Peptides with skin condition improving activity and their uses

Abstract

The present invention relates to a peptide having skin condition improving activity and its uses, and provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1, and a cosmetic composition for improving skin condition containing the peptide as an active ingredient.

Description

Technical Field

[0001] The present invention relates to a peptide having an activity of improving skin condition and its use.

Background Art

[0002] Human skin constantly experiences changes, and the most typical ones are the decline in skin function due to aging and the reduction in visual beauty. Aging causes skin wrinkles, and typical factors for wrinkle formation include ultraviolet exposure and decreased biosynthesis of collagen. Skin aging is broadly classified into intrinsic aging due to genetic factors and extrinsic aging due to external environmental factors such as sunlight. In the case of extrinsic aging, it is known to prevent, treat or delay aging through removal of reactive oxygen species, promotion of fibroblast proliferation and collagen biosynthesis.

[0003] On the other hand, collagen, which is a major component of the extracellular matrix, is the main matrix protein produced by skin fibroblasts. Collagen forms most of the organic substances in skin, tendon, bone and teeth, and particularly has a high content in bone and skin (dermis). Such collagen decreases due to photoaging caused by age and ultraviolet irradiation, and this is known to be closely related to skin wrinkle formation. Also, collagen plays an important role in wound healing, promoting the synthesis of collagen in damaged epithelium and enabling the wound to recover quickly and without scarring. At the same time, it has been reported that by promoting the biosynthesis of collagen, if the density of the basal layer etc. increases, the melanin pigment concentration per unit skin density decreases, and the skin tone can be expected to become brighter.

[0004] Under such a technical background, extensive research for improving skin condition has been carried out through mechanisms such as promotion of collagen biosynthesis, proliferation and enhanced activity of fibroblasts etc. (Korean Patent Publication No. 10-1043081), but the actual situation is still imperfect.

Summary of the Invention

[0005] One embodiment provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0006] Another embodiment provides a cosmetic composition for improving skin condition that contains the peptide as an active ingredient.

[0007] Other objects and advantages of the present invention will become even clearer from the following detailed description, along with the appended claims and drawings. Any matters not described herein are readily apparent and inferable to those skilled in the art of the present invention or similar art, and are therefore omitted from this description. [Means for solving the problem]

[0008] Each description and embodiment disclosed in this invention may also apply to other descriptions and embodiments. That is, all combinations of the various elements disclosed in this invention fall within the scope of this invention. Furthermore, the scope of this invention is not considered to be limited by the following specific descriptions.

[0009] One embodiment provides a peptide consisting of the amino acid sequence of SEQ ID NO: 1.

[0010] As used herein, the term "peptide" means a linear molecule formed by the bonding of amino acid residues to one another via peptide bonds. Such peptides can be produced by chemical synthesis methods known in the art, particularly by solid-phase synthesis techniques (Merrifield, J. Amer. Chem. Soc. 85:2149-54 (1963)) or liquid-phase synthesis techniques (US Patent No. 5,516,891).

[0011] The inventors, in their efforts to develop a peptide with biologically effective activity, have identified a peptide consisting of the amino acid sequence of Sequence ID No. 1. Here, the biologically effective activity exhibits one or more properties selected from (a) promotion of human dermal fibroblast proliferation and (b) increased expression of extracellular dermal matrix selected from collagen type 1, fibronectin, or elastin. Therefore, the peptide can be utilized for applications in improving skin conditions.

[0012] The peptide is fitted with a protecting group at its N or C terminus to acquire chemical stability, enhanced pharmacological properties (half-life, absorption, potency, efficacy, etc.), altered specificity (e.g., broad biological activity spectrum), and reduced antigenicity. Therefore, the peptide can be used as encompassing the peptide itself or its modified derivatives. In one specific example, the N-terminus of the peptide may be bonded to any one protecting group selected from the group consisting of acetyl, fluorenylmethoxycarbonyl, formyl, palmitoyl, myristyl, stearyl, butoxycarbonyl (Boc), allyloxycarbonyl (Alloc), and polyethylene glycol (PEG), and / or the C-terminus of the peptide may be bonded to any one protecting group selected from the group consisting of amino (-NH2), tertiary alkyl (-NH2), and azide (-NHNH2). The peptide may also selectively further include targeted sequences, tags, labeled residues, and amino acid sequences manufactured for specific purposes to increase half-life or peptide stability.

[0013] The peptides are artificially synthesized, non-naturally occurring, or engineered, where “non-naturally occurring or engineered” means a state produced by artificial modification rather than being in its natural state. Here, such artificial modification may include artificially synthesizing an amino acid sequence by mimicking a plurality of amino acid structures, or, as described above, being engineered to acquire chemical stability, enhanced pharmacological properties, altered specificity, or reduced antigenicity.

[0014] As used herein, the term "stability" refers not only to in vivo stability, which protects the peptide from attack by endogenous protein-cleaving enzymes, but also to storage stability (e.g., room temperature storage stability).

[0015] Another embodiment provides a cosmetic composition for improving skin condition that contains a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0016] Any terms or elements mentioned in the description of the aforementioned peptide that are the same as those already mentioned are as previously stated.

[0017] As used herein, the term "improvement" means all actions that reduce, for example, the degree of symptoms, which are parameters related to the alleviation or treatment of a condition.

[0018] As used herein, the term "improvement of skin condition" comprehensively means the process or effect of treating, reducing, or alleviating skin damage caused by intrinsic or extrinsic factors of the skin, and is interpreted to include, but is not limited to, wrinkle reduction, improved skin elasticity, or wound healing.

[0019] Here, "wrinkle improvement," "improvement of skin elasticity," and "wound healing" refer to all effects that increase the total amount of extracellular matrix factors, including the promotion of collagen synthesis.

[0020] Conventional functional peptides, despite their effective biological activity, have drawbacks such as not being effectively absorbed into target tissues or cells due to the size of the peptide itself, or being eliminated from the body in a short period due to their short half-life. On the other hand, the cosmetic composition according to one example contains a peptide consisting of 10 or fewer amino acids as an active ingredient, resulting in excellent skin penetration of the active ingredient. For example, when applied topically to the skin, it can effectively improve skin condition.

[0021] According to one example, the peptide demonstrated the effect of promoting the proliferation of human dermal fibroblasts and increasing the expression of extracellular dermal matrix selected from collagen type 1, fibronectin, or elastin. Therefore, the peptide can be used as an active ingredient in cosmetic compositions for improving skin condition.

[0022] The cosmetic composition contains, but is not limited to, a cosmetically effective amount of the peptide and / or a cosmetically acceptable carrier.

[0023] As used herein, the term "cosmetically effective amount" means an amount sufficient to achieve the skin condition improvement efficacy of the cosmetic composition.

[0024] The weight ratio between the peptide and the cosmetically acceptable carrier is, for example, 500:1 to 1:500, and is not limited to, but may include, for example, 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2.

[0025] The cosmetic composition is manufactured in any dosage form that is commonly manufactured in the industry. For example, it can be formulated into solutions, suspensions, emulsions, pastes, gels, creams, lotions, powders, soaps, surfactant-containing cleansers, oils, powder foundations, emulsion foundations, wax foundations, and sprays, etc., but is not limited thereto. For example, it can be manufactured in dosage forms such as softening lotions, nourishing lotions, nourishing creams, massage creams, essences, eye creams, cleansing creams, cleansing foams, cleansing waters, packs, sprays, or powders.

[0026] When the dosage form of the cosmetic composition is a paste, cream or gel, animal oils, vegetable oils, waxes, paraffins, starches, tragacanths, cellulose derivatives, polyethylene glycols, silicones, bentonites, silicas, talcs or zinc oxides, etc. can be used as the carrier component.

[0027] When the dosage form of the cosmetic composition is a powder or a spray, lactose, talc, silica, aluminum hydroxide, calcium silicate or polyamide powder is used as the carrier component. For example, when it is a spray, it may additionally contain a propellant such as chlorofluorohydrocarbon, propane / butane or dimethyl ether.

[0028] When the dosage form of the cosmetic composition is a solution or an emulsion, a solvent, a solubilizer or an emulsifier is used as the carrier component. For example, it may contain water, ethanol, isopropanol, ethyl carbonate, ethyl acetate, benzyl alcohol, benzyl benzoate, propylene glycol, 1,3-butyl glycol oil, glycerol fatty acid ester, polyethylene glycol or sorbitan fatty acid ester.

[0029] When the dosage form of the cosmetic composition is a suspension, the carrier component may be a liquid diluent such as water, ethanol, or propylene glycol; a suspension agent such as ethoxylated isostearyl alcohol, polyoxyethylene sorbitol ester, or polyoxyethylene sorbitan ester; or microcrystalline cellulose, aluminum methhydroxyl, bentonite, aga, or tracant.

[0030] When the dosage form of the cosmetic composition is a surfactant-containing cleanser, the carrier component may be an aliphatic alcohol sulfate, an aliphatic alcohol sulfate ether, a sulfosuccinate monoester, isethionate salt, imidazolinium derivative, methyl taurate, sarcosinate salt, fatty acid amide ether sulfate, alkylamide betaine, aliphatic alcohol, fatty acid glyceride, fatty acid diethanolamide, vegetable oil, lanolin derivative, or ethoxylated glycerol fatty acid ester.

[0031] The peptide may be contained in nanosomes or nanoparticles to further improve skin penetration or stability issues. For example, the nanosomes may be produced in a microfluidizer using lecithin as a raw material and contained within lecithin particles. Any known method for producing the nanosomes can be used. The size of the nanosome particles is preferably 30 to 200 nm. If the size of the nanosome particles is less than 30 nm, skin penetration proceeds very quickly, causing skin side effects, and if it exceeds 200 nm, skin penetration is not easy, making it difficult to obtain the benefits of using the nanosome structure.

[0032] The components contained in the cosmetic composition include, in addition to the peptide and carrier components as active ingredients, components commonly used in cosmetic compositions, such as antioxidants, stabilizers, solubilizers, vitamins, pigments, and fragrances.

[0033] The content of peptides as active ingredients in the cosmetic composition can be appropriately and non-restrictively selected depending on the shape of the product, the desired use, etc., and can be added in amounts of 0.01 to 15% by weight of the total weight of the cosmetic composition. Alternatively, for example, the cosmetic composition may contain peptides in amounts of 1.0% to 3.0% by weight, preferably 2.0% to 3.0% by weight, based on the total weight.

[0034] Another embodiment provides a method for improving skin condition, comprising the step of applying a cosmetic composition containing a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient to the skin of an individual.

[0035] The terms or elements mentioned in the description of the cosmetic composition that are the same as those already mentioned are as described above.

[0036] As used herein, the terms “apply,” “administer,” and “coat” are interchangeable and may mean bringing a composition according to one embodiment to a desired site at least partially, or placing a composition according to one embodiment into an individual via an administration route.

[0037] Another embodiment provides a pharmaceutical composition for improving skin condition, comprising a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0038] Any terms or elements mentioned in the description of the aforementioned peptide that are the same as those already mentioned are as previously stated.

[0039] According to one example, the peptide demonstrated the effect of promoting the proliferation of human dermal fibroblasts and increasing the expression of extracellular dermal matrix selected from collagen type 1, fibronectin, or elastin. Therefore, the peptide can be utilized as an active ingredient in pharmaceutical compositions for improving skin conditions.

[0040] The pharmaceutical composition comprises, but is not limited to, a pharmaceutically effective amount of the peptide and / or a pharmaceutically acceptable carrier.

[0041] As used herein, the term "pharmaceutical effective amount" means an amount sufficient to achieve the preventive or therapeutic efficacy of the pharmaceutical composition for a skin disease.

[0042] The pharmaceutically acceptable carriers mentioned above are those commonly used in formulation and include, but are not limited to, lactose, dextrose, sucrose, sorbitol, mannitol, starch, acacia gum, calcium phosphate, alginate, gelatin, calcium silicate, fine crystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methyl hydroxybenzoate, propyl hydroxybenzoate, talc, magnesium stearate, and mineral oil. Suitable pharmaceutically acceptable carriers and formulations are described in detail in Remington's Pharmaceutical Sciences (19th ed., 1995).

[0043] The weight ratio between the peptide and the pharmaceutically acceptable carrier is, for example, 500:1 to 1:500, and is not limited to, but may be 450:1 to 1:450, 400:1 to 1:400, 350:1 to 1:350, 300:1 to 1:300, 250:1 to 1:250, 200:1 to 1:200, 150:1 to 1:150, 100:1 to 1:100, 80:1 to 1:80, 60:1 to 1:60, 40:1 to 1:40, 20:1 to 1:20, 10:1 to 1:10, 8:1 to 1:8, 6:1 to 1:6, 4:1 to 1:4, or 2:1 to 1:2.

[0044] The aforementioned pharmaceutical composition may further include, but is not limited to, lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspension agents, preservatives, and the like, in addition to the aforementioned components.

[0045] The aforementioned pharmaceutical composition is administered orally or parenterally, preferably parenterally, and in the case of parenteral administration, by methods such as intramuscular injection, intravenous injection, subcutaneous injection, intraperitoneal injection, local administration, or transdermal administration, but is not limited to these methods.

[0046] The dosage of the pharmaceutical composition may be 0.0001 to 1000 μg (micrograms), 0.001 to 1000 μg, 0.01 to 1000 μg, 0.1 to 1000 μg, or 1.0 to 1000 μg per day, but is not limited to these, and can be prescribed in various ways depending on factors such as the formulation method, administration method, patient's age, weight, sex, medical condition, diet, administration time, route of administration, excretion rate, and response sensitivity.

[0047] The pharmaceutical composition may be manufactured in unit dose form or encapsulated in a multi-dose container by formulating it using pharmaceutically acceptable carriers and / or excipients in a manner readily carried out by a person with ordinary skill in the art to which the invention pertains.

[0048] The dosage form may be in the form of an oil or aqueous medium, such as a solution, suspension or emulsion, and may be in the form of an extract, powder, granules, tablet or capsule, and may further contain a dispersant and / or stabilizer.

[0049] Another embodiment provides a food composition for improving skin condition that contains a peptide consisting of the amino acid sequence of SEQ ID NO: 1 as an active ingredient.

[0050] As previously stated, any terms or elements mentioned in the description of the peptides that are identical to those already mentioned are as described above.

[0051] The content of the peptide as an active ingredient contained in the food composition can be appropriately and non-restrictively selected depending on the shape of the food, the desired use, etc., and can be added in an amount of 0.01 to 15% by weight of the total food weight. Also, for example, in a health drink composition, it can be added in a ratio of 0.02 to 10 g, preferably 0.3 to 1 g, based on 100 ml. [Effects of the Invention]

[0052] One aspect of peptides suggests that they can be applied to improve skin conditions, including wrinkle reduction, improved skin elasticity, and wound healing, by promoting the proliferation of fibroblasts (human dermal fibroblasts) or increasing the expression of extracellular matrix components such as collagen, fibronectin, or elastin. [Brief explanation of the drawing]

[0053] [Figure 1] This is the result of measuring the cell growth promoting effect of a peptide consisting of the amino acid sequence of Sequence ID No. 1 on fibroblasts. [Figure 2] This is the result of measuring the effect of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on increasing the expression of extracellular matrix cells in skin cells. [Modes for carrying out the invention]

[0054] The present invention will be described in more detail below through examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

[0055] Example 1: Peptide synthesis Using an automated peptide synthesizer (Milligen 9050, Millipore, USA), peptides having the amino acid sequence of SEQ ID NO: 1, as shown in Table 1 below, were synthesized. These synthesized peptides were then separated into pure forms using C18 reversed-phase high-performance liquid chromatography (HPLC) (Waters Associates, USA). The column used was ACQUITY UPLC BEH300 C18 (2.1 mm × 100 mm, 1.7 μm, Waters Co, USA).

[0056] [Table 1]

[0057] Example 2: Confirmation of fibroblast proliferation effect We aimed to confirm the fibroblast proliferation-promoting effect of a peptide consisting of the amino acid sequence of SEQ ID NO: 1. Specifically, we used 5 × 10⁶ human dermal fibroblasts. 3 Cells were seeded into 96-well plates at a cell / well density and cultured for 24 hours. After changing to serum-free mesenchymal stem cell medium, they were cultured for another 24 hours. The peptide was treated at concentrations of 10 μM, 50 μM, or 100 μM, respectively, and cultured at 37°C for 72 hours. A positive control group was treated with 50 nM of human recombined bFGF protein. After the culture was complete, the culture supernatant was removed and the cells were immobilized using ethanol. After washing with PBS, staining was carried out by treating with SRB solution. After washing with 1% acetic acid, the cells were observed under a microscope and destained by treating with 20 mM tris solution. Cell viability was measured by measuring the absorbance at a wavelength of 560 nm using a spectrophotometer (SpectraMax iD3, Molecular Devices (USA, CA)).

[0058] The results are shown in Figure 1. From the results in Figure 1, it can be confirmed that proliferation was induced in a concentration-dependent manner in the peptide-treated group, increasing up to a maximum of 1.62 times.

[0059] Example 3: Confirmation of the effect of increasing the expression of extracellular matrix in skin cells We aimed to confirm the effect of a peptide consisting of the amino acid sequence of SEQ ID NO: 1 on increasing the expression of the dermis extracellular matrix. Specifically, we used 3 × 10⁶ human dermal fibroblasts. 5 Cells were seeded into 6-well plates at a cell / well density and cultured for 24 hours. After changing to serum-free mesenchymal stem cell medium, they were cultured for another 24 hours. Peptides were treated at concentrations of 10 μM, 50 μM, or 100 μM, respectively, and cultured at 37°C for 48 hours. Human recombined bFGF protein was treated at 100 nM. After completion of culture, the culture supernatant was removed and fixed with 4% paraformaldehyde for 30 minutes. After washing with PBS, the cells were reacted with 0.5% Triton X-100 for 15 minutes. After reacting with 3% BSA for 1 hour, primary antibodies against collagen type 1, fibronectin, and elastin were diluted 1:100 and reacted overnight at 4°C. The secondary antibody-FITC was diluted to 1:500 and reacted at room temperature for 2 hours, after which DAPI staining and mounting were performed. The expression images of each marker were observed using a confocal microscope (Leica, Germany). The green fluorescence intensity was measured using ImageJ.

[0060] The results are shown in Figure 2. From the results in Figure 2, it can be confirmed that the expression of collagen type 1, fibronectin, and elastin, which are extracellular matrix components of the skin, increases upon peptide treatment. In the case of collagen, the expression increased by up to 1.32 times compared to the control group; in the case of fibronectin, it increased by up to 1.38 times; and in the case of elastin, it increased by up to 2 times.

[0061] Dosage Form Example 1: Production of Peptide Nanosomes 50 mg of the peptide from Example 1 was dissolved in 500 ml of distilled water by thorough stirring. The mixture was then mixed with 5 g of lecithin, 0.3 ml of sodium oleate, 50 ml of ethanol, and a small amount of oil. After adjusting the volume with distilled water to a total of 1 L, the mixture was emulsified using a microfluidizer under high pressure to produce peptide nanosomes with a size of approximately 100 nm.

[0062] Dosage form example 2. Softening lotion A softening lotion containing a peptide according to one embodiment and having the following composition was manufactured using a method known in the industry.

[0063] [Table 2]

[0064] Dosage form example 3: Nutritional cream A nutritional cream containing a peptide according to one embodiment and having the following composition was manufactured using a method known in the industry.

[0065] [Table 3]

[0066] Dosage form example 4. Nourishing lotion A nourishing lotion containing a peptide according to one embodiment and having the following composition was manufactured using a method known in the industry.

[0067] [Table 4]

[0068] Dosage Form Example 5: Essence An essence containing a peptide according to one embodiment and having the following composition was prepared using a method known in the industry.

[0069] [Table 5]

[0070] The above-mentioned description of the present invention is illustrative, and a person with ordinary skill in the art to which the present invention pertains will understand that it can be easily modified into other specific forms without altering the technical idea or essential features of the present invention. Therefore, the above-mentioned embodiments should be understood to be illustrative in all respects and not limiting.

Claims

1. A peptide consisting of the amino acid sequence of SEQ ID NO:

1.

2. The peptide according to claim 1, wherein the N-terminus of the peptide is bonded to one protecting group selected from the group consisting of an acetyl group, a fluoreonylmethoxycarbonyl group, a formyl group, a palmitoyl group, a myristyl group, a stearyl group, a butoxycarbonyl group, an allyloxycarbonyl group, and polyethylene glycol (PEG).

3. The C-terminus of the peptide is an amino group (-NH ２ ), tertiary alkyl group and azide (-NHNH) ２ The peptide according to claim 1, which is bonded to any one protecting group selected from the group consisting of ).

4. The peptide according to claim 1, wherein the peptide exhibits one or more of the following characteristics: (a) Promotion of human dermal fibroblast proliferation, (b) Increased expression of extracellular matrix selected from collagen type 1, fibronectin, or elastin.

5. A cosmetic composition for improving skin condition, comprising the peptide described in any one of claims 1 to 4 as an active ingredient.

6. The cosmetic composition according to claim 5, wherein the improvement of skin condition is wrinkle reduction, improvement of skin elasticity, and wound healing.

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