Novel applications for peptides derived from the CPNE7 protein

Abstract

The composition for inhibiting skin aging or treating skin wounds according to the present invention comprises a peptide consisting of the following amino acid sequence of general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1). In general formula 1, R1 is arginine (R), lysine (K), or glutamine (Q); R2 is arginine (R) or glutamine (Q); R3, R4, and R5 are arginine (R) or lysine (K), respectively; R6 is asparagine (N) or serine (S); and R7 and R8 are lysine (K) or tyrosine (Y).

Description

Technical Field

[0001] The present invention relates to a novel use of a peptide, and more particularly, to a novel use of a peptide derived from CPNE7 protein that promotes skin regeneration, enhances elasticity, and moisturizing ability.

Background Art

[0002] The skin is the outermost layer of our body and at the same time the widest tissue distributed throughout the body. Therefore, it is exposed to various stimuli including external pressure, impact, friction, etc. The skin is an important tissue that protects the internal organs in the body from such external environments, regulates body temperature, and senses sensations.

[0003] The structure of the skin is divided into the epidermis, dermis, and subcutaneous fat layer. The subcutaneous fat layer is a fat layer composed of fat cells under the dermis, a layer in which the excess of the ingested food is accumulated in the form of fat, located between the skin and muscles, having a heat insulation effect, and is distinguished from the dermis layer in order to perform the function of maintaining body temperature using this. The epidermis observed from the outside is the outermost layer of the skin that directly contacts the external environment and is mainly composed of the stratum corneum. Since the stratum corneum functions as a barrier to protect our body, it functions so that a drop of water, bacteria, virus, etc. cannot enter from the outside. The dermis is a layer that exists immediately below the epidermis and mainly contains fiber components such as collagen fibers and elastic fibers, and there are blood vessels, hair follicles, nerves, and sweat glands between them. The dermis is an important part that gives force (tension) to the skin and forms the shape of the skin. When collagen fibers, elastic fibers, etc. in the dermis layer become weak or thin, wrinkles can occur and cause a decrease in skin elasticity.

[0004] The skin is a protective barrier with various physiological functions, and at the same time, it is an important tissue for gaining aesthetic appeal from others. Therefore, aging skin can be accompanied by functional impairment as well as changes in appearance. Furthermore, severe skin aging can have a psychological impact on vitality in life, and in severe cases, can even lead to depression. The importance of preventing and treating skin aging is gradually increasing in modern society. In particular, the increase in average life expectancy and the decline in the birth rate are leading to an increase in the elderly population, and South Korea is projected to reach the criteria for a super-aged society designated by the United Nations by 2025. Therefore, efforts to prevent skin aging cannot be overlooked from the perspective of social costs.

[0005] One of the defining characteristics of skin aging is a decrease in skin thickness. This is particularly pronounced in the epidermis, and it has been reported that the decrease in epidermal thickness gradually accelerates after adulthood, with an average decrease of approximately 6.4% over a 10-year period. This decrease in epidermal thickness can be a direct pathological cause of xerosis (dry skin), as the water content on the skin and the water content of the stratum corneum within the epidermis decrease, and the overall lipid content also decreases by approximately 65%. Furthermore, as we age, the proliferation of keratinocytes (keratin-forming cells) decreases, so in older adults, the rate of recovery after keratin removal is significantly slower than in younger people.

[0006] On the other hand, the most prominent structural change that occurs internally with skin aging is the flattening of the dermo-epidermal junction due to the loss of dermal papillae. This flattening of the epidermo-epidermal junction reduces resistance to external friction and makes the skin more vulnerable to wound formation. Internally, the supply of nutrients and oxygen between the dermis and epidermis decreases, disrupting various intercellular signaling systems and inducing separation of the dermis and epidermis. This process is known to be one of the main mechanisms of wrinkle formation.

[0007] The causes of skin aging can be divided into intrinsic aging and extrinsic aging. Extrinsic aging mainly refers to photoaging caused by ultraviolet (UV) radiation. However, recent research suggests that various extrinsic factors other than UV radiation, such as cigarette smoke and pollution, also promote skin aging in conjunction with intrinsic factors. Intrinsic factors that promote skin aging include genome instability, epigenetic alteration, hormonal changes, and oxidative stress. It is not yet clear at what point these mechanisms are activated and interact with each other at the genetic and hormonal levels. On the other hand, reactive oxygen species (ROS) have been pointed out as a major cause of overall aging. Many studies have shown that reactive oxygen species can cause changes in the extracellular matrix (ECM) of the dermis in conjunction with intrinsic and extrinsic factors of the skin. External factors such as ultraviolet radiation and reactive oxygen species naturally generated after cellular metabolism bind to cysteine ​​located at the enzyme locus within receptor protein tyrosine phosphatase (RPTP), inhibiting RPTP's inhibitory ability to receptor tyrosine kinase (RTK). Phosphorylation of RTK affects various intracellular signaling pathways, inducing the activity of mitogen-activated protein kinase (MAPK), NF-κB (Nuclear Factor-κB), and AP-1 (Activator Protein-1) transcription factors. Activation of NF-κB and AP-1 inhibits collagen production, increases MMP gene transcription, reduces collagen levels in skin tissue, and contributes to the development of abnormal structural characteristics.

[0008] The treatment and prevention of skin aging has steadily become a major area of ​​interest throughout human history, and research into its mechanisms and therapeutic agents has been continuously conducted. However, basic research on the treatment and prevention of skin aging has yielded relatively few results compared to research on therapeutic agents for other diseases. This is partly due to the fact that fundamental answers have not been found regarding the theme of "cellular senescence," which is a higher-level concept than skin aging. There are countless studies on fibroblasts in vitro, but there are also countless studies that have not been able to link their results to measures to prevent the aging of human skin tissue. This suggests that the effects of systemic aging at the organism level on the skin may be more complex than previously thought, and that research related to skin aging must be viewed from a broader perspective, including not only the skin itself, but also subcutaneous fat, the vascular system, the nervous system, or the immune system. [Overview of the Initiative] [Problems that the invention aims to solve]

[0009] The present invention aims to provide a peptide that promotes skin elasticity and prevents aging by synthesizing type I and type III collagen in fibroblasts within the dermis layer of the skin, as well as its applications.

[0010] Another objective of this invention is to provide a peptide that promotes skin elasticity and prevents aging by stimulating type IV collagen synthesis in the basement membrane at the epidermal-dermal junction of the skin, and to provide the same peptide and its uses.

[0011] Another objective of the present invention is to provide a peptide and its uses that promote cell division and differentiation, thereby increasing the expression of Ki-67 in keratinocytes within the epidermal layer of the skin, thereby promoting epidermal regeneration and preventing aging.

[0012] Another objective of this invention is to provide a peptide and its applications that enhance the bonding function between keratinocytes by synthesizing filaggrin and involucrin within keratinocytes in the epidermal layer of the skin, thereby promoting skin hydration and preventing aging.

[0013] Another objective of the present invention is to provide a peptide that inhibits collagen degradation and prevents skin aging by reducing the expression of MMP-1 in the epidermal layer, keratinocytes in the dermal layer, and dermal cells, and also to provide applications for the same peptide.

[0014] The objects of the present invention are not limited to those mentioned above, and any other objects not mentioned should be clearly understood by a person with ordinary skill in the art to which the present invention pertains from the following description. [Means for solving the problem]

[0015] As a result of various studies conducted to solve the aforementioned technical problems, the inventors have developed a composition containing a CPNE7 protein-derived peptide for inhibiting skin aging or treating wounds.

[0016] As one embodiment to achieve the objectives of the preceding paragraph, the present invention provides a composition for inhibiting skin aging or treating wounds, comprising a CPNE7 protein-derived peptide having the amino acid sequence of the following general formula 1. KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the above general formula 1, R1 is arginine (R), lysine (K), or glutamine (Q). R2 is either arginine (R) or glutamine (Q), R3, R4, and R5 are arginine (R) or lysine (K), respectively. R6 is either asparagine (N) or serine (S). R7 and R8 are either lysine (K) or tyrosine (Y).

[0017] The peptide provided in this invention does not exhibit cytotoxicity and can provide excellent collagen synthesis ability.

[0018] The peptides provided in this invention include CPNE7 protein-derived peptides, and mutant peptides having a sequence that differs from the amino acid sequence of the CPNE7 protein-derived peptide by one or more amino acid residues, as long as they can exert an effect of inhibiting skin aging or treating wounds.

[0019] In general, amino acid exchanges in proteins and polypeptides that do not alter the overall molecular activity are well known in the field. The most common exchanges are between amino acid residues Ala / Ser, Val / Ile, Asp / Glu, Thr / Ser, Ala / Gly, Ala / Thr, Ser / Asn, Ala / Val, Ser / Gly, Thy / Phe, Ala / Pro, Lys / Arg, Asp / Asn, Leu / Ile, Leu / Val, Ala / Glu, and Asp / Gly. Furthermore, mutations or modifications in the amino acid sequence may increase the structural stability of the peptide against heat, pH, etc., or increase the efficacy of the peptide in inhibiting skin aging or treating wounds.

[0020] For example, glutamine, the acidic amino acid located at the third position of the peptide of SEQ ID NO: 1 provided in the present invention, can be replaced with lysine or arginine, both basic amino acids, while still exhibiting the effects of the peptide provided in the present invention; arginine, the basic amino acid located at the fourth or fifth position of the peptide of SEQ ID NO: 1, can be replaced with glutamine, an acidic amino acid, or lysine, a basic amino acid, while still exhibiting the effects of the peptide provided in the present invention; lysine, the basic amino acid located at the sixth, seventh, or ninth position of the peptide of SEQ ID NO: 1, can be replaced with arginine, a basic amino acid, or tyrosine, an aromatic amino acid, while still exhibiting the effects of the peptide provided in the present invention; asparagine, the acidic amino acid located at the eighth position of the peptide of SEQ ID NO: 1, can be replaced with serine, a neutral amino acid, while still exhibiting the effects of the peptide provided in the present invention; and tyrosine, the aromatic amino acid located at the tenth position of the peptide of SEQ ID NO: 1, can be replaced with lysine, a basic amino acid, while still exhibiting the effects of the peptide provided in the present invention.

[0021] Thus, even if the acidic amino acids, basic amino acids, or aromatic amino acids constituting the peptide of the present invention are substituted with different acidic amino acids, basic amino acids, neutral amino acids, or aromatic amino acids, the effects of the peptide provided by the present invention can still be observed. Therefore, it is obvious that mutant peptides having a sequence that differs from the amino acid sequence constituting the peptide of the present invention by one or more amino acid residues are also included in the category of peptides provided by the present invention.

[0022] In addition, the peptide of the present invention may have a form in which any amino acid is added to its N-terminus or C-terminus, and can still exhibit the effects of the peptide provided by the present invention as it is, and thus is included in the scope of the peptide provided by the present invention. As an example, it may be in a form in which 1 to 300 amino acids are added to the N-terminus or C-terminus of the peptide. As another example, it may be in a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide. As yet another example, it may be in a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0023] In another aspect, the present invention provides a polynucleotide encoding the peptide.

[0024] The polynucleotide can be mutated by substitution, deletion, insertion, or a combination thereof of one or more bases. When chemically synthesizing and manufacturing a nucleotide sequence, synthetic methods known in the art, for example, the methods described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988) can be used, and it can be synthesized using the phosphotriester, phosphite, phosphoramidite, and H-phosphonate methods, PCR and other autoligation methods, oligonucleotide synthesis methods on solid supports, etc. For example, the polynucleotide encoding the peptide of the present invention may contain the nucleotide sequence of SEQ ID NO: 4.

[0025] In yet another aspect, the present invention provides an expression vector containing the polynucleotide, a transformant containing the expression vector, and a method for producing the peptide using the transformant.

[0026] In this invention, the term "expression vector" refers to a recombinant vector capable of expressing a target peptide in target host cells, and is a gene crop containing essential regulatory elements operably linked to the expression of a gene insert. The expression vector includes expression regulatory elements such as a start codon, a termination codon, a promoter, and an operator, wherein the start and termination codons are generally considered to be part of the nucleotide sequence encoding the polypeptide, and must exert their effect in the organism when the gene crop is administered, and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inductive.

[0027] In this invention, the term "operably linked" refers to a state in which a nucleic acid expression regulatory sequence and a nucleic acid sequence encoding a target protein or RNA are functionally linked to perform a general function. For example, a promoter and a nucleic acid sequence encoding a protein or RNA can be operably linked to influence the expression of the coding sequence. Operational linking with an expression vector can be produced using genetic engineering techniques known in the art, and site-specific DNA cleavage and linking can be performed using enzymes or other methods commonly known in the art.

[0028] Furthermore, the expression vector may include a signal sequence for peptide efflux to facilitate the separation of the peptide from the cell culture medium. Specific start signals may also be necessary for efficient translation of the inserted nucleic acid sequence. These signals include an ATG start codon and adjacent sequences. In some cases, an exogenous translational regulatory signal, which may include an ATG start codon, may be required. These exogenous translational regulatory signals and start codons can come from a variety of natural and synthetic sources. Expression efficiency may be increased by introducing appropriate transcription or translational enhancers.

[0029] In addition, the expression vector may further include a protein tag that can be optionally removed using an endopeptidase to facilitate the detection of the peptide.

[0030] In this invention, the term "tag" refers to a molecule that exhibits quantifiable activity or properties, and may be a fluorescent molecule containing a chemical fluorescent substance (fluoracer) such as fluorescein, a polypeptide fluorescent substance such as green fluorescent protein (GFP) or an associated protein, or an epitope tag such as a Myc tag, Flag tag, histidine tag, leucine tag, IgG tag, or streptavidin tag. In particular, when using an epitope tag, a peptide tag consisting of 6 or more amino acid residues is preferably used, and more preferably 8 to 50 amino acid residues can be used.

[0031] In this invention, the term "photodermatitis" refers to a condition in which ultraviolet light or certain substances combine with sunlight to trigger an inflammatory response in the skin. One of the main causes of skin damage due to ultraviolet exposure is oxidative stress. Oxidative stress affects dermal fibroblasts, which can accelerate collagen breakdown and inflammatory responses. Such processes can alter the properties of the skin and worsen the symptoms of photodermatitis.

[0032] The term "atopic dermatitis" in this invention refers to a chronic dermatitis characterized by dry, itchy skin and recurrent inflammation. It is known to be associated with genetic factors, environmental factors, and immune system abnormalities, and stress on dermal fibroblasts may be associated with the onset and progression of atopic dermatitis. Oxidative stress generates free radicals that damage cells, leading to inflammatory responses and impaired cellular function. Dermal fibroblasts are located in the dermis layer of the skin and play a crucial role in maintaining skin structure and function. Therefore, impaired dermal fibroblast function due to oxidative stress can weaken the skin's protective function. This makes the skin more vulnerable to external stimuli, potentially worsening the symptoms of atopic dermatitis. This oxidative stress can also promote inflammatory responses, which are one of the main symptoms of atopic dermatitis and can cause dermal fibroblasts to malfunction. Furthermore, cellular damage due to oxidative stress can exacerbate damage to the skin barrier. When the skin barrier is damaged, allergens can penetrate the skin more easily, which can further worsen the symptoms of atopic dermatitis.

[0033] In the present invention, the expression vector may contain a nucleotide sequence encoding a peptide consisting of amino acids, such as the CPNE7 protein or SEQ ID NO: 1. However, the vector used is not particularly limited as long as it can produce the peptide, but is preferably plasmid DNA, phage DNA, etc., and more preferably commercially developed plasmids (pUC18, pBAD, pIDTSAMRT-AMP, etc.), plasmids derived from Escherichia coli (pYG601BR322, pBR325, pUC118, pUC119, etc.), plasmids derived from Bacillus subtilis (pUB110, pTP5, etc.), plasmids derived from yeast (YEp13, YEp24, YCp50, etc.), phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), animal virus vectors (retrovirus, adenovirus, vaccinia virus). These can be insect virus vectors (such as baculoviruses), etc. Since the expression levels and modifications of the aforementioned expression vectors vary depending on the host cell, it is desirable to select and use the host cell that best suits the purpose.

[0034] The transformants provided in the present invention are produced by introducing the expression vector provided in the present invention into a host and performing transformation, and can be used to express the polynucleotides contained in the expression vector and produce the peptide. The transformation can be carried out by various methods, but is not particularly limited as long as the peptide can be produced. Possible methods include CaCl2 precipitation, the Hanahan method which improves efficiency by using a reducing agent called DMSO (dimethyl sulfoxide) in addition to CaCl2 precipitation, electroporation, calcium phosphate precipitation, plasmofusion, stirring method using silicon carbide fibers, agrobacteria-mediated transformation, PEG-mediated transformation, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation. Furthermore, the host used to produce the transformants is not particularly limited as long as it can produce the peptide, but may be bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium; yeast cells such as Saccharomyces cerevisiae and Schizosaccharomyces pombe; fungal cells such as Pichia pastoris; insect cells such as Drosophila and Spodoptera frugiperda Sf9 cells; animal cells such as CHO, COS, NSO, 293, and Bowes melanoma cells; or plant cells.

[0035] The transformant may also be used in a method for producing a peptide consisting of amino acids according to Sequence ID No. 1 of the present invention. Specifically, a method for producing a peptide consisting of amino acids according to Sequence ID No. 1 of the present invention may include (a) culturing the transformant to obtain a culture, and (b) recovering the peptide of the present invention from the culture.

[0036] In this invention, the term "culture" refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions. In this invention, the method of culturing the transformants can be carried out by methods known in the art. Specifically, the culture is not particularly limited as long as it can be produced by expressing a peptide consisting of amino acids as shown in Sequence ID No. 1 of this invention, but it can be carried out continuously by batch processing, fed-batch culture, or repeated fed-batch process.

[0037] The culture medium used for cultivation must meet the requirements of the specific strain by adjusting the temperature, pH, etc., under aerobic conditions within a standard culture medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc., using appropriate methods. Possible carbon sources include a mixture of glucose and xylose as the main carbon source, as well as sugars and carbohydrates such as sucrose, lactose, fructose, maltose, starch, and cellulose; oils and fats such as soybean oil, sunflower oil, castor oil, and coconut oil; fatty acids such as palmitic acid, stearic acid, and linoleic acid; alcohols such as glycerol and ethanol; and organic acids such as acetic acid. These substances can be used individually or in mixtures. Furthermore, possible nitrogen sources include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate, and organic nitrogen sources such as amino acids and peptones, NZ-amines, meat extracts, yeast extracts, malt extracts, corn maceration, casein hydrolysates, fish or their degradation products, defatted soy cake or its degradation products. These nitrogen sources may be used individually or in combination. The culture medium may contain monopotassium phosphate, dispotassium phosphate, and corresponding sodium-containing salts as phosphorus sources. Possible phosphorus sources include potassium dihydrogen phosphate or dipotassium hydrogen phosphate, or corresponding sodium-containing salts. Inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate may be used. Finally, in addition to the above substances, growth-essential substances such as amino acids and vitamins may be used.

[0038] Furthermore, appropriate precursors may be used in the culture medium. The aforementioned raw materials may be added to the culture in batch, fed-batch, or continuous mode by an appropriate method during the culture process, but are not limited to these methods. The pH of the culture may be adjusted by using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acidic compounds such as phosphoric acid or sulfuric acid by an appropriate method.

[0039] Furthermore, antifoaming agents such as fatty acid polyglycol esters can be used to suppress bubble formation. To maintain aerobic conditions, oxygen or oxygen-containing gas (e.g., air) is injected into the culture medium. The culture temperature is typically 27°C to 37°C, preferably 30°C to 35°C. Culturing is continued until the maximum amount of peptide is produced. For this purpose, this is usually achieved in 10 to 100 hours.

[0040] Furthermore, the step of recovering the peptide from the culture can be carried out by methods known to the art. Specifically, the recovery method is not limited to these, as long as it can be used to recover the produced peptide, but preferably methods such as centrifugation, filtration, extraction, spraying, drying, evaporation, precipitation, crystallization, electrophoresis, fractional dissolution (e.g., ammonium sulfate precipitation), and chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

[0041] According to the present invention, a pharmaceutical composition for the treatment of wounds containing a CPNE7 protein-derived peptide can be used in the form of a pharmaceutical composition for the treatment of skin diseases caused by oxidative stress on dermal fibroblasts. Such compositions may be further prepared to include a suitable carrier (natural or non-natural carrier), excipient, or diluent that is commonly used. Specifically, each of the pharmaceutical compositions may be used in the form of a sterile injectable solution that can be administered to the site of skin disease by conventional methods. Examples of carriers, excipients, and diluents that may be included in the pharmaceutical composition in the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia gum, alginic acid, gelatin, calcium phosphate, calcium silicate, cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, and the like. When formulated, the product may be prepared using commonly used fillers, bulking agents, binders, wetting agents, disintegrants, surfactants, and other diluents or excipients. In particular, sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized agents, suppositories, and ointments may be used. Non-aqueous solvents and suspensions may include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases may include witepsol (registered trademark), macrogol (registered trademark), tween (registered trademark) 61, cocoa butter, lauric acid butter, and glycerol gelatin.

[0042] The amount of the peptide contained in the pharmaceutical composition of the present invention is not particularly limited, but may be 0.0001 to 50% by weight, more preferably 0.01 to 20% by weight, based on the total weight of the final composition.

[0043] The pharmaceutical compositions of the present invention may be administered in pharmaceutically effective amounts, where the term “pharmaceutically effective amount” in the present invention means an amount sufficient to treat or prevent a disease with a reasonable benefit / risk ratio applicable to medical treatment or prevention. The effective dose level may be determined based on the severity of the disease, the activity of the drug, the patient’s age, weight, health status, sex, the patient’s sensitivity to the drug, the time of administration, route of administration, and elimination ratio of the composition of the present invention used, the duration of treatment, the drugs used in combination with or concurrently with the composition of the present invention, and other factors known in the medical field. The pharmaceutical compositions of the present invention may be administered alone or in combination with known pharmaceutical compositions for the treatment of skin diseases caused by oxidative stress of dermal fibroblasts. Taking all of the above factors into consideration, it is important to administer an amount that provides the maximum effect with the minimum amount without side effects.

[0044] The dosage of the pharmaceutical composition of the present invention can be determined by a person skilled in the art, taking into consideration the intended use, the severity of the disease, the patient's age, weight, sex, medical history, or the type of substance used as the active ingredient. For example, the pharmaceutical composition of the present invention can be administered to an adult at a dose of about 0.1 ng / kg to about 100 mg / kg, preferably 1 ng / kg to about 10 mg / kg. The frequency of administration of the composition of the present invention is not particularly limited, but it may be administered once a day or divided into doses and administered several times a day. The aforementioned dosage does not limit the scope of the present invention in any way.

[0045] In another aspect, the present invention provides a method for treating a skin disease caused by oxidative stress on dermal fibroblasts, comprising the step of administering the pharmaceutical composition in a pharmaceutically effective amount to an individual other than a human who has developed a skin disease caused by oxidative stress on dermal fibroblasts.

[0046] In this invention, the term "individual" may include, without limitation, mammals such as rats and livestock that require treatment for skin diseases caused by oxidative stress on dermal fibroblasts, but excludes humans from among the individuals in which the disease has developed.

[0047] The pharmaceutical composition for the treatment of skin diseases caused by oxidative stress on dermal fibroblasts of the present invention can be administered via any common route, as long as it can reach the target tissue. The pharmaceutical composition of the present invention may be administered via routes such as oral administration, cutaneous administration, intramuscular injection, intravenous injection, subcutaneous injection, respiratory administration, rectal administration, or site administration, depending on the purpose, but is not limited to these.

[0048] Oral administration (intraoral administration) is a method of taking drugs by mouth, and drugs can generally be used in the form of tablets, capsules, or liquids. Skin administration is a method of applying or patching drugs to the skin, and drugs can be used in the form of creams, gels, patches, etc. Intramuscular injection is a method of injecting drugs directly into muscle tissue, and syringes and needles can be mainly used. Intravenous injection is a method of injecting drugs directly into blood vessels, and syringes and needles or intravenous cannulas can be used. Subcutaneous injection is a method of injecting drugs into the fatty tissue under the skin, and drugs such as insulin can be administered. Respiratory administration is a method of delivering drugs to the respiratory system via the mouth or nose, and drugs can be used in the form of inhalers, gases, vapors, etc. Rectal administration is a method of injecting drugs into the rectum, and drugs can be used in the form of rectal medications, rectal drops, etc. Site administration is a method of directly applying drugs to the area where treatment is needed, and drugs can be used in the form of creams, gels, patches, etc., depending on the characteristics of the disease.

[0049] In another embodiment, the present invention provides a quasi-drug composition containing the peptide for preventing or improving skin diseases caused by oxidative stress on dermal fibroblasts.

[0050] In this invention, the term "improvement" means all actions that at least reduce parameters related to the condition being treated, such as the severity of symptoms.

[0051] In the present invention, the term "improvement" can be interpreted as meaning all actions that involve administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to an individual seeking treatment for a skin disease caused by oxidative stress on dermal fibroblasts, thereby promoting the removal of reactive oxygen species within the dermal fibroblasts, and thereby improving or benefiting the symptoms of the skin disease caused by oxidative stress on dermal fibroblasts.

[0052] In this invention, the term "quasi-drug" refers to articles used for the purpose of diagnosing, treating, improving, alleviating, managing, or preventing diseases in humans or animals, which have a milder effect than pharmaceuticals. For example, according to the Pharmaceutical Affairs Law, quasi-drugs are articles excluding those used for pharmaceutical purposes, and include textile and rubber products used for the treatment or prevention of diseases in humans and animals, items that have a mild or no direct effect on the human body and are not instruments or machines, as well as similar items, and disinfectants and insecticides for preventing infectious diseases.

[0053] In the present invention, the type and dosage form of the quasi-drug composition containing the peptide are not particularly limited, but examples include ointments, patches, powders, gelling agents, tablets, or sprays.

[0054] In another embodiment, the present invention provides a health functional food composition containing the peptide for preventing or improving skin diseases caused by oxidative stress on dermal fibroblasts.

[0055] In this invention, the term "food" includes all foods in the ordinary sense, such as meats, sausages, bread, chocolates, candies, snacks, confectionery, pizzas, ramen noodles, other noodle products, gums, dairy products including ice cream, various soups, drinking water, tea, energy drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods.

[0056] The term "functional food" refers to a food with high medical and therapeutic effects that has been processed to efficiently exhibit biological regulatory functions in addition to nutrient supply. "Functionality" here refers to effects useful for health purposes, such as regulating nutrients or physiological effects, on the structure and function of the human body. The food of the present invention can be manufactured by methods commonly used in the industry, and can be manufactured by adding raw materials and components commonly used in the industry. Furthermore, the dosage form of the food can also be manufactured without restriction, as long as it is a dosage form recognized as a food. The food composition of the present invention can be manufactured in various dosage forms, and unlike general pharmaceuticals, it has the advantage of using food as a raw material, thus avoiding side effects that can occur with long-term use of pharmaceuticals, and is highly portable. The food of the present invention can be taken as an adjunct to enhance the effect of preventing or improving skin diseases caused by oxidative stress on dermal fibroblasts.

[0057] The term "health food" refers to foods that have a more active effect on maintaining or promoting health compared to general foods, while "health supplement food" refers to foods intended for health supplementation. In some cases, the terms "health functional food," "health food," and "health supplement food" may be used interchangeably.

[0058] Specifically, the aforementioned health functional foods are foods in which the peptide of the present invention is added to food ingredients such as beverages, teas, spices, gums, and confectionery, or manufactured as encapsulated, powdered, or suspended, and which, when consumed, produce specific health effects. However, unlike general pharmaceuticals, they have the advantage of being made from food ingredients and not having the side effects that can occur with long-term use of pharmaceuticals.

[0059] Since the food composition of the present invention can be consumed on a daily basis, it is expected to have a high effect in preventing or improving skin diseases caused by oxidative stress on dermal fibroblasts, and therefore can be used very effectively.

[0060] The food composition may further contain a physiologically acceptable carrier, but the type of carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0061] Furthermore, the food composition may contain additional ingredients commonly used in food compositions that can improve smell, taste, appearance, etc. For example, it may contain vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. It may also contain minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), and copper (Cu). It may also contain amino acids such as lysine, tryptophan, cysteine, and valine.

[0062] Furthermore, the food composition may contain food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), disinfectants (bleaching powder and high-grade bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar dyes, etc.), color fixatives (sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (monosodium glutamate (MSG), etc.), sweeteners (dulcin, cyclamic acid, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, potassium bitartrate, etc.), fortifiers, emulsifiers, thickeners, coating agents, gum bases, defoamers, solvents, and improvers. The additives may be selected and used in appropriate amounts depending on the type of food.

[0063] The peptide of the present invention can be added as is or used in combination with other foods or food components, and can be used as appropriate in the usual manner. The amount of active ingredient mixed can be suitably determined according to its intended use (prevention, health, or therapeutic treatment). In general, when manufacturing food or beverages, the food composition of the present invention can be added in an amount of 50 parts by weight or less, specifically 20 parts by weight or less, per 100 parts by weight of food or beverage. However, for long-term intake for health and hygiene purposes, the active ingredient may be included in amounts below the above range, and there are no safety concerns, so it can also be used in amounts above the above range.

[0064] An example of the food composition of the present invention may be used as a health beverage composition, in which case, like ordinary beverages, it may contain various flavorings or natural carbohydrates as additional ingredients. The aforementioned natural carbohydrates may be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; or sugar alcohols such as xylitol, sorbitol, and erythritol. Sweeteners may include natural sweeteners such as thaumatin and stevia extract, or synthetic sweeteners such as saccharin and aspartame. The ratio of the aforementioned natural carbohydrates is typically about 0.01g to 0.04g per 100mL of the health beverage composition of the present invention, specifically about 0.02g to 0.03g.

[0065] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavorings, colorings, pectin acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonation agents. It may also contain fruit pulp for the production of natural fruit juices, fruit juice beverages, or vegetable beverages. Such components may be used individually or in combination. While the proportion of such additives is not particularly important, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the health beverage composition of the present invention.

[0066] The food composition of the present invention may contain CPNE7 protein-derived peptides in various weight percentages, provided that they can exert an effect of inhibiting skin aging or treating wounds. Specifically, the peptides of the present invention may be contained in 0.00001 to 100% by weight or 0.01 to 80% by weight relative to the total weight of the food composition, but are not limited thereto. [Effects of the Invention]

[0067] The compositions for inhibiting skin aging or treating wounds, according to the embodiments of the present invention, which contain CPNE7 protein-derived peptides, can promote the synthesis of type I and type III collagen in fibroblasts within the dermis, which are involved in promoting skin elasticity and preventing aging.

[0068] Another embodiment of the present invention provides a composition for inhibiting skin aging or treating wounds, which contains a CPNE7 protein-derived peptide and may promote the synthesis of type IV collagen, which is involved in promoting skin elasticity and preventing aging in the basement membrane at the epidermal-dermal junction.

[0069] Another embodiment of the present invention provides a composition for inhibiting skin aging or treating wounds, comprising a CPNE7 protein-derived peptide, which may provide a peptide and its uses for promoting epidermal regeneration and preventing aging within keratinocytes in the epidermal layer of the skin.

[0070] Another embodiment of the present invention provides a composition for inhibiting skin aging or treating wounds, comprising a CPNE7 protein-derived peptide, which may promote the synthesis of filaggrin and involucrin, which are involved in promoting skin hydration and preventing aging within keratinocytes in the epidermal layer of the skin.

[0071] Another embodiment of the present invention provides a composition for inhibiting skin aging or treating wounds, which contains a CPNE7 protein-derived peptide and inhibits collagen degradation in the epidermal and dermal layers of the skin, thereby reducing the expression of MMP-1, which is involved in preventing skin aging.

[0072] The effects of the present invention are not limited to those mentioned above, and any other effects not mentioned will be clearly understood by a person with ordinary skill in the art to which the present invention pertains from the following description. [Brief explanation of the drawing]

[0073] [Figure 1] Figure 1 shows the results after treating the peptides of the present invention at different concentrations for 48 hours, dispensing the WST-8 solution into culture medium in a 10:1 ratio, protecting it from light, reacting it in a 37°C incubator for 2 hours, and then measuring the absorbance at 450 nm using a microplate. [Figure 2] Figure 2(A) shows the degree of type I collagen protein formation after treating with the peptide of the present invention (SEQ ID NO: 96) at various concentrations for 7 days, and (B) shows the EC50 value confirmed by type I collagen protein formation after treating with 100 μM of the peptide of the present invention (SEQ ID NO: 96) 7 times for 7 days. [Figure 3a] Figure 3a is a graph comparing the expression levels of type I collagen protein in relation to the peptides listed in Table 13. [Figure 3b] Figure 3b shows the degree of collagen deposition after treating the peptide of the present invention (SEQ ID NO: 96) at a concentration of 100 μM at 24-hour intervals for 7 days, compared with the control group. (A) is the result of confocal microscopy (Type I collagen) analysis, and (B) is the result of analysis using the confocal microscopy Z-Stack Imaging program. [Figure 3c] Figure 3c shows the efficacy of the peptide of the present invention in synthesizing α-smooth muscle actin (α-SMA) in hDFs (N=3). [Figure 4] Figure 4 shows the results of confirming the collagen formation ability of human-derived dermal fibroblasts after multiple treatments with the peptide of the present invention. [Figure 5] Figure 5 shows the results of confirming the permeability of the peptide of the present invention to human-derived three-dimensional skin tissue. [Figure 6a]Figure 6a shows the results of confirming the efficacy of the peptide of the present invention on human-derived three-dimensional skin tissue. [Figure 6b] Figure 6b shows the collagen-forming ability of the peptide in Neoderm-ED skin tissue (ex vivo). [Figure 7] Figure 7 shows the results of confirming the keratinocyte proliferation efficacy of the peptide of the present invention. [Figure 8] Figure 8 shows the results of confirming the efficacy of the peptide of the present invention through skin treatment. [Figure 9] Figure 9 shows the results of confirming the (A) reactive oxygen species reduction effect and (B) anti-photodamage effect by treatment with the peptide of the present invention. [Figure 10] Figure 10 shows the results of confirming the melanin secretion reduction effect of the peptide of the present invention at different concentrations. [Figure 11] Figure 11 shows the results of confirming the migration efficacy of the peptide of the present invention in keratinocytes and hDFs. [Modes for carrying out the invention]

[0074] The objectives and effects of the present invention, as well as the technical configurations for achieving them, will become clear by referring to the embodiments described in detail below, along with the accompanying drawings. In the description of the present invention, if it is determined that a specific description of a known function or configuration would be unnecessary and obscure the gist of the invention, such detailed description will be omitted. Furthermore, the terms used below are defined for the purpose of describing the embodiments of the present invention and may change depending on the intent or conventions of the user or operator.

[0075] However, the present invention is not limited to the embodiments disclosed below and can be realized in a variety of different forms. These embodiments are provided solely to ensure the complete disclosure of the present invention and to fully inform those skilled in the art of the invention of the category of invention, which is defined only by the claims. Therefore, the definition should be based on the content throughout this specification.

[0076] The following describes specific embodiments of the present invention.

[0077] (Example 1: Synthesis of CPNE7 protein-derived peptide) The inventors synthesized a peptide derived from the CPNE7 protein (SEQ ID NO: 1) using 9-fluorenylmethyloxycarbonyl (Fmoc), and then synthesized peptides for each group by substituting amino acids in the synthesized peptide (Tables 1-12). N-KYQRRKKNKY-C (Sequence ID 1)

[0078] First, the peptides in Group 1 were synthesized by substituting the peptide of SEQ ID NO: 1 or the 5th to 7th amino acids of the peptide of SEQ ID NO: 1 with lysine or arginine (Table 1).

[0079] [Table 1]

[0080] Next, the peptides in Group 2 were synthesized by substituting the 5th to 7th amino acids of the peptide of Sequence ID No. 1 with lysine or arginine, and substituting the 8th amino acid with serine (Table 2).

[0081] [Table 2]

[0082] Next, the peptides in Group 3 were synthesized by substituting the 5th to 7th amino acids of the peptide of Sequence ID No. 1 with lysine or arginine, and substituting the 9th amino acid with tyrosine (Table 3).

[0083] [Table 3]

[0084] Next, the peptides of Group 4 were synthesized by substituting the 5th to 7th amino acids of the peptide of Sequence ID No. 1 with lysine or arginine, substituting the 8th amino acid with serine, substituting the 9th amino acid with tyrosine, and substituting the 10th amino acid with lysine (Table 4).

[0085] [Table 4]

[0086] Next, the peptides in Group 5 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with arginine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 5).

[0087] [Table 5]

[0088] Next, the peptides in group 6 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 6).

[0089] [Table 6]

[0090] Next, the peptides in Group 7 were synthesized by substituting the third amino acid of the peptide of Sequence ID No. 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 7).

[0091] [Table 7]

[0092] Next, the peptides in group 8 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with arginine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 8).

[0093] [Table 8]

[0094] Next, the peptides in group 9 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, and the fifth to seventh amino acids with lysine or arginine (Table 9).

[0095] [Table 9]

[0096] Next, the peptides of group 10 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, and the eighth amino acid with serine (Table 10).

[0097] [Table 10]

[0098] Next, the peptides of group 11 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 11).

[0099] [Table 11]

[0100] Finally, the peptides in group 12 were synthesized by substituting the third amino acid of the peptide of SEQ ID NO: 1 with lysine, the fourth amino acid with glutamine, the fifth to seventh amino acids with lysine or arginine, the eighth amino acid with serine, the ninth amino acid with tyrosine, and the tenth amino acid with lysine (Table 12).

[0101] [Table 12]

[0102] (Example 2. WST-8 assay (Cellrix, B1007-500)) Human dermal fibroblasts (hDFs) were placed in 96-well plates (in Fibroblast Growth Medium (Lonza, CC-4126) containing 2% FBS, 0.1% insulin, 0.1% FGF, and 0.1% gentamicin sulfate-Amphotericin) in a ratio of 0.3 × 10⁶ cells. 5 The solution was dispensed into a single well and incubated for 24 hours in an incubator at 37°C and 5% CO2.

[0103] WST-8 analysis was performed to confirm the cytotoxicity of the peptide (SEQ ID NO: 96) in dermal fibroblasts. The peptide was treated with dermal fibroblasts at concentrations of 10 μM, 100 μM, 1 mM, 5 mM, and 10 mM, respectively. After 48 hours of treatment at each concentration, the WST-8 solution was dispensed into culture medium in a 10:1 ratio, incubated in a 37°C incubator under light-shielding conditions for 2 hours, and then measured by 450 nm absorbance using a microplate.

[0104] Figure 1 shows the results after treating the peptide of the present invention (SEQ ID NO: 96) at different concentrations for 48 hours, dispensing WST-8 solution into culture medium in a 10:1 ratio, protecting it from light, reacting it in a 37°C incubator for 2 hours, and then measuring the absorbance at 450 nm using a microplate.

[0105] Dermal fibroblasts were treated with the peptide of the present invention (SEQ ID NO: 96) at concentrations of 10 μM, 100 μM, 1 mM, 5 mM, and 10 mM, respectively. After 48 hours, cell viability was quantified by WST-8 analysis. The results showed that the peptide of the present invention (SEQ ID NO: 96) did not show cytotoxicity to dermal fibroblasts up to a concentration of 5 mM, but showed significant cytotoxicity at a concentration of 10 mM.

[0106] (Example 3-1. Confirmation of EC50 value for type I collagen formation by multiple peptide treatments in hDFs) To confirm the type I collagen synthesis ability achieved by multiple treatments with the peptide according to the present invention (SEQ ID NO: 96), dermal fibroblasts were placed in a 60 mm dish in a 1 × 10⁶ arrangement. 5 The samples were dispensed into 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. These samples were then treated with peptide (SEQ ID NO: 96) at 24-hour intervals for 7 days at concentrations of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 100 nM, 1 μM, 10 μM, 100 μM, and 1 mM (11 groups in total).

[0107] Subsequently, after washing the cells with PBS, proteins were separated, and the changes in type I collagen protein were confirmed by Western blotting (WB). The results were then normalized and compared based on the amount of β-actin protein.

[0108] Figure 2(A) shows the degree of type I collagen protein formation after treating with the peptide of the present invention (SEQ ID NO: 96) at various concentrations for 7 days, and (B) shows the EC50 value after treating with 100 μM of the peptide of the present invention (SEQ ID NO: 96) 7 times for 7 days, confirming the EC50 value due to type I collagen protein formation. Referring to Figure 2, it can be seen that when the peptide of the present invention (SEQ ID NO: 96) was treated multiple times, the EC50 value was confirmed to be 8.621 pM, indicating a significant increase in type I collagen expression compared to the control group.

[0109] (Example 3-2. Confirmation of type I collagen synthesis ability of peptides from groups 1-12 in human-derived dermal fibroblasts) To confirm the type I collagen synthesis ability of each peptide group, dermal fibroblasts were placed in a 60 mm dish in a 1 × 10⁶ arrangement. 5 The cells were dispensed into 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. Each peptide was then treated with 100 μM and incubated for another 24 hours. After 24 hours, the cells were washed with PBS, and the proteins were separated. The protein changes in type I collagen were confirmed by Western blotting (WB), and the results were normalized and compared based on the amount of β-actin protein.

[0110] Table 13 shows the results of single-treatment of dermal fibroblasts with 100 μM concentrations of each of the 1 to 12 peptides of the present invention, and the change in type I collagen protein levels after 24 hours. Figure 3a is a graph comparing the expression levels of type I collagen protein for the peptides in Table 13. As can be seen from this, the peptides of the present invention exhibit excellent type I collagen synthesis ability.

[0111] [Table 13]

[0112] (Example 3-3. Confirmation of type I collagen deposition in hDFs after multiple peptide administrations) To confirm the type I collagen synthesis ability achieved by multiple treatments with the peptide according to the present invention (SEQ ID NO: 96), dermal fibroblasts were placed in a 60 mm dish in a 1 × 10⁶ arrangement. 5 The samples were dispensed into 6-well plates and incubated at 37°C in a 5% CO2 incubator for 24 hours. These samples were then treated with peptide (SEQ ID NO: 96) and vitamin C (positive control group) at a concentration of 100 μM at 24-hour intervals for 7 days.

[0113] Afterward, the cells were washed with PBS, and the proteins were separated. The degree of protein change in type I collagen was then confirmed using a confocal microscope (type I collagen).

[0114] Figure 3b shows the degree of collagen deposition after treating the peptide of the present invention (SEQ ID NO: 96) at a concentration of 100 μM at 24-hour intervals for 7 days, compared with a control group. (A) is the result of confocal microscopy (type I collagen) analysis, and (B) is the result of analysis using the confocal microscopy Z-Stack Imaging program.

[0115] This confirmed that, on day 7, the amount of type I collagen in the ECM (Extracellular Matrix) was significantly increased in the group treated with the peptide of the present invention (SEQ ID NO: 96) compared to the control group.

[0116] (Examples 3-4. Efficacy confirmation test for peptide α-SMA synthesis in hDFs) To confirm the potential for promoting wound healing by administering the peptide according to the present invention (SEQ ID NO: 96), dermal fibroblasts were subjected to a study involving 1.5 × 10⁶ cells. 5 The samples were dispensed into confocal dishes and incubated at 37°C in a 5% CO2 incubator for 24 hours. These were then treated with peptide (SEQ ID NO: 96) and 100 μM vitamin C (positive control group) for 48 hours.

[0117] Afterward, the cells were washed with PBS, and the changes in α-SMA (alpha-smooth muscle actin) protein levels were examined using a confocal microscope.

[0118] Alpha-smooth muscle actin (α-SMA) is expressed in fibroblasts in the dermis of the skin and plays a role in providing structural support to skin tissue and maintaining skin elasticity. When a wound occurs, fibroblasts at the wound site express α-SMA, which plays a role in contracting the wounded skin. This reduces the size of the wound and protects the wounded skin from tissue damage during the wound healing process. Therefore, when inflammation occurs in the skin, α-SMA increases at the site of inflammation and plays a role in regulating the inflammatory response, thus α-SMA plays an important role in maintaining and stabilizing skin tissue.

[0119] Figure 3c shows the efficacy of peptide (SEQ ID NO: 96) in α-smooth muscle actin (α-SMA) synthesis in hDFs (N=3). As can be seen from Figure 3c, even a single treatment with 100 μM of peptide (SEQ ID NO: 96) significantly increased α-SMA, demonstrating its efficacy in promoting wound healing.

[0120] (Example 4. Observation of cellular changes using an optical microscope) In vitro, the collagen synthesis efficacy of vitamin C, known as a representative substance that increases the collagen synthesis capacity of dermal fibroblasts, and the peptide of the present invention (SEQ ID NO: 96) was compared by applying them to human-derived dermal fibroblasts. The cells were divided into a single application group (total 1 application) and a multiple application group (total 7 applications), and each group was treated for a total of 7 days with 100 μM of the peptide of the present invention (SEQ ID NO: 96) and 100 μM of vitamin C, respectively.

[0121] Specifically, dermal fibroblasts are placed in a 96-well plate in a quantity of 0.3 × 10⁶. 5 The mixture was dispensed into one well and incubated at 37°C in a 5% CO2 incubator for 24 hours.

[0122] Picro Sirius Red staining is one of the representative collagen staining methods that primarily targets type I and type III collagen. For a total of seven days, samples were treated with 100 μM of the peptide of the present invention (SEQ ID NO: 96) and 100 μM of vitamin C in single applications (1 application total) and multiple applications (7 applications total), followed by Picro Sirius Red staining analysis. Quantification of collagen positive for Picro Sirius Red showed that in the single-application group, the peptide of the present invention (SEQ ID NO: 96) treatment group showed significantly higher levels compared to the control group (Figure 4). In the multiple-application group, the peptide of the present invention (SEQ ID NO: 96) treatment group and the vitamin C treatment group showed a clear increase in collagen positive for Picro Sirius Red compared to the control group, and the quantitative results also showed statistical significance (Figure 4).

[0123] (Example 5-1. Results of confirmation of skin permeability of the peptide of the present invention in human-derived 3D skin tissue) To confirm the potential skin permeability of the peptide of the present invention, a three-dimensional skin tissue composed of epidermis and dermis was prepared using human-derived keratinocytes and dermal fibroblasts, and then treated with 1 mM of the peptide of the present invention (SEQ ID NO: 96) conjugated with a fluorescent substance. After treatment, the location of the fluorescence was continuously observed using a confocal microscope at 1 hour, 6 hours, 12 hours, and 24 hours.

[0124] One hour after treatment, the peptide of the present invention (SEQ ID NO: 96) was observed in the stratum corneum, and six hours later it was observed in the epidermis (Figure 5). It began to be observed in the dermis after 12 hours, and after 24 hours, the peptide of the present invention (SEQ ID NO: 96) was clearly observed in the dermis (Figure 5). This confirmed that the peptide of the present invention (SEQ ID NO: 96) can penetrate through the stratum corneum and epidermis to the dermis.

[0125] (Example 5-2. Confirmation of peptide skin permeability in human-derived 3D skin tissue) FITC fluorescence was added to the lysine residue of the peptide of the present invention (SEQ ID NO: 96), and peptide-FITC was prepared using the peptide of the present invention (SEQ ID NO: 96). 1 mM peptide-FITC was applied to human-derived 3D skin tissue (Tego Science, Neoderm-ED), and the skin permeability of peptide-FITC was confirmed by confocal microscopy at 1 hour, 6 hours, 12 hours, and 24 hours.

[0126] Specifically, in human-derived three-dimensional skin tissue, to confirm histological and cytological changes upon treatment with the peptide of the present invention (SEQ ID NO: 96), tissue sections were stained with hematoxylin and eosin (HE) 24 hours after treatment with 1 mM of the peptide of the present invention (SEQ ID NO: 96) and analyzed.

[0127] A normal epidermis consists of the following layers from the outside in: Stratum Corneum, Stratum Lucidum, Stratum Granulosum, Stratum Spinosum, and Stratum Basale. Millions of keratinocytes are formed in the basal layer every day, and these do not remain in one place but are constantly pushed to the outermost layer of the epidermis. As keratinocytes are pushed up, these cells gradually transform into hardened keratin. On the surface of human skin, aged keratinocytes continue to flake off, but in aged skin, the formation of new keratinocytes is not efficient, and it takes longer for the stratum corneum to shed, resulting in a thicker stratum corneum. Therefore, a decline in keratinocyte function leads to an increase in dead keratinocytes, which can be a direct cause of fine wrinkles and rough skin.

[0128] Histological analysis revealed that in the control group, keratinocytes were observed in the basal layer, but the spinous layer, granular layer, slender layer, and stratum corneum were not yet properly formed (Figure 6a). On the other hand, in the peptide-treated group of the present invention, the histological characteristics of normal epidermis were clearly observed (Figure 6a). This indicates that the peptide of the present invention can promote the keratinization process by enhancing the function of keratinocytes.

[0129] (Example 5-3. Confirmation of the collagen-forming ability of peptides in human-derived 3D skin tissue (Ex vivo)) The collagen synthesis efficacy of vitamin C and the peptide of the present invention was compared by applying them to human-derived dermal fibroblasts. To confirm the collagen-forming ability of the peptide of the present invention in skin tissue, three-dimensional skin tissue composed of epidermis and dermis was prepared using human-derived keratinocytes and dermal fibroblasts (Neoderm-ED). Then, vitamin C (1000 μM) and the peptide of the present invention (SEQ ID NO: 96) were treated twice at concentrations of 10 μM, 100 μM, 300 μM, and 1000 μM, respectively. After treatment, the collagen-forming ability of the negative control group, positive control group, and treated group was confirmed at 48-hour intervals using Masson trichrome staining.

[0130] Figure 6b shows the collagen-forming ability of the peptide in Neoderm-ED skin tissue (ex vivo). As can be seen in Figure 6b, the excellent collagen-forming ability of the peptide treatment of the present invention can be confirmed. Specifically, compared to the negative control group (NC group), the amount of collagen (Blue) deposition increased in a dose-dependent manner in the peptide (SEQ ID NO: 96) treatment group.

[0131] (Example 5-4. Peptide skin efficacy confirmation test in human-derived 3D skin tissue) To confirm the proliferation efficacy of the peptide of the present invention on basal layer keratinocytes, immunofluorescence staining analysis of the representative cell division and proliferation marker Ki-67 was performed. For this purpose, 1 mM of the peptide of the present invention (SEQ ID NO: 96) was treated with human-derived 3D skin tissue (Neoderm-ED, Tego Science Co., Ltd.) and cultured for 24 hours at 37°C in a 5% CO2 incubator. After fixing each human-derived 3D skin tissue, frozen sections were prepared. The tissue sections were stained with hematoxylin and eosin and comparatively analyzed using a light microscope (confocal microscope).

[0132] Referring to Figure 7, it can be seen that after 24 hours of treatment of human-derived 3D skin tissue with 1 mM of the peptide of the present invention (SEQ ID NO: 96), the number of Ki-67-positive cells in basal layer keratinocytes increased compared to the control group. Quantitative results of the ratio of Ki-67-positive cells showed that the group treated with the peptide of the present invention (SEQ ID NO: 96) had 64.6% (±11.1%), a 5.3-fold increase compared to 12.2% (±3.6%) in the control group (Figure 7). This can be interpreted as the peptide of the present invention promoting cell division and proliferation of keratinocytes.

[0133] (Example 5-5. Peptide skin efficacy confirmation test in human-derived 3D skin tissue) To confirm the skin efficacy of the peptide of the present invention, the expression patterns of type IV collagen, MMP-1, filaggrin, and involucrin were compared by immunofluorescence analysis 24 hours after treatment of human-derived three-dimensional skin tissue with 1 mM of the peptide of the present invention.

[0134] Type IV collagen is primarily expressed in the epidermal basement membrane at the epidermal-dermal junction. The basement membrane has an anatomical function of connecting the epidermis to the underlying dermis, facilitating the permeability of fluids between the epidermis and dermis, while also acting as a protective membrane that controls the permeability of inflammatory and tumor cells. Recent studies have revealed that changes occur in the basement membrane in aging skin, and that type IV collagen decreases during this process. Such changes can lead not only to deformation of keratinocytes but also to an increase in collagen-degrading enzymes (MMPs).

[0135] Referring to Figure 8, it can be confirmed that the peptide of the present invention (SEQ ID NO: 96) reduces the expression of MMP-1 in the epidermis and dermis of human-derived three-dimensional skin tissue and increases the expression of type IV collagen in the basement membrane. Maintaining skin moisture is a fundamental condition for maintaining healthy skin, and the stratum corneum maintains moisture through natural moisturizing factors such as filaggrin and involucrin, as well as the lipid layer present between keratinocytes and sebum secreted from the sebaceous glands.

[0136] Filaggrin acts as an adhesive, connecting the outer membrane of keratinocytes with keratin intermediate microfibers, while involucrin plays a crucial role in the formation and maintenance of the keratinocyte cell membrane, which acts as a physical barrier for the skin. A decrease in the expression of these proteins can lead to skin diseases such as psoriasis and atopic dermatitis due to impaired skin barrier function. In this regard, referring to Figure 8, it can be confirmed that the peptide of the present invention increases the expression levels of filaggrin and involucrin, which are natural moisturizing factors, in the epidermis of human-derived three-dimensional skin tissue (Figure 8).

[0137] (Example 6-1. Confirmation of peptide anti-photodamage efficacy in Neoderm-ME tissue) To confirm the anti-photodamage efficacy of the peptide of the present invention, human-derived three-dimensional skin tissue consisting of keratinocytes and melanocytes was irradiated with UV-B light. Subsequently, 48 hours after treatment with 100 μM of the peptide of the present invention, the expression patterns of DCFDA staining, filaggrin, and involucrin were compared by immunofluorescence analysis.

[0138] Figure 9 shows the results of confirming the (A) reactive oxygen species reduction effect and (B) anti-photodamage effect of treatment with the peptide (SEQ ID NO: 96). It can be confirmed that the group treated with the peptide (SEQ ID NO: 96) provides a significant reduction in skin damage caused by UV-B irradiation.

[0139] (Example 6-2. Confirmation of skin whitening efficacy using Neoderm-ME) To confirm the skin whitening efficacy of the peptide of the present invention, human-derived three-dimensional skin tissue consisting of keratinocytes and melanocytes was irradiated with UV-B light. Subsequently, after 48 hours of treatment with 30 μM, 100 μM, 300 μM, and 1000 μM of the peptide of the present invention, the tissue was compared using the Melanin Content Assay and Fontana Masson Staining analysis methods.

[0140] Figure 10 shows the efficacy of peptide (SEQ ID NO: 96) in reducing melanin secretion at different concentrations. It was confirmed that a significant reduction in melanin secretion occurred at concentrations of 30 μM or higher.

[0141] (Example 7. Efficacy confirmation study of peptide (SEQ ID NO: 96) migration in keratinocytes and hDF) To confirm the potential for cell migration and wound healing promotion by administration of the peptide according to the present invention (SEQ ID NO: 96), keratinocytes and dermal fibroblasts were subjected to a study involving 1.5 × 10⁻⁶ cells. 5 The mixture was dispensed into a transwell, treated with 100 μM of peptide (SEQ ID NO: 96), and incubated at 37°C in a 5% CO2 incubator for 48 hours.

[0142] Subsequently, the cells were washed with PBS, stained with crystal violet, and the changes in cell migration were observed using a light microscope.

[0143] Figure 11 shows the migration efficacy of the peptide of the present invention in keratinocytes and hDFs, and confirms that the peptide of the present invention promotes cell migration in human dermal fibroblasts and keratinocytes, thereby promoting wound healing.

[0144] This specification and drawings disclose preferred embodiments of the present invention, and although specific terms are used, these are merely general terms used to illustrate the technical content of the invention and to aid in understanding the invention, and are not intended to limit the scope of the invention. It will be obvious to those ordinary skill in the art to which the present invention pertains that other modifications based on the technical idea of ​​the present invention are also possible, in addition to the embodiments disclosed herein.

Claims

1. A composition for inhibiting skin aging or treating skin wounds, comprising a peptide consisting of the amino acid sequence of the following general formula 1: KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1) In the above general formula 1, R1 is arginine (R), lysine (K), or glutamine (Q). R2 is either arginine (R) or glutamine (Q), R3, R4, and R5 are arginine (R) or lysine (K), respectively. R6 is either asparagine (N) or serine (S). R7 and R8 are lysine (K) or tyrosine (Y).

2. The composition according to claim 1, characterized in that it promotes the activity of type I collagen, type III collagen, type IV collagen, Ki-67, filaggrin, or involucrin, or inhibits the activity of MMP-1.

3. The composition according to claim 1, characterized in that the peptide is any of the amino acid sequences of SEQ ID NOs: 1 to 96.

4. A polynucleotide encoding the peptide described in claim 1.

5. An expression vector comprising the polynucleotide described in claim 4.

6. The composition according to claim 1, characterized in that the composition comprises a polypeptide in which the peptide is repeatedly linked.

7. A composition for preventing, improving, or treating skin aging by promoting skin regeneration, elasticity, and moisture retention, comprising a peptide consisting of any of the amino acid sequences of Sequence ID No. 1 to 96.

8. A quasi-drug composition for preventing and improving skin aging by promoting skin regeneration, elasticity, and moisture retention, comprising a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 96.

9. A cosmetic composition for preventing and improving skin aging by promoting skin regeneration, elasticity, and moisture retention, comprising a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 96.

10. A health functional food composition for preventing and improving skin aging by promoting skin regeneration, elasticity, and moisture retention, comprising a peptide consisting of any of the amino acid sequences of SEQ ID NOs: 1 to 96.

11. The composition according to claim 7, further comprising a pharmaceutically acceptable carrier, excipient, or diluent.

12. A method for preventing, improving, or treating skin aging by administering the composition according to claim 7 to an individual other than a human, thereby promoting skin regeneration, elasticity, and moisturizing.

13. The composition according to claim 1, wherein the skin injury is due to a wound, diabetes, or pressure ulcer.

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