psoriasis was induced by application.
  • the peptide according to the present invention was treated by SC injection every day for 3 days at 0.1 and 10 ug/kg, respectively, from the next day (Day 2) after IMQ application.
  • Control mice were coated with Vaseline (Vaselin Lanette cream; Fagron) as vehicle control cream instead of IMQ, and PBS was injected instead of the peptide.
  • Positive control mice were injected with 2 mg/kg SC of methotrexate (MTX) instead of the peptide.
  • MTX methotrexate
  • mice were sacrificed to obtain lesional skin tissue, fixed with paraformaldehyde to make paraffin sections, and hematoxylin & eosin (H&E) staining results were analyzed.
  • H&E hematoxylin & eosin
  • Epidermis thickness was measured in H&E tissue with Image scope program. The results of measuring 5 random sites for each tissue were plotted as mean values and standard deviations were indicated.
  • lymph nodes were removed from the mice, and Th17 cells were stained for comparison.
  • the removed lymph node tissue was lightly ground and the cells were harvested and counted. It was spread on a 24-well plate at a concentration of 1x10 6 /mL and a number of 2x10 6 /2 mL per well.
  • PMA 25 ng/mL and Ionomycin 1 ug/mL were treated together and incubated at 37° C., CO 2 for 2 hours.
  • Monensin was treated at 5 ug/mL and incubated for 2 hours more.
  • the cells were washed with PBS to remove the remaining PMA, Ionomycin, and Monensin, and then the CD4-Alexa 488 antibody was added and surface staining was performed at room temperature for 10 minutes. After washing the cells to remove the remaining antibody, the cells were fixed with Fixation buffer (BD bioscience) at 4°C for 30 minutes. After washing the cells to remove the remaining fixation buffer, permeabilization was performed with perm/wash buffer (BD bioscience) to prepare for intracellular staining. IL-17-APC antibody was added and intracellular IL-17 was stained for 1 hour at 4°C. All stained cells were washed to remove all remaining antibodies, and CD4+/IL-17+ Th17 cells were analyzed using a flow cytometer (BD, FACS Calibur).
  • Fixation buffer BD bioscience
  • Example 1 Histological analysis of psoriasis mouse model
  • FIG. 1 shows the results of comparing the degree of psoriasis treatment according to the peptide treatment of the present invention through histological staining.
  • the psoriasis-induced mice had a thickened epidermal layer, and a large amount of scaling was generated, indicating that psoriasis was well induced by IMQ.
  • mice treated with MTX which is a positive control, the epidermal layer became thinner again and the scaling fell to a normal level compared to PBS.
  • mice treated with the peptide according to the present invention it was confirmed that the thickness of the epidermal layer became thinner and scaling was reduced to a level similar to that of MTX.
  • the peptide according to the present invention was treated, the lesions of psoriasis were remarkably reduced and the therapeutic effect of psoriasis was excellent.
  • Figure 2 shows the results of scoring the degree of reduction of erythema (Erythema) according to the peptide treatment of the present invention.
  • the score of the mice treated with the peptide according to the present invention compared to the PBS-treated group (control group), in which the Erythema score increases as psoriasis is induced it was confirmed that the score of the MTX-treated group (positive control group) fell to the level of the MTX-treated group (positive control group). .
  • the Erythema score of the group treated with the peptide according to the present invention at 10 ug/kg was much lower than that of MTX at the beginning of the experiment (Day 1 and Day 2), but at the end of the experiment (Day 3 and Day 4), MTX or It was confirmed that the Erythema level was similar to that of the group treated with the peptide 0.1 ug/kg. That is, by the peptide treatment according to the present invention, the degree of Erythema, which is one measure of the PASI score, fell to a level similar to or lower than that of the MTX-treated group, which is a positive control, and it was confirmed that the treatment effect for psoriasis was very excellent.
  • FIG. 3 shows the results of measuring the thickness of the epidermis according to the peptide treatment of the present invention.
  • the thickness was similar to that of the MTX-treated group, which is a positive control, or decreased to a normal level. That is, since the thickness of the epidermal layer increased by psoriasis was significantly reduced by the peptide treatment according to the present invention, it was confirmed that the treatment effect for psoriasis was excellent.
  • CD4+/IL-17+ cells in the lymph node were stained and compared to investigate the changes in Th17 cells by peptide treatment.
  • FIG. 4 shows the results of measuring the Th17 cell population decreased according to the peptide treatment of the present invention.
  • the Th17 cell population increased by the PBS treatment was reduced to a level similar to that of the MTX-treated group, which is a positive control, by the peptide treatment according to the present invention.
  • the peptide according to the present invention was treated, the Th17 cell population was remarkably decreased and the treatment effect of psoriasis was excellent.
  • the present invention relates to a peptide for the treatment of inflammatory skin disease and its use.
  • the novel synthetic peptide according to the present invention is a very small-scale peptide consisting of 9 amino acids, low molecular weight and biodegradable, so there is little possibility of inducing hypersensitivity reaction to foreign substances Since it minimizes the side effects of administration, it can be usefully used as an active ingredient in a composition for preventing, treating or improving inflammatory skin diseases.

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Abstract

The present invention relates to a peptide for treating inflammatory skin diseases and a use thereof. A novel synthetic peptide according to the present invention exhibits excellent treatment effects on inflammatory skin diseases. Moreover, because the peptide is an extremely small peptide composed of nine amino acids, and thus a small molecule and biodegradable, the peptide has a low probability of causing a hypersensitivity reaction, and thus can minimize side effects caused by the administration of foreign substances. Thus, the novel synthetic peptide is expected to be utilized as an active ingredient which can replace existing therapeutic agents for inflammatory skin diseases.

Description

염증성 피부질환 치료용 펩타이드 및 이의 용도Peptides for the treatment of inflammatory skin diseases and uses thereof
본 발명은 염증성 피부질환 치료용 펩타이드 및 이의 용도에 관한 것이다.The present invention relates to a peptide for the treatment of inflammatory skin diseases and uses thereof.
건선(psoriasis)은 피부에서 염증성 세포 침윤의 만성적 활성화와 표피 각질세포의 조절 장애에 의해 유도된 면역-매개 자가면역 피부질환이다. Psoriasis is an immune-mediated autoimmune skin disease induced by chronic activation of inflammatory cell infiltration and dysregulation of epidermal keratinocytes in the skin.
현재까지 건선의 발명 메커니즘이 완전히 규명되지는 않았으나, 염증 유발 사이토카인이나 T 세포와 같은 면역 세포 침윤 간의 상호작용을 포함하는 복잡한 메커니즘과 관련되어 있는 것으로 보고되고 있으며, 특히, Th17 cells과 Th17 cells에서 분비되고 IL-23에 의해 분비가 촉진되는 IL-17은 건선을 유발시키는데 중요하게 관여하는 것으로 알려져있다. Although the mechanism of the invention of psoriasis has not been fully elucidated so far, it is reported that it is related to a complex mechanism including the interaction between inflammatory cytokines and immune cell infiltration such as T cells. IL-17, which is secreted and stimulated by IL-23, is known to be importantly involved in inducing psoriasis.
이러한 건선의 일반적인 치료로는 국소 투약, 광선 요법 및 내복 투약법 등이 있다. 국소 투약은 스테로이드, 콜타르, 안트랄린, 비타민 D3 및 그의 유사체, 레티노이드 및 햇볕 등을 포함하는 치료이며, 이러한 국소 투약은 피부 얇아짐, 튼살, 화상, 자극 및 광과민증의 부작용이 있다. 또한, 스테로이드는 내성을 유도할 수 있어 이후의 치료에 영향을 미칠 수 있다는 문제가 있다. 광선 요법은 자외선 B 또는 자외선 A와 함께 소랄렌의 투여를 포함하는 것으로, 피부 노화를 촉진하고 피부암 발병률을 증가시키는 단점이 있다. 또한, 증상이 심각한 건선에 적용하는 내복 투여는 사이클로스포린과 같은 면역조절제를 투여하는 것으로 현재 널리 사용되고 있으나, 장시간 사용에 따른 신장독성(nephrotoxicity)이나 고혈압 발생의 위험성이 존재한다.Common treatments for psoriasis include topical administration, phototherapy, and oral administration. Topical administration is a treatment including steroids, coal tar, anthralin, vitamin D3 and its analogues, retinoids and sunburn, etc., and such topical administration has side effects of skin thinning, stretch marks, burns, irritation, and photosensitivity. In addition, there is a problem that steroids may induce tolerance, which may affect subsequent treatment. Phototherapy, which involves the administration of psoralen along with ultraviolet B or ultraviolet A, has the disadvantage of promoting skin aging and increasing the incidence of skin cancer. In addition, oral administration applied to psoriasis with severe symptoms is currently widely used as administration of an immunomodulatory agent such as cyclosporine, but there is a risk of nephrotoxicity or hypertension due to prolonged use.
따라서, 상기와 같은 부작용을 최소화하면서도 향상된 치료 효능을 보이는 새로운 치료제의 개발이 요구되고 있다.Therefore, there is a demand for the development of a new therapeutic agent that exhibits improved therapeutic efficacy while minimizing the side effects as described above.
본 발명은 상기와 같은 문제점을 해결하기 위해 안출된 것으로서, 본 발명자들은 건선을 비롯한 염증성 피부질환 치료용 제제에 대해서 예의 연구한 결과, 9개의 아미노산으로 이루어진 작은 사이즈의 펩타이드를 제조하였으며, 상기 펩타이드가 염증성 피부질환 치료에 우수한 효과가 있음을 확인하고, 이에 기초하여 본 발명을 완성하였다.The present invention was devised to solve the above problems, and as a result of intensive research on the preparation for the treatment of inflammatory skin diseases including psoriasis, the present inventors prepared a small-sized peptide consisting of 9 amino acids, and the peptide was It was confirmed that there was an excellent effect in the treatment of inflammatory skin diseases, and based on this, the present invention was completed.
이에, 본 발명의 목적은, 서열번호 1로 표시되는 아미노산으로 이루어진, 염증성 피부질환 치료용 펩타이드를 제공하는 것이다.Accordingly, it is an object of the present invention to provide a peptide for treating inflammatory skin diseases, which consists of an amino acid represented by SEQ ID NO: 1.
또한, 본 발명의 다른 목적은, 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 치료용 약학적 조성물을 제공하는 것이다.Another object of the present invention is to provide a pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising the peptide or a polynucleotide encoding the same as an active ingredient.
또한, 본 발명의 또 다른 목적은, 상기 펩타이드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 개선용 건강기능식품/화장품 조성물을 제공하는 것이다.In addition, another object of the present invention is to provide a health functional food / cosmetic composition for preventing or improving inflammatory skin disease, comprising the peptide as an active ingredient.
또한, 본 발명의 또 다른 목적은, 약학적으로 유효한 양의 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 개체에 투여하는 단계를 포함하는 염증성 피부질환의 예방, 치료 또는 개선방법을 제공하기 위한 것이다.In addition, another object of the present invention is to provide a method for preventing, treating or improving inflammatory skin diseases, which includes administering to an individual a pharmaceutically effective amount of the peptide or a polynucleotide encoding the same.
아울러, 본 발명의 또 다른 목적은, 염증성 피부질환의 예방, 치료 또는 개선용 조성물로 사용하기 위한 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드의 용도를 제공하기 위한 것이다.In addition, another object of the present invention is to provide the use of the peptide or a polynucleotide encoding the same for use as a composition for the prevention, treatment or improvement of inflammatory skin diseases.
본 발명은 상기 과제를 해결하기 위하여, 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드를 제공한다.In order to solve the above problems, the present invention provides a peptide consisting of amino acids represented by SEQ ID NO: 1.
또한, 본 발명은 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 치료용 약학적 조성물을 제공한다.In addition, the present invention provides a pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising a peptide or a polynucleotide encoding the same as an active ingredient, consisting of the amino acid represented by SEQ ID NO: 1.
이때, 상기 염증성 피부질환은 건선(psoriasis), 과각화증(hyperkeratosis), 어린선(ichthyosis), 아토피 피부염(atopic dermatitis), 모공성각화증(keratosis pilaris), 광선각화증(actinic keratoses), 지루각화증(seborrheic keratoses), 천포창(pemphigus), 티눈(corns), 사마귀(warts), 및 편평태선(lichen planus)으로 이루어진 군에서 선택될 수 있다.In this case, the inflammatory skin disease is psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratoses ), pemphigus, corns, warts, and lichen planus.
또한, 본 발명은 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 개선용 화장품 조성물을 제공한다.In addition, the present invention provides a cosmetic composition for preventing or improving inflammatory skin disease, comprising a peptide as an active ingredient, consisting of an amino acid represented by SEQ ID NO: 1.
이때, 상기 조성물은 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 왁스 또는 스프레이 형태일 수 있다.In this case, the composition may be in the form of a suspension, emulsion, paste, gel, cream, lotion, powder, wax or spray.
또한, 본 발명은 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 개체에 투여하는 단계를 포함하는 염증성 피부질환 예방 또는 개선방법을 제공한다.In addition, the present invention provides a method for preventing or improving inflammatory skin disease comprising administering the peptide or a polynucleotide encoding the same to an individual.
또한, 본 발명은 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 개체에 투여하는 단계를 포함하는, 염증성 피부질환 치료방법을 제공한다.In addition, the present invention provides a method for treating inflammatory skin disease, comprising administering the peptide or a polynucleotide encoding the same to an individual.
또한, 본 발명은 상기 펩타이드의 염증성 피부질환 치료 용도를 제공한다.In addition, the present invention provides the use of the peptide for treating inflammatory skin diseases.
또한, 본 발명은 염증성 피부질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드의 용도를 제공한다.In addition, the present invention provides the use of the peptide or a polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating inflammatory skin diseases.
아울러, 본 발명은 염증성 피부질환 예방 또는 개선용 화장품 조성물로 사용하기 위한 상기 펩타이드의 용도를 제공한다.In addition, the present invention provides the use of the peptide for use as a cosmetic composition for preventing or improving inflammatory skin disease.
본 발명에 따른 신규 합성 펩타이드는 우수한 염증성 피부질환 치료 효과뿐만 아니라, 9개의 아미노산으로 구성된 매우 작은 규모의 펩타이드로 저분자이며 생분해성이기 때문에 과민반응 유발 가능성이 적어 외부 물질의 투여에 따른 부작용을 최소화시킬 수 있는바, 기존의 염증성 피부질환 치료용 제제를 대체할 수 있는 유효 물질로 활용될 수 있을 것으로 기대된다.The novel synthetic peptide according to the present invention has not only an excellent therapeutic effect on inflammatory skin diseases, but also a very small-scale peptide consisting of 9 amino acids, low molecular weight and biodegradable, so it is less likely to cause hypersensitivity reaction, so side effects caused by the administration of external substances can be minimized. As such, it is expected that it can be used as an effective substance that can replace the existing agents for treating inflammatory skin diseases.
도 1은 본 발명의 펩타이드 처리에 따른 건선 치료 정도를 조직학적 염색을 통해 비교한 결과를 나타낸 것이다.1 shows the results of comparing the degree of psoriasis treatment according to the peptide treatment of the present invention through histological staining.
도 2는 본 발명의 펩타이드 처리에 따른 홍반(Erythema)의 감소 정도를 스코어링한 결과를 나타낸 것이다.Figure 2 shows the results of scoring the degree of reduction of erythema (Erythema) according to the peptide treatment of the present invention.
도 3은 본 발명의 펩타이드 처리에 따른 표피의 두께를 측정한 결과를 나타낸 것이다.3 shows the results of measuring the thickness of the epidermis according to the peptide treatment of the present invention.
도 4는 본 발명의 펩타이드 처리에 따라 감소하는 Th17 cells population을 측정한 결과를 나타낸 것이다. 4 shows the results of measuring the Th17 cell population decreased according to the peptide treatment of the present invention.
이하, 본 발명을 더욱 구체적으로 설명하기로 한다.Hereinafter, the present invention will be described in more detail.
본 발명은 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드를 제공한다. The present invention provides a peptide, consisting of amino acids represented by SEQ ID NO: 1.
본 발명에서, "펩타이드 (peptide)"는 아마이드 결합 (또는 펩타이드 결합)으로 연결된 2개 이상의 아미노산으로 이루어진 폴리머를 의미하며, 본 발명의 목적상, 건선을 비롯한 건조성 피부질환의 발생억제, 개선 및 치유 활성을 가지는 펩타이드를 의미한다. 펩타이드 치료제에 대한 다양한 연구에도 불구하고, 펩타이드 자체 크기가 너무 커서 표적 조직 또는 세포에 효과적으로 유입되지 못하거나 반감기가 짧아 단기간에 체내에서 소멸되는 단점을 드러내었는바, 본 발명은 유효한 치료 효과를 가짐과 동시에 9개의 아미노산으로 이루어진 펩타이드를 개발하였다는 점에 기술적 의의가 있다.In the present invention, "peptide" refers to a polymer consisting of two or more amino acids linked by an amide bond (or peptide bond), and for the purpose of the present invention, inhibiting, improving and improving the occurrence of dry skin diseases including psoriasis and It refers to a peptide having a therapeutic activity. Despite various studies on peptide therapeutics, the peptide itself is too large to effectively enter the target tissue or cell, or has a short half-life, which reveals disadvantages that it disappears in the body in a short period of time. The present invention has an effective therapeutic effect and It has technical significance in that it developed a peptide consisting of 9 amino acids at the same time.
본 발명의 펩타이드는 서열번호 1로 표시되는 아미노산으로 이루어질 수 있고, 상기 서열번호 1로 표시되는 아미노산 서열과 각각 75% 이상, 바람직하게는 80% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상의 서열 상동성을 가지는 아미노산 서열을 포함할 수 있으며, 표적화 서열, 태그 (tag), 표지된 잔기, 반감기 또는 펩타이드 안정성을 증가시키기 위한 특정 목적으로 제조된 아미노산 서열도 추가적으로 포함할 수 있다.The peptide of the present invention may consist of the amino acid represented by SEQ ID NO: 1, and each of the amino acid sequence represented by SEQ ID NO: 1 and at least 75%, preferably at least 80%, more preferably at least 90%, most preferably may include an amino acid sequence having 95% or more sequence homology, and may additionally include a targeting sequence, a tag, a labeled residue, an amino acid sequence prepared for a specific purpose to increase half-life or peptide stability. .
또한, 본 발명의 펩타이드에 대한 기능성 변이체도 포함할 수 있다. 상기 기능성 변이체는 본 명세서에 기재된 펩타이드 서열 (서열번호 1)의 생물학적 균등물들을 포함한다. 예를 들면, 펩타이드의 결합 친화도 및/또는 기타 생물학적 특성을 보다 개선시키기 위하여 펩타이드의 아미노산 또는 폴리뉴클레오티드 서열에 추가적인 변화를 줄 수 있다. 이러한 변형은 펩타이드의 아미노산 서열 잔기의 결실. 삽입, 및/또는 치환을 포함하며, 아미노산 곁사슬 치환체의 상대적인 유사성, 예컨대, 소수성, 친수성, 전하 크기 등에 기초하여 이루어질 수 있다.In addition, functional variants for the peptides of the present invention may be included. The functional variants include biological equivalents of the peptide sequences described herein (SEQ ID NO: 1). For example, additional changes may be made to the amino acid or polynucleotide sequence of the peptide to further improve the binding affinity and/or other biological properties of the peptide. Such modifications are deletions of amino acid sequence residues in the peptide. insertions, and/or substitutions, and may be made based on the relative similarity of amino acid side chain substituents, such as hydrophobicity, hydrophilicity, charge magnitude, and the like.
또한, 본 발명의 펩타이드는 당해 분야에서 널리 공지된 다양한 방법으로 획득할 수 있다. 일례로서, 폴리뉴클레오타이드 재조합과 단백질 발현 시스템을 이용하여 제조하거나 펩타이드 합성과 같은 화학적 합성을 통하여 시험관 내에서 합성하는 방법, 및 무세포 단백질 합성법 등으로 제조될 수 있다.In addition, the peptide of the present invention can be obtained by various methods well known in the art. As an example, it may be prepared using polynucleotide recombination and protein expression systems, or synthesized in vitro through chemical synthesis such as peptide synthesis, and cell-free protein synthesis.
또한, 보다 나은 화학적 안정성, 강화된 약리 특성 (반감기, 흡수성, 역가, 효능 등), 변경된 특이성 (예를 들어, 광범위한 생물학적 활성 스펙트럼), 감소된 항원성을 획득하기 위하여, 펩타이드의 N- 또는 C-말단에 보호기가 결합되어 있을 수 있다. 바람직하게, 상기 보호기는 아세틸기, 플루오레닐 메톡시 카르보닐기, 포르밀기, 팔미토일기, 미리스틸기, 스테아릴기 또는 폴리에틸렌글리콜(PEG)일 수 있으나, 펩타이드의 개질, 특히 펩타이드의 안정성 증진시킬 수 있는 성분이라면, 제한없이 포함할 수 있다. 본 발명에서 사용되는 용어, "안정성"은 생체 내 단백질 절단효소의 공격으로부터 본 발명의 펩타이드를 보호하는 인 비보 안정성 뿐만 아니라, 저장 안정성 (예컨대, 상온 저장 안정성)도 의미한다. 본 발명에서, "폴리뉴클레오타이드 (polynucleotide)"는 뉴클레오티드가 결합된 중합체로서, 유전 정보를 전달하는 역할을 한다. 본 발명의 목적상, 서열번호 1의 펩타이드를 코딩하며, 상기 펩타이드를 코딩하는 폴리뉴클레오타이드 서열과 각각 75% 이상, 바람직하게는 85% 이상, 더욱 바람직하게는 90% 이상, 가장 바람직하게는 95% 이상 서열 상동성을 가지는 서열을 포함할 수 있다. 상기 "상동성"은 야생형 아미노산 서열 또는 폴리뉴클레오타이드 서열과의 유사한 정도를 나타내기 위한 것으로서, 이러한 상동성의 비교는 당업계에서 널리 알려진 비교 프로그램을 이용하여 수행할 수 있으며, 2개 이상의 서열간 상동성을 백분율 (%)로 계산할 수 있다.In addition, to obtain better chemical stability, enhanced pharmacological properties (half-life, absorption, potency, potency, etc.), altered specificity (e.g., broad spectrum of biological activity), reduced antigenicity, N- or C of the peptide -A protecting group may be attached to the terminal. Preferably, the protecting group may be an acetyl group, a fluorenyl methoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, or polyethylene glycol (PEG), but the modification of the peptide, particularly the stability of the peptide As long as it is a possible component, it may be included without limitation. As used herein, the term "stability" refers to storage stability (eg, room temperature storage stability) as well as in vivo stability that protects the peptide of the present invention from attack by proteolytic enzymes in vivo. In the present invention, "polynucleotide (polynucleotide)" is a polymer to which nucleotides are bound, and serves to transmit genetic information. For the purpose of the present invention, encoding the peptide of SEQ ID NO: 1, and each of the polynucleotide sequence encoding the peptide at least 75%, preferably at least 85%, more preferably at least 90%, most preferably at least 95% It may include a sequence having aberrant sequence homology. The "homology" is intended to indicate a degree of similarity with a wild-type amino acid sequence or a polynucleotide sequence, and the comparison of such homology can be performed using a comparison program well known in the art, and homology between two or more sequences can be calculated as a percentage (%).
본 발명의 다른 양태로서, 본 발명은 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 치료용 약학적 조성물; 염증성 피부질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드의 용도; 및 치료상 유효량의 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드를 개체에게 투여하는 단계를 포함하는, 염증성 피부질환 치료방법을 제공한다.As another aspect of the present invention, the present invention is a pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising as an active ingredient a peptide or a polynucleotide encoding the same, consisting of amino acids represented by SEQ ID NO: 1; Use of the peptide or polynucleotide encoding the same for use as a pharmaceutical composition for preventing or treating inflammatory skin diseases; And it provides a method for treating inflammatory skin disease, comprising administering to an individual a therapeutically effective amount of the peptide or a polynucleotide encoding the same.
본 발명에서 사용되는 용어, "치료"는 본 발명에 따른 약학적 조성물의 투여에 의해 상처에 대한 증세가 호전되거나 이롭게 변경되는 모든 행위를 의미한다.As used herein, the term “treatment” refers to any action in which the symptoms of a wound are improved or beneficially changed by administration of the pharmaceutical composition according to the present invention.
본 발명에서, "개체"는 상처의 치료를 필요로 하는 대상을 의미하며, 보다 구체적으로 인간 또는 비-인간인 영장류, 생쥐(mouse), 개, 고양이, 말 및 소 등의 포유류를 의미한다.In the present invention, "individual" refers to a subject in need of treatment of a wound, and more specifically, refers to mammals such as human or non-human primates, mice, dogs, cats, horses and cattle.
본 발명에서, 상기 염증성 피부질환은 건선(psoriasis), 과각화증(hyperkeratosis), 어린선(ichthyosis), 아토피 피부염(atopic dermatitis), 모공성각화증(keratosis pilaris), 광선각화증(actinic keratoses), 지루각화증(seborrheic keratoses), 천포창(pemphigus), 티눈(corns), 사마귀(warts), 및 편평태선(lichen planus)으로 이루어진 군에서 선택된 하나 이상일 수 있고, 바람직하게는 건선일 수 있으나, 이에 한정되는 것은 아니다.In the present invention, the inflammatory skin disease is psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratosis ( seborrheic keratoses), pemphigus, corns, warts, and may be at least one selected from the group consisting of lichen planus, preferably psoriasis, but is not limited thereto.
한편, 본 발명의 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드는 콜로이드 현탁액, 분말, 식염수, 지질, 리포좀, 미소구체 microspheres), 또는 나노 구형입자와 같은 약학적으로 허용될 수 있는 담체에 운반될 수 있다. 이들은 운반 수단과 복합체를 형성하거나 관련될 수 있고, 지질, 리포좀, 미세입자, 금, 나노입자, 폴리머, 축합 반응제, 다당류, 폴리아미노산, 덴드리머, 사포닌, 흡착 증진 물질 또는 지방산과 같은 당업계에 공지된 운반 시스템을 사용하여 생체 내 운반될 수 있다.On the other hand, the peptide of the present invention or a polynucleotide encoding the same may be delivered in a colloidal suspension, powder, saline, lipid, liposome, microsphere microspheres), or a pharmaceutically acceptable carrier such as nano spherical particles. They may form complexes with, or be associated with, vehicles and are known in the art such as lipids, liposomes, microparticles, gold, nanoparticles, polymers, condensation reagents, polysaccharides, polyamino acids, dendrimers, saponins, adsorption enhancing substances or fatty acids. It can be delivered in vivo using known delivery systems.
이 외에도, 약학적으로 허용되는 담체는 제제시 통상적으로 이용되는 락토스, 덱스트로스, 수크로스, 솔비톨, 만니톨, 전분, 아카시아, 고무, 인산칼슘, 알기네이트, 젤라틴, 규산 칼슘, 미세 결정성 셀룰로스, 폴리비닐 피로리돈, 셀룰로스, 물, 시럽, 메틸 셀룰로스, 메틸히드록시벤조에이트, 프로필 히드록시벤조에이트, 활석, 스테아르산 마그네슘 및 미네랄 오일 등을 포함할 수 있으나, 이에 한정되는 것은 아니다. 또한, 상기 성분들 이외에 윤활제, 습윤제, 감미제, 향미제, 유화제, 현탁제, 보존제 등을 추가로 포함할 수 있다.In addition, pharmaceutically acceptable carriers include lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia, gum, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, which are commonly used in the formulation. polyvinyl pyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate and mineral oil, and the like. In addition, it may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. in addition to the above components.
본 발명의 약학적 조성물은 목적하는 방법에 따라 경구 투여하거나 비경구투여 (예를 들어, 근육 내, 정맥 내, 복강 내, 피하, 피내, 또는 국소에 적용)할 수 있으며, 투여량은 환자의 상태 및 체중, 질병의 정도, 약물형태, 투여경로 및 시간에 따라 다르지만, 당업자에 의해 적절하게 선택될 수 있다.The pharmaceutical composition of the present invention may be administered orally or parenterally (eg, intramuscularly, intravenously, intraperitoneally, subcutaneously, intradermally, or topically applied) according to a desired method, and the dosage may be determined by the patient. Although it varies depending on the condition and weight, the degree of disease, the drug form, the administration route and time, it may be appropriately selected by those skilled in the art.
특히, 본 발명의 약학적 조성물을 피부 외용제로 사용하는 경우, 추가로 지방 물질, 유기 용매, 용해제, 농축제 및 겔화제, 연화제, 항산화제, 현탁화제, 안정화제, 발포제(foaming agent), 방향제, 계면활성제, 물, 이온형 유화제, 비이온형 유화제, 충전제, 금속이온 봉쇄제, 킬레이트화제, 보존제, 비타민, 차단제, 습윤화제, 필수 오일, 염료, 안료, 친수성 활성제, 친유성 활성제 또는 지질 소낭 등 피부 외용제에 통상적으로 사용되는 임의의 다른 성분과 같은 피부 과학 분야에서 통상적으로 사용되는 보조제를 함유할 수 있다. 또한 상기 성분들은 피부 과학 분야에서 일반적으로 사용되는 양으로 도입될 수 있다. 또한, 상기 약학적 조성물이 피부 외용제로 제공될 경우, 이에 제한되는 것은 아니나, 연고, 패취, 겔, 크림 또는 분무제 등의 제형일 수 있다.In particular, when the pharmaceutical composition of the present invention is used as an external preparation for skin, a fatty substance, an organic solvent, a solubilizer, a thickening agent and a gelling agent, an emollient, an antioxidant, a suspending agent, a stabilizer, a foaming agent, a fragrance , surfactant, water, ionic emulsifier, non-ionic emulsifier, filler, sequestering agent, chelating agent, preservative, vitamin, blocker, wetting agent, essential oil, dye, pigment, hydrophilic active agent, lipophilic active agent or lipid vesicle It may contain adjuvants commonly used in the field of dermatology, such as any other ingredients commonly used in external preparations for skin. In addition, the above ingredients may be introduced in an amount generally used in the field of dermatology. In addition, when the pharmaceutical composition is provided as an external preparation for skin, it may be in the form of an ointment, patch, gel, cream or spray, but is not limited thereto.
본 발명의 약학적 조성물은 약학적으로 유효한 양으로 투여한다. 본 발명에 있어서 "약학적으로 유효한 양"은 의학적 치료에 적용 가능한 합리적인 수혜/위험 비율로 질환을 치료하기에 충분한 양을 의미하며, 유효 용량 수준은 환자의 질환의 종류, 중증도, 약물의 활성, 약물에 대한 민감도, 투여 시간, 투여 경로 및 배출 비율, 치료기간, 동시 사용되는 약물을 포함한 요소 및 기타 의학 분야에 잘 알려진 요소에 따라 결정될 수 있다. 본 발명에 다른 약학적 조성물은 개별 치료제로 투여하거나 다른 치료제와 병용하여 투여될 수 있고 종래의 치료제와는 동시에, 별도로, 또는 순차적으로 투여될 수 있으며, 단일 또는 다중 투여될 수 있다. 상기 요소들을 모두 고려하여 부작용 없이 최소한의 양으로 최대 효과를 얻을 수 있는 양을 투여하는 것이 중요하며, 이는 당업자에 의해 용이하게 결정될 수 있다.The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. In the present invention, "pharmaceutically effective amount" means an amount sufficient to treat a disease at a reasonable benefit/risk ratio applicable to medical treatment, and the effective dose level is determined by the type, severity, drug activity, and type of the patient's disease; Sensitivity to the drug, administration time, administration route and excretion rate, treatment period, factors including concurrent drugs and other factors well known in the medical field may be determined. The pharmaceutical composition according to the present invention may be administered as an individual therapeutic agent or may be administered in combination with other therapeutic agents, may be administered simultaneously with, separately, or sequentially from a conventional therapeutic agent, and may be administered single or multiple. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect with a minimum amount without side effects, which can be easily determined by those skilled in the art.
구체적으로 본 발명의 약학적 조성물의 유효량은 환자의 연령, 성별, 상태, 체중, 체내에 활성 성분의 흡수도, 불활성율, 배설 속도, 질병 종류, 병용되는 약물에 따라 달라질 수 있으며, 투여 경로, 비만의 중증도, 성별, 체중, 연령 등에 따라 증감될 수 있다.Specifically, the effective amount of the pharmaceutical composition of the present invention may vary depending on the patient's age, sex, condition, weight, absorption of the active ingredient into the body, inactivation rate, excretion rate, disease type, and drugs used in combination, the route of administration, It can increase or decrease according to the severity of obesity, sex, weight, age, etc.
또한, 본 발명의 다른 양태로서, 본 발명은 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 개선용 건강기능식품/화장품 조성물; 염증성 피부질환 예방 또는 개선용 건강기능식품/화장품 조성물로 사용하기 위한 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드의 용도; 및 치료상 유효량의 상기 펩타이드 또는 이를 코딩하는 폴리뉴클레오타이드를 개체에게 투여하는 단계를 포함하는, 염증성 피부질환 예방 또는 개선방법을 제공한다.In addition, as another aspect of the present invention, the present invention is a health functional food / cosmetic composition for preventing or improving inflammatory skin disease, comprising a peptide or a polynucleotide encoding the same as an active ingredient, consisting of an amino acid represented by SEQ ID NO: 1; Use of the peptide or polynucleotide encoding the same for use as a health functional food / cosmetic composition for preventing or improving inflammatory skin disease; And it provides a method for preventing or improving inflammatory skin disease, comprising administering to an individual a therapeutically effective amount of the peptide or a polynucleotide encoding the same.
본 발명에서 사용되는 용어, "개선"이란 치료되는 상태와 관련된 파라미터, 예를 들면, 증상의 정도를 적어도 감소시키는 모든 행위를 의미한다. 이때, 상기 건강기능식품/화장품 조성물은 염증성 피부질환 예방 또는 개선을 위하여, 관련 질환의 발병 단계 이전 또는 발병 후, 치료를 위한 약제와 동시에 또는 별개로서 사용될 수 있다.As used herein, the term “improvement” refers to any action that at least reduces a parameter related to the condition being treated, for example, the severity of symptoms. In this case, the health functional food/cosmetics composition may be used simultaneously with or separately from a drug for treatment before or after the onset of a related disease in order to prevent or improve inflammatory skin disease.
본 발명의 건강기능식품 조성물에서, 유효성분을 식품에 그대로 첨가하거나 다른 식품 또는 식품 성분과 함께 사용될 수 있고, 통상적인 방법에 따라 적절하게 사용될 수 있다. 유효 성분의 혼합량은 그의 사용 목적 (예방 또는 개선용)에 따라 적합하게 결정될 수 있다. 일반적으로, 식품 또는 음료의 제조시에 본 발명의 조성물은 원료에 대하여 바람직하게 15 중량% 이하, 바람직하게는 10 중량% 이하의 양으로 첨가될 수 있다. 그러나 건강 및 위생을 목적으로 하거나 또는 건강 조절을 목적으로 하는 장기간 섭취의 경우에는 상기 양은 상기 범위 이하일 수 있다.In the health functional food composition of the present invention, the active ingredient may be added to food as it is or used together with other food or food ingredients, and may be appropriately used according to a conventional method. The mixing amount of the active ingredient may be appropriately determined depending on the purpose of its use (for prevention or improvement). In general, in the production of food or beverage, the composition of the present invention may be added in an amount of preferably 15% by weight or less, preferably 10% by weight or less, based on the raw material. However, in the case of long-term ingestion for health and hygiene or health control, the amount may be less than or equal to the above range.
본 발명의 건강기능식품 조성물은 상기 유효성분을 함유하는 것 외에 특별한 제한없이 다른 성분들을 필수 성분으로서 함유할 수 있다. 예를 들면, 통상의 음료와 같이 여러 가지 향미제 또는 천연 탄수화물 등을 추가 성분으로서 함유할 수 있다. 상술한 천연 탄수화물의 예는 모노사카라이드, 예를 들어, 포도당, 과당 등; 디사카라이드, 예를 들어 말토스, 슈크로스 등; 및 폴리사카라이드, 예를 들어 덱스트린, 시클로덱스트린 등과 같은 통상적인 당, 및 자일리톨, 소르비톨, 에리트리톨 등의 당 알코올일 수 있다. 상술한 것 이외의 향미제로서 천연 향미제 (타우마틴, 스테비아 추출물 (예를 들어, 레바우디오시드 A, 글리시르히진등)) 및 합성 향미제 (사카린, 아스파르탐 등)를 유리하게 사용할 수 있다. 상기 천연 탄수화물의 비율은 당업자의 선택에 의해 적절하게 결정될 수 있다.The health functional food composition of the present invention may contain other ingredients as essential ingredients without any particular limitation in addition to containing the active ingredient. For example, as in conventional beverages, various flavoring agents or natural carbohydrates may be contained as additional ingredients. Examples of the above-mentioned natural carbohydrates include monosaccharides such as glucose, fructose and the like; disaccharides such as maltose, sucrose and the like; and polysaccharides, for example, conventional sugars such as dextrin, cyclodextrin, and the like, and sugar alcohols such as xylitol, sorbitol, and erythritol. As flavoring agents other than those described above, natural flavoring agents (taumatin, stevia extract (eg, rebaudioside A, glycyrrhizin, etc.)) and synthetic flavoring agents (saccharin, aspartame, etc.) may be advantageously used can The ratio of the natural carbohydrate may be appropriately determined by the selection of those skilled in the art.
상기 외에 본 발명의 건강기능식품 조성물은 여러 가지 영양제, 비타민, 광물 (전해질), 합성 풍미제 및 천연 풍미제 등의 풍미제, 착색제 및 중진제 (치즈, 초콜릿 등), 펙트산 및 그의 염, 알긴산 및 그의 염, 유기산, 보호성 콜로이드 증점제, pH 조절제, 안정화제, 방부제, 글리세린, 알코올, 탄산음료에 사용되는 탄산화제 등을 함유할 수 있다. 이러한 성분은 독립적으로 또는 조합하여 사용할 수 있으며, 이러한 첨가제의 비율 또한 당업자에 의해 적절히 선택될 수 있다.In addition to the above, the health functional food composition of the present invention includes various nutrients, vitamins, minerals (electrolytes), flavoring agents such as synthetic and natural flavoring agents, coloring agents and thickeners (cheese, chocolate, etc.), pectic acid and salts thereof, Alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohols, carbonates used in carbonated beverages, and the like may be contained. These components may be used independently or in combination, and the proportion of these additives may also be appropriately selected by those skilled in the art.
본 발명의 화장품 조성물은 당업계에서 통상적으로 제조되는 어떠한 제형으로도 제조될 수 있으며, 예를 들어, 용액, 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 비누, 계면활성제-함유 클린싱, 오일, 분말 파운데이션, 유탁액 파운데이션, 왁스 파운데이션 및 스프레이 등으로 제형화될 수 있으나, 이에 한정되는 것은 아니다. 보다 상세하게는, 유연 화장수, 영양 화장수, 영양 크림, 마사지 크림, 에센스, 아이 크림, 클렌징 크림, 클렌징폼, 클렌징 워터, 팩, 스프레이 또는 파우더의 제형으로 제조될 수 있다.The cosmetic composition of the present invention may be prepared in any formulation conventionally prepared in the art, for example, a solution, suspension, emulsion, paste, gel, cream, lotion, powder, soap, surfactant-containing cleansing agent , oil, powder foundation, emulsion foundation, wax foundation, spray, etc., but is not limited thereto. More specifically, it may be prepared in the form of a flexible lotion, a nourishing lotion, a nourishing 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.
본 발명의 화장품 조성물에 함유된 유효한 담체는 제형에 따라, 당업계에서 통상적으로 이용되는 담체가 이용될 수 있다. 본 발명의 제형이 페이스트, 크림 또는 겔인 경우에는 담체 성분으로서 동물성유, 식물성유, 왁스, 파라핀, 전분, 트라칸트, 셀룰로오스 유도체, 폴리에틸렌 글리콜, 실리콘, 벤토나이트, 실리카, 탈크 또는 산화아연 등이 이용될 수 있다.The effective carrier contained in the cosmetic composition of the present invention may be a carrier commonly used in the art, depending on the formulation. When the formulation of the present invention is a paste, cream or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc or zinc oxide may be used as a carrier component. can
본 발명의 제형이 파우더 또는 스프레이인 경우에는 담체 성분으로서 락토스, 탈크, 실리카, 알루미늄 히드록시드, 칼슘 실리케이트 또는 폴리아미드 파우더가 이용될 수 있고, 특히 스프레이인 경우에는 추가적으로 클로로플루오로히드로카본, 프로판/부탄 또는 디메틸 에테르와 같은 추진체를 포함할 수 있다.When the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder may be used as a carrier component, and in particular, in the case of a spray, additional chlorofluorohydrocarbon, propane /may contain propellants such as butane or dimethyl ether.
본 발명의 제형이 용액 또는 유탁액인 경우에는 담체 성분으로서 용매, 용해화제 또는 유탁화제가 이용되고, 예컨대 물, 에탄올, 이소프로판올, 에틸 카보네이트, 에틸 아세테이트, 벤질 알코올, 벤질 벤조에이트, 프로필렌 글리콜, 1,3-부틸글리콜 오일, 글리세롤 지방족 에스테르, 폴리에틸렌 글리콜 또는 소르비탄의 지방산 에스테르가 있다.When the formulation of the present invention is a solution or emulsion, a solvent, solubilizer or emulsifier is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1 ,3-butylglycol oil, glycerol fatty esters, fatty acid esters of polyethylene glycol or sorbitan.
본 발명의 제형이 현탁액인 경우에는 담체 성분으로서 물, 에탄올 또는 프로필렌 글리콜과 같은 액상의 희석제, 에톡실화 이소스테아릴 알코올, 폴리옥시에틸렌 소르비톨 에스테르 및 폴리옥시에틸렌 소르비탄 에스테르와 같은 현탁제, 미소결정성 셀룰로오스, 알루미늄 메타히드록시드, 벤토나이트, 아가 또는 트라칸트 등이 이용될 수 있다.When the formulation of the present invention is a suspension, as a carrier component, a liquid diluent such as water, ethanol or propylene glycol, a suspending agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester and polyoxyethylene sorbitan ester, microcrystals Adult cellulose, aluminum metahydroxide, bentonite, agar or tracanth may be used.
본 발명의 제형이 계면-활성제 함유 클린징인 경우에는 담체 성분으로서 지방족 알코올 설페이트, 지방족 알코올 에테르 설페이트, 설포숙신산 모노에스테르, 이세티오네이트, 이미다졸리늄 유도체, 메틸타우레이트, 사르코시네이트, 지방산 아미드 에테르 설페이트, 알킬아미도베타인, 지방족 알코올, 지방산 글리세리드, 지방산 디에탄올아미드, 식물성 유, 라놀린 유도체 또는 에톡실화 글리세롤 지방산 에스테르 등이 이용될 수 있다.When the formulation of the present invention is a surfactant-containing cleansing agent, aliphatic alcohol sulfate, aliphatic alcohol ether sulfate, sulfosuccinic acid monoester, isethionate, imidazolinium derivative, methyl taurate, sarcosinate, fatty acid amide as carrier components Ether sulfate, alkylamidobetaine, fatty alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative or ethoxylated glycerol fatty acid ester and the like can be used.
본 발명의 화장품 조성물에 포함되는 성분은 유효 성분과 담체 성분 이외에, 화장품 조성물에 통상적으로 이용되는 성분들을 포함하며, 예컨대 항산화제, 안정화제, 용해화제, 비타민, 안료 및 향료와 같은 통상적인 보조제를 포함할 수 있다.The ingredients included in the cosmetic composition of the present invention include ingredients commonly used in cosmetic compositions in addition to the active ingredient and carrier ingredient, for example, conventional adjuvants such as antioxidants, stabilizers, solubilizers, vitamins, pigments and fragrances. may include
[실시예][Example]
이하, 실시예를 통하여 본 발명을 더욱 상세히 설명하고자 한다. 이들 실시예는 오로지 본 발명을 예시하기 위한 것으로, 본 발명의 범위가 이들 실시예에 의해 제한되는 것으로 해석되지 않는 것은 당업계에서 통상의 지식을 가진 자에게 있어서 자명할 것이다. 따라서 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.Hereinafter, the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and it will be apparent to those of ordinary skill in the art that the scope of the present invention is not to be construed as being limited by these examples. Accordingly, the substantial scope of the present invention will be defined by the appended claims and their equivalents.
펩타이드의 제조Preparation of peptides
먼저, 서열번호 1로 표시되는 아미노산 서열(RDGRDGPSP)로 이루어진 펩타이드를 제조하였다. 이후, 고성능액체크로마토그래피 (SHIMADZU Prominence HPLC)를 이용하여 합성된 펩타이드를 순수 분리하였으며, 컬럼은 Shiseido capcell pak C18 Column (4.6 x 50mm)을 이용하였다. 또한, 질량분석기 (HP 1100 series LC/MSD)를 이용하여, 합성된 펩타이드의 질량을 확인하였다.First, a peptide consisting of the amino acid sequence (RDGRDGPSP) represented by SEQ ID NO: 1 was prepared. Thereafter, the synthesized peptide was purely separated using high performance liquid chromatography (SHIMADZU Prominence HPLC), and a Shiseido capcell pak C18 column (4.6 x 50 mm) was used as the column. In addition, the mass of the synthesized peptide was confirmed using a mass spectrometer (HP 1100 series LC/MSD).
마우스 동물모델의 확립 및 펩타이드 처리Establishment of mouse animal model and peptide treatment
건선 마우스 동물 모델을 확립하기 위하여, Imiquimod (IMQ)를 이용하여 건선과 유사한 피부 염증반응을 유도했다(Journal of Investigative Dermatology 2015;135:2764-2774.). 먼저, 6주령 female 마우스 (C57BL/6)의 등을 IMQ 도포하기 하루 전(Day 0)에 쉐이빙한 후, 다음날(Day 1)부터 5% IMQ (3M, aldara cream) 62.5 mg씩 매일 4일 동안 도포하여 건선을 유발시켰다. 본 발명에 따른 펩타이드를 IMQ 도포한 다음날(Day 2)부터 각각 0.1, 10 ug/kg씩 매일 3일 동안 SC injection하여 처리하였다. Control 마우스는 IMQ 대신 vehicle control 크림으로 바세린 (Vaselin Lanette cream; Fagron)을 도포하고, 상기 펩타이드 대신 PBS를 주입하였다. 양성 대조군(Positive control) 마우스는 상기 펩타이드 대신 methotrexate (MTX)를 2 mg/kg SC injection 하였다.To establish an animal model for psoriasis, Imiquimod (IMQ) was used to induce a skin inflammatory response similar to psoriasis (Journal of Investigative Dermatology 2015;135:2764-2774.). First, after shaving the back of a 6-week-old female mouse (C57BL/6) one day before IMQ application (Day 0), from the next day (Day 1), 62.5 mg of 5% IMQ (3M, aldara cream) 62.5 mg daily for 4 days Psoriasis was induced by application. The peptide according to the present invention was treated by SC injection every day for 3 days at 0.1 and 10 ug/kg, respectively, from the next day (Day 2) after IMQ application. Control mice were coated with Vaseline (Vaselin Lanette cream; Fagron) as vehicle control cream instead of IMQ, and PBS was injected instead of the peptide. Positive control mice were injected with 2 mg/kg SC of methotrexate (MTX) instead of the peptide.
조직 분석tissue analysis
Day 5일째 마우스를 희생시켜 lesional skin tissue를 확보하고, paraformaldehyde로 고정하여 paraffin section을 만들고, hematoxylin & eosin (H&E) staining 결과를 분석하였다.On Day 5, mice were sacrificed to obtain lesional skin tissue, fixed with paraformaldehyde to make paraffin sections, and hematoxylin & eosin (H&E) staining results were analyzed.
Erythema scoringErythema scoring
Clinical 변화를 수치화하는 Psoriasis area and severity index (PASI) 측정방법 중 하나인 redness를 관찰하여 Erythema score로 나타내었다. Day 5일째 마우스의 사진을 찍고 육안 관찰을 통하여 redness를 scoring 하였다. 점수는 다음과 같이 0점부터 4점까지 나타내었다(0점, no symptoms: 1점, mild: 2점, moderate: 3점, severe: 4점, very severe)Redness, one of the measurement methods of Psoriasis area and severity index (PASI) to quantify clinical changes, was observed and expressed as the Erythema score. On Day 5, pictures of mice were taken and redness was scored through visual observation. Scores were expressed from 0 to 4 points as follows (0 points, no symptoms: 1 point, mild: 2 points, moderate: 3 points, severe: 4 points, very severe)
표피 두께 측정Epidermal thickness measurement
H&E 조직을 Image scope 프로그램으로 epidermis 두께를 측정하였다. 각 조직별로 random 부위 5곳을 측정한 결과를 mean값으로 그래프를 그리고 standard deviation을 표시하였다.Epidermis thickness was measured in H&E tissue with Image scope program. The results of measuring 5 random sites for each tissue were plotted as mean values and standard deviations were indicated.
Th17 cells 염색Th17 cells staining
Day 5일째 마우스로부터 lymph node를 제거하여 Th17 cells을 염색하여 비교하였다. 제거한 lymph node 조직을 가볍게 갈아서 세포를 수확한 후 counting 하였다. 1x106/mL의 농도로 well당 2x106/2 mL의 number로 24-well plate에 깔아주었다. 이때 T 세포의 활성화를 유도하기 위하여 PMA 25 ng/mL과 Ionomycin 1 ug/mL을 함께 처리하여 2시간 동안 37℃, CO2의 조건하에서 인큐베이션 시켰다. PMA와 Ionomycin에 의해 세포 밖으로 분비되는 IL-17을 세포 내에 가두기 위해 Monensin을 5 ug/mL로 처리하고 2시간 더 인큐베이션 시켰다. 이후, PBS로 세포를 워싱하여 남아있는 PMA, Ionomycin, Monensin을 제거한 후, CD4-Alexa 488항체를 넣고 10분 동안 상온에서 표면 염색(surface staining)을 하였다. 세포를 워싱하여 남아있는 항체를 제거한 뒤, 4℃에서 30분 동안 Fixation buffer (BD bioscience)로 세포를 고정하였다. 세포를 워싱하여 남아 있는 fixation buffer를 제거한 뒤, perm/wash buffer (BD bioscience)로 permeabilization 시켜 intracellular staining 준비를 하였다. IL-17-APC 항체를 넣고 4℃에서 1시간 동안 intracellular IL-17을 염색하였다. 염색이 모두 끝난 세포를 워싱하여 남아 있는 항체를 모두 제거하고 유세포 분석기 (BD, FACS Calibur)를 이용하여 CD4+/IL-17+ Th17 세포를 분석하였다.On day 5, lymph nodes were removed from the mice, and Th17 cells were stained for comparison. The removed lymph node tissue was lightly ground and the cells were harvested and counted. It was spread on a 24-well plate at a concentration of 1x10 6 /mL and a number of 2x10 6 /2 mL per well. At this time, in order to induce T cell activation, PMA 25 ng/mL and Ionomycin 1 ug/mL were treated together and incubated at 37° C., CO 2 for 2 hours. In order to trap IL-17 secreted out of the cell by PMA and Ionomycin, Monensin was treated at 5 ug/mL and incubated for 2 hours more. Thereafter, the cells were washed with PBS to remove the remaining PMA, Ionomycin, and Monensin, and then the CD4-Alexa 488 antibody was added and surface staining was performed at room temperature for 10 minutes. After washing the cells to remove the remaining antibody, the cells were fixed with Fixation buffer (BD bioscience) at 4°C for 30 minutes. After washing the cells to remove the remaining fixation buffer, permeabilization was performed with perm/wash buffer (BD bioscience) to prepare for intracellular staining. IL-17-APC antibody was added and intracellular IL-17 was stained for 1 hour at 4°C. All stained cells were washed to remove all remaining antibodies, and CD4+/IL-17+ Th17 cells were analyzed using a flow cytometer (BD, FACS Calibur).
실시예 1. 건선 마우스 모델의 조직학적 분석Example 1. Histological analysis of psoriasis mouse model
도 1은 본 발명의 펩타이드 처리에 따른 건선 치료 정도를 조직학적 염색을 통해 비교한 결과를 나타낸 것이다. 도 1에 나타난 바와 같이, 정상 마우스와 비교하여 PBS를 처리한 건선을 유발한 마우스에서는 표피층의 두께가 두꺼워지고, scaling이 많이 생성되었는바, IMQ에 의해 건선이 잘 유발되었음을 알 수 있다. 다음으로, 양성 대조군인 MTX를 처리한 마우스에서는 PBS에 비해 표피층이 다시 얇아지고 scaling도 정상 수준으로 떨어지는 것으로 확인되었다. 마찬가지로, 본 발명에 따른 펩타이드를 처리한 마우스에서도 MTX와 비슷한 수준으로 표피층의 두께가 얇아지고 scaling이 적어지는 것을 확인하였다. 이러한 결과를 통해 본 발명에 따른 펩타이드를 처리할 경우, 건선의 병변이 현저하게 줄어들어 건선의 치료 효과가 우수함을 확인하였다. 1 shows the results of comparing the degree of psoriasis treatment according to the peptide treatment of the present invention through histological staining. As shown in FIG. 1 , compared to normal mice, in the mice treated with PBS, the psoriasis-induced mice had a thickened epidermal layer, and a large amount of scaling was generated, indicating that psoriasis was well induced by IMQ. Next, it was confirmed that in mice treated with MTX, which is a positive control, the epidermal layer became thinner again and the scaling fell to a normal level compared to PBS. Similarly, in mice treated with the peptide according to the present invention, it was confirmed that the thickness of the epidermal layer became thinner and scaling was reduced to a level similar to that of MTX. Through these results, it was confirmed that, when the peptide according to the present invention was treated, the lesions of psoriasis were remarkably reduced and the therapeutic effect of psoriasis was excellent.
실시예 2. 건선 마우스의 Erythema scoring 결과Example 2. Erythema scoring results in psoriasis mice
도 2는 본 발명의 펩타이드 처리에 따른 홍반(Erythema)의 감소 정도를 스코어링한 결과를 나타낸 것이다. 도 2에 나타난 바와 같이, 건선이 유발될수록 Erythema score가 높아지는 PBS 처리군(대조군)과 비교하여 본 발명에 따른 펩타이드를 처리한 마우스의 스코어가 MTX 처리군(양성 대조군)의 수준으로 떨어지는 것을 확인하였다. 특히, 본 발명에 따른 펩타이드를 10 ug/kg으로 처리한 그룹의 Erythema score는 실험 초반(Day 1과 Day 2)에는 MTX 보다 훨씬 낮은 수치를 기록했으나, 실험 후반(Day 3과 Day4)에는 MTX 혹은 펩타이드 0.1 ug/kg을 처리한 그룹과 비슷한 수준의 Erythema를 보이는 것으로 확인되었다. 즉, 본 발명에 따른 펩타이드 처리에 의해, PASI score중 하나의 척도인 Erythema 정도가 양성 대조군인 MTX 처리군과 비슷하거나 더 낮은 수준으로 떨어지는 바, 건선의 치료 효과가 매우 우수함을 확인하였다. Figure 2 shows the results of scoring the degree of reduction of erythema (Erythema) according to the peptide treatment of the present invention. As shown in Figure 2, the score of the mice treated with the peptide according to the present invention compared to the PBS-treated group (control group), in which the Erythema score increases as psoriasis is induced, it was confirmed that the score of the MTX-treated group (positive control group) fell to the level of the MTX-treated group (positive control group). . In particular, the Erythema score of the group treated with the peptide according to the present invention at 10 ug/kg was much lower than that of MTX at the beginning of the experiment (Day 1 and Day 2), but at the end of the experiment (Day 3 and Day 4), MTX or It was confirmed that the Erythema level was similar to that of the group treated with the peptide 0.1 ug/kg. That is, by the peptide treatment according to the present invention, the degree of Erythema, which is one measure of the PASI score, fell to a level similar to or lower than that of the MTX-treated group, which is a positive control, and it was confirmed that the treatment effect for psoriasis was very excellent.
실시예 3. 건선 마우스 표피 두께 측정 결과Example 3. Psoriasis mouse epidermal thickness measurement result
도 3은 본 발명의 펩타이드 처리에 따른 표피의 두께를 측정한 결과를 나타낸 것이다. 도 3에 나타난 바와 같이, 실제 조직에서 건선 마우스의 표피 두께를 측정한 결과, 본 발명에 따른 펩타이드를 처리할 경우, 양성 대조군인 MTX 처리군과 비슷하거나 정상 수준까지 두께가 줄어드는 것으로 확인되었다. 즉, 본 발명에 따른 펩타이드 처리에 의해 건선에 의해 증가한 표피층의 두께가 현저하게 줄어드는바, 건선의 치료 효과가 우수함을 확인하였다. 3 shows the results of measuring the thickness of the epidermis according to the peptide treatment of the present invention. As shown in FIG. 3 , as a result of measuring the epidermal thickness of psoriasis mice in actual tissues, it was confirmed that when the peptide according to the present invention was treated, the thickness was similar to that of the MTX-treated group, which is a positive control, or decreased to a normal level. That is, since the thickness of the epidermal layer increased by psoriasis was significantly reduced by the peptide treatment according to the present invention, it was confirmed that the treatment effect for psoriasis was excellent.
실시예 4. 건선 마우스의 Th17 cells population 변화Example 4. Changes in Th17 cell population in psoriasis mice
건선이 유발되면 Th17 cells의 활성화에 의해 IL-17 분비가 증가하고, 약물 처리에 의해 건선 증상이 완화될수록 IL-17도 함께 감소하는 것은 잘 알려져 있다. 따라서, 본 발명에서는 펩타이드 처리에 의한 Th17 cells 변화를 알아보고자, lymph node내 CD4+/IL-17+ cells을 염색하여 비교하였다. It is well known that when psoriasis is induced, IL-17 secretion increases by activation of Th17 cells, and IL-17 decreases as psoriasis symptoms are alleviated by drug treatment. Therefore, in the present invention, CD4+/IL-17+ cells in the lymph node were stained and compared to investigate the changes in Th17 cells by peptide treatment.
도 4는 본 발명의 펩타이드 처리에 따라 감소하는 Th17 cells population을 측정한 결과를 나타낸 것이다. 도 4에 나타난 바와 같이, PBS 처리에 의해 증가한 Th17 cells population이 본 발명에 따른 펩타이드 처리에 의해 양성 대조군인 MTX 처리군과 유사한 수준으로 감소하는 것으로 확인되었다. 이러한 결과를 통해 본 발명에 따른 펩타이드를 처리할 경우, Th17 cells population이 현저하게 감소하여 건선의 치료 효과가 우수함을 확인하였다. 4 shows the results of measuring the Th17 cell population decreased according to the peptide treatment of the present invention. As shown in FIG. 4 , it was confirmed that the Th17 cell population increased by the PBS treatment was reduced to a level similar to that of the MTX-treated group, which is a positive control, by the peptide treatment according to the present invention. Through these results, it was confirmed that when the peptide according to the present invention was treated, the Th17 cell population was remarkably decreased and the treatment effect of psoriasis was excellent.
이상으로 본 발명 내용의 특정한 부분을 상세히 기술하였는 바, 당업계의 통상의 지식을 가진 자에게 있어서 이러한 구체적 기술은 단지 바람직한 실시태양일 뿐이며, 이에 의해 본 발명의 범위가 제한되는 것이 아닌 점은 명백할 것이다. 따라서, 본 발명의 실질적인 범위는 첨부된 청구항들과 그것들의 등가물에 의하여 정의된다고 할 것이다.As described above in detail a specific part of the content of the present invention, for those of ordinary skill in the art, this specific description is only a preferred embodiment, and it is clear that the scope of the present invention is not limited thereby. something to do. Accordingly, it is intended that the substantial scope of the present invention be defined by the appended claims and their equivalents.
본 발명은 염증성 피부질환 치료용 펩타이드 및 이의 용도에 관한 것으로, 구체적으로 본 발명에 따른 신규 합성 펩타이드는 9개의 아미노산으로 구성된 매우 작은 규모의 펩타이드로 저분자이며 생분해성이기 때문에 과민반응 유발 가능성이 적어 외부 물질의 투여에 따른 부작용을 최소화시키므로, 염증성 피부질환 예방, 치료 또는 개선용 조성물의 유효성분으로 유용하게 이용할 수 있다.The present invention relates to a peptide for the treatment of inflammatory skin disease and its use. Specifically, the novel synthetic peptide according to the present invention is a very small-scale peptide consisting of 9 amino acids, low molecular weight and biodegradable, so there is little possibility of inducing hypersensitivity reaction to foreign substances Since it minimizes the side effects of administration, it can be usefully used as an active ingredient in a composition for preventing, treating or improving inflammatory skin diseases.

Claims (9)

  1. 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드.Consisting of amino acids represented by SEQ ID NO: 1, a peptide.
  2. 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 치료용 약학적 조성물.A pharmaceutical composition for preventing or treating inflammatory skin diseases, comprising as an active ingredient a peptide or a polynucleotide encoding the same, consisting of amino acids represented by SEQ ID NO: 1.
  3. 제2항에 있어서,3. The method of claim 2,
    상기 염증성 피부질환은 건선(psoriasis), 과각화증(hyperkeratosis), 어린선(ichthyosis), 아토피 피부염(atopic dermatitis), 모공성각화증(keratosis pilaris), 광선각화증(actinic keratoses), 지루각화증(seborrheic keratoses), 천포창(pemphigus), 티눈(corns), 사마귀(warts), 및 편평태선(lichen planus)으로 이루어진 군에서 선택되는 것을 특징으로 하는, 약학적 조성물.The inflammatory skin disease is psoriasis, hyperkeratosis, ichthyosis, atopic dermatitis, keratosis pilaris, actinic keratoses, seborrheic keratoses, A pharmaceutical composition, characterized in that it is selected from the group consisting of pemphigus, corns, warts, and lichen planus.
  4. 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드를 유효성분으로 포함하는, 염증성 피부질환 예방 또는 개선용 화장품 조성물.A cosmetic composition for preventing or improving inflammatory skin disease, comprising a peptide as an active ingredient, which consists of an amino acid represented by SEQ ID NO: 1.
  5. 제4항에 있어서, 5. The method of claim 4,
    상기 조성물은 현탁액, 유탁액, 페이스트, 겔, 크림, 로션, 파우더, 왁스 또는 스프레이 형태임을 특징으로 하는, 화장품 조성물.The composition is a suspension, emulsion, paste, gel, cream, lotion, powder, wax or spray form, characterized in that the cosmetic composition.
  6. 약학적으로 유효한 양의 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 개체에 투여하는 단계를 포함하는 염증성 피부질환 예방 또는 개선방법.A method for preventing or improving inflammatory skin disease comprising administering to an individual a pharmaceutically effective amount of an amino acid represented by SEQ ID NO: 1, a peptide or a polynucleotide encoding the same.
  7. 약학적으로 유효한 양의 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드를 개체에 투여하는 단계를 포함하는 염증성 피부질환 치료방법.A method for treating inflammatory skin disease comprising administering to an individual a pharmaceutically effective amount of a peptide or a polynucleotide encoding the same, consisting of the amino acid represented by SEQ ID NO: 1.
  8. 염증성 피부질환 예방 또는 치료용 약학적 조성물로 사용하기 위한 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드 또는 이를 코딩하는 폴리뉴클레오티드의 용도.Use of a peptide or a polynucleotide encoding the amino acid represented by SEQ ID NO: 1 for use as a pharmaceutical composition for preventing or treating inflammatory skin diseases.
  9. 염증성 피부질환 예방 또는 개선용 화장품 조성물로 사용하기 위한 서열번호 1로 표시되는 아미노산으로 이루어진, 펩타이드의 용도.Use of a peptide comprising the amino acid represented by SEQ ID NO: 1 for use as a cosmetic composition for preventing or improving inflammatory skin disease.
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KR20140005110A (en) * 2012-07-03 2014-01-14 일양약품주식회사 Novel peptides and use thereof
KR101897122B1 (en) * 2016-03-09 2018-09-10 주식회사 바이오펩 Peptides for treating inflammatory diseases and use thereof
KR20200057592A (en) * 2018-11-16 2020-05-26 주식회사 카인사이언스 Peptide for treating inflammatory skin diseases and use thereof

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