Novel use of cpne7 protein-derived peptide

Abstract

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

Description

Novel uses of peptides derived from CPNE7 protein

[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, elasticity enhancement, and moisturizing ability.

[0002]

[0003] The skin is the outermost layer of our body and is also the largest tissue distributed throughout the entire body. Therefore, it is exposed to various external stimuli, including pressure, impact, and friction. Skin is a vital tissue that protects internal organs from the external environment, regulates body temperature, and allows us to sense sensations.

[0004] The skin's structure is divided into the epidermis, dermis, and subcutaneous fat layers. The subcutaneous fat layer, located beneath the dermis and composed of fat cells, is where excess dietary fat accumulates. Located between the skin and muscles, it provides insulation and maintains body temperature, distinguishing it from the dermis. The epidermis, visible from the outside, is the outermost layer of the skin and directly in contact with the external environment. It is primarily composed of the stratum corneum. This layer acts as a protective barrier, preventing water, bacteria, and viruses from penetrating. The dermis, located directly beneath the epidermis, is composed primarily of collagen and elastic fibers, with blood vessels, hair follicles, nerves, and sweat glands nestled between them. The dermis provides strength (tension) to the skin and is crucial for its shape. Weakening or thinning of collagen or elastic fibers within the dermis can lead to wrinkles and loss of skin elasticity.

[0005] Skin is a protective barrier with diverse physiological functions and is also a crucial tissue for aesthetic appeal from others. Therefore, aging skin not only manifests as visible changes but also carries functional impairments. Furthermore, severe skin aging can have a psychological impact on vitality, even leading to depression in severe cases. The prevention and treatment of skin aging are increasingly important in modern society. In particular, increasing life expectancy and declining birth rates have led to a growing elderly population. Korea is projected to reach the UN's criteria for a super-aged society by 2025. Therefore, efforts to prevent skin aging cannot be overlooked from a socially costly perspective.

[0006] A hallmark of skin aging is a decrease in skin thickness. This decrease is particularly noticeable in the epidermis, and it has been reported that epidermal thickness decreases gradually 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 dry skin, as it reduces moisture content on the skin and in the stratum corneum within the epidermis, and overall lipid content is also known to decrease by approximately 65%. Furthermore, keratinocyte proliferation decreases with age, so the recovery rate after exfoliation is significantly slower in older individuals compared to younger individuals.

[0007] Meanwhile, the most prominent structural change internally associated with skin aging is the flattening of the dermo-epidermal junction (DJ) due to the loss of dermal papillae. This flattening reduces resistance to external friction, making the skin more susceptible to wound formation. Internally, it reduces the supply of nutrients and oxygen between the dermis and epidermis, disrupts various intercellular signaling pathways, and causes dermal-epidermal separation, a process known to be a key mechanism of wrinkle formation.

[0008] The causes of skin aging can be divided into intrinsic aging and extrinsic aging. Extrinsic aging primarily refers to photoaging caused by ultraviolet rays. However, recent studies suggest that various extrinsic factors, such as cigarette smoke and pollution, in addition to ultraviolet rays, may also interact with intrinsic factors to accelerate skin aging. Intrinsic factors known to accelerate skin aging include genomic instability, epigenetic alterations, hormonal changes, and oxidative stress. However, it is not clear when these mechanisms are activated and linked by genetic or hormonal changes. On the other hand, reactive oxygen species (ROS) have been identified as a major cause of overall aging. Many studies have shown that reactive oxygen species can induce changes in the extracellular matrix (ECM) within the dermis in conjunction with internal and external factors of the skin. External factors such as ultraviolet rays and naturally generated reactive oxygen species after cellular metabolism bind to cysteine ​​at the enzyme site of Receptor Protein Tyrosine Phosphatase (RPTP), thereby inhibiting RPTP's ability to inhibit Receptor Tyrosine Kinase (RTK). Phosphorylation of RTK affects various signaling pathways within the cell and induces the activation of Mitogen-Activated Protein Kinase (MAPK), Nuclear Factor-κB (NF-κB), and Activator Protein-1 (AP-1) transcription factors. Activation of NF-κB and AP-1 inhibits collagen production and increases MMP gene transcription, which reduces the amount of collagen in skin tissue and contributes to abnormal structural features.

[0009] The treatment and prevention of skin aging have consistently been a topic of great interest throughout human history, and research into its mechanisms and therapeutics has continued. However, basic research on skin aging treatment and prevention has not achieved as remarkable results as research on treatments for other diseases. This is related to the lack of fundamental answers to the topic of "cellular aging," a broader concept of skin aging. While numerous studies on in vitro fibroblasts exist, many have failed to translate their findings into anti-aging strategies for human skin tissue. This suggests that the impact of systemic aging on the skin at the organism level may be more complex than previously thought, and that research on skin aging should be viewed from a broader perspective, encompassing not only the skin but also subcutaneous fat, the vascular system, the nervous system, and the immune system.

[0010] The purpose of the present invention is to provide a peptide that promotes skin elasticity and prevents aging by synthesizing Type I Collagen and Type III Collagen within fibroblasts in the dermal layer of the skin, and a use thereof.

[0011] Another object of the present invention is to provide a peptide that promotes skin elasticity and prevents aging by synthesizing Type IV Collagen in the basement membrane of the dermal-epidermal boundary of the skin, and a use thereof.

[0012] Another object of the present invention is to provide a peptide and its use that promotes regeneration of the epidermal layer and prevents aging by increasing the expression of Ki-67 in keratinocytes within the epidermal layer of the skin, thereby promoting cell division and differentiation.

[0013] Another object of the present invention is to provide a peptide that promotes skin moisturization and prevents aging by synthesizing Filaggrin and Involucrin within keratinocytes in the epidermal layer of the skin, thereby enhancing the adhesion function between keratinocytes, and a use thereof.

[0014] Another object of the present invention is to provide a peptide and its use that prevents skin aging by inhibiting collagen decomposition by reducing the expression of MMP-1 in keratinocytes and dermal cells in the epidermal layer and dermal layer of the skin.

[0015]

[0016] The purpose of the present invention is not limited to what has been mentioned above, and other purposes not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the description below.

[0017]

[0018] In order to solve the above technical problem, the inventors of the present invention conducted various studies and developed a composition for inhibiting skin aging or treating wounds containing a peptide derived from CPNE7 protein.

[0019] As one embodiment for achieving the above-described purpose, the present invention provides a composition for inhibiting skin aging or treating wounds, comprising a peptide derived from CPNE7 protein, comprising an amino acid sequence of the following general formula 1.

[0020] KY-R1-R2-R3-R4-R5-R6-R7-R8 (general formula 1)

[0021] In the above general formula 1,

[0022] R1 is arginine (R), lysine (K), or glutamine (Q);

[0023] R2 is arginine (R) or glutamine (Q);

[0024] R3, R4 and R5 are arginine (R) or lysine (K), respectively;

[0025] R6 is asparagine (N) or serine (S); and

[0026] R7 and R8 are lysine (K) or tyrosine (Y).

[0027]

[0028] The peptide provided in the present invention can provide excellent collagen synthesis ability without exhibiting cytotoxicity.

[0029] The peptide provided in the present invention includes a peptide derived from CPNE7 protein, and as long as it can exhibit an anti-aging or wound healing effect, a mutant peptide having a sequence that differs in one or more amino acid residues from the amino acid sequence constituting the peptide is also included in the category of the peptide provided in the present invention.

[0030] In general, amino acid exchanges in proteins and polypeptides that do not alter the overall activity of the molecule are well known in the art. 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, Asp / Gly. In addition, peptides with increased structural stability against heat, pH, etc., or enhanced skin aging inhibition or wound healing effects may be included due to mutations or modifications in the amino acid sequence.

[0031] For example, even if glutamine, an acidic amino acid located at position 3 of the peptide having sequence number 1 provided in the present invention, is replaced with lysine or arginine, which are basic amino acids, the effect of the peptide provided in the present invention can be maintained; even if arginine, a basic amino acid located at position 4 or 5 of the peptide having sequence number 1, is replaced with glutamine, an acidic amino acid, or lysine, which is a basic amino acid, the effect of the peptide provided in the present invention can be maintained; even if lysine, a basic amino acid located at position 6, 7, or 9 of the peptide having sequence number 1, is replaced with arginine, a basic amino acid, or tyrosine, which is an aromatic amino acid, the effect of the peptide provided in the present invention can be maintained; even if asparagine, an acidic amino acid located at position 8 of the peptide having sequence number 1, is replaced with serine, which is a neutral amino acid, the effect of the peptide provided in the present invention can be maintained; Tyrosine, an aromatic amino acid located at position 10 of the peptide of sequence number 1, can be replaced with lysine, a basic amino acid, and still exhibit the effect of the peptide provided by the present invention.

[0032] In this way, even if the acidic amino acid, basic amino acid, or aromatic amino acid constituting the peptide of the present invention is replaced with another acidic amino acid, basic amino acid, neutral amino acid, or aromatic amino acid, the effect of the peptide provided by the present invention can be exhibited as is, so it is clear that a mutant peptide having a sequence that differs by one or more amino acid residues from the amino acid sequence constituting the peptide of the present invention is also included in the category of the peptide provided by the present invention.

[0033] In addition, since the peptide of the present invention can exhibit the effect of the peptide provided by the present invention even if it has a form in which any amino acid is added to its N-terminus or C-terminus, it is included in the category of the peptide provided by the present invention. As an example, it may be 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 a form in which 1 to 100 amino acids are added to the N-terminus or C-terminus of the peptide, and as yet another example, it may be a form in which 1 to 24 amino acids are added to the N-terminus or C-terminus of the peptide.

[0034] In another aspect, the present invention provides a polynucleotide encoding the peptide.

[0035] The polynucleotide may be mutated by one or more base substitutions, deletions, insertions, or a combination thereof. When producing a nucleotide sequence by chemical synthesis, synthetic methods widely known in the art, such as the method described in the literature (Engels and Uhlmann, Angew Chem IntEd Engl., 37:73-127, 1988), may be used, and may be synthesized using triester, phosphite, phosphoramidite, and H-phosphate methods, PCR and other autoprimer methods, oligonucleotide synthesis on a solid support, etc. For example, a polynucleotide encoding a peptide of the present invention may include the base sequence of SEQ ID NO: 4.

[0036] In another aspect, the present invention provides an expression vector comprising the polynucleotide, a transformant comprising the expression vector, and a method for producing the peptide using the transformant.

[0037] The term "expression vector" of the present invention refers to a recombinant vector capable of expressing a target peptide in a target host cell, and refers to a genetic construct containing essential regulatory elements operably linked to enable expression of a gene insert. The expression vector contains expression regulatory elements such as an initiation codon, a stop codon, a promoter, and an operator. The initiation codon and the stop codon are generally considered to be part of a nucleotide sequence encoding a polypeptide, and must be functional in a subject when the genetic construct is administered and must be in frame with the coding sequence. The promoter of the vector may be constitutive or inducible.

[0038] The term "operably linked" in the present invention refers to a state in which a nucleic acid expression control 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 are operably linked to affect the expression of the coding sequence. The operably linked sequence with the expression vector can be produced using genetic recombination techniques well known in the art, and site-specific DNA cleavage and ligation can be performed using enzymes generally known in the art.

[0039] Additionally, the expression vector may include a signal sequence for the release of the peptide to facilitate its release from the cell culture medium. A specific initiation signal may also be required for efficient translation of the inserted nucleic acid sequence. These signals include the ATG initiation codon and adjacent sequences. In some cases, an exogenous translational control signal, which may include the ATG initiation codon, must be provided. These exogenous translational control signals and initiation codons may come from a variety of natural and synthetic sources. Expression efficiency may be increased by the introduction of appropriate transcriptional or translational enhancing factors.

[0040] In addition, the expression vector may additionally include a protein tag that can be optionally removed using an endopeptidase to facilitate detection of the peptide.

[0041] The term "tag" in the present invention means a molecule exhibiting a quantifiable activity or characteristic, and may be a fluorescent molecule including a chemical fluorophore such as fluorescein, a polypeptide fluorophore such as a fluorescent protein (GFP) or a related protein, or an epitope tag such as a Myc tag, a Flag tag, a histidine tag, a leucine tag, an IgG tag, or a streptavidin tag. In particular, when an epitope tag is used, a peptide tag preferably composed of 6 or more amino acid residues, and more preferably composed of 8 to 50 amino acid residues, can be used.

[0042] The term "photodermatitis" in the present invention can refer to a condition in which ultraviolet rays or certain substances combine with sunlight to induce an inflammatory response in the skin. One of the main causes of skin damage caused by UV exposure is oxidative stress. Oxidative stress can affect dermal fibroblasts, leading to collagen breakdown and inflammatory responses. This process can alter skin properties and exacerbate the symptoms of photodermatitis.

[0043] The term "atopic dermatitis" used herein refers to a chronic dermatitis characterized by dry, itchy skin and recurrent inflammation. Genetic, environmental, and immune system abnormalities are known to be involved. Dermal fibroblast stress may be associated with the onset and progression of atopic dermatitis. Oxidative stress generates free radicals that damage cells, leading to inflammatory responses and decreased cell function. Dermal fibroblasts are located in the dermal layer of the skin and play a crucial role in maintaining the structure and function of the skin. Therefore, if dermal fibroblast function is impaired due to oxidative stress, the skin's protective function may be weakened. This makes the skin more vulnerable to external stimuli and may worsen the symptoms of atopic dermatitis. This oxidative stress can promote inflammatory responses. This inflammatory response is a major symptom of atopic dermatitis and causes dermal fibroblasts to malfunction. Furthermore, cellular damage caused by oxidative stress can worsen skin barrier damage. When the skin barrier is damaged, substances that cause allergic reactions can more easily penetrate the skin, which can further worsen the symptoms of atopic dermatitis.

[0044] In the present invention, the expression vector may include a nucleotide sequence encoding a CPNE7 protein or a peptide composed of amino acids having sequence number 1. The vector used here is not particularly limited thereto as long as it can produce the peptide, but may preferably be a plasmid DNA, phage DNA, etc., and more preferably a commercially developed plasmid (pUC18, pBAD, pIDTSAMRT-AMP, etc.), an E. coli-derived plasmid (pYG601BR322, pBR325, pUC118, pUC119, etc.), a Bacillus subtilis-derived plasmid (pUB110, pTP5, etc.), a yeast-derived plasmid (YEp13, YEp24, YCp50, etc.), a phage DNA (Charon4A, Charon21A, EMBL3, EMBL4, λgt10, λgt11, λZAP, etc.), an animal virus. It can be a vector (retrovirus, adenovirus, vaccinia virus, etc.), or an insect virus vector (baculovirus, etc.). Since the amount of protein expression and the number of modifications of the expression vector vary depending on the host cell, it is desirable to select and use the host cell most suitable for the purpose.

[0045] The transformant provided in the present invention can be produced by introducing the expression vector provided in the present invention into a host and transforming it, and can be used to produce the peptide by expressing the polynucleotide included in the expression vector. The transformation can be performed by various methods, and is not particularly limited thereto, as long as the peptide can be produced, but may include, but is not limited to, the CaCl2 precipitation method, the Hanahan method which increases efficiency by using a reducing substance called DMSO (dimethyl sulfoxide) in the CaCl2 precipitation method, electroporation, calcium phosphate precipitation, protoplast fusion, stirring using silicon carbide fibers, Agrobacterium-mediated transformation, transformation using PEG, dextran sulfate, lipofectamine, and drying / inhibition-mediated transformation methods. In addition, the host used for producing the transformant is not particularly limited thereto, as long as it can produce the peptide, and may be a bacterial cell such as Escherichia coli, Streptomyces, or Salmonella Typhimurium; a yeast cell such as Saccharomyces cerevisiae or Schizosaccharomyces pombe; a fungal cell such as Pichia pastoris; an insect cell such as Drosophila or Spodoptera Sf9 cells; an animal cell such as CHO, COS, NSO, 293, or Bow melanoma cells; or a plant cell.

[0046] The transformant can also be used in a method for producing a peptide comprising an amino acid having sequence number 1 of the present invention. Specifically, the method for producing a peptide comprising an amino acid having sequence number 1 of the present invention can include the steps of (a) culturing the transformant to obtain a culture; and (b) recovering the peptide of the present invention from the culture.

[0047] The term "cultivation" in the present invention refers to a method of growing microorganisms under appropriately artificially controlled environmental conditions. In the present invention, the method of culturing the transformant can be performed using a method widely known in the art. Specifically, the culturing is not particularly limited to the above, as long as it can express and produce a peptide composed of amino acids consisting of SEQ ID NO: 1 of the present invention, but can be continuously cultured in a batch process or a fed batch or repeated fed batch process.

[0048] The medium used for cultivation should meet the requirements of a specific strain in an appropriate manner while controlling temperature, pH, etc. under aerobic conditions in a general medium containing appropriate carbon sources, nitrogen sources, amino acids, vitamins, etc. The carbon sources that can be used include mixed sugars such as glucose and xylose as the main carbon source, and other 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 as a mixture. Nitrogen sources that can be used include inorganic nitrogen sources such as ammonia, ammonium sulfate, ammonium chloride, ammonium acetate, ammonium phosphate, ammonium carbonate, and ammonium nitrate; Organic nitrogen sources such as amino acids such as glutamic acid, methionine, and glutamine, and peptone, NZ-amine, meat extract, yeast extract, malt extract, corn steep liquor, casein hydrolysate, fish or its decomposition product, defatted soybean cake or its decomposition product can be used. These nitrogen sources can be used alone or in combination. The medium can contain potassium phosphate monobasic, potassium phosphate dibasic, and the corresponding sodium-containing salts as phosphorus. Potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus. In addition, inorganic compounds such as sodium chloride, calcium chloride, iron chloride, magnesium sulfate, iron sulfate, manganese sulfate, and calcium carbonate can be used. Finally, in addition to the above substances, essential growth substances such as amino acids and vitamins can be used.

[0049] Additionally, suitable precursors may be used in the culture medium. The above-mentioned raw materials may be added to the culture in a batch, fed-batch, or continuous manner during the culture process, but are not particularly limited thereto. The pH of the culture may be adjusted using basic compounds such as sodium hydroxide, potassium hydroxide, or ammonia, or acid compounds such as phosphoric acid or sulfuric acid, in an appropriate manner.

[0050] Additionally, foaming can be suppressed using a defoaming agent, such as a fatty acid polyglycol ester. Oxygen or an oxygen-containing gas (e.g., air) is injected into the culture to maintain aerobic conditions. The culture temperature is typically between 27°C and 37°C, preferably between 30°C and 35°C. Cultivation is continued until the maximum production of the peptide is achieved. This is typically achieved within 10 to 100 hours.

[0051] In addition, the step of recovering the peptide from the culture can be performed by a method known in the art. Specifically, the recovery method is not particularly limited thereto, as long as it can be used to recover the produced peptide, but preferably, a method such as centrifugation, filtration, extraction, spraying, drying, enrichment, precipitation, crystallization, electrophoresis, differential dissolution (e.g., ammonium sulfate precipitation), chromatography (e.g., ion exchange, affinity, hydrophobicity, and size exclusion) can be used.

[0052] According to the present invention, a pharmaceutical composition for wound healing containing a peptide derived from CPNE7 protein can be used in the form of a pharmaceutical composition for treating skin diseases caused by oxidative stress of dermal fibroblasts. Such a composition can be prepared by additionally including a suitable carrier (natural or unnatural carrier), excipient, or diluent commonly used. Specifically, the pharmaceutical composition can be formulated and used in the form of a sterile injection solution that can be administered to an area where a skin disease has occurred according to a conventional method. In the present invention, carriers, excipients, and diluents that can be included in the pharmaceutical composition include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol,

[0053] Examples include erythritol, maltitol, starch, acacia gum, alginate, gelatin, calcium phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil, collagen, etc. When formulated, it can be prepared using diluents or excipients such as fillers, bulking agents, binders, wetting agents, disintegrants, and surfactants that are commonly used. In particular, sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, lyophilized preparations, suppositories, ointments (e.g., pulp lining materials), etc. can be included. Non-aqueous solvents and suspending agents can include propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable esters such as ethyl oleate. Suppository bases that can be used include witepsol, macrogol, tween 61, cocoa butter, laurin butter, and glycerogelatin.

[0054] The content of the peptide included in the pharmaceutical composition of the present invention is not particularly limited thereto, but may be included in an amount of 0.0001 to 50 wt%, more preferably 0.01 to 20 wt%, based on the total weight of the final composition.

[0055] The pharmaceutical composition of the present invention may be administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount" as used herein means an amount sufficient to treat or prevent a disease at a reasonable benefit / risk ratio applicable to medical treatment or prevention, and the effective dosage level may be determined according to factors including the severity of the disease, the activity of the drug, the patient's age, weight, health, sex, the patient's sensitivity to the drug, the time of administration of the composition of the present invention used, the route of administration and the excretion rate, the treatment period, drugs used in combination or simultaneously with the composition of the present invention used, and other factors well known in the medical field. The pharmaceutical composition of the present invention may be administered alone or in combination with a known pharmaceutical composition for treating skin diseases caused by oxidative stress of dermal fibroblasts. It is important to take all of the above factors into consideration and administer an amount that can achieve the maximum effect with the minimum amount without side effects.

[0056] The dosage of the pharmaceutical composition of the present invention can be determined by those skilled in the art in consideration of the intended use, the degree of toxicity 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 at about 0.1 ng to about 100 mg / kg, preferably 1 ng to about 10 mg / kg, per adult, and the frequency of administration of the composition of the present invention is not particularly limited thereto, but can be administered once a day or administered in divided doses several times. The above dosage does not limit the scope of the present invention in any way.

[0057] In another aspect, the present invention provides a method for treating a skin disease caused by oxidative stress of dermal fibroblasts, comprising administering a pharmaceutical composition in a pharmaceutically effective amount to a subject other than a human suffering from a skin disease caused by oxidative stress of dermal fibroblasts.

[0058] The term "subject" of the present invention may include, without limitation, mammals including rats, livestock, etc., which require treatment for skin diseases caused by oxidative stress of dermal fibroblasts, but excludes humans among subjects suffering from the disease.

[0059] The pharmaceutical composition of the present invention for treating skin diseases caused by oxidative stress in dermal fibroblasts may be administered via any common route as long as it can reach the target tissue. The pharmaceutical composition of the present invention is not particularly limited thereto, but may be administered via oral administration, dermal administration, intramuscular injection, intravenous injection, subcutaneous injection, respiratory administration, rectal administration, or administration to the affected area, depending on the intended purpose.

[0060] Oral administration (intraoral administration) involves taking medication through the mouth. Medications are typically administered in the form of tablets, capsules, or liquids. Transdermal administration involves applying or attaching medication to the skin, and can be administered in the form of creams, gels, or patches. Intramuscular injection involves directly injecting medication into muscle tissue, typically using a syringe and needle. Intravenous injection involves directly injecting medication into a blood vessel, typically using a syringe and needle or an intravenous cannula. Subcutaneous injection involves injecting medication into fatty tissue beneath the skin, typically used for insulin. Inhaled medications reach the respiratory tract through the mouth or nose, and can be administered in the form of inhalers, gases, or vaporizers. Rectal administration involves injecting medication into the rectum, and can be administered in the form of rectal drops or rectal instillations. Topical administration involves directly applying medication to the affected area. Depending on the nature of the disease, these can be administered in the form of creams, gels, or patches.

[0061] In another aspect, the present invention provides a pharmaceutical composition for preventing or improving skin diseases caused by oxidative stress of dermal fibroblasts, comprising the peptide.

[0062] The term "improvement" in the present invention means any action that at least reduces a parameter related to the condition being treated, for example, the severity of a symptom.

[0063] In the present invention, the improvement may be interpreted to mean all acts that improve or benefit the symptoms of a skin disease caused by oxidative stress of dermal fibroblasts by promoting the removal of active oxygen in dermal fibroblasts by administering a pharmaceutical composition containing the peptide of the present invention as an active ingredient to a subject in need of treatment of a skin disease caused by oxidative stress of dermal fibroblasts.

[0064] The term "quasi-drug" in the present invention refers to products that are used for the purpose of diagnosing, treating, improving, alleviating, managing or preventing diseases of humans or animals, and have a milder effect than drugs. For example, according to the Pharmaceutical Affairs Act, quasi-drugs are products other than those used for the purpose of drugs, and include fiber and rubber products used for the treatment or prevention of diseases of humans or animals, products that have a mild or no direct effect on the human body, are not instruments or machines and similar thereto, and sterilizers and insecticides for preventing infectious diseases.

[0065] In the present invention, the type or formulation of the pharmaceutical composition containing the peptide is not particularly limited, but may be, for example, an ointment, a patch, a powder, a gelling agent, a spray, a tablet, or a spray.

[0066] In another aspect, the present invention provides a health functional food composition for preventing or improving skin diseases caused by oxidative stress of dermal fibroblasts, comprising the peptide.

[0067] The term "food" of the present invention includes dairy products including meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gum, ice cream, various soups, beverages, tea, drinks, alcoholic beverages, vitamin complexes, health functional foods, and health foods, and includes all foods in the conventional sense.

[0068] The above functional food is the same term as food for special health use (FoSHU), and refers to a food with high medical and healthcare effects that is processed to efficiently exhibit a bioregulatory function in addition to providing nutrition. Here, "functionality" means regulating nutrients for the structure and function of the human body or obtaining a useful effect for health purposes such as physiological action. The food of the present invention can be manufactured by a method commonly used in the art, and can be manufactured by adding raw materials and ingredients commonly added in the art during the manufacturing process. In addition, the formulation of the food can be manufactured without limitation as long as it is a formulation recognized as a food. The food composition of the present invention can be manufactured in various forms of formulations, and unlike general drugs, it has the advantage of not having side effects that may occur with long-term administration of drugs by using food as a raw material, and has excellent portability, so the food of the present invention can be consumed as a supplement to enhance the effect of preventing or improving skin diseases caused by oxidative stress of dermal fibroblasts.

[0069] The above-mentioned "health food" refers to foods that have a more active health maintenance or promotion effect than regular 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.

[0070] Specifically, the health functional food means a food product manufactured by adding the peptide of the present invention to food materials such as beverages, teas, spices, gums, and confectionery, or by manufacturing it in the form of encapsulation, powder, suspension, etc., and which has a specific health effect when consumed, but unlike general drugs, it has the advantage of not having side effects that may occur with long-term use of drugs made from food as a raw material.

[0071] The food composition of the present invention can be used very usefully because it can be consumed on a daily basis and is expected to be highly effective in preventing or improving skin diseases caused by oxidative stress of dermal fibroblasts.

[0072] The above food composition may additionally include a physiologically acceptable carrier. The type of the carrier is not particularly limited, and any carrier commonly used in the art may be used.

[0073] In addition, the food composition may include additional ingredients commonly used in food compositions to improve odor, taste, sight, etc. For example, it may include vitamins A, C, D, E, B1, B2, B6, B12, niacin, biotin, folate, pantothenic acid, etc. In addition, it may include minerals such as zinc (Zn), iron (Fe), calcium (Ca), chromium (Cr), magnesium (Mg), manganese (Mn), copper (Cu), chromium (Cr), etc. In addition, it may include amino acids such as lysine, tryptophan, cysteine, and valine.

[0074] In addition, the food composition may include food additives such as preservatives (potassium sorbate, sodium benzoate, salicylic acid, sodium dehydroacetate, etc.), sterilizers (bleaching powder and high-purity bleaching powder, sodium hypochlorite, etc.), antioxidants (butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT), etc.), colorants (tar colorants, etc.), color developers (sodium nitrite, sodium nitrite, etc.), bleaching agents (sodium sulfite), seasonings (MSG, monosodium glutamate, etc.), sweeteners (dulcin, cyclamate, saccharin, sodium, etc.), flavorings (vanillin, lactones, etc.), leavening agents (alum, D-potassium hydrogen tartrate, etc.), reinforcing agents, emulsifiers, thickeners (glucose fillers), film agents, gum bases, foam suppressants, solvents, and improvers. The above additives can be selected according to the type of food and used in an appropriate amount.

[0075] The peptide of the present invention can be added as is or used together with other foods or food ingredients, and can be used appropriately according to a conventional method. The amount of the active ingredient mixed can be appropriately determined depending on its purpose of use (prevention, health, or therapeutic treatment). Generally, when manufacturing a food or beverage, the food composition of the present invention can be added to the food or beverage in an amount of 50 parts by weight or less, specifically 20 parts by weight or less. However, when consumed for a long period of time for health and hygiene purposes, the content below the above range can be included, and since there is no problem in terms of safety, the active ingredient can also be used in an amount above the above range.

[0076] As an example, the food composition of the present invention can be used as a health beverage composition, and in this case, it can contain various flavoring agents or natural carbohydrates as additional ingredients like a regular beverage. The above-mentioned natural carbohydrates can be monosaccharides such as glucose and fructose; disaccharides such as maltose and sucrose; polysaccharides such as dextrin and cyclodextrin; sugar alcohols such as xylitol, sorbitol, and erythritol. The sweetener can be a natural sweetener such as thaumatin and stevia extract; a synthetic sweetener such as saccharin and aspartame, etc. The proportion of the natural carbohydrate can be generally about 0.01 to 0.04 g, specifically about 0.02 to 0.03 g per 100 mL of the health beverage composition of the present invention.

[0077] In addition to the above, the health beverage composition may contain various nutrients, vitamins, electrolytes, flavoring agents, coloring agents, pectic acid, salts of pectic acid, alginic acid, salts of alginic acid, organic acids, protective colloid thickeners, pH adjusters, stabilizers, preservatives, glycerin, alcohol, or carbonating agents. In addition, the health beverage composition may contain fruit pulp for the production of natural fruit juice, fruit juice drinks, or vegetable drinks. These ingredients may be used independently or in combination. The proportion of these additives is not particularly important, but is typically 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.

[0078] The food composition of the present invention may contain a peptide derived from CPNE7 protein in various weight % as long as it can exhibit an anti-aging or wound healing effect, but specifically, the peptide of the present invention may be contained in an amount of 0.00001 to 100 wt % or 0.01 to 80 wt % relative to the total weight of the food composition, but is not limited thereto.

[0079]

[0080] A composition for inhibiting skin aging or treating wounds, comprising a peptide derived from CPNE7 protein according to an embodiment of the present invention, can promote the synthesis of Type I Collagen and Type III Collagen, which are involved in promoting skin elasticity and preventing aging, within fibroblasts in the dermal layer of the skin.

[0081] A composition for inhibiting skin aging or treating wounds, comprising a peptide derived from CPNE7 protein according to another embodiment of the present invention, can promote the synthesis of Type IV Collagen, which is involved in promoting skin elasticity and preventing aging, in the basement membrane of the dermal-epidermal boundary of the skin.

[0082] According to another embodiment of the present invention, a composition for inhibiting skin aging or treating a wound, comprising a peptide derived from CPNE7 protein, is provided, which promotes regeneration of the epidermal layer and prevents aging within keratinocytes in the epidermal layer of the skin, and its use.

[0083] A composition for inhibiting skin aging or treating wounds, comprising a peptide derived from CPNE7 protein according to another embodiment of the present invention, can promote the synthesis of Filaggrin and Involucrin, which are involved in promoting skin moisturizing and preventing aging, within keratinocytes in the epidermal layer of the skin.

[0084] According to another embodiment of the present invention, a composition for inhibiting skin aging or treating wounds, comprising a peptide derived from CPNE7 protein, can reduce the expression of MMP-1, which is involved in preventing skin aging, by inhibiting collagen decomposition in the epidermal layer and dermal layer of the skin.

[0085]

[0086] The effects of the present invention are not limited to those mentioned above, and other effects not mentioned will be clearly understood by those skilled in the art to which the present invention pertains from the description below.

[0087]

[0088] Figure 1 shows the results of measuring the absorbance at 450 nm using a microplate after treating the peptide of the present invention for 48 hours at different concentrations, distributing the WST-8 solution in a 10:1 ratio to the culture medium, and reacting for 2 hours under light-shielding conditions in a 37°C incubator.

[0089] (A) of Fig. 2 shows the degree of Type I Collagen protein formation after treating with the peptide (#96) of the present invention at various concentrations for 7 days, and (B) of Fig. 4b shows the result of confirming the EC50 value according to Type I Collagen protein formation after treating with 100 uM of the peptide (#96) of the present invention 7 times for 7 days.

[0090] Figure 3a is a graph comparing the Type I Collagen protein expression levels for the peptides in Table 13.

[0091] Figure 3b shows the degree of collagen deposition compared to the control group after treatment with the peptide (#96) of the present invention at a concentration of 100 uM at 24-hour intervals for 7 days. (A) is the result of Confocal Microscope (Type I Collagen) analysis, and (B) is the result of analysis using the Confocal Microscope Z-Stack Imaging program.

[0092] Figure 3c shows the confirmation of the alpha-smooth muscle actin (α-SMA) synthesis efficacy of the peptide of the present invention in hDFs (N=3).

[0093] Figure 4 shows the results of confirming the collagen formation ability of human-derived dermal fibroblasts when treated multiple times with the peptide of the present invention.

[0094] Figure 5 shows the results of confirming the permeability of the peptide of the present invention through human-derived three-dimensional skin tissue.

[0095] Figure 6a shows the results confirming the efficacy of the peptide of the present invention on human-derived three-dimensional skin tissue.

[0096] Figure 6b shows the confirmation of the collagen formation ability of the peptide in Neoderm-ED skin tissue (ex vivo).

[0097] Figure 7 shows the results confirming the keratinocyte proliferation efficacy of the peptide of the present invention.

[0098] Figure 8 shows the results of confirming the efficacy of the peptide of the present invention according to skin treatment.

[0099] Figure 9 shows the results of confirming (A) the effect of reducing active oxygen and (B) the anti-photodamage effect according to treatment with the peptide of the present invention.

[0100] Figure 10 shows the results of confirming the melanin secretion reduction effect according to the concentration-specific treatment of the peptide of the present invention.

[0101] Figure 11 shows the results of confirming the migration efficacy of the peptide of the present invention in keratinocytes and hDF.

[0102]

[0103] The purposes and effects of the present invention, as well as the technical configurations for achieving them, will become clearer with reference to the embodiments described in detail below, along with the accompanying drawings. In describing the present invention, detailed descriptions of known functions or configurations will be omitted if they are deemed to unnecessarily obscure the gist of the invention. Furthermore, the terms described below are defined in consideration of the contributions made to the present invention, and may vary depending on the intentions or practices of the user or operator.

[0104] However, the present invention is not limited to the embodiments disclosed below and can be implemented in various different forms. These embodiments are provided solely to ensure complete disclosure of the present invention and to fully convey the scope of the invention to those skilled in the art. The present invention is defined solely by the scope of the claims. Therefore, such definitions should be based on the contents of this specification.

[0105] Hereinafter, embodiments of the present invention will be described in detail.

[0106]

[0107] Example 1. Synthesis of a peptide derived from CPNE7 protein

[0108]

[0109] The present inventors synthesized a peptide derived from CPNE7 protein (SEQ ID NO: 1) using the 9-fluorenylmethyloxycarbonyl (Fmoc) method, and substituted amino acids of the synthesized peptide to synthesize peptides of each group (Tables 1 to 12).

[0110] N-KYQRRKKNKY-C (SEQ ID NO: 1)

[0111] First, the peptide of group 1 was synthesized by substituting amino acids 5 to 7 of the peptide of sequence number 1 or the peptide of sequence number 1 with lysine or arginine (Table 1).

[0112] Group 1 Peptide Sequence Number Amino Acid Sequence (NC) 12345678 KYQRRKKNKYKYQRRKRNKYKYQRRRKNKYKYQRRRRNKYKYQRKKKNKYKYQRKRKNKYKYQRKKRNKYKYQRKRRNKY

[0113] Next, the peptide of group 2 was synthesized by substituting amino acids 5 to 7 of the peptide of sequence number 1 with lysine or arginine and substituting amino acid 8 with serine (Table 2).

[0114] Group 2 Peptide Sequence Number Amino Acid Sequence (NC) 910111213141516KYQRRKKSKYKYQRRKRSKYKYQRRRKSKYKYQRRRRSKYKYQRKKKSKYKYQRKRKSKYKYQRKKRSKYKYQRKRRSKY

[0115] Next, the peptide of group 3 was synthesized by substituting amino acids 5 to 7 of the peptide of sequence number 1 with lysine or arginine and substituting amino acid 9 with tyrosine (Table 3).

[0116] Group 3 Peptide Sequence Number Amino Acid Sequence (NC) 1718192021222324KYQRRKKNYKKYQRRKRNYKKYQRRRKNYKKYQRRRRNYKKYQRKKKNYKKYQRKRKNYKKYQRKKRNYKKYQRKRRNYK

[0117] Next, the peptide of group 4 was synthesized by substituting amino acids 5 to 7 of the peptide of sequence number 1 with lysine or arginine, amino acid 8 with serine, amino acid 9 with tyrosine, and amino acid 10 with lysine (Table 4).

[0118] Group 4 peptide sequence number amino acid sequence (NC) 2526272829303132KYQRRKKSYKKYQRRKRSYKKYQRRRKSYKKYQRRRRSYKKYQRKKKSYKKYQRKRKSYKKYQRKKRSYKKYQRKRRSYK

[0119] Next, the peptide of group 5 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with arginine, amino acid 4 with glutamine, and amino acids 5 to 7 with lysine or arginine (Table 5).

[0120] Group 5 Peptide Sequence Number Amino Acid Sequence (NC) 3334353637383940KYRQRKKNKYKYRQRKRNKYKYRQRRKNKYKYRQRRRNKYKYRQKKKNKYKYRQKRKNKYKYRQKKRNKYKYRQKRRNKY

[0121] Next, the peptide of group 6 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with arginine, amino acid 4 with glutamine, amino acids 5 to 7 with lysine or arginine, and amino acid 8 with serine (Table 6).

[0122] Group 6 Peptide Sequence Number Amino Acid Sequence (NC) 4142434445464748KYRQRKKSKYKYRQRKRSKYKYRQRRKSKYKYRQRRRSKYKYRQKKKSKYKYRQKRKSKYKYRQKKRSKYKYRQKRRSKY

[0123] Next, the peptide of group 7 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with arginine, amino acid 4 with glutamine, amino acids 5 to 7 with lysine or arginine, amino acid 9 with tyrosine, and amino acid 10 with lysine (Table 7).

[0124]

[0125] Group 7 peptide sequence number amino acid sequence (NC) 4950515253545556KYRQRKKNYKKYRQRKRNYKKYRQRRKNYKKYRQRRRNYKKYRQKKKNYKKYRQKRKNYKKYRQKKRNYKKYRQKRRNYK

[0126] Next, the peptide of group 8 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with arginine, amino acid 4 with glutamine, amino acids 5 to 7 with lysine or arginine, amino acid 8 with serine, amino acid 9 with tyrosine, and amino acid 10 with lysine (Table 8).

[0127] Group 8 peptide sequence number amino acid sequence (NC) 5758596061626364KYRQRKKSYKKYRQRKRSYKKYRQRRKSYKKYRQRRRSYKKYRQKKKSYKKYRQKRKSYKKYRQKKRSYKKYRQKRRSYK

[0128] Next, the peptide of group 9 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with lysine, amino acid 4 with glutamine, and amino acids 5 to 7 with lysine or arginine (Table 9).

[0129] Group 9 peptide sequence number amino acid sequence (NC) 6566676869707172 KYKQRKKNKYKYKQRKRNKYKYKQRRKNKYKYKQRRRNKYKYKQKKKNKYKYKQKRKNKYKYKQKKRNKYKYKQKRRNKY

[0130] Next, the peptide of group 10 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with lysine, amino acid 4 with glutamine, amino acids 5 to 7 with lysine or arginine, and amino acid 8 with serine (Table 10).

[0131] Group 10 Peptide Sequence Number Amino Acid Sequence (NC) 7374757677787980KYKQRKKSKYKYKQRKRSKYKYKQRRKSKYKYKQRRRSKYKYKQKKKSKYKYKQKRKSKYKYKQKKRSKYKYKQKRRSKY

[0132] Next, the peptide of group 11 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with lysine, amino acid 4 with glutamine, amino acids 5 to 7 with lysine or arginine, amino acid 9 with tyrosine, and amino acid 10 with lysine (Table 11).

[0133] Group 11 Peptide Sequence Number Amino Acid Sequence (NC) 8182838485868788KYKQRKKNYKKYKQRKRNYKKYKQRRKNYKKYKQRRRNYKKYKQKKKNYKKYKQKRKNYKKYKQKKRNYKKYKQKRRNYK

[0134] Finally, the peptide of group 12 was synthesized by substituting amino acid 3 of the peptide of sequence number 1 with lysine, amino acid 4 with glutamine, amino acids 5 to 7 with lysine or arginine, amino acid 8 with serine, amino acid 9 with tyrosine, and amino acid 10 with lysine (Table 12).

[0135] Group 12 Peptide Sequence Number Amino Acid Sequence (NC) 8990919293949596 KYKQRKKSYKKYKQRKRSYKKYKQRRKSYKKYKQRRRSYKKYKQKKKSYKKYKQKRKSYKKYKQKKRSYKKYKQKRRSYK

[0136] Example 2. WST-8 Assay (Cellrix, B1007-500)

[0137]

[0138] Human dermal fibroblasts (hDFs) were seeded at 0.3 x 10 in a 96-well plate (using Fibroblast Growth Medium (Lonza, CC-4126) containing 2% FBS, 0.1% Insulin, 0.1% FGF, and 0.1% Gentamycin sulfate-Amphotericin). 5 / well and cultured for 24 hours in an incubator at 37°C and 5% CO2 conditions.

[0139]

[0140] To confirm the cytotoxicity of the peptide (SEQ ID NO: 96) in dermal fibroblasts, a WST-8 assay was performed. The peptide was treated with dermal fibroblasts at concentrations of 10, 100 uM, 1, 5, and 10 mM, respectively. After 48 hours of treatment at each concentration, the WST-8 solution was dispensed into culture medium at a ratio of 10:1, and incubated in a 37°C incubator under light-shielding conditions for 2 hours. The absorbance was then measured at 450 nm using a microplate.

[0141]

[0142] Figure 1 shows the results of measuring the absorbance at 450 nm using a microplate after treating the peptide (#96) of the present invention for 48 hours at different concentrations, distributing the WST-8 solution in a 10:1 ratio to the culture medium, and reacting for 2 hours under light-shielding conditions in a 37°C incubator.

[0143]

[0144] Dermal fibroblasts were treated with the peptide (#96) of the present invention at concentrations of 10, 100 uM, 1, 5, and 10 mM, respectively. After 48 hours, cell viability was quantified using the WST-8 assay. As a result, the peptide (#96) of the present invention did not exhibit cytotoxicity toward dermal fibroblasts up to a concentration of 5 mM, and exhibited significant cytotoxicity at a concentration of 10 mM.

[0145]

[0146] Example 3-1. Confirmation of EC50 values ​​for Type I collagen formation according to multiple peptide treatments in hDFs.

[0147]

[0148] In order to confirm the Type I collagen synthesis ability according to multiple treatments of the peptide (#96) of the present invention, dermal fibroblasts were seeded in a 60 mm dish at a density of 1x105 / The cells were dispensed into 6-well plates and cultured for 24 hours in a 37°C, 5% CO2 incubator. Here, the peptide (SEQ ID NO: 96) was treated at concentrations of 1 pM, 10 pM, 100 pM, 1 nM, 10 nM, 1000 nM, 1 uM, 10 uM, 100 uM, and 1 mM at 24-hour intervals for 7 days (11 groups in total).

[0149] Afterwards, the cells were washed with PBS, the proteins were separated, and the protein changes in Type I Collagen were confirmed through Western blot, and compared by normalization through the amount of β-Actin protein.

[0150] Figure 2 (A) shows the degree of Type I Collagen protein formation after treating with the peptide (#96) of the present invention at various concentrations for 7 days, and Figure 4b (B) shows the results of confirming the EC50 value according to Type I Collagen protein formation after treating with 100 uM of the peptide (#96) of the present invention 7 times for 7 days. Referring to Figure 2, the EC50 value was confirmed to be 8.621 pM when treating with the peptide (#96) of the present invention multiple times, and it can be seen that the expression of Type I Collagen significantly increases compared to the control group.

[0151]

[0152] Example 3-2. Confirmation of the Type I collagen synthesis ability of peptides of groups 1 to 12 in human-derived dermal fibroblasts.

[0153]

[0154] To determine the Type I collagen synthesis ability of each peptide group, dermal fibroblasts were seeded at 1x10 in a 60 mm dish. 5 / The cells were dispensed into 6-well plates and cultured for 24 hours in a 37°C, 5% CO2 incubator. 100 μM of each peptide was treated and cultured for 24 hours. After 24 hours, the cells were washed with PBS, and the protein was isolated. The protein amount of Type I Collagen was confirmed through Western blot, and the amount was normalized and compared with the amount of β-Actin protein.

[0155] Table 13 shows the results of examining changes in the amount of Type I Collagen protein 24 hours after a single treatment of dermal fibroblasts with 100 uM concentrations of each group of peptides 1 to 12 of the present invention. Figure 3a is a graph comparing the amount of Type I Collagen protein expression for the peptides of Table 13. As can be seen from this, the peptides of the present invention exhibit excellent Type I collagen synthesis ability.

[0156]

[0157] SEQ ID NO Amino Acid Sequence Relative Protein Expression levels of Type I CollagenControl-11KYQRRKKNKY1.335168KYQRKRRNKY2.311519KYQRRKKSKY1.518216KYQRKRRSKY2.8300617KYQRRKKNYK1.9985724KYQRKRRNYK2.8985 25KYQRRKKSYK1.8852132KYQRKRRSYK3.2917833KYRQRKKNKY2.0341640KYRQKRRNKY3.5052441KYRQRKKSKY1.657548KYRQKRRSKY3.1583449KYRQRKKNYK1 .2733456KYRQKRRNYK2.8084557KYRQRKKSYK1.4006764KYRQKRRSYK3.6240465KYKQRKKNKY1.6199472KYKQKRRNKY3.0821873KYKQRKKSKY1.9176480KYKQ KRRSKY4.6899681KYKQRKKNYK3.2870488KYKQKRRNYK4.4088589KYKQRKKSYK2.3189993KYKQKKKSYK2.6312795KYKQKKRSYK3.4318296KYKQKRRSYK2.14839

[0158] Example 3-3. Confirmation of Type I collagen deposition following multiple administrations of peptides in hDFs

[0159]

[0160] In order to confirm the Type I collagen synthesis ability according to multiple administrations of the peptide (#96) of the present invention, dermal fibroblasts were seeded in a 60 mm dish at a density of 1x10 5 / The cells were dispensed into 6-well plates and cultured for 24 hours in a 37°C, 5% CO2 incubator. Here, the peptide (SEQ ID NO: 96) and Vitamin C (positive control) were treated at a concentration of 100 uM at 24-hour intervals for 7 days.

[0161] Afterwards, the cells were washed with PBS, the proteins were separated, and the protein changes in Type I Collagen were confirmed using a Confocal Microscope (Type I Collagen).

[0162] Figure 3b shows the degree of collagen deposition compared to the control group after treatment with the peptide (#96) of the present invention at a concentration of 100 uM at 24-hour intervals for 7 days. (A) is the result of Confocal Microscope (Type I Collagen) analysis, and (B) is the result of analysis using the Confocal Microscope Z-Stack Imaging program.

[0163] Through this, it was confirmed that on the 7th day, the amount of Type I Collagen in the ECM (Extracellular Matrix) significantly increased in the group treated with the peptide (#96) of the present invention compared to the control group.

[0164]

[0165] Example 3-4. Confirmation of the α-SMA synthesis efficacy of peptides in hDFs

[0166] In order to confirm the possibility of promoting wound healing by administering the peptide (#96) of the present invention, dermal fibroblasts were administered at a density of 1.5x10 5 / Dispensed into confocal dishes and cultured in a 37 ℃ 5% CO2 incubator for 24 hours. Here, peptide (#96) and 100 uM Vitamin C (positive control) were treated for 48 hours.

[0167] Afterwards, the protein changes in alpha-smooth muscle actin (α-SMA) were confirmed using a confocal microscope after washing the cells with PBS.

[0168] α-Smooth muscle actin (α-SMA) is expressed by fibroblasts in the dermis, providing structural support for skin tissue and maintaining skin elasticity. Furthermore, when a wound occurs, fibroblasts at the wound site express α-SMA, which acts to contract the wounded skin. This can reduce the size of the wound during the wound healing process and protect the wounded skin from tissue damage. 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 playing a crucial role in the maintenance and stability of skin tissue.

[0169]

[0170] Figure 3c shows the confirmation of the alpha-smooth muscle actin (α-SMA) synthesis efficacy of peptide (#96) in hDFs (N=3). Referring to Figure 3c, it can be seen that a single treatment with 100 uM of peptide (#96) significantly increases α-SMA and has a wound healing promoting effect.

[0171]

[0172] Example 4. Observation of cell changes using an optical microscope

[0173]

[0174] The collagen synthesis efficacy of Vitamin C, known as a representative substance that increases the collagen synthesis capacity of dermal fibroblasts in vitro, and the peptide (#96) of the present invention were compared by applying them to human-derived dermal fibroblasts. The cells were divided into a single application group (total of 1 application) and a multiple application group (total of 7 applications), and were treated with 100uM of the peptide (#96) of the present invention and 100uM of Vitamin C, respectively, for a total of 7 days.

[0175]

[0176] Specifically, dermal fibroblasts were seeded at 0.3x10 in a 96-well plate. 5 ​ / well and cultured for 24 hours in a 37 ℃ 5% CO2 incubator.

[0177]

[0178] Picro Sirius Red staining is one of the representative collagen staining methods that mainly targets Type I Collagen and Type III Collagen. After single application (total of 1 application) and multiple application (total of 7 applications) of 100 uM of the peptide (#96) of the present invention and 100 uM of Vitamin C for a total of 7 days, Picro Sirius Red staining analysis was performed. When the collagen positive for Picro Sirius Red was quantified, it was observed that the peptide (#96) treatment group of the present invention was higher than the control group in the single application group (Fig. 4). In the multiple application group, the collagen positive for Picro Sirius Red was significantly increased in the peptide (#96) treatment group of the present invention and the Vitamin C treatment group compared to the control group, and the quantitative results were also significant (Fig. 4).

[0179]

[0180] Example 5-1. Results of skin penetration of the peptide of the present invention in human-derived three-dimensional skin tissue

[0181]

[0182] To confirm the skin penetration potential of the peptide of the present invention, three-dimensional skin tissue consisting of the epidermis and dermis was created using human-derived keratinocytes and dermal fibroblasts, and then treated with 1 mM of the peptide of the present invention (#96) conjugated to a fluorescent substance. After treatment, the location of fluorescence was observed continuously for 1, 6, 12, and 24 hours using a confocal microscope.

[0183]

[0184] After 1 hour of treatment, the peptide (#96) of the present invention was observed in the stratum corneum, and after 6 hours, in the epidermis (Fig. 5). After 12 hours, it began to be observed in the dermis, and after 24 hours, the peptide (#96) of the present invention was clearly observed to be present in the dermis (Fig. 5). This confirmed that the peptide (#96) of the present invention was able to penetrate through the stratum corneum and epidermis to the dermis.

[0185]

[0186] Example 5-2. Confirmation of skin permeability of peptides in human-derived three-dimensional skin tissue.

[0187]

[0188] Peptide-FITC was produced using the peptide (#96) of the present invention by attaching FITC fluorescence to the Lysine residue of the peptide (#96). 1 mM of peptide-FITC was treated on human-derived 3D skin tissue (Tegoscience, Neoderm-ED), and the skin permeability of peptide-FITC was confirmed using a confocal microscope every 1 hour, 6 hours, 12 hours, and 24 hours.

[0189]

[0190] Specifically, in order to confirm the histological and cytological changes upon treatment with the peptide (#96) of the present invention in human-derived three-dimensional skin tissue, analysis was performed by staining tissue sections with Hematoxylin & Eosin (H&E) 24 hours after treatment with 1 mM of the peptide (#96) of the present invention.

[0191]

[0192] The normal epidermis is composed of the stratum corneum, stratum lucidum, stratum granulosum, stratum spinosum, and stratum basale, from the outside in. Millions of new keratinocytes are formed in the stratum basale every day, and these cells do not remain in one place but are continuously pushed up to the outermost layer of the epidermis. As the keratinocytes are pushed up, they gradually harden into keratin. On the surface of human skin, aged keratinocytes are continuously shed. However, in aged skin, new keratinocyte formation is slow, so it takes longer for the stratum corneum to shed, causing it to thicken. Therefore, the decline in keratinocyte function leads to an increase in dead keratinocytes, which can directly cause fine wrinkles and rough skin.

[0193]

[0194] Histological analysis revealed that in the control group, keratinocytes were observed in the basal layer, but the stratum spinosum, stratum granulosum, stratum lucidum, and stratum corneum had not yet been properly formed (Fig. 6a). In contrast, in the group treated with the peptide of the present invention, histological characteristics of normal epidermis were clearly observed (Fig. 6a). This suggests that the peptide of the present invention can promote keratinization by enhancing keratinocyte function.

[0195]

[0196] Example 5-3. Confirmation of the Collagen-forming Ability of Peptides in Human-Derived 3D Skin Tissue (Ex vivo)

[0197]

[0198] The collagen synthesis efficacy of Vitamin C and the peptide of the present invention was compared by applying it to human-derived dermal fibroblasts. In order to confirm the collagen formation ability of the peptide of the present invention in skin tissue, 3D skin tissue consisting of the epidermis and dermis was created using human-derived keratinocytes and dermal fibroblasts (Neoderm-ED), and then treated twice with 1,000 uM of Vitamin C and the peptide of the present invention (#96) at 10 uM, 100 uM, 300 uM, and 1,000 uM, respectively. After treatment, the collagen formation ability of the negative control group, positive control group, and treatment group was confirmed at 48-hour intervals using the Masson Trichrome Staining method.

[0199] Figure 6b confirms the collagen formation ability of peptides in Neoderm-ED skin tissue (ex vivo). Figure 6b demonstrates the excellent collagen formation ability achieved by the peptide treatment of the present invention. Specifically, it can be seen that the amount of collagen (Blue) deposition increases in a dose-dependent manner in the peptide (#96) treatment group compared to the negative control group (NC group).

[0200]

[0201] Example 5-4. Peptide skin efficacy test in human-derived 3D skin tissue

[0202]

[0203] To confirm the proliferation efficacy of the peptide of the present invention in keratinocytes within the basal layer, immunofluorescence staining analysis of Ki-67, a representative cell division and proliferation marker, was performed. To this end, human-derived 3D skin tissues (Tegoscience, Neoderm-ED) were treated with 1 mM of the peptide of the present invention (#96) and cultured in a 5% CO2 incubator at 37°C for 24 hours. Afterwards, each human-derived 3D skin tissue was fixed and frozen sections were prepared. The sectioned tissues were stained with hematoxylin & eosin and comparatively analyzed using an optical microscope (confocal microscope).

[0204]

[0205] Referring to Fig. 7, it can be confirmed that 24 hours after 1 mM treatment of human-derived 3D skin tissue with the peptide (#96) of the present invention, the number of Ki-67 positive cells in keratinocytes in the basal layer increased compared to the control group. As a result of quantitative analysis of the ratio of Ki-67 positive cells, the peptide (#96) treatment group of the present invention showed a 5.3-fold increase compared to 12.2 (±3.6)% of the control group, which was 64.6 (±11.1%) (Fig. 7). This can be interpreted that the peptide of the present invention promotes cell division and proliferation of keratinocytes.

[0206]

[0207] Example 5-5. Peptide skin efficacy test in human-derived 3D skin tissue

[0208]

[0209] In order to confirm the skin efficacy of the peptide of the present invention, 24 hours after treatment of 1 mM of the peptide of the present invention on human-derived three-dimensional skin tissue, the expression patterns of Type IV Collagen, MMP-1, Filaggrin, and Involucrin were compared using immunofluorescence analysis.

[0210]

[0211] Type IV collagen is primarily expressed in the epidermal basement membrane (BMB) at the dermal-epidermal junction. The BMB serves an anatomical function, connecting the epidermis to its underlying structure, the dermis. It facilitates the permeation of fluid between the two layers, while simultaneously acting as a barrier to control the penetration of inflammatory and tumor cells. Recent studies have shown that changes in the BMB occur in aged skin, accompanied by a decrease in Type IV collagen. These changes can lead to not only keratinocyte deformation but also an increase in matrix metalloproteinases (MMPs), collagen-degrading enzymes.

[0212]

[0213] Referring to Fig. 8, it can be confirmed that the peptide (#96) of the present invention reduced the expression of MMP-1 in the epidermis and dermis of human-derived three-dimensional skin tissue and increased the expression of Type IV Collagen in the basement membrane. Maintaining skin moisture is a basic condition for maintaining healthy skin, and the stratum corneum maintains moisture through natural moisturizing factors such as filaggrin and involucrin, a lipid layer existing between keratinocytes, and sebum secreted from sebaceous glands.

[0214]

[0215] Filaggrin acts as an adhesive that connects the outer membrane of keratinocytes and keratin intermediate filaments, and involucrin plays an important role in the formation and maintenance of the function of keratinocyte cell membranes, which serve as a physical barrier to the skin. A decrease in their expression can cause skin diseases such as psoriasis and atopic dermatitis due to a decline in skin barrier function. In this regard, referring to Fig. 8, it can be confirmed that the peptide of the present invention increases the expression levels of natural moisturizing factors Filaggrin and Involucrin in the epidermis of human-derived three-dimensional skin tissue (Fig. 8).

[0216]

[0217] Example 6-1. Confirmation of anti-photodamage efficacy of peptides in Neoderm-ME tissues

[0218] To confirm the skin anti-photodamage efficacy of the peptide of the present invention, UV-B light was irradiated on human-derived three-dimensional skin tissue composed of keratinocytes and melanocytes. 48 hours after treatment with 100 uM of the peptide of the present invention, the expression patterns of DCFDA staining, Filaggrin, and Involucrin were compared using immunofluorescence analysis.

[0219]

[0220] Figure 9 shows the results of confirming the (A) active oxygen reduction effect and (B) anti-photodamage effect according to treatment with peptide (#96), and it can be confirmed that the peptide (#96) treatment group provides a significant skin damage reduction effect according to UV-B irradiation.

[0221]

[0222] Example 6-2. Skin whitening efficacy using Neoderm-ME

[0223] To confirm the skin whitening efficacy of the peptide of the present invention, human-derived three-dimensional skin tissue composed of keratinocytes and melanocytes was irradiated with UV-B rays. After 48 hours of treatment with 30 uM, 100 uM, 300 uM, and 1000 uM of the peptide of the present invention, the results were compared using Melanin Content Assay and Fontana Masson Staining analysis.

[0224]

[0225] Figure 10 shows the efficacy of reducing melanin secretion according to the concentration of peptide (#96), and it was confirmed that a significant reduction in melanin secretion occurred at a concentration of 30 uM or higher.

[0226]

[0227] Example 7. Migration Efficacy Test of #96 in Keratinocytes and hDF

[0228] In order to confirm the possibility of promoting cell migration and wound healing by administration of the peptide (#96) of the present invention, keratinocytes and dermal fibroblasts were administered at a density of 1.5x10 5 / Dispensed into Transwell and treated with 100 uM of peptide (#96) and cultured in a 37°C, 5% CO2 incubator for 48 hours.

[0229] Afterwards, the cells were washed with PBS, stained with Crystal violet, and the change in cell movement was confirmed using a light microscope.

[0230]

[0231] Figure 11 confirms the cell migration efficacy of the peptide of the present invention in keratinocytes and hDF, and confirms that the peptide of the present invention promotes cell migration in human dermal fibroblasts and keratinocytes and has a wound healing promoting effect.

[0232]

[0233] This specification and drawings have disclosed preferred embodiments of the present invention. Although specific terms have been used, they are used in a general sense only to easily explain the technical content of the present invention and to aid in understanding the invention, and are not intended to limit the scope of the present invention. It will be apparent to those skilled in the art that other modifications based on the technical concept of the present invention are possible in addition to the embodiments disclosed herein.

Claims

1. A composition for treating skin aging or skin wounds, comprising a peptide having an 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 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).

2. A composition according to claim 1, characterized in that the composition 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. A composition according to claim 1, characterized in that the peptide has an amino acid sequence of any one of SEQ ID NOs: 1 to 96.

4. A polynucleotide encoding the peptide of paragraph 1.

5. An expression vector comprising the polynucleotide of clause 6.

6. A 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 moisturizing, comprising a peptide consisting of any one of the amino acid sequences of sequence numbers 1 to 96.

8. A pharmaceutical composition for preventing and improving skin aging by promoting skin regeneration, elasticity, and moisturizing, comprising a peptide consisting of any one of the amino acid sequences of sequence numbers 1 to 96.

9. A cosmetic composition for preventing and improving skin aging by promoting skin regeneration, elasticity and moisturizing, comprising a peptide consisting of any one of the amino acid sequences of sequence numbers 1 to 96.

10. A health functional food composition for preventing and improving skin aging by promoting skin regeneration, elasticity, and moisturizing, comprising a peptide consisting of any one of the amino acid sequences of sequence numbers 1 to 96.

11. A composition for skin treatment, characterized in that the composition further comprises a pharmaceutically acceptable carrier, excipient or diluent in the 7th paragraph.

12. A method for preventing, improving or treating skin aging by administering the composition of Article 7 to a non-human subject to promote skin regeneration, elasticity and moisturizing.

13. In paragraph 1, A composition wherein the above skin wound is caused by a wound, diabetes, or bedsore.

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