Fusion protein comprising CD300c protein or fragment thereof and immunoglobulin fc region and use thereof for improving skin condition

Abstract

The present invention relates to: a cosmetic composition for improving skin condition, a cosmetic composition for strengthening skin barrier, a pharmaceutical composition for preventing or treating atopic dermatitis, and a quasi-drug composition for preventing or ameliorating atopic dermatitis, each composition comprising, as active ingredient, a fusion protein comprising a CD300c protein or a fragment thereof and an immunoglobulin Fc region; and a method for treating atopic dermatitis by using the pharmaceutical composition. The compositions of the present invention have excellent effects of alleviating skin inflammation, reducing dead skin cells, strengthening skin barrier, alleviating xeroderma, and alleviating atopic dermatitis, and thus can be used as an effective therapeutic agent for atopic dermatitis.

Description

A fusion protein comprising CD300C protein or a fragment thereof and an immunoglobulin FC region and its use for improving skin condition

[0001] The present invention relates to a cosmetic, quasi-drug, and pharmaceutical composition comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region, and a method for treating atopic dermatitis using the same.

[0002] The skin is the primary organ that functions as a physical barrier protecting the body against various environmental factors. Meanwhile, dry skin, considered one of the major diseases of modern society, is a symptom caused by dysfunction of the skin barrier. Recently, it has been on the rise due to various factors such as environmental pollution, the increase in dry environments like apartments and high-rise buildings, rising social stress, and skin aging; consequently, cases requiring treatment due to severe symptoms are also continuously increasing.

[0003] Atopic dermatitis, one of the various skin diseases, is known to be fundamentally caused by abnormalities in skin barrier function. Atopic dermatitis is a chronic and recurrent skin disease that generally occurs in infants and children, characterized by symptoms such as severe itching and skin eczema that worsen, improve, and recur due to various causes. The incidence of atopic dermatitis continues to increase in industrialized countries, reaching 1-5% in adults and 10-20% in infants and children, leading to active research worldwide to address this issue.

[0004] Accordingly, the development of various formulations exhibiting therapeutic effects for atopic dermatitis is underway (Korean Patent Publication 10-2016-0137035 A, etc.). In particular, active research is being conducted on non-steroidal and steroidal substances with excellent anti-inflammatory effects; however, due to various reasons such as high manufacturing costs, the therapeutic effect of atopic dermatitis being one-time, unverified side effects, or serious side effects, atopic dermatitis treatments are not being commercialized.

[0005] The problem to be solved by the present application is to provide a cosmetic composition for improving skin condition, comprising as an active ingredient a fusion protein including a CD300c protein or a fragment thereof and an immunoglobulin Fc region; a cosmetic composition for strengthening the skin barrier; a pharmaceutical composition for preventing or treating atopic dermatitis; a quasi-drug composition for preventing or improving atopic dermatitis; and a method for treating atopic dermatitis using the said pharmaceutical composition.

[0006]

[0007] One object of the present invention is to provide a cosmetic composition for improving skin condition, comprising as an active ingredient a fusion protein comprising a CD300c protein or a fragment thereof; and an immunoglobulin Fc region.

[0008] Another objective of the present invention is to provide a cosmetic composition for strengthening the skin barrier, comprising as an active ingredient a fusion protein comprising a CD300c protein or a fragment thereof; and an immunoglobulin Fc region.

[0009] Another objective of the present invention is to provide a pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0010] Another objective of the present invention is to provide a method for treating atopic dermatitis, comprising the step of administering the pharmaceutical composition to an individual suspected of having atopic dermatitis.

[0011] Another objective of the present invention is to provide a quasi-drug composition for preventing or improving atopic dermatitis, comprising as an active ingredient a fusion protein comprising a CD300c protein or a fragment thereof; and an immunoglobulin Fc region.

[0012]

[0013] A composition comprising the CD300c protein or a fragment thereof and a fusion protein comprising an immunoglobulin Fc region as an active ingredient has excellent effects in alleviating skin inflammation, improving skin exfoliation, strengthening the skin barrier, alleviating skin dryness, and alleviating atopic dermatitis, and is highly useful as a cosmetic composition, pharmaceutical composition, and quasi-drug composition that is safe for the skin and has excellent effects in improving skin condition. In particular, since it has been confirmed that the fusion protein of the present invention exhibits effective preventive or therapeutic effects on atopic symptoms, it can be widely used as a preventive or therapeutic agent for atopic dermatitis.

[0014]

[0015] FIG. 1 is a figure showing the structure of a CD300c-Ig fusion protein according to one embodiment of the present invention.

[0016] Figure 2 is a figure showing the SDS-PAGE electrophoresis results of a CD300c-Ig fusion protein according to one embodiment of the present invention.

[0017] FIG. 3 is a figure confirming the immunosuppressive effect of a CD300c-Ig fusion protein in a mouse macrophage (Raw264.7) according to one embodiment of the present invention.

[0018] Figure 4 is a figure confirming the immunosuppressive effect of a CD300c-Ig fusion protein in human monocytes (THP-1) according to one embodiment of the present invention.

[0019] Figure 5 is a figure showing the results of measuring the secretion amounts of IL-8 and IL-1b, respectively, in order to confirm the ability of the CD300c-Ig fusion protein to regulate human skin keratinocyte activity according to one embodiment of the present invention.

[0020]

[0021] This is explained in detail as follows. Meanwhile, each description and embodiment disclosed in the present invention may be applied to other descriptions and embodiments. That is, all combinations of the various elements disclosed in the present invention fall within the scope of the present invention. Furthermore, the scope of the present invention should not be considered limited by the specific descriptions provided below. Additionally, numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated by reference into this specification in their entirety to more clearly explain the level of the art to which the present invention pertains and the content of the present invention.

[0022]

[0023] Against the aforementioned background, the inventors have made diligent research efforts to develop a formulation capable of safely and more effectively strengthening the skin barrier or treating atopic dermatitis. As a result, they have confirmed the excellent ability to improve skin condition and atopic dermatitis of a fusion protein comprising the CD300c protein or a fragment thereof and the immunoglobulin Fc region, and have completed the present invention.

[0024]

[0025] One aspect of the present invention provides a cosmetic composition for improving skin condition comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0026] In the present invention, the term "CD300c" refers to a type of CD300 family cell surface protein involved in immune regulation, also named CLM-6 (CMRF35-like molecule 6). CD300c is encoded by the CD300c gene and may be expressed in antigen-presenting cells, cancer cells, or immune cells, but is not limited thereto.

[0027] In the present invention, the term "fragment" of a protein refers to a fragment from a full-length protein from which some amino acid residues have been deleted, and which is an analogue that retains the intrinsic activity and function of the said protein. For the purposes of the present invention, a fragment of CD300c refers to a fragment from the full-length protein of CD300c from which some amino acid residues have been deleted, and which may refer to a functional partial fragment in which the activity and function of CD300c are preserved.

[0028] In one embodiment, the CD300c protein of the present invention may be human CD300c or mouse CD300c, but is not limited thereto.

[0029]

[0030] Specifically, the fragment of CD300c may include the full length or a portion of the CD300c protein, and specifically may consist of or include the extracellular domain of the CD300c protein.

[0031] In one embodiment, the extracellular domain of CD300c of the present invention may comprise the amino acid sequence of SEQ ID NO. 4, 10, 16 or 22 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO. 4, 10, 16 or 22.

[0032] In one embodiment, the extracellular domain of CD300c of the present invention may be encoded by the polynucleotide sequence of SEQ ID NO. 3, 9, 15 or 21 or a polynucleotide sequence having 90% or more sequence identity with the polynucleotide sequence of SEQ ID NO. 3, 9, 15 or 21, but is not limited thereto.

[0033] In one embodiment, the CD300c protein or fragment thereof of the present invention may comprise a sequence of a natural form derived from humans, or may be an analog in which one or more amino acid residues are substituted, added, deleted, or modified compared to the natural form and retain the function of CD300c, but is not limited thereto.

[0034]

[0035] Specifically, the immunoglobulin Fc region may be selected from the group consisting of (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of one or more domains among the CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain with an immunoglobulin hinge region or a part of a hinge region; and (f) a dimer of each domain of the heavy chain constant region and the light chain constant region. In the present invention, the immunoglobulin Fc region may also be interchangeably referred to as "Fc," "Fc region," and "Fc site," etc.

[0036] In one embodiment, the immunoglobulin Fc region is characterized by undergoing a modification selected from the group consisting of substitution, addition, deletion, modification, and combinations thereof at least one amino acid in the natural immunoglobulin Fc region.

[0037] In one embodiment, the immunoglobulin Fc region may have a site capable of forming disulfide bonds removed, some amino acids at the N-terminus of the natural form Fc removed, a methionine residue added to the N-terminus of the natural form Fc, a complement binding site removed, or an ADCC (antibody dependent cell mediated cytotoxicity) site removed.

[0038] As an example of implementation, the immunoglobulin Fc region may be non-glycolytic.

[0039] In one embodiment, the immunoglobulin Fc region may be derived from IgG, IgA, IgD, IgE, or IgM. In one example, the immunoglobulin Fc region may be derived from IgG4.

[0040] As another example of implementation, the immunoglobulin Fc region may be a hybrid of domains of different origin derived from immunoglobulins selected from the group consisting of IgG, IgA, IgD, IgE, and IgM.

[0041] In one embodiment, the immunoglobulin Fc may comprise the amino acid sequence of SEQ ID NO. 28 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO. 28, but is not limited thereto.

[0042]

[0043] The fusion protein of the present invention comprises a CD300c protein or a fragment thereof; and an immunoglobulin Fc region, and may be a protein artificially synthesized such that the CD300c protein or a fragment thereof; and the immunoglobulin Fc region are combined, but is not limited thereto. In this specification, the fusion protein of the present invention may also be referred to by names such as "CD300c-Fc" or "CD300c-Ig".

[0044] The fusion protein of the present invention may comprise, but is not limited to, a CD300c protein or a fragment thereof; and an immunoglobulin Fc region directly linked, linked via a linker, or additionally include other protein moiety. The linking method and linking location of the fusion protein of the present invention may utilize methods and locations practiced in the art without limitation, provided that such modification does not alter the structure or activity of the linked protein.

[0045] Specifically, the direct connection may be a covalent connection, but is not limited thereto.

[0046] Specifically, the linker may be a peptide linker or a non-peptide linker, but is not limited thereto.

[0047] For example, the peptide linker may include one or more amino acids, for instance, from 1 to 1000, specifically from 1 to 100, more specifically from 1 to 50 amino acids, but is not particularly limited thereto. Any peptide linker known in the art may be included, for example, [GS]x linker, [GGGS]x linker, and [GGGGS]x linker, etc., where x may be a natural number greater than or equal to 1 (e.g., 1, 2, 3, 4, 5, or more).

[0048] The above-mentioned nonpeptide linker is not limited to any type as long as it can link the CD300c protein or a fragment thereof and the immunoglobulin Fc region. For example, the above-mentioned nonpeptide linker may be selected from lipid polymers, biodegradable polymers, chitins, and oligonucleotides, but is not limited thereto.

[0049] For example, the above linker may include the amino acid of SEQ ID NO. 30, but is not limited thereto.

[0050] The fusion protein of the present invention may be prepared by methods such as conjugating a substance capable of increasing the half-life of the CD300c protein or a fragment thereof, the immunoglobulin Fc region, or a combination thereof, or by introducing a mutation to prevent degradation in the body, and any method known in the art that can act on the protein and increase persistence is included in the scope of the present invention. The substance capable of increasing the half-life may be selected from the group consisting of, but is not limited to, polymers, fatty acids, cholesterol, albumin and fragments thereof, albumin conjugates, antibodies, antibody fragments, FcRn conjugates, in vivo connective tissue, nucleotides, fibronectin, transferrin, saccharides, heparin, and elastin.

[0051] In one embodiment, the fusion protein of the present invention may comprise the amino acid sequence of SEQ ID NO. 6, 12, 18, or 24 or an amino acid sequence having 90% or more sequence identity with the amino acid sequence of SEQ ID NO. 6, 12, 18, or 24, but is not limited thereto.

[0052] In one embodiment, the fusion protein of the present invention may be encoded by the polynucleotide sequence of SEQ ID NO. 5, 11, 17 or 23 or by an amino acid sequence having 90% or more sequence identity with the polynucleotide sequence of SEQ ID NO. 5, 11, 17 or 23, but is not limited thereto.

[0053] As an example of one embodiment, the fusion protein of the present invention may have the structure of FIG. 1, but is not limited thereto.

[0054] In one embodiment, the fusion protein of the present invention may be included in an amount of 0.00001% to 99.99% by weight based on the total weight of the cosmetic composition, specifically 0.1% to 90% by weight, more specifically 0.1% to 70% by weight, and even more specifically 0.1% to 50% by weight, but is not limited thereto and may be varied in many ways, and if necessary, may also be included as the total content of the cosmetic composition.

[0055]

[0056] The present invention has one major technical significance in that it has identified that the fusion protein has an excellent skin condition improvement effect.

[0057] In the present invention, the term "improvement of skin condition" refers to any act that alleviates the skin condition or at least reduces parameters related to skin treatment, such as the degree of skin-related symptoms. Specifically, it may be one or more selected from the group consisting of alleviation of skin inflammation, improvement of skin exfoliation, strengthening of the skin barrier, alleviation of skin dryness, and alleviation of atopic dermatitis, but is not limited thereto.

[0058] The above improvement in skin condition may be achieved through an increase in stratum corneum structural proteins, inhibition of inflammatory responses, strengthening of skin barrier function, and regulation of immune responses, but is not limited thereto.

[0059] Specifically, the inventors confirmed that a fusion protein comprising the CD300c protein or a fragment thereof and an immunoglobulin Fc region possesses excellent skin condition improvement activity. More specifically, in one embodiment of the present invention, it was confirmed that the fusion protein comprising the CD300c protein or a fragment thereof and an immunoglobulin Fc region exhibits excellent effects such as alleviating skin inflammation, strengthening the skin barrier, improving skin exfoliation, alleviating atopic dermatitis, and regulating immune responses.

[0060]

[0061] The term "inflammatory response" in this invention refers to a type of in vivo response to damage or infection of a specific tissue, and the main mediator is immune cells. In response to various external stimuli, the skin produces inflammatory substances such as nitric oxide (NO) and pro-inflammatory cytokines, including interleukin-1 beta (IL-1β), interleukin-6 (IL-6), and tumor necrosis factor-alpha (TNF-α), due to excessive immune cell activity such as dryness, itching, allergic skin diseases, and atopic dermatitis.

[0062] The term "immune response regulation" in this invention refers to the regulation of a series of reactions triggered by immune cells against foreign and endogenous foreign substances (antigens). It is known that damage to the skin barrier and abnormal immune response are involved in skin barrier dysfunction and the process of inducing itching.

[0063] In one embodiment of the present invention, effects such as inhibiting the increase of TNF-α, regulating the activity of immune cells, and inducing a degranulation reaction were confirmed, thereby confirming that the fusion protein of the present invention possesses excellent activity in alleviating skin inflammation and regulating immune responses.

[0064]

[0065] In the present invention, the term "skin barrier" refers to the epidermis of the stratum corneum, which is the outermost layer of the skin. The stratum corneum is about 10 to 20 μm thick and plays an important role in preventing moisture loss within the body and blocking the intrusion of external harmful substances. During the stage of keratinization, where keratinocytes differentiate in the stratum corneum, structural proteins important for keratinocyte formation, such as filaggrin, involucrin, loricrin, and envoflakin, are important elements that can strengthen the skin barrier function by providing physical rigidity.

[0066] In this invention, the term "keratin" refers to a major structural substance that constitutes the epithelial cells of the skin, hair, and nails. It enhances the cells' ability to withstand trauma and is a foreign substance produced during the process of cells dying and sloughing off from the epidermal layer of the skin; keratin increases in cases of psoriasis, warts, acne, dandruff, calluses, keratosis, seborrheic dermatitis, etc.

[0067] In the present invention, the term "skin dryness" refers to a condition in which skin moisture is insufficient. Skin dryness can affect atopic dermatitis, dry skin, itching, exacerbation of keratinization, and skin irritation.

[0068] In one embodiment of the present invention, it was confirmed that the fusion protein of the present invention has the ability to regulate activity on skin keratinocytes and has the ability to inhibit the phosphorylation of Stat 6 of skin keratinocytes, and thus has an effect on improving skin keratinization and skin dryness.

[0069]

[0070] In the present invention, the term "atopic dermatitis" refers to a chronic and recurrent inflammatory skin disease that begins primarily in infancy or childhood, and is an allergic immune disease characterized by an increase in Th2 cells, mast cells, and basophils in skin lesions, accompanied by symptoms of dry skin, pruritus (itching), and characteristic eczema. In the present invention, the term atopic dermatitis may be referred to interchangeably with "AD." "Xeroderma" refers to a skin condition in which discomfort may be felt due to dryness, or a condition in which the moisture in the skin is deficient to less than 10% of normal levels; clinically, it refers to a skin condition with a rough surface that exhibits scales and has slight red spots and fissures. "Pruritus" refers to an unpleasant sensation that triggers an urge to scratch or rub the skin; it is a highly subjective sensation that varies greatly depending on the body part or individual, and the causes are also diverse.

[0071] The cause of atopic dermatitis is not yet clearly known, and since clinical symptoms such as dry skin and eczema appear in various ways, it is difficult to specify the cause of the disease; however, it is suspected that environmental factors, genetic predisposition, immunological reactions, and abnormalities in the skin barrier are complex causes. In one embodiment of the present invention, the effect of the fusion protein of the present invention on alleviating atopic dermatitis in an atopic mouse model was directly confirmed.

[0072]

[0073] Since the "cosmetic composition" of the present invention is basically applied to the skin, it can be prepared in any formulation conventionally manufactured by referring to cosmetic compositions in the art. For example, it can be prepared in a formulation selected from the group consisting of solutions, topical ointments, creams, foams, nourishing lotions, softening lotions, softening waters, packs, emulsions, makeup bases, foundations, essences, soaps, liquid cleansers, bath additives, sunscreens, creams, sun oils, suspensions, emulsions, pastes, gels, lotions, powders, surfactant-containing cleansing products, oils, powder foundations, emulsion foundations, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, patches, and sprays, but is not limited thereto.

[0074] In addition to the fusion protein of the present invention, the above cosmetic composition may further include conventional auxiliary agents and carriers such as antioxidants, stabilizers, solubilizers, vitamins, pigments, fragrances, etc., which are commonly used in cosmetic compositions. For example, the above cosmetic composition may further include auxiliary ingredients such as glycerin, butylene glycol, polyoxyethylene hydrogenated castor oil, tocopheryl acetate, citric acid, panthenol, squalane, sodium citrate, and allantoin.

[0075] The above cosmetic composition may be formulated as a softening lotion, astringent lotion, nourishing lotion, nourishing cream, massage cream, essence, pack, skin adhesive patch, skin adhesive gel, powder, ointment, paste, gel, suspension, emulsion, spray, cosmetic liquid, or capsule, but is not particularly limited thereto.

[0076] The cosmetic composition of the present invention may additionally include one or more cosmetically acceptable carriers that are incorporated into general skin cosmetics, and may appropriately incorporate, for example, oils, water, surfactants, moisturizers, lower alcohols, thickeners, chelating agents, colorants, preservatives, fragrances, etc., as conventional ingredients, but is not limited thereto. The cosmetically acceptable carriers included in the cosmetic composition of the present invention vary depending on the formulation.

[0077] In the case where the formulation of the present invention is an ointment, paste, cream, or gel, animal oil, vegetable oil, wax, paraffin, starch, tracanth, cellulose derivative, polyethylene glycol, silicone, bentonite, silica, talc, zinc oxide, or a mixture thereof may be used as a carrier component.

[0078] In the case where the formulation of the present invention is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate, polyamide powder, or a mixture thereof may be used as a carrier component, and in particular in the case of a spray, it may additionally include a propellant such as chlorofluorohydrocarbon, propane / butane, or dimethyl ether.

[0079] When the formulation of the present invention is a solution or an emulsion, a solvent, a solubilizing agent, or an emulsifying agent is used as a carrier component, such as water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, and in particular, cottonseed oil, peanut oil, corn germ oil, olive oil, castor oil and sesame oil, glycerol aliphatic ester, polyethylene glycol or fatty acid ester of sorbitan may be used.

[0080] In the case where the formulation of the present invention is a suspension, liquid diluents such as water, ethanol, or propylene glycol, ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, and polyoxyethylene sorbitan ester, microcrystalline cellulose, aluminum metahydroxide, bentonite, agar, or tracant, etc. may be used as carrier components.

[0081] Meanwhile, when the formulation of the present invention is a capsule, it may be formulated in the form of an alginate capsule, an agar capsule, a gelatin capsule, a wax capsule, or a double capsule, but is not particularly limited thereto.

[0082]

[0083] One aspect of the present invention provides a cosmetic composition for strengthening the skin barrier comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0084] The above "CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region," "skin barrier strengthening," and "cosmetic composition" are defined in the same manner as previously described.

[0085]

[0086] One aspect of the present invention provides a pharmaceutical composition for the prevention or treatment of atopic dermatitis, comprising as an active ingredient a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0087] The above "CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region" and "atopic dermatitis" are defined as previously stated.

[0088] The term "prevention" in this invention refers to any act of suppressing or delaying atopic dermatitis by administering a composition containing the fusion protein.

[0089] The term "treatment" in this invention refers to any act in which the symptoms of atopic dermatitis are improved or beneficially altered by the administration of a composition containing the fusion protein.

[0090] In one embodiment of the present invention, it was confirmed that a fusion protein comprising the CD300c protein or a fragment thereof and an immunoglobulin Fc region of the present invention inhibits immune cell activity, inhibits the phosphorylation of Stat 6 in skin keratinocytes, and reduces the secretion of inflammatory cytokines. Most importantly, based on this, when the fusion protein of the present invention was administered to an atopic mouse model, the alleviation of atopic symptoms and the reduction of inflammation were confirmed (Example 4), suggesting that the pharmaceutical composition of the present invention can be utilized as an effective treatment for atopic dermatitis.

[0091] In one embodiment, the fusion protein of the present invention may be included in an amount of 0.00001% to 99.99% by weight based on the total weight of the pharmaceutical composition, specifically 0.1% to 90% by weight, more specifically 0.1% to 70% by weight, and even more specifically 0.1% to 50% by weight, but is not limited thereto and may be varied depending on the condition of the subject to administration, specific symptoms, degree of progression, etc. If necessary, it may also be included as the total content of the pharmaceutical composition.

[0092] The above pharmaceutical composition may further include a pharmaceutically acceptable carrier, excipient, or diluent commonly used in the manufacture of pharmaceutical compositions, and the carrier may include a non-naturally occurring carrier. Examples of the carrier, excipient, and diluent include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil.

[0093] In one embodiment, the pharmaceutical composition may be formulated and used in the form of tablets, pills, powders, granules, capsules, suspensions, liquids, emulsions, syrups, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, transdermal absorption agents, gels, lotions, ointments, creams, patches, cataplasmas, pastes, sprays, skin emulsions, skin suspensions, transdermal delivery patches, drug-containing bandages, or suppositories, respectively, according to conventional methods. Specifically, when formulating, it may be prepared using diluents or excipients such as fillers, weighting agents, binders, humectants, disintegrants, and surfactants that are commonly used. Solid dosage forms for oral administration include, but are not limited to, tablets, pills, powders, granules, and capsules. These solid dosage forms may be prepared by mixing at least one excipient, for example, starch, calcium carbonate, sucrose, lactose, gelatin, etc. In addition, lubricants such as magnesium stearate and talc may be used in addition to simple excipients. In addition to liquids and liquid paraffin for oral administration, various excipients such as humectants, sweeteners, flavorings, and preservatives may be added. Preparations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate may be used as non-aqueous solvents and suspensions. Witepsol, Macrogol, Tween 61, cocoa dough, laurin dough, glycerogelatin, etc. may be used as bases for suppositories.

[0094]

[0095] One aspect of the present invention provides a method for treating atopic dermatitis comprising the step of administering the pharmaceutical composition to an individual suspected of having atopic dermatitis.

[0096] The above "pharmaceutical composition," "atopic dermatitis," and "treatment" are defined in the same way as previously stated.

[0097] The term "administration" in the present invention refers to the act of introducing a pharmaceutical composition containing the fusion protein to an individual by an appropriate method.

[0098] The term "individual" in this invention refers to all animals, including humans, rats, mice, and livestock, that have developed or may develop atopic dermatitis. Specific examples may include mammals, including humans, or individuals excluding humans.

[0099] The pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceuticalally effective amount" means an amount sufficient to treat a disease with a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level may be determined based on factors including individual type and severity, age, sex, drug activity, sensitivity to the drug, time of administration, route of administration and elimination rate, duration of treatment, concurrently used drugs, and other factors well known in the medical field. For example, the pharmaceutical composition may be administered at a dose of 0.01 to 500 mg / kg per day, but is not limited thereto, and the administration may be divided into once or several doses per day. The pharmaceutical composition may be administered as an individual therapeutic agent or in combination with other therapeutic agents, additional agents, or adjuvants, and may be administered sequentially or simultaneously with conventional therapeutic agents. Furthermore, the pharmaceutical composition may be administered as a single or multiple doses. It is important to administer an amount that obtains maximum effect with a minimum amount without side effects, taking all of the above factors into consideration, and this can be easily determined by a person skilled in the art.

[0100] In one embodiment, the composition may be administered in combination with one or more agents selected from the group consisting of interleukin inhibitors and Janus kinase (JAK) inhibitors, but is not limited thereto.

[0101] In addition, the above pharmaceutical composition may be administered orally or parenterally (e.g., intravenously, subcutaneously, intraperitoneally, or topically) depending on the intended method, and the dosage may be appropriately selected by a person skilled in the art, although it varies depending on the patient's condition and body weight, the severity of the disease, the form of the drug, the route of administration, and the time.

[0102]

[0103] Another embodiment of the present invention provides a quasi-drug composition for preventing or improving atopic dermatitis, comprising as an active ingredient a fusion protein comprising a CD300c protein or a fragment thereof and an immunoglobulin Fc region.

[0104] The above "CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region", "atopic dermatitis", and "prevention" are defined in the same way as previously described.

[0105] The term "improvement" in the present invention refers to any act that at least reduces parameters related to the condition treated by the administration of the pharmaceutical composition, such as the degree of symptoms.

[0106] The term "quasi-drug" in the present invention may be selected from the group consisting of, but is not limited to, body cleansers, disinfectants, detergents, kitchen detergents, cleaning detergents, toothpaste, mouthwash, wet wipes, detergents, soaps, hand washes, hair cleansers, hair softeners, humidifier fillers, masks, ointments, and filter fillers.

[0107] The quasi-drug composition of the present invention may further include, in addition to the above components, a pharmaceutically acceptable carrier, excipient, or diluent as needed. The pharmaceutically acceptable carrier, excipient, or diluent is not limited as long as it does not impair the effects of the present invention, and may include, for example, fillers, extenders, binders, wetting agents, disintegrants, surfactants, lubricants, sweeteners, fragrances, preservatives, etc.

[0108] Representative examples of pharmaceutically acceptable carriers, excipients, or diluents of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, maltitol, starch, gelatin, glycerin, acacia gum, alginate, calcium phosphate, calcium carbonate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, propylene glycol, polyethylene glycol, vegetable oil, injectable esters, Witepsol, macrogol, Tween 61, cocoa dough, laurize, etc.

[0109] When the fusion protein of the present invention is used as a quasi-drug, it may additionally contain one or more active ingredients exhibiting the same or similar functions. For example, it may include known ingredients for promoting beneficial skin bacteria, inhibiting harmful skin bacteria, soothing the skin, improving skin inflammation, whitening the skin, regenerating the skin, and healing wounds. If additional ingredients for wrinkle improvement, skin whitening, improvement of skin troubles, and skin moisturization are included, the wrinkle improvement, elasticity improvement, skin moisturization, and anti-aging effects of the composition of the present invention may be further enhanced. When adding the above ingredients, skin safety due to combined use, ease of formulation, and stability of the active ingredients may be considered.

[0110] The quasi-drug composition of the present invention may additionally include one or more ingredients selected from the group consisting of skin anti-aging ingredients known in the art, such as retinoic acid, TGF, protein derived from animal placenta, betulinic acid, and Chlorella extract; non-steroidal anti-inflammatory ingredients known in the art, such as flufenamic acid, ibuprofen, benzydamine, indomethacin, prednisolone, dexamethasone, allantoin, azuene, and hydrocortisone; and derivatives thereof and various plant extracts. The additional ingredients may be included in an amount of 0.0001% to 10% by weight based on the total weight of the composition, and the said content range may be adjusted according to requirements such as skin safety and ease of formulation of the fusion protein of the present invention.

[0111] The formulation method, dosage, method of use, components, etc. of quasi-drugs can be appropriately selected from the ordinary technology known in the technical field.

[0112]

[0113] Another embodiment of the present invention provides a use for improving skin condition of a composition comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0114] Another embodiment of the present invention provides a skin barrier strengthening use of a composition comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0115] Another embodiment of the present invention provides a composition for the prevention or treatment of atopic dermatitis comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

[0116] At this time, the "CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region," "skin barrier strengthening," "improvement of skin condition," "atopic dermatitis," "treatment," and "prevention" are defined in the same way as described above.

[0117] The present invention will be explained in more detail below through examples. These examples are intended to explain the invention more specifically, and the scope of the invention is not limited by these examples.

[0118]

[0119] Example 1: Fabrication of CD300c-Ig

[0120] Example 1.1. Preparation of CD300c-Ig expression vector

[0121] To evaluate the effect of CD300c administration on the treatment of atopic dermatitis, a vector was constructed by combining the extracellular domain of CD300c with the structure of immunoglobulin (Ig). More specifically, the extracellular domain sequences were separated to maintain the function of CD300c. A total of four types of CD300c extracellular domain sequences (Human CD300c ECD (SEQ No. 4), Mouse CD300c A2 ECD (SEQ No. 10), Mouse CD300c D3 ECD (SEQ No. 16), Mouse CD300c AA ECD (SEQ No. 22)) and the Fc domain of immunoglobulin (SEQ No. 27) were combined via a linker (SEQ No. 30) to construct a vector. The gene sequences were produced through oligonucleotide synthesis and bound to the pcDNA 3.1 vector (source: Invitrogen). Through this, a vector expressing CD300c-Ig was constructed, and an exemplary structure of Fig. 1 was shown.

[0122]

[0123] Example 1.2 Preparation and Purification of CD300c-Ig

[0124] After transfecting HEK293 cells with the vector prepared in Example 1.1, the vector for expressing the atopic dermatitis treatment was mixed with PEI (polyethylenimine) at a mass ratio of 1:1 and transfected into 293T cells to induce expression. On day 8, the culture medium was centrifuged to remove the cells, and the culture medium was obtained. The obtained culture medium was filtered and then resuspended using a solution containing a mixture of 0.1 M NaH2PO4 and 0.1 M Na2HPO4 (pH 7.0). The resuspended solution was then purified using affinity chromatography with protein A beads (source: GE healthcare) and finally eluted using elution buffer (source: Thermofisher). Subsequently, it was dialyzed with PBS at 4°C.

[0125] To verify the synthesized CD300c-Ig, 5 μg of purified CD300c-Ig was added to both the reducing sample buffer and the non-reducing sample buffer, respectively, followed by electrophoresis using pre-made SDS-PAGE (source: Invitrogen), and then the proteins were stained with Coomassie blue. The results are shown in Figure 2.

[0126]

[0127] Example 2. Confirmation of the immunosuppressive effect of CD300c-Ig

[0128] Example 2.1. Confirmation of the immunosuppressive effect of CD300c-Ig on mouse macrophage (Raw264.7)

[0129] To confirm whether CD300c-Ig produced by the method of Example 1 exhibits an inhibitory effect on mouse macrophage activation, the production of TNF-α (Tumor necrosis factor-α) was determined. More specifically, 1.5 x 10⁶ cells / well of RAW264.7 cells in a 96-well plate were pretreated with 5 μg / mL CD300c-Ig for 24 hours, and 1 ng / mL LPS was added to each well and treated for 24 hours. The production of TNF-α was confirmed using an ELISA kit (mouse TNF-α Quantikine kit, R&D systems). The results are shown in Figure 3.

[0130] As shown in Figure 3, it was confirmed that both CD300c-Fc using Mouse CD300c A2 ECD (SEQ No. 10) and Mouse CD300c D3 ECD (SEQ No. 16) inhibit the increase of TNF-α caused by LPS. These results suggest that the CD300c-Ig antigen of the present invention can be used to treat skin diseases such as atopic dermatitis by inhibiting immune activation.

[0131]

[0132] Example 2.2. Confirmation of the immunosuppressive effect of CD300c-Ig on human monocytes (THP-1)

[0133] To determine whether CD300c-Fc produced by the method of Example 1 exhibits an inhibitory effect on human monocyte activation, the production of TNF-α (Tumor necrosis factor-α) was checked. More specifically, 4 x 10⁶ in a 96-well plate 4THP-1 cells in cells / well were pretreated with 10 μg / mL of human CD300c-Fc for 18 hours, and 100 ng / mL of LPS was added to each well and treated for 48 hours. The amount of TNF-α produced was confirmed using an ELISA kit (human TNF-α Quantikine kit, R&D systems). The results are shown in Figure 4.

[0134] As shown in Figure 4, it was confirmed that CD300c-Fc inhibits the increase in TNF-α caused by LPS. These results suggest that the CD300c-Fc antigen of the present invention can inhibit immune activation and thus possess therapeutic ability for skin diseases such as atopic dermatitis.

[0135]

[0136] Example 3. Confirmation of the skin condition improvement effect of CD300c-Ig

[0137] Example 3.1. Confirmation of the ability of CD300c-Ig to regulate human skin keratinocyte activity

[0138] We aimed to determine whether CD300c-Ig regulates the activity of human keratinocytes. Specifically, using HaCaT cells, the secretion levels of IL-8, IL-1β, MDC, and TARC were measured using an ELISA kit (R&D systems). More specifically, 1.5 x 10 4 HaCaT cells were seeded into a 96-well plate and stabilized for 18 hours. Then, 1.1 µg / ml of CD300c-Ig was treated for 6 hours, followed by treatment with 10 ng / ml, 30 ng / ml, and 90 ng / ml of TNF-α and INF-γ, respectively, for 24 hours. Subsequently, the secretion levels of IL-8, IL-1β, MDC, and TARC in each cell culture medium were measured using an ELISA kit (R&D systems). The measurement results for IL-8 and IL-1β are shown in Figure 5.

[0139] As shown in Figure 5, it was confirmed that IL-8 and IL-1b were reduced by treatment with CD300c-Ig, and additionally, MDC and TARC were reduced by treatment with CD300c-Ig. This suggests that the CD300c-Ig of the present invention can provide an effect of improving skin condition by regulating skin keratinocytes and can be usefully utilized in the treatment of dry skin conditions, including atopic dermatitis.

[0140]

[0141] From the results of the above examples, the excellent skin condition improvement effect, skin barrier strengthening effect, and atopic dermatitis treatment effect of the composition containing CD300c-Ig of the present invention were confirmed, and it was determined that the present invention can be usefully utilized as a skin improvement agent and a treatment for related diseases.

[0142]

[0143] From the foregoing description, those skilled in the art to which the present invention pertains will understand that the present invention may be implemented in other specific forms without altering its technical concept or essential features. In this regard, the embodiments described above should be understood as illustrative in all respects and not restrictive. The scope of the present invention should be interpreted as including all modifications or variations derived from the meaning and scope of the claims set forth below and their equivalents, rather than from the detailed description above.

Claims

1. A cosmetic composition for improving skin condition comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

2. A cosmetic composition for improving skin condition according to claim 1, wherein the improvement of the skin condition is achieved through one or more selected from the group consisting of alleviation of skin inflammation, improvement of skin exfoliation, strengthening of the skin barrier, alleviation of skin dryness, and alleviation of atopic dermatitis.

3. A cosmetic composition for strengthening the skin barrier, comprising as an active ingredient a fusion protein comprising a CD300c protein or a fragment thereof and an immunoglobulin Fc region.

4. A pharmaceutical composition for the prevention or treatment of atopic dermatitis comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

5. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, wherein the fragment of CD300c according to claim 4 comprises the extracellular domain of the CD300c protein.

6. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, wherein the extracellular domain of CD300c comprises the amino acid sequence of SEQ ID NO. 4, 10, 16, or 22 in claim 5.

7. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 4, wherein the fusion protein is a CD300c protein or a fragment thereof and an immunoglobulin Fc region fused by a linker or covalent bond.

8. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, wherein the fusion protein comprises the amino acid sequence of SEQ ID NO. 6, 12, 18, or 24.

9. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 4, wherein the immunoglobulin Fc region is selected from the group consisting of (a) a CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain; (b) a CH1 domain and a CH2 domain; (c) a CH1 domain and a CH3 domain; (d) a CH2 domain and a CH3 domain; (e) a combination of one or more domains among the CH1 domain, a CH2 domain, a CH3 domain, and a CH4 domain with an immunoglobulin hinge region or a part of a hinge region; and (f) a dimer of each domain of the heavy chain constant region and the light chain constant region.

10. A pharmaceutical composition for the prevention or treatment of atopic dermatitis according to claim 4, wherein the immunoglobulin Fc region is an immunoglobulin Fc fragment derived from IgG, IgA, IgD, IgE, or IgM.

11. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, wherein the CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region modulate the activity of skin keratinocytes and reduce inflammatory responses.

12. In Paragraph 4, A pharmaceutical composition for the prevention or treatment of atopic dermatitis, wherein the above composition further comprises a pharmaceutically acceptable carrier, excipient, or diluent.

13. A pharmaceutical composition for the prevention or treatment of atopic dermatitis, wherein, in paragraph 4, the composition is administered in combination with one or more agents selected from the group consisting of interleukin inhibitors and Janus kinase (JAK) inhibitors.

14. A method for treating atopic dermatitis comprising the step of administering a pharmaceutical composition of any one of claims 4 to 13 to an individual suspected of having atopic dermatitis, excluding humans.

15. A quasi-drug composition for preventing or improving atopic dermatitis, comprising as an active ingredient a fusion protein comprising a CD300c protein or a fragment thereof and an immunoglobulin Fc region.

16. Use of a composition for improving skin condition comprising, as an active ingredient, a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

17. Use of a composition for the treatment of atopic dermatitis comprising as an active ingredient a CD300c protein or a fragment thereof; and a fusion protein comprising an immunoglobulin Fc region.

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